Pediococcus acidilactici aislado del rumen de ovinos by Alejandro Ley de Coss

Research in Veterinary Science 90 (2011) 26–30

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Pediococcus acidilactici isolated from the rumen of lambs with rumen acidosis, 16S rRNA identiﬁcation and sensibility to monensin and lasalocid M.A. Cobos a,*, A. Ley de Coss a, N.D. Ramirez b, S.S. Gonzalez a, R. Ferrera Cerrato a a b

Programa de Ganadería, Campus Montecillo, Colegio de Postgraduados, km 36.5 Carretera México-Texcoco, Montecillo, Texcoco, Estado de México 56230, Mexico Facultad de Medicina, Universidad del Estado de México, Toluca, Mexico

a r t i c l e

i n f o

Article history: Received 6 September 2008 Accepted 5 May 2010

Keywords: Rumen acidosis Pediococcus acidilactici Rumen bacteria Streptococcus bovis

a b s t r a c t A lactic-acid producing bacterium was isolated from the rumen of lambs with rumen acidosis. The cells were Gram-positive, nonmotile, nonsporing, catalase negative spherical, 1.5–2.0 lm in diameter, and occur in pairs and tetrads. Analysis of 16S ribosomal RNA indicated that the rumen bacterium was a strain of Pediococcus acidilactici with 99% of nucleotide homology. This bacterium was sensible to monensin and lasalocid at the unique dose tested of 300 ppm. The concentration of lactic acid and DM degradation decreased (P < 0.05) when monensin or lasalocid were added to the culture media after 24, 48 and 72 h of incubation. In contrast, total VFA concentration and pH were higher (P < 0.05) in the culture media added with the ionophores. Up to now S. bovis is considered the main ruminal bacterium related with rumen acidosis, but the importance of P. acidilactici should be also reconsidered in experimental studies focused on the control rumen acidosis. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction

2. Materials and methods

When ruminants are fed high-cereal grain diets, the rate of lactic acid and VFA production can be so fast that ruminal pH decreases. Ruminal acidosis depresses food intake and animal efficiency, and in extreme cases even causes death of the animal (Asanuma and Hino, 2002). Rumen acidosis is initiated by the proliferation of lactate-producing bacteria (Tung and Kung, 1993). The rumen bacterium S. bovis is of particular interest because of its role in the development of lactic acidosis in cattle and sheep fed an excess of starch (Whitford et al., 2001). However, it has been suggested that other rumen bacteria may be more important sources of lactate (Owens et al., 1998). In recent years, some rumen bacteria have been re-classified based on their 16S rRNA sequences. For example, Bacteroides amylophilus is now Ruminobacter amylophilus and Bacteroides succinogenes is now Fibrobacter succinogenes (Krause and Russell, 1996). The sensitivity of 16S rRNA methodology has been enhanced by the polymerase chain reaction, and new kits and equipments that make genetic methodologies for bacteria classification easier to use than phenotypic methodologies (i.e. gram stain, cell shape, motility, nutritional requirements and fermentation products). In the present study, we isolated a rumen bacterium from lambs with rumen acidosis; this bacterium shares phenotypical characteristics of the species Streptococcus bovis, but its 16S rRNA revealed a different classification.

2.1. Source of lactate-producing rumen bacteria

Ten male lambs were fed a diet high in carbohydrates of easy fermentation (see Table 1). These animals had an average rumen pH of 6.4 before fed them with this diet. Rumen acidosis was considered as a rumen pH iqual or below 5.8 after 3 h post feeding. Most of the lambs (nine) started to present rumen acidosis 5 d after the exposure to the experimental diet. After 30 d, five of these lambs were selected as the source of rumen fluid for isolation of lactate-producing rumen bacteria. The rumen fluid (300 mL) was obtained with an esophagus probe 3 h after morning feeding. The ruminal fluid was strained through three layers of cheesecloth and immediately used for inoculation of selective media for lactate-producing bacteria. Streptocel broth and solid media (Dioxon) were used as enrichment media for isolation. These media contain (g/L): peptone from casein, 15.0 g; peptone from soy meal, 5.0; sodium chloride, 4.0; sodium citrate, 1.0; L-cystine,0.2; sodium sulfite, 0.2; D(+) glucose, 5.0; sodium azide, 0.2; crystal violet, 0.0002; the solid medium has also agar–agar (13.0 g). The media were prepared according with the company instructions; 30 g/L of distilled water were autoclaved 15 min at 121 °C and it was dispensed (4.5 mL) in 13 100 mm culture tubes while CO2 was bubbled into the culture tubes. Culture tubes containing 9 mL of Streptocel broth were inoculated by triplicate with 1.0 mL of rumen fluid of each lamb with ru-

