Lactobacillus plantarum - Wiley Online Library

Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Isolation of bovine intestinal Lactobacillus plantarum and Pediococcus acidilactici with inhibitory activity against Escherichia coli O157 and F5 A. Rodriguez-Palacios1, H.R. Staempfli1, T. Duffield2 and J.S. Weese1 1 Department of Clinical Studies, University of Guelph, Guelph, Ontario, Canada 2 Department of Population Medicine, University of Guelph, Guelph, Ontario, Canada

Keywords calves, coliforms, ileum, intestinal colonization, lactic acid bacteria, probiotics, silage. Correspondence A. Rodriguez-Palacios: Food Animal Health Research Program, Department of Veterinary Preventive Medicine, The Ohio State University, 1680 Madison Ave, Wooster, OH 44691, USA. E-mail: [email protected]

2008 ⁄ 0209: received 5 February 2008, revised 20 May 2008 and accepted 5 June 2008 doi:10.1111/j.1365-2672.2008.03959.x

Abstract Aims: The growth rate of bovine lactic acid bacteria (LAB) in five different culture conditions, and their inhibitory activity against Escherichia coli O157 and F5 in two assays was assessed to identify LAB for potential prophylactic use in cattle. Methods and Results: 106 bovine-derived faecal ⁄ intestinal LAB were tested in vitro for tolerance to pH 2Æ0, pH 4Æ0, 0Æ15% and 0Æ3% bile, aerobic incubation, and for inhibitory activity against E. coli O157 (n = 3) and F5 (n = 1). While no LAB grew at pH 2Æ0, LAB survivability varied between 35% and 100% on the other tests. Exactly 7Æ6% (8 ⁄ 106) of LAB supernatants inhibited the growth of E. coli in two assays, whereas 6Æ6% (7 ⁄ 106) of isolates enhanced the growth of all E. coli strains. Partial 16s rRNA gene sequencing of six best isolates (95th percentile) revealed that five were Lactobacillus plantarum and one Pediococcus acidilactici. Conclusion: Lactobacillus plantarum with acid ⁄ bile and aerobic resistance and inhibitory activity against E. coli O157 and F5 inhabit the intestinal tract of healthy cattle. Some LAB may enhance E. coli growth. Significance and Impact of the Study: Lactobacillus plantarum and P. acidilactici are natural plant micro-organisms and studied silage inoculants. Their identification from gastrointestinal samples of healthy cattle is prophylactically promising.

Introduction Lactic acid bacteria (LAB) represent a clade of grampositive micro-organisms capable of producing lactic acid from carbohydrate fermentation. LAB genera include Lactobacillus, Pediococcus, Leuconostoc, Lactococcus, Streptococcus and Enterococcus, among others (Ljungh and Wadstrom 2006). LAB constitute a major component of the intestinal microflora protecting against colonization with pathogenic micro-organisms (Filho-Lima et al. 2000; Collado et al. 2007). Known protective mechanisms for beneficial LAB include local production of broad-spectrum antimicrobials (Brink et al. 2006), hydrogen peroxide (Hawes et al. 1996), and the enhancement of mucosal

and systemic immunological responses (Ljungh and Wadstrom 2006; Fuentes et al. 2008) but these mechanisms vary across strains. To date, various attempts have been made to select LAB for therapeutic and prophylactic (probiotic) use to enhance health beyond nutritional benefits; however, despite their theoretical potential, only a limited number of LAB have shown specific health benefits in humans (Alvarez-Olmos and Oberhelman 2001; Salminen and Arvilommi 2001; Heczko et al. 2006). In animals, perhaps with the exception of Lactobacillus acidophilus strain NP51 (NPC 747) able to modify faecal shedding of Escherichia coli O157 in steers (Elam et al. 2003; Peterson et al. 2007; Sargeant et al. 2007) no LAB with proven probiotic health benefits are available for cattle.

ª 2009 The Authors Journal compilation ª 2009 The Society for Applied Microbiology, Journal of Applied Microbiology 106 (2009) 393–401

