International Microbiology

CONTENTS International Microbiology (2016) 19:1-68 ISSN (print): 1139-6709. e-ISSN: 1618-1095 www.im.microbios.org

Volume 19, Number 1, March 2016 RESEARCH REVIEW

Núñez A, Amo de Paz G, Rastrojo A, García AM, Alcamí A, Gutiérrez-Bustillo AM, Moreno DA Monitoring of the airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios

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RESEARCH ARTICLES

Lara-Severino RC, Camacho-López MA, Casanova-González E, Gómez-Oliván LM, Sandoval-Trujillo AH, Isaac-Olivé K, Ramírez-Durán N Haloalkalitolerant Actinobacteria with capacity for anthracene degradation isolated from soils close to areas with oil activity in the State of Veracruz, Mexico

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Contreras-Cordero JF, Romo-Sáenz CI, Menchaca-Rodríguez GE, Infante-Ramírez R, Villarreal-Treviño L, Hernández-Luna CE, Rodríguez-Padilla C, Tamez-Guerra RS Genetic and serologic surveillance of rotavirus with P[8] and P[4] genotypes in feces from children in the city of Chihuahua, northern Mexico

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Devi A, Gunjilac AS, Wilkinson JM, Vanniasinkam T, Mahony TJ Prevalence of Campylobacter spp. in diarrhoea samples from patients in New South Wales, Australia

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Srivastava A, McMahon KD, Stepanauskas R, Grossart H-P De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany)

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Almarza O, Valderrama K, Ayala M, Segovia C, Santander J A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis

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Dec M, Puchalski A, Nowaczek A, Wernicki A Antimicrobial activity of Lactobacillus strains of chicken origin against bacterial pathogens

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PIONEERS IN MICROBIOLOGY: Paulina Beregoff (1902–1989), Colombia

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Journal Citations Reports 5-year Impact Factor of International Microbiology is 2,17.

The journal is covered in several leading abstracting and indexing databases, including the following ones: Agricultural & Environmental Bio­­technology Abstracts; ASFA/Aquatic Sciences & Fisheries Abstracts; BIOSIS; CAB Abstracts; Chemical Abstracts; SCOPUS; Current Contents/Agriculture, Biology & Environmental Sciences; EBSCO; EMBASE/Elsevier Bibliographic Databases; Food Science & Technology Abstracts; ICYT/CINDOC; IBECS/ BNCS; ISI Alerting Services; MEDLINE/Index Medicus; Latindex; MedBioWorld; PubMed; SciELO-Spain; Science Citation Index Expanded; SciSearch.

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Front cover legends century architecture. It is declared of Cultural Interest since 1962. [See article by Núñez et al. pp. 1-14 this issue.]

Upper left. Papillomavirus, the causal agent of several human diseases, some of them developing as cancers. Several Spanish groups perform outstanding research on this virus and on the illnesses that it causes. The definitive link between the presence of the papillomavirus and cervix cancer in women was established by Colombian physician and researcher Nubia Muñoz, in Lyon, France. (Magnification, 600,000×)

Center. Octagonal 42-m high dome of the Technical University of Madrid’s School of Industrial Engineering. The building, where the School has been located since 1907, was originally the Palace of Arts and Industry of Madrid, built in 1882–1886. The large building, which also holds the Spanish Natural History Museum, has a structure of iron, bricks and glass, and is an excellent example of the late 19th-

the left. Photo by Rubén Duro, CIM, Barcelona.

(Mag­­ni­fication, 3000×) Lower right. Macrophotograph of a growing colony of the mold Aspergillus sp. The colony is growing in a Petri dish. Note the whitish, button -like structure formed by a drop of liquid secreted by the sector on the left. Photo by Rubén Duro, CIM, Barcelona. (Magnification, 1.4×)

Upper right. Dark field micrograph of the cyanobacterium Chroococcus sp., isolated from a freshwater pond. Note the envelope surrounding the paired cells. Photo by Rubén Duro, Center for Microbiological Research (CIM), Barcelona. (Magnification, 1500×) Lower left. Dark field micrograph of the predator ciliate Pseudoprorodon sp., isolated from a freshwater lake. Note the pieces of food inside the large digestive vacuoles and the small ciliate being engulfed near the cytostome of the cell on

Back cover: Pioneers in Microbiology Paulina Beregoff (1902–1989), Colombia Paulina Beregoff was the first woman to obtain a degree in medicine in Colombia.

She was born in 1902 in Kiev—by then a city of the Russian Empire—, in an aristocratic family of Jewish descent. Due to the political situation in her country, she was educated in the United States, where, in 1921, she graduated in Bacteriology and Parasitology and Pharmacy and Chemistry at the University of Pennsylvania. She started working at the laboratory of Pathology of that university and became a member of the Rivas Bacteriological Society of the University of Pennsylvania. In 1922, the Dean of the School of Medicine of the University of Cartagena, Colombia, asked the University of Pennsylvania for an expert in tropical diseases, including yellow fever. This disease was a great concern in Cartagena due to the high mortality rates it caused and because of the implications on the image of the city, which was a major commercial and harbor center. The University needed a qualified advisor that could also train local physicians, and the University of Pennsylvania chose Beregoff for that task. Once in Cartagena, she had to identify an epidemic outbreak that had been causing many fatalities, mostly among indigenous peoples living in the Magdalena River shores. Colombian phys­ icians were not familiar with symptoms and causal agents of diseases such as yellow fever, typhoid fever and malaria, but thought that the epidemic outbreak could be due to one of them. Beregoff sent samples of cultures

from corpses of people killed by the disease to be analyzed at the University of Pennsylvania. The disease turned out to be fiebre tifomalárica and not simply malaria, as they first had considered. Beregoff thought that the infection depended mostly on the deficiencies or resistance of the immune system and proposed that physicians should work to prevent the disease. Once she had achieved her task, she intended to go back to Philadelphia to study medicine at Temple University, but she was asked to remain in Cartagena, where she could also study medicine. In 1922 she enrolled at the University of Cartagena under special conditions. Due to her previous studies and qualification, she could be waived the first two years of the studies of medicine. She set up the first laboratories of bacteriology and parasitology in Cartagena, with microscopes and other equipment donated by the University of Pennsylvania.

Her thesis director recognized her great contribution, she having been able to differentiate the various species of Laveran’s haematozoa, to observe the treponema causing yaws, to find the Piroplasma Donovani, the parasite of KalaAzar (visceral leishmaniasis) in the blood, and having been the first to isolate the “typhoid bacillus”, confirming thus the presence of typhoid fever in town.

She could also to properly perform the Wassermann technique on syphilis.

The fact that she was a foreign woman and the she had had some privileges in her medicine studies was criticized by some people. In 1933 she married bacteriologist Arthur Stanley Gillow and they moved to Canada. Since then she signed her publications as Pauline Beregoff-Gillow. After her husband’s death, in 1964, she returned to Colombia and dedicated his husband’s legacy to set up a foundation under his name that should work on preventive medicine. She died on September 20, 1989 and left her fortune to the foundation.

Front cover and back cover design by MBerlanga & RGuerrero

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RESEARCH REVIEW International Microbiology (2016) 19:1-13 doi:10.2436/20.1501.01.258. ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Monitoring of airborne biological particles in outdoor atmosphere. Part 1: Importance, variability and ratios Andrés Núñez,1 Guillermo Amo de Paz,2 Alberto Rastrojo,3 Ana M. García,1 Antonio Alcamí,3 A. Montserrat Gutiérrez-Bustillo,2 Diego A. Moreno1* 1 Higher Technical School of Industrial Engineering, Technical University of Madrid, Madrid, Spain. 2Departament of Plant Biology II, Faculty of Pharmacy, Complutense University of Madrid, Madrid, Spain. 3Center of Molecular Biology Severo Ochoa, CSIC-UAM, Madrid, Spain

Received 10 February 2016 · Accepted 10 March 2016 Summary. The first part of this review (“Monitoring of airborne biological particles in outdoor atmosphere. �������������� Part 1: Importance, variability and ratios”) describes the current knowledge on the major biological particles present in the air regarding their global distribution, concentrations, ratios and influence of meteorological factors in an attempt to provide a framework for monitoring their biodiversity and variability in such a singular environment as the atmosphere. Viruses, bacteria, fungi, pollen and fragments thereof are the most abundant microscopic biological particles in the air outdoors. Some of them can cause allergy and severe diseases in humans, other animals and plants, with the subsequent economic impact. Despite the harsh conditions, they can be found from land and sea surfaces to beyond the troposphere and have been proposed to play a role also in weather conditions and climate change by acting as nucleation particles and inducing water vapour condensation. In regards to their global distribution, marine environments act mostly as a source for bacteria while continents additionally provide fungal and pollen elements. Within terrestrial environments, their abundances and diversity seem to be influenced by the land-use type (rural, urban, coastal) and their particularities. Temporal variability has been observed for all these organisms, mostly triggered by global changes in temperature, relative humidity, et cetera. Local fluctuations in meteorological factors may also result in pronounced changes in the airbiota. Although biological particles can be transported several hundreds of meters from the original source, and even intercontinentally, the time and final distance travelled are strongly influenced by factors such as wind speed and direction. [Int Microbiol 2016; 19(1):1-13] Keywords: airborne biological particles · airbiota · bioaerosols · meteorological factors · air-genome ratios

Airborne biological par­ticles and their importance in the atmosphere According to Després et al. [26], bioaerosol particles can include viruses, bacteria, fungi and their spores, pollen, fragments of lichen, plants or invertebrates. In a recent study, Corresponding author: D.A. Moreno Escuela Técnica Superior de Ingenieros Industriales Universidad Politécnica de Madrid (ETSII-UPM) José Gutiérrez Abascal, 2 28006 Madrid, Spain Tel. +34-913363164. Fax +34-913363007 *

E-mail: diego.moreno@upm.es

Fröhlich-Nowoisky et al. [38] described also the diversity of airborne archaea, adding up new organisms to the list. The attention and impact of research on airborne microbiota have diversified and grown as the knowledge has improved. Initially focused on global human airborne diseases such as viral infections (influenza or meningitis), bacterial diseases (Legionnaires’ disease) and fungal infections (aspergillosis) [32], current studies have extended the importance of the biological aerosols to more particular situations. For instance, respiratory diseases developed by farmers and livestock workers have been related to inhalation of organic dust carrying particle-associated bacteria, archaea, fungi and viruses [7], with subsequent exposure to bioaerosols containing antibiotic re-

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sistant bacteria [3]. It has been also demonstrated that the exposure to high concentrations of certain species of airborne pollen and fungal propagules can cause allergy-related diseases including hay fever, asthma, rhinitis and atopic eczema to a significant percentage of citizens [4,92], affecting 18– 20% of the European population [9]. Thus, monitoring pollen and fungal spores in the air, as aerobiological networks of different countries around the world do (Fig. 1), is relevant for the diagnosis, treatment and prevention of these allergic diseases, whose prevalence is now three times higher than 30 years ago [105]. More recently, research has focused on health risks to pets and other animals inhabiting human environments that could potentially act as disease vectors [29].

In addition to her role in human health, bioaerosols have relevance in global economy too. The concern about crosspollination between genetically modified crops and natural plant varieties has led to develop procedures to track pollen particles [35]. Many airbone fungal species can cause severe plant and animal diseases resulting in large losses in agriculture and cattle raising; and porcine epidemic diarrhea and foot-and-mouth disease viruses, also transmitted by aerosols, are a major concern for animal farming [1,34]. Another interesting aspect related to the study of airborne microbiota is the biological deterioration or biodeterioration, triggered when certain microorganisms colonize the surfaces of materials such as those in monuments and buildings of important cultural heritage. The risks of biodeterioration are not restricted only to external stone surfaces in buildings such as cathedrals [85], but also to cave paintings [83], paper-based historical archives [8] and cinematographic films stock [106].

Recent studies have suggested that air is not merely a dissemination path for the biota (bacteria, viruses, fungi, spores and pollen) coming from other environments (soil, water or plant/animal microenvironments) but that it also has its particular communities of organisms (airbiota), so it should be considered as an ecosystem in itself [40,110]. The atmosphere can be seen as an extreme environment due to its chemical and physical characteristics, such as extremes of temperature (both low and high), solar irradiation (especially the UV part of the spectrum), desiccation, and the presence of strong chemical oxidants (e.g., ozone, hydroxyl and nitrate radicals), low nutrient availability and its large dispersing potential [39,40]. Under such conditions, the main activity in the airbiota ecosystem is expected to be bacteria-related. Because of their small sizes and ease of reproduction, bacteria can remain in the airspace for days or weeks, long enough to complete reproductive cycles. Accordingly, fog and cloud droplets have been proposed as an atmospheric niche for bacterial growth,

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providing nutrients as well as protection against desiccation and UV radiation [104]. However, the knowledge on the distribution of bacteria in the atmosphere is still limited in contrast to other environments to confirm this theory. For instance, Tamames et al. [101] studied the environmental distribution of prokaryotic taxa using 16S rDNA sequences from 3,502 sampling experiments in natural and artificial sources.

They identified selective environmental factors that could explain bacterial distribution in most cases: particular conditions in animal tissues and in thermal locations, or salinity were found to be major constraints on prokaryotic diversity, while soil and freshwater habitats would be far less restrictive environments. Unfortunately, data for air habitats in that study were inadequate due to insufficient sampling, and more studies are needed to draw any conclusion. Thus, so far, it is still unclear whether airborne bacteria are an actual ecological community or only a pool of organisms passively gathered together from different sources.

Last but not least is the ability of biological particles as agents to cloud condensation nuclei (CNN) and/or ice nuclei (IN). Nucleating particles are necessary to induce water vapour condensation to form liquid water (CCN) or to trigger crystallization of supercooled water (IN). For a long time, it was assumed that only inorganic particles such as those making up dust were responsible for CCN and IN in the atmosphere. However, in the 1970s the presence of biological IN was discovered.

Maki et al. [61] identified Pseudomonas syringae as a responsible agent for IN, and it has been used as model organism of atmospherically relevant IN active bacteria. The contribution of pollen, fungi, and bacteria to atmospheric CCN and IN has been studied in more detail over the last few decades. Some works have characterized bacteria isolated from rainwater using specialized and sterile devices. Joly et al. [53] found that 2.7% of cultivable strains were ice-active at ≤ –8 ºC, and ŠantlTemkiv et al. [86] observed that approximately 12% of cultivable bacteria caused ice formation at ≤ –7 ºC. These bacteria had probably been emitted to the atmosphere from vegetation or terrestrial surfaces, e.g., by convective transport. In both studies the main bacteria isolated were Pseudomonas spp., which is consistent with other reports from aerosols, clouds and fog [2,11]. Steiner et al. [100] and O’Sullivan et al. [70] have shown that small pollen particles (SPP) can contribute to CCN and IN, influenced by pollen concentrations and the number of SPP generated from a single pollen grain. With regards to fungi, Spracklen and Heald [99] found that fungal spores and bacteria contributed very little (0.003%) to global average immersion freezing IN rates, which were dominated by soot and dust. This findings concur with global modeling studies that found bio-

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Fig. 1. Air collector placed on the roof of the Higher Technical School of Industrial Engineering, Madrid. The octagonal dome, 42 m high, was built in 1881–1887, originally to host the Palace of Arts and Industry of Madrid. The School was located in the building in 1907. (See cover and page A2 of this issue.)

logical particles to be non-significant as a source of IN at global scale [51]. Even so, biological particles may be important at altitudes between 4 and 7 km, where the contribution of IN of biological origin becomes dominant at temperatures warmer than –15 ºC whereas mineral dust particles are typically considered to be the major contributor below this temperature [24,66].

Huffman et al. [52] and Schumacher et al. [89] suggested that bioaerosols might also play a major role in midlatitude semi-arid forest ecosystems, which is consistent with the observation that biogenic emissions significantly impact CCN in the region [58]. Accordingly, deforestation and changes in land use and biodiversity might have a significant influence on the abundance of IN, the microphysics and dynamic of clouds and precipitation in these regions, and thus on regional and global climate [25].

Thus, far from being airborne inert matter, biological particles have a relevant role in the atmosphere with consequences for health, economy and meteorology, although further studies are necessary to clarify their role.

Variability of biological communities in atmospheric aerosols Geographic patterns, global dispersion and spatial variability. We have a limited understanding of both how airborne communities vary across different geographical regions and what are the factors that determine their

patterns across large scales [110]. The nature of the biological particles found above terrestrial areas is likely to be different from that of biological particles found above oceans. For example, bioaerosols in marine environments can be expected to be rich in bacteria while, over land, they should be rich in pollen and fungi, in addition to bacteria. Oceans cover more than 70% of the global surface and have a bacterial concentration of 104 ml–1 in surface waters [16]. It is assumed that bacteria coming from this source, where sea spray aerosols are primarily formed at the air/sea interface through bubble-mediated processes, may have a major contribution [6]. The spatial distribution of bacterial populations in marine bioaerosol samples has been investigated using culture-independent techniques by Seifried et al. [90] and Xia et al. [112]. They have reported that the bacterial community in marine aerosols is more diverse than previously thought and the overall biological community is markedly different. Seifried et al. [90] have detected a strikingly abundance of Proteobacteria, which represent ca. 50%, with the genus Sphingomonas being the major representative. Xia et al. [112] have also suggested that the composition of the bacterial community would depend on whether the air masses came from the adjacent terrestrial areas or from marine environments. In agreement to this, different studies have concluded that terrestrial bioaerosol samples mainly contain Gram-positive and spore-forming bacteria, whereas marine bioaerosols comprise high abundances of Gram-negative bacteria and lower concentrations of fungal spores than land samples [37,44]. Continental environments

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have also their own particularities. According to Shaffer and Lighthart [91], bacterial concentrations in air samples from terrestrial regions are under the influence of the land-use, they having observed differences between forests, rural and coastal areas. In contrast, Bowers et al. [13] have found similar levels of bacteria independently of the land-use types when analyzing samples from agriculture fields, suburban areas and forest. However, their results indicate a wider diversity in rural areas than in cities.

Within metropolitan areas, bacterial concentrations have particularly high spatial variation because they are released form strong points sources in contrast to the more spatially homogeneous release in agriculture areas [26]. We will discuss the state-of-the-art of the knowledge about airborne biological particles in urban areas more extensively in the Part 2 of this review [69].

Some studies have been performed in remote places including Antarctica, such as the one carried out by Pearce et al.

[73] using culture-independent techniques. In accordance with the harsh conditions there, the results showed low bacterial biodiversity and many of the DNA sequences belonged to uncultivated microorganisms. Those that could be identified were associated to local origin (research station), but they also found bacteria from distant sources (either marine or terrestrial). These results highlight the strong influence of the meteorology to spread biological communities from their real source.

While airborne bioaerosols have been sampled from diverse locations around the world for many years, many questions remain about the nature of biological “pollution,” particularly on the topic of global dispersion and how far bioaerosols can travel from the points of origin [97]. Some aerobiological studies indicate that pollen can be transported up to 100–1000 km [88,98], and evidences for long-range transport of pollen and fungal spore bioaerosols have been also described by Mandrioli et al. [63].

A recent work of Prussin et al. [80] simulated the global transport of atmospheric particles, considering a scenario in which a small virus and a large fungal spore were released at the same time from the top of a 10-m tall building. According to their model, they concluded that the spore would be transported a horizontal distance of less than 150 m before settling to the ground, while the viral particle would be transported nearly 200,000 km. Although too simple, the study leads to remark two important ideas regarding biological particles in the atmosphere: (i) the potential for long-distance transport of very small particles, especially viruses; (ii) the distance transport can be significantly modified by different factors such as

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aggregation between different biological particles (and also with non-biological matter), adaptations for aerial transport (as some fungal spores or pollen have), and the influence of meteorological factors (wind speed, precipitation). Thus, it is difficult to interpret whether a particular airborne community is representative of the local area where it has been found or a combination of more distant sources.

Additional information can be found in a review of bacterial distribution in the global atmosphere published by Burrows et al. [16], revealing the complexity of the matter.

Abundance vs. altitude. Of all the different layers that form the atmosphere, it is in the troposphere (the lowest layer, reaching a maximum height of around 18–20 km above ground) where biological particles are to be found and where studies on aerobiology are carried out. Nonetheless, some microorganisms have been found also in the stratosphere (the second layer of Earth’s atmosphere, between 20 and 50 km above ground).

Within the troposphere, there is a first layer, known as the near-surface or lower atmosphere in which all of the aforementioned biological particles are found (i.e., viruses, fungi, bacteria, pollen, et cetera). Physics suggests that the concentration of these particles will gradually decrease towards zero as altitude increases, the largest ones, such as pollen grains, being the first to disappear. At higher altitudes, in the free troposphere, only smaller biological particles (such as fungi and bacteria and probably viruses) are expected to be found. However, there is no information about the point at which they shall no longer be found and note that pollen grains have been observed up to 2 km [68], supporting the influence of many factors, including atmospheric turbulences promoting vertical transport.

Human exposure to aeroallergens usually occurs at ground level. However, the stations of the aerobiological networks usually operate with samplers placed on the roof of buildings, around 10–30 m over ground level. Several studies, including those performed by Fernández-Rodríguez et al. [31] for pollen and Khattab and Levetin [57] for fungi have examined the abundances of these particles using spore traps set at ground level and rooftops at 12–16 m. Both authors conclude, and it is widely accepted, that pollen and fungal spore counts do not significantly differ between the two locations but some divergences have been observed when different pollen types or fungal spores are separately analyzed. In this regard, Hart et al. [50] analyzed three different heights (12, 24 and 34 m) and reported that the differences in the pollen abundance between the different levels were highly correlated with weather condi-

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tions, especially wind speed and rainfall. These results corroborate the hypothesis that recurring meteorological factors favor vertical exchange and long-range transport of pollen grains suggested by Mandrioli et al. [63]. Unfortunately, there is not enough information correlating pollen distribution in height and weather variables to set a conclusion.

For altitudes above towers, the use of light aircrafts (Fig. 2), air balloons and more recently unmanned aerial vehicles (UAVs), have been proposed. UAVs can fly below 1000 feet, altitude at which light aircrafts can be operated; hot air balloons have been used for some studies at higher altitudes. Fungal spores have been detected by culture and microscopy in air samples collected using UAVs at 25–45 m [103] and 320 m [87]. West and Kimber [107] recently reviewed the use of UAVs for agronomic studies for plant pathogens monitoring.

At altitudes of 10 km, DeLeon-Rodriguez et al. [22] have found, by quantitative PCR (qPCR), that bacterial abundance at this height (5.1 × 103 cells/m3) is at least two orders of magnitude greater than that of fungi, which suggests that particles with sizes similar to those of bacterial cells (0.1–3 µm) tend to stay longer in the atmosphere than larger particles, such as fungal cells and spores (typically >3 µm in diameter). These conclusions have been intensely questioned by Smith and

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Fig 2. (A) Airborne particle collector designed for the Program AIRBIOTA-CM to sample high volumes of air for metagenomic studies. This prototype can be attached to UAVs or any other vehicle as shown in the picture (wing of an airplane). (B) Agarose gel showing the results of the PCR to detect the DNA from different groups of organisms obtained with such collector after sampling in the specified locations during 1 h in 2015 summer session (unpublished data). Flights were conducted by Airestudio Geoinformation Technologies S.Coop and Ingeniería Medio Ambiental S.L. (IMA) in La Rioja and Murcia (Spain), respectively.

Griffin [94], and replied by DeLeon-Rodriguez et al. [23].

At altitudes of 20 km, at the interface between the troposphere and the stratosphere, Griffin [42] and Smith et al. [96] identified the presence of viable microorganisms by using culture techniques. They found bacteria of the genus Bacillus and the fungal genus Penicillium, which are common in terrestrial and aquatic environments. All of the isolates identified were spore-forming pigmented fungi or bacteria of terrestrial origin, which suggested that the presence of viable microorganisms in the Earth’s upper atmosphere might not be uncommon. In fact, spores can protect microorganisms from physical stresses such as UV radiation induced DNA damage, dryness, and extreme temperatures. Although both studies provide evidence for the long-distance, stratospheric level transport of microbes across the open oceans, this does not prove the existence of an independent airborne microbial ecosystem.

Lastly, Smith [93] reviewed the presence of microorganisms in the upper atmosphere (from 21 km to 77 km), noting the lack of publications about stratospheric microbiology missions, mainly due to the difficulties in obtaining sufficient biomass to study (morphology, culture or DNA analysis). He remarked that while astrobiology experiments in space are expensive and occasional, many fundamental questions about life in the uni-

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verse could be studied in our own atmosphere. At the very least, studying life in the limits of our atmosphere can provide a better comprehension about the diversity, distribution, and evolution of life on Earth. Furthermore, radiation-resistant cells found in the upper atmosphere could help us to identify genes or enzymes that provide such great endurance [93].

