AFAB Volume 3 Issue 4

ISSN: 2159-8967 www.AFABjournal.com

Volume 3, Issue 4 2013

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EDITORIAL BOARD Sooyoun Ahn

Hae-Yeong Kim

University of Florida, USA

Kyung Hee University, South Korea

Walid Q. Alali

Woo-Kyun Kim

University of Georgia, USA

University of Georgia, USA

Kenneth M. Bischoff

M.B. Kirkham

NCAUR, USDA-ARS, USA

Kansas State University, USA

Debabrata Biswas

Todd Kostman

University of Maryland, USA

University of Wisconsin, Oshkosh, USA

Claudia S. Dunkley

Y. M. Kwon

University of Georgia, USA

University of Arkansas, USA

Lawrence Goodridge

Maria Luz Sanz

Colorado State University, USA

MuriasInstituto de Quimica Organic General, Spain

Leluo Guan

Melanie R. Mormile

University of Alberta, Canada

Missouri University of Science and Tech., USA

Joshua Gurtler

Rama Nannapaneni

ERRC, USDA-ARS, USA

Mississippi State University, USA

Yong D. Hang

Jack A. Neal, Jr.

Cornell University, USA

University of Houston, USA

Armitra Jackson-Davis

Benedict Okeke

Alabama A&M University, USA

Auburn University at Montgomery, USA

Divya Jaroni

John Patterson

Oklahoma State University, USA

Purdue University, USA

Weihong Jiang Shanghai

Toni Poole

Institute for Biol. Sciences, P.R. China

FFSRU, USDA-ARS, USA

Michael Johnson

Marcos Rostagno

University of Arkansas, USA

LBRU, USDA-ARS, USA

Timothy Kelly

Roni Shapira

East Carolina University, USA

Hebrew University of Jerusalem, Israel

William R. Kenealy

Kalidas Shetty

Mascoma Corporation, USA

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EDITORIAL STAFF EDITOR-IN-CHIEF Steven C. Ricke University of Arkansas, USA

EDITORS Todd R. Callaway FFSRU, USADA-ARS, USA Philip G. Crandall University of Arkansas, USA Janet Donaldson Mississippi State University, USA

MANAGING and LAYOUT EDITOR Ellen J. Van Loo Ghent, Belgium

TECHNICAL EDITOR Jessica C. Shabatura Fayetteville, USA

ONLINE EDITION EDITOR C.S. Shabatura Fayetteville, USA

Ok-Kyung Koo Korea Food Research Institute, South Korea

ABOUT THIS PUBLICATION Mailing Address: 2138 Revere Place . Fayetteville, AR . 72701

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TABLE OF CONTENTS ARTICLES 268 The Role of Cellular Prion Proteins (PrPC) on Neuronal Brucella Infections M. Aydin, D. F. Gilmore, S. Erdogan, V. Duzguner, and S. Ahn

281 Prevalence of Foodborne Pathogens and Spoilage Microorganisms and their Drug Resistant Status in Different Street Foods of Dhaka

Z. Tabashsum, I. Khalil, Md. N. Uddin, A.K.M. M. Mollah, Y. Inatsu and Md. L. Bari

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Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Producing Bacteria In Vitro H. D. Edwards, R. C. Anderson, T. M. Taylor, R. K. Miller, M. D. Hardin, N. A. Krueger, D. J. Nisbet

303 Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens

J. G. Wilson, T. C. McLaurin, J. A. Carroll, S. Shields-Menard, T. B. Schmidt, T. R. Callaway, and J. R. Donaldson

312 Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over time A. R. Mantz, D. N. Miller, M. J. Spiehs, B. L. Woodbury, and L. M. Durso

Introduction to Authors 327 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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NEW EDITORIAL STAFF Biography of new AFAB Editor: Dr. Janet Donaldson It is the pleasure of the AFAB editorial staff to welcome Dr. Janet Donaldson as a newly appointed editor beginning in January, 2014. Dr. Janet Donaldson is currently an Associate Professor in the Department of Biological Sciences at Mississippi State University. She is a microbiologist with special interests in determining mechanisms by which bacteria are able to grow and adapt to conditions within the gastrointestinal tract. Her research is primarily focused upon identifying these mechanisms in Listeria monocytogenes and Escherichia coli O157:H7. Her work has identified variations in survival as related to strain diversity, which sheds light on the mechanisms by which these dangerous pathogens survive and cause disease. She has also identified a novel probiotic that provides additional energy to the host. Other research interests include probiotics mechanisms of actions and applicability. She also has research interests related to improving bioenergy sources through microbial community manipulations. She has been the recipient of the National Pork Board Innovations in Research Award in 2013 and also the Randall Lectureship award for her research. Dr. Donaldson has published 21 peer-reviewed journal articles. She has been a PI or Co-PI on several grants, with funded research totaling over $12.5 million. She and her students have given over 50 presentations since 2008 at both national and international conferences and venues. She is an associate editor for five journals in her field, has been an invited reviewer for 23 journals, and is currently the president elect of the South Central Branch of the American Society for Microbiology.

Biography of new AFAB Editor: Ok-Kyung Koo Ok-Kyung Koo, PhD, is a senior scientist in Food Safety Research Group at Korea Food Research Institute since 2012. She completed both bachelor’s and master’s degrees in the Department of Food and Animal Biotechnology from Seoul National University in South Korea. Then Koo joined PhD program in Dr. Arun K. Bhunia’s Molecular Food Microbiology lab at Purdue University in 2006. Her doctoral dissertation was on “Listeria adhesion protein and heat shock protein 60: Application in pathogenic Listeria detection and implication in listeriosis prevention”. After the degree, she moved to Fayetteville, Arkansas to work as a postdoctoral research associate in Center for Food Safety at the University of Arkansas in 2010. With Dr. Steven Ricke and Dr. Philip Crandall in UA, she conducted research on understanding the microbial ecology of food processing environment and application of probiotics as well as different chemical and physical methods to control the contamination of L. monocytogenes and Salmonella spp.. Her current research focuses on food safety epidemiology, pathogenesis of foodborne pathogens and their interaction with background bacteria in the food system, and natural antimicoribal agents including probiotics. She is also an assistant professor at the University of Science and Technology in South Korea.

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Dr. Armitra Jackson-Davis appointed to AFAB editorial Board Dr. Armitra Jackson-Davis is an Assistant Professor of Food Microbiology at Alabama A&M University. At Alabama A&M University, she has teaching and research responsibilities in addition to the advisement of undergraduate and graduate students. She earned her Bachelor of Science degree in Animal Science from the University of Arkansas-Pine Bluff and her Master of Science and Doctor of Philosophy degrees from Iowa State University in the area of Meat Science with a food microbiology emphasis. While at Iowa State University, she received the Iowa State University Teaching Excellence Award. Dr. Jackson-Davis believes that children should be educated at an early age when it comes to safe food handling practices. As a result, she authored “The Birthday to Remember Forever”, which is the first in the series “Eating safe with Ace and Mace”. The series is designed to teach safe food-handling practices to children in a storytelling manner. Her research interests include investigating the microbiological safety of food products labeled as “natural” and organic. Her work related to this research area has been published in the Journal of Food Protection and Meat Science. She was recently awarded the 2013-2014 Grant for Minority Serving Institutions to conduct research that evaluates multiple-hurdle antimicrobial technologies on the inactivation of Escherichia coli O157:H7 in beef trim.

She has travelled internationally to learn more about different food systems around the world.

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www.afabjournal.com Copyright © 2013 Agriculture, Food and Analytical Bacteriology

The Role of Cellular Prion Proteins (PrPC) on Microglial Brucella Infections M. Aydin1, D. F. Gilmore2, S. Erdogan3, V. Duzguner4, and S. Ahn5* Molecular Biosciences Program, Arkansas State University, Jonesboro, AR 72401 Department of Biological Sciences, Arkansas State University, Jonesboro, AR 72401 3 Department of Medical Biochemistry, Emine-Bahaeddin Nakiboglu Medical School, Zirve University, 27260 Gaziantep, Turkey 4 Ardahan University, Health Services Vocational School, Ardahan, Turkey 5 Food Science and Human Nutrition Department, University of Florida, Gainesville, FL 32611 1

2

ABSTRACT Brucella species can settle and proliferate in microglial cells of host animals. The primary focus of this mini-review is to discuss the biochemical and pathogenic processes that could potentially develop between the host and the agent. Brucella’s antioxidant responses to host’s oxidative reactions, which are one of the defense systems in neuronal cells against the Brucella infection, are believed to be an important element in its pathogenicity; however their exact mechanisms to exert pathogenicity are not fully understood. In this review, the effects of cellular prion proteins (PrPC) on entrance of Brucella into host cells and on development of oxidative defenses in the host cells will be discussed. Additionally, we will discuss the potential of utilizing small interference RNA or short interference RNA to suppress the expression of PrPC and determine the subsequent effect on Brucella infection on microglial cells. Finally the effects of PrPC on oxidative events, and roles of the Brucella virulence factors during the entrance into the host cells will also be discussed.

Keywords: Brucella, prions, microglial, infections

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INTRODUCTION Although brucellosis has been eradicated in most developed countries, it is still an endemic disease in many regions in the Middle East and Mediterranean countries (Al-Sekait, 2000; Boschiroli et al., 2001; PapCorrespondence: Soohyoun Ahn, sahn82@ufl.edu Tel: +1 352-392-1991 Ext. 310 Fax: +1 352-392-9467

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pas et al., 2006; Mantur et al., 2007). Consequently brucellosis remains an important public and animal health problem in many countries in these regions.

This results in tremendous economic losses in these respective geographical regions. Brucellosis is a zoonotic disease that can easily be transmitted to humans from raw or inadequately heated milk and products derived from raw milk such as cream, butter, and cheese. Brucellosis can be considered an occu-

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pational disease as it is mostly observed in farmers, livestock keepers, veterinarians, butchers, or employees of meat and dairy production facilities (Corbel, 1997). Direct contact with sick animals and their urine, blood, etc. could transmit Brucella from sick animals to humans (Megid et al., 2010). Additionally, oral route, respiratory tract, eyes, and open wounds are other important ways for Brucella to enter the body.

Brucellosis causes abortions or stillbirths in female ruminants and orchitis in male ruminants as well as cerebral peroxidation which can lead to tremendous economic losses (Corbel, 1990; Orozco et al., 2003; Melek et al., 2006; Kataria et al., 2010). Humans who have been infected by Brucella can develop chronic

It has been thought that the virulence factors of the bacteria consist of outer membrane lipopolysaccharides (LPS) and it has been speculated that proteins involved in signalling, gene regulation, and transmembrane transportation may also be involved(Hong et al., 2000; Jimenez de Bagues et al., 2005; Franco et al., 2007). It has also been established that the LPS-O side chain in particular may be an important feature of the bacteria that allows them to protect themselves from the host defense systems that they may encounter (Freer et al., 1996; Giambartolomei et al., 2004). Brucella species, however, contrary to what is considered typical for most other Gram-negative bacterial pathogens, do not possess

symptoms such as undulant fever, loss of appetite, extreme sweating, and arthritis (Franco et al., 2007; Christopher et al., 2010). Due to its resistance to simple treatments and its potential as a biological weapon, Brucella is considered one of the most important pathogens (Leitenberg, 2001).

Cellular events underlying the development of brucellosis have not yet been fully revealed. Therefore the focus of this mini-review is to discuss the potential infection mechanism of Brucella and various experimental approaches to gain a better understanding of the infection mechanism. In particular, this review will discuss host oxidative processes that might serve as defense mechanisms and the potential roles for cellular prion proteins (PrPC) in Brucella infection. To the best of our knowledge there are currently few published studies in the literature that have examined oxidative processes as a host defensive system and the roles of PrPC on the development of the disease. In addition, the potential for a small interference RNA-based transfection approach for determining the role of PrPC in neurobrucellosis will be discussed.

exotoxins, invasive proteases, capsules, or virulence plasmids. Therefore, they can easily enter the host’s reticuloendothelial system (RES) and eventually mononuclear phagocytes, and remain viable while replicating themselves in the intracellular environment of the host cells (Ficht, 2003; Gross et al., 2003).

Brucella ensure their intracellular survival by avoiding fusion of the phagosome in which they are contained with lysosomes in macrophages (Celli and Gorvel, 2004). Thus, these bacteria can gain entry into the intracellular environment of the host via the host’s phagocytic cells, and after successful entry, may be carried to and remain latent in a variety of organ tissues such as the spleen, brain, heart, and bone marrow for years after the initial exposure (Leitenberg, 2001; Gross et al., 1998, 2003). This obviously makes them problematic over an extended period of time and creates an ongoing risk for the host and susceptible individuals exposed to the organism.

The next section addresses the virulence properties of Brucella and the standard assays that can be used to assess their virulence.

BRUCELLA – GENERAL CHARACTERISTICS

BRUCELLA – GENERAL PATHOGENESIS AND STANDARD ASSAYS

Brucella spp. are Gram-negative, facultative, nonmotile, and non-spore forming bacteria that can be either intracellular or extracellular pathogens (Mantur and Amarnath, 2008; Christopher et al., 2010).

In most Brucella infection studies, Brucella melitensis M16 strain has been used to infect tissue culture cells because this strain is known for its virulence in humans. The main differences between Brucella

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and other pathogens such as Yersinia, Salmonella, and Listeria are its survival ability and reproducibility within the immune defense system of the host cells (i.e. microglia). These abilities are due to a variety of virulence factors that have been identified over the years. For example, Brucella species can activate the virB operon and in turn initiate the synthesis of Type IV secretion systems (T4SS; O’Callaghan et al., 1999).

By utilizing this virulence factor, Brucella species can invade the host cell and live in it without causing any reaction within the endocytic vacuole (phagosome) (O’Callaghan et al., 1999). It has also been reported that Brucella infection prevents the synthesis of inflammatory cytokines tumor necrosis factor alpha

any invasion assay, gentamicin has been shown not to affect internalized bacteria (Durant et al.,1999, 2000a,b; Ficht, 2003; Howard et al., 2005).

To assess internalized cells as well as intracellular metabolites requires preparation of cell homogenates. After incubation of host cells with Brucella, trypsin is typically added to remove the tissue culture cells from the bottom of the flasks. After incubation with trypsin for 1 to 2 minutes with gentle shaking, suspended cells are collected by pipette and centrifuged to remove them from the culture medium. At this point, a tissue culture cell lysis buffer is added and cytoplasmic contents are collected by centrifugation. The generated cellular homogenates then

(TNF-α), interferon-gamma (IFN-γ), and interleukin-1 (IL-1β) in humans and domestic animals (Gross et al., 2003; Erdogan et al., 2007, 2008). However, some recent studies reported that Brucella species could cause secretion of IL-1β and TNF-α in mouse macrophages and microglia cells (Covert et al., 2009; Samartino et al., 2010).

The classical way to assess and quantitate pathogenesis for any microorganism is invasion assay, also known as the gentamicin protection assay, in which the organism of interest is incubated with the corresponding type of tissue cell culture to determine the ability of pathogenic bacteria to invade the target eukaryotic cells. In an invasion assay with B. melitensis, the number of the bacterial cells can simply be determined after serial dilution by colony counting or by a spectrophotometer to arrive at a specified inoculation level. Subsequently the resulting bacterial suspension is typically washed several times with a phosphate buffered saline (PBS) solution, and an aliquot of this suspension is introduced to the tissue cell culture. In most studies the preferred bacteria/ tissue culture cell ratio is set at 20:1. The flasks containing these bacterial/tissue culture cells are incubated at 37°C, with a 5% CO2 atmosphere in a sterile incubator for 30 minutes to allow for phagocytosis

can be subjected to either direct culture to quantify the internalized bacterial cells or biochemical analyses to measure the effect of Brucella invasion on the production of specific metabolite effects by tissue culture cells.

Production of reactive oxygen species (ROS) such as superoxide, nitric oxide, and hydroxyl ions are among the most important defense mechanisms that are developed by infected host cells (Fang, 1997; Kaymak et al., 2011). Bactericidal and apoptosis-stimulating properties of these molecules can help eliminate pathogens. Nitric oxide is an antimicrobial molecule produced in the macrophages from L-arginine by inducible nitric oxide synthase (iNOS) that serves as an immune defense mechanism during inflammations and infections (Fang, 1997). To determine the level of nitric oxide, nitrite and nitrate content can be measured by the Griess reaction after deproteinization (Green et al., 1982; Sun et al., 2003). Typically when conducting the assay, coppercoated cadmium granules and glycine buffer (pH 9.7) are incubated for 90 minutes with test samples and the reduction of nitrate to nitrite are measured by spectrophotometry at 545 nm. This reduction results in the generation of a pink color, which is formed by diazotization of sulfanilamide and related N-naph-

of the bacteria. The bacteria that are not phagocytized by tissue culture cells are initially washed away with PBS containing 30 µg/mL gentamicin to ensure elimination of all remaining bacteria on the cell surfaces. At this concentration that is routinely used in

thylethylene diamine (NNDA).

To counter host defense mechanisms, Brucella can produce antioxidant enzymes such as superoxide dismutase and catalase. Therefore, assessing the levels of these enzymes in infected cells can be

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a critical component of evaluating Brucella pathogenicity. Superoxide dismutase is an antioxidant enzyme, which strongly inhibits phagocytosis-associated nitroblue tetrazolium (NBT) reduction by removing superoxide anion (O2-) radical produced by xanthine/xanthine oxidase reaction (Johnston et al., 1975; Choi et al., 2006). When estimating the activity of this enzyme in homogenate samples, a mixture of chloroform/ethanol is added to the sample, mixed by vortexing and subsequently centrifuged. After centrifugation, the upper aqueous phase can be used for the test by adding this collected supernatant to a mixture that contains xanthine, NBT, bovine serum albumin and xanthine oxidase enzymes (Sun et al.,

infrequent, but are clinically important for their severity and high morbidity (Ceran et al., 2011). The most common symptom of neurobrucellosis is headache with meningeal irritation (Mousa et al., 1986; Shakir et al., 1987). It is still not clear how Brucella gain entry into the central nervous system. Two proteins that have been shown to have a role in intracellular pathogenicity and invasion are BvrR and BvrS ((Brucella virulence, Sola-Landa et al., 1998; Gándara et al., 2001). These proteins regulate production of the Type IV secretion system in B. abortus and may be involved in allowing Brucella spp. to enter central nervous system cells (Nunez-Martinez et al., 2010). It has been previously shown that the entrance of Bru-

1988). Following incubation at 25°C, CuCl2 is added to stop the reaction and each sample can be read on a spectrophotometer at 560 nm. Catalase activity can be measured directly from homogenates using a spectrophotometric assay based on the absorbance of hydrogen peroxide at a wavelength of 240 nm. A decrease in absorbance can be used as an indicator of catalase production. With the production of these enzymes, Brucella can eliminate the free radicals that may be produced by the host cell (Kim et al., 2000).

For example, it has been suggested that B. abortus after early infection may survive by either expressing genes to counteract the impact of a high nitric oxide environment or activate genes that allow it to use nitric oxide as a potential nitrogen source (Wang et al., 2001). Consequently, Brucella spp. may be able to resist the host oxidative defense system and protect themselves from host’s bactericidal defensive attacks (Orozco et al. 2003; Gross et al. 2004). The following sections describe the interaction between the central nervous system and Brucella.

cella to macrophages and intracellular replication is reduced by inactivation of the BvrR-BvrS system in Brucella mutants (Sola-Landa et al., 1998; Gándara et al., 2001; Guzmán-Verri et al., 2002). In a later study, Guzmán-Verri et al. (2002) determined that the BvrRBvrS system regulates the expression of outer membrane proteins (Omp), especially Omp3a (Omp25) and Omp3b, in B. abortus. Another study reported that Brucella were able to enter and invade the host cells by a sialic acid-mediated lectin recognition receptor (Del Carmen Rocha-Gracia et al., 2002).