M.A. Cobos et al. / Research in Veterinary Science 90 (2011) 26–30 Table 1 Composition of the experimental diet. Ingredient

g per kg (DM basis)

Sorghum Corn Soy bean meal Corn silage Corn stover Mineral mixa EM (Mcal kg 1)b PC (%)b

448 275 139 57 61 20 2.9 12.0

a Contains (per 1000 g) calcium, 130 g; phosphorus, 50 g; sodium, 109 g, chlorine, 200 g; iron, 4.3 g; magnesium, 3.3 g; manganese, 0.2 g; copper, 80 mg; cobalt, 67 mg; iodine, 4 mg; zinc, 8 mg. b According to NRC, Nutrient Requirements of Sheep, Sixth Revised Edition, 1985.

men acidosis. The tubes were incubated at 39 °C for 3 d, and then 1.0 mL were transferred to fresh Streptocel broth media and incubated in the same conditions. This procedure was repeated one more time in order to enhance the enrichment procedure. All inoculations were done inside a Class II biosafety cabinet (vertical flow, Labconco) under a CO2 phase. Petri dishes with Streptocel solid medium (15 mL) were inoculated (by the pour-plate method) in triplicate with 0.1 mL of the third enrichment culture obtained. The Petri dishes were placed in an anaerobiosis jar with GasPack (hydrogen plus carbon dioxide generator) and incubated at 39 °C for 48 h. 2.2. Isolation of lactate-producing bacterium By using disposable inoculating loops, colonies were isolated from Petri dishes with well-spaced colonies and placed in culture tubes containing 4.5 mL of Streptocel broth medium, and incubated at 39 °C for 48 h. Smears of isolates were stained using the gram method and observed microscopically in an Olympus contrast microscope at magnification 100X. 2.3. Lyophilization procedure The pure bacteria obtained (on the basis of cell morphology and size, and Gram reaction) were pour-plated on solid medium and incubated at 39 °C for 48 h, the biggest colonies were transferred, with an inoculating loop into serum bottles containing 100 mL of Streptocel broth medium. The inoculated serum bottles were incubated at 39 °C for 72 h; then they were frozen at 10 °C for 48 h before freeze-drying. The samples were lyophilized in a freeze dry system (Labconco, Freezone, 6 L model) for 72 h at 40 °C under a pressure vacuum of 133 10 3 mBar. From here, when samples of the isolated bacterium were required (for chemical or genetic analyses), they were obtained from the samples preserved by lyophilization. In order to find out the required rehydration time of the lyiophilizedresp freeze-dried bacterium before start metabolic or genetic study; the lyophilized bacterium (0.1 g) was rehydrated during 4, 12 and 24 h at 39 °C in 9.9 mL of GCS–RF anaerobic broth medium, following the procedure described by Cobos et al. (2007) and Hungate (1969). After each period the viable bacteria concentration was estimated by the most probable number method (Harrigan and McCance, 1979) with three repetitions and dilution from 10 1 to 10 13. Molecular characterization of the rumen bacterium isolated. A pure culture of the rumen bacteria isolated and conserved by lyophilization, was hydrated (0.1 g in 9.9 mL of Streptocel broth medium, 24 h at 39 °C). This culture (0.1 mL) was pour plated in Petri dish containing Streptocel solid medium and incubated 48 h at 39 °C. The colonies obtained were used for DNA extraction.

DNA extraction was performed following the instructions of the ‘‘Wizard Genomic DNA Puriﬁcation Kit” (Promega A 1120). Ampliﬁcation of the 16S rRNA was carried out using the polymerase chain reaction method. The sequences of the primers were:

8F : AGAGTTTGATCMTGGCTCAG; and 1492r : TACGGYTACCTTGTTACGACTT The amplification reaction was carried out using Taq DNA Polymerase in Storage Buffer B (Promega M1661). Reaction parameters were: 5 min cycle of pre-denaturation at 94 °C; denaturation 30 s at 94 °C; annealing 20 s at 52 °C; extension 90 s at 72 °C. After 34 cycles a final cycle of post-extension for 7 min at 72 °C. The amplified fragments were stained with ethidium bromide and observed by an agarose gel electrophoresis at 1% (Sambrook et al., 1989). 2.4. Phylogenetic tree The purified PCR products were sequenced at the Service of Sequence of the Universidad Autonoma Metropolitana-Iztapalapa. The 800 bases sequence obtained was compared with sequences of the genusPediococcus, Lactobacillus, Aerococcus y Tetragenococcus, Enterococcus y Streptococcus from the data base of the Gen Bank using the Blast program (Altschul et al., 1997). Finally, the identified bacterium was phylogenetically analyzed using the Neighbor-Joining y Kimura 2 method (Saitou and Imanishi, 1989) and the Phylo_win program (Galtier et al., 1996), the grouping stability was estimated using a 1000 bootstrap replicates (Efron and Gong, 1983; Felsenstein, 1985). 2.5. Metabolic characteristics with or without ionophores This study was performed in culture tubes (18 150 mm) containing 9 mL of glucose-rumen fluid broth medium (G-RF). The GFR broth was prepared under anaerobic conditions following the procedures described by Cobos et al. (2007).The medium G-RF contains (each 100 mL): distilled water, 52.1 mL; clarified rumen fluid, 30 mL, mineral solution I, 5 mL; mineral solution II, 5 mL; sodium carbonate solution (0.8 g/L), 5 mL; resazurin solution (0.01 mL/L), 0.1 mL; tripticase-peptone, 0.2 g; yeast extract, 0.1 g; cystein–sulfide solution (0.25 g/L), 2 mL; and glucose, 0.5 g. The treatments evaluated were: T1 = (control), inoculation of 0.5 mL of the isolated lactate-producing bacteria by triplicate in culture tubes (18 150 mm) containing 9.0 mL of G-FR broth medium and 0.25 g (dry matter basis) of the diet used to fed the lambs with ruminal acidosis; T2 = T1 + monensin, 30 ppm, and T3 = T1 + lasalocid, 40 ppm. The variables measured after 0, 24, 48 and 72 h of incubation were: dry matter degraded, pH, and volatile fatty acids (VFA), lactic acid, ammonia and bacteria concentration. At the end of each incubation period; non degraded DM was recovered by filtration on Whatman paper No. 41, dried 24 h at 60 °C, and weighted to estimate percent of DM degraded. For VFA analysis, 5 mL of the incubated culture was centrifugated at 10,000 rpm for 10 min to precipitate small feed particles and bacteria, 2 mL of the supernatant was transferred to disposable microcentrifuge tubes (2.5 mL capacity) containing 0.5 mL 25% methaphosporic acid (4:1proportion), the sample was mixed with a vortex, after 30 min, the samples were centrifugated at 14,000 rpm for 10 min. Two mL of the supernatant was transferred to chromatographic vials and analyzed without further preparation. The VFA (acetate, propionate and butyrate) analysis was done in a Clarus 500 (Perkin–Elmer) chromatograph with flame ionization detector (FID) and auto sampler, with the following conditions: injection volume, 1 lL; column, 15m 0.32 mm Elite capillary FFAP; oven, injector and column temperature, 80, 250

M.A. Cobos et al. / Research in Veterinary Science 90 (2011) 26–30

and 140 °C (respectively); carrier gas, nitrogen 60 psi; H2 and O2, 45 and 450 mL/min; and total run time, 8 min. The concentration of lactate-producing bacteria was measured with the Petroff– Hausser chamber, the sample (5 mL) was mixed with 1 mL formaldehyde (18% solution) and the bacteria were observed and counted in an Olympus microscope at 100x. 2.6. Statistical analyses Data obtained pH, VFA, and lactic acid were statistically analyzed by least squares analyses of variance in a completely randomized design with three treatments and three replications (SAS, 1999; Steel and Torrie, 1988). 3. Results A single bacteria colony was obtained after three transfers in Streptocell broth media. The cells were spherical, 1.5–2.0 lm in diameter, and occur in pairs, tetrads and single cells were rare. Gram-positive, nonmotile, nonsporing and catalase negative, according with Holth et al. (1993) this bacterium belongs to the group 17, Gram-positive cocci. The effects of the rehydration time on lyophilized-bacterium viability. The results obtained were different (P < 0.05) and the lyophilized bacteria samples had a viable bacteria concentration (per mL of broth medium) of 1.4 104, 1.5 105 and 4.5 1011 after rehydration of the cell mass for 4, 12 and 24 h, respectively. It was decided to use a 24 h of rehydration period from lyophilized samples in order to achieve a considerable bacteria concentration for metabolic and genetic studies. The survival and stable shelf-life of lactate-producing bacteria after freeze drying has been improved with addition of individual and combined compounds used as starters cryoprotectants including: lactose, skim milk, ascorbic acid and sodium glutamate (Zarate