So far, most studies at different altitudes have been conducted using culture methods. The biases introduced by culturing have been well documented for other environments and ecosystems, but the limitations are even more pronounced with air samples since microbes can be damaged or inactivated by desiccation and irradiation during atmospheric transport [95]. In the next few years, next-generation sequencing will confirm the biodiversity and relative abundances of the different biological particles as a function of altitude, although new devices and procedures must be developed to face this challenge.

Meteorological factors As exposed above, air samples collected from different locations may differ with respect to the relative abundances of specific biological particles. Moreover, samples from a same location may vary significantly in abundances and biodiversity throughout a given season but also in a short time (days or even hours). These differences are probably caused by changes in regional and local meteorological factors such as temperature, wind speed, and rainfall. Temperature, set by global location and time of the year, is probably one of the most influential factors. An extensive review about the influence of meteorological factors on atmospheric bioaerosols was published by Jones and Harrison in 2004 [54]. Here, we review more recent publications, mostly based on DNA-sequence analyses, to extract some common conclusions.

Temporal variability. Many works have been conducted that studied the seasonal changes of bioaerosols in different locations, especially by analyzing bacterial diversity. In general, microorganisms are less abundant in the air during winter [5,12,14], which can be explained by the unfavorable weather conditions for bacterial growth and changes in the emission sources (plants, soil and water), as suggested by Bowers et al. [12,14]. Nevertheless, not only changes in bacterial abundances but also in diversity between seasons have been observed by Tanaka et al. [102] in a survey over oneyear period. Franzetti et al. [36] studied the bacterial communities associated with airborne particulate matter, finding

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large seasonal variations, with plant-associated bacteria dominating in summer and spore-forming bacteria in winter.

Those findings were supported by other authors including Brodie et al. [15], who suggested that warm temperatures might increase dehydration in soil/plant-related bacteria, inducing spore formation and their aerosolization.

Regarding fungal variability, Pashley et al. [72] studied by restriction fragment length polymorfism (RFLP) analyses the fungal variation in the atmosphere in three selected days representing dry and wet summer periods. They found differences not only in the fungal composition but also in the abundances of clones, which suggested a clear influence of the meteorology.

Oliveira et al. [71] conducted a 2-year study to analyze the influence of several environmental factors on fungal spores composition in the air in two cities of Portugal. They found higher abundances of spores in summer and autumn, with the lowest peak during winter months. Their results classified the fungal spores in three types based on their peak of abundance: summer (with Alternaria and Cladosporium as main representatives), spring-autumn, and late spring-early summer spores. Within the selected spore types under study, they concluded that summer spores correlated positively with temperature and negatively with relative humidity and rainfall. The opposite was determined for spring-autumn spores, and no correlations with meteorological factors were found for Aspergillus/Penicillum spore type. These results agree with the study performed by Grinn-Gofroń and Bosiacka [45], in Szczecin, Poland. They reported that air temperature was the most influential variable to explain the variation in airborne spore composition, the allergens Alternaria and Cladosporium being the most abundant at high temperatures, while there was a negative correlation with relative humidity. These examples show the complexity to study the influence of meteorological factors, since different fungi react inversely to variations of the same factor. Nonetheless, they clearly show the influence of environmental factors on fungal spore dispersion.

The most seasonal variation in biological particles, however, is probably represented by plants. Pollen spectra change dramatically over the year: while some pollen types appear, some others disappear completely because of different phenological stages. For instance, in Madrid (Spain), poplar and elm trees (genera Populus and Ulmus, respectively) have a peak of pollination in winter, and pollen grains of these two genera are not present in the regional atmosphere at a different times of the year [47]. Current studies about the influence of meteorological factors in pollen spreading usually focus on one particular pollen type, specially those with allergenic effects,

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concluding that the pollination calendar can extend or shorten depending on factors such as temperature and rainfall, and the abundances of pollen in a particular area are highly influenced by wind speed and direction [81,84].Changes in meteorological factors over the seasons correlate with fluctuations of several concomitant factors such as temperature, relative humidity, and solar radiation [54]. Consequently, the single influence of any of these factors on the airborne biological particle is difficult to analyze.

In addition to seasonal changes and fluctuations in shorter periods of time have been observed. Lighthart and Shaffer [60] suggested that the bacterial abundance could be split into five spans during the day, the lowest concentrations occurring before dawn and the highest during the morning. Diurnal variations were also observed by Fang et al. [30] in China, but with the opposite pattern, and Fierer et al. [33] described significant changes in bacterial and fungal relative abundances in a 10-day span. However, Polymenakou [76] and Womack et al. [110] concluded that, despite short-term fluctuations, the microbial composition tended to remain steady throughout the year.

The relationships between the different biological particles have been scarcely explored. Whon et al. [108] studied the presence of airborne viruses and bacteria over several months, always observing higher abundances of viruses. Bowers et al.

[10] studied, by culture-independent analyses, the relative abundance of bacteria, fungi and pollen associated to particulate matter over a year. They found that bacteria were always the dominant organisms in two different locations, with fungal relative abundances peaking during spring and summer months.

In addition, Fierer et al. [33] detected significant daily changes in bacterial and fungal abundances, inverting their relative importances. All these results were probably influenced by weather conditions and local sources of organisms. Therefore, additional studies are needed to reach a conclusion.

Wind as responsible for transport of biological particles and dust. Wind is the main factor responsible for releasing and transporting biological particles from terrestrial environments and also promotes the formation and spread of marine bioaerosols. Wind speed initially contributes to the release of spores and bacteria from the surfaces of plants and soil, and to their dispersion. The typical threshold wind speed required to remove biological particles from the ground (3.0–5.4 m/s) is greater than that necessary to remove them from plants (0.5–2.0 m/s) [21]. However, for the vast majority of fungi, there is no information about the strength of the spore attachment and the values of the threshold wind speed required [65].

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Local wind direction was largely responsible for changes in the concentrations of different airborne biological particles.

Di Giorgio et al. [27] described that bacterial concentrations increased with wind speed, and Rojo et al. [84] found that pollen spectra were governed by the location of pollen source and wind direction.

High wind speeds favor that marine bacteria are ejected into the air along with sea spray aerosol particles [67]. Moreover, it has been observed that the wind speed is related with the wind direction. Polymenakou and Mandalakis [77] identified higher numbers of marine bacteria-associated sequences when south winds crossed the inland of Crete, while the opposite was observed when north winds passed over the Aegean Sea. This discrepancy could be partly explained by the fact that north winds were blowing at very low speed (11 ± 6 km/h), which constrained the formation of sea-spray aerosol and the ejection of marine microbes from sea surface to the atmosphere. In a similar way, Fahlgren et al. [28] described, in a sea coast of Sweden, higher abundances of airborne bacteria in winter, contrary to other authors, who observed the opposite [5,12,14]. However, they explained that this controversial results might be due to the wind speed over the sea source regions (upwind of the sampling site), which was higher in winter than in summer in that particular location.

Consequently, aerobiological studies must take into account variables such as wind speed and direction, which can change the results and conclusions dramatically.

Strong winds over arid lands can lift dust above the boundary layer and transport it several kilometers or even more than 5000 km before settling it, depending on the particle characteristics (size, chemical composition) and the air-mass properties (e.g., velocity, density, height). The intercontinental transport of millions of tons of desert dust per year has been studied for decades, but research on the biological particles traveling between continents with the dust started recently (reviewed by Kellogg and Griffin [56]). Along with organic and inorganic nutrients bound to dust minerogenic particles, biological particles are mobilized from the arid soil and transported over long distances [44,78], and might include allergens and pathogens as well as pollutants. Therefore, transport of desert dust is believed to play a major role in many geochemical, climatological, environmental and health processes.

In general, most studies comparing dust events with nondusty events describe higher abundances of the biological particles and a greater diversity of the microbial community structure. Thus, Griffin et al. [43] found that the abundances of airborne microorganisms can be 2–3 times those found during “African dust-events” in the Caribbean, observing an

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order of magnitude increase in dust-associated viruses versus background. Correspondingly, concentrations of bacterial cells and mineral particles were ten-fold higher during Kosa (Asian dust) event than in non-Kosa event days [62].

Even Katra et al. [55] found higher numbers of bacterial and eukaryotic OTUs that were unique when the organismal diversities of dust from two storm events separated by 18 days in southern Israel were analyzed, also implying different origins of the biota.

Besides viruses and bacteria, dust storms can contribute with foreign fungal and pollen diversity. Because of their sizes, it is improbable that they are carried attached to dust particles; they must be dragged by the air mass and transported along with the storm. Similar to the results for bacteria, it is widely accepted that these events lead to an increase in fungal concentration and diversity [46,111]. Cariñanos et al. [18] also detected pollen from five non-native plants exclusively during dust events from North Africa to Spain via Saharan dust, which supported that not only microorganisms would be submitted to dust storm transportation.

The study conducted by Polymenakou et al. [75] found a correlation between particle sizes and microbial community structure during a dust storm in Crete, Greece, which suggests the existence of a preferable particle-selection by microorganisms to travel. Additionally, they observed that a large fraction of microorganisms at respiratory particle sizes (<3.3 µm) were phylogenetic neighbors to human pathogens.

Because of the character extremophile of the atmosphere, most bacteria may not survive during their long-range transports. In agreement, Hara and Zhang [49] found that the bacterial viability in long-range transported dust was less than 40%, whereas in non-dusty air it was more than 76%. Despite such differences, the final viable bacterial concentrations were comparable or even higher in long-range transported dust than in non-dusty air because of the great input. Note that they found a quantitative relation between coarse particles (diameter >1.0 µm) and viable bacterial cells for dust samples, which suggested a protective effect of the large particles towards the microbial community.

In parallel, Cao et al. [17] studied the microbiota associated to air pollutants PM2,5 and PM10 (particulate matter with diameters less than 2.5 and 10 µm, respectively) during a severe smog event in Beijing, China. They found that the relative abundances of bacteria, archaea, fungi and viruses seemed to rise with the increase in PM concentration. The authors suggested that this could represent a recently evolved transfer-mechanism supporting and increasing their natural dustmediated dispersion.

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Ratios of airborne biological particles and air genome Ratios of airborne biological particles. Ratios or proportions between different microorganisms are frequently used in environmental studies. The most common are the bacterial/fungal ratio (BFR) and the virus/bacterial ratio (VBR). The BFR for air samples has been estimated based on the microbial abundances of several works. Di Giorgio et al. [27] observed that BFRs were different in urban and natural areas. In the metropolitan spaces, BFR held steady at 4 during the whole year, while in the rural areas this value was around 2 from October to March (fall and winter) and ranged from 1 to 0.4 from March to October (spring and summer). However, Haas et al. [48] found that the levels of fungi in an urban environment (the city of Graz, Austria) changed from equal to higher than those of bacterial levels (BFR ranged from 1 to 0.06). Even a higher variation was described by Fierer et al. [33], with bacterial/fungal ratios that ranged from 8 to 0.08 throughout the five sampling days in the University of Colorado.

The BFR also varies with altitude and the studies performed at a height of 10 km by DeLeon-Rodriguez et al. [22] yielded results that implied that this altitude was much more favorable for bacteria (BFR = 29). Overall, BFRs for airborne samples seems to vary between 0.06 and 29, clearly influenced by location, altitude and seasonal factors. We calculated a fungi/pollen ratio (FPR) estimation of 100, indicating higher concentrations of fungal spores than of pollen grains in a boreal forest in Finland, based on data from Manninen et al. [64] .

The VBR is usually employed to describe the relative abundance between viruses and bacteria. By using fluorescence microscopy, Prussin et al. [79] determined a VBR value of 1.4 from outdoor air samples collected during September and October in (Blacksburg) Virginia (USA), indicating that ca. 40% more viruses than bacteria were present in the air.

Whon et al. [108] examined outdoor air in Korea and found an average VBR of 2.2 while Griffin et al. [43] observed a VBR of 1.3 in the Caribbean air. Considering that these values are not directly comparable because different methodologies were applied, and that the VBR in other environments can range dramatically from 0.2 (in the human gut) [82] to 2750 (in agricultural soil) [109], the VBRs obtained from air samples could be a very good approximation to the real value, although further research is required to confirm it.

DNA in the atmosphere, or air genome. Studies on the biodiversity and abundances of biological particles in the atmosphere have been hampered by the low concentra-

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Table 1. Estimation of air volumes required for air genome studies Biological abundance in the outdoor atmosphere

Genome size (Mb)

DNA concentration (pg/m3)

Air required (m3) to sample for 1 ng DNA

Pollen

10–104 grains/m3 (Gutiérrez-Bustillo et al., 2001) [47]

5.8 × 102–1.2 × 105 (Gregory et al., 2007) [41]

5.8–1.2 × 106

8.3 × 10–4 –1.7 × 102

Fungi

103 –104 spores/m3 (Codina et al., 2008) [20]

9.0–8.1 × 102 (Gregory et al., 2007) [41]

9.0–8.1 × 103

0.12–1.1 × 102

Bacteria

104–106 cells/m3 (Lighthart, 2000) [59]

0.58–10 (Claverie et al., 2006) [19]

5.8–1.0 × 104

0.10–1.7 × 102

Viruses

1.7 × 106–4.0 × 107 VPLs/m3 (Whon et al., 2012) [108]

0.002–2.5 (Whon et al., 2012) [108] (Philippe et al., 2013) [74]

3.4–1.0 × 105

0.01–2.9 × 102

tions of biological particles per m3 of air, especially for culture-dependent methods. Among the main airborne biological particles, i.e. pollen, fungi, bacteria and viruses, the latter are the most difficult to study because (a) their sizes are orders of magnitude smaller than those of the other airborne biological particles, and (b) they are obligate intracellular parasites and therefore the use of culture-dependent techniques are limited by our knowledge about the host of each specific virus—the viability of the viruses during collection turns a major issue.

In the case of a metagenomic approach, viruses do not share a common marker gene, such as 16S ribosomal DNA in bacteria, or internal transcribed spacer 2 (ITS2) in fungi. So, a “shotgun” sequencing procedure is required to study viral community structure. Additionally, the extraction efficiency of nucleic acid from viral particles is usually low, making it even more difficult to obtain enough DNA. Former NGS technologies required 1–5 µg of DNA to prepare a shotgun-sequencing library, while current kits have reduced remarkedly this amount to ca. 1 ng of viral genomic material [80].

Assuming high concentrations of airborne viruses such as those found by Whon et al. [108] in Korea, (1.7 × 106– 4.0 × 107 viruses/m3), and according to our estimations (Table 1), it would be necessary to sample 290 m3 of air to recover 1 ng of DNA to perform a viral metagenomic study.

Obviously, it is possible to find lower concentration values in air than those listed in Table 1 due to meteorological factors, seasonal variations, height, location, et cetera. In these cases, sampling larger volumes of air would be required to obtain enough DNA but fast advances in sequencing platforms lead to require less and less genetic material for the analyses.

Conclusions Outdoor air contains plenty of biological particles from different sources, mostly bacteria, fungi and spores thereof, pollen

and viruses. Some of these organisms, or just parts of them, can cause allergies, disorders and diseases in humans and other animals, with important health and economical consequences. They have been also suggested to influence weather conditions and have a role in climate change. Therefore, monitoring and broadening our knowledge about their distribution and global and local patterns should be a priority. It is a complex matter and so are the challenges to face. Oceans, forests, urban spaces, et cetera have different point sources of bacteria, fungi, pollen and viruses, which vary substantially from one environment to another, not only in concentration but also in the diversity of each group of organisms. In addition, meteorological factors such as temperature or wind affect dramatically to their air transport, and they may undertake major temporal changes. As a consequence, sampling organization is crucial to obtain valid representative results.

Ideally, all the biological particles should be analyzed together. So far, most studies analyzing the diversity and/or distribution of airborne biological particles focus on one only type of them, e.g. bacteria, fungi or pollen, separately. Works considering two or more usually provide ratio information (VBR, BFR, FPR), which is a good advance to consider the atmosphere as a particular ecosystem as has been proposed by some authors. The limitation to confront an integral study of all the biological particles has been set mainly by methodology. Traditional methods in aerobiology and microbiology applied to the study of air samples are based on microscopy and culture-dependent techniques; they are time-consuming, require great expertise for visual identification, and the bias of culturing must be assumed. Moreover, the low concentrations of airborne biological particles require the analysis of high volumes of air, which is limited by the samplers, time and the collection surface. Nevertheless, new tools and devices, such as the above mentioned UAVs, offer new alternatives.

Thus, metagenomics emerges as a promising solution for

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many of these difficulties. The development of DNA sequencing technologies has made it possible to detect all the organisms in a sample independently of their viability. The great sensitivity of such techniques allow to detect even minor representatives in the samples. The application of high-throughput DNA sequencing to study airborne biological particles will be addressed in Part 2 of this review [69].

Acknowledgements. This study was funded by the Community of Madrid, Spain, under the AIRBIOTA-CM Program (S2013/MAE-2874).

Competing interests. None declared.

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RESEARCH ARTICLE International Microbiology (2016) 19:15-26 doi:10.2436/20.1501.01.259 ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Haloalkalitolerant Actinobacteria with capacity for anthracene degradation isolated from soils close to areas with oil activity in the State of Veracruz, Mexico Reyna del C. Lara-Severino,1 Miguel A. Camacho-López,1 Edgar Casanova-González,2 Leobardo M. Gómez-Oliván,3 Ángel H. Sandoval-Trujillo,4 Keila Isaac-Olivé,1 Ninfa Ramírez-Durán1* Faculty of Medicine, Autonomous University of the State of Mexico, Toluca, Mexico. 2National Coordination of the de Preservation of Cultural Heritage, National Institute of Anthropology and History, Mexico DF, Mexico. 3Faculty of Chemistry, Autonomous University of the State of Mexico, Toluca, Mexico. 4Department of Biological Systems, Autonomous University Metropolitana-Xochimilco, Mexico DF, Mexico

1

Received 28 January 2016 · Accepted 3 March 2016

Summary. The use of native strains of microorganisms from soils is an excellent option for bioremediation. To our knowledge, until now there has been no other group working on the isolation of Actinobacteria from contaminated soils in Mexico. In this study, samples of soils close to areas with oil activity in the State of Veracruz, Mexico, were inoculated for the isolation of Actinobacteria. The strains isolated were characterized morphologically, and the concentrations of NaCl and pH were determined for optimal growth. Strain selection was performed by the detection of a phylogenetic marker for Actinobacteria located at the 23S rRNA gene, followed by species identification by sequencing the 16S rRNA gene. Several haloalkalitolerant Actinobacteria were isolated and identified as: Kocuria rosea, K. palustris, Microbacterium testaceum, Nocardia farcinica and Cellu­ lomonas denverensis. Except for C. denverensis, the biomass of all strains increased in the presence of anthracene. The strains capacity to metabolize anthracene (at 48 h), determined by fluorescence emission, was in the range of 46–54%. During this time, dihydroxy aromatic compounds formed, characterized by attenuated total reflectance Fourier transform infrared spectroscopy bands of 1205 cm–1 and 1217 cm–1. Those Actinobacteria are potentially useful for the bioremediation of saline and alkaline environments contaminated with polycyclic aromatic hydrocarbon compounds. [Int Microbiol 2016; 19(1):15-26] Keywords: Kocuria · Microbacterium · haloalkalitolerant Actinobacteria · anthracene degradation · State of Veracruz, Mexico

Introduction Contamination by crude oil spills is among the most concerning environmental problems worldwide. Oil is a complex Corresponding author: N. Ramírez-Durán Facultad de Medicina Universidad Autónoma del Estado de México Paseo Tollocan esquina Jesús Carranza, s/n 50180 Toluca, México Tel. & Fax +52-7222173552 *

E-mail: ninfard@hotmail.com

mixture of hydrocarbons and related compounds classified as aliphatics, asphaltenes and polycyclic aromatic hydrocarbons (PAH) [39]. PAHs represent more than 10% of organic compounds in oil [52], and have been found to be widely distributed in the atmosphere because of their moderate vapor pressure, low solubility and low reactivity [22]. These hydrophobic organic compounds have two or more linear, angular or branched benzene rings [30]. Those with three or more rings tend to be strongly adsorbed to the soil [32].

Anthracene is a 3-ring PAH, which is among the priority environmental pollutants by the US Environmental Protection

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Agency (EPA). According to EPA, there is insufficient information to classify the anthracene as a substance that causes cancer [http://www.epa.gov/osw/hazard/wastemin/priority. htm] but the fact that its structure resembles to carcinogenic PAHs such as benzo[a]pyrene and benzo[a]anthracene arises concern [37]. Therefore, anthracene is usually used as a model PAH in studies of degradation [5,19,31]. During the last decades, bacteria capable of degrading several hydrocarbons, including anthracene, naphthalene, phenanthrene and/or pyrene, have been isolated from PAH-contaminated soils. Most of these bacteria belong to the genera Burkholderia [44], Di­ etzia [2] and Sphingomonas [8].

Depending on the physiological conditions regarding salinity and pH required for the growth of microorganisms, they are classified as halophile, halotolerant, alkaliphile or alkalitolerant. With respect to salinity, halophile microorganisms are those that require NaCl for living and growth [34]. Halotolerant microorganisms can grow in media with NaCl from concentrations as low as zero up to as high as 25% [20]. With respect to pH tolerance, a microorganism is classified as alkaliphile or alkalitolerant when it grows at pH values higher than 9 [17]. The classification of haloalkaliphile is given to microorganisms that require both NaCl (in concentrations up to 30%) and an alkaline pH (pH 9) for growth [17]. In this work, we call haloalkalitolerant to those microorganisms that can live and grow either in the absence or the presence of salt; they can even tolerate high NaCl concentrations (up to 25%) and grow optimally in a wide range of pH (8–9, or higher).

Due to the salinity and pH ranges tolerated by haloalkalitolerant microorganisms, they are a good prospect for the bioremediation of soils contaminated with PAH. Among bacteria, Actinobacteria can produce several extracellular enzymes that metabolize various complex organic compounds, and also produce biosurfactants [13,35]. These two characteristics make them good candidates for the bioremediation of sites contaminated with organic pollutants [13].

Several Actinobacteria, such as Kocuria rosea, K. flava, Mi­ crobacterium marinilacus, Nocardia pneumoniae and Cellu­ lomonas bogoriaensis, have been isolated from soils and water contaminated with oil in Pakistan [1] and Kuwait [2], and their capacity to degrade naphthalene, phenanthrene and/or fluoranthene has been demonstrated.

The bioremediation of PAH-contaminated environments has been reported with native halotolerant or alkalitolerant Actinobacteria, as shown by studies performed with Arthro­ bacter crystallopoietes and Arthrobacter arilaitensis [36] and Micrococcus sp., Dietzia sp. and Rhodococcus sp. [14]. However, there are no reports about the use of Actinobacteria clas-

LARA-SEVERINO ET AL.

sified as haloalkalitolerant able to grow in polluted environments such as those affected by PAHs. Because PAHs have a relatively high quantum yield, fluorescence spectroscopy has been successfully used to monitor the processes of biodegradation of PAHs dissolved in aqueous solutions [54].

Mexico has several areas with oil activity. Therefore, it is expected that (i) the soils close to these areas are contaminated with oil and its derivatives, and (ii) some of the physicochemical conditions of these soils are usually found in contaminated environments. One option for bioremediation could be the use of local microbial strains adapted to live under these conditions, such as haloalkalitolerant Actinobacteria. To our knowledge, however, there have been no previous reports related to the isolation and characterization of such microorganisms from soils close to areas with oil activity, and we have published a preliminary study on the comparison based on sensitivity, linearity, and detection limits of the excitation, emission, and synchronous fluorescence methods, during the quantification of the residual anthracene concentration from the haloalkalitolerant Actinobacteria cultures [21]. The aim of this work was to isolate and genetically identify haloalkalitolerant Actinobacteria strains from soils close to areas with oil activity, and to assess their capacity to degrade PAHs by using the anthracene model and fluorescence emission.

Materials and methods Sample collection. A random sampling was performed on soils close to 4 areas with oil activity in the State of Veracruz, Mexico (Fig. 1). Soil samples were taken from the surface, at a maximum depth of 15 cm, maintaining sterile conditions, placed in sterile polyethylene bags and transported to the lab under refrigeration conditions. Major oil activities close to the sampling area included oil drilling, extraction of liquids, refining and petrochemical production. Facilities where oil activities are carried out have risks of leaking oil, diesel and gasoline either by pipeline rupture, or oily water filtration.

These activities potentially contaminate the soil mainly by discharges and spills of oily water or flooding in the rainy season.

Physicochemical characteristics of the soil samples. Soil samples were separately placed on aluminum plates and dried in an oven at 40 ºC (Scientific, H-71) for 7 days. The lumps present were grounded in a porcelain mortar and in a pestle and passed through a 2-mm-pore sieve to standardize the particle sizes. Physicochemical variables measured for each sample were NaCl concentration and pH. One gram of dried soil was placed in an assay tube, 9 ml of distilled water was added, and the sample was agitated in a vortex for 10 min and filtered overnight through Whatman 2 filter paper. Both NaCl concentration and pH were measured from the filtered solution using a refractometer (Hann, HI931100) and a pH-meter (Hanna, HI98128), respectively.