BRUCELLA INTERACTION WITH THE CENTRAL NERVOUS SYSTEM It has been reported that in 1.3 to 11% of brucellosis cases, Brucella settle and cause disease in the central nervous system (Mousa et al., 1986; McLean et al., 1992; Gul et al., 2008, 2009; Buzgan et al., 2010; Erdem et al., 2012). Neurological complications are

BRUCELLA INFECTION AND PRION PROTEINS Cellular prion proteins (PrPC) and scrapie-type prion proteins (PrPSC) are also called sialoglycoproteins because they contain sialic acid in their structures (Prusiner, 1991, 1995). PrPSC is morphologically different than PrPC. Although the exact three dimensional structure of PrPSC is unknown, it has a higher proportion of β-sheet structure in place of the normal α-helix structure (Pan et al., 1993). PrPSC is an infectious protein that is responsible for several prion diseases including bovine spongiform encephalopathy (mad cow disease), Creutzfeldt-Jakob disease (CJD), and scrapie (Schreuder et al., 1994; Prusiner et al., 1995; Foster et al., 2000). PrPC is a glycoprotein that contains a disulfide bond structure, two N-glycosylation sites, and a glycosyl-phosphatidyl anchor (Biasini et

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al., 2012). These proteins are localized on clathrincoated membrane rafts on the cell surface (Vey et al., 1996). Neurotransmitter metabolism of the PrPC has been associated with several important roles in biological functions including such functions as cell adhesion, signal transmission, copper metabolism (Cashman et al., 1990; Mazzoni et al., 2005), and programmed cell death (apoptosis) (Kim et al., 2004).

Watarai et al. (2003) determined that these prion proteins also play an important role in the entrance of B. abortus to macrophages particularly with the presence of heat shock protein (Hsp60). However, in a later study Fontes et al. (2005) disagreed with Watarai et al. (2003) and reported that PrPC did

by binding Cu++ to themselves and transmitting it to the superoxide dismutase enzyme (McMahon et al., 2001). In brucellosis, the roles of antioxidative superoxide dismutase and its activator (PrPC) have not yet been elucidated. In the current review, it is speculated that the PrPC protein can be silenced with the use of small interference RNA, and this in turn would impact the central nervous system cell viability as well as the number of bacteria that enter the host cells. The following sections discuss central nervous system tissue culture approaches and small interference siRNA (small interference RNA or short interference RNA; from here on through the remainder of the text will be referred to as small interference RNA)

not seem to play a noticeably effective role during the entrance and uptake of the Brucella into macrophages. Nevertheless, in B. melitensis infected hosts, the role of the PrPC during the invasion of the host cells and the following oxidative-antioxidative metabolic reactions still remains largely unknown.

The involvement of the host cells and B. melitensis needs to be further characterized before distinctive roles of the PrPC can be identified.

During Brucella infection, host defense mechanisms are initiated and as part of these mechanisms, nitric oxide reacts with superoxide anion and generates hydroxyl (OH-) ion and peroxynitrite (ONOO-) radical to kill the invading bacteria. However, in this process, Brucella weakens the corresponding defense of the host cell by increasing their superoxide dismutase enzyme activity and in doing so scavenging the free radicals which would have been lethal to the bacterial cells (Kim et al., 2000). As discussed previously, Brucella possesses both superoxide dismutase and catalase activities that are also capable of dissipating these free radicals produced by the host cells. These antioxidative enzyme activities can in turn lead to ineffectiveness in oxidative capacity in the host cells (Kim et al., 2000). In addition, an increase in the intracellular concentration

characteristics and methodology that could be used to elucidate these mechanisms.

of Cu++ directly up-regulates the expression of the PrPC proteins and as a result increases the activity of superoxide dismutase (Brown et al., 1999). Octarepeats that are on the N-terminal of PrPC activate superoxide dismutase on the endoplasmic reticulum

gies Corp., Grand Island, NY), a cationic liposome which binds nucleic acid and delivers it to the cell cytoplasm. Based on the mechanism of small interference RNA, it is hypothesized that these molecules can be used to suppress the expression of the cel-

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TRANSIENT TRANSFECTION OF SMALL INTERFERENCE RNA The small interference RNA molecules are a class of double-stranded RNA molecules that usually consist of 19 to 25 base pairs in length (Elbashir et al., 2001). In a series of steps, small interference RNA are incorporated into RNA-induced silencing complexes (RISC) which are capable of binding specifically to mRNA molecules, leading to their destruction, thus blocking expression of that gene. Although first discovered as a natural phenomenon, small interference RNA are mostly seen as effective tools for knocking out expression of specific genes. Small interference RNA can be introduced into a cell as small interference RNA DNA in a vector. Transcription of this DNA eventually leads to stably-produced active small interference RNA. More commonly, cells are transiently transfected by bringing small interference RNA directly into the cell using electroporation or by mixing the small interference RNA with a reagent such as Lipofectamine® 2000 (Life Technolo-

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lular prion proteins. This becomes particularly useful for characterizing Brucella infection as a means to sort out the possible effects of PrPC transition from the host and corresponding oxidative responses (Erdogan et al., 2013). Using small interference RNA to selectively degrade the mRNA would in turn allow for the PrPC levels to be reduced or completely silenced in a controlled fashion. The following sections describe some of the tissue culture cellular assay approaches that can be used to differentiate the mechanisms associated with Brucella interactions with the central nervous system.

staining using a hemocytometer or other means to enumerate cells.

It is critical in any tissue culture assay to retain consistent levels of viable cells for the duration of the study or series of experiments. For viability assessment of tissue culture cells, an MTT (Thiazolyl Blue Tetrazolium Bromide) assay is one of several colorimetric systems that have been used (Mosmann, 1983; Watts et al., 1989; Nikš and Otto. 1990; Ciapetti et al., 1993; Vega-Avila and Pugsley, 2011). With the MTT assay the ratio of live cells in a cell community (containing both live and dead cells) can quantitatively be determined with a spectrophotometer. This approach is based on being able to detect cleavage

CENTRAL NERVOUS SYSTEM-ORIENTED TISSUE CULTURE APPROACH

of the MTT’s tetrazolium ring by mitochondrial reductases resulting in change of dye color from yellow to the blue-purple of the formazan product. This cleavage reaction is dependent on the activity of mitochondrial succinate dehydrogenase and therefore can only occur in healthy viable cells.

Astrocytes, oligodendrocytes, and microglia are found in the central nervous system (Guillemin and Brew, 2004; Hanisch and Kettenmann, 2007; Bertrand and Venero, 2013). Microglial cells belong to the macrophage defense system and are able to phagocytize pathogens in the central nervous system (Bertrand and Venero, 2013; Norden, and Godbout, 2013). For central nervous system-oriented tissue culture studies, microglial cell lines are widely used as a model for functional studies with a number of in vitro systems having been developed and evolved over the years (Guillemin and Brew, 2004; Ponomarev et al., 2005; Flode and Combs, 2007; Moussaud and Draheim, 2010; Bertrand and Venero, 2013; Erdogan et al., 2013). These will not be discussed in detail but rather a brief overview of generalized methods will be described as follows. Generally, microglia cells have the ability to adhere to and grow on the bottoms of flasks or plates. Microglial cells are typically incubated at 37°C with 5% CO2 in Dulbecco’s Modified Eagle’s Medium (DMEM) liquid medium in the presence of variations of the following components: inactivated fetal bovine serum (FBS), sodium pyruvate, sodium bicarbonate, HEPES, glutamine, glucose, and along with the antibiotics penicillin and streptomycin. Microglia cells are typically passaged every 2 to 3 days and during this time the number of microglia cells can be determined with trypan blue

BRUCELLA INFECTION MICROGLIA

OF

HUMAN

Apoptosis is an important host defense system against intracellular infections; however, apoptosis is not stimulated in Brucella infections. Samartino et al.

(2010) infected astrocytes and microglia with B. abortus. Their study suggested that in neurobrucellosis, although proinflammatory mediators were induced in both astrocytes and microglia, apoptosis was induced only in astrocytes but not in microglia. Other studies have shown that PrPC prevents apoptosis by inhibiting apoptotic caspase 3 and 9 (Sakudo et al., 2003; Kim et al., 2004; Erdogan et al., 2013). Silencing the prion protein gene prnp results in stimulation of apoptosis. It is still unknown why apoptosis is suppressed in brucellosis, and whether PrPC has a role in this process (Sakudo et al., 2003). PrPC also acts in clearing superoxide from the environment. Sakudo et al. (2003) have speculated that PrPC takes an intermediary place in transfer of Cu++ to superoxide dismutase.

One approach to dissecting out the role of PrPC

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would be to degrade and thus silence the PrPC mRNA by administering siRNA prior to Brucella infection occurring in the host cells. Once PrPC mRNA is silenced, the resulting impacts on Brucella infection can be assessed based on analysis of cellular metabolism and the corresponding metabolites (i.e. antioxidative metabolism, proinflammatory mediators’ levels, cytokine levels etc.) that are produced.

The role of PrPC could be further determined by directly enumerating invasive bacterial cells during and after the infection process.

To answer these questions, the human microglia C13-NJ cell line can be infected with B. melitensis for various times (for example, 0, 15 minutes, 3 hours

For the actual PCR reactions, a proportion of RNA can be removed from the corresponding samples after synthesis of the complementary DNA (cDNA) by reverse transcriptase (RT). After an aliquot of cDNA is taken and combined with the commercial PCR reaction mix, the previously designed and constructed primer set (specific for each reaction) for the amplification of iNOS, nNOS and PrPC genes can be added. PCR products are then analyzed by gel electrophoresis. Normalizations can be done using a constitutive gene such as β-actin gene as a control. Although semi-quantitative evaluations can be achieved by determining the intensity of the bands, direct quantitative PCR approaches are considered

and 24 hours). After this infection period of the cell line, the cellular viability can be monitored by the enzymatic MTT test discussed in the previous section as well as measuring nitric oxide levels in cellular supernatants, and superoxide dismutase, catalase and glutathione peroxidase activity. For example, glutathione peroxidase activity is typically determined by using a commercial kit. In the presence of hydrogen peroxide, glutathione peroxidase produces oxidized glutathione and the oxidized glutathione is in turn reduced by glutathione reductase to NADPH and reduced glutathione. The determination of the glutathione peroxidase activity is usually calculated as the decline in the absorbance measured on a spectrophotometer at a wavelength of 340 nm during the oxidation of NADPH to NADP+.

Transcriptional analyses of PrPC, and induced and neuronal nitric oxide synthase (iNOS and nNOS) mRNA can be easily performed using a reverse transcriptase polymerase chain reaction (RT-PCR). Recovering mRNA for RT-PCR analysis typically involves the addition of reagents for RNA isolation to a set quantity of cells, using protocols pre-described by the commercial manufacturers (Bustin, 2002; Hanna et al., 2005; Maciorowski et al. 2005; Sirsat et al. 2010; Erdogan et al., 2013) and yet it is important to assess

more precise and have been used in a multitude of studies (Bustin, 2002; Hanna et al., 2005; Maciorowski et al., 2005; Saengkerdsub etal, 2007a,b; Jarquin et al., 2009; Sirsat et al., 2010; Dunkley et al., 2007, 2008, 2012; Park et al., 2009, 2011a,b, 2013).

Cellular prion proteins levels can also be assessed directly with the use of Western Blotting approaches involving specific monoclonal antibodies that have been generated to the protein(s) of interest. The details of a variety of methodologies and approaches for producing specific monoclonal antibodies to PrPC and perspectives on their specificity are described elsewhere (Barry and Prusiner, 1986; Bodemer, 1999; Furuoka et al., 2007; Liu et al., 2010) and will only be discussed in general terms here. Briefly, in order to analyze PrPC, an aliquot of the respective protein containing sample is typically removed from the cell homogenates and subsequently denatured in Laemmli Buffer, which contains sodium dodecyl sulfate (SDS) as the denaturing agent. After the proteins have been sufficiently denatured, the resulting preparations are applied to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) to separate out the respective proteins and allow for further characterization. Once the SDS-PAGE has been completed, the separated proteins are typically transferred from the

the amount and purity of the RNA. This is usually done by using a spectrophotometer at two different wavelengths (260/280 nm) where RNA/DNA ratios that are greater than 1.7 are considered acceptable for RT-PCR application.

gel to a nitrocellulose membrane via blotting. Antibodies (usually monoclonal generated antibodies) that are specific to PrPC, are followed by the corresponding secondary (typically polyclonal generated antibodies) antibodies, are subsequently applied to

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the nitrocellulose membrane. Once the antibodies have been added and allowed to bind to their corresponding proteins, the presence of PrPC and their densities can be determined by staining the membrane with a chromogenic substance.

SUMMARY AND CONCLUSIONS Brucellosis is a zoonotic disease caused by Brucella spp. and is characterized by acute and chronic symptoms. The pathogen prevents phagosomelysosome fusion and oxidative bursts, and does not induce sufficient activity of the immune defense sys-

PrPC, which possess antioxidative properties and is thought to be associated in some fashion with the engulfment of the pathogen through phagocytosis.

The potential of using PrPC-specific small interference RNA molecules to silence PrPC mRNA before Brucella infection in microglial cells was also discussed as means to more precisely delineate the sequence of events that occur during the expression of the pathogenesis phenotype. Pathogenesis can be assessed by quantitating the number of invasive bacterial cells, determining host viability, and measuring oxidative events that occur during the infection and invasion process. Further characterization of Brucella infection in microglial cells using biochemi-

tem. Therefore, it is able to survive within the cellular compartments of the host without any disturbance for a long period of time. The relapse rate is observed in 5 to 10% of the patients and the nervous system involvement is approximately 1 to 11% in humans. The invasion of the Brucella species in the central nervous system and the response of the host such as oxidative events, and other defense mechanisms against the pathogen are not well known yet.

The overall aim of this review was to describe the oxidative events that occur against Brucella infection and the antioxidative responses that follow this initial event and potential approaches for studying this pathogenesis mechanism. In particular, Brucella’s antioxidant responses that ensure their survival from host defense system are certainly factors that must be considered in the pathogenic mechanism. With the advent of more sophisticated genetic tools the potential for human neuronal microglia cells to serve as a neurobrucellosis model offer opportunities to further explore this relationship at the molecular level. It has been established that the cellular prion protein has beneficial roles such as protecting nerve cells from oxidative stress, but this may also serve to help bacteria for entry into the cell cytoplasm. However, in neurobrucellosis, the interaction between

cal and molecular biology techniques should reveal the interaction between the host neuronal cells and the pathogen. In addition, the possible involvement of PrPC in neurobrucellosis would potentially be revealed if small interference RNA molecules can be applied to block particular steps of the infection.

This ability to target certain steps will allow for an assessment of how each of the singular infection events that occur contribute to overall pathogenesis of the microorganism and where effective control measures might be most optimally targeted.

the host and the corresponding Brucella virulence factors that engage the oxidative host defense system and the concomitant effect of PrPC on these events still remain virtually unknown. Despite these unknowns there is believed to be a potential role for

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Prevalence of Foodborne Pathogens and Spoilage Microorganisms and Their Drug Resistant Status in Different Street Foods of Dhaka city Z. Tabashsum1,6, I. Khalil2, Md. Nazimuddin3 , A.K.M. M. Mollah4 , Y. Inatsu5 and Md. L. Bari1 Center for advanced Research in Sciences, University of Dhaka, Dhaka-1000, Bangladesh Bangladesh Standards and Testing Institution, Tejgaon Industrial Area, Dhaka-1205, Bangladesh 3 Bangladesh Agricultural Research Institute, Gazipur 1701, Bangladesh 4 Faculty of Life Sciences, Asian University for Women, Chittagong 4000, Bangladesh 5 National Food Research Institute, 2-1-12 Kannondai, Tsukuba-shi, Japan 6 Faculty of Life Science, Independent University Bangladesh, Dhaka-1229, Bangladesh 1

2

ABSTRACT The street foods play an important socio-economic role in meeting food and nutritional requirements of city consumers at affordable prices. This study was designed to evaluate the detailed microbial status including foodborne pathogen and spoilage bacteria and their drug sensitivity status in different street foods of Dhaka city. For this assessment, 39 street foods samples of 13 kinds were collected from Motijheel area, the busiest part of the Dhaka city area. These samples were analyzed for foodborne pathogens including, Salmonella spp., Escherichia coli O157, O111, O26 and other E. coli, other coliforms, Cronobacter sakazakii, Yersinia spp., Listeria spp., Staphylococcus spp., and spoilage microorganisms including Enterococcus spp., Pseudomonas spp., Bacillus spp., and lactic acid fermenting bacteria (LAB). The average natural aerobic bacterial population varied from 3.0 ± 0.04 log CFU/g to 8.8 ± 0.02 log CFU/g and the average coliform count varied from 2.0 ± 0.01 log CFU/g to 7.5 ± 0.02 log CFU/g. In addition, Salmonella spp. and Escherichia coli (O157, O111, O26) were identified in 2 street food samples, other E. coli were found in 5 samples, coliform bacteria was found in 28 samples and Enterococcus spp. in 10 samples, out of 39 food sample analyzed. Moreover, Listeria spp. were detected in 15 samples, Yersinia spp. in 10 samples, Enterobacter sakazakii in 8 samples, and Staphylococcus spp. in all 39 samples. Among the spoilage organisms, Bacillus spp. were identified in 12 food samples, Pseudomonas spp. in 15 food samples and lactic acid fermenting bacteria (LAB) in 24 samples, out of the 39 samples tested. The isolated pathogens were then checked for antibiotic sensitivity and the results revealed that all the Salmonella spp. exhibited multi drug resistance (at least 7 antibiotics), all Escherichia coli O157, O111, O26 and other E. coli were multi drug resistant (at least 6 antibiotics), Enterobacter sakazakii (at least 6 drugs) and the similar results were found for all the coliform (at least 5 antibiotics), Listeria spp., Pseudomonas spp. and lactic acid fermenting bacteria (LAB). In addition, Staphylococcus spp., Bacillus spp., isolates were resistant to most of the antibiotics and some isolates were resistant to all the antibiotics tested. Enterococcus spp. was found to be sensitive to vancomycin. These study result demonstrated that foods sold in the street of Dhaka City constitutes a potential microbial hazard to human health.

Agric. Food Anal. Bacteriol. 3: 281-292, 2013

Correspondence: Md. Latiful Bari, latiful@univdhaka.edu Tel: 8801971560560 Fax: : 8802-8615583

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INTRODUCTION Foodborne diseases are now becoming a great concern involving a wide range of illnesses caused by bacterial, viral, parasitic or chemical contamination of food. In addition, resistance of these microorganisms to multi-drugs made this situation more of a concern to public health. Approximately, 30 million people in Bangladesh suffer from food borne illnesses each year (FAO, 2012). Diarrheal diseases are the most common food poisoning cases in Bangladesh and in some cases, these can cause death. The diseases are caused by either the toxin produced by the microorganism, or by the human body’s reac-

18 hours a day without having toilet facilities. Most of the vending shops (68%) were located on the footpath irrespective of areas surveyed and 30% vending carts were placed near municipal drains and 18% near sewage. A microbiological study on different foods items, drinking water and hand swab samples revealed the prevalence of overwhelmingly high numbers of aerobic bacteria and coliform bacteria. The study also indicated that a significant portion of drug resistant bacteria are spreading in the community through the street foods. This study also suggested the need to conduct a detailed microbial study and profile their drug resistance characteristics to assess the potential public health hazards. There-

tions to the microorganism.

Street foods are described as a wide range of ready-to-eat foods and beverages, or prepared at home and consumed on the streets without further preparation (Dardano, 2003). The food items are sold by vendors and hawkers especially in the streets and other similar public places. While street-vended foods are appreciated for their unique flavors as well as their convenience, they are also important in contributing to the nutritional status of the people.