and Nader-Macias, 2006; Borrego et al., 2001). In the present study there was not added any cryoprotectant, but the viable bacteria concentration was high and only dependent on the rehydration period applied. The results obtained indicated that some components of the G-RFculture medium may act as protecting agents during the lyophilization process. This result may be due to the trypticase and glucose contents of the G-RF medium; according with Gherna (1994) this compounds reduce the cellular damage caused during freezing. Using a 800 bp fragment from the rumen bacterium isolated, the 16S rRNA gene sequencing gives 99% homology with the specie Pediococcus acidilactici (strains L200, B1104, Ro17, KLB69–2, NCDO2767, Uga146-3 and E-7), and 97% homology with P. pentosaceus. The genetic distance was 0.0 for most P. acidilactici strains and 0.4 for P. pentosaceus. The phylogenetic tree of some P. acidilactici species shows clearly the position of the rumen bacterium strain isolated in the P. acidilactici cluster (see Fig. 1). Effect of monensin and lasalocid on P. acidilactici (DM degradation and metabolic activity). The percent of DM degradation was highest (P > 0.05) in the pure culture of P. acidilactici compared with the cultures with lasalocid or monensin after 24, 48 and 72 of incubation (Table 2). The pH on the culture medium at the beginning of the experiment was 6.8, but after 24 h of incubation all treatments developed an acidic pH. In general, rumen bacteria require a neutral pH for their metabolic activity and below 6.1 these bacteria decrease their activity (Wales et al., 2004). The results obtained suggested a great tolerance of P. acidilactici to low pH values (4.71–4.98) without a negative effect on its capacity to degrade DM. There was a higher (P < 0.05) concentration of total VFA when the P. acidilactici culture was cultivated with monensin or lasalocid (Table 3). The ionophores also altered the molar proportions of VFA. Acetate and propionate were signiﬁcantly increased (P < 0.05) and butyrate was similar (P > 0.05) when the P. acidilac-

Fig. 1. Phylogenetic tree showing the position of the rumen bacterium isolated in the Pediococcus acidilactici cluster.

M.A. Cobos et al. / Research in Veterinary Science 90 (2011) 26–30 Table 2 In vitro DM degradation (%) and pH in Pediococcus acidilactici cultures with or without ionophores. Incubation P. time, h acidilactici

24 48 72 24 48 72

DM degraded,% 38.76a 48.70a 51.73a pH 4.96b 4.53c 4.70b

P. P. SEM acidilactici + monensin acidilactici + lasalocid

32.86b 37.26b 38.93b

27.83c 33.90b 39.66b

2.21 2.87 3.35

5.73a 5.63a 5.46a

5.69a 5.31b 5.30ab

0.12 0.12 0.26

a,b,c Means in same row with different letters differ (P < 0.05). SEM, standard error of mean.

tici culture was added with monensin or lasalocid. Considering that butyric acid concentration remains low and constant from 24 to 72 h of incubation in all treatments, it can be suggested that P. acidilactici does not produce butyric acid. The mean lactic acid concentration in the culture medium inoculated with P. acidilactici without ionophores increased signiﬁcantly after 24 h of incubation (P < 0.05), compared with that of the culture media added with monensin or lasalocid (Table 3). The fresh culture medium had an average lactic acid concentration (before incubation) of 1.52 mM, and increased to 17.07, 2.92 and 2.74 mM after 24 h of incubation in the culture media inoculated with P. acidilactici alone, with monensin or lasalocid, respectively. Similar results were observed at 48 and 72 h of incubation among treatments. The medium inoculated with P. acidilactici alone maintains its high lactic acid concentration, while the media inoculated with P. acidilactici with monensin or lasalocid had a light increase in its lactic acid concentration.