Culture media. Six cultures recommended for the isolation of halophilic bacteria and Actinobacteria were initially used: culture media for moderate halophilic microorganisms (MH) [38], SAUTON-UAM [42], Czapek agar,

ANTHRACENE DEGRADATION BY ACTINOBACTERIA

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Fig. 1. Two locations near the area with oil activity in the State of Veracruz, Mexico where the samples for the isolation of the strains included in this study were obtained. soil extract agar [16], NZ amine A [28], GA agar [33], and yeast and malt extract agar (YME) [45]. MH medium was used as reported by Quesada et al.

[38]: 10 g/l yeast extract, 5 g/l proteose peptone; 1 g/l glucose, 18 g/l agar; 10% NaCl, pH 7. This medium was also adjusted to pH 10 before use. Other two variations were to replace 10% NaCl for 3% NaCl and to adjust the pH to either 7 or 10. YME agar was used as reported by Shirling and Gottlieb [45]: 4 g/l yeast extract; 10 g/l malt extract; 4 g/l glucose; 20 g/l agar; pH 7.

This medium was also used with 10% NaCl added and after adjusting the pH to 7. All media were sterilized in an autoclave at 121 ºC for 15 min, cooled to 45 ºC and poured in petri dishes.

Isolation, purification and morphological characterization of Actinobacteria. One gram of soil from each sample was weighed and placed individually in assay tubes, after which 9 ml of 10% NaCl solution was added. The mixture was agitated in a vortex for 30 s and subsequently diluted until obtaining a final concentration of 8-orders lower than the initial concentration. A 200-µl aliquot of this dilution was inoculated in a series of 6 petri dishes containing the culture media previously described. The mixture was homogenized until dried, and the culture media were incubated at 37 ºC for 7 days. The selection of strains was performed according to the morphological characteristics reported for Actinobacteria in the Bergey’s Manual [4].

The strains selected were purified in the culture medium from which they were isolated. The colonies were described according to their size, color, shape, texture, aspect, height, the presence of vegetative or aerial mycelium and diffusible pigment in the culture medium. To confirm the purity of the strains, Gram staining was performed. The stained strains were observed under a microscope (Leica 5605) to identify the presence of filamentous, branched, coccoid, Gram-positive cells.

Genetic identification. DNA extraction was performed according to the protocol of the Promega Wizard Genomic DNA Purification kit (Promega, A1120). A phylogenetic marker, 250/350 base pairs (bp) in length, located in the 23S rRNA gene was amplified from the isolated strains using polymerase chain reaction PCR. The sequences of the primers used were as follows: 23 insF: 5′-(AC)AGCGTAG(AGCT)CGA(AT)GG-3′ and 23S insR: 5′-GTG(AT)CGGTTT(AGCT)(GCT)GGTA-3′ [40]. The reaction was performed using commercial Taq DNA polymerase (Dongsheng Biotech, P1082). PCR amplification of the 16S rRNA gene was performed on strains

that showed amplification of the 350-bp phylogenetic marker located in the 23S rRNA gene. For this amplification, the sequences of the primers used were as follows: 8f: 5′-AGAGTTTGATCMTGGCTCAG5′- and 1492r: 5′-TACGGYTACCTTGTTACGACTT5′. The reaction was performed using commercial Taq DNA polymerase (Dongsheng Biotech, P1082). The amplified fragments of the 16S rRNA gene were filtered using PCR Amicon Ultra 0.5 ml purification equipment (Merck Millipore, UFC503096) following the methodology provided by the manufacturer. The amplified products were sent to a sequencing service (Macrogen, USA), and the sequences obtained were checked and corrected. Consensus sequences were built from the forward and reverse fragments using BioEdit software version 7.0.9 [15]. To determine the percentage of similarity of the consensus sequences, they were compared with sequences already deposited in the GenBank database (National Center for Biotechnology Information–NCBI) using Basic Local Alignment Search Tool (BLAST) [3].

Determination of the concentration of NaCl and pH value for the optimal growth of the isolated strains (physiological characterization). The range and optimal concentration of NaCl for the growth of purified strains were determined by inoculating each strain in the medium from which they were isolated. The media were adjusted to the following NaCl concentrations: 0, 0.5, 3, 5, 10, 15, 20, 25 and 30% at pH 7.0.

The inoculum was streaked and incubated at 37 ºC. The plates were checked for growth every day for 10 days. The liquid medium, from which the strain was isolated, with the corresponding NaCl concentration previously determined for optimal growth, was used for pH determinations. The pH was adjusted to values of 5, 6, 7, 8, 9, 10, 11 and 12. The strain was inoculated from solid medium in the culture medium, and the flasks were incubated at 37 ºC. Microbial growth was determined by measuring the optical density at 600 nm [11] in a spectrophotometer (Perkin-Elmer UV-Vis, model 551S). Readings were taken at 0 h and subsequently every 24 h for 10 days. The group of pH values in which an optical density equal to or higher than 0.2 was obtained was established as the pH range for growth. The pH value at which the highest optical density was obtained was considered as the optimal pH for growth.

Phylogenetic analysis. Sequences from well-known collections, such as the Deutsche Sammlung von Mikroorganismen (DSM), the American Type Culture Collection (ATCC), the Biological Resource Center of the Na-

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tional Institute of Technology and Evaluation (NBRC), and the Centers for Disease Control and Prevention (CDC), were obtained, corresponding to the species that showed the highest percentage of similarity. The consensus sequences and sequences from GenBank collections DSM, ATTC, NBRC and DCD were aligned using BioEdit Sequence Alignment Editor version 7.0.9 [15]. The phylogenetic analysis was performed with the maximum parsimony method using Molecular Evolutionary Genetics Analysis (MEGA) software version 5.2 [49]. The bootstrap was calculated with 1000 repetitions.

(The nucleotide sequence data reported are available in GenBank database under the accession numbers from KP100512 to KP100519.)

Growth kinetics in Czapeck and minimum salts media with anthracene. To characterize the tolerance of the isolated strains to growth in the presence of anthracene, all strains were cultivated in two Czapeck media containing 0.01% of glucose as growth starter and 100 mg/L or 200 mg/L of anthracene respectively. Czapeck media were supplemented with 0.5%, 3% or 10% NaCl for allowing the optimal growth of the strains.

Strains M2C, M3H, M3I and M5B were inoculated in this medium supplemented with 0.5% NaCl. Strains M1B, M4A and M10A were inoculated in the medium supplemented with 3% NaCl, and strain M11A was inoculated in the medium supplemented with 10% NaCl. In all cases, pH was adjusted at 8. Each solution was sterilized in an autoclave at 121 ºC for 15 min. The inoculated flasks were maintained in an incubator with agitation at 150 rpm in the dark. Microbial growth was determined by measuring optical density at 600 nm [11] using a spectrophotometer. The capacity of each strain to use anthracene was related to the increase in the medium’s turbidity.

Once the exponential phase of the growth in Czapeck was determined, a volume corresponding to an OD600 of 0.25 at the 50% of this phase was taken and inoculated in two minimum salt medium, one without anthracene and another with 1 µg/ml of anthracene respectively, both supplemented with NaCl (as described for Czapeck medium). As in the previous cases, the inoculated flasks were maintained in an incubator with agitation at 150 rpm in the dark. Microbial growth was determined by measuring the OD600 [11] using a spectrophotometer. The capacity of each strain to use anthracene was related to the increase of the medium’s turbidity.

The composition of the minimal salts medium (MSM) [43] was: anthracene, 0 or 1 µg/ml; (NH4)2SO4, 1000 mg/l; Na2HPO4, 800 mg/l; K2HPO4, 200 mg/l; MgSO4∙7H2O, 200 mg/l; CaCl2∙2H2O, 200 mg/l; FeCl3∙H2O, 5 mg/l; (NH4)6Mo7O24∙H2O, 0.5 mg/l. This MSM was supplemented with NaCl in three different variants, adding 0.5%, 3% and 10% NaCl, respectively. In all cases the pH was adjusted to 8. Each solution was sterilized in an autoclave at 121 ºC for 15 min.

Analysis of the concentration of anthracene by fluorescence emission and identification of functional groups by ATR-FTIR. Primary inocula were prepared by transferring the strain from solid medium into the corresponding flasks containing 60 ml of MSM with 1 mg/ml of anthracene and the percentage of NaCl and pH required for their growth, as described previously. Screw-cap flasks were covered with aluminum foil and were maintained in an incubator with agitation at 150 rpm in the dark until reaching half the exponential growth phase. The exponential growth phase was already known from the kinetics growth curves.

At the time corresponding to half of the exponential growth phase from each primary inoculum, the volume corresponding to an OD600 of 0.25 was transferred to 60 ml of MSM with 270 ng/ml of anthracene and the required percentage of NaCl and pH as previously indicated. This optical density corresponds to 0.5 nephelometric turbidity units (NTU). The flasks were incubated in the dark at 150 rpm. Five-ml aliquots of each culture were taken at 0, 1, 24 and 48 h and were centrifuged at 10,000 rpm for 5 min to separate biomass. The concentration of anthracene was measured in the supernatant by fluorescence emission. In order to identify the functional groups of the of anthracene degradation, the infrared spectrum by attenuated total reflec-

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tance–Fourier transform infrared spectroscopy (ATR-FTIR) of the supernatant was recorded. The strains were also inoculated in the MSM medium supplemented with the required NaCl concentration for optimal growth without anthracene in order to verify that metabolites that could interfere with the anthracene signal had not been produced during the incubation time of cultures. These control cultures were analyzed in the same manner as the culture samples.

The fluorescence emission spectra of the supernatants were read in a spectrofluorometer (Horiba, Fluoromax-3). In all cases, the recording conditions of the spectra were as follows: excitation wavelength of 340 nm, 2-nm resolution, integration time of 0.5 s and 2-nm slits. An emission signal of 401 nm was used for strains M1B, M10A, M2C, M5B, M3H and M3I; for strain M11A, the emission signal was 419 nm. Infrared spectra were obtained in an ATR-FTIR spectrometer (Brucker, Alpha) equipped with a diamond ATR module for individual reflection (Platinum ATR single reflection diamond ATR module) in a range of 500–4000 cm–1, with a resolution of 4 cm–1. Each spectrum was obtained from 5 µl of supernatant and is the mean of 24 scans.

All assays of fluorescence emission and ATR-FTIR were repeated 5 times.

Statistical analysis. The residual anthracene concentrations in the media inoculated with microorganisms were statistically compared by a bivariate analysis. The two studied variables were strains (7 levels) and time (3 levels). Each experiment was performed five times (five independent inoculations for each strain).

Results Isolation and characterization of Actinobacteria.

The eleven soil samples analyzed contained from 1 to 3% NaCl and their pH ranged from 7.8 to 8.6. No other analyses of the samples collected were conducted.

From the six culture media employed, only MH and YME allowed the isolation of Actinobacteria and eubacteria, with no growth of fungi or yeasts, indicating that the nutrients present in these media, the NaCl concentration and the pH were appropriate. From the isolated strains, eight were Actinobacteria. These strains were identified as M1B, M10A, M2C, M3H, M3I, M5B, M4A and M11A. From MH medium at pH 7 supplemented with 3% NaCl, strains M1B, M10A, M4A, M2C and M5B were isolated. From MH medium at pH 7 and supplemented with 10% NaCl, M11A was isolated, and from YME medium at pH 7 with 0% NaCl, M3H and M3I were isolated. The eight isolated strains formed small or medium border-raised, soft colonies, which were cream or yellow and in some cases orange; circular or irregular; opaque or brilliant; with vegetative mycelium; and without pigment diffused into the medium. Under the microscope, they could be seen as unique cocci and in tetrads, small bacilli, or branched filamentous cells. They were Gram-positive.

Genetic and physiological identification. Eight strains showed amplification of a 350‑bp fragment, indicating

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Table 1. Identification and physiological characterization of the eight strains of Actinobacteria isolated in this work. All of them are haloalkalitolerant and have an optimal growth at pH of 8. Major similitude with known strains (DSM or ATCC) is shown as percentage (%) Strain

bp of the amplified fragment

Species

Strains with higher similitude

%

g/100 ml NaCl range

g/100 ml NaCl for optimal growth

pH range

M1B

1360

Kocuria rosea

DSM 20447

99.0

0‒10

3

5‒11

M11A

1415

Kocuria palustris

DSM 11925

98.0

0‒20

10

5‒12

M10A

1372

Microbacterium testaceum

DSM 20166

98.0

0‒10

3

5‒11

M2C

1350

Nocardia farcinica

ATCC 3318/DSM 43665

99.0

0‒5

0.5

5‒10

M3H

1356

Nocardia farcinica

ATCC 3318/DSM 43665

99.0

0‒5

0.5

5‒10

M3I

1325

Nocardia farcinica

ATCC 3318/DSM 43665

99.0

0‒5

0.5

5‒10

M5B

1363

Nocardia farcinica

ATCC 3318/DSM 43665

98.0

0‒5

0.5

5‒10

M4A

1355

Cellulomonas denverensis

DSM 15764

99.0

0‒3

3

5‒10

that they were Gram-positive bacteria with high G+C content.

The comparative analysis of 16S rRNA sequencing resulted in the identification of 2 strains belonging to the Kocuria genus, of which, strain M1B was similar to the Kocuria rosea species and strain M11A to Kocuria palustris. Strain M10A was identified as Microbacterium testaceum. Strains M2C, M3H, M3I and M5B were identified as Nocardia farcinica.

Strain M4A was identified as Cellulomonas denverensis. The similarity percentages obtained from BLAST analysis are shown in Table 1. The table also shows the % NaCl range, the % NaCl for optimal growth, and the pH range.

All strains were capable of growing within a range of NaCl concentrations which was as narrow as 0–3% up to as wide as 0–20%. In addition, they could also grow within a wide range of pH. Therefore all these eight Actinobacteria species were classified as haloalkalitolerant. Within the haloalkalitolerant category, they were classified, according to Russell [41], as low haloalkalitolerant (M2C, M3H, M3I, M5B and M4A), moderate haloalkalitolerant (M1B and M10A) and extreme haloalkalitolerant (M11A).

Phylogenetic analysis. The phylogenetic tree shows the formation of solid groups for genera and species (Fig. 2).

The locations of the strains identified inside of the groups confirmed their identification and phylogenetic relationships, meaning that changes over time in their nucleotide sequences have maintained them in their corresponding genera.

Growth kinetics in MSM with anthracene: Figure 3 shows the growth over time of K. rosea, K. palustris, M. tes­ taceum, N. farcinica and C. denverensis in Czapeck medium containing 100 and 200 mg/ml (Fig. 3A,B), as well as in MSM

without and with anthracene (1 µg/ml) (Fig. 3C,D). Except for C. denverensis, all strains could grow in these culture media.

The growth of the strains in the Czapeck medium with anthracene was accelerated, the stationary phase was reached between 1 and 1.5 h with 100 and 200 mg/l of anthracene, the stationary phase was stable up to at least 48 h. The bacterial population (log DO/DO0) increased slightly with anthracene (200 mg/l), showing that anthracene is tolerated and utilized by the bacteria. To confirm quantitatively this result, the fluorescence experiment was designed (see results below). Czapeck medium had a high background signal in fluorescence.

For this reason, fluorescence experiments were carried out in MSM medium and the kinetic growth curves in MSM were determined.

To study the growth kinetics in MSM medium, strains were transferred from Czapeck to MSM medium without anthracene to cultivate a primary inoculum, which was subsequently transferred to MSM medium with 1 mg/ml anthracene. From Czapeck to MSM without anthracene, and from this to MSM with 1 mg/ml anthracene, the volume transferred used as primary inoculum corresponded to an OD of 0.25 of culture strains taken at 50% of the logarithmic phase. In MSM medium without anthracene (Fig. 3C), the growth of the strains was due to the carbon source that bacteria accumulated as a reserve at the time that were cultured in medium Czapeck (Fig. 3A,B).

In the MSM medium, the growth of bacteria was lower than in the Czapeck medium, which was an expected result because MSM has a lower nutrient content. However, growth behavior was similar in both media. In three cases (Fig. 3A–C), a rapid growth was observed during the first h, and the stationary phase began at some tim between 1.5 and 2 h. In MSM media with 1

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mg/ml anthracene (Fig. 3D), the growth curve for K. rosea, M. testaceum and N. farcinica strains showed an abrupt growth within the first hour, followed by a slower growth until 7.5 h, when the stationary phase began, and, from then, it was constant for at least 24 h (Fig. 3D), (measurements are not continued after this time). In the case of K. palustris, the exponential growth phase finished at 10 h. Therefore, the primary inocula used for the spectrofluorimetry and ATR-FTIR assays were obtained at 4 and 5 h, respectively.

Analysis of the concentration of anthracene by fluorescence emission and identification of functional groups by ATR-FTIR. The concentrations of anthracene at 0, 1, 24 and 48 h present in the culture medium of K. rosea, K. palustris, M. testaceum and N. far­ cinica strains were determined by fluorescence emission.

Fig. 2. Phylogenetic tree of haloalkalitolerant Actino­ bacteria.

The results obtained (Fig. 4) show steep decreases in anthracene concentration (40–55%) during the first hour for all strains. This reduction was not further increased during the 1 to 24 h and 24 to 48 h intervals, respectively. This result agrees with the abrupt bacteria growth (Fig. 3) within the first hour that was later stabilized for at least 48 h. A bivariate analysis of the anthracene residual concentration shows statistically differences (P < 0.05) among the strains and the studies time interval.

The ATR-FTIR spectra of all strains were very similar and are shown in Fig. 5. The MSM with anthracene at 0 h and 48 h, as well as the cultures with anthracene after 48 h of incubation presented the band at 1237 cm–1 which decreased in intensity over time and did not appear in the medium inoculated for 48 h without anthracene. However, this band did not correspond to any vibration of the anthracene molecule; instead,

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ANTHRACENE DEGRADATION BY ACTINOBACTERIA

Fig. 3. Kinetic growth curves in Czapeck and MSM media with anthracene of the haloalkalitolerant identified Actinobacteria.

pected to be due to some interaction or direct effect of the solvent (medium) on the anthracene molecule, either solvating or hydroxylation. The decrease of this band over time indicates a molecular transformation in the anthracene. Another transformation of the anthracene is the emergence of bands at 1205 cm–1 and 1217 cm–1, which are not initially present in either the MSM with anthracene at 0 h or the microorganism solution incubated with anthracene for 1 and 24 h, nor they

Int Microbiol

it was related to the decrease in its concentration, as shown in Fig. 5. The reduction of the 1237 cm–1 signal at 48 h is in agreement with the reduction in the residual anthracene concentration (Fig. 4). The anthracene was dissolved in MSM at pH 8. This solution had both high salt content and high ionic force.

Therefore, this band at 1237 cm–1, which corresponds to a C–C–O–phenol asymmetric stretching vibration [46], is ex-

Fig. 4. Residual anthracene concentration in the Actinobacteria cultures by emission fluorescence (n = 5).

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Fig. 5. ATR-FTIR spectra of the Actinobacteria species incubated with and without anthracene at t = 48 h, and the MSM medium with anthracene at t = 0 h and t = 48h.

are present in the microorganism solution incubated for 48 h without anthracene. These bands only appear in the samples inoculated with anthracene for 48 h. These last two bands correspond to the C–C–O–phenol asymmetric stretching vibration [46]. This band indicates that the anthracene is being hydroxylated, and the emergence of 2 bands indicates that the anthracene in fact is being dihydroxylated.

Discussion The main objective of this study was to identify genetically haloalkalitolerant Actinobacteria isolated from soils close to oil activity and to evaluate their potential PAH degradation capacities. Bioremediation of PAH-contaminated environments is possible when using native halotolerant and alkalitolerant microorganisms [14,36]. Therefore, microorganisms with these two physiological properties should be able to grow in polluted environments presenting a wide range of NaCl concentrations and pH. Soils contain the highest numbers of existing phylogenetic groups; there are more than 109

bacterial cells per gram of soil [12]. However, it have been estimated that only 1% of the microbial soil populations can be cultured using traditional methods [50], which makes isolation a very difficult task. Therefore, in this study, we initially prepared MH culture medium [38], SAUTON-UAM [42], Czapek agar, soil extract agar [16], NZ amine A [28], GA agar [33] and YME [45], which are the most used media for the isolation of halophilic microorganisms and Actinobacteria. By modifying pH and NaCl concentration in these media, we obtained 22 different combinations (data not shown).

Under these conditions, 45 strains were isolated, of which only 8 were haloalkalitolerant Actinobacteria according to the morphological and physiological characterization, as well as the genetic identification.

The M1B strain, identified as K. rosea, had been previously classified inside the Micrococcus genus and recently has been re-classified in the Kocuria genus [47]. It has been mainly isolated from water and soil samples. Mahjoubi et al.

[23] reported its isolation in Bushnell Hass mineral salts (BHMS) medium supplemented with 1% petroleum from sediments and seawater collected near a refinery in Tunisia.

ANTHRACENE DEGRADATION BY ACTINOBACTERIA

The authors also evaluated its capacity for the production of biosurfactants and concluded that it could be a potential candidate for bioremediation. Ahmed et al. [1] isolated Kocuria rosea from artificial seawater (ASW) medium from water and soil samples contaminated with hydrocarbons in Pakistan.

They found and reported K. rosea capacity to grow on naphthalene, phenanthrene and fluoranthene and to degrade the first. In this study, K. rosea was isolated from a soil sample containing 1% NaCl at pH 8.3.

Strain M11A was identified as K. palustris. This species was described by Kovács et al. [18] after being originally isolated from Typha angustifolia. In 2007, Mariano et al. [25] isolated Kocuria palustris from the soil of a gas station in PCA medium (plate count agar), and one year later, Mariano et al. [24] reported the degradation of commercial diesel using this strain. To our knowledge, there have no other reports on its isolation from other environments. In this study, K. palus­ tris was isolated from a soil containing 3% NaCl at pH 8.4.

Strain M10A was identified as M. testaceum. This species was first isolated from Chinese rice paddies and classified in the Aureobacterium genus but subsequently re-classified in the Microbacterium genus [48]. De Vasconcellos et al. [9] isolated the genus Microbacterium sp. from coastal oil wells in nutritive broth and reported its capacity to degrade dihydrophenanthrene. To our knowledge, there have no other reports on the isolation of Microbacterium testaceum from contaminated environments. In this study, Microbacterium testaceum was isolated from a soil containing 3% NaCl at pH 8.6.

Strains M2C, M3H, M3I and M5B correspond to Nocar­ dia farcinica. This species was originally isolated from a lesion on a horse by Trevisan [51]. Al-Awadhi et al. [2] isolated it along with N. pneumoniae in mineral medium from samples of soils contaminated with hydrocarbons in Kuwait. N. farci­ nica was isolated particularly from a soil with 1.6% of salinity and pH 6.4. Additionally, Zeinali et al. [53] reported the isolation of N. otitidiscaviarum from soils of different oil-industrial sites. This species is capable of degrading naphthalene. In this study, N. farcinica was isolated from four soil samples containing 2%, 3%, 3% and 2% NaCl at pH values of 7.8, 7.8, 7.8 and 8.1, respectively. The salinity and pH values from our soil samples are slightly higher than the salinity and pH of the soil from which Al-Awadhi et al. isolated N. farci­ nica [2]. These results agree with the physiological characterization carried out in this work (Table 1), which showed that N. farcinica tolerated % NaCl in the range 0–3 and pH in the range 5–10.

Strain M4A corresponds to C. denverensis. This new species was first isolated from a blood sample of a patient with

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endocarditis [7]. Brito et al. [6] suggested that Cellulomonas sp., isolated from sediments of mangroves in Brazil, could degrade pyrene in ASW medium. In this case, the species was isolated from a soil sample containing 3% NaCl at pH 7.8.

Note that this is the first time that the isolation of C. denveren­ sis has been reported from soil.

Considering the interest in determining the residual concentration of anthracene present in the culture medium over time using fluorescence emission, initially Czapeck medium was employed but later MSM was used for this purpose. MH and YME media, used for the strains’ isolation and purification, are not colourless and could affect the analytical determination. Czapeck is colourless but showed high fluorescence background.

Except for C. denverensis, which did not grow in Czapeck and MSM media, the remaining seven strains increased the OD600 when anthracene was present (Fig. 3). This indicates that the strains increased their bacterial biomass so that anthracene could be used as the sole carbon source. The results of our study do not discard the possibility that C. denverensis could grow in the presence of anthracene given that Brito et al. [6] reported the capacity of Cellulomonas to degrade pyrene. This strain simply did not grow under our study conditions.