Street food vending assures food security for lowincome urban populations and provides a livelihood for a large number of workers who would otherwise be unable to establish a business for want of capital.

In contrast to these potential benefits, it is also recognized that street-food vendors are often poor, uneducated and lack knowledge in safe food handling practices, environment, sanitation and hygiene, mode of food display, food service and hand washing, sources of raw materials, and use of portable water. Consequently, street foods are perceived to be a major public health risk.

A study of the socio-economic conditions and determination of the hygienic and sanitary practices of street food vendors in Dhaka City Corporation was carried out by FAO 2010. The study result demon-

fore, this study was designed to evaluate the detailed microbial status including foodborne pathogen and spoilage bacterial content and their drug sensitivity status from different street foods of Dhaka city.

strated that 25% street food vendors are illiterate and cannot write their names and have no formal education. As street food business requires low investment, most of the vendors (88%) were found to own their business. They reportedly work for 13 to

Twenty five (25) g of each sample were homogenized in 225 milliliters of saline water (0.85% NaCl).

Decimal dilutions were prepared upto 10-6 and appropriate dilutions were spread plated on Tryptic soy agar (Oxoid Ltd., Hampshire, England) and incu-

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MATERIALS AND METHODS Sample collection Street food samples (36) of thirteen categories were purchased from the vendors at Motijheel area of Dhaka between October 15 and November 15, 2012. All the samples were transported to the Food Analysis and Research Laboratory, Center for Advanced Research in Sciences (CARS) of University of Dhaka at the earliest convenience for processing and further assessment. All the analysis was carried out according to the standard methods described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual and the schematic diagram is presented in Figure 1.

Total aerobic count and total coliform count

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Figure 1. Flow diagram for the identification of foodborne pathogens and food spoilage bacteria using conventional, immunological, and molecular methods.

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bated at 35ºC for 24 hours for total aerobic bacterial count and on MacConkey agar (Oxoid Ltd., Hampshire, England) and incubated at 35ºC and 42ºC for 24 hours for total coliform counts. Total aerobic counts were used to indicate the quality and shelf life of the products and total coliform counts to indicate the unhygienic condition of the food preparation surfaces.

Escherichia coli O157, O111, O26 Twenty five (25) g of each samples were homogenized in 225 milliliters mEC medium (Nissui Co., Ltd., Tokyo, Japan) and incubated at 42ºC for 20 hours. The enriched cultures were streaked onto Sorbitol MacConkey agar (Oxoid Ltd., Hampshire, England) complemented with Cefixime and potassium tellurite supplement (Fluka, Sigma-Aldrich, Bangalore, India) and characteristic colonies were subjected to biochemical tests (IMViC). Biochemically confirmed isolates were screened through Rainbow agar (Biolog, France) and CHROM agar (Kanto Co. Ltd., Kyoto, Japan). The colonies which gave characteristic color were subsequently serotyped by O157, O111 and O26 specific antisera. The isolates were subsequently tested for stx1 and stx2

by NH-Immunochromato VT1/2 and by PCR using primer 5’-CAGTTAATGTGGTGGCGAAGG-3’ and 5’-CACCAGACAAATGTAACCGCTC-3’ for stx1 and 5’-ATCCTATTCCCGGGAGTTTACG-3’ and 5’-GCGTCATCGTATACACAGGAGC-3’ for stx2 (Vidal et al., 2004).

Escherichia coli, Coliform bacteria, Enterobacter sakazakii Twenty five (25) g of each sample were homogenized in 225 milliliters Enterobacteria enrichment broth-Mossel pre-enrichment medium (Oxoid Ltd., Hampshire, England) and incubated at 35ºC for 20 hours. One milliliter aliquots of pre-enriched cultures were mixed with nine milliliters of 2x EC medium (Nissui Co., Ltd., Tokyo, Japan) and incubated at 35ºC for 20 hours. To confirm the existence of fecal coliforms, 284

one loopful of the culture was inoculated into 10 milliliters 1x EC medium with Durham fermentation tubes and incubated at 42ºC for 20 hours. Gas production in the tubes were used to indicate the presence of fecal coliforms. To isolate E. coli, one loopfull of gas produced 1x EC culture broth was streaked on EMB agar plates (Nissui Co., Ltd., Tokyo, Japan) and developed typical colonies were then confirmed using biochemical characterization (IMViC) and API 20E kit (bioMérieux, Durham, NC, USA). The same pre-enrichment culture was also used for isolation and characterization of coliform bacteria on Sorbitol MacConkey agar (Nissui Co., Ltd., Tokyo, Japan) and isolated strains were subjected to further characterization using an API 20E kit. Pre-enriched culture was streaked onto Chromocult Enterobacter sakazakii (Merck, Darmstadt, Germany) agar plates to isolate Cronobacter sakazakii. The typical colonies were further characterized using an API 20E kit (BioMérieux, Durham, NC, USA). Presence of Escherichia coli or fecal coliform bacteria was used to indicate that the food had become contaminated with fecal material in some fashion.

Salmonella spp.

Twenty five (25) g of each sample were homogenized in 225 milliliters of buffered peptone water (Merck, Darmstadt, Germany) and incubated at 35ºC for 20 hours. One milliliter pre-enrichment cultures were mixed with nine milliliters of Hanja Tetrathionate Broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and incubated at 35ºC for 20 hours and nine milliliters of Rappaport-Vassiliadis Broth (Eiken Chemical Co. Ltd., Tokyo, Japan) and incubated at 42ºC for 20 hours. The culture broths were subsequently streaked onto DHL and MLCB and characteristics of isolates from candidate colonies was determined through biochemical tests (TSI and LIM). Biochemically confirmed isolates were re-confirmed using Salmonella LA latex agglutination test and API 20E kits.

Yersinia spp.

Twenty five (25) g of each sample were homog-

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enized in 225 milliliters of 0.85% NaCl and incubated at 10ºC for 7 days. These enriched cultures were streaked on Yersinia selective agar (Fluka, SigmaAldrich, Bangalore, India) and incubated at 30ºC for 20 hours. The candidate colonies were subsequently confirmed by API 20E (BioMérieux, Durham, NC, USA).

Bacillus spp., Staphylococcus spp. and Pseudomonas spp.

Twenty five (25) g each samples were homogenized in 225 milliliters of buffered peptone water (Merck, Darmstadt, Germany) and incubated at 30ºC for 20 hours. The respective pre-enrichment cultures were streaked on NaCl Glycine Kim Goepfert (NGKG) agar (Nissui Co., Ltd., Tokyo, Japan) with 20% egg yolk, mannitol salt agar (Nissui Co., Ltd., Tokyo, Japan) and NAC agar (Nissui Co., Ltd., Tokyo, Japan) to isolate Bacillus spp., Staphylococcus spp. and Pseudomonas spp. Typical colonies were subjected to biochemical characterization tests using suitable API kits. API 50CH with API CHB and API Staph (BioMérieux, Durham, NC, USA) were used for the identification of Bacillus spp. and Staphylococcus spp., respectively. The isolates of Bacillus spp. were subsequently checked for CRS gene by polymerase chain reaction (PCR) using the sense strand primer 5’-GGTGAATTGTGTCTGGGAGG-3’ and antisense strand primer 5’-ATTTTTATTAAGAGGCAATG-3’. Typical colonies of Pseudomonas spp. were further checked by Oxidase and Catalase tests and API 20NE (BioMérieux, Durham, NC, USA) diagnostic kits.

Enterococcus spp.

Twenty five (25) g of each sample were homogenized in 225 milliliters of buffered peptone water (Merck, Darmstadt, Germany) and incubated at 30ºC for 20 hours. One milliliter aliquots of these pre-enrichment cultures were added to 9 milliliters of 2x AC medium (HI media, Mumbai, India) and incubated at 35ºC for 20 hours. After incubation, one loopfull aliquots of these cultures were inoculated into nine

milliliters of 1x AC medium and incubated at 42ºC for 20 hours, and then, a loopfull of each respective culture was streaked onto EF agar (Nissui Co., Ltd., Tokyo, Japan.). Vancomycin containing EF (VR-EF) antibiotic agar plates (Nissui Co., Ltd., Tokyo, Japan) were also used for the isolation of vancomycinresistant Enterococci spp. (VRE). Developed typical colonies on the EF agar plates were confirmed by biochemical tests by using an API Strep (BioMérieux, Durham, NC, USA) kit.

Listeria spp.

Twenty five (25) g of each samples was homogenized in 225 milliliters of DifcoTM Listeria enrichment broth (Difco, Detroit, Michigan, USA) and incubated at 30ºC for 40 hours. The enriched cultures were streaked on Listeria selective agar base (Oxoid Ltd., Hampshire, England) with selective supplement SR0206E (Oxoid Ltd., Hampshire, England) and incubated at 30ºC for 20 hours or for extended incubation times if needed. Characteristic colonies were confirmed by NH-immunochromato Listeria and API Listeria kits.

Lactic acid bacteria Twenty five (25) g of each sample was homogenized in 225 milliliters of de Man, Rogosa and Sharpe (MRS) broth (Difco, Detroit, Michigan, USA) and incubated at 30ºC for 20 hours under anaerobic conditions. A loopfull of culture was streaked on MRS agar and incubated at 30ºC for 20 to 40 hours under anaerobic condition. Typical catalase negative colonies were subjected to biochemical tests by using API 50CH kit with API CHL (BioMérieux, Durham, NC, USA).

Antibiotic Susceptibility Test All isolated strains were then tested for antibiotic susceptibilities. The ranges of antibiotic susceptibility of the isolates were measured using commercially purchased discs (Oxoid Ltd., Hampshire, England) by the disc diffusion method on Mueller-Hinton Agar

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(Oxoid Ltd., Hampshire England). Antibiotics used in this experiment included gentamicin 10 µg (CN), vancomycin 30 µg (VA), amoxicillin 10 µg (AML), erythromycin 15 µg (E), streptomycin 10 µg (S), novobiocin 30 µg (NV), kanamycin 30 µg (K), ampicillin 10 µg (AMP), tetracycline 30 µg (TE), cephalexin 30 µg (CL), azithromycin 15 µg (AZM), ciprofloxacin 5 µg (CIP), cefixime 5 µg (CFM), chloramphenicol 30 µg (C), rifampicin 5 µg (RD), nalidixic acid 30 µg (NA).

Statistical Analysis Each category of street foods was taken three times from the same vendor. Reported plate count data represent the mean values obtained from three individual trials, with each of these values being obtained from duplicated samples. Data were subjected to analysis of variance using the Microsoft Excel program (Redmond, Washington DC, USA). Significant differences in plate count data were established by the least-significant difference at the 5% level of significance.

the ICMSF standard limit in most of the street food samples. The total aerobic bacterial and total coliform populations were the highest in the shingara and chatpoti samples, and jar water and vegetable rolls yielded the lowest levels of these bacteria. The total aerobic bacterial populations and total coliform populations are presented in Table 1.

Almost every sample contained different serotypes of coliforms and after confirmation using API 20E diagnostic kits these strains were identified as Enterobacter cloacae, E. aerogenes, Klebsiella oxytoca, K. pneumoniae, Kluyvera spp., Citrobacter spp., Erwinia spp., Aeromonas spp., Pantoea spp., Serratia odorifera, Raoultella ornithinolytica, R. terrigena,

In this study, thirteen different kinds of street food including, singara, jhal-muri, chatpati, chetoi pitha, sola, jilapi, drinking water, pickles, amra, tehari, vegetables roll, sugarcane juice and cucumber were assessed for total aerobic bacterial populations, total coliform and some specific food spoilage and pathogenic bacteria.

Singara (also called samosa) is a deep fried pastry with a savory filling, such as spiced potatoes, onions, peas, lentils, ground lamb, ground beef or ground chicken. The size, shape and consistency may vary, but typically, they are distinctly triangular. Chatpati is a type of snack. The main ingredients are boiled yellow chickpeas and potatoes. It is spicy, savory

Serratia odorifera.

Thirteen presumptive Salmonella spp. were isolated from selective plates (Table 2), and after biochemical, serological and API 20E confirmation tests, only two isolates were confirmed as Salmonella Choleraesuis (from chatpati and jilapi). Sola is boiled bengal gram/chickpeas mixed with spice, onion slices, green chili slices and boiled potato slices and served on used newspaper or on a plate with salad.

From three street food samples (sola, vegetable roll and cucumber), four presumptive pathogenic E. coli serotype O157, O111 or O26 strains were detected (Table-2) and after further investigation using biochemical, serological and API 20E tests, these three isolates were confirmed as either E. coli serotypes O157, O111 or O26. Six E. coli other than O157, O111, O26 serotype were isolated from five street food samples including chetoi pitha, jilapi, tehari, amra and cucumber (Table 2).

Fourteen presumptive Enterobacter skazakii strains were isolated from singara, sola, chitoi pitha, vegetable roll, sugarcane juice, and water samples and after confirmation using API 20E kits, eight isolates were confirmed as Enterobacter skazakii. Jhal Muri is a savoury snack. It is made of puffed rice and mixed with potatoes, onions, chili, chat masala. Oth-

and tangy, combining the ingredients onion slices, chili slices, egg slices, coriander leaves, tomato slices, cucumber slices and tamarind sauce. In this study, total aerobic bacterial populations and total coliform populations were found to be greater than

er commonly used ingredients include slices of tomatoes, onions and green chilies added to the base.

Chetoi pitha is baked ground rice or rice flour mixed with salt and served with green chili paste or dried fish paste. From ten street food samples (singara,

RESULTS

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Table 1. Total aerobic bacterial and total coliform populations in street food samples*

Total Aerobic Bacterial population (log CFU/g)

Total Coliform Population (log CFU/g)

Singara

8.8 ± 0.02

7.5 ± 0.02

Muri

7.5 ± 0.05

5.0 ± 0.08

Chatpati

8.0 ± 0.06

5.8 ± 0.05

Chetoi pitha

7.2 ± 0.05

4.0 ± 0.01

Sola

7.5 ± 0.04

4.8 ± 0.09

Jilapi

4.9 ± 0.03

3.3 ±0.06

Jar water

3.0 ± 0.04

2.5 ± 0.08

Achar

6.5 ± 0.03

2.0 ± 0.01

Amra

6.3 ± 0.04

2.7± 0.04

Tehari

6.8 ± 0.05

2.0 ± 0.01

Vegetable Roll

5.7 ± 0.06

2.6 ± 0.05

Sugarcane juice

6.0 ± 0.04

5.1 ± 0.05

Slice Cucumber

6.2 ± 0.01

2.7 ± 0.01

Street food Samples

* Results are expressed in average of three replicate samples ± SD, which were calculated from duplicate plates.

jhal-muri, chatpati, chetoi pitha, sola, drinking water, amra, vegetables rolls, sugarcane juice and cucumber), twenty presumptive Yersinia spp strains were isolated and further confirmed by API 20E diagnostic kits (Table 2). None of the isolated strains were confirmed as Yersinia enterocolitica. Jilapi is a type of sweet and made by deep-frying a wheat-flour batter in pretzel or circular shapes, which are subsequently soaked in sugar syrup. The sweets are served warm or cold. From 11 street food samples, 21 Enterococcus spp were isolated, however, after confirmation

and E. faecalis. Except for achar and tehari, Listeria spp. was identified from all other street food sample tested. Fifteen Listeria spp were isolated and confirmed using API Listeria kits. Further identification revealed that the isolated Listeria spp. were L. ivanovii, L. grayi, L. welshimeri, L. seeligeri and L. monocytogenes. From sola sample, the isolated strains were determined to be L. monocytogenes. Twenty four Staphylococcus spp. isolates were isolated from selective plates of 39 street food samples (table-2) and only ten isolates were confirmed as Staphylococcus

using API step. kits, only ten isolates were identified as Enterococcus spp. that had originally been isolated from chitoi pitha, sola, jilapi, drinking water, sugarcane juice, and cucumber samples. These species were identified as E. avium, E. solitaries, E. faecium

spp by API Staph kits. (singara, chatpati, muri, boiled motor, jilapi, cucumber). The species of Staphylococcus were found as S. lentus, S. xylosus, S. sciuri and S. aureus. Staphylococcus aureus was found in jilapi food samples only.

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Table 2. Presence of pathogenic and spoilage bacteria on street food samples as detected on selective microbiological medium.

E.coli O157,O111, O26

E.coli

Coliform

Cronobacter sakazakii

Yersinia spp.

Bacillus spp.

Staphylococcus spp.

Pseudomonas spp.

Enterococcus spp.

Listeria spp.

LAB

Singara (3)*

0

0

0

3

0

1

3

3

1

2

1

3

Muri (3)

1

0

0

3

2

1

1

3

1

1

1

3

Chatpati (3)

2

0

0

3

3

3

3

3

3

1

1

3

Pitha (3)

1

0

1

3

3

2

2

3

1

2

3

2

Boiled motor (3)

2

2

0

3

3

3

2

2

3

3

3

3

Jilapi (3)

2

0

1

3

1

0

2

1

1

3

2

1

Water (3)

2

0

0

2

0

3

2

1

3

3

1

1

Achar (3)

0

0

0

1

0

0

2

1

0

0

0

2

Amra (3)

0

0

1

0

0

1

2

1

0

1

1

1

Tehari (3)

0

0

1

2

0

0

1

1

0

0

0

0

Roll (3)

0

1

0

2

0

2

1

2

0

2

2

1

Sugarcane juice (3)

2

0

0

2

1

2

0

1

0

2

1

2

Cucumber (3)

1

1

2

2

1

2

2

2

2

1

2

2

Street food Samples

Salmonella spp.

Number of bacterial isolates

*parenthesis: number of samples

Twenty three Bacillus spp. were isolated from 33 street food samples (Table 2) and twelve isolates were identified as Bacillus spp. by API 50 CHB. These

confirmed by biochemical tests and API 20NE diagnostic kits. From 39 food samples, 24 Lactic acid fermenting bacteria (LAB) were isolated. Different

isolates were identified as Bacillus atrophaeus, B. globigii, B. licheniformis. No B. cereus strains were detected throughout the study.

Fifteen Pseudomonas spp. were isolated from 39 street food samples, all Pseudomonas spp. were

species of LAB identified using API 50 CHL included Lactobacillus brevis, L. pentosus, L. plantarum, L. collinoides, L. salivarius, Lactococcus lactis, L. raffinolactis, Weissella confusa, Pediococcus pentosaceus, Leuconostoc mesenteroiodes.