393

Bovine intestinal L. plantarum and P. acidilactici

Two independent literature reviews have documented inconsistent results for the oral administration of probiotics to cattle (Yoon and Stern 1995; Rodriguez-Palacios et al. 2004). The most recent review revealed that 45% (20 ⁄ 44) of published clinical trials (1973–2004) investigating the effect of LAB on calf diarrhoea reported no benefits or the increase in the incidence and severity of diarrhoea (Rodriguez-Palacios et al. 2004). Recent clinical trials in foals also demonstrated that LAB strains initially considered beneficial may unexpectedly induce gastrointestinal side effects (Weese and Rousseau 2005). Hence, further characterization studies of LAB are needed for cattle. Unfortunately, the lack of adequate scientific support for existing bovine LAB strains is in part due to the limited number of studies assessing any given strain, and the improper identification of studied strains to trace supporting literature for specific isolates (RodriguezPalacios et al. 2004). Biochemical identification was common in previous bovine studies, but it is now clear that there are discrepancies between biochemical (API 50 CH) and genetic (16s rRNA gene sequencing) speciation, including bovine L. acidophilus (Brashears et al. 2003). Proper identification is necessary as not all isolates within a given LAB species exert the same clinical effects (Song et al. 1999; Salminen and Arvilommi 2001). This is particularly true for L. acidophilus, the most commonly studied species in bovine clinical trials (Rodriguez-Palacios et al. 2004). Marked metabolic and dietary differences between humans and cattle, and recent studies illustrating inadequate and transient intestinal colonization when human LAB strains are fed to cattle (i.e. Lactobacillus rhamnosus strain GG; Ewaschuk et al. 2004, 2006) indicate the need to further identify resident bovine-adapted LAB. Alternative approaches to antimicrobial therapy practices in food animals and the need for preharvest reduction of foodborne pathogens including E. coli O157 (Sargeant et al. 2007) also warrant further studies in search of potentially beneficial LAB. This study selected and tested the in vitro resistance of bovine-derived LAB to various culture conditions as potential predictor for intestinal survivability, and assessed their in vitro inhibitory effect against four strains of E. coli O157 and F5. Two culture protocols for the isolation of LAB from faecal ⁄ intestinal specimens were also compared. Materials and methods

A. Rodriguez-Palacios et al.

adult cows and 21 < 1-month-old dairy calves (total, n = 54). Samples were collected in winter from three University of Guelph dairy farms in Ontario, Canada. Animals had not received antimicrobials for >1 month or had signs of diarrhoea for >1 week. Composite mucosal scrapings and intestinal contents (e.g. ileum, cecum, rectum; 5 ml) were obtained from 15 calves that had been used in approved terminal research and teaching protocols by the University of Guelph Animal Care Committee. Ruminal samples (50 ml) were collected through fistulas from three teaching cows. Cows were fed grass hay and concentrate, while calves were bottle-fed 4 l of antibioticfree fresh whole cow milk per day. The samples were processed within 2 h of collection. Isolation of LAB from samples Samples were cultured using two methods, a direct plating method (DPM) and a series of selective broths method (SBM) to identify 50 and 56 LAB isolates, respectively. The DPM consisted of direct inoculation of the sample onto deMan, Rogosa and Sharpe agar (MRSa; Oxoid) and anaerobic incubation at 37C for 48 h (de Man et al. 1960). Based on previously reported selection principles after sequential exposure to hydrochloric acid and bile salts (Chou and Weimer 1999), we used a modified extended acid-bile-aerobic SBM. The selection consisted of a stepwise incubation process where 100 ll of the sample were anaerobically incubated into modified MRS media (37C) to select for resistant LAB. The samples were first enriched in 9 ml of straight MRS broth (MRSb) for 6 h, and then, serial transfers of 100-ll aliquots were incubated into pH 2Æ0 MRSb (hydrochloric acid) for 6 h, and later into 0Æ3% bile MRSb (dried unfractionated bovine bile; Sigma) for 16 h. Finally, two loopful of the homogenized broth were streaked onto MRSa plates, which were consecutively incubated for 8 and 24 h under aerobic and anaerobic conditions, respectively. LAB colonies were presumptively identified on MRSa based on gram-positive staining, and negative-catalase reaction (de Man et al. 1960). Up to two colonies per plate were cryopreserved at )80C (MRSb ⁄ 50% glycerol, 1 : 1, v ⁄ v) until processing. LAB isolates were revived on MRSa to re-assess purity. Testing was performed in batches of 20 isolates, and a strong inhibitory laboratory strain, Lactobacillus pentosus WE7 (Weese and Rousseau 2005), was used as a control in all runs.

Animals

Growth of LAB under acid, bile, and aerobic conditions

Using a convenience-sampling scheme, faecal, intestinal, and ⁄ or ruminal samples were obtained from 33 healthy

Isolated LAB were tested under five different growth conditions to determine their tolerance to acid, bile, and