The fluorescence emission study demonstrated that K. ro­ sea, K. palustris, M. testaceum and N. farcinica, isolated from soils near areas with oil activity, were capable of using and transforming anthracene (Figs. 4,5). The residual anthracene values measured in this study were in the range of those reported for other PAHs (naphthalene, phenanthrene, dihydrophenanthrene) [1,2,9]. For example, Ahmed et al. [1] reported residual naphthalene and phenanthrene concentrations after 10 days of incubation in the presence of K. rosea and Kocuria flava of 64% and 91% and 47% and 91%, respectively. In our study, residual anthracene concentrations after 48 h of incubation with K. rosea and K. palustris were 49% and 54%, respectively. De Vasconcellos et al. [9] reported residual dihydrophenanthrene concentrations from 40-day cultures of the genera Microbacterium sp., Bacillus sp., and Halomonas sp. of 56%, 59%, and 79%, respectively. In our study, the residual anthracene concentration after 48 h of incubation with M. testaceum was 36%. Al-Awadhi et al. [2] reported, after 15 days of incubation of Nocardia pneumoniae and Streptomyces cellulosae with phenanthrene, residual concentrations of 85% and 30%, respectively. In our case, residual anthracene concentrations after 48 h of incubation with 4 different N. farci­ nica samples were 48%, 52%, 51% and 53%.

The decrease in the fluorescence signal of the residual an-

24

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thracene during the first hour indicated a rapid transformation, which coincided with the accelerated bacteria growth. From this point until 48 h, the observed fluorescence signal stabilized. The emergence of signals at 1205 cm–1 and 1217 cm–1 in the ATR-FTIR spectra at 48 h were associated with the formation of dihydroxy-aromatic intermediate compounds. In fact, these compounds have been reported as the initial phase of the anthracene degradation mechanism for Actinobacteria [10, 29]. They were probably formed from the first hour but the FTIR detection limit in our conditions (no chemical separation of anthracene was involved) did not allow their detection.

The initial anthracene concentration in the cultures was 270 ng/ml. In 1 h it was reduced to about 135 ng/ml. The ATRFTIR analysis was carried out with 5 ml of culture containing about 0.675 ng (135 ng/ml × 0.005 ml), which was a very small mass to be detected by ATR-FTIR without chemical separation. The determination of PAH (including anthracene) by the SW-846 Test Method 8410 EPA by GC-FTIR has a detection limit of tenths ng [27], and it involves the separation of PAH by capillary gas chromatography and subsequent detection by FTIR. The kinetic growth curves showed that the accelerated growth occurred during the first hour, but the stationary phase was present at least up to 48 h. Therefore metabolic activity continued in the culture and made it possible the detection of the dihydroxy-aromatic compounds at 48 h.

The reported anthracene degradation by Actinobacteria continues with the formation of COOH and COH groups [10,29]. In our ATR-FTIR spectra, however, there was no evidence of this step, indicating that this process might occur after 48 h. The total PAH oxidation by microorganisms is a slow process that requires several days or even weeks, as demonstrated by Martin et al. [26]. In our study, the concentration of residual anthracene at 48 h, was in the order of other reported studies of PAH biodegradation by Actinobacteria at 10, 15 and 40 days [1,2,9].

The capacity to transform anthracene by N. farcinica, K. rosea, M. testaceum and K. palustris strains was demonstrated at their optimal growth conditions (0.5%, 3% and 10% NaCl, respectively, and pH 8). However, they could growi at a wide range of pH and NaCl concentrations. Thus, they are potential candidates for the treatment of saline and alkaline environments contaminated with polycyclic aromatic hydrocarbons compounds. Note that N. farcinica should be used for biodegradation purposes only in places where it is native; it should not be seeded in foreigner locations due to its pathogenic nature.

To sum up, in this study eight strains of haloalkalitolerant Actinobacteria were isolated from soils close to areas with oil

LARA-SEVERINO ET AL.

activity in Mexico. Analysis of the 16S rRNA gene revealed the presence of species K. rosea, K. palustris, M. testaceum, N. farcinica and C. denverensis. The first four species had already been isolated from soils in other parts of the world, but, to our knowledge, this report describes the first isolation of C. denverensis from this kind of environment. These strains were classified as haloalkalitolerant (based on the NaCl % and pH range), although K. palustris was classified as extreme haloalkalitolerant. Their biomass could increase in the presence of anthracene, this increase correlates with the decrease in the residual anthracene concentration in the culture. ATR-FTIR spectroscopy indicated that K. rosea, K. palustris, M. testa­ ceum and N. farcinica transformed anthracene in dihydroxyaromatic compounds during the first 48 h. These seven strains had the capacity to transform anthracene; thus, they have the potential to be employed for biodegradation processes using native haloalkalitolerant Actinobacteria in saline and alkaline environments contaminated with PAH. Acknowledgements. The authors acknowledge financial assistance from: (i) the Secretary of Research and Advanced Studies of Autonomous University of the State of Mexico 3690/2014/CID, and the 1039/2014RIF network, (ii) Laboratory of Preservation, Diagnostic, and Spectroscopical characterization of materials, National Institute of Anthropology and History for the support in recording ATR-FTIR spectra through the grant CONACyTINFRA-225845, (iii) to the PRODEP 2015 “Red-Hispano-Mexicana para la búsqueda y aprovechamiento de microorganismos extremófilos con aplicaciones ambientales y biomédicas” network through the research project “Estudio de la degradación de hidrocarburos policíclicos aromáticos por actinomicetos halo-alcalotolerantes utilizando métodos de espectrofluorimetría.”

(This work is based on the PhD thesis of Reyna del C. Lara-Severino, student in the Health Sciences PhD program of the Autonomous University of the State of Mexico, registered in the PNPC-CONACYT.)

Competing interests. None declared.

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RESEARCH ARTICLE International Microbiology (2016) 19:27-32 doi:10.2436/20.1501.01.260 ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Genetic and serologic surveillance of rotavirus with P[8] and P[4] genotypes in feces from children in the city of Chihuahua, northern Mexico Juan F. Contreras-Cordero,1* César I. Romo-Sáenz,1 Griselda E. Menchaca-Rodríguez,1 Rocío Infante-Ramírez,3 Licet Villarreal-Treviño,1 Carlos E. Hernández-Luna,2 Cristina RodríguezPadilla,1 Reyes S. Tamez-Guerra1 1 Department of Microbiology and Immunology, Faculty of Biological Sciences, Autonomous University of Nuevo Leon, San Nicolas de los Garza, Nuevo Leon, Mexico. 2Department of Chemistry, Faculty of Biological Sciences, Autonomous University of Nuevo Leon, San Nicolas de los Garza, Nuevo Leon, Mexico. 3Faculty of Chemical Sciences, Autonomous University of Chihuahua, Chihuahua, Mexico

Received 7 February 2016 · Accepted 7 March 2016 Summary. Rotavirus vaccine was developed using the most prominent G and P genotypes circulating in children population.

Therefore, severe gastroenteritis has been reduced around the world. This study investigated the G and P rotavirus genotypes circulating in children from two hospitals in the city of Chihuahua, Mexico. Additionally, polyclonal antibodies against Rotavirus Wa strain were used to determine their homotypic and heterotypic reactivity to both P[8] and P[4] genotypes. G1, G2, and G3 VP7 genotypes and P[8] and P[4] VP4 genotypes were detected in common and uncommon combinations as well as mixed infectious. The predominant combination was G1P[8]. Phylogenetic analysis of VP4 gene revealed the presence of P[8]-1 and P[8]-3 lineages of P[8] genotype and P[4]-5 lineage of P[4] genotype. All but five G1P[8] rotavirus were detected by polyclonal anti-Rotavirus Wa strain. Mutation analysis revealed differences in three of the four neutralizing epitopes previously reported to VP8* subunit of VP4 protein. Results of this study offer insights over genetic variants of field rotavirus that could be detected in a homotypic and heterotypic way by antibodies elicited to rotavirus with P[8] genotype. [Int Microbiol 2016; 19(1):27-32] Keywords: rotavirus · viral genotypes · lineages of virus · epitopes · Chihuahua, Mexico

Introduction Rotavirus is the most important cause of viral gastroenteritis in humans. Annually, rotavirus is responsible for 197,000233,000 deaths in children less than five years of age mainly Corresponding author: Juan F. Contreras-Cordero Department of Microbiology and Immunology Faculty of Biological Sciences Autonomous University of Nuevo Leon San Nicolas de los Garza, Nuevo Leon, Mexico Tel. +52-8183294115 *

E-mail: juan.contrerascr@uanl.edu.mx; contrerasjfco@gmail.com

in developing countries [25]. Rotavirus (family Reoviridae, subfamily Sedoreovirinae, genus Rotavirus, species Rotavirus A) is a nonenveloped virion composed by six structural proteins that form three concentric layers enclosing a genome of 11 segments of double-stranded RNA. The outer layer is composed by VP7 and VP4 proteins [9]. Based on these proteins, rotavirus type A has been classified in a binary system.

So far 27 G (VP7) and 37 P (VP4) genotypes have been identified [19,27].

Surveillance studies around the world, before and after the introduction of rotavirus vaccines have shown that P[8] is the main genotype circulating in human populations. To date,

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P[8] genotype can be detected in levels of 70% of cases of infantile diarrhea, followed by P[4] that account nearly 15% [22]. Frequently, P[8] genotype is detected in combination with G1, G3, G4, G8 or G9 genotypes, whereas P[4] mainly is detected in combination with G2 genotype. In less extension, these P[8] and P[4] genotypes can also be detected in combination with other G genotypes [3,13,17].

P[8] and P[4] genotypes have been extensively studied in nucleotide and amino acid sequence. These studies have showed that genetic diversity is frequent among rotavirus circulating around the world. As a consequence, four lineages of P[8] (P[8]-1 to P[8]-4) and five lineages of P[4] (P[4]-1 to P[4]-5) have been detected with considerable intralineage and interlineage diversity [5,8,29]. Although P[8]-3 and P[4]-5 lineages are prevalent around the world, new lineages could emerge through different mechanisms including mutations, reassortment or recombination [1,20,23]. Nine antigenic regions have been established on the VP4 outer capsid protein that are responsible to elicit neutralizing antibodies. Four of these regions have been mapped on VP8* subunit (8–1 to 8–4) and five on VP5* subunit (5–1 to 5–5) [6,7]. Genetic studies have revealed changes in amino acid sequence on these epitopes among rotavirus strains with the same genotype and few studies focused on the combined effect of these changes on the detection of rotavirus by antibodies [1,15,29].

In this study, G and P genotypes of rotavirus circulating in the city of Chihuahua, Mexico, were detected. In addition, polyclonal antibodies elicited against Rotavirus Wa strain were used to test the reactivity of the lineages detected and to determine whether changes in the amino acid sequence of P[4] and P[8] genotypes could influence the antigen-antibody reaction.

Materials and methods Stool specimens. A total of 140 positive samples for rotavirus detected by polyacrylamide gel electrophoresis (PAGE) collected from 2004 to 2011 from children younger than five years of age were used in this study. All samples were collected from Chihuahua General Hospital Dr. Salvador Zubiran and the Children’s Hospital from the city of Chihuahua, State of Chihuahua, northern Mexico. The fecal specimens were stored at –20 °C. Reverse transcription polymerase chain reaction (RT-PCR).

Viral RNA was purified from 20% stools suspension in phosphate-buffered saline (PBS) by using TRI reagent (Molecular Research Center Inc., Cincinnati, OH, USA) following the manufacturer’s protocol. G and P genotypes were detected using the set of specific primers for G1-G4, P[8] and P[4] genotypes previously reported [10,11].

Sequencing and phylogenetic analysis of VP8* subunit. A PCR product of 762 bp of gene 4 using PA1 and PC2 primers was cloned and sequenced in the plasmid pGEM-T using the pGEM®-T Vector System I

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(Promega, Madison, WI, USA) [4]. The nucleotide sequence was determined using an automated system (Perkin Elmer/Applied Biosystem, CA, USA) at the Institute of Biotechnology of the National Autonomous University of Mexico (IBT-UNAM). The sequence analysis was done using CLUSTALW/ BioEdit sequence alignment Version 7.0 [12] and MEGA Version 6.0 software [24]. The VP4 nucleotide and amino acid sequences included in this study were submitted to the GenBank database under accession numbers JX012330 to JX012347. The GenBank accesion numbers of VP4 gene used in sequence analysis were HQ585864 to HQ585866, FJ665380 to FJ6653091, M96825, JN849113, EU045252, and DQ492672.

Polyclonal antibodies. The whole Rotavirus Wa strain were used to produce polyclonal antibodies. Polyclonal antibodies against a mixture of whole Rotavirus strains Wa, DS-1, ST3, SA11 and YM, here denominated anti-RVs (kindly provided by Carlos F. Arias, IBT-UNAM, Mexico) were used to detect rotavirus in specimen samples.

Enzyme-linked immunsorbent assay (ELISA). Microtiter plates (Costar, NY, USA) were coated with goat polyclonal anti-RVs diluted in carbonate-bicarbonate buffer pH 9.6 and incubated overnight at 4 °C. After two washing with PBS, the plates were blocked with 5% non-fat dry milk in PBS at 37 °C for 1.5 h. After four washes with PBS-Tween 0.1% (washing buffer), 20% fecal suspensions from children with gastroenteritis containing P[8] or P[4] rotavirus were added in duplicate. Fecal samples without rotavirus were used as negative controls. The plates were then incubated at 37 °C for 2 h, and after washing four times, rabbit polyclonal anti-Rotavirus Wa strain was added and incubated at 37 °C for 1 h. After four washes, peroxidase-conjugated protein A (Amersham, Buckinghamshire, UK) was added and incubated at 37 °C for 1 h. The plates were then washed four times and the ABTS (2,2′-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid]) peroxidase substrate (KPL, Gaithersburg, MD, USA) was added and incubated at 37 °C for 30 min. The absorbance at 405 nm was measured using an automatic microplate reader (Digital and Analog Systems, Roma, Italy). The cutoff value was defined as the mean of the control negative OD 405 nm values plus three standard deviations.

Results G and P genotyping. Of the total samples, two specimens positive by electrophoresis were typed as rotavirus group C, and 138 (98.5%) were assigned as G and P genotypes, respectively. Of the latter, both genotypes were detected in 128 (92.7%). The most prevalent combination was G1P[8] with 57.85%, and G2P[4] genotype was detected in a small proportion (2.86%) of the specimens tested. However, either P[8] or P[4] were found in 133 specimens including samples with mixed infections of both genotypes (Table 1).

VP4 lineages. To determine the genetic diversity of rotavirus belonging to the VP4 genotypes detected, 33 VP4 genes of the VP8* subunit were analyzed. Of these, 25 samples of P[8] genotype grouped into two lineages; 5 of them had 97.2– 98% identity with the Wa strain of P[8]-1 lineage, and 20 had 98–99.6% identity with the Dhaka 16-03 Bangladesh strain of P[8]-3 lineage. Identity of P[8]-1 and P[8]-3 lineages used in

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Table 1. Prevalence of VP7 and VP4 rotavirus genotypes in feces from 140 positive samples from children under five from two hospitals in the city of Chihuahua, Mexico G and P-type

Strain

Common types

N Â

% Â

G1P[8]

81

57.85

G3P[8]

5

3.57

G2P[4]

4

2.86

G1P[4]

12

8.57

G2P[8]

1

0.71

G3P[4]

1

0.71

G1P[8]P[4]

17

12.14

G2P[8]P[4]

2

1.43

G3P[8]P[4]

4

2.86

G1G3P[8]P[4]

1

0.71

NEGP[8]P[4]

1

0.71

NEG P[8]

4

2.86

G1 NEG

5

3.57

NEG NEG

2

1.43

140

100

Uncommon types

Mixed infections

G or P non typable

Group C Total

this study ranged from 93.6 to 91.6% (Fig. 1). In turn, eight P[4] rotavirus grouped into the P[4]-5 lineage with high identity to each other, ranging from 98.8 to 100% identity with the PY05SR1297 Paraguay strain of P[4]-5 lineage (Fig. 1). Identity of amino acids between P[8] and P[4] lineages was in order of 85.6 to 90%.

ELISA of field rotavirus. To evaluate the antigenicity of genotypes detected, 94 fecal samples positive to Rotavirus type A with sufficient material were further subjected to ELISA. Both P[4] and P[8] genotypes were present in the fecal specimens. Of the 94 fecal specimens, P[8] was present in 63 (67%), P[4] in 18 (19.1%), and mixed P[4] and P[8] in 13 (13.9%). All specimens carried P[4] were detected, however, all but 5 (5.3%) specimens carried G1P[8] genotype were detected by these antibodies (Table 2).

Comparison of P[8] and P[4] antigenic regions of VP8* subunit. The genetic variation into rotavirus was made using 33 samples randomly selected. This comparison

was made with the prototype Rotavirus Wa strain and the Rotarix vaccine. The study of the 33 sequences analyzed revealed 58 variations in the VP8* subunit of VP4 when they were compared with VP8* sequence of Rotavirus Wa strain. The analysis of all changes showed that 37 amino acid substitutions were between residues with similar properties. In addition, 15 charge amino acid changes (Y19H, E28K, T75K, N89D, N113T/D/S, D116N, S131R/E, D133E/S, D135N, N160D, N193D, C215R, K245R/T/N, N250K and E251K) were identified as well as 6 changes of residues with possible structural implications (conformational flexibility or constrain) on the proteins (G38S, P71S, P114Q, G145S, G195N and P236S). Specific comparison of the amino acid residues in antigenic epitopes of VP8* subunit between Rotavirus Wa and P[8] and P[4] genotypes of field rotavirus showed differences on 3 of the 4 epitopes. In addition, the analyses on the epitope 8-1 and 8-3 identified 5 and 8 changes respectively, while rotavirus with P[4] genotype had differences on the epitope 8-4. The alignment between Rotavirus Wa and Rotarix vaccine revealed consensus in the antigenic domains of the VP8* subunit.

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Fig. 1. Phylogenetic tree constructed from VP8* subunit amino acid sequences of the P[8] and P[4] rotavirus genotypes using neighbor-joining method (MEGA v6.0) with bootstrap analysis of 1000 replicates. Stool specimens selected from 140 positive samples for rotavirus from children under five from two hospitals in the city of Chihuahua, Mexico. Bar indicates 1% amino acid difference.

Discussion Traditionally, epidemiological studies based on surface proteins VP7 and VP4 have classified rotavirus in a binary sys-

tem, identifying multiple genotypes combinations G/P [19].

Despite the great variability of this pathogen, G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] genotypes have been frequently detected around the world and G12P[8] genotype in a lesser extension [13,17,21]. In this study, P[8] and P[4] genotypes

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Table 2. Rotavirus genotyped detected by polyclonal antibodies anti-Rotavirus Wa strain and enzyme-linked immunosorbent assay (ELISA) Anti-Rotavirus Wa strain Genotype

Positive

Negative

G1P[8]

56

5

G1P[4]

11

0

G2P[4]

5

0

G3P[8]

2

0

G3P[4]

1

0

G1P[8]P[4]

12

0

G2P[8]P[4]

1

0

NEGP[4]

1

0

89 (94.68 %)

5 (5.32 %)

Total

were detected in more than 95% of the rotavirus analyzed, indicating the same prevalence of these genotypes worldwide [1,13,23]. In addition, phylogenetic analysis of P[8] and P[4] genotypes have shown four and five lineages respectively. Of them, P[8]-3 and P[4]-5 lineages are prominent around the world [5,8,15,23]. In this study lineage P[8]-3 was detected in 80% of rotavirus with P[8] genotype and, to a lesser extent, in the lineage P[8]-1, which corresponds to the lineage present in the Rotarix vaccine. Moreover, lineage P[4]-5 was identified in all samples with P[4] genotype, which corresponds to the lineage of this genotype with more incidence worldwide [8,26]. After the introduction of the vaccine, several reports have revealed the need of continued epidemiological surveillance of rotavirus to understand the impact of genetic variation in the efficiency of the vaccine [2,16,18,23,29].

In this study, 94.7% of the samples analyzed were recognized by antibodies against Rotavirus Wa strain. These results are in accordance with previous reports that showed homotypic and heterotypic antibody reactivity between strains with different genotype [28]. Note that all rotavirus with P[4] genotype were recognized by these antibodies. However, five with P[8] genotype did not show reactivity, which suggests that amino acid changes in regions of VP8* subunit could interfere with antibody recognition and consequently could generate strains of rotavirus that elude the immunity generated by vaccination. The sequence analysis conducted in this study revealed both amino acid changes with similar properties and amino acid changes that could be related to physicochemical and structural alterations in the VP8* subunit.

Likewise, the analysis of variations between the sequenc-

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31

es analyzed revealed that residues from 106 to 199 had the majority of amino acid changes of VP8*. Note that, in this region, there are epitopes responsible for neutralizing antibodies previously reported [6,7]. However, despite amino acid changes present in the VP8* subunit, these antibodies maintained the reactivity in the majority of the specimens. Sequence analysis of two negative samples by ELISA showed one specific amino acid substitution (N113T), when they were compared with vaccine strain or genetic variants (N113D/S) of positive samples, specifically into the epitope 8-3 on the VP8* subunit. This result is in contrast with previous reports that have revealed that single amino acid substitutions in these regions can alter the antigenic characteristics of VP4 [14].

Therefore, it is necessary to extend the studies of antigenic variability in field rotavirus to establish the possible role of putative surface residues as N/D/S at 113 position as well as genetic variants as N113T or other residues with conformational implications to elude antibody recognition.

Acknowledgements. This work was supported in part by grant CN1074-11 PAICYT from the Autonomous University of Nuevo Leon, Mexico. César I. Romo-Sáenz is recipient of a fellowship from the CONACYT, Mexico.

Competing interests. None declared.

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19. Matthijnssens J, Ciarlet M, McDonald SM, Attoui H, Bányai K, Brister JR, Buesa J, Esona MD, Estes MK, et al (2011) Uniformity of Rotavirus Strain Nomenclature Proposed by the Rotavirus Classification Working Group (RCWG). Arch Virol 156:1397-1413 20. Matthijnssens J, Van Ranst M (2012) Genotype constellation and evolution of group A rotaviruses infecting humans. Curr Opin Virol 2:426-433 21. Mijatovic-Rustempasic S, Teel EN, Kerin TK, Hull JJ, Roy S, Weinberg GA, Payne DC, Parashar UD, Gentsch JR, Bowen MD (2014) Genetic analysis of G12P[8] rotaviruses detected in the largest U.S. G12 genotype outbreak on record. Infect Genet Evol 21:214-219 22. Santos N, Hoshino Y (2005) Global distribution of rotavirus serotypes/ genotypes and its implication for the development and implementation of an effective rotavirus vaccine. Rev Med Virol 15:29-56 23. Steyer A, Sagadin M, Kolenc M, Poljšak-Prijatelj M (2014) Molecular characterization of rotavirus strains from pre- and post-vaccination periods in a country with low vaccination coverage: The case of Slovenia. Infect Genet Evol 28:413-425 24. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30:2725-2729 25. Tate JE, Burton AH, Boschi-Pinto C, Parashar UD, World Health Organization-Coordinated Global Rotavirus Surveillance Network (2016) Global regional, and national estimates of rotavirus mortality in children >5 years of age, 2000–2013. Clin Infect Dis 62 Suppl 2:S96-S105 26. Tatte V, Chitambar SD (2011) Intragenotypic diversity in the VP4 encoding genes of rotavirus strains circulating in adolescent and adult cases of acute gastroenteritis in Pune, Western India: 1993 to 1996 and 2004 to 2007. J Gen Mol Virol 3:53-62 27. Trojnar E, Sachsenröder J, Twardziok S, Reetz J, Otto PH, Johne R (2013) Identification of an avian group A rotavirus containing a novel VP4 gene with a close relationship to those of mammalian rotaviruses. J Gen Virol 94:136-142 28. Wen X, Cao D, Jones RW, Hoshino Y, Yuan L (2015) Tandem truncated rotavirus VP8* subunit protein with T cell epitope as non-replicating parenteral vaccine is highly immunogenic. Hum Vaccin Immunother 11:2483-2489 29. Zeller M, Patton JT, Heylen E, De Coster S, Ciarlet M, Van Ranst M, Matthijnssens J (2011) Genetic analyses reveal differences in the VP7 and VP4 antigenic epitopes between human rotaviruses circulating in Belgium and rotaviruses in Rotarix and RotaTeq. J Clin Microbiol 50:966-976

RESEARCH ARTICLE International Microbiology (2016) 19:33-37 doi:10.2436/20.1501.01.261 ISSN (print): 1139-6709. e-ISSN: 1618-1095

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Prevalence of Campylobacter spp. in diarrhoea samples from patients in New South Wales, Australia Aruna Devi,1 Adam S. Gunjilac,2 Jenny M. Wilkinson,1 Thiru Vanniasinkam,1 Timothy J. Mahony2,* School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, Australia.