288

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Table 3. Antibiotic sensitivity pattern of the isolated bacteria

C (30 µg)

S (10 µg)

CL (30 µg)

VA (30 µg)

AML (10 µg)

CIP (5 µg)

K (30 µg)

AZM (15 µg)

NV (30 µg)

NA (30 µg)

CN (10 µg)

RD (5 µg)

AMP (10 µg)

CFM (5 µg)

TE (30 µg)

E (15 µg)

Number of isolates resistant to antibiotics

Salmonella spp. (2)

0

1

0

2

1

0

0

2

2

0

1

2

1

0

0

2

Escherichia coli other than O157, 0111, 026 (5)

0

1

1

1

1

0

0

1

1

0

1

1

1

1

1

1

Escherichia coli O157,0111,026 (3)

2

3

2

3

2

3

2

3

3

2

0

3

3

0

3

3

Enterobacter sakazakii (8)

0

3

3

4

4

0

0

5

5

0

1

4

4

1

0

4

Klebsiella spp. (5)

0

4

1

5

4

0

0

5

5

0

0

5

4

0

0

5

Enterobacter cloacae (11)

3

6

3

7

6

0

1

6

7

2

1

7

6

1

2

7

Enterobacter aerogenes (9)

1

4

3

6

5

0

1

5

6

0

1

6

5

0

1

6

Erwinia spp. (7)

2

2

5

6

5

0

3

4

6

3

1

6

6

2

4

6

Aeromonas spp. (5)

2

3

3

3

3

0

2

3

3

2

2

3

3

2

3

3

Kluyvera spp. (4)

1

2

1

2

1

0

1

2

2

2

0

2

1

1

2

2

Serratia spp. (6)

4

2

3

5

5

1

2

5

5

2

0

5

5

1

1

5

Pantoea spp.(6)

3

1

2

4

3

1

1

3

4

2

0

3

3

2

2

5

Citrobacter spp. (7)

1

5

1

6

6

0

0

5

6

1

0

6

5

2

0

6

Raoultella spp.(7)

0

3

2

5

4

0

1

5

5

0

1

5

4

0

0

5

Enterococcus spp. (10)

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Yersinia spp. (20)

4

6

13 17

9

8

15 12

8

7

11

2

4

18

3

6

Bacillus spp. (12)

0

4

2

4

0

1

4

6

5

8

4

3

0

8

3

3

Staphylococcus spp.(10)

0

3

1

3

0

0

2

5

3

8

2

1

0

6

1

2

Pseudomonas spp. (15)

10

9

10 10 10

0

10

9

10 10

0

10 10

10

9

10

Listeria spp.( 15)

10 12 11 13 10

2

5

13 12 15 11

8

11

15

13

12

Lactobacillus spp. (6)

5

6

6

6

5

5

6

4

5

6

6

6

6

6

6

5

Weissalla spp. (8)

3

8

4

8

4

3

7

3

3

8

4

7

4

8

3

3

Lactococcus spp. (7)

7

7

5

7

7

5

7

6

7

7

7

7

7

7

7

5

Pediococcus spp. (2)

0

1

0

0

0

0

0

0

0

1

1

1

0

1

0

0

Leuconostoc spp. (1)

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

Isolates

*parenthesis: number of isolates Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013

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Table 3 Abbreviation: chloramphenicol(C), streptomycin (S), cephalexin (CL), vancomycin (VA), amoxicillin (AML), ciprofloxacin (CIP), kanamycin (K), azithromycin (AZM), novobiocin (NV), nalidixic acid (NA) gentamicin (CN), rifampicin (RD), ampicillin (AMP), cefixime (CFM), tetracycline (TE), erythromycin (E).

Antibiotic sensitivity pattern of Pseudomonas spp. isolates revealed that these strains were resistant to multiple antibiotics (minimum 10 antibiotics and maximum 14 antibiotics) (Table 3). However, antibiotic sensitivity patterns of Enterococcus isolates revealed that these strains were not resistant to any of the 16 antibiotics tested (Table 3). In contrast, isolated Enterococcus spp. was sensitive to vancomycin (30 Âľg/milliliter). Antibiotic sensitivity pattern of E. coli O157, Salmonella Listeria spp and Yersinia spp

higher than the ICMSF recommended (less than 106 CFU/g) levels of aerobic bacterial populations and coliform bacterial (less than 11 CFU/g) populations were observed in most of the samples tested. Moreover potential pathogenic bacteria including E. coli O157, O111 or O26, Salmonella, Listeria monocytogenes, Staphylococcus aureus were detected in some street food samples. These findings demonstrate that street foods sold in Dhaka constitute a likely potential hazard to human health. The presence of

isolates revealed that these strains were resistant to multiple antibiotics (minimum 4 antibiotics and maximum 12 antibiotics) (Table 3). Furthermore, antibiotic sensitivity patterns of LAB isolates revealed that these strains were resistant to multiple antibiotics (minimum 9 antibiotics and maximum 16 antibiotics) (Table 3).

higher numbers of Enterobacteriaceae in street food samples that were cooked or deep oil fried appears to be a good indicator of post-processing contamination. Contamination of food by enteric pathogens can occur from inadequate cooking or use of contaminated water during preparation and processing, or improper washing and handling or lack of hygiene of the vendors. Sometimes the source of the food or water may also be contaminated. Therefore, access to running water and health education to the vendors on personal hygiene, food safety and proper disposal of waste would improve food quality thereby reducing food borne incidences.

DISCUSSION Food borne illnesses of microbial origin are a major health problem associated with street foods (Kaneko et al., 1999; Mensah et al., 1997, 2001, 2002).

The traditional processing methods that were used in the preparation, inappropriate holding temperature and poor personal hygiene of food handlers are some of the main causes of contamination of street foods (Barro et al., 2006; Mensah et al., 2002). In addition the foods were not effectively protected from flies and dust (Bryan et al., 1997; Bryan et al., 1992).

In Bangladesh, street foods are mostly prepared and processed manually and sold to the public at various lorry terminals, by the roadside or by itinerant vendors (Mensah et al., 2002). Researchers have investigated the microbiological quality of street vended foods in different countries and high bacterial counts and a high incidence of food borne pathogens in such foods typically have been reported (Jayasuriya, 1994; Mosupye and Von, 2000; Kubheka et al., 2001; Hanashiro, 2005; Tendekayi et al., 2008). In this study, 290

CONCLUSIONS Of the samples analyzed, almost all the street foods were found to be heavily contaminated with coliforms, fecal coliform bacteria and other pathogens. Therefore, the inspection authorities are required to take the necessary steps to make these products safe for consumers. There are alarming levels of multi drug resistant (MDR) pathogens present in some street food samples. This is a great concern for human health and the regulatory agencies should take the necessary measures to improve the food hygiene conditions of street food. The deep fried street food including shingara, samocha and jilapi were found microbiologically safe when served immediately. However, the holding bowls and the

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013

adjacent areas including the vendor’s personal hygiene were not found suitable for providing food that would be considered safe as these products were found to be moderately contaminated with coliforms, fecal coliforms and other pathogenic bacteria. However, the raw sugercane juice and raw fresh-cut fruits were contaminated with fecal coliforms and other pathogens and pose serious health problem; because these items are usually eaten raw.

Street foods pose risks of both a physical and chemical nature given that they are exposed to the road side and traffic pollution caused from different kinds of vehicles. It is necessary to investigate the physical and chemical contamination of the street food

ACKNOWLEDGEMENTS

samples and there is a need to develop awareness in order to avoid physical and chemical hazards.

Street food vendors practiced minimal hygienic and sanitary practices. The hygienic practices in question included food preparation, handling of utensils; a place for food preparation, personal hygiene and methods of storing cooked food. Due to lack of proper knowledge and guidance on street food vending, vendors prepared their foods in explicitly unhygienic and unsanitary conditions.

Improving the safety of street-vended foods remains a tremendous challenge. The research data presented here suggested the need of every vendor, helper or food handler to undergo basic training in food hygiene before being involved in food retail as street vendors. The food inspectors role is to ensure that these vendors follow the required rules for proper hygiene and sanitation. Mass awareness using electronic media, food hygiene trainers training, regular monitoring, and Hazard Analysis and Critical Control Point (HACCP) control measures should be developed in reducing the safety of street foods. Nevertheless, the HACCP system is the most cost-effective approach for assuring food safety at all stages of the food supply. It will enable the systematic identification of potential hazards and their

and A. Traoré. 2006. Hygienic status and assessment of dishwashing waters, utensils, hands, and pieces of money from street food processing sites in Ouagadougou, Burkina Faso. African J. Biotech.

5:1107-1112.

Bryan, F., M. Jermini, R. Schmitt, E. Chilufya, M. Mwanza, A. Matoba, E. Mfume, and E. Chibiya.

1997. Hazards associated with holding and reheating foods at vending sites in a small town in Zambia. J. Food Prot. 60:391-398.

Bryan, F., P. Teufel, S. Riaz, S. Roohi, F. Qadar, and Z. Malik.1992. Hazards and critical control points of street-vending operations in a mountain resort town in Pakistan. J Food Prot. 55:701-707.

Kaneko, K., I. Hayashidani, O. Hideki, and K. Yoshimitsu. 1999. Bacterial contamination of ready-to- eat foods and fresh products in retail shops and food factories. J. Food Prot. 62:644-649.

FAO. 2012. National food safety policy and seminar entitled Towards a National Food Safety and Quality Policy - a key note speeches. Available at http://bdfoodsafety.org/news.php?NewsId=22, accessed on May 19, 2013.

FAO. 2010. Institutionalization of healthy street food system in Bangladesh: A pilot study with three wards of Dhaka City Ccrporation as a model”. Fi-

control measures. A HACCP approach also provides guidance in the selection of enforcement and education priorities, rather than general sanitation and superficial improvements.

nal Report PR #7/07. Pages 1-93.

Hanashiro, A., M. Morita, G. R. Matte, M. H. Matte, and E. A. F. S. Torres. 2005. Microbiological quality of selected street foods from a restricted area of Sao Paulo city, Brazil. Food Control 16:439–444.

The authors would like to thank Mr. Arafat-alMamun for technical assistance, and Mr. Harun-ur Rashid and Ms. Emon Sharmin for the laboratory assistance required to complete this task. The authors would also like to thank the United Nations University, Tokyo, Japan (UNU-ISP) for financial support in this work.

REFERENCES Barro, N., A. Bello, S. Aly, C. Ouattara, A, Ilboudo,

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Jayasuriya, D.C. (1994). Street food vending in Asia: Some policy and legal aspects. Food Control 5:222-226.

Kubheka, L.C., F.M. Mosupye and H. A. Von 2001.

Microbiological survey of the food vended salad and gravy in JHB, South Africa. Food Control 2:127-131.

Mensah, P., M. Amar-Klemesu, A. Hammond, and A. Haruna., .2001. Bacterial contamination on lettuce, tomatoes, beef and goat meat from metropolitan Accra. Ghana Medical Journal 35:1-6.

Mensah, P., K. Owusu-Darko D. Yeboah-Manu, A. Ablordey, F. Nkrumah, and H. Kamiya, 1997. The role of street vended foods in the transmission of enteric pathogens. Ghana Medical Journal 33:19-29.

Mensah, P., D. Yeboah-Manu, K. Owusu-Darku, and A. Ablordey. 2002. Street food in Accra, Ghana: how safe are they? Bulletin of the World Health Organization 80:546-556.

Mosupye F.M. and H. A. Von. 2000. Microbiological hazard identification and exposure assessment of street food vending in JHB, South Africa. Int. J. Food Microbial. 61:137-145.

Tambekar, D., V. Jaiswal, D. Dhanorkar, P. Gulhane, and M. Dudhane. 2008. Identification of microbiological hazards and safety of ready-to-eat food vended streets of Amravati City, India. J. Appl.

Biosci. 7:195 - 201.

Tendekayi, H. G., K. S. Bernard, M. Cabinet, and C. Dombo. 2008. The microbiological quality of informally vended foods in Harare, Zimbabwe. Food Control 19:829-832.

Vidal, R., M. Vidal, R. Lagos, M. Levine and V. Prado.

2004. Multiplex PCR for diagnosis of enteric infections associated with diarrheagenic Escherichia coli. J. Clin. Microbiol. 42:1787-1789.

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www.afabjournal.com Copyright © 2013 Agriculture, Food and Analytical Bacteriology

Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Producing Bacteria In Vitro‡ H. D. Edwards1, R. C. Anderson2*, T. M. Taylor1, R. K. Miller1, M. D. Hardin3, N. A. Krueger2, D. J. Nisbet2 Texas A&M University, College Station, TX USDA/ARS, Southern Plains Agricultural Research Center, College Station, TX 3 IEH Laboratories & Consulting Group, Lake Forest Park, WA 1

2

Mention of trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does

‡

not imply its approval to the exclusion of other products that may be suitable.

ABSTRACT Lipids are often used as substrates when measuring growth and lipolytic activity of lipase-producing bacteria, however, the introduction of non-miscible lipid substrates into aqueous in vitro culture systems is problematic for generating accurate and consistent results. The objective of the present study was to develop a digesta-free method for culturing and assaying lipolytic activity of mixed as well as pure populations of known ruminal lipase-producing bacteria. Accordingly, the inclusion of 0, 11, and 21 g of glass beads as a solid support matrix in place of digesta was examined. Results showed a significant increase (P < 0.05) in rate of lipolysis in the incubations containing 11 or 21 g glass beads compared to the non bead incubations. Activity was also increased (P < 0.05) in a separate study when tubes containing beads were incubated horizontally rather than vertically. These results indicate that glass beads are a suitable substitute for rumen digesta when examining lipolytic activity of mixed rumen cultures in vitro. When tested against pure cultures of the ruminal lipase-producing bacteria Anaerovibrio lipolyticus 5s, Butyrivibrio fibrisolvens 49, Propionibacterium avidum and acnes, addition of glass beads did not significantly increase rates of free fatty acid release; however, results showed that there was substantial variation between triplicate sets incubated without glass beads. Standard deviations suggest that the use of glass beads had a tendency to reduce variability within triplicate sets. Thus, inclusion of glass beads provides a clean and consistent incubation system for examining lipase activity in vitro.

Keywords: lipolysis, lipase, glass beads, digesta, support matrix, lipid, rumen, enzyme, interfacial activation, microbes Agric. Food Anal. Bacteriol. 3: 293-302, 2013

Correspondence: Robin C. Anderson, Robin.Anderson@ars.usda.gov Tel:+1-979-260-9317 Fax:+1-979-260-9332

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INTRODUCTION Currently there is a need for the advancement and development of methods for culturing ruminal lipaseproducing bacteria in order to gain an enhanced insight of the functional role(s) these organisms have in the rumen. The lipid substrates generally used to study these bacteria are problematic due to their uneven dispersion when added to water based media, generally leading to inconsistent results. Ruminantproduced foods contain high proportions of saturated fats, a result of microbial biohydrogenation within the rumen which rapidly saturates and thus limits the availability of free unsaturated fatty acids for absorp-

would serve as an acceptable replacement for rumen digesta. We hypothesize that the glass beads will increase lipase activity to a greater extent than digesta by providing a more homogenous support matrix allowing a consistent dispersion of the oil substrate and therefore improved interaction between lipase and oil substrate.

MATERIALS AND METHODS Mixed culture handling procedures The mixed bacterial populations used in this study

tion and assimilation (Harfoot and Hazlewood, 1997).

Biohydrogenation of mono- or polyunsaturated fatty acids by ruminal microbes cannot occur unless free fatty acids (FFA) are first hydrolyzed from their lipid precursors, a process known as lipolysis.

As reviewed by Lourenço and colleagues (2010) the ability of ruminal microbes to hydrolyze triglycerides was reported more than 50 years ago (Garton et al., 1958). Since then, numerous studies have been conducted to characterize the biological and physical factors affecting ruminal lipolysis by mixed or pure populations of ruminal bacteria. For instance, Hawke and Silcock (1970) have shown that more than 50% of ruminal lipase activity is contained within the particulate fraction of freshly collected ruminal fluid.

It has been recognized that the enzymatic activity of lipases is markedly increased in environments that stabilize the lipid/water interface that occurs at the point of contact between oil and water (Paiva et al., 2000). Thus, studies using rumen contents as incubation materials likely provided a solid support that served to stabilize the lipid/water interface, a phenomenon referred to as interfacial activation (Rao and Damodaran, 2002). A major limitation to studies conducted with particulate matter and digesta is that these materials are not homogenous in size or mi-

were obtained from fresh rumen contents collected from a cannulated cow grazing on predominantly ryegrass (Lolium multiflorum Lam.) pasture. Rumen fluid and digesta were separated by straining though a nylon paint strainer (Leyendecker et al., 2004) into separate pre-warmed insulated containers that had been flushed with warm water prior to sample collection. The containers were filled completely (approximately 500 mL), capped and transported to the lab. Upon arrival at the laboratory, CO2 was bubbled through the rumen fluid to keep it in an anaerobic state until its use as a source of bacterial inoculum (within 30 min of collection). The digesta was kept in its closed container until distributed under a continuous stream of CO2 to its respective incubation tubes.

The cow was cared for according to procedures approved by U.S. Department of Agriculture – Agricultural Research Service (USDA-ARS) Southern Plains Agricultural Research Center’s Animal Care and Use Committee (Protocol #2010005).

crobial composition, which can lead to considerable variation and experimental error during incubation.

The main objective of this study is to examine the use of glass beads as a solid support matrix at varying levels (0, 11, and 21 g) in vitro to determine if they

and Bryant, 1981), 22.5 mg each of K2HPO4 and 22.5 mg KH2PO4 (JT Baker, Mallinckrodt Baker Inc., Phillipsburg, NJ), 45.0 mg (NH4)2SO4, 45.0 mg NaCl, 4.5 mg MgSO4.7H2O, 4.5 mg CaCl2.6H2O and 22.5 mg CaCl2; 1.0 mL of 0.1% resazurin, 4,000 mg NaHCO3

294

Media preparation Mixed bacterial populations in fresh ruminal fluid were cultured in a standard rumen fluid medium containing per liter: 100 mL clarified rumen fluid (Hespell

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013

and 500.0 mg cysteine hydrochloride (JT Baker). For cultivation of pure cultures of lipase-producing bacteria, a standard pure culture medium was prepared and contained per liter: 292 mg each of K2HPO4 and KHPO4, 480 mg (NH4)2SO4, 480 mg NaCl, 100 mg MgSO4•7H2O, 64 mg CaCl2•2H20, 4,000 mg Na2CO3, 600 mg cysteine-HCl, 10 g trypticase (BBL Microbiology Systems, Cockeysville, MD), 2.5 g yeast extract (Difco; Becton, Dickinson and Company, Sparks, MD), branched-chain fatty acids (1 mmol each of isobutyrate, isovalerate, and 2-methylbutyrate), 15.0 mL of 2.0% glucose stock dissolved in water, hemin, vitamin mix (20 mg each thiamine, pantothenate, nicotinamide, pryridoxine HCl, riboflavin, 1 mg p-aminobenzoic acid, 0.5 mg biotin, 0.5 mg folic acid, 0.2 mg vitamin B-12, and 0.5 mg lipoic acid) and trace minerals (Cotta and Russell, 1982). All chemicals were purchased from Sigma-Aldrich (Milwaukee, WI) unless otherwise noted. Media were prepared by boiling to remove dissolved O2 and then saturated with O2-free CO2 gas by cooling on ice while under a continuous flow of 100% CO2. The cooled media were distributed (6 mL/tube unless otherwise specified) using the anaerobic Hungate technique as described by Bryant (1972) into 18 x 150 mm glass tubes pre-loaded with 0, 11, or 21 g soda lime glass beads as indicated (Fisher Scientific, Pittsburgh, PA) and with 0.1 mL olive oil for the standard rumen fluid medium and 0.2 mL olive oil for the standard pure culture medium.

The tubes were immediately closed with crimp tops.

After sterilization, the tubes were cooled and stored at room temperature until inoculation.

Determination of lipolytic activity of mixed rumen bacterial populations in the presence of varying levels of glass beads

were used to determine presence of FFA that may have been introduced during inoculation, and provide for proper baseline correction for subsequent FFA analyses. Biological activity was terminated immediately upon collection of tubes by the addition of 0.5 mL of concentrated (37%) HCl. Lipolytic activity was determined by extraction and colorimetric measurement of FFA accumulation using methods described by Kwon and Rhee (1986). All subsequent studies herein described used the same incubation procedures and measurement of FFA accumulation unless otherwise specified.

Comparison of incubation orientations and their effects on enzyme activity Differing tube incubation orientations were compared to identify tube orientation most conducive to obtaining the highest rate of lipolytic activity.

Standard rumen fluid medium was prepared as previously described and 6.0 mL distributed to tubes preloaded with 21 g of glass beads and 0.1 mL olive oil. Following sterilization, two sets of tubes for each tube orientation were inoculated with 1.0 mL freshly collected rumen fluid and then incubated horizontally or vertically under 100% CO2 for 0 or 48 h while agitating at 40 rpm. Concluding incubation, biological activity was stopped and accumulation of FFA measured.

Comparison of glass beads as a support matrix versus rumen digesta

Two separate triplicate sets of incubation tubes for each treatment (0, 11 or 21 g glass beads) were inoculated (1 mL/tube) with freshly collected ruminal

Five g of freshly collected and squeezed (to eliminate residual rumen fluid) rumen digesta or 21 g sterile glass beads were added to sterile 18 x 150 mm glass tubes preloaded with 0.1 mL olive oil.