4. Discussion To our knowledge, this is the ﬁrst report so far were other bacteria than Streptococcus bovis have been isolated from ruminants

Table 3 VFA and lactic acid concentration in P. acidilactici cultures with or without ionophores. Incubation time, h

P. acidilactici

P. acidilactici + monensin

P. acidilactici + Lasalocid

SEM

24 48 72

Acetic acid, mM L 1 23.56b 23.32b 24.37c 26.34bc 27.99b 39.81a

26.35a 36.31a 34.64a

1.56 2.26 1.42

24 48 72

Propionic acid, mM L 1 2.84b 3.92a 2.81c 5.22b 3.11c 7.39b

4.39a 8.38a 11.76a

0.22 0.61 0.50

24 48 72

Butyric acid, mM L 1 2.13b 1.69a 1.82 1.85 1.95 2.15

1.81a 2.12 1.83

0.15 0.19 0.15

24 48 72

Total VFA, mM L 1 28.54b 28,94b 29.01b 33.41b 33.06b 49.36a

32.55a 46.82a 48.24a

1.84 2.98 2.01

24 48 72

Lactic acid, mM L 1 17.07a 2.92b 17.19a 5.71b 15.63a 5.57b

2.74b 6.07b 5.33b

2.23 1.74 1.90

a,b,c Means in same row with different letters differ (P < 0.05). SEM, standard error of mean.

with rumen acidosis. Some P. acidilactici species reported in the Gen Bank has been isolated from vaginal samples (Jin et al., 2007), cocoa fermentations (Nielsen et al., 2007), paddy rice silage (Ennahar et al., 2003), and from beer, milk and meat products (Dobson et al., 2002). To our knowledge, no Pediococcus acidilactici species has been isolated so far from the rumen of healthy animal or with rumen acidosis, and it is therefore unknown to which extent this strain is widespread in the rumen microbiota and contributes to the rumen acidosis. In general Streptococcus bovis is considered the main or unique ruminal bacterium related with ruminal acidosis (Owens et al., 1998; Whitford et al., 2001). However, considering the metabolic and morphological similarities between P. acidilactici and Streptococcus bovis (Garvie, 1986; Hardie, 1986; Holth et al., 1993; Asanuma and Hino, 2002), and that they are able to growth in the same selective media used for isolation of lactate-producing bacteria; it is possible that P. acidilactici has been misidentified with S. bovis in some studies reported on rumen acidosis. Now with the use of PCR techniques it is possible to confirm if S. bovis or other rumen bacteria, like P. acidilactici are important in the development of rumen acidosis, we based this idea on the P. acidilactici 16S rRNA sequence obtained and its lactic acid production in the rumen of lambs fed a diet high in carbohydrates of easy fermentation. The P. acidilactici bacterium isolated showed a great tolerance to low pH values (4.71–4.98) without a negative effect on its capacity to degrade DM. According with Fitzsimons et al. (1992) the results obtained may be considered normal, since P. acidilactici has an optimum pH for activity between 4.5 and 8.0. However, the addition of ionophores affects negatively the DM degradation. The results obtained are similar to other reports where monensin and lasalocid have decreased the production of lactic acid in vitro and in vivo experiments (Russell and Strobel, 1989). This effect is desirable in ruminants feeding high carbohydrates diets, because there will be less production of lactic acid and lower acidification of the rumen which in turns will reduces the incidence of rumen acidosis (Yang and Beauchemin, 2004). Ionophores like monensin and lasalocid reduce acidosis in ruminants by directly inhibiting lactate-producing bacteria like S. bovis (Callaway et al., 2003). These ionophores had a bacteriostatic effect on P. acidilactici, similar with that reported in S. bovis (Ipharraguerre and Clark, 2003). The ionophores tested affected both total production and molar proportion of VFA. According with Thomas et al. (1985) bacteria from the genus Pediococcus rapidly ferment glucose to lactic acid with small production of acetate. In contrast, the P. acidilactici isolated produce more acetate than lactic acid after 72 h of incubation, and when monensin or lasalocid were added to the culture medium the production of lactate by P. acidilactici was clearly depressed. The results are similar to in vivo experiments where the addition of monensin (300 mg anim 1 d 1) decreased the rumen lactate concentration from 19.4 mM to 1.0 mM (Coe et al., 1999; Mutsvangwa et al., 2002).

5. Conclusions Using a 16S rRNA gene analysis, it was possible to identify the isolated rumen bacterium from the rumen of lambs with acidosis as Pediococcus acidilactici with 99% of nucleotide homology. This bacterium was sensible to monensin and lasalocid at the regular dose used to control of rumen acidosis in dairy cattle. Although the characterization of P. acidilactici was limited, it is clear that this rumen bacterium has important metabolic and morphological similarities with Streptococcus bovis and they can be confused. The traditional diagnostic methods for identiﬁcation of microorganisms are inefﬁcient in comparison with the new molecular biology tech-

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