Queensland Alliance for Agriculture and Food Innovation, University of Queensland, Brisbane, Australia 1

2

Received 23 January 2016 · Accepted 7 March 2016

Summary. Campylobacteriosis is a leading cause of bacterial foodborne disease in many industrialized countries including Australia. New South Wales (NSW) is the most populous state in Australia yet the lack of any Campylobacter species surveillance programs has led to a knowledge gap in the importance of these pathogens as causes of diarrhoea. The data collected in this study demonstrated a need for such programs. In this study, 400 human clinical fecal samples were collected from two NSW locations, Western Sydney and Wagga Wagga, and tested for the presence of Campylobacter spp. Patients were clustered by location, age and gender to assess Campylobacter spp. prevalence within these groups between the two regions. The frequency of Campylobacter spp. was higher in males compared to females in the age groups 0–4 and 5–14 years; 6.4% and 1.0%, and 8.2% and none, respectively (P < 0.05). A second peak was noted in elderly adults compared with those in younger age groups. Based on the findings of the quantitative PCR analysis it was estimated that the age-adjusted prevalence of Campylobacter spp. associated diarrhoea was 159 cases per 100,000 persons. [Int Microbiol 2016; 19(1):33-37] Keywords: Campylobacter species · campylobacteriosis · foodborne diseases · prevalence of pathogens · New South Wales, Australia

Introduction In Australia, approximately 5.4 million cases of foodborne diseases are reported annually resulting in an estimated cost to the health system of $1.2 billion per year. Campylobacter species have been reported to be the most common enteric pathoCorresponding author: T.J. Mahony Queensland Alliance for Agriculture and Food Innovation Queensland Bioscience Precinct University of Queensland 306 Carmody Road Brisbane, QLD 4072 Australia Tel. +61-733466505. Fax +61-733466555 *

E-mail: t.mahony@uq.edu.au

gens amongst all known foodborne pathogens in Australia.

The annual report produced by “OzFood Net” [21], an Australian government initiative which reports on the incidence of foodborne diseases, reported 16,968 cases of Campylobacter spp. In 2010. This report, however, excluded the most populous state, New South Wales (NSW) [13].

Campylobacteriosis is the leading cause of bacterial foodborne disease in industrialized countries, including Australia.

The most prevalent species seen in human infection are Campylobacter jejuni and C. coli [15]. Human campylobacteriosis is generally a self-limiting disease with typical enteric symptoms including vomiting, fever, diarrhoea and abdominal pain [13,25]. Chronic diseases such as reactive arthritis and Guillain-Barré syndrome may occur sporadically as a

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consequence of campylobacteriosis [19]. Campylobacter spp. infections are widely prevalent in Australia and infection rates have shown a steady increase in recent years [13]. Most campylobacteriosis cases in Australia are thought to be sporadically acquired [29], showing a similar infection pattern to studies conducted in North America [8,23], Europe [10,12] and New Zealand [26].

The most important route of Campylobacter infection is the consumption and handling of poultry [3]. In fact, in Australia, the United States and Europe, 50–70% of all Campylobacter spp. infections have been attributed to the consumption of undercooked or contaminated chicken [1,18]. Furthermore, improper food handling or inadequate hygiene techniques, as well as household pets and domestic chickens are thought to be linked to campylobacteriosis in children and adults [28].

However, there has been no direct investigation of the wide range of potential human exposure sources or systematic exploration of possible ecological cycles of Campylobacter spp. in Australia. Human Campylobacter spp. infection is a communicable disease in all Australian states and territories, apart from NSW, and the data collected can be used to inform prevention and control measures. The exclusion of NSW from this system means there is no accurate information on either the prevalence or incidence of Campylobacter spp. infections in this state, which makes the development of more effective control polices and measures problematic [13].

The aim of this study was to estimate the prevalence of Campylobacter spp. in patients with symptoms of diarrhoea living in two areas of NSW who sort medical care. Prevalence of Campylobacter spp. in different study population categories were also compared to determine if factors such as patient age or gender influenced the Campylobacter spp. infection prevalence.

DEVI ET AL.

were refrigerated after routine testing was completed by the Microbiology Departments at both locations. Samples from western Sydney were placed on ice and transported to Charles Sturt University for DNA extraction within 24 h of collection. Samples collected at the Wagga Wagga Base Hospital were transported to the university laboratory within one hour and processed immediately.

DNA extraction. Total DNA was extracted from human faecal samples using QIAamp DNA Stool Mini Kit (QIAGEN Pty Ltd, Australia). An aliquot of the sample (200 µl) was transferred to a 2-ml Eppendorf tube and heated to 70 oC for 10 min. Total DNA was extracted using the manufacturer’s protocol. The final elution volume of DNA was 200µL. The DNA samples were stored at –20 oC prior to PCR analysis.

Real time PCR assay (qPCR). The presence or absence of DNA from Campylobacter spp. in the clinical sample extracts was determined by using quantitative real-time PCR (qPCR). The qPCR primers and the probe used were modified from previously published studies [4,6]. The qPCR assay was designed to detect all Campylobacter spp. by targeting the 16S rRNA gene using the primer pair Lund_16S_F 5´-CGT GCT ACA ATG GCA TAT ACA ATG A-3´ and Lund_16S_R 5´-CGA TTC CGG CTT CAT GCT C-3´. A novel 16S probe, Campy_16S-LNACy5 Campylobacter Cy5-5´-ATA [+G] AT [+T] TC [+C] AC C-3´-BHQ3 (Sigma-Aldrich, Australia) was designed using Beacon Designer version 8.12 (Premier Biosoft International). The nucleotide residues designated as [+N] have been modified with locked nucleic acid technology [5,14]. Briefly, the LNA probe was designed by importing the forward and reverse oligonucleotides into the software, and the default settings used to identify the optimal probe sequence within the 44bp amplicon. To confirm that the optimal probe sequence was identical to Campylobacter spp. 16S sequences and to assess the likelihood of cross-reaction with bacterial species that might be present in clinical samples, a Primer-BLAST search was performed [30].

All qPCR assay runs included control templates from strains; C. jejuni NCTC 11168, C. jejuni ATCC 29428, C. jejuni ATCC 49943, C. coli NCTC 11366, and C. coli ATCC 33559. Each qPCR reaction volume of 20 µl contained 10 µl of 2 × TaqMan master mix (Applied Biosystems), 16S forward primer 0.2 µM, 16S reverse primer 0.2 µM, 16S probe 0.2 µM. DNA template 1 µl was used with the following conditions; Taq Hot Start for 15 min at 95 oC, one cycle, 40 cycles of denaturation for 1 min at 95 oC, annealing and extension for 1 min at 60 oC in the Rotor-Gene Q (QIAGEN).

Study design and locations. A cross-sectional study was conducted to investigate the prevalence of Campylobacter spp. infection at the Microbiology Department of the Westmead Hospital in Western Sydney and the Microbiology Department of the Wagga Wagga Base Hospital from October 2012 to February 2014. A total of 400 (n = 200 Wagga Wagga, n = 200 Western Sydney) diarrhoeal samples, as defined according to World Health Organization guidelines, were opportunistically collected from the faecal samples submitted for routine analysis and culture at these two laboratories for the duration of the sampling period. The Australian Bureau of Statistics has described Sydney and Wagga Wagga as a major city and an inner regional city respectively.

Data analysis. The qPCR results were analyzed using Rotor-Gene Q Series software. The cycle threshold values determined based on the run parameters with software default setting. Population denominators were obtained for the relevant NSW regions from the Australian Bureau of Statistics data, with 2011 being the most recent data collection year. The Western Sydney population for the year 2011 was 846,001 and for Wagga Wagga was 237,338.

For the estimation of prevalence per 100,000, the NSW 2011 standard population data of 7,218,529 was used [20,22]. Based on the age distribution of cases used in previous studies [7,9], the following age groups were identified for data analysis and age–specific prevalence of Campylobacter spp.: 0–4, 5–14, 15–24, 25–34, 35–44, 45–54, 55–64, 65–74 and ≥75 years were calculated. Samples from all age groups were included in this study. Samples from both genders were also included in the study.

The sample results were tabulated using the package software Statistical Package for the Social Sciences (SPSS, version 20). An unconditional logistic regression model was used in multivariate analysis. Chi-square analysis was performed to compare results in various groups with P < 0.05, deemed to be statistically significant.

Sample collection. Clinical faecal samples were aliquoted in sterile containers and an appropriate code was assigned to each sample. The samples

Ethics approval. This study was approved by the Charles Sturt University Ethics in Human Research Committee, with approval for sample collec-

Materials and methods

tion also granted by the Pathology Department managers at each of the hospital sites used in this study. The approvals permitted the collection of basic patient information on age and gender, in a de-identified manner to protect patient confidentiality.

Results and Discussion Prevalence of infection. This study reports a Campylobacter spp. population adjusted prevalence of Campylobacter spp of 159 cases per 100,000 population in NSW (Fig. 1). The estimate was determined by testing, in diarrhoea samples (n = 400) collected from the two locations, the presence of Campylobacter spp DNA by qPCR. Campylobacter spp. were detected in 209 of the samples tested, giving a Campylobacter spp. prevalence of 52.3% (95%; confidence interval 47.8–58.3%) (Table 1). The NSW prevalence estimate was higher than the Australian case rate of 93.5 cases per 100,000 population, without including NSW data during 2013 when the majority of the study samples were collected [2]. Regarding the various Australian states, the data collected in this study suggested that NSW and Tasmania the most similar rates of human Campylobacter spp. infections, with 158.9 and 135.7 cases per 100,000 population respectively for 2013 (Fig. 1). Somewhat surprisingly, the Australian Capital Territory, located within NSW, had a much lower case rate of 98.4 cases per 100,000 for this year compared to the jurisdictions (Fig. 1) [2]. Differences in population size, demographics and health polices within each juris-

Fig. 1. Comparative prevalence of Campylobacter species infection per 100,000 population in NSW (this study) and other Australian states and territories in 2013; Australian Capital Territory (ACT), New South Wales (NSW), Northern Territory (NT), Queensland, (Qld), South Australia (SA), Tasmania (Tas), Victoria, (Vic), and Western Australia (WA). The 2011 popu­ la­ tion census NSW HealthStats from the Australia Bureau of Statistics were used to calculate rate per 100,000 population.

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diction suggest that additional research is required to establish if risk factors for infection are also similar.

The higher prevalence of Campylobacter spp. in NSW reported in this study compared to other states may have resulted from the detection methodologies used. Culture is often considered the gold standard technique for Campylobacter spp. and it is used in most microbiology laboratories across Australia, while the current study has used qPCR to determine the results for NSW. It is well established in the literature that qPCR is more sensitive than culture for the detection of fastidious [16,17,24]. Due to the fastidious and liable nature of Campylobacter spp., culture-based detection might underestimate the true prevalence in a population. In contrast, qPCR would be more likely to overestimate the true prevalence of Campylobacter spp. as it could detect both live and dead bacteria in the samples. This study utilised qPCR as the detection method to ensure that the results from samples collected at different locations were comparable. In fact, samples from Western Sydney could not be tested for at least 24 h after collection while those from Wagga Wagga could be processed just one hour after having been collected. Therefore, the use of culture could have dramatically underestimated the true prevalence in Western Sydney. Moreover, the estimated prevalence for NSW in this study is consistent with, albeit higher, analogous data from other Australian states determined using culture (Fig. 1), which suggests that qPCR detection is a rapid, sensitive and cost-effective method of detecting this pathogen.

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DEVI ET AL.

Table 1. Categorisation of Campylobacter spp. positive diarrhoeal specimens by age and gender determined using quantitative real time PCR (n = 394*) Patient sex All Samples

Male

Female

Age group (years)

Tested

Positive (%)

Positive (%)

Positive (%)

0-4

12

8 (3.8)

7 (6.4)

1 (1.0)

5-14

15

9 (4.3)

9 (8.2)

0

15-24

24

10 (4.8)

6 (5.5)

4 (4.0)

25-34

34

20 (9.5)

9 (8.2)

11 (11.1)

35-44

23

16 (7.7)

5 (4.5)

11 (11.1)

45-54

40

20 (9.6)

9 (8.2)

11 (11.1)

55-64

58

35 (16.7)

21 (19.0)

14 (14.1)

65-74

76

41 (19.6)

21 (19.0)

20 (20.2)

>75

112

50 (23.9)

23 (20.9)

27 (27.3)

Total

394

209

110

99

*

* Information on patient age and gender for six samples, of which two were positive from Wagga Wagga, were missing and therefore not included in this table.

Distribution of infection by age. The distributions of Campylobacter spp. positive samples by age and gender are shown in Table 1. The highest prevalence of Campylobacter spp. infections were found in age groups 55–64, 65–74 and ≥75 years (16.7%, 19.6% and 23.9%) respectively. The lowest numbers of cases were in children and youngsters aged 0–4, 5–14 and 25–34 (3.8%, 4.3% and 4.8% respectively (Table 1). Age details were not available for six samples two of which were positive for Campylobacter spp. and as a result these could not be included in the analysis. A previous cohort study reported a higher prevalence for the 0–4year-old group [11]. In comparison the current study included samples from all age groups as it was reliant on the opportunistic sampling of the study population. Another reason for the lower prevalence and reduced number of samples in the 0–4-yearold group of the current study could be the limited pediatric services provided at the Westmead hospital where the sampling was conducted; therefore, patients in this age group might have been treated in another hospital such as Childrens’ hospital for specialised treatment.

Prevalence of infection by gender. When comparing males and females, no significant differences in the Campylobacter spp. prevalence were found, although the frequency was slightly higher for males (52.6%) than for females (47.4%) (Table 1). In the age groups 0–4 and 5–14 years, statistically significant differences were noted in the number of Campylobacter spp. infection in males (6.4%) compared with females (1.0%), and 8.2% in males

and none in females, respectively (P < 0.05). In the 35–44 age range, significantly higher prevalences of Campylobacter spp. were detected in females with 11.1% compared to 4.5% in males (P < 0.05, Table 1). For the age groups 25–34 and ≥75, the prelences were higher in females than in males (P < 0.05, Table 1). The gender differences in Campylobacter spp. prevalence identified in the present study were largely in agreement with rates reported in another similar study [27].

We can conclude that this study provides significant data on selected demographic determinants, as well as trends of Campylobacter spp. infections in NSW, Australia. The data collected in this study suggest that NSW might have the highest prevalence of Campylobacter spp. infections among all of the Australian health jurisdictions. To obtain more detailed data on the prevalences of Campylobacter spp. infections in the whole country, mandatory reporting in NSW would be highly desirable as this would effectively establish a national surveillance program. The results reported in this study suggest that more research into the role of Campylobacter spp. in the cases of diarrhoea could help to determine the effects of a wide range of risk factors in NSW and all Australia and underpin better control measures.

Acknowledgements. The authors would like to thank the staff of the Microbiology Department, Westmead Hospital in Western Sydney and the Wagga Wagga Base Hospital in Wagga Wagga for their continuous support during sampling.

Competing interests: None declared.

CAMPYLOBACTER SPP. IN NEW SOUTH WALES

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RESEARCH ARTICLE International Microbiology (2016) 19:39-47 doi:10.2436/20.1501.01.262 ISSN (print): 1139-6709. e-ISSN: 1618-1095

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De novo synthesis and functional analysis of the phosphatase-encoding gene acI-B of uncultured Actinobacteria from Lake Stechlin (NE Germany) Abhishek Srivastava,1 Katherine D McMahon,2 Ramunas Stepanauskas,3 Hans-Peter Grossart1,4,5* Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Stechlin, Germany. 2Departments of Civil and Environmental Engineering, and Bacteriology, University of Wisconsin at Madison, Madison, WI, USA. 3Bigelow Laboratory for Ocean Sciences, East Boothbay, ME, USA. 4Potsdam University, Institute for Biochemistry and Biology, Potsdam, Germany.

5 Berlin-Brandenburg Institute of Advanced Biodiversity Research, Berlin, Germany

1

Received 29 January 2016 · Accepted 7 March 2016

Summary. The National Center for Biotechnology Information [http://www.ncbi.nlm.nih.gov/guide/taxonomy/] database enlists more than 15,500 bacterial species. But this also includes a plethora of uncultured bacterial representations. Owing to their metabolism, they directly influence biogeochemical cycles, which underscores the the important status of bacteria on our planet. To study the function of a gene from an uncultured bacterium, we have undertaken a de novo gene synthesis approach.

Actinobacteria of the acI-B subcluster are important but yet uncultured members of the bacterioplankton in temperate lakes of the northern hemisphere such as oligotrophic Lake Stechlin (NE Germany). This lake is relatively poor in phosphate (P) and harbors on average ~1.3 x 106 bacterial cells/ml, whereby Actinobacteria of the ac-I lineage can contribute to almost half of the entire bacterial community depending on seasonal variability. Single cell genome analysis of Actinobacterium SCGC AB141P03, a member of the acI-B tribe in Lake Stechlin has revealed several phosphate-metabolizing genes. The genome of acI-B Actinobacteria indicates potential to degrade polyphosphate compound. To test for this genetic potential, we targeted the exoPannotated gene potentially encoding polyphosphatase and synthesized it artificially to examine its biochemical role. Heterologous overexpression of the gene in Escherichia coli and protein purification revealed phosphatase activity. Comparative genome analysis suggested that homologs of this gene should be also present in other Actinobacteria of the acI lineages. This strategic retention of specialized genes in their genome provides a metabolic advantage over other members of the aquatic food web in a P-limited ecosystem. [Int Microbiol 2016; 19(1):39-47] Keywords: acI-B in Actinobacteria · phosphatases · single cell genomics · phosphate limitation · Lake Stechlin, NE Germany

Introduction Only 1% of all bacteria on Earth are readily cultivated and there is information on ca. 60 estimated bacterial phyla Corresponding author: H.P. Grossart Leibniz-Institute of Freshwater Ecology and Inland Fisheries Alte Fischerhuette 2 16775 Stechlin, Germany Tel. +49-3308269991 *

E-mail: hgrossart@igb-berlin.de

[46]. Of those, at least 31 bacterial phyla have no cultured representation [17]. These bacteria have been detected in diverse habitats including aquatic and terrestrial environments [34]. In particular, lake ecosystems consist of a diverse and greatly uncultured microbiota [1,2,27]. In planktonic food webs, heterotrophic bacteria play an important role in pelagic energy flow and nutrient cycling within the microbial loop and thereby compete with phytoplankton for inorganic and organic nutrition sources such as phosphorus (P) [4,9].

Culture-independent approaches have revealed a lot of in

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silico information on microbial genome, but the function of any given gene cannot be determined without testing for it experimentally. Therefore, we chose an uncultured freshwater Actinobacteria, since they occur in diverse habitats and are globally distributed, making them one of the most successful bacterial phyla world-wide [47]. This universal group also dominates the microbial community in oligotrophic, temperate, and P-limited Lake Stechlin [30] in northeastern Germany [1]. 16S rRNA-gene sequence analyses and bacterial cell counts via CARD-FISH (catalyzed reporter deposition fluorescence in situ hybridization) of epilimnic bacteria, it was revealed that up to 50% of the bacterioplankton in this lake consists of uncultured Actinobacteria of the acI-B and acI-A sub-clusters [1,3].

Phosphorous dynamics in freshwaters have been well studied and it has been demonstrated that this is the most important limiting element for phytoplankton in oligotrophic freshwater ecosystems, a fact that has been termed as “the phosphorus limitation paradigm” [37,39]. Phosphorous is also considered to be the element with the lowest supply and demand ratio in many oligotrophic freshwater ecosystems [21]. Note that 80% of the whole P pool in such habitats is consumed by the bacterioplankton and solely 20% of the P pool remains left for other food web members, e.g., algae [20,22,44].

Consequently, P uptake in the epilimnion of oligotrophic lakes is to a large extent linked to bacterial communities and their remineralization via death and protozoan grazing provides substantial amounts of P to the phytoplankton [9,44].

Substrate preferences of heterotrophic bacteria, however, are largely dependent upon their genetic and metabolic potentials [11,33]. Therefore, in order to get a deeper insight into the P utilization capabilities of the dominant but yet uncultured freshwater Actinobacteria in a P-limited ecosystem, we explored the ‘single-cell genome’ of Actinobacterium SCGC AB141-P03 (representing an abundant, uncultured phylotype within the acI-B clade in Lake Stechlin). Single-cell genomics approach requires isolation of unculturable bacteria by fluorescence-activated cell sorting (FACS) followed with whole-genome amplification to conduct genome analysis at single-cell level [12]. Based on the presence of several phosphate-metabolizing genes in the genome we hypothesized that Actinobacteria of the acI-B clade should be superior in P uptake. To test the function of one of the P-utilizing genes and predict its beneficial role to the host bacterium in a P-poor environment, we overexpressed, purified and functionally characterized the putative phosphatase-encoding gene (exoP).

To our knowledge, this is the first time, that a de novo gene synthesis approach in conjunction with single cell genomics

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has been used to study how bacteria can adapt and thrive in a P-limited environment and thereby may interfere with primary producers.

Materials and methods Location and sampling. Lake Stechlin is a well-studied lake located in the Mecklenburg Lake District region, which originated after the latest ice age (Weichselian Stage) ca. 12,000 years ago. Oligotrophic Lake Stechlin has a maximum depth of 69.5 m and generally has high hypolimnetic oxygen concentrations, i.e., up to 60% O2 saturation [24]. Lake Stechlin was sampled monthly from 2003 to 2015 by collecting water at various depths, except during periods of unstable ice coverage. Here, we only show 5-m depth data since the epilimnic zone is usually well mixed. In addition, the single amplified genome (SAG) Actinobacterium SCGC AB141-P03 of the clade acI-B was obtained from this depth. To generate a SAG library from this sample, 1 ml sample aliquots were amended with 6% (final concentration) betaine and stored at –80 °C. The SAGs were generated and identified at the Bigelow Laboratory Single Cell Genomics Center (https://scgc.bigelow.org) following previously described procedures [40]. Limnetic factors such as temperature, oxygen and pH were measured by electrodes (WTW, Weilheim, Germany). While unfiltered water samples were used for determining total phosphorus (TP), soluble reactive phosphorus (SRP) was measured after filtration of samples through 0.45-µm cellulose acetate membrane filters (Sartorius AG, Göttingen, Germany). Samples were incubated with 5% (w/v) K2S2O8 for 30 min at a constant temperature of 134 °C in a steam autoclave.

Phosphorus concentrations were then analyzed photometrically (FIA compact analyzer MLE, Dresden, Germany) following the molybdenum-blue method [23].

Phylogenetic relatedness. We conducted a phylogenetic analysis of exoP sequences predicted from all available acI draft genomes and members of another abundant lineage of freshwater ultramicrobacteria, the LD12 Alphaproteobacteria. Protein sequences were previously generated and described elsewhere [13,49]. The draft genomes are available via Genbank and “The Integrated Microbial Genomes (IMG) system” of the Joint Genome Institute (http://www.jgi.doe.gov/) [28]. Protein sequences were retrieved from IMG and aligned using the software Geneious version 7.1.4, (Biomatters, New Zealand). Homologs from Streptomyces sviceus ATCC 29083 and Candidatus Pelagibacter ubique were used as reference sequences for acI and LD12 proteins, respectively. A total of 308 positions were used to infer a maximum likelihood phylogeny using RAxML version 8.0.9 with default settings [38], implemented at the CIPRES Science Gateway.

Bootstrap analysis was conducted with 1000 resamplings.

Bacterial strain, plasmid and growth condition. Escherichia coli Bl21(DE3) bacterial strain and pET-28a_exoP plasmid were used in this study. The E. coli strain was maintained and grown on lysogeny broth (LB) medium at 37 °C [36]. The antibiotic kanamycin was added to the medium at a concentration of 25 µg/ml.

Molecular genetic techniques and protein detection.

Isolation of plasmid DNA, restriction enzyme digestion, agarose gel electrophoresis, purification of DNA fragments from agarose gels, electroporation, ligation of DNA fragments and several other routine molecular methods were performed using standard protocols [36]. The 918bp putative exoP gene from Actinobacterium SCGC AB141-P03 was de novo synthesized by Eurofins MWG Operon (Ebersberg, Germany) and delivered in pEX-A vector. exoP_Fwd and exoP_rev primers were used to PCR amplify

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the exoP gene and subsequently cloned in Novagen pET-28a(+) expression vector (Merck KGaA, Darmstadt, Germany) using Nde1 at 5′-end and EcoR1 restriction site at 3′-end yielding pET-28a_exoP with N-terminal fusion to Hisx6. From the plasmid preparation, 200 ng were electroporated by using BioRad GenePulser (Bio-Rad Laboratories Inc., USA) at 25 mF, 200 Ohms and 2.5 kV into the electro-competent cells of E. coli BL21(DE3) (New England Biolabs GmbH, Frankfurt am Main, Germany). The transformants were grown at 37 °C for 1 h in SOC broth and plated on LB agar media containing kanamycin.