Tubes were then each inoculated with 6 mL of freshly collected, strained ruminal fluid. Transfer of digesta and ruminal fluid were done while flushing with 100%

fluid. One set of tubes for each treatment was collected immediately after inoculation to serve as 0 h controls and the other set was collected after 48 h incubation at 39°C while being agitated at 40 rpm in an Innova™ 4000 – incubator shaker. Zero h controls

CO2. All tubes were subsequently closed with rubber stoppers and then collected as either 0 h controls (three each of tubes prepared with digesta or glass beads) or after 48 h of horizontal incubation at 39°C while being agitated at 40 rpm. Immediately upon

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collection, biological activity was terminated and accumulation of FFA was measured.

Free fatty acid accumulation by pure cultures of ruminal lipase-producing bacteria in the presence or absence of glass beads Pure cultures of Anaerovibrio lipolyticus 5s and Butyrivibrio fibrisolvens 49 were obtained from Dr. Jay Yanke, Agriculture-Agri Food Canada. Strains of Propionibacterium avidum and Propionibacterium acnes were previously isolated from the rumen of a pastured cow (Krueger et al., 2008). For long-term preservation of pure cultures, bacteria were stored in 20% anaerobic glycerol at -80ºC. Upon removal from storage, each bacterial isolate was revived by two consecutive 24 to 48 h culture transfers, each in 10 mL standard pure culture medium supplemented with 2.0% pre-sterilized olive oil. Revived cultures were each inoculated (0.2 mL) into four triplicate sets of 18 x 150 mm crimp top tubes containing 6 mL standard pure culture medium plus 0.2 mL added olive oil and either no (2 sets of triplicate tubes) or 21 g of glass beads (other two sets of triplicate tubes).

Immediately following inoculation two sets of the triplicate tubes from each bacterial isolate, one set with and the other without added beads, were acidified with 0.5 mL of concentrated (37%) HCl to stop growth and enzyme activity prior to each incubation series, thus serving as 0 h controls. The remaining sets of tubes were incubated horizontally for 48 h under above-described conditions and accumulation of FFA measured.

Statistical analysis

RESULTS Lipolytic activity of mixed rumen bacterial populations in the presence of varying levels of glass beads When mixed ruminal populations were tested during batch culture, a main effect (P = 0.0048) of bead inclusion was observed on rates of FFA release after 48 h culture. For instance, rates of FFA accumulation (mean ± SD) were higher (P < 0.05) for broth cultures grown in tubes containing 11 g of glass beads completely immersed (88.59 ± 12.93 nmol/mL per h) or 21 g of glass beads just barely covered by the meniscus of the broth in the upright tube (174.34 ± 64.48 nmol/mL per h) than for cultures grown in broth without added glass beads (4.49 ± 7.77 nmol/mL per h).

Comparison of reaction tube orientation and its effect on enzyme activity observed during incubation Different tube orientations were compared during incubation of mixed culture containing 21 g glass beads (Figure 1, Study 1). There was approximately a 9.5 fold increase in the observed rate of FFA release by ruminal microorganisms incubated in horizontally oriented reaction tubes as compared to microbes incubated in vertically oriented tubes. Mean rates of FFA release significantly increased from approximately 12.39 nmol/mL per h to approximately 130.54 nmol/mL per h when tubes were incubated horizontally as compared to vertically (P < 0.05).

Lipolytic activity during incubation of strained rumen fluid containing either glass bead or digesta

Tests for the effects of the different treatments were done using a general analysis of variance

The inclusion of glass beads or rumen digesta was compared to determine if the use of glass beads

(ANOVA) (Statistix v.9.0, Analytical Software, Tallahassee, FL). Significant differences between means were separated and identified by least squares differences (LSD) analysis (P < 0.05).

would prove to be sufficient to replace rumen digesta as a support matrix for ruminal-lipase producing bacteria. Results show that FFA release by mixed bacterial populations in freshly collected and strained ruminal fluid was higher (P < 0.05) following

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Figure 1. Comparison of rates of FFA accumulation by mixed populations of ruminal microbes incubated 48 h at 39°C in two separate incubation studies. Study 1, culture tube sets were incubating vertically or horizontally containing 21 g glass beads and 6.0 mL mixed culture rumen fluid; Study 2, culture tube sets were incubating with 5.0 g rumen digesta or 21.0 g glass beads (each occupying approximately the same volume within the tubes) in the presence of 6.0 ml of mixed culture rumen fluid; values are means ± standard deviation (n = 3). Unlike letters indicate that means differ (P < 0.05).

Accumulation of Free Fatty Acids (nmol/mL per h)

450

Study 1

Study 2

400

a

350 300 250 200

a

150

b

100 50

b

0 Horizontal Incubation

Vertical Incubation

48 h incubation on a bed of glass beads than on a bed of fresh squeezed rumen digesta (Figure 1, Study 2). Mean rates of FFA release significantly increased from approximately 90.32 nmol/mL per h to approximately 336.36 nmol/mL/h when tubes were incubated with glass beads as compared to rumen digesta (P < 0.05).

Growth of ruminal lipase-producing bacteria in pure culture incubated in the presence and absence of glass beads

Beads (21 g) Digesta (5 g)

statistically significant differences in observed FFA release amongst tested lipase-producing microorganisms, the presence of the glass beads did result in numerically higher rates of FFA release for all of the bacterial organisms tested with the exception of P. avidum. The variability in rate measurements, whether expressed as standard deviations or as the coefficient of variation, also were numerically higher for tubes not containing glass beads as compared to the glass bead treatments, with the exception of B. fibrisolvens 49 (Table 1).

Glass beads as a support matrix for enzyme activity was used to characterize major contributors to ruminal lipolytic activity from amongst tested

DISCUSSION

bacterial isolates. Results showed that the presence or absence of glass beads did not (P > 0.05) affect lipolytic activity of A. lipolyticus 5s, B. fibrisolvens 49, P. avidum, and P. acnes (Table 1). Nevertheless, although inclusion of glass beads did not result in

It is recognized that the insolubility of lipid substrates and the lack of interfacial activation are major limitations to the study of lipolytic enzymes in aqueous media. For instance, lipolytic activity by mixed populations of ruminal microbes is known to be in-

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0.0715

169.43 ± 49.57 (29%)

89.98 ± 27.08 (30%)

Butyrivibrio fibrisolvens 49

0.0560

422.22 ± 59.91 (14%)

797.98 ± 237.31 (30%)

Propionibacterium avidum

0.7043

47.05 ± 14.84 (36%)

54.87 ± 29.69 (54%)

Propionibacterium acnes

19%

33%

Average coefficient of variation

a

Values depict least square means ± SD calculated from cultures incubated in triplicate. Coefficient of variation (CV) is presented within parenthesis. b Cultures were incubated in 18 x 150 mm crimp top culture tubes containing 6 mL standard anaerobic medium (100% CO2) without or with 21 g glass beads and 0.2 mL added olive oil at 39°C for 48 h with constant agitation (40 rpm).

0.0817

107.52 ± 0.85 (1%)

With glass beads

P value

86.78 ± 15.50 (18%)

Anaerovibrio lipolyticus 5s

Without beads

Incubation conditionsb

Rate of FFA accumulation (nmol/mL per h (CV)a)

Table 1. Comparison of rates of FFA accumulation and associated variability by pure cultures of lipase-producing ruminal microbes incubated without and with addition of glass beads.

creased when using rumen microbes associated with digesta or added forage substrate than when using strained ruminal fluid alone (Dohme et al., 2003; Garton et al., 1958; Hawke and Silcock, 1970; Krueger et al., 2010; Shorland et al., 1955; Van Nevel and Demeyer, 1995). However, the heterogeneous makeup of these contents introduces considerable variability into the conduct of such studies as differences in particle size, chemical composition, stage of digestion and microbial colonization can markedly affect the amount of surface area available for contact with the lipid substrate. For instance, Krueger et al. (2010) reported rates of ruminal lipolysis to be approximately 5,060 nmol FFA liberated/g of undiluted rumen con-

present study demonstrated that rates of FFA accumulation during incubation of 1 mL freshly collected rumen fluid in 6 mL of a standard aqueous medium supplemented with 0.1 mL olive oil were lower than previous research discussed above.

It is possible that the glass beads may provide a solid support matrix that promotes secretion of the extracellular lipases by microorganisms growing in medium by providing surfaces for microbial attachments and/or lipid adsorption. Thus the glass beads may allow microorganisms and lipids to come into proximity of one another and their subsequent interfacial activation but also provide an environment conducive to the growth of lipase-producing bacte-

tents per h during a 24 h incubation of 5 g freshly collected rumen digesta with 0.5 g added olive oil.

Conversely, based on estimates of amounts of lipid degraded following 24 h incubation of 25 mL freshly collected strained ruminal fluid (lacking particleassociated bacteria), with 0.4 g ground forage and 0.125 g added soy oil, approximately 640 nmol FFA would have been liberated/mL of rumen contents per h (Dohme et al., 2003). In contrast, based on accumulations of FFA reported by Van Nevel and Demeyer (1995) during a 6 h incubation of 10 mL freshly collected and filtered rumen fluid diluted with 50 mL buffer containing 0.5 g of a ground concentrate diet and 0.08 g soy oil, the rate of lipolysis was calculated to be approximately 170 nmol FFA/mL per h.

In attempt to reduce the variability of FFA liberation observed between the studies, the present study examined the use of glass beads in several experiments as a potential replacement for rumen digesta. Glass beads serve as a more homogenous support matrix and thus it would be reasonable to hypothesize that they would provide a more consistent dispersion of lipids in aqueous media than the heterogeneous, rumen digesta. When mixed ruminal populations were tested during batch culture, rates of FFA release after 48 h incubation were 20- and 39-

ria by allowing for their attachment. In support of this Martinez and Nudel (2002) demonstrated that secretion of lipase produced by Acinetobacter calcoaceticus was stimulated by glass beads. Similarly, adsorption of lipases to siliconized or hydrocarbon-coated glass beads has been used to cause interfacial activation in a variety of lipases (Fernandez-Lafuente et al., 1998; Ferrato et al., 1997).

Rates of FFA release were also higher for cultures grown in tubes where beads (11 g) were completely immersed within the broth medium than for cultures grown in broth alone. This result was unexpected because approximately 2 cm of aqueous medium, on which most but not all of the added oil floated, remained above the bed-level for tubes where the beads were completely immersed. This result provides further support that even though the glass beads were not necessarily in contact with the oil their contact with the bacteria may have provided an environment optimal for bacterial growth and/or lipase secretion as discussed prior.

Different tube orientations were compared during incubation and tubes were agitated in attempt to increase contact between the medium and lipid substrate, and microorganisms to increase rates of observed lipolytic activity. Tube sets containing 21 g

fold higher (P < 0.05) for cultures incubated in tubes where the broth medium was in contact with a bed of glass beads (11 and 21 g beads, respectively) versus cultures grown in broth medium not containing beads (4.49 ± 7.77 nmol/mL per h). Results from the

of glass beads were incubated vertically or horizontally and results showed that there was an increase (P < 0.05) in rates of FFA accumulation (nmol/mL per h) in reaction tubes incubated horizontally (Figure 1, Study 1). It is likely that the horizontal incubation

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provided a greater surface area, free movement of enzyme and dispersion of oil allowing for increased interaction resulting in the higher rates of FFA release for horizontally incubated tubes.

Glass beads and rumen digesta were directly compared by examining both matrices in liquid medium during incubation. Lipolytic activity was significantly higher (P < 0.05) for mixed populations of ruminal microbes incubated in liquid medium containing glass beads as compared to those incubated in medium containing rumen digesta (Figure 1, Study 2).

The rates of FFA release observed in samples containing digesta-supported microbes in this study were comparable to those reported by Van Nevel

Henderson (1971) demonstrated that the lipase of A. lipolyticus 5s appears in the medium early in the life of the culture and the lipase activity was not associated with the bacterial cell or fragmented bacteria.

Similar to A. lipolyticus 5s, the lipase for P. avidum and P. acnes have been shown to also be produced extracellularly (Greenman et al., 1983). Furthermore, one function of the lipase secreted by P. acnes has been shown to possibly aid in colonization, by promoting cell adherence to components such as oleic acid (Gribbon et al., 1993), thus the glass beads likely aid in increasing this adhesion and contact with the oil substrate. However, B. fibrisolvens, differs from the extracellular lipase-producing rumen bacterium

and Demeyer (1995), thus demonstrating that the glass beads served as a sufficient replacement for rumen digesta.

The glass beads were also used in this study to determine their applicability for use in supporting the characterization of in vitro lipase activity from pure cultures of ruminal lipolytic microorganisms. Regarding the bacteria used in this study, Anaerovibrio lipolyticus and Butyrivibrio fibrisolvens have long been recognized as important contributors to ruminal lipolysis. Propionibacterium avidum and P. acnes are also known to express lipase activity, though less is known regarding their contribution to ruminal lipolysis. Each of the different bacterial strains were cultured individually in the presence and absence of glass beads for 48 h. Different from ruminal mixed cultures, the presence or absence of beads did not influence (P > 0.05) observed lipase activity by any of these bacterial strains although the rate of FFA accumulation by P. avidum was numerically higher when cultured without beads (Table 1). However, the standard deviation for the P. avidum cultures incubated without the glass beads was quite high which lessens the level of confidence in the measured rate.

Conversely, the variability was numerically lower for the bead than the non bead treatments for all the

A. lipolyticus 5s, P. avidum, and P. acnes in that they produce esterases exhibiting lipase activity that are cell bound (Lanz and Williams, 1973). The production of cell bound esterases instead of an extracellular lipase may suggest that B. fibrisolvens does not require a support matrix due to the esterases being already supported by the cell itself. This is consistent with results in Table 1, showing that unlike the other bacteria the standard deviation is higher for B. fibrisolvens 49 with the bead treatment than the fluid treatment. This suggests that the glass beads may be effective at reducing variability between replicates providing a consistent incubation system for obtaining reproducible results for extracellularly produced lipase but not when the enzyme is cell bound.

Conversely, the inclusion of glass beads in mixed cultures did have a marked effect on lipolytic activity for mixed ruminal microbes. The differences in characterization between pure cultures and mixed cultures with and without the glass beads may suggest that while species of ruminal microbes grown in pure culture contribute appreciably to cumulative lipolytic activity in the rumen, the identities of highly active, extracellular lipase producing, rumen bacterial genera/species has yet to be made.

bacteria except B. fibrisolvens 49 and coefficients of variation were numerically lower for all strains.

When averaged across all four strains, the coefficient of variation was 42% lower for the bead treatments than for the non bead treatment.

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CONCLUSIONS The introduction of glass beads markedly increased in vitro lipolytic activity in mixed culture

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incubation over rumen digesta, most likely due to increased interfacial activation due to a more homogeneous support matrix. However, the presence of beads as a support matrix for individual cultures of lipase-producing bacteria examined in this study did not appear to have a significant effect on lipolytic activity, leaving potential for the identification of other highly active contributors to rumen lipolysis.

The results of these studies may be utilized both for the development or improvement of methods for culturing lipase-producing bacteria, and for gaining further insight into the bacteria responsible for the majority of lipolytic activity in the rumen.

terfacial activiation. In: B. Rubin and E. A. Dennis (eds.) Methods in Enzymology No. 286 Part B. Academic Press, New York.

Garton, G. A., P. N. Hobson, and A. K. Lough. 1958.

Lipolysis in the rumen. Nature 182: 1511-1512.

Greenman, J., K. T. Holland, and W. J. Cunliffe.

1983. Effects of pH on biomass, maximum specific growth rate and extracellular enzyme production by three species of cutaneous propionibacteria grown in continuous culture. J. Gen. Microbiol.

129: 1301-1307.

Gribbon, E. M., W. J. Cunliffe, and K. T. Holland. 1993.

Interaction of Propionibacterium acnes with skin lipids in vitro. J. Gen. Microbiol. 139: 1745-1751.

Bryant, M. P. 1972. Commentary on the Hungate technique for culture of anaerobic bacteria. Am. J. Clin. Nutr. 25: 1324-1328.

Cotta, M. A., and J. B. Russell. 1982. Effect of peptides and amino acids on efficiency of rumen bacterial protein synthesis in continuous culture. J. Dairy Sci. 65: 226-234.

Dohme, F., V. Fievez, K. Raes, and D. I. Demeyer.

2003. Increasing levels of two different fish oils lower ruminal biohydrogenation of eicosapentaenoic and docosahexaenoic acid in vitro. Anim. Res. 52: 309-320.

Fernandez-Lafuente, R., P. Armisen, P. Sabuquillo, G.

Harfoot, C. G., and G. P. Hazlewood. 1997. Lipid metabolism in the rumen. In: P. N. Hobson and C. S. Stewart (eds.) The Rumen Microbial Ecosystem p 382-426. Chapman & Hall, London, UK. Hawke, J. C., and W. R. Silcock. 1970. The in vitro rates of lipolysis and biohydrogenation in rumen contents. Biochim. Biophys. Acta 218: 201-212.

Henderson, C. 1971. A study of the lipase produced

by Anaerovibrio lipolytica, a rumen bacterium. J. Gen. Microbiol. 65: 81-89.

Hespell, R. B., and M. P. Bryant. 1981. The genera Butyrivibrio, Succinvirbio, Succinimonas, Lachnospira, and Selenomonas. In: M. P. Starr, H. Stolp, H. G. Trüper, A. Barlows and H. G. Schlegel (eds.) The Prokaryotes. A Handbook on Habitats, Isolation, and Identification of Bacteria No. II. p 1002-1021.

Springer-Verlag, Berlin.

Krueger, N. A., R. C. Anderson, T. R. Callaway, T. S. Edrington, and D. J. Nisbet. 2008. Isolation of prominent lipolytic rumen bacteria. J. Anim. Sci. 86, E-Suppl. 2/J. Dairy Sci., Vol. 91, E. Suppl. 1: 87.

Krueger, N. A., R. C. Anderson, L. O. Tedeschi, T. R. Callaway, T. S. Edrington, and D. J. Nisbet. 2010.

Evaluation of feeding glycerol on free-fatty acid production and fermentation kinetics of mixed ruminal microbes in vitro. Bioresour. Technol. 101:

Frenandez-Lorente, and J. M. Guisan. 1998. Immobilization of lipase by selective adsorption on hydrophobic supports. Chem. Phys. Lipids 93: 185-197.

Ferrato, F., F. Carrlere, L. Sarda, and R. A. Verger.

1997. Critical reevaluation of the phenomenon in-

8469-8472.

Kwon, D., and J. Rhee. 1986. A simple and rapid colorimetric method for determination of free fatty acids for lipase assay. J. Am. Oil. Chem. Soc. 63: 89-92.

ACKNOWLEDGEMENTS This research was supported in part by USDA, Cooperative State Research, Education and Extension Service (CSREES) grant number 2009-51110-05852.

The expert technical assistance of Jackie Kotzur (USDA/ARS, College Station, TX) is greatly appreciated.

REFERENCES

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Lanz, W. W., and P. P. Williams. 1973. Characterization of esterases produced by a ruminal bacterium identified as Butyrivibrio fibrisolvens. J. Bacteriol.

113: 1170-1176.

Leyendecker, S. A., T. R. Callaway, R. C. Anderson, and D. J. Nisbet. 2004. Technical note on a much simplified method of collecting ruminal fluid using a nylon paint strainer. J. Sci. Food Agri. 84: 387-389.

Lourenço, M., E. Ramos-Morales, and R. J. Wallace.

2010. The role of microbes in rumen lipolysis and biohydrogenation and their manipulation. Animal 4: 1008-1023.

Martinez, D. A., and B. C. Nudel. 2002. The improvement of lipase secretion and stability by addition of inert compounds into Acinetobacter calcoaceticus cultures. Can. J. Microbiol. 48: 1056-1061.