Protein purification. For overexpression of pET-28a_exoP in E. coli BL21, one liter of E. coli cell culture was grown at 37 °C. When the culture reached an OD600 of 0.5, IPTG (isopropyl b-d-thiogalactoside) was added to the end concentration of 1 mM. The culture was grown overnight at 18 °C, and then cells were harvested by centrifugation at 6000 rpm for 10 min, washed with 1 volume of TN buffer (50 mM NaCl, 50 mM Tris-HCl [pH 7.6]), resuspended in 6 ml of disruption buffer (50 mM Tris-HCl [pH 7.6], 1 mM DTT, 10 mM MgCl2, 1 mM EDTA, 10% glycerol), and disrupted by sonication. Cellular debris were removed by centrifugation at 45,000 ×g and 4 °C for 60 min. Total protein amount in the crude extracts was determined using a Nanodrop apparatus (Thermo scientific, Wilmington, USA). Ni-NTA agarose resin was used to elute His6-fusion ExoP protein (Qiagen, Hilden, Germany). Protein fractions were loaded equally (5mg/lane) and separated by 10% SDS-PAGE. Electrophoresis and Coomassie brilliant blue staining was conducted by standard procedures [36].

Phosphatase activity assay. Phosphatase activity was determined using the EnzChek Phosphatase Assay kit (Life Technologies GmbH, Darmstadt, Germany) according to the supplier’s recommendation. As a substrate for purified ExoP, 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) was used, and the fluorescent product was detected by BioTek Synergy 2 Microplate Reader (BioTek Instruments, Inc., Bad Friedrichshall, Germany), at excitation of 360 nm and emission at 460 nm. The protein sample was replaced by buffer in the negative control experiment.

Results Lake data. Monthly sampling of the epilimnion (5 m) of Lake Stechlin between 2003 and 2015 showed that summer water temperatures at the sampling site reached up to 21 °C, while winter temperatures were as low as 2.5 °C (data not shown). Our routine data revealed that soluble reactive phosphorus (SRP) was low, on average ~2 mg/l. However, during the winter season, when the lake was fully mixed and often covered by an ice sheet, SRP levels increased to up to ~20 mg/l. Total phosphorus (TP) level remained in the annual range of ~13 mg/l, except for the winter season (Fig. 4A). This indicated that only ~15% of TP at 5 m depths was freely available for epilimnic organisms. According to the European Environment Agency [http://www.eea.europa.eu], mean annual total phosphorus content-based categorization of European lakes exists between class1 (TP < 0.02 mg/l) to class 6 (TP ≥ 0.50 mg/l). With our current data, Lake Stechlin would fall in class1 that is minimal TP content reported.

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Bioinformatics analyses. Phylogenetic analysis obtained from amino acid sequences of putative phosphatases from several acI lineages showed that the ExoP protein from Actinobacterium SCGC AB141-P03 was closely associated with other ExoP proteins found in acI-B tribes. The acI SAGs had up to two open reading frames (ORF) annotated as ExoPcoding genes, and both copies are included in the dendrogram.

This was also the case for the Streptomyces sviceus reference genome, which contains two ExoP sequences that are presumably paralogs. ExoP from the LD12 tribe clustered with the ExoP from the Candidatus P. ubique genome, as expected (Fig. 1).

Further examination of the Actinobacterium SCGC AB141-P03 and other acI genomes revealed that the two putative exoP genes are in the same neighborhood as other P-associated genes, e.g. phosphatase- and kinase-coding genes (Fig. 4B). Genomes of the acI lineage contain several putative but specific genes potentially involved in P metabolism, e.g., exopolyphosphatase-, serine/threonine phosphatase prpC, polyphosphate kinase-, phosphatase phoE, phosphohydrolaseand phosphate-starvation inducible protein PhoH-coding genes.

Overexpression, purification and enzyme kinetics. The de-novo synthesized 918-bp putative exoP gene from Actinobacterium SCGC AB141-P03 was successfully overexpressed heterologously in E. coli.

N-terminal His6-fusion ExoP was purified and a ~34-kDa protein band was visualized on a denaturing polyacrylamide gel (Fig. 2A). The purified protein activity data were plotted in the double reciprocal format (reciprocal of increasing velocity vs. varying substrate concentrations), which was first mentioned by Lineweaver and Burk [26]. The Michaelis constant (Km) value was determined to be 125 µM at saturated substrate concentration, by using the formula (Fig. 2B):

Km 1 1 1 = × + V Vmax [ S ] Vmax

Discussion This study aims at gaining advantage of the de novo gene synthesis approach in the field of aquatic microbial ecology by determining the function of a gene in an uncultured bacterium and its potential role for the bacterium’s ecology in a P-limited environment. Therefore, a putative P-utilizing

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gene was tested for its potential ecological role in phosphate assimilation in oligotrophic and P-poor Lake Stechlin.

Uncultured Actinobacterium SCGC AB141-P03 is a member of the acI-B clade within the highly abundant acI lineage of Actinobacteria in oligotrophic Lake Stechlin. Its genome reconstruction was conducted with the single cell genome sequencing approach and the genome sequence is now available at [http://www.jgi.doe.gov/] [13]. Note that Actinobacterium SCGC AB141-P03 has two copies of the putative exoP gene in its genome (estimated size of 2.0 Mbp), which indicates an important ecological function. This notion is supported by the fact that homologs of this gene are found within many acI SAGs, suggesting a key function for P uptake in a P-limited environment. Phosphate transporter subunit-, polyphosphate kinase- and alkaline phosphatase-encoding genes could be other potential candidate marker genes to explore phosphate utilization capability in unculturable acI Actinobacteria. But ABC-type phosphate transporter is composed of several subunits and therefore may increase the complexity in handling of full-reconstituted multimeric protein for functional in vitro testing with this approach. In

Fig. 1. Dendrogram showing the phylogenetic relatedness of amino acid sequences of putative; exoP genes from Actinobacteria belonging to the acI lineage and LD12 Alphaproteobacteria. Grey shade refers to the ExoP of ac1B SCGC AB141-P03 from Lake Stechlin, Germany.

ExoP amino acid sequences from Streptomyces sviceus ATCC 29083 and Candidatus Pelagibacter ubique were included as reference sequences. Bootstrap values above 50% are shown next to the appropriate nodes.

addition, targeting polyphosphate kinase gene (ppk1) would answer a potential for polyphosphate granule synthesis within the cell instead of direct utilization of phosphate available in environment. An alkaline phosphatase-encoding gene is not well represented in ac1 Actinobacteria. Thus, it was tempting for us to test for functionality of the putative phosphatase protein that is not only monomeric but also present in many ac1 clades of Actinobacteria. A plethora of freshwater limnological reports demonstrate that the majority of P uptake and utilization is attributed to bacteria, but information on bacterial genes related to P metabolism, particularly of purified enzymes, and their functional role is rather limited [6,8,15,35,43]. Yet, the role of phosphatases originating from limnic microplankton, e.g., algae and bacteria, has been experimentally determined by either size fractionation experiments of natural water samples or by using various bacterial isolates. Specifically, phosphatase activities have been used as indicators for the nutrient status in a given environment or at given experimental conditions [10,45,48].

To our knowledge, this study represents a first attempt to evaluate key metabolic processes of uncultured and abundant

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Fig. 2. (A) Polyacrylamide gel showing ~34-kDa purified ExoP. (B) Lineweaver–Burk plot representing a relationship between varying substrate concentration and reaction velocity of ExoP enzyme.

aquatic bacteria, e.g., actinobacterial phosphatase activity in environments with low P availability, by using the de novo gene synthesis approach.

Thereby, we demonstrate the value and feasibility of a new technical approach in microbial ecology, where a gene of interest is targeted from the information gathered from single-cell genomics, followed by de novo synthesis, heterologous expression and functional characterization. This represents a straightforward way of determining the function of protein-coding genes in uncultured microorganisms. The kinetic data of ExoP clearly proved that the putative enzyme ExoP was functional, suggesting that ExoP (together with other phosphatases coded from acI Actinobacteria genomes) should be central for exploiting phosphate in a P-poor environment. This suggestion is supported by the fact that phosphatase affinity (Km value) of natural size-fractionated, particle-associated or sediment bacterial communities as well as plankton isolates ranges from 0.1 µM to ~3 mM [5,19,25,31,32,50]. Although the measured Km value of 125 mM fits well within the range of reported Km values, it hints to the fact that ExoP of acI-B Actinobacteria may be a weak affinity enzyme. However, heterologous expression has been performed in E. coli and ExoP overexpression could not be conducted in the native Actinobacterium to determine its true affinity. Alternatively, acI Actinobacteria in Lake Stechlin might experience situations when concentrations of SRP are low, but those of TP are high. One of these events has occurred in Lake Stechlin during the breakdown of an under ice bloom of Aphanizomenon flos-aquae in March 2010 [42].

Later, Bižić-Ionescu et al., have reported that large amounts of polyphosphate were released from the lysing cyanobacterial cells, and acI Actinobacteria contributed to the majority of the free-living bacterioplankton during the initial phase of bloom in Lake Stechlin [7]. During that time TP values were high, but SRP values were found to be low (see Fig. 4A) indicating that bacterioplankton with functional phosphatases have a potential growth advantage.

Several putative P-utilizing genes are widely distributed in the available acI and LD12 SAGs indicating their important role in P-sequestering of freshwater ultramicrobacteria [http://www.jgi.doe.gov/], [13,49] (Fig. 3). Exoenzyme phosphatases are capable of releasing phosphate residues from polyphosphates and a variety of biomolecules. Import of phosphorylated molecules into the bacterial cell is possible due to the presence of the partial or entire operon of genes potentially encoding phosphate translocation ABC transporters. Genes encoding phytase and pyrophosphatase (maximum up to three copies) are present in some acI SAGs, but absent in the LD12 SAGs (Fig. 3). These enzymes allow the release of soluble inorganic P, thereby providing an instant Pi repertoire for lipid metabolism and nucleic acid synthesis.

Similarly, the presence of phosphonate utilization genes seems to be important for ocean-dwelling microorganisms.

Several ABC transporter-coding genes potentially involved in phosphonate-P import are present in the genome of marine Actinobacteria SCGC AAA015 D07 sampled from the South Atlantic Ocean, but major genes required for phosphonate catabolism have not been found in the genome [40] (Fig. 3).

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Fig. 3. Depiction of phosphate / phosphonate metabolizing protein distribution in freshwater acI and LD12 lineages in comparison to a marine Actinobacterium.

Square symbols represent the number of occurrence of genes in the genome, i.e., not present not present (□), one (■), two (■), and three (■) Y-axis represents the important and potential proteins involved in phosphate utilization. Top X-axis represents the ultramicrobacteria harboring P-utilizing genes in the following order: acI tribes, LD12 groups and a marine Actinobacteria candidate.

Genes exoP and ppk (polyphosphate kinase) were frequently found in close proximity in acI and LD12 genomes (Fig. 4B).

This could be significant for a bacterium, since phosphate concentrations can vary considerably in a limnic system.

P‑utilization genes in the LD12 genome were distributed in a more conserved manner than in acI genomes, which might be explained by the overall greater genetic diversity within acI lineages (Fig. 4B). The ecological importance of the exoP gene can be underpinned by the fact that freshwater acI Actinobacteria sometimes has two paralogs of the exoP gene when compared with other ultramicrobacteria such as LD12 and Polynucleobacter necessarius subspecies asymbioticus strain QLW-P1DMWA-1T, which both carry only one copy of this gene (Fig. 4B), [29]. Polynucleobacter necessarius

strains are known to be cosmopolitan and found in many diverse climatic zones and hence they have a much wider range of adaptations [16]. The frequent presence of P‑utilizing genes in bacterial genomes demonstrates the great potential of bacteria to successfully compete with other organisms, e.g., primary producers, when P-availability is limited.

In conclusion, the presence of several phosphatase-coding genes in ultramicrobacterial genomes retrieved from P-limited lakes indicates a concerted focus on degradation of available inorganic and organic P sources in temporally fluctuating environments. With our current approach, we can determine the function of a protein with the help of prior annotation that indicates a potential role of a gene-of-interest. In this study, we demonstrated that the exoP gene indeed coded for a functional

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Fig. 4. (A) Graph representing concentrations of total phosphorus (TP) and soluble reactive phosphorus (SRP) from Lake Stechlin between 2003 and 2015.

(B) Genetic distribution of P utilizing genes in freshwater acI Actinobacteria and LD12 Alphaproteobacteria. Gene names are abbreviated: exoP (putative exopolyphosphatase), ppk1 (putative polyphosphate kinase), sixA (putative phosphohistidine phosphatase), ppa (putative inorganic pyrophosphatase), phoB (putative phosphate regulon transcriptional regulatory protein), phoU (putative ABC-type phosphate transport system, regulatory protein), phoH (putative phosphate starvation-inducible protein PhoH), phoR (putative signal transduction histidine kinase), pstA (putative ABC-type phosphate transport system, permease protein), pstB (putative ABC-type phosphate transport system, ATP-binding protein), pstC (putative ABC-type phosphate transport system, permease protein), and pstS (putative ABC-type phosphate transport system, phosphate-binding periplasmic component).

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phosphatase. However, a phosphatase family might contain several members, e.g. exopolyphosphatase, pyrophosphatase, alkaline phosphatase, histidine phosphatase, lipid phosphatase etc. Whereas we did not carry out further refinement, we showed that a de novo gene synthesis approach can be successfully used in the field of aquatic microbial ecology. In addition to SRP, polyphosphate can substantially contribute to P sources available for bacteria in such aquatic environments, especially when SRP concentrations are low during the growing season. In Lake Stechlin, it has been shown that almost 20% of the total phosphorus from the sediment consists of polyphosphate [18], and that polyphosphate storing bacteria are abundant in the water column and sediment [14].

Hence, the presence of P-utilizing genes by ecologically successful bacteria, particularly acI Actinobacteria, indicates a valuable adaptation to a P-poor aquatic ecosystem. In order to understand the genetic potential of any uncultured microbe, de novo gene synthesis approach appear to be relevant to address a multitude of microbial metabolism related hypotheses in ecology. Caution, however, needs to be taken since a wrong pre-annotation of a gene might lead to an undesirable experimental outcome. Apart from annotation biases, heterologous expression in another host might result in a failure of functional protein synthesis. Nevertheless, our study demonstrates that the de novo gene synthesis approach can be successfully used to evaluate gene function from whole gene sequences of so far uncultivated bacteria and thereby circumvents the often labor-intensive cultivation step of environmental microbes.

Acknowledgements. We thank Elke Mach, Ute Mallok and Michael Sachtleben for technical assistance during sampling and gathering measurements of various limnetic variables, and Mark Gessner and the Department Experimentelle Limnologie of IGB Stechlin for providing data on water chemistry. This study was supported by a grant of the German Research Foundation (DFG; GR 1540/17-1) and by the Leibniz Foundation.

This material is based upon work supported by the National Institute of Food and Agriculture, United States Department of Agriculture, ID WIS01516 (to KDM). KDM also acknowledges funding from the United States National Science Foundation Long Term Ecological Research program (NTL-LTER DEB-0822700). RS acknowledges support by the United States National Science Foundation grants EF 0633142, OCE-821374 and DEB-0841933.

Competing interests. None declared

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RESEARCH ARTICLE International Microbiology (2016) 19:49-55 doi:10.2436/20.1501.01.263 ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

A functional ferric uptake regulator (Fur) protein in the fish pathogen Piscirickettsia salmonis Oscar Almarza,1 Katherine Valderrama,2 Manuel Ayala,1 Cristopher Segovia,1 Javier Santander1,3* Universidad Mayor, Faculty of Sciences, Huechuraba, Chile; 2PhD Program in Aquaculture, Universidad Catolica del Norte; 3 Memorial University of Newfoundland, Department of Ocean Sciences, Faculty of Sciences, Canada

1

Received 18 January 2016 · Accepted 25 March 2016

Summary. Piscirickettsia salmonis, a Gram-negative fastidious facultative intracellular pathogen, is the causative agent of the salmonid rickettsial septicemia (SRS). The P. salmonis iron acquisition mechanisms and its molecular regulation are unknown. Iron is an essential element for bacterial pathogenesis. Typically, genes that encode for the iron acquisition machinery are regulated by the ferric uptake regulator (Fur) protein. P. salmonis fur sequence database reveals a diversity of fur genes without functional verification. Due to the fastidious nature of this bacterium, we evaluated the functionality of P. salmonis fur in the Salmonella Δfur heterologous system. Although P. salmonis fur gene strongly differed from the common Fur sequences, it restored the regulatory mechanisms of iron acquisition in Salmonella. We concluded that P. salmonis LF-89 has a conserved functional Fur protein, which reinforces the importance of iron during fish infection. [Int Microbiol 2016; 49-55] Keywords: Piscirickettsia salmonis · ferric uptake regulator protein (Fur) · transcriptional regulatory element · iron acquisition · fish pathogens

Introduction Piscirickettsia salmonis is the bacterial pathogen with the most significant impact in the Chilean aquaculture industry and the causative agent of the salmonid rickettsial septicemia (SRS) or piscirickettsiosis [25]. Since its first description [4,7], several SRS outbreaks have occurred. Currently, 60% of the Chilean salmon farming centers are positive for P. salmonis detection [29]. P. salmonis is the only member of the Piscirickettsia genus, having the virulent strain LF-89 (ATCC Corresponding author: J. Santander Memorial University of Newfoundland Department of Ocean Sciences, Faculty of Sciences.

Ocean Science Centre; 0 Marine Lab Rd, Logy Bay, Canada Tel. + 1-709-8643268 *

E-mail: jasantanderm@asu.edu

VR-1361) as type strain [11,12]. This Gram-negative intracellular facultative bacteria replicates in infected fish cells, specifically in P. salmonis containing vesicles [13,24]. Despite the significant advancement in P. salmonis in vitro culture [14,16,36], there is a lack of knowledge about its pathogenic regulatory mechanisms due to its fastidious nature.

Salmon susceptibility to P. salmonis infection correlates with the expression of iron withholding genes [20]. For instance, salmon macrophages infected with P. salmonis trigger the expression of ferritin and transferrin [21,22]. This indicates that salmonid fish sequester iron from invading pathogens, and P. salmonis has unknown mechanisms to get this essential nutrient from the host. Iron is both a nutritional and regulatory element, determining the adaptation of bacteria to host by adjusting the expression of functional genes as well as virulence factors [27]. Bacteria capture iron from host tissues

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but also it must prevent toxic iron overloads [19]. Typically, the ferric uptake regulator (Fur) protein regulates the iron acquisition and also several virulence factors in Gram-negative bacteria [3,8,9,10]. Fur protein is a homodimer of 17-kDa subunits, originally described as a repressor upon interaction with its co-repressor Fe2+ [1,34].

Sequence analysis of the P. salmonis genome has led to the finding of several putative genes encoding for proteins related to iron metabolism (HemH, TonB, TbpB), siderophore utilization, and the ferric uptake regulator protein Fur [20,35].

Despite the relevance of this bacterial pathogen, and the wellknown relation between iron homeostasis and virulence related genes, there is no experimental verification of the functionality of P. salmonis fur gene. In addition, there are several different sequences of P. salmonis fur gene at the genome databases without functional verification. This implies no information regarding the consensus sequence necessary for DNAbinding and set of genes regulated through this interaction.

The aim of this study was to verify the functionality of P. salmonis LF-89 Fur as an iron-dependent regulator and validate the Pfur and fur sequence of the P. salmonis LF-89 type strain as a conserved regulatory mechanism.

Materials and methods Bacterial strains, media and reagents. Bacterial strains and plasmids used in this study are described in Table 1. Piscirickettsia salmonis LF89 (ATCC VR-1361) was grown on CHSE-214 monolayer cell culture, inoculated in CHAB agar plates (brain heart infusion supplemented with lcysteine 1 g/l and 5% ovine blood) and incubated at 15 °C for 20 days. A single colony was inoculated onto Austral-SRS broth [35,16] and incubated at 15 °C for another 10 days with gentle shaking (100 rpm) until ~108 cells/ ml (OD600 ~1.0). Escherichia coli DH5a and Salmonella enterica serovar Typhimurium c11143 Dfur-44 strain were grown at 37 °C in LB broth (tryptone 10 g/l; yeast extract 5 g/l and NaCl 5 g/l) or LB agar (tryptone 10 g/l; yeast extract 5 g/l; NaCl 5 g/l and 1.5% agar). Nutrient media were supplemented with ampicillin (Amp, 100 µg/ml), kanamycin (Km, 50 mg/ml) and/or chloramphenicol (Cm, 25 mg/ml) as indicated in Table 1.

In silico sequence analysis. Gene sequences were obtained from the National Center for Biotechnology Information (NCBI). Aligned amino acid sequences of Fur were obtained with EMBL-EBI Clustal Omega [29] and visualized using ESPript v.3.0 [23]. Fur three-dimensional structures were predicted by PSI-BLAST alignment and HHpred [31]. P. salmonis fur genetic context was analyzed with Softberry web-base software, using the Bprom algorithm [32].

The evolutionary history of P. salmonis Fur was inferred by using the maximum likelihood method based on the JTT matrix-based model [15]. An initial tree for the heuristic search was obtained automatically by applying neighbor-join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6 [33].

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A total of 24 Fur proteins were analyzed, comprising those of Aeromonas salmonicida subsp. salmonicida A449 (ABO90840.1), Bacillus cereus (WP_002010356.1), Coxiella burnetii RSA 493 (NC_002971.3), Cycloclasticus sp. PY97M (EPD14371.1), Edwardsiella ictaluri 93-146 (NC_012779.2), Edwardsiella tarda strain PPD 130/91 (AEO72442.1), Escherichia coli str. K-12 substr. MG1655 (NP_415209.1), Flavobacterium columnare ATCC 49512 (AEW85616.1), Flavobacterium psychrophilum JIP02/86 (CAL43969.1), Francisella tularensis subsp. tularensis SCHU S4 (YP_169106.1), Haemophilus influenzae Rd KW20 (NP_438359.1), Hydrogenovibrio marinus (WP_029909427.1), Legionella pneumophila (NZ_ CCZV01000002.1), Methylophaga nitratireducenticrescens (AFI85361.1), Piscirickettsia salmonis LF-89 = ATCC VR-1361 (KJ804204.1), Pseudomonas aeruginosa PAO1 (NP_253452.1), Pseudomonas putida (WP_003249922.1), Salmonella enterica subsp. enterica serovar Typhimurium str. LT2 (NP_459678.1), Staphylococcus aureus subsp. aureus NCTC 8325 (YP_500494.1), Streptococcus mutans UA159 (NP_721026.1), Thioalkalimicrobium cyclicum ALM1 (AEG31828.1), Thiomicrospira chilensis (WP_028486797.1), Vibrio cholerae (AAA27519.1), and Yersinia pestis strain KIM5 (WP_002210357.1).

Cloning procedures. Genomic DNA extraction was performed using Wizard® Genomic DNA purification kit (Promega, Madison, WI, USA).

PCR amplifications of P. salmonis Pfur-fur and Plac-fur were performed using Vent DNA polymerase (New England, Biolabs). The products were purified with a purification kit from Qiagen (Valencia, CA, USA) and submitted for sequencing at Macrogen (Seoul, South Korea). The Pfur-fur fragment contains 200 bp upstream of the fur starting codon and PCR amplified using the forward primer 5′-GACTTGTGTTTGTGTCATGCC-3′. The Plac-fur sequence was PCR amplified using the forward primer5′-TTTACACTTTATGCTTCC GGCTCGTATGTTTAAAGGAGAGGTAACCTATGTCTCAACAAG-3′.

The reverse primer utilized was 5′-CAGCCATGCCAATCACAACG-3′. The PCR fragments were cloned into TOPO-TA (Invitrogene). The obtained plasmids were named pEZ287 (Pfur) and pEZ283 (Plac) (Table 1).

Heterologous fur complementation in c11143 Salmonella enterica subsp. enterica serovar Typhimurium Dfur-44. The Dfur-44 strain was complemented with P. salmonis fur expressed under its promoter and Plac, independently. Edwardsiella ictaluri fur gene was used as positive control (pEZ116) [27]. Complemented S. enterica subsp. enterica serovar Typhimurium Dfur-44 cells were transformed with each plasmid using the CaCl2 method [26]. Transformant colonies were used to evaluate P. salmonis Fur synthesis and functionality.

Growth under iron-restricted conditions. Fifty ml of LB supplemented with iron (FeSO4 μM) and LB supplemented with 2, 2-dipyridyl (150 mM and 250 mM) were inoculated with 50 ml of early log-phase culture. The cultures were incubated at 37ºC with aeration (180 rpm). Bacterial growth was monitored by optical density (A600nm). Samples were measured in triplicate and identical experiments were performed twice.

SDS-PAGE and western blotting. Synthesis of P. salmonis Fur in χ11143 S. enterica subsp. enterica serovar Typhimurium Dfur-44 harboring pEZ283, pEZ287 or pEZ116 was evaluated by Western blot. One ml of middle log culture, grown at 37 °C with aeration (180 rpm), were harvested. After centrifugation, bacterial cells were resuspended in 100 ml sample buffer [18] and heat denatured for 10 min. Total protein were separated in 12% SDSPAGE transferred onto a nitrocellulose membrane and blocked using a fatfree milk solution (5% wt/vol) in phosphate-buffered saline with 0.05% Tween 20 (PBS-T). The membranes were incubated overnight at 4 °C with 1:10,000 dilution of anti-Fur primary rabbit polyclonal antibody [19]. After washing with PBS-T, the membranes were incubated with a 1:10,000 dilution of the secondary antibody (alkaline phosphatase-conjugated anti-rabbit im-

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Table 1. Strains and plasmids used in this study Strains Piscirickettsia salmonis LF-89

Relevant characteristics

Reference

ATCC VR 1361 Type culture strain

ATCC; [13]

Escherichia coli DH5α

F–Φ80lacZ∆M15 ∆(lacZYA-argF) U169 recA1 endA1 hsdR17 (rK–, mK+) phoA supE44 λ– thi-1 gyrA96 relA1

Invitrogen

Salmonella enterica serovar Typhimurium c3761

UK-1; wild-type

[27]

Salmonella enterica serovar Typhimurium c11143

UK-1; ∆fur44

[27]

Plasmids

TOPO vector

3.9 kb, pUC ori, Km , Amp

pEZ283

4.3 kb, pUC ori, Km , Amp , P fur (P. salmonis fur), TOPO

This study

pEZ287

4.3 kb, pUC ori, Kmr, Ampr, Pfur–fur (P. salmonis fur), TOPO

This study

pEZ116

4.6 kb, Pfur-fur (E. ictaluri fur), Cmr, pSC101ori, pACYC184

[27]

r r

munoglobulin G, Sigma). Immune reactive proteins were detected using a chromogenic substrate for alkaline phosphatase (5-bromo-4-chloro-3-indolylphosphate and nitroblue tetrazolium). A primary rabbit serum against GAPDH was used as control.

Detection of secreted siderophores. Siderophore synthesis was evaluated in chromoazurol S (CAS) agar plates [6,28]. One ml of an overnight culture of S. enterica subsp. enterica serovar Typhimurium χ11143 Dfur-44 complemented strain was pelleted, washed two times and resuspended in 50 µl of PBS. A 10-µl aliquot of each bacterial culture was spotted on CAS agar plates, and incubated overnight at 37 °C. Siderophore production was visualized as a yellow-orange halo around the bacterial growth.

Detection of iron-free siderophores by thin layer chromatography (TLC). Salmonella enterica iron-free siderophores were obtained by the following method. Bacterial cultures grown overnight in 10 ml of LB broth for 18–24 h at 37 °C (OD600 of 1.0) supplemented with FeSO4 (150 mM) or with 2, 2′-Dipyridyl (150 µM) were harvested by centrifugation at 5,000 ×g for 10 min. The supernatants were passed through 0.2 µm poresize membrane filter to remove residual bacteria, acidified with 50 ml of 10 N HCl and extracted twice with 4 ml of ethyl-acetate to obtain catechol siderophores [17]. The aqueous phase was dried with gaseous N2 and resuspended in 80 ml of methanol. Twenty µl were spotted onto 225-mm-layer (20 × 20 cm) TLC Silica gel 60 F254 aluminum sheets and the chromatography was developed with benzene-glacial acetic acid-water (125:72:3, vol./vol./vol.) in a closed chamber. The aluminum sheets were removed, dried and immersed in 0.1% FeCl3 to visualize the sideropheres.

Outer membrane protein analysis. Sarkosyl-insoluble outer membrane proteins (OMPs) were obtained as previously described [5]. OMP proteins were isolated from S. enterica subsp. enterica serovar Typhimurium Dfur-44 grown in iron-replete conditions (LB supplemented with 150 μM FeSO4) and iron-regulated outer membrane proteins (IROMPs) were isolated from Salmonella enterica subsp. enterica serovar Typhimurium Dfur-44 grown in LB supplemented with 2′2′ -dipyridyl (150 mM) (iron-limited con-

Promega

r r

– lac

ditions). The total proteins were normalized to 25 mg/μl by using the nano Genova spectrophotometer (Jenway) and separated by 10% (wt/vol) SDSPAGE. Coomassie blue staining was performed to visualize proteins.

Results Piscirickettsia salmonis fur sequence analysis.

Piscirickettsia salmonis fur gene has a 444 bp length, similar to S. Typhimurium (453 bp), E. coli (447 bp), E. ictaluri (450 bp) and A. salmonicida (429 bp). However, the G+C content of P. salmonis fur (40.7%) is lower compared to S. Typhimurium (47.2%), E. coli (47.9%), E. ictaluri (53.8%) and A. salmonicida (54.1%). Also, P. salmonis fur gene has a low percentage of identity compared to E. coli and Salmonella fur genes, with 29% and 52% respectively. The low G+C content of P. salmonis fur is shared with phylogenetic relatives from the genera Coxiella and Legionella (Fig. 1). Piscirickettsia salmonis Fur contains 147 amino acid residues, which 71 residues are identical to E. ictaluri (149 aa) and S. enterica subsp. enterica serovar Typhimurium (150 aa) (Fig. 2A). In terms of structure, we found that P. salmonis Fur has two putative functional domains: a DNA-binding domain in the N-terminal region, and a C-terminal domain involved in dimerization (Fig. 2B). According to its metalloprotein nature, the amino acid residues related to Fe2+ and Zn2+ binding pockets are perfectly aligned in all the compared sequences (Fig. 2A). The differences in the amino acid residues between P. salmonis Fur and

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Iron-free catechol siderophore synthesis in S. Typhimurium Dfur-44 complemented with P. salmonis Fur was evaluated by TLC analysis. All bacterial cultures grown under irondeprived conditions were able to synthesize siderophores. In contrast, when the bacterial cultures were supplemented with iron (FeSO4, 150 mM) the wild type and the complemented Dfur-44 strains did not synthesize siderophores (Fig. 3C). In contrast, the non-complemented Dfur-44 strain synthesized siderophores constitutively (Fig. 3C).

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Outer membrane protein profile. Wild-type S. Typhimurium expressed IROMPs (iron regulated outer membrane proteins) only under iron-deprived condition. In contrast, Dfur-44 strain showed a constitutive IROMPs expression (Fig. 3D).

IROMPs regulation was restored in S. Typhimurium Dfur-44 complemented with P. salmonis fur gene (Fig. 3D).

Growth under iron-restricted conditions. Growth curves were determined in LB, LB supplemented with 150 μM FeSO4 and LB supplemented with 150 mM and 250 mM of 2,2′-dipyridyl at 37 °C. As shown in Fig. 3E, S. enterica subsp. enterica serovar Typhimurium Dfur-44 complemented with P. salmonis Pfur-fur (pEZ287) showed a longer generation time (Fig. 3E).

Fig. 1. Evolutionary relationships based on Fur protein sequence.

Discussion other Fur proteins have no evident impact on the secondary and tertiary structure (Figs. 2A-2B).

Piscirickettsia salmonis fur gene cloning and protein expression. Piscirickettsia salmonis fur was expressed in χ11143 Dfur-44 under control of its promoter (Fig. 3A) and under control of Plac promoter. P. salmonis Fur showed a molecular weight of 16 kDa, similar to the 16.9 kDa predicted in silico (Fig. 3A).

Detection of siderophores synthesis regulated

by Piscirickettsia salmonis Fur. Salmonella Typhimurium Dfur-44 displays a constitutive secretion of siderophores, forming an orange-yellow halo around the bacterial colony on CAS agar plates [28]. Complementation with a functional Fur protein restores the iron-mediated gene repression, precluding the constitutive synthesis of siderophores, as observed in the strain transformed with pEZ116 (positive control). S. Typhimurium Dfur-44 transformed with pEZ283 (P. salmonis, Plac-fur) or pEZ287 (P. salmonis, Pfur-fur) restores the iron-mediated gene repression, evidencing that P. salmonis fur gene encodes a functional Fur (Fig. 3B).

The NCBI and UNIPROT databases have a diversity of P. salmonis fur genes without functional validation. Here, we analyzed the fur genes of the virulent P. salmonis LF-89 type strain. The phylogenetic analysis showed that P. salmonis Fur grouped with Francisella, Coxiella and Legionella, which are closely related genera (Fig. 1). Despite the nucleotide differences of P. salmonis fur genes, the structure of P. salmonis Fur protein was very similar to other Gram-negative Fur proteins (Fig. 2). The structural identity and amino acid similarity at the active sites of Fur, supported the P. salmonis Fur functionality. For instance, the amino acids related to Zn2+ and Fe2+ binding were conserved, in agreement with the iron-dependent regulation found in the complementation assays. Sequence analysis and comparisons showed that P. salmonis Fur was prone to dimerization since residues required for dimerization and meta binding were present in P. salmonis Fur (Fig. 2B). Cysteines in positions 92 and 95 are essential for the activity of Fur proteins family [6]. These cysteine residues are found in a CXYCG motif [2], which is also present in P. salmonis Fur (Fig. 2A).

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Piscirickettsia salmonis fur functionality was confirmed in S. Typhimurium Dfur-44, revealing that, despite the differences in G+C content of P. salmonis genome, the predicted fur promoter (with standard -10 and -35 sequences) was functional when tested in S. Typhimurium Dfur-44. However, the expression of P. salmonis fur in S. Typhimurium Dfur-44 under control of its promoter caused a longer generation time, in contrast to P. salmonis fur under Plac control (Fig. 3E). These results suggest that P. salmonis Fur might recognize similar consensus sequences than Salmonella and E. coli Fur. However, the promotor of P. salmonis fur gene seems to have a pleiotropic effect on S. Typhimurium growth (Fig. 3E).

The fastidious nature of P. salmonis and its facultative intracellular life cycle precluded the study of basic aspects of its biology, however utilization of heterologous systems, like S. Typhimurium, can be utilized to understand virulent mechanisms of P. salmonis.

In summary, we identified the fur gene of P. salmonis and demonstrated the functionality of the encoded protein in a heterologous complementation system. The analysis also showed that P. salmonis Fur transcriptional regulation was

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Fig. 2. Piscirickettsia salmonis Fur alignment and structure. (A) Fur alignment and secondary structure. The secondary structure is displayed as spirals (representing a-helix) and arrows (representing b-sheets). The structure on the top of the alignment is P. salmonis Fur protein and the structure at the bottom is from Salmonella enterica subsp. enterica serovar Typhimurium Fur protein. DNA-binding protein domain is marked with black stars, and the dimerization domains are double underlined. The white and black circles indicate the amino acid residues related with Zn2+ or Fe2+ binding pocket respectively. Black triangles indicate the cysteine residues related to Escherichia coli Fe2+ and Zn2+ binding pockets. (B) Three dimensional structure predictions of Fur proteins.

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iron-dependent. This study provided a useful starting point for the analysis of the Fur-regulated genes in P. salmonis, the elucidation of its Fur-box sequence and the unveiling of novel mechanisms of bacterial infection in fish.

Acknowledgements. This work was funded by Grant No. 1140330 from FONDECYT-Chile, and by Grant COPEC−UC 2014.J0.71.

Competing interests: None declared.

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Fig. 3. Fur protein expression and complementation assays. (A) Synthesis of Fur in Salmonella enterica subsp. enterica serovar Typhimurium Dfur-44 verified by western blot analysis. GAPDH was used as control; pEZ116 (Escherichia ictaluri Fur); pEZ287 (Piscirickettsia salmonis Fur). (B) Detection of siderophore in CAS agar plates. (C) Detection of secreted siderophores in S. enterica subsp. enterica serovar Typhimurium grown under iron-rich (+) and iron-limited (–) conditions by TLC. (D) Outer membrane protein profile of S. enterica subsp. enterica serovar Typhimurium grown under iron supplemented (+) or iron deprived (–) conditions. The arrowhead indicate Fur-regulated IROMP proteins. (E) Growth of S. enterica subsp. enterica serovar Typhimurium Dfur-44 growth at 37 °C with aireation (180 rpm).

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26. Sambrook J, Russell W (2001) Molecular cloning: a laboratory manual.

Cold Spring Harbor Laboratory Press, NY 27. Santander J, Golden G, Wanda SY, Curtiss III R (2012) The Fur regulated iron uptake system of Edwardsiella ictaluri and its influence on pathogenesis and immunogenicity in the catfish host. Infect Immun 80:2689-2703 doi:10.1128/IAI.00013-12 28. Schwyn B, Neilands JB (1987) Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160:47-56 doi:10.1016/0003-2697(87)90612-9 29. SERNAPESCA (2014) Informe sanitario de salmonicultura en centros marinos, año 2013. Servicio Nacional de Pesca y Agricultura. Gobierno de Chile. Santiago de Chile 30. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, Lopez R, McWilliam H, Remmert M, Soding J, Thompson JD, Higgins DG (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol 7:539 doi:10.1038/msb.2011.75

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31. Söding J, Biegert A, Lupas AN (2005) The HHpred interactive server for protein homology detection and structure prediction. Nucleic Acids Res 33:W244-248 doi:10.1093/nar/gki408 32. Solovyev V, Salamov A (2011) Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW (ed.) Metagenomics and its application in agriculture, biomedicine and environmental studies.

Nova Science Publishers, New York, pp 61-78 33. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: Molecular Evolutionary Genetics Aanalysis version 6.0. Mol Biol Evol 30:2725-2729 doi:10.1093/molbev/mst197 34. Troxell B, Hassan HM (2013) Transcriptional regulation by ferric uptake regulator (Fur) in pathogenic bacteria. Front Cell Infect Microbiol 3:59 doi:10.3389/fcimb.2013.00059 35. Yáñez AJ, Molina C, Haro RE, Sánchez P, Isla A, Mendoza J, RojasHerrera M, Trombert A, Silva AX, Cárcamo JG, Figueroa J, Polanco P, Manque P, Maracaja-Coutinho V, Olavarría VH (2014) Draft genome sequence of virulent strain AUSTRAL-005 of Piscirickettsia salmonis, the etiological agent of piscirickettsiosis. Genome Announc. 2:e0099014 doi:10.1128/genomeA.00990-14 36. Yáñez AJ, Silva H, Valenzuela K, Pontigo JP, Godoy M, Troncoso J, Romero A, Figueroa J, Cárcamo JG, Avendaño-Herrera R (2013) Two novel blood-free solid media for the culture of the salmonid pathogen Piscirickettsia salmonis. J Fish Dis 36:587-591 doi:10.1111/jfd.12034

RESEARCH ARTICLE International Microbiology (2016) 19:57-67 doi:10.2436/20.1501.01.264 ISSN (print): 1139-6709. e-ISSN: 1618-1095

www.im.microbios.org

Antimicrobial activity of Lactobacillus strains of chicken origin against bacterial pathogens Marta Dec,* Andrzej Puchalski, Anna Nowaczek, Andrzej Wernicki Sub-Department of Veterinary Prevention and Avian Diseases, Institute of Biological Bases of Animal Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, Lublin, Poland Received 16 February 2016 · Accepted 16 March 2016

Summary. This study was conducted to identify and evaluate the antimicrobial activity of some Lactobacillus isolates of chicken origin. Among 90 isolates 14 Lactobacillus species were distinguished using MALDI-TOF mass spectrometry and 16S-ARDRA. The dominant species was L. salivarius (34.4%), followed by L. johnsonii (23.3%), L. crispatus (13.3%) and L. reuteri (11.1%). All lactobacilli were screened for antimicrobial activity against wild-type strains of Salmonella enterica, Escherichia coli, and Clostridium perfringens. Results from the agar slab method showed that all Lactobacillus isolates were able to produce active compounds on solid media with antagonistic properties against these pathogens. The highest sensitivity to lactobacilli was observed in C. perfringens strains, and the lowest in E. coli. Lactobacillus salivarius exhibited particularly strong antagonism towards all of the indicator bacteria. Strains of L. ingluviei and L. johnsonii and one strain of L. salivarius (10d) selectively inhibited the growth of C. perfringens. No antimicrobial activity of many Lactobacillus isolates was observed when cell-free culture supernatant was used in a well diffusion assay. All Lactobacillus isolates exhibited the ability to produce H2O2 and proved to be hydrophobic (excluding one of L. salivarius). [Int Microbiol 19(1):57-67 (2016)] Keywords: Lactobacillus spp. · avian lactobacilli · antimicrobial activity · gut health · poultry pathogens

Introduction The poultry industry is one of the fastest growing segments of the livestock sector in the world. At the same time, however, due to high production efficiency, the dietary and health needs of poultry require particular care. Among aspects that should be taken into account for optimum poultry performance, the

Corresponding author: M. Dec Sub-Department of Veterinary Prevention and Avian Diseases Institute of Biological Bases of Animal Diseases Faculty of Veterinary Medicine University of Life Sciences in Lublin Akademicka 12, 20-033 Lublin, Poland Tel. +48-814456965. Fax +48-814456032 *

E-mail: marta.dec@up.lublin.pl; martde16@gmail.com

overall health and proper functioning of the avian gastrointestinal tract (GIT) is crucial [41]. Gut health is maintained by complex mechanisms in which the commensal microflora seems to have a pivotal role. It is involved in host physiology, metabolism and absorption of nutrients, and recent studies on gut microbiota function have highlighted its importance in health and disease. The protective potency of beneficial gut microflora is of particular interest and knowledge of its composition is critical to understanding the function of members of the microbiota [37].

Enteric disorders are one of the most important problems in the poultry industry, with necrotic enteritis, salmonellosis and colibacillosis regarded as the major bacterial diseases occurring in chicken. Clostridium perfringens, Salmonella spp. and Esch­ erichia coli infections range from severe acute disease to mild infections of a chronic nature. They cause substantial economic

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losses worldwide due to increased mortality rates and treatment costs, decreased rates of body weight gain, poor feed conversion efficiency, and increased risk of contamination of poultry products for human consumption. Enteric disorders can also result in reduced egg production, reductions in fertility, and low hatchability of infected eggs [27,30].

In Europe, the number of diagnosed gastrointestinal tract (GIT) infections in poultry, particularly those induced by C. perfringens, increased after 2006, when antibiotic growth promoters were removed from the list of allowed feed additives [6]. This has prompted the search for alternative methods for preventing intestinal infections in chickens. One strategy is based on the use of probiotics —live microorganisms which when administered in adequate amounts confer a health benefit on the host. The fundamental role of probiotics is to maintain the bacterial balance in the gut by eliminating unfavourable microflora, and antimicrobial activity is a key criterion in the selection of probiotic strains [26]. Administration of probiotic feed additives is particularly advisable in chickens whose intestinal microflora is not yet formed and whenever its stability is at risk, e.g., during antibiotic treatment, when their diet is changed, or when the birds are exposed to stress factors (e.g., overcrowding, inappropriate ventilation and temperature, insufficient water or feed). Stress lowers immune resistance and disrupts the balance of the intestinal microflora, which facilitates colonization of the GIT by pathogens, leading to the development of infections [4,26]. Hence ensuring the appropriate composition of the intestinal microflora is the best means of improving the immunity of the organism and the health of the animals.

Bacteria of the genus Lactobacillus are recognized candidates for probiotics. They are non-pathogenic Gram-positive rods that naturally inhabit the mucous membranes of humans and animals. In chicken, Lactobacillus colonization of the alimentary tract takes place soon after hatching, during food ingestion. From the crop they pass through successive parts of the chicken intestine and become important members of the microbial population [3]. Lactobacilli are lactic acid bacteria that play an important beneficial role in the physiology of their host by providing a protective barrier in the gut. In addition, they improve digestion and assimilation of nutrients, remove toxic substances, and enhance immunity [11]. The use of selected Lactobacillus strains as feed additives for poultry can reduce infections caused by intestinal pathogens such as Salmonella [34,44], C. perfringens [5,23], E. coli [18], Cam­ pylobacter sp. [14] and Brachyspira pilosicoli [28]. Administration of probiotics prevents pathogenic bacteria from colonizing the intestinal epithelium and passing into the internal

DEC ET AL.

organs and eggs [8]. Through elimination of unwanted microflora and other beneficial activity in the gut, selected Lactoba­ cillus strains can also promote weight gain in birds [19] and increase egg production [33].

Probiotic lactobacilli may protect animals from intestinal pathogens by several possible mechanisms, including production of inhibitory substances, such as organic acids, hydrogen peroxide, bacteriocins and carbon peroxide, blocking of adhesion sites on intestinal epithelial surfaces, competition for nutrients, and stimulation of immunity [24]. These health-benefiting properties of lactobacilli are largely dependent on their prolonged residence in the GIT and are dictated by adherence to the intestinal mucosa. The adhesion mechanism involves passive forces and electrostatic and hydrophobic interaction, as well as specific binding dependent on bacterial surface adhesins [15].

The objective of this study was to identify native lactobacilli of chicken origin and evaluate their probiotic potential, expressed as in vitro ability to suppress the growth of C. per­ fringens, S. enterica and E. coli. In addition, the adhesive properties of Lactobacillus isolates were assessed by determination of their hydrophobicity.

Materials and methods Bacteria and growth conditions. Lactobacillus isolates were from the fresh faeces or cloacae of 30 healthy chickens (broilers and Green-legged Partridge hens) from eight large-scale poultry farms in Poland. The age of the birds ranged from 2 days to 7 weeks. Samples were inoculated into MRS medium (BTL, Poland) supplemented with 0.05% (w/v) cysteine hydrochloride (Sigma-Aldrich, Poland) (MRS-cys). The plates were incubated at 37 °C for 48 h in 5% CO2. Only catalase-negative Gram-positive rods were considered as presumtively belonging to the genus Lactobacillus and were stored at –80 °C until further analysis.

Strains of Salmonella enterica subsp. enterica (3 strains, serovars Enteritidis, Newport and Typhimurium), E. coli (1 isolate) and Clostridium per­ fringens (3 isolates), used in the experiment to evaluate the antibacterial activity of Lactobacillus sp., were isolated from chickens (intestinal contents) with symptoms of infection affecting the digestive tract (salmonellosis, colibacteriosis and necrotic enteritis). Species identification of all pathogenic isolates was confirmed using matrix-assisted laser desorption⁄ionization timeof-flight mass spectrometry (MALDI-TOF MS, Bruker, Germany) according to the procedure described below.

Identification of Lactobacillus strains using MALDI-TOF MS. Bacteria were identified using an UltrafleXtreme MALDI-TOF mass spectrometer (Bruker, Germany) as previously described [9]. The mass spectra were processed with the MALDI Biotyper 3.0 software package (Bruker, Germany), and the results were shown as the top 10 identification matches along with confidence scores ranging from 0.00 to 3.00. The identification result was considered reliable when at least the two best matches having a log(score) of 1.70–3.00 with the MALDI Biotyper database indicated the same species. For samples for which the top two matches indicated different

ANTIMICROBIAL ACTIVITIES OF LACTOBACILLUS STRAINS

species, we took into account the first match, provided that the log(score) was ≥ 0.25 greater than the value for the second match [9]. If the scores from two independent runs were <1.7 or a sample yielded a MALDI mass spectrum with no peaks, ethanol/formic acid extraction was performed (according to the protocol recommended by the manufacturer, Bruker, Germany).

Differentiation of L. johnsonii and L. gasseri strains by 16S-ARDRA. Twenty-one isolates for which definitive species identification was not obtained using MALDI-TOF MS (L. johnsonii/L. gasseri) were identified using 16S-ARDRA with the MseI restriction enzyme, as described in our previous publication [9].

Detection of antibacterial activity of Lactobacillus strains.

Agar slab method. The Lactobacillus sp. isolates grown on MRS-cys broth were centrifuged and suspended in 0.9% NaCl so that the optical density of the suspension at 600 nm (OD600) was 0.5. Plates 4 cm in diameter containing 15 ml MRS agar were inoculated with 200 μl of lactobacilli and incubated at 37 °C in 5% CO2 for 24 h. Then agar slabs 9 mm in diameter were cut and placed on Müller-Hinton agar inoculated with 0.5 ml of the target indicator strain suspended in 0.9% NaCl (OD600 = 0.1 for Salmonella sp. and E. coli, OD600 = 0.8 for C. perfringens). For initial diffusion of the substance from the agar slabs, the plates were first refrigerated for 3 h at 4 °C and then kept for 24 h at 37 °C in aerobic conditions for Salmonella and E. coli or in anaerobic conditions for C. perfringens. After incubation, the plates were checked for inhibition zones. The results are presented as the mean diameter of the inhibition zone ± SD for two independent experiments.

Detection of antibacterial activity of Lactobacillus strains.

Well diffusion method. Lactobacillus isolates were grown in a 1.2 ml volume of MRS-cys broth for 24 h (37 °C, 5% CO2). The bacteria were separated from the medium by centrifugation and then each sample of medium was divided into 2 equal volumes. In half of the samples the pH was adjusted to 6.5–7.0 using NaOH (to eliminate the effect of organic acids), and an equal volume of water was added to the remaining samples, with pH 3.5–5.0.

The indicator bacteria were inoculated on Müller-Hinton agar according to the protocol described above. Cylindrical metal wells 8 mm in diameter were placed on the plates and filled with 100 μl of the cell-free supernatant.