Paiva, A. L., V. M. Balcão, and F. X. Malcata. 2000.

Kinetics and mechanisms of reactions catalyzed by immobilized lipases. Enzyme Microb. Technol. 27: 187-204.

Rao, C. S., and S. Damodaran. 2002. Is interfacial activation of lipases in lipid monolayers related to thermodynamic activity of interfacial water? Langmuir 18: 6294-6306.

Shorland, F. B., R. O. Weenink, and A. T. Johns. 1955.

Effect of the rumen on dietary fat. Nature 175: 1129-1130.

Van Nevel, C., and D. I. Demeyer. 1995. Lipolysis and biohydrogenation of soybean oil in the rumen in vitro: inhibition by antimicrobials. J. Dairy Sci. 78: 2797-2806.

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www.afabjournal.com Copyright © 2013 Agriculture, Food and Analytical Bacteriology

Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens † J. G. Wilson1, T. C. McLaurin1, J. A. Carroll2, S. Shields-Menard1, T. B. Schmidt3, T. R. Callaway4, and J. R. Donaldson1 Department of Biological Sciences, Mississippi State University, Mississippi State, MS Livestock Issues Research Unit, U. S. Department of Agriculture, Agriculture Research Service, Lubbock, TX 3 Animal Science Department, University of Nebraska, Lincoln, NE 4 Food and Feed Safety Research Unit, U. S. Department of Agriculture, Agriculture Research Service, College Station, TX 1

2

†Mandatory Disclaimer: “Proprietary or brand names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or exclusion of others that may be suitable.” USDA is an equal opportunity provider and employer.

ABSTRACT Saccharomyces cerevisiae boulardii is frequently used as a dietary supplement to promote intestinal health and reduce the impact of growth of enteric pathogens in livestock, including cattle and swine. Citrus by-products are also fed as dietary supplements that have the additional benefit of inhibiting the growth of enteric pathogens. Previous research identified that supplementation of Saccharomyces boulardii to feed containing citrus pulp significantly reduced the average daily gain of weanling pigs challenged with Salmonella enterica, suggesting citrus pulp reduces the effectiveness of Saccharomyces boulardii. To investigate this possibility, an in vitro analysis was conducted on the activity of Saccharomyces boulardii in swine fecal microbial media supplemented with citrus pulp. Citrus pulp inclusion reduced (P < 0.01) populations of Saccharomyces boulardii within 48 h post-exposure, suggesting that this product may exhibit antifungal properties. Co-incubation of Salmonella with Saccharomyces boulardii reduced populations of both microbes; inclusion of citrus pulp did not lead to a further reduction of yeast populations in the coculture. The cell lysate from Saccharomyces boulardii was also found to provide a carbon source that was utilizable by Escherichia coli, but not Salmonella. Together, these results suggest that citrus pulp reduces the viability of Saccharomyces boulardii and that the subsequent effects of this interaction on enterics are varied. Though further research is needed to determine how citrus pulp influences the activity of Saccharomyes boulardii in vivo, these data strongly suggest caution should be exercised in providing citrus pulp to livestock being fed diets supplemented with live yeast probiotics.

Keywords: probiotics, citrus pulp, Salmonella Typhi, Escherichia coli, E. coli O157:H7, Saccharomyces boulardii, enterics, swine, feed supplement, antifungal Agric. Food Anal. Bacteriol. 3: 303-311, 2013

Correspondence: Janet R. Donaldson, donaldson@biology.msstate.edu, Tel: +1 662 325 9547; Fax +1 662 325 7582

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INTRODUCTION The microbial communities associated with the gastrointestinal (GI) tract of animals can be altered in response to changes in environment, food consumption or exercise (Chaucheyras-Durand and Durand, 2010). This can cause severe distress, which is problematic particularly in livestock. Abrupt changes in the gastrointestinal microbial community due to lifestyle or environmental changes can lead to acidosis, increased colonization of pathogens, and other harmful effects. In order to reduce these deleterious effects, probiotics have often been administered to livestock (Chaucheyras-Durand and Durand 2010;

product feeds also act as antimicrobial agents against the enteric pathogens Escherichia coli O157:H7 and Salmonella enterica (Callaway et al., 2008, Fett and Cooke 2003). This antimicrobial activity is likely attributed to the essential oils associated with these citrus products, including, but not limited to citrullene, linalool, and limonene (Nannapaneni et al., 2008).

Citrus products have been reported to promote the growth of Bacillus subtilis (Sen et al., 2011), which indicates that supplementation of diets with citrus by-products may promote growth of certain microorganisms within the GI tract. However, depending upon the source, citrus by-products can also have inhibitory effects on the probiotic Bifidobacterium

Siragusa and Ricke, 2012). Probiotics are microorganisms that provide a benefit to the host by improving health and growth. The mechanisms by which probiotics function are varied and debated, but are primarily attributed to a competitive ability to prevent pathogens from having access to colonization sites within the host and also to prevent pathogens from acquiring nutrients (Boirivant and Strober, 2007; Rolfe, 2000). These changes alter the GI population and influence immune parameters and responses, which ultimately improve growth efficiency (Isolauri et al., 2001; Vanbelle et al.,1990).

Saccharomyces cerevisiae subtype boulardii is a probiotic yeast that has been extensively studied in relation to preventing or alleviating intestinal distress (Rolfe, 2000). Along with pathogen inhibitory effects, evidence suggests Saccharomyces cerevisiae helps to stabilize the rumen microbial community, which may decrease the risk of acidosis in ruminants (Chaucheyras-Durand et al., 2005, Newbold et al., 1996; Nisbet and Martin, 1991). Furthermore, weanling pigs provided a diet supplemented with Saccharomyces boulardii had an improved average daily weight gain (ADG) and reduced mortality associated with endotoxemia (Collier et al., 2011).

Citrus pulp is a by-product produced from citrus

bifidum (Sendra et al., 2008). Carroll and colleagues have reported that weanling pigs provided a diet supplemented with both Saccharomyces boulardii and citrus by-products experienced a decline in ADG post-exposure to Salmonella (unpublished results), suggesting an undesirable interaction occurred between the yeast and pathogen in the gut. The aim of the current study was to analyze the interaction between Saccharomyces boulardii and enteric bacteria to determine if the viability of Saccharomyces boulardii is altered in the presence of citrus pulp using an in vitro swine fecal microbial fermentation system.

processing and is used as a low cost alternative carbohydrate source in livestock diets, particularly in citrus producing regions of the United States and South America (Ariza et al., 2001, Bampidis and Robinson 2006). Previous studies have reported that citrus by-

ssp. boulardii was obtained from a commercial supplier (Saccharomyces cerevisiae I-1077, Lallemand Animal Nutrition). Saccharomyces boulardii was routinely cultured in yeast peptone dextrose media (YPD, Sigma-Aldrich) at 37°C.

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MATERIALS AND METHODS Microbial strains and growth conditions Escherichia coli O157:H7 (ATCC 43895) and Salmonella enterica ssp. Typhi (ATCC 6539) were routinely cultured in the general culture medium tryptic soy broth (TSB) at 37°C. E. coli and S. Typhi were transformed with the plasmid pXEN-13 to allow for selection onto TSB supplemented with 100 μg/ml ampicillin (TSB amp) as previously described by our group (Free et al., 2012). Saccharomyces cerevisiae

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Survival in fecal growth medium Fecal samples were collected from pigs at the Leveck Animal Research Center at Mississippi State University (Mississippi State, MS). Fecal medium was prepared essentially as previously described (Free et al., 2012; Russell and Martin, 1984). Briefly, 33.3g of fresh feces were vortex-mixed in 33.3mL of sterile water prior to addition to 1L of base medium. The fecal medium was incubated overnight at 39°C in a shaker incubator. The following day 5.0 g of ground citrus pulp (Texas Citrus Exchange, Mission, TX) was added to 100 mL of fecal growth medium.

Bacterial cultures were grown overnight at 39°C in 5 mL TSB amp. Cultures were then diluted 1:100 and allowed to grow for 4 h to reach log phase (Optical density, OD600 approximately 0.4), at which time cultures were centrifuged for 5 min at 10,000 x g to remove antibiotics. Resulting cell pellets were resuspended in an equal volume of freshly prepared swine fecal fluid media supplemented with either 0% or 5% citrus by-products. Cultures were subsequently incubated at 39°C for 48 h. For enumeration of yeast, aliquots were diluted in 1X phosphate buffered saline (1X PBS) and plated on YPD agar supplemented with 100 U/mL of penicillin and 100 μg/mL streptomycin and 0.25 μg/mL fungizone (Invitrogen).

Cultivation trials confirmed that this medium did not inhibit the growth of Saccharomyces boulardii.

For enumeration of bacteria, samples were serially diluted in 1X PBS and subsequently plated onto nutrient agar supplemented with 100 μg/mL ampicillin (NA amp). Plates were incubated at 37°C and colony forming units (CFU) were enumerated after 24 to 48 h of incubation. A minimum of three independent replicates was performed for each strain and condition tested.

Scanning electron microscopy Cultures of Saccharomyces boulardii were grown for 24 h at 37°C in YPD. Cultures were allowed to incubate for an additional 48 h at 39°C in the presence (or absence) of 5% citrus pulp. Cells were pelleted at 10,000 x g for 5 min and fixed in 2.5% glutaraldehyde

in 0.1M sodium cacodylate buffer for a minimum of 16 h. Samples were then prepared for observation as previously described (Merritt et al. 2010). Samples were viewed using a JOEL 6500F field emission scanning electron microscope (JOEL Ltd, Tokyo, Japan).

A minimum of 20 cells was examined.

Survival in Saccharomyces boulardii lysate Cultures of E. coli, S. Typhi, and Saccharomyces boulardii were grown overnight at 37°C as described in the previous section. Cells were then pelleted, washed with 1X PBS, and resuspended in mineral salts media (MSM) lacking a carbon source (Alvarez et al., 1996). Saccharomyces boulardii cultures were lysed via sonication (Fisher Scientific Sonic Dismembrator Model 120; setting 3, 30 sec pulse; Pittsburgh, PA) and filtered through a 0.22 μm syringe filter.

Salmonella and E. coli were diluted 1:100 in 0.2mL of fresh MSM supplemented with either 20% filtrate from Saccharomyces boulardii or 2% glucose.

Growth was monitored by OD600 readings over a 24 h period with a Biotek Synergy HT microplate reader (Biotek, Winooski, VT). A minimum of three independent replicates was performed.

Statistical analysis The fold change (log10 Ntreated CFU/mL / log10 Noriginal CFU/mL) and log10 CFU/mL of yeast and bacterial populations were analyzed as means across each treatment. Data were analyzed by analysis of variance (ANOVA) using the Glimmix procedures of SAS (version 9.2, 2013, Institute, Inc, Cary, NC), with significance declared at P < 0.05.

RESULTS AND DISCUSSION Citrus pulp reduces the viability of Saccharomyces boulardii in vitro Saccharomyces boulardii was grown in swine fecal microbial fluid and viability was assessed over a

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Table 1. Fold change of Saccharomyces cerevisiae boulardii (SCB) populations (Log10 CFU/mL) cultured with citrus pulp (CP), Salmonella typhi and/or Escherichia coli O157:H7

12 h

24 h

48 h

0.99 a, x

0.96 a, x

0.89 a, y

+CP

0.95 a, c, x

0.89 a, b, x

0.79 b, y

+Salmonella

0.99 a, x

0.86 b, y

0.80 b, z

+Salmonella +CP

0.99 a, x

0.84 b, y

0.83 a, b, y

+E. coli

1.14 b, x

1.09 c, x

0.96 c, y

+E. coli +CP

1.11 b, c, x

0.99 a, y

0.84 a, b, d, z

SCB

a,b,c x,y,z

Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.

Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.

Figure 1. Citrus pulp introduces alterations into the cell surface of S. boulardii. S. boulardii was cultured in the absence (A) or presence (B) of citrus pulp for 24 h and samples were subsequently analyzed by scanning electron microscopy. Scale bars represent 1μm.

48 h growth period. Viability decreased by 0.85 log10 CFU/mL (from 7.95 to 7.10 log10 CFU/mL) after 48 h in this cultivation medium (P = 0.005; Table 1). In the presence of 5% citrus pulp, viability was reduced by 1.65 log10 after 48 h (7.97 to 6.31 log10 CFU/mL, P < 0.0001). This was an approximate 10% reduction be-

broth in the presence (or absence) of 5% citrus pulp and the integrity of the cell walls were assessed by scanning electron microscopy (Figure 1). Alterations in the cell wall morphology were evident in yeast treated with citrus pulp, indicating that citrus pulp introduces damage into the cell wall of Saccharo-

yond what was attributed to the medium alone. This suggests that citrus pulp can directly impact yeast viability.

To further analyze this interaction with citrus pulp, Saccharomyces boulardii was incubated in YPD

myces boulardii. Together, these data suggest that citrus by-products may exhibit slight fungicidal activity, or that the mechanism by which the products were processed confers this activity to the product.

The essential oils from citrus products are known to

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Table 2. Fold change of Salmonella typhi populations (Log10 CFU/mL) cultured with citrus pulp (CP) and/or Saccharomyces cerevisiae boulardii (SCB).

12 h

24 h

48 h

0.94

0.92

0.88 a

+CP

1.01 x

0.94 y

0.84 a, b, z

+SCB

0.99 x

0.91 y

0.83 a, b, z

+SCB+CP

0.94

x

0.88 x

0.79 b, y

Salmonella

a,b

Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.

Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.

x,y,z

exhibit antifungal, as well as antibacterial activities (Caccioni et al., 1998; Cvetnia and Vladimir-Kneevia, 2004). A study using Saccharomyces cerevisiae reported that while not all of the oils from citrus products eradicated Saccharomyces cerevisiae, all seemed to have an inhibitory effect (Belletti et al., 2004). This suggests that the presence of any citrus essential oils may directly alter the overall effectiveness of live yeast probiotics.

To investigate the possibility that the reduced viability of Saccharomyces boulardii in the presence of citrus pulp would alter the competitive activity of this probiotic against Salmonella, Saccharomyces boulardii and S. Typhi were cultured concurrently in a swine fecal microbial fermentation system and viability for both microbes was assessed over a 48 h period. The addition of S. Typhi reduced the viability of Saccharomyces boulardii by 14% within 24 h (8.23 to 7.05 log10 CFU/mL, P = 0.005) and by 20% within 48 h (8.23 to 6.55 log10 CFU/mL, P = 0.005; Table 1). Co-

tions in populations of Saccharomyces boulardii were more severe than those of S. Typhi within 24 h (P = 0.04), it is possible that S. Typhi utilizes nutrients in the fecal fluid media first or may be more efficient at utilization of nutrients in a mixed culture. This data warrants further investigation.

The addition of citrus pulp decreased populations of Salmonella as expected based on a previous study (Callaway et al., 2008). Within 48 h post exposure, populations of S. Typhi were reduced by 16% in the presence of citrus pulp (6.80 to 5.73 log10 CFU/mL, P < 0.0001). Populations also decreased by 17% within 48 h of cultivation in the presence of Saccharomyces boulardii (7.04 to 5.87 log10 CFU/mL, P < 0.0001). However, in the presence of both citrus pulp and Saccharomyces boulardii, populations of S. typhi were reduced by 12% within 24 h (P = 0.0585) and by 21% within 48 h (P < 0.0001; Table 2).

These data indicate that a combination of citrus pulp and Saccharomyces boulardii might lead to an enhanced lysis of S. Typhi. Though this is a promising result, it does not necessarily correlate with a beneficial synergy in vivo. A previous study found that the combination of Saccharomyces bou-

cultivation of S. Typhi and Saccharomyces boulardii reduced populations of S. Typhi by 8% within 24 h (7.03 to 6.43 log10 CFU/mL, P = 0.003) and by 17% within 48 h (7.03 to 5.86 log10 CFU/mL, P < 0.0001; Table 2). Since in a co-culture condition the reduc-

lardii and citrus pulp reduced the ADG of weanling pigs following Salmonella infections (Carroll et al., unpublished results). Therefore, an alternative interpretation of these data could suggest that the enhanced lysis of S. Typhi from the combination of

Interactions between Saccharomyces boulardii and Salmonella

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Table 3. Fold change of Escherichia coli O157:H7 populations (Log10 CFU/mL) cultured with citrus pulp (CP) and/ or Saccharomyces cerevisiae boulardii (SCB).

12 h

24 h

48 h

1.02

0.99

0.98 a

+CP

1.03 x

0.98 x

0.77 b, y

+SCB

1.00

0.99

0.98 a

+SCB+CP

0.93 x

0.87 x, y

0.78 b, y

E. coli

a,b x,y

Means within a column sharing a common superscript are not different. Significance declared at P < 0.05.

Means within a row sharing a common superscript are not different. Significance declared at P < 0.05.

Saccharomyces boulardii and citrus pulp may lead to an increase in cytotoxin release. This must be taken into account when analyzing potential antimicrobial compounds in vivo and warrants further investigation.

Cell lysate of Saccharomyces boulardii as a potential carbon source for other microorganisms

Interactions between Saccharomyces boulardii and E. coli O157:H7

Variations in the growth analysis of Saccharomyces boulardii populations may be due to the reduced viability of Saccharomyces boulardii in the presence of citrus pulp. This reduction in viability could have po-

Since enhanced reductions of S. Typhi populations were observed in the presence of citrus pulp and Saccharomyces boulardii, Escherichia coli O157:H7 was also examined to determine whether this effect would extend to other gram-negative bacteria. Populations of E. coli O157:H7 remained stable in the fecal growth medium during the 48 h (P = 0.7; Table 3). Reductions in E. coli populations in the presence of citrus pulp were only observed after 48 h (7.78 to 6.01 log10 CFU/mL reduction, P < 0.001). Saccharomyces boulardii did not reduce populations of E. coli, but the combination of Saccharomyces boulardii and citrus pulp reduced populations of E. coli by 22% within 48 h (8.43 to 6.60 log10 CFU/mL reduction, P = 0.0076; Table 3). These results indicate that the presence of Saccharomyces boulardii does not affect the viability of E. coli O157:H7 and that even in a mixed culture the effects are due to the presence of citrus pulp-related factors.

tentially two effects on the other microorganisms in the system: 1) removes competition for nutrients, or 2) provides an additional source of nutrients that can be utilized by other microorganisms in the system.

To determine whether it was possible that lysed Saccharomyces boulardii could provide an additional source of nutrients to enteric bacteria, Saccharomyces boulardii cells were lysed and the filter-sterilized lysate was analyzed as a potential carbon source.

Cultures of E. coli O157:H7 or S. Typhi were grown in MSM supplemented with either glucose or Saccharomyces boulardii lysate. MSM without the addition of a carbon source did not support growth of either E. coli or S. Typhi; the addition of glucose to this medium did allow for growth of both microorganisms (data not shown). Surprisingly, E. coli O157:H7, but not S. Typhi, utilized the lysate from Saccharomyces boulardii as a carbon source (Figure 2). Though the growth was minimal, this could potentially allow for sustainability of the population as Saccharomyces boulardii are reduced by citrus pulp.

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Figure 2. Escherichia coli O157:H7, but not Salmonella typhi, can utilize the cell lysate from S. boulardii as a sole carbon source. Salmonella typhi and E. coli O157:H7 were cultured in minimal media lacking carbon supplemented with S. boulardii cell lysate. Growth was monitored over a 24 h period by the OD600. Values represent the average of three independent replicates.

0.5

0.4

OD600

0.3

E. coli Salmonella typhi

0.2

0.1

0 0

3

6

9

12

15 Time (h)

18

21

24

In co-cultures with Saccharomyces boulardii and E. coli O157:H7, citrus pulp may affect E. coli initially. This is evident by an increase in populations of Saccharomyces boulardii and a decrease in E. coli populations (Tables 1 and 3). However, as exposure increased, the populations of Saccharomyces boulardii decreased (through lysis with citrus pulp by-products). The populations of E. coli did not decrease to the same level as cultures in the presence of citrus pulp alone. The co-culture data, along with the ability of E. coli O157:H7 to utilize Saccharomyces boulardii lysate as a carbon source, suggests that extended exposure to citrus pulp would decrease populations of Saccharomyces boulardii, which may potentially lead to a stabilization of populations of E. coli O157:H7.

of enteric pathogen populations. Further research is needed to determine how this relationship alters the gastrointestinal microbiome in vivo.

CONCLUSIONS

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www.afabjournal.com Copyright © 2013 Agriculture, Food and Analytical Bacteriology

Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over time‡ A. R. Mantz 1, D. N. Miller2, M. J. Spiehs3, B. L. Woodbury3, and L. M. Durso2 1

Department of Biological Systems Engineering, University of Nebraska, 223 L. W. Chase Hall, P. O. Box 830726, Lincoln, NE 68583, USA 2 USDA, ARS, 137 Keim Hall, UNL-East Campus, Lincoln, NE 68583, USA 3 USDA, ARS, Meat Animal Research Center, State Spur 18D, Clay Center, NE 68933, USA

Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not

‡

imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

ABSTRACT Antibiotic resistance in food animals has become an important issue for public health safety. The genes that code antibiotic resistance often enter the feedlot environment via feces and have the potential to be transferred through agroecosystems and into the food chain, either directly in their original bacterial host or via horizontal gene transfer. The objective of this study was to determine the distribution of erythromycin resistance genes associated with beef cattle excretions and ascertain whether these genes are enriched in areas of feedlot pens with high deposition of fecal material over time. The spatial distribution of manure accumulation was determined using georeferenced electromagnetic induction (EMI) readings at two times and EMI directed soil sampling. Feedlot surface samples from high- and low-manure accumulation zones were compared. The data indicated that 14 months of manure accumulation did not result in an increase in erm(B) positive feedlot soils, and the distribution of erm(B) genes was not correlated with areas of high manure deposition within the pens.

Keywords: Antibiotic resistance, resistance, antibiotic resistance gene, manure, erythromycin, ermB, feedlot pen, cattle, PCR, food animals Agric. Food Anal. Bacteriol. 3: 312-320, 2013

INTRODUCTION Erythromycin, a macrolide antibiotic commonly used to treat infections in humans, is on the World Health Organization’s list of antimicrobial agents that are critical to human health (World Health OrCorrespondence: Lisa Durso, lisa.durso@ars.usda.gov Tel: +1 -402-472-9622 Fax: +1-402-437-5712

312

ganization, 2007). Related macrolides (Tulathromycin (Draxxin), Tilmicosin (Micotil), and Tylosin (Tylan)) have been used in cattle to treat respiratory disease, pneumonia, metritis, mastitis, and foot rot (Smith Thomas, 2009). Tylosin is also used as a feed additive for cattle to prevent liver abscesses, and as part of a mineral supplement to help control pinkeye (Smith Thomas, 2009). Bacteria can develop resistance to macrolide antibiotics by encoding a suite of more

Agric. Food Anal. Bacteriol. • AFABjournal.com • Vol. 3, Issue 4 - 2013

than 30 erythromycin ribosomal methylase (erm) genes (Roberts et al., 1999). The erm genes can be found in both commensal and pathogenic bacteria, including Gram-positive and -negative species (De Leener et al., 2004; Roberts, 2004; Bae et al., 2005; Chen et al., 2007; Dogan et al., 2005).

In bacteria from cattle, resistance to tylosin is encoded by erm genes and erm(B) is the most common (Jost et al., 2004). For example, in a survey of U.S. livestock systems, the erm(B) gene was found to represent 56% of the total erm genes in bovine manure samples (Chen et al., 2007). The erm(B) gene has been reported in Campylobacter from cattle feedlots (Bae et al., 2005), Enteroccocci from pastured cattle (Anderson et al., 2008), in livestock manure and pre-harvest production systems (Chen et al., 2007), and pen floor fecal samples from feedlot heifers (Jacob et al., 2008). Many studies screen for the erm(B) gene from pathogenic and commensal bacterial isolates, but this strategy does not allow for the assessment of non-target, unculturable bacteria. Since one of the primary concerns associated with antibiotic resistance in agricultural settings is the horizontal gene transfer from animals to humans, whole community DNA needs to be screened in order to assess the entire reservoir of antibiotic resistance genes present in a sample (Isaacson and Torrence, 2002). One element that contributes to human health risk associated with antibiotic resistance genes from agricultural settings is the persistence of the genes over time (Unc and Goss, 2004). A longitudinal study demonstrated that the erm(B) gene could persist in fecal samples from cattle in field conditions for over 150 days (Alexander et al., 2011).

In commercial cattle feedlot operations, feces are continually deposited onto the pen surface and accumulate until they are removed by scraping, typically once a year. Identification of zones within the feedlot that are enriched for antibiotic resistance genes would allow for targeted sampling and remediation efforts. The large size and spatial heterogeneity of the feedlot pen presents challenges for sample collection. Typically, cattle in pens tend to congregate in certain areas, resulting in zones of high manure accumulation in the pen. Previous studies identi-

fied correlations between electromagnetic induction (EMI) readings and areas of high manure deposition (Woodbury et al., 2009; Eigenberg et al., 2010).

We hypothesized that the incidence of erm(B) genes in the feedlot were a consequence of excretion from the animal and would be concentrated in areas with high manure enrichment. To test this hypothesis we examined cattle feedlot pens that were allowed to accumulate manure for 14 months. Feedlot pen surface samples were collected based on differences in manure accumulation, delineated using EMI sampling methods and were evaluated using a conventional PCR-based erm(B) assay of total community DNA samples.

MATERIALS AND METHODS Sampling In order to ensure that samples were collected from areas representing the continuum of manure deposition, pens were mapped for EMI and sample sites were co-located with selected EMI values using the spatial response surface sampling design (RSSD) program contained in the USDA-ARS ESAP (ECe Sampling Assessment and Prediction) software package (Lesch et al., 2000).

Feedlot surface material samples were taken from ten feedlot pens (each 30 m by 60 m) at the U.S. Meat Animal Research Center, in conjunction with a previously described study (Spiehs et al., 2012).

Half of the pens contained animals receiving a normal, controlled diet of dry-rolled corn and half of the pens contained animals receiving a diet containing 14 â&#x20AC;&#x201C; 35% wet distillers grains plus solubles (WDGS) (levels changed based upon the age of the cattle in the pens). All pens have a concrete apron adjacent to the feeding area and water areas along the lateral sides of the pens with a mound in the center.

Following EMI mapping, twelve sample sites were identified in each pen, as described above, and GPS coordinates were recorded. In general, the feedlot pens have a gradient slope at 2% declination from the feeding area down to the bottom of the pen

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where aged manure and liquids accumulate (Woodbury et al., 2009). During the 13-month study, the pens were not cleaned and two separate groups of cattle were fed in the pens, each containing 32 mixed-breed finishing steers per pen. The first set was exchanged for the second between September 18th and 22nd, 2009.

A total of 240 feedlot surface material samples were collected (120 from June 2009, 120 from August 2010). Grab samples of the feedlot surface material were collected from the surface (0 to 10 cm depth), placed in 3.8-L plastic bags, and held on ice during transport to the laboratory. Aliquots of the samples were stored at -20ºC until DNA extractions could be performed. The remaining feedlot surface material was immediately dried in a forced-air oven at 100ºC for 24 hours, ground, and analyzed for moisture content, volatile solids content, nutrients, and soil pH as previously described by Spiehs et al. (2012).

DNA Extraction and Quantification Feedlot surface soil samples were extracted as previously described by Miller et al. (1999), and purified using a Wizard® DNA Purification System (Promega, Madison, WI). DNA concentrations were determined using fluorometry. Calibration standards were created using diluted λ DNA (Quant-iT™ PicoGreen® dsDNA Assay Kit) at concentrations of 1 µg mL-1, 10 µg mL-1, 100 µg mL-1, and 1000 µg mL1 , and PicoGreen® was diluted to 1:200 with 1xTE. The standards, mixed with the diluted PicoGreen®, were used to make a linear standard curve for calibration. Samples were prepared by mixing 5 µL of sample, 45 µL of 1xTE, and 50 µL of diluted PicoGreen®. Samples were allowed to rest under aluminum foil for 5 minutes and then the fluorescence was measured. To verify fluorometric results, a subset of samples (three samples from each set of 30) was also screened on 1.5% agarose gels using established mass standards. Gels were stained for 10 minutes in an ethidium bromide solution, destained for 25 minutes in distilled water, and visualized on a UV transilluminator (Ultraviolet Productions, Upland, CA).

314

Polymerase Chain Reaction A polymerase chain reaction (PCR) assay was performed for the detection of the erm(B) gene using primers developed by Böckelmann et al.

(2009). The forward and reverse primer sequences were 5’-GGATTCTACAAGCGTACCTTGGA-3’ and 5’-GCTGGCAGCTTAAGCAATTGCT-3’, respectively.

The amplification reactions were made with 0.25 µL each of forward and reverse primers (1:100 concentration), 11 µL PCR grade water, 12.5 µL Jumpstart Red TAQ ReadyMix (Sigma-Aldrich, St. Louis, MO), and 1 µL diluted sample (1:100 concentration). Positive and negative controls were run for every assay.

Thermocycling was performed using a PTC-100 Peltier thermocycler (Bio-Rad, Hercules, CA). The cycles were set at 95°C for 2 minutes, then 35 cycles repeating through 95°C for 30 seconds, 60°C for 45 seconds and 72°C for 1 minute, then finally 72°C for 7 minutes. Samples were run on an agarose gel for 45 minutes on 145 V, stained with ethidium bromide for 10 minutes, and then destained with distilled water for 20 minutes. Gels were photographed using a Kodak Gel Logic 100 Imaging System (Carestream Health, Inc., Rochester, NY).

Statistical Analysis The ANOVA and Logistic procedures available in SAS Analysis program version 9.2 (SAS Inst., Cary, NC) were used to determine the effect of diet treatment, date of sampling, and pen location on erm(B) prevalence and used to determine differences between soil parameters related to erm(B) status. Differences were considered significant at P ≤ 0.05 and were considered tendencies when the P-values ranged from P = 0.05 to P < 0.10.

RESULTS AND DISCUSSION The persistence and distribution of the antibiotic resistance gene erm(B) was examined in cattle feedlot pens over a 14 month period. Data indicate no differences in the incidence of erm(B) over the

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Table 1. Prevalence of erm(B) positive samples based upon diet fed, pen location, and sample date June 2009

August 2010

Diet*

Pen

Mound†

Edge

Mound

Edge

0% WDGS

307

60.0% (n = 5)‡

71.4% (n = 7)

83.3% (n = 6)

50.0%, (n = 6)

309

50.0% (n = 4)

100.0% (n = 8)

100.0% (n = 4)

87.5% (n = 8)

311

80.0% (n = 5)

85.7% (n = 7)

50.0% (n = 2)

70.0% (n = 10)

313

50.0% (n = 2)

90.0% (n = 10)

75.0% (n = 4)

62.5% (n = 8)

315

71.4% (n = 7)

100.0% (n = 3)

100.0% (n = 9)

100.0% (n = 5)

Ave

62.3%

89.4%

308

40.0% (n = 5)

85.7% (n = 7)

100.0% (n = 3)

88.9% (n = 9)

310

25.0% (n = 4)

62.5% (n = 8)

100.0% (n = 4)

87.5% (n = 8)

312

80.0% (n = 5)

71.4% (n = 7)

75.0% (n = 4)

62.5% (n = 8)

314

75.0% (n = 4)

75.0% (n = 8)

100.0% (n = 3)

88.9% (n = 9)

316

83.3% (n = 6)

100.0% (n = 6)

50.0% (n = 4)

87.5% (n = 8)

Ave

60.7%

78.9%

85.0%

83.1%

0% vs 35% WDGS

P diff

0.905

0.244

0.813

0.404

Overall

Ave

61.5%A§

84.2%B

83.3%B

78.5%B

35% WDGS

81.7%

74.0%

*Diet indicates either a corn-based diet excluding wet distillers grains plus solubles (0% WDGS) or a diet including up to 35% WDGS. †Mound indicates sample from the central mound and edge indicates the lower area surrounding the mound. ‡Number of samples in each cell classified as either mound or edge. Twelve total samples per pen. §Means with different letters within a row are significantly different at P < 0.05.

course of the study, regardless of animal diet (Table 1). Initial samples from June 2009 revealed a high prevalence of the gene in the pens, with 76% (n=91) of the samples testing positive for the erm(B) gene.

change.

Since the initial source of erm(B) genes detected in feedlot pen soil is likely to be the fecal deposition, the locations of high manure deposition within the

In August 2010, fourteen months later, 81% (n=97) of the feedlot surface material samples were positive for the erm(B) gene. Thus, despite fourteen months of manure deposition, the prevalence of the gene in the feedlot soil samples showed no statistical

pen were assumed to be the locations where erm(B) would most likely be detected. Mapping of the pens with EMI allows for the identification of regions in the pen with signatures characteristic of high manure deposition (Woodbury et al., 2009). Thus, if

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the erm(B) genes were concentrated in areas of high manure deposition, we would have expected to find more erm(B) positive samples in areas with high EMI readings. Our data did not support this theory at either the initial or final sample collection (Figure 1), as erm(B) positive and negative results were scattered indiscriminately across low and high EMI reading samples. Pen size and orientation may impact cattle behavior (Wilson et al., 2010), and therefore manure accumulation, so further evaluation of other pens with different designs is warranted.

Next, the ecology of the feedlot pen was considered, including pen design and animal behavior.

Cattle feedlot pens are generally outdoors and ex-

ences between erm(B) positive and erm(B) negative samples. Furthermore, when a significant difference was observed between erm(B) positive and negative samples in one set of circumstances (pen location and date), that difference was not significant for any of the other set of date and location combinations.

For instance, surface pH for June 2009 in pen edge samples was lower in erm(B) positive compared to erm(B) negative samples, but there were no differences in pH for these mound or edge samples in August 2010 or in the mound samples for June 2009.

There were no clear linkage between erm(B) and abiotic environmental parameters of the feedlot surface material such as temperature and pH (Table 2).

posed to the elements. Often there is a mound located in the pen to provide a dry area for the cattle during wet weather (Woodbury et al., 2001). Cattle are non-randomly distributed in the pens and even though cattle movement can mix surface material across the feedlot pen, distinct zones can develop where fecal organisms are fortified (Woodbury et al., 2009). Subtle differences were detected in erm(B) gene prevalence between pen sites (mound versus edge) based upon the date (Table 1). Initial prevalence of erm(B) on the mound in June 2009 was less than the prevalence of erm(B) in pen edge samples in June 2009, and the prevalence differed (P = 0.016) from both mound and pen edge samples in August 2010. The prevalence of erm(B) in the mound versus the pen edge, however, did not differ from one another in August 2010. A comparison of the overall prevalence in 2009 to 2010 (75.8% and 80.8%, respectively) showed no difference (P= 0.271).

A variety of feedlot surface properties were evaluated to determine if any had an effect on erm(B) distribution in the cattle feedlot pen (Table 2). Our data did not support the idea that the erm(B) genes are distributed across the entire feedlot pen over time. Both erm(B) positive and negative surface samples were compared for each sampling date and

In this study, results are based on PCR assays and therefore are not capable of detecting whether viable antibiotic resistant microorganisms are present in the environment, only whether a specific gene is present in the environment. However, since there is concern that antibiotic resistance genes from animal production settings may impact human health via horizontal gene transfer (Brabban et al., 2005; Colomer-Lluch et al., 2011a; b; Hawkey and Jones, 2009), the gene-based information is relevant when considering issues of public health. An organism does not need to be alive to contribute an antibiotic resistance gene. The mechanism used by genes to move through the environment to impact humans remains unclear.

The addition of antibiotic resistant bacteria to the feedlot surface is attributed to animal feces, but after the fecal bacteria leave the gastrointestinal tract (GIT), they are exposed to a drier, more oxygenated soil environment that quickly inactivates or kills many gut microorganisms. The bacterial community found on the feedlot surface material has been shown to be very distinct from the composition of the individual animalâ&#x20AC;&#x2122;s GIT (Durso et al., 2011). So, even though the original source of the erm(B) genes is assumed to be fecal bacteria, once excreted from animals the

location (mound versus edge) within the pen. Significant differences were observed between erm(B) positive and negative samples for VS, total N, pH, and ECa. However, for most surface parameters on a particular date and location, there were no differ-

biological components of feces, such as the erm(B) genes, display distribution and persistence patterns that are different from those of the chemical and physical components of the fecal material.

Finally, it must be noted that antibiotic resistance

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June 2009

Figure 1. Mapping of feedlot pens at two time points. Results of electromangnetic induction (EMI) maps of feedlot pens are displayed on a color scale. High EMI readings have been previously correlated with areas of high manure deposition (Woodbury et al., 2009; Eigenberg et al., 2010). Results of the erm(B) screening locations are displayed using red dots to indicate of erm(B) positive samples and black dots to indicate erm(B) negative samples.

307

308

309

310

311

312

313

314

315

316

308

309

310

311

August 2010

307

erm(B) positive

312

313

314

315

316

erm(B) negative

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Table 2. Feedlot pen surface composition at the edge and mound areas based upon the detection of erm(B) at the beginning and end of the feedlot feeding trial. Bold numbers indicate data that was found to be statistically relevant.

June 2009 erm(B) Positive

erm(B) Negative

August 2010 erm(B) Positive

erm(B) Negative

Pen Edge

Moisture, %

19.7

17.3

20.3

19.4

Volatile Solids, %

20.1

17.6

17.0

15.1

Total S, g/kg DM

2.4

2.6

2.5

2.3

Total N, g/kg DM

6.7

5.8

8.4

7.2†

Total P, g/kg DM

3.2

3.1

3.8

3.5

Total K, g/kg DM

9.8

10.3

9.2

8.5

Soil temperature, ºC

30.0

28.9

33.2

33.4

Surface temperature, ºC

43.3

39.2

43.2

41.3

Soil pH

7.4

7.5

Shallow ECa‡, mS/m

171.2

192.0

171.1

138.8*

Deep ECa, mS/m

165.1

173.6

174.4

149.6

15.2

7.7

8.1*

Pen Mound

Moisture, %

12.3

10.7

14.6

Volatile Solids, %

13.6

13.0

13.0

9.5*

Total S, g/kg DM

1.4

1.3

1.7

1.3†

Total N, g/kg DM

5.3

4.4

6.1

4.3*

Total P, g/kg DM

2.3

2.1

2.8

2.2†

Total K, g/kg DM

8.8

8.2

8.0

6.8

Soil temperature, ºC

30.0

30.8

33.1

33.6

Surface temperature, ºC

43.5

44.9

42.7

42.6

Soil pH

7.5

7.5

7.3

7.3

Shallow ECa, mS/m

124.5

121.3

119.2

102.0

Deep ECa, mS/m

135.0

134.6

127.6

107.5

*Means with a different letter within a row for a particular sample time differ at P < 0.05. †Indicates a tendency (0.05<P < 0.1) for the erm(B) positive and negative samples to differ for that particular sample date. ‡Apparent electrical conductivity as measured by Woodbury et al. (2009)

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is complex, encompassing many different classes of drugs and mechanisms of resistance. The dynamics of erythromycin resistance, as coded for by erm(B), is not necessarily the same as the dynamics of other macrolide antibiotic resistance genes. There is not currently enough information to determine how different kinds of antibiotic resistance genes persist and move through agroecosystems, or how the data collected here for erm(B) relates to distribution and persistence of other antibiotic resistance genes.