After 24 h of incubation in conditions appropriate for the indicator bacteria (described above), the plates were checked for inhibition zones. The results are presented as the mean diameter of the inhibition zone ± SD from two independent experiments.

Detection of H2O2 production by Lactobacillus isolates.

The lactobacilli were plated on MRS-cys supplemented with TMB substrate (0.25 mg/ml, Sigma-Aldrich) and horseradish peroxidase (0.01 mg/ml, Sigma-Aldrich) and grown for 48 h at 37 °C, 5% CO2. Blue colour in the colonies indicated H2O2 production by the bacteria. Colour intensity was designated as follows: –, +, ++, +++ [10].

Measurement of bacterial hydrophobicity. Hydrophobicity of the investigated bacteria was determined on the basis of microbial adhesion to hydrocarbon as described by Rosenberg [38]. Lactobacilli grown in MRS-cys broth were centrifugated and resuspended in 0.02 M PBS, pH 6.8, to an optical density (OD600) of 0.8–1.0 (A0), 1,7 ml xylene was added to glass test tubes containing 5 ml of bacterial suspension. The mixtures were vortexed vigorously for 90 s. After phase separation of about 15 min the optical density of the aqueous phase (A) was again measured and compared with the initial value. The percentage of cell surface hydrophobicity (%H) was calculated using the following equation: %H= [(A0 – A)/A0] × 100.

Strains with hydrophobicity equal to or more than 50% were considered hydrophobic.

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Statistical analysis. The mean diameters of the inhibition zones for indicator microorganisms that were determined to be sensitive to various Lactobacillus species were compared by one-way analysis of variance (with species as a categorical predictor and zone as a dependent variable adjusted for the pathogen), with the Tukey HSD (honest significant difference) post hoc test, with modification for unequal N, as a correction for multiple comparisons. Normal distribution of data was tested using the Shapiro–Wilk test and the equality of variance was tested by the Brown–Forsythe test. Due to a lack of normal distribution and/or unequal variance of data, Kruskal–Wallis analysis of variance was used to analyse the differences between means. A level of P < 0.05 was considered statistically significant. All statistical analyses were carried out using Statistica 10.0 software (StatSoft, Inc., Tulsa, OK, USA).

Results Identification of Lactobacillus isolates. Lacto­ bacillus bacteria were isolated from all samples tested, and 2–7 strains of varying colony morphology were isolated from each sample. A total of 90 isolates was identified as bacteria of the genus Lactobacillus using MALDI-TOF mass spectrometry. For 32 (35.5%) of the strains the log(score) was 1.70– 1.99, for 50 (55.5%) strains it was 2.00–2.29, and for 8 (8.8%) it was 2.30–3.00. For 69 (76.7%) strains either at least the two best matches in Biotyper indicated the same species or the difference between the first and second best matches indicating different species was greater than 0.25. Identification of these isolates was considered to be reliable. For 21 strains (23.3%) the first and second best matches indicated different species, and the differences between their log(score) values were less than 0.25. For these isolates the best match indicated L. johnsonii and the second best match indicated L. gas­ seri.

The 90 isolates identified belonged to 8 Lactobacillus species: L. salivarius 31 strains (34.4%), L. johnsonii/L. gasseri, 21 (23.3%), L. crispatus 12 (13.3%), L. reuteri 10 (11.1%), L. ingluviei 8 (8.9%), L. agilis 3 (3.3%), L. saerimneri 3 (3.3%) and L. oris 2 strains (2.2%).

Differentiation of L. johnsonii/L. gasseri strains by 16S-ARDRA. Analysis of the electrophoretic profiles obtained by digestion of 16S amplicon with MseI showed that all the strains previously identified in MALDI-TOF MA as L. johnsonii/L. gasseri belonged to the species L. johnsonii. The electrophoretic profiles of these wild isolates and the reference strain L. johnsonii LMG 9436 contained five bands of molecular size 940, 256, 145, 130 and 90 bp. The electrophoretic profile of the reference strain L. gasseri ATCC 19992 differed from the profiles of the remaining strains and comprised 5 restriction fragments of 680, 450, 237, 130 and 90 bp (Fig. 1).

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Fig. 1. Agarose gel image of 16S amplicons digested with MseI. Lines: L.g. – Lactobacillus gasseri ATCC 19992; L.j. – L. johnsonii LMG 9436; 1–3 – examples of wild Lactobacillus strains identified in MALDI-TOF MS as L johnsonii /L.gasseri.

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Agar slab method. The diameter of the growth inhibition zones of the test bacteria induced by the lactobacilli ranged from 9 ± 0.0 to 27 ± 2.83 mm, where the diameter of the slab

was 9 mm (Fig. 2). Individual indicator strains exhibited varied susceptibility to the lactobacilli. All 90 Lactobacillus isolates inhibited the growth of all three strains of C. perfringens.

The Salmonella Typhimurium ST strain was inhibited by 60 (33.3%) strains of Lactobacillus, Salmonella Typhimurium A by 58 (64.4%), Salmonella Enteritidis by 48 (53.3%), Salmo­ nella Newport by 47 (52.2%), and E. coli by 34 (37.7%) lactobacilli. The largest mean inhibition zones (16.7–17.3 mm) were observed in the case of C. perfringens isolates, and ANOVA of the mean diameters showed that the C. perfringens strains were more sensitive (P < 0.05) than other indicator bacteria to the antagonistic substances produced by lactobacilli (Fig. 3, Table 1). More detailed analysis showed that the average inhibition zones of C. perfringens strains were significantly higher (P < 0.05) than the zones obtained for Salmonella (all serovars) and E. coli due to the antagonistic effect of L. sali­ varius, L. ingluviei, L. johnsoni, L. crispatus and L. reuteri (Table 1). The E. coli, Salmonella Enteritidis and Salmonella Newport strains were found to be the least susceptible to Lac­ tobacillus activity. The average inhibition zone of these pathogens (10.3±1.8 – 11.0±2.1 mm) caused by the lactobacilli (when all lactobacilli were considered as one group) differed (P < 0.05) from the average zone of inhibition obtained for Salmonella Typhimurium and C. perfringens. Moderately large zones were observed for Salmonella Typhimurium strains (12.7 ± 3.6 – 12.8 ± 3.5 mm) (Table 1, Fig. 3).

Fig. 2. Anatagonistic activity of Lactobacillus sp. against indicator bacteria in the agar slab method. (A) Salmonella Typhimurium ST. (B) Clostridium perfringens A. (C) C. per­ fringens PW1. (D) C. perfringens L3.

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1The results are presented as mean diameter of the growth inhibition zone (mm) for two independent experiments; the diameter of the agar slab was 9 mm.

2n = number of strains. a–d Different superscript lower-case letters indicate differences between mean values in columns (comparison of the sensitivity of the indicator microorganism to the antagonistic substances produced by strains of each Lactobacillus species); P < 0.05.

A–E Different superscript capital letters indicate differences between mean values in rows (comparison of the inhibitory effect of various Lactobacillus species on each strain of indicator bacteria); P < 0.05.

10.9 ± 2.3A 11.0 ± 2.1A Mean

12.7 ± 3.6B

12.8 ± 3.5B

10.3 ± 1.8A

16.7 ± 3.0C

16.8 ± 3.4C

17.3 ± 3.2C

10.5 ± 1.2bd 11.1 ± 1.9b,AB 11.0 ± 0.8b,AB 11.9 ± 0.8b,A 9.0 ± 0.0b,B 10.9 ± 0.8abc,AB 9.7 ± 0.9ac,AB 9.4 ± 0.5abc,AB L. oris (n=2)

10.7 ± 0.5acb,AB

11.3 ± 3.0bd 15.2 ± 1.1b,BC 14.6 ± 2.4b,AC 14.6 ± 2.4b,AC 9.0 ± 0.0b,A 10.3 ± 1.7bc,AC 9.0 ± 0.0b,A 9.0 ± 0.0bc,A L. saerimneri (n=3)

9.2 ± 0.4bc,A

12.3 ± 2.8bcd 16.2 ± 0.8a,CE 14.4 ± 0.8b,BE 15.6 ± 1.2ab,CDE 9.2 ± 0.4b,A 11.8 ± 1.6abc,ABDE 9.7 ± 1.2ac,AB 9.7 ± 1.2bc,AB L. agilis (n=3)

11.9 ± 1.3acb,ABC

12.0 ± 4.2bd 16.5 ± 3.6b,B 17.0 ± 3.0ab,B 16.3 ± 3.1ab,B 9.1 ± 0.3b,A 9.4 ± 1.5cd,A 9.0 ± 0.0b,A 9.2 ± 0.9bc,A L. ingluviei (n=8)

9.4 ± 1.4bc,A

12.4 ± 3.3cd 16.1 ± 2.2b,B 15.0 ± 3.4b,BC 15.7 ± 2.6b,B 9.1 ± 0.4b,A 12.0 ± 1.9b,CD 9.8 ± 1.3b,AE 9.9 ± 1.3bc,AD L. reuteri (n=10)

11.8 ± 1.6b,CDE

13.3 ± 3.8c 17.0 ± 2.7b,B 17.3 ± 3.5ab,B 16.4 ± 2.2b,B 9.9 ± 1.4b,A 11.7 ± 2.4bd,A 10.7 ± 2.0c,A 11.2 ± 2.1c,A L. crispatus (n=12)

12.0 ± 3.3b,A

11.3 ± 3.3b 15.4 ± 2.3b,B 14.8 ± 2.6b,B 14.7 ± 2.4b,B 9.0 ± 0.0b,A 9.0 ± 0.1c,A 9.0 ± 0.0bc,A 9.0 ± 0.0b,A L. johnsonii (n=21)

9.1 ± 0.4c,A

16.0 ± 3.5a 19.6 ± 2.5a,B 19.0 ± 2.4a,B 18.8 ± 2.5a,B 12.1 ± 1.6a,A 16.4 ± 1.9a,C 16.3 ± 2.4a,C 13.1 ± 1.8a,A 13.2 ± 1.2a,A

C. perfringens PW1 C. perfringens L3 C. perfringens A E. coli D7 Salmonella Typhimurium A Salmonella Typhimurium ST Salmonella Enteritidis Salmonella Newport

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L. salivarius (n=31)

Well diffusion method. The pH of the supernatant obtained from the 24 h culture of Lac­ tobacillus strains ranged from 3.0 to 4.5. The inhibition of growth of pathogenic bacteria by na-

Table 1. ANOVA of the growth inhibition of indicator microorganisms by Lactobacillus isolates, as determined by the agar slab method1

Lctobacillus salivarius exerted particularly strong antagonism against all of the pathogens, as the mean growth inhibition zone diameter for the indicator strains (when all the indicator strains were considered as one group) induced by isolates of this species was 16.0 ± 3.5 mm and differed significantly (P < 0.05) from the mean growth inhibition zones caused by the remaining Lactobacillus species (≤ 13.3 ± 3.8 mm) (Table 1, Fig. 4). Such a remarkable potent antimicrobial activity of L. salivarius strains was also observed when the sensitivity of each indicator microorganism was considered individually (Table 1).

Strains of the species L. oris, L. johnsonii, L. saerimneri and L. ingluviei exhibited weak antagonistic properties. The average diameters of the growth inhibition zones of the pathogenic bacteria (considered as one group) induced by these species of Lactobacillus were ≤ 12.0 ± 4.3 mm (Table 1, Fig. 4). However, the assays in which each indicator strain was considered individually showed that the activity of Lactobacillus species varied depending on the species of an indicator bacterium. The isolates of L. ingluviei and L. johnsonii were ineffective towards Salmonella and E. coli, but they selectively inhibited (14.7 ± 2.7–17.0 ± 3.0 mm) the growth of C. perfringens (Figs. 5C and 5D).

Based on the experiment, the strains with the strongest antagonism towards the indicator strains were chosen. Among 15 selected isolates, 13 belonged to the species L. salivarius and 2 to the species L. ingluviei (Table 2). Particularly noteworthy is the strong selective inhibitory activity of the strains L. salivarius 10d and L. inglu­ viei 9a and 18b against growth of C. perfringens.

Average diameters for the inhibition of the growth of C. perfringens caused by these strains ranged from 20.0 ± 0.9 mm (9e) to 21.2 ± 2.2 mm (10d), while the growth of other indicator strains was inhibited only slightly (isolate10d inhibited the growth of Salmonella, up to 10.2 ± 1.3 mm) or not all (9e, 18b).

Mean

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Fig. 3. The susceptibility of the indicator strains to Lactobacillus species, as determined by the agar slab method. The results are presented as mean diameter of the growth inhibition zone (mm) for two independent experiments; the diameter of the agar slab was 9 mm; Different capital letters (A–C) indicate significant differences (P < 0.05) between mean size of growth inhibition zones of pathogenic bacteria caused by Lactobacillus isolates (when all Lactobacillus strains where considered as one group); the vertical bars denote 0.95 CI.

Fig. 4. The effect of strains of different Lactobacillus species on the growth of indicator strains, as determined by the agar slab method. The results are presented as mean diameter of the growth inhibition zone (mm) of all indicator bacteria for two independent experiments; the diameter of the agar slab was 9 mm. Different small letters (a-e) indicate significant differences (P < 0.05) between mean diameter of growth inhibition zones caused by various Lactobacillus species; the vertical bars denote 0.95 CI.

tive cell-free broth was generally very weak or absent, even in the case of many Lactobacillus strains that showed a strong inhibitory effect in the agar slab method. The size of the inhibition zones caused by native acidified supernatants was up to 14.5 ± 0.7 mm, where the well diameter was 8 mm.

The highest susceptibility to the antagonistic activity of an acidic environment was exhibited by the C. perfringens strains, as their growth was inhibited by 90–92% (depending on the C. perfringens strain) of the media with acidic pH. The inhibition zones for these indicator strains ranged from 8.00 ± 0.0 to 14.5 ± 0.7 mm, and the ANOVA of the mean diameters showed that the C. perfringens strains were more sensitive (P < 0.05) than the other indicator microorganisms to the antagonistic substances present in the cell-free Lactobacillus media (Fig. 6). In the case of Salmonella strains (all serovars), very small inhibition zones of up to 9.0 ± 0.0 mm were observed, and inhibition was caused only by 8–12% (depending on the indicator strain) of unneutralized cell-free supernatants. None of the native media was able to inhibit the growth of the E. coli indicator strain.

Cell-free supernatants with neutralized acids (pH 6.5–7.0) did not exhibit antagonistic activity towards the indicator strains, with the exception of ten supernatants (from the cultures of different Lactobacillus species) which exhibited a

slight inhibitory effect towards C. perfringens (∅ 8.5–11 mm). Statistical analysis (Kruskal-Wallis test) showed no significant difference between the inhibitory effect of the cellfree culture supernatants of different species of Lactobacillus.

Production of H2O2. All Lactobacillus strains tested produced H2O2. The highest rate of production (+++) was observed in 59 isolates, including all strains belonging to the species L. johnsonii, L. ingluviei, L. agilis, L. saerimnerii and L. oris and some strains of L. salivaius, L. crispatus and L. reuteri. Moderate hydrogen peroxide production (++) was noted in 14 strains, most of which were of the species L. sali­ varius. The group with the lowest H2O2 production (+) comprised 17 strains, including 15 strains of L. salivarius and 2 strains of L. crispatus.

The bacteria capable of producing H2O2, grown on MRS medium supplemented with TMB and horseradish peroxidase, varied not only in colour intensity, but also in the manner in which the colonies were coloured. In some strains of L. salivarius the blue colour appeared only on the periphery of the bacterial colonies, while in other lactobacilli, e.g., L. john­ sonii, L. ingluviei and L. agilis, entire colonies were blue. In some L. salivarius strains only the middle of the colony and their periphery was blue, resembling an eye.

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Table 2. Lactobacillus strains inducing the largest inhibition zones of pathogenic indicator bacteria in the agar slab method Pathogen

Size of inhibition zone

Lactobacillus isolates

Clostridium perfringens (A, L3, PW1)

≥20 mm

L. salivarius: 6a, 8b, 10d, 17a, 23a, 24b, 27e, 40a L. ingluviei: 9e, 18b

Salmonella Typhimurium (ST, A)

≥18 mm

L. salivarius: 5a, 21b, 22a, 24b, 30b

Salmonella Enteritidis and Newport

≥14 mm

L. salivarius: 6b, 17a, 21a, 24b, 27e, 40a

Escherichia coli (D7)

≥14 mm

L. salivarius: 6b, 17a, 27e

Hydrophobicity. The %H of all lactobacilli tested, except one strain of L. salivarius (50d), was ≥50%, and therefore these isolates were considered hydrophobic. The vast majority of the strains tested showed high affinity towards xylene for 65.5% Lactobacillus isolates the %H was 90–100% (Table 3). The strains of L. johnsonii showed relatively low hydrophobicity compared to the other lactobacilli tested; only 38.1% of isolates displayed hydrophobicity at 90–100% and as many as 33.3% exhibited %H in the range of 50–69%.

Discussion In the present work, we successfully identified chicken lactobacilli to the species level using MALDI-TOF mass spectrometry, and for some strains additionally by 16S-ARDRA. The reliability and effectiveness of these methods in typing lactobacilli has been confirmed in our previous research [9].

The Lactobacillus species identified in this study from broiler chickens and laying hens raised in Poland are similar to those identified from broiler chickens around the world, supporting the notion that these species are autochthonous inhabitants

within the chicken GIT. The occurrence of L. salivarius, L. crispatus, L. johnsonii and L. reuteri in the GIT of broilers have been also observed by Wang [47] and Vidanarachchi et al. [45]. Some other reports have pointed out the predominance of Lactobacillus crispatus, L. reuteri and L. salivarius, but not L. johnsonii among intestinal chicken lactobacilli [3,7,16,17]. The occurrence of L. ingluviei, L. agilis and L. sarimneri strains in the chicken GIT has been also described [31,44]. Differences in the frequency of isolation of some Lactobacillus species from the chicken GIT reported by various authors may be the result of different breeding conditions, the diet of the birds, and the procedures for isolating and identifying bacteria.

The results of the agar slab method showed that Lactoba­ cillus bacteria originating in chickens have growth-inhibiting properties for bacterial poultry pathogens and that this antagonistic effect depends on the type of pathogen and is due to the production of antimicrobial substances by lactobacilli.

Our findings are in agreement with those of Kizerwetter-Swida and Binek [21] who observed a greater antibacterial in vitro effect of chicken lactobacilli against C. perfringens than against E. coli and Salmonella Enteritidis. Other authors

Table 3. Percentage hydrophobicity of Lactobacillus strains 90–100%

70–89%

50–69%

<50%

L. salivarius (n = 31)

17 (54.8%)

7 (22.6%)

6 (19.3%)

1 (3.2%)

L. johnsonii (n = 21)

8 (38.1%)

6 (28.6%)

7 (33.3%)

–

L. crispatus (n = 12)

11 (91.7%)

1 (8.3%)

–

–

L. reuteri (n = 10)

7 (70.0%)

3 (30.0%)

–

–

L. ingluviei (n = 8)

8 (100%)

–

–

–

L. agilis (n = 3)

3 (100%)

–

–

–

L. saerimneri (n = 3)

3 (100%)

–

–

–

L. oris (n = 2)

2 (100%)

–

–

–

Total (n = 90)

59 (65.5%)

17 (18.9%)

13 (14.4%)

1 (1.1%)

species

%H

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Fig. 5. The susceptibility of the indicator strains to selected Lactobacillus species, as determined by the agar slab method. Different capital letters (Aâ&#x20AC;&#x201C;C) indicate significant differences (P < 0.05) between mean diameter of growth inhibition zones caused by the strains of L. salivarius (A), L. crispatus (B), L. ingluviei (C) and L. johnsonii (D). Different capital letters (Aâ&#x20AC;&#x201C;C) indicate significant differences (P < 0.05) between mean size of growth inhibition zones of pathogenic bacteria caused by the strains of individual Lactobacillus species; the vertical bars denote 0.95 CI.

[18,43] showed that chicken lactobacilli were more effective in inhibiting the growth of Salmonella than E. coli, but contrary to our findings they did not observe greater sensitivity of Salmonella Typhimurium compared to Salmonella Enteritidis.

Antimicrobial in vitro activity of L. salivarius strains and some other Lactobacillus species isolated from chickens against Salmonella, E. coli and C. perfringens has also been observed by many other authors [2,25,32,42,48], and some of them [13] concluded that organic acids produceb by lactobacilli are resposible for inhibityory effect. It was also shown that Lactobacillus strains of chicken origin exert a protective effect in vivo, especially L. salivarius against Salmonella in chickens [20,34,35,40]. Kizerwetter-Ĺ&#x161;wida and Binek [20] and La Ragione et al. [23] demonstrated antimicrobial effect of selected strains of L. salivarius and L. johnsonii against C. perfringens in chickens, but there are no reports of anti-clostridial activity of L. ingluviei strains.

The results of the well diffusion method indicated that the reduced pH of the supernatant (probably due to lactic acid) might play a role in inhibiting pathogenic bacteria. However we were unable to clearly identify which substances produced by lactobacilli growing on agar inhibited the growth of pathogenic bacteria. This was because the antagonistic activity of the native cell-free broth was very weak or absent in the case of most Lactobacillus strains, including those that showed a strong inhibitory effect in the agar slab method. This phenomenon, also observed by some other authors [25,36], can be explained by the fact that the release of antimicrobial molecules by lactobacilli is influenced by culture conditions, cell density and population kinetics [1]. Moreover, Lactobacillus bacteria grown on agar medium are able to synthesize bacteriocins in significantly greater amounts than in a liquid culture [39]. In the case of C. perfringens, which was grown in anaerobic conditions, it should be taken into account that after

ANTIMICROBIAL ACTIVITIES OF LACTOBACILLUS STRAINS

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Int. Microbiol. Vol. 19, 2016

the antagonistic bacteria were transferred onto the agar slab they were able to grow simultaneously with the indicator bacteria. Anaerobic culture conditions may have stimulated production of bacteriocins or other antibacterial substances by lactobacilli.

The results of the present study showed that Lactobacillus sp. strains originating in chicken produce H2O2. However, production of this reactive oxygen species was not clearly correlated with the antimicrobial activity of lactobacilli observed in the slab method. All strains of L. johnsonii, L. ingluviei and L. oris exhibited strong production of hydrogen peroxide, but they were generally inactive towards Salmonella and E. coli.

Moreover, some L. salivarius strains that most strongly inhibited the growth of pathogens exhibited intermediate (++) or even weak (+) H2O2 production. Correlations between Lacto­ bacillus species and the ability to produce H2O2 and a lack of relationship between antimicrobial activity of lactobacilli and the intensity of H2O2 production were also observed in our previous work on goose lactobacilli [10]. The ability of chicken intestinal lactobacilli to produce H2O2 was also reported by Heravi et al. [17] and Mota et al. [29], but contrary to our findings, these authors recognised L. salivarius strains as the best producers of H2O2, while the isolates of L. johnsonii were considered weak producers (+) or H2O2-negative (–).

Hydrophobicity of bacteria is dependent on cell surface components and generally reflects the adhesive ability of bacteria. Several researchers have reported a high degree of correlation between hydrophobicity of Lactobacillus strains and

65

Fig. 6. The susceptibility of the pathogenic bacteria to Lactobacillus species, as determined by the well diffusion method. The results are presented as mean diameter of the growth inhibition zone (mm) for two independent experiments (the media obtained after cultivation of all Lactobacillus strains were considered as one group); the diameter of the metal well was 8 mm; the vertical bars denote 0.95 confidence intervals. Different capital letters (A-B) means significant differences (P < 0.05) between mean diameter of growth inhibition zones caused by native cell free-media.

their adhesion to epithelial cells [12,22,46]. The results of our study showing high hydrophobicity of lactobacilli tested are in line with data obtained by Heravi et al. [17], who reported that the adhesion of 8 chicken strains of L. salivarius, L. cris­ patus, L. johnsonii and L. reuteri to xylene ranged from 78.2% to 93.2%. Mota et al. [29] found that almost 80% of chicken intestinal lactobacilli had hydrophobic surfaces (H > 50%).

In summary, gut health challenges are currently the most important issue for poultry production. Knowledge of the composition of the intestinal microflora is critical for understanding the contribution of microbiota members to the wellbeing of the avian host and for selection of probiotics. The results presented here demonstrate that Lactobacillus isolates from chickens may have probiotic potential in reducing intestinal infections. The study made it possible to select strains of Lactobacillus characterized by antagonistic properties towards bacterial pathogens resulting from the production of growth inhibitory compounds and adhesive properties. They can be considered for use as prophylactic agents or as an alternative to antibiotic therapy for infections with Salmonella, E. coli or C. perfringens in chickens.

Acknowledgements. The authors thank Dr Elżbieta Kukier of the National Veterinary Research Institute in Puławy for supplying strains of Clos­ tridium perfringens, Marcin Markiewicz, DVM, for his assistance in collecting the Lactobacillus strains and Tomasz Banach, MSc, for his technican assistance during identification of bacteria using MALDI-TOF MS. Competing interests. None declared.

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