Previous studies strongly support the idea that the composition of resistance genes in any particular habitat is a reflection of the species of bacteria that are commonly found in each environment (Durso et

serve compared to that of enterococci in pastured cattle. Appl. Environ. Microbiol. 74:1726-1730.

Bae, W., K.N. Kaya, D.D. Hancock, D.R. Call, Y.H. Park, and T.E. Besser. 2005. Prevalence and antimicrobial resistance of thermophilic Campylobacter spp. from cattle farms in Washington state.

Appl. Environ. Microbiol. 71:169-174.

Böckelmann, U., H.H. Dörries, M.N. Ayuso-Gabella, M. Salgot de Marçay, V. Tandoi, C. Levantesi, C. Masciopinto, E. Van Houtte, U. Szewzyk, T. Wintgens, and E. Grohman. 2009. Quantitative PCR monitoring of antibiotic resistance genes and bacterial pathogens in three European artificial groundwater recharge systems. Appl. Environ. Mi-

al., 2012; Patterson et al., 2007).

In conclusion, erm(B) genes were not enriched in feedlot soils despite 14 months of manure accumulation. Locations of high manure deposition were not the same as the locations of the erm(B) gene and the gene was not associated with specific feedlot pen zones. The dynamics of antibiotic resistance in cattle feedlot pens is likely dependent on the specific antibiotic resistance gene being studied, and is likely influenced by a number of biological, physical, and chemical parameters of the soil.

Alexander, T.W., J.L. Yanke, T. Reuter, E. Topp, R.R. Read, B.L. Selinger, and T.A. McAllister. 2011. Longitudinal characterization of antimicrobial resis-

crobiol. 75:154-163.

Brabban, A.D., E. Hite, and T.R. Callaway. 2005. Evolution of foodborne pathogens via temperate bacteriophage-mediated gene transfer. Foodborne Pathog. Dis. 2, 287-303.

Chen, J, Z. Z. Yu, F.C. Michel, Jr., T. Wittum, and M. Morrison. 2007. Development and application of Real-time PCR assays for quantification of erm genes conferring resistance to macrolides-lincosamides-sterptogramin B in livestock manure and manure management systems. Appl. Environ. Microbiol. 73:4407-4416.

Colomer-Lluch, M., L. Imamovic, J. Jofre, and M. Muniesa. 2011a. Bacteriophages carrying antibiotic resistance genes in fecal waste from cattle, pigs, and poultry. Antimicrob. Agents Chemother.

55:4908–4911. doi: 10.1128/AAC.00535-11.

Colomer-Lluch, M., J. Jofre, and M. Muniesa. 2011b.

Antibiotic resistance genes in the bacteriophage DNA fraction of environmental samples. PLoS One 6, e17549. doi: 10.1371/journal.pone.0017549.

De Leener, E., A. Martel, A. Decostere, and F. Haesebrouck. 2004. Distribution of the erm (B) gene, tetracycline resistance genes, and Tn1545-like transposons in macrolide- and lincosamide-resistant enterococci from pigs and humans. Microb. Drug

tance genes in feces shed from cattle fed different subtherapeutic antibiotics. BMC Microbiol. 11:19.

Anderson, J.F., T.D. Parrish, M. Akhtar, L. Zurek, and H. Hirt. 2008. Antibiotic resistance of enterococci in American Bison (Bison bison) from a nature pre-

Resist. 10:341-345.

Dogan, B., Y. H. Schukken, C. Santisteban, and K. J. Boor. 2005. Distribution of serotypes and antimicrobial resistance genes among Streptococcus agalactiae isolates from bovine and human hosts.

ACKNOWLEDGEMENTS Thanks are extended to Jennifer McGhee for providing technical assistance and guidance, as well as Alan Kruger, Todd Boman, John Holman, Dale Janssen, and Sue Wise for data collection and processing.

REFERENCES

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Durso, L.M., G.P. Harhay, T.P. Smith, J.L. Bono, T.Z. DeSantis, and M.L. Clawson. 2011. Bacterial community analysis of beef cattle feedlots reveals that pen surface is distinct from feces. Foodborne Pathog. Dis. 8:647-649. doi: 10.1089/fpd.2010.0774.

Durso, L.M., D.N. Miller, and B.J. Wienhold. 2012.

Distribution and quantification of antibiotic resistant genes and bacteria across agricultural and non-agricultural metagenomes. PLOS ONE 10.1371/journal.pone.0048325 Eigenberg,, R.A., B.L. Woodbury, J.A. Nienaber, M.J. Spiehs, D.B. Parker, and V.H. Varel. 2010. Soil conductivity and multiple linear regression for preci-

Environ. Microbiol. 9: 703–715. doi: 10.1111/j.14622920.2006.01190.x Roberts, M.C., J. Sutcliffe, P. Courvalin, L.B. Jenses, J. Rood, and H. Seppala. 1999. Nomenclature for macrolide and macrolide-lincosamide-stretogramin B resistance determinants. Antimicrob.

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Roberts, M.C. 2004. Distribution of macrolide, lincosamide, streptogramin, ketolide and oxazolidinone (MLSK) resistance genes in Gram-negative bacteria. Curr. Drug Targets Infect. Disord. 4:207-215.

Smith Thomas, H. 2009. The Cattle Health Handbook: preventative care, disease, treatments and emergency procedures. Editors R. Boyd-Owens,

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Hawkey, P.M. and A.M. Jones. 2009. The changing epidemiology of resistance. J Antimicrob Chermother 64:i3-i10. doi: 10.1093/jac/dkp256.

Isaacson, R.E. and M.E. Torrence. 2002. The role of antibiotics in agriculture. American Academy of Microbiology, Washington, DC. Jacob, M.E., Z.D. Paddock, D.G. Renter, K.F. Lechtenberg, and T.G. Nagaraja. 2010. Inclusion of dried or wet distillers’ grains at different levels in diets of feedlot cattle affects fecal shedding of Escherichia coli O157:H7. Appl. Environ. Microbiol.

76:7238-7242. doi: 10.1128/AEM.01221-10.

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Lesch, S.M., J.D. Rhoades, and D.L. Corwin. 2000.

ESAP-95 version 2.10R: User manual and tutorial guide. Research Rep. 146. USDA–ARS, G.E. Brown, Jr. Salinity Laboratory, Riverside, CA. Miller, D.N., J.E. Bryant, E.L. Madsen, and W.C. Ghiorse. 1999. Evaluation and optimization of DNA extraction and purification procedures for soil and sediment samples. Appl. Environ. Microbiol.

S. Guare, D. Burns. Storey Publishing, North Adams, MA , pp38-39.

Spiehs, M.J., D.N. Miller, B.L. Woodbury, R.A. Eigenberg, V.H. Varel, and D.B. Parker. 2012. Effect of feeding wet distillers grains with solubles to beef cattle on air and manure quality. Appl. Eng. Agricul. 28:423-430.

Unc, A. and M.J. Goss. 2004. Transport of bacteria from manure and protection of water resources.

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Wilson, S.C., R.C. Dobos, and L.R. Fell. 2010. Spectral analysis of feeding and lying behavior of cattle kept under different feedlot conditions. J. Appl.

Anim. Welfare Sci. 8:13-24.

Woodbury, B.L., D.N. Miller, J.A. Nienaber, and R.A. Eigenberg. 2001. Seasonal and spatial variations of denitrifying enzyme activity in feedlot soil. Trans.

ASAE 44:1635-1642.

Woodbury, B.L., S.M. Lesch, R.A. Eigenberg, D.N. Miller, and M.J. Spiehs. 2009. Electromagnetic induction sensor data to identify areas of manure accumulation on a feedlot surface. Soil Sci. Soc. Am. J. 73:2068-2077.

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Patterson, A. J., R. Colangeli, P. Spigaglia, and K.P. Scott. 2007. Distribution of specific tetracycline and erythromycin resistance genes in environmental samples assessed by macroarray detection.

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VOLUME 3 ISSUE 1 REVIEW 17

Greenhouse Gas Emissions from Livestock and Poultry C. S. Dunkley and K. D. Dunkley

30

Can Salmonella Reside in the Human Oral Cavity?

S. A. Sirsat

39

Shiga Toxin-Producing Escherichia coli (STEC) Ecology in Cattle and Management Based Options for Reducing Fecal Shedding T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, D. J. Nisbet

ARTICLES 6

Growth of Acetogenic Bacteria In Response to Varying pH, Acetate Or Carbohydrate Concentration R. S. Pinder, and J. A. Patterson

70

Independent Poultry Processing in Georgia: Survey of Producers’ Perspective E. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, S. C. Ricke

Introduction to Authors 79

Instructions for Authors

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VOLUME 3 ISSUE 2 ARTICLES 94

Consumers’ Interest in Locally Raised, Small-Scale Poultry in Georgia E. J. Van Loo, W. Q. Alali, S. Welander, C. A. O’Bryan, P. G. Crandall, and S. C. Ricke

129 Isolation and Initial Characterization of Acetogenic Ruminal Bacteria Resistant to Acidic Conditions

P. Boccazzi and J. A. Patterson

145 Linoleic Acid Isomerase Expression in Escherichia coli BL21 (DE3) and Bacillus spp S. Saengkerdsub

REVIEW 103 Current and Near-Market Intervention Strategies for Reducing Shiga Toxin-Producing Escherichia coli (STEC) Shedding in Cattle.

T. R. Callaway, T. S. Edrington, G. H. Loneragan, M. A. Carr, and D. J. Nisbet

121 Potential for Rapid Analysis of Bioavailable Amino Acids in Biofuel Feed Stocks D. E. Luján-Rhenals, and R. Morawicki

Introduction to Authors 162 Instructions for Authors

The publishers do not warrant the accuracy of the articles in this journal, nor any views or opinions by their authors.

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VOLUME 3 ISSUE 3 BRIEF COMMUNICATIONS 186 Vibrio Densities in the Intestinal Contents of Finfish from Coastal Alabama J.L. Jones, R.A. Benner Jr., A. DePaola, and Y. Hara-Kudo

ARTICLES 176

Antimicrobial Activity of Red Clover (Trifolium pratense L.) Extract on Caprine Hyper-Ammonia-Producing Bacteria M. D. Flythe, B. Harrison, I. A. Kagan, J. L. Klotz, G. L. Gellin, B. M. Goff, G. E. Aiken

230 Suitability of Various Prepeptides and Prepropeptides for the Production and Secretion of Heterologous Proteins by Bacillus megaterium or Bacillus licheniformis S. Saengkerdsub, R. Liyanage, J. O. Lay Jr.

REVIEW 195 Utility of Egg Yolk Antibodies for Detection and Control of Foodborne Salmonella P. Herrera, M. Aydin, S. H. Park, A. Khatiwara and S. Ahn

218 Potential for Dry Thermal Treatments to Eliminate Foodborne Pathogens on Sprout Seeds T. Hagger and R. Morawicki

Introduction to Authors 252 Instructions for Authors

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VOLUME 3 ISSUE 4 ARTICLES 268 The Role of Cellular Prion Proteins (PrPC) on Neuronal Brucella Infections M. Aydin, D. F. Gilmore, S. Erdogan, V. Duzguner, and S. Ahn

281 Prevalence of Foodborne Pathogens and Spoilage Microorganisms and their Drug Resistant Status in Different Street Foods of Dhaka

Z. Tabashsum, I. Khalil, Md. N. Uddin, A.K.M. M. Mollah, Y. Inatsu and Md. L. Bari

293

Development of Non-Forage Based Incubation System For Culturing Ruminal Lipase-Producing Bacteria In Vitro H. D. Edwards, R. C. Anderson, T. M. Taylor, R. K. Miller, M. D. Hardin, N. A. Krueger, D. J. Nisbet

303 Effect of Citrus Pulp on the Viability of Saccharomyces boulardii in the Presence of Enteric Pathogens

J. G. Wilson, T. C. McLaurin, J. A. Carroll, S. Shields-Menard, T. B. Schmidt, T. R. Callaway, and J. R. Donaldson

312 Persistence of erythromycin resistance gene erm(B) in cattle feedlot pens over time A. R. Mantz, D. N. Miller, M. J. Spiehs, B. L. Woodbury, and L. M. Durso

Introduction to Authors 327 Instructions for Authors

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INSTRUCTIONS TO AUTHORS MANUSCRIPT SUBMISSION

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We invite you to consider submitting your research and review manuscripts to AFAB. The journal serves as a peer reviewed scientific forum for to the latest advancements in bacteriology research on Agricultural and Food Systems which includes the following fields:

• • • • • • • • • • • • • • • •

Aerobic microbiology Aerobiology Anaerobic microbiology Analytical microbiology Animal microbiology Antibiotics Antimicrobials Bacteriophage Bioremediation Biotechnology Detection Environmental microbiology Feed microbiology Fermentation Food bacteriology Food control

• • • • • • • • • • • • • • • •

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Variability, Replication, and Statistical Analysis To properly assess biological systems independent replication of experiments and quantification of variation among replicates is required by AFAB. Reviewers and/or editors may request additional statistical analysis depending on the nature of the data and it will be the responsibility of the authors to respond appropriately. Statistical methods commonly used in the bacteriology do not need to be described in detail, but an adequate description and/or appropriate references should be provided.

The statistical model and experimental unit must be designated when appropriate. The experimental unit is the smallest unit to which an individual treatment is imposed. For bacterial growth studies, the average of replicate tubes per single study per treatment is the experimental unit; therefore, individual studies must be replicated. Repeated time analyses of the same sample usually do not constitute independent experimental units. Measurements on the same experimental unit over time are also not independent and must not be considered as independent experimental units. For analysis of time effects, assess as a rate of change over time. Standard deviation refers to the variability in the biological response being measured and is presented as standard deviation or standard error according to the definitions described in statistical references or textbooks.

Results Results represent the presentation of data in words and all data should be described in same fashion. No discussion of literature is included in the results section.

Discussion The discussion section involves comparing the current data outcomes with previously published work in this area without repeating the text in the results section. Critical and in-depth dialogue is encouraged.

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Results and Discussion Results and discussion can be under combined or separate headings.

Conclusions State conclusions (not a summary) briefly in one paragraph.

Acknowledgments Acknowledgments of individuals should include institution, city, and state; city and country if not U.S.; and City or Province if in Canada. Copies being reviewed shall have authors’ institutions omitted to retain anonymity.

References a) Citing References In Text Authors of cited papers in the text are to be presented as follows: Adams and Harry (1992) or Smith and Jones (1990, 1992). If more than two authors of one article, the first author’s name is followed by the abbreviation et al. in italics. If the sentence structure requires that the authors’ names be included in parentheses, the proper format is (Adams and Harry, 1982; Harry, 1988a,b; Harry et al., 1993). Citations to a group of references should be listed first alphabetically then chronologically. Work that has not been submitted or accepted for publication shall be listed in the text as: “G.C. Jay (institution, city, and state, personal communication).” The author’s own unpublished work should be listed in the text as “(J. Adams, unpublished data).” Personal communications and unsubmitted unpublished data must not be included in the References section. Two or more publications by the same authors in the same year must be made distinct with lowercase letters after the year (2010a,b). Likewise when multiple author citations designated by et al. in the text have the same first author, then even if the other authors are different these references in the text and the references section must be identified by a letter. For example

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“(James et al., 2010a,b)” in text, refers to “James, Smith, and Elliot. 2010a” and “James, West, and Adams. 2010b” in the reference section. b) Citing References In Reference Section In the References section, references are listed in alphabetical order by authors’ last names, and then chronologically. List only those references cited in the text. Manuscripts submitted for publication, accepted for publication or in press can be given in the reference section followed by the designation: “(submitted)”, “(accepted)’, or “(In Press), respectively. If the DOI number of unpublished references is available, you must give the number. The year of publication follows the authors’ names. All authors’ names must be included in the citation in the Reference section. Journals must be abbreviated. First and last page numbers must be provided. Sample references are given below. Consult recent issues of AFAB for examples not included in the following section.

Journal manuscript: Author(s). Year. Article title. Journal title [abbreviated].

Volume number:inclusive pages.

Book Chapter: Examples: Author(s) of the chapter. Year. Title of the chapter. In: author(s) or editor(s). Title of the book. Edition or volume, if relevant. Publisher name, Place of publication.

Inclusive pages of chapter.

O’Bryan, C. A., P. G. Crandall, and C. Bruhn. 2010.

Assessing consumer concerns and perceptions of food safety risks and practices: Methodologies and outcomes. In: S. C. Ricke and F. T. Jones. Eds. Perspectives on Food Safety Issues of Food Animal Derived Foods. Univ. Arkansas Press, Fayetteville, AR. p 273-288.

Dissertation and thesis: Author. Date of degree. Title. Type of publication, such as Ph.D. Diss or M.S. thesis. Institution, Place of institution. Total number of pages.

Maciorowski, K. G. 2000. Rapid detection of Salmonella spp. and indicators of fecal contamination in animal feed. Ph.D. Diss. Texas A&M University, College Station, TX.

Examples: Chase, G., and L. Erlandsen. 1976. Evidence for a complex life cycle and endospore formation in the attached, filamentous, segmented bacterium from murine ileum. J. Bacteriol. 127:572-583.

Donalson, L. M. 2005. The in vivo and in vitro effect of a fructooligosacharide prebiotic combined with alfalfa molt diets on egg production and Salmonella in laying hens. M.S. thesis. Texas A&M University, College Station, TX.

Jiang, B., A.-M. Henstra, L. Paulo, M. Balk, W. van Doesburg, and A. J. M. Stams. 2009. A typical one-carbon metabolism of an acetogenic and hydrogenogenic Moorella thermioacetica strain.

Arch. Microbiol. 191:123-131.

Van Loo, E. 2009. Consumer perception of ready-toeat deli foods and organic meat. M.S. thesis. University of Arkansas, Fayetteville, AR. 202 p.

Book: Author(s) [or editor(s)]. Year. Title. Edition or volume (if relevant). Publisher name, Place of publication. Number of pages.

Examples: Hungate, R. E. 1966. The rumen and its microbes Academic Press, Inc., New York, NY. 533 p.

Web sites, patents: Examples: Davis, C. 2010.

Salmonella. Medicinenet.com. http://www.medicinenet.com/salmonella /article. htm. Accessed July, 2010.

Afab, F. 2010, Development of a novel process. U.S. Patent #_____

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Abstracts and Symposia Proceedings: Fischer, J. R. 2007. Building a prosperous future in which agriculture uses and produces energy efficiently and effectively. NABC report 19, Agricultural Biofuels: Tech., Sustainability, and Profitability. p.27 Musgrove, M. T., and M. E. Berrang. 2008. Presence of aerobic microorganisms, Enterobacteriaceae and Salmonella in the shell egg processing environment.

IAFP 95th Annual Meeting. p. 47 (Abstr. #T6-10) Vianna, M. E., H. P. Horz, and G. Conrads. 2006. Options and risks by using diagnostic gene chips. Program and abstracts book , The 8th Biennieal Congress of the Anaerobe Society of the Americas. p.

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Data Presentation in Tables and Figures Figures and tables to be published in AFAB must be constructed in such a fashion that they are able to “stand alone” in the published manuscript. This

means that the reader should be able to look at the figure or table independently of the rest of the manuscript and be able to comprehend the experimental approach sufficiently to interpret the data.

Consequently, all statistical analyses should be very carefully presented along with variation estimates and what constitutes an independent replication and the number of replicates used to calculate the averages presented in the table or figure.

Each table and figure must be on a separate page in the submitted paper. In addition, you will need to submit all data for charts, tables and figures in native format when possible (e.g., Microsoft Excel, Powerpoint). Photographs should be submitted as high-resolution (600 dpi) .jpg or tif. files. All figures should be clearly presented with well defined axis and units of measurement. Symbols, lines, and bars must be made distinct as “stand alone” black and white presentations. Stippling, dashed lines etc. are encouraged for multiple comparison but shades of gray are discouraged. Color images, micrographs, pictures are recommended and there is no additional fee for their submission.

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