394



aerobic incubation. Growth in each test MRSb tube was compared with the concurrent control growth in unmodified MRSb. Bacterial growth was assessed based on turbidity changes of the broth. Tolerance (% of control growth) was calculated based on changes of optical density (OD620) of the broth after 24 h of anaerobic incubation at 37C. Acid and bile tolerance assays were performed by incubating 100 ll of McFarland 2Æ0 suspensions of LAB [phosphate-buffered saline (PBS); 24-h-old colonies] into 9 ml of modified MRSb. For acid tolerance, MRSb was adjusted to pH 2Æ0 and 4Æ0 with hydrochloric acid, and for bile tolerance with 0Æ15% and 0Æ3% of dried unfractionated bovine bile. Testing was performed once for each LAB except the first 10 isolates that were tested in triplicates showing adequate repeatability (data not shown). To assess overall bacterial survivability to continuous incubation at pH 2Æ0, 5–10 ll of pH 2Æ0 MRSb was streaked onto MRSa after 24 h of incubation. Tolerance to aerobic incubation was subjectively determined by comparing the density and size of macroscopic LAB colonies in paired MRSa plates after 24 h of incubation at 37C. Briefly, McFarland 2Æ0 suspensions of LAB (30 ll) were inoculated onto two MRSa plates; one of each was incubated under aerobic and anaerobic conditions, respectively. When evaluating aerobically incubated plates, aerobic tolerance was considered absent if no growth was present, poor if the density and size of the colonies were clearly reduced compared with the anaerobically incubated plates, or optimal if density and colony size were comparable in both paired MRSa plates. Inhibition of Escherichia coli O157 and F5 by LAB The in vitro inhibitory activity of bovine LAB against four pathogenic E. coli strains was determined using two separate assays, a tryptose soya broth turbidity test (TSBT), and a well diffusion in tryptose soya agar test (TSAD). Both assays used cell-free LAB supernatants and four E. coli strains (F5 strain 3312; O157 strains E318, 438897, and E32511; Laboratory of Foodborne Zoonoses, Health Canada, Guelph, Canada). Each LAB was tested once against each of the four E. coli monocultures using two inhibition assays. The LAB supernatants, including control L. pentosus WE7, were prepared as follows. After incubating 100 ll of McFarland 4Æ0 suspensions of LAB isolates (PBS; 24-h-old colonies) into 35 ml of MRSb at 37C for 24 h, the broths were centrifuged (4000 g · 10 min), filtered (0Æ2lm syringe filters; Fisherbrand, Ireland), and treated with 0Æ1 ml of catalase (Sigma Chemical Co.) for removal of cells and LAB-produced hydrogen peroxide, which may exert unspecific antimicrobial effects (Sookkhee et al.


2001). After recording the pH of the supernatants, three 10-ml aliquots were frozen at )70C. Two of these were 10-fold concentrated by a 24-h cycle of freeze-drying. Freeze-dried supernatants were adjusted with distilled sterile water to achieve 1-ml volumes. Supernatant sterility was assessed onto 5% sheep blood agar (Oxoid). Aliquots of MRS broth adjusted to various pH ranges were also prepared and 10-fold concentrated to be used as controls. Escherichia coli suspensions were prepared by diluting 24-h-old colonies in PBS until OD620 absorbance was adjusted to 0Æ645 (95% CI = 0Æ638, 0Æ653); equivalent to 6 ± 1 · 108 CFU ml)1 (n = 10). The TSBT (turbidity) inhibition assay tested the effect of 1 ml of nonconcentrated LAB supernatants on the growth of 200 ll of E. coli suspension incubated into 4 ml of TSB at 37C for 24 h. Paired controls were prepared by adding 1 ml of MRSb adjusted to the pH ±0Æ1 of the LAB supernatant (hydrochloride acid) instead of the LAB supernatant. Escherichia coli growth (%) was calculated based on optical density changes (OD620) before and after incubation. Adjusted inhibition for the pH of each supernatant was calculated comparing the OD changes of the paired LAB supernatant and pH-adjusted control tubes (% of growth). LAB supernatants were considered to have an inhibitory effect beyond the effect of the pH if E. coli growth in the LAB tube was less than that of the control tube (i.e. 100%). The TSAD (well diffusion) assay tested the inhibitory effect of 1 ml of the concentrated supernatants on the growth of 100 ll of E. coli suspensions spread onto the surface of TSA plates. After spreading the suspensions and drying, the agar was perforated to create six 6-mm wells, where 90-ll aliquots of concentrated LAB culture supernatants were poured. Paired control wells were inoculated with pH-matching concentrated MRSb. Supernatant from a strong inhibitory L. pentosus WE7 (Weese and Rousseau 2005) was also inoculated in the central well to ensure that there was no plate-to-plate variability with regards to the diffusion characteristics of the agar. Clear zones around the wells (recorded in millimetres) were considered zones of E. coli inhibition after 24 h of incubation. Selection of isolates for 16s rRNA sequencing Tested LAB were selected (95th percentile) based on their average growth in pH 4Æ0 MRSb and 0Æ3% bile MRSb, and the degree of E. coli inhibition in the two assays. Selected isolates were biochemically (API 50 CH; bioMe´rieux sa, France; Song et al. 1999) and genotypically identified (Cai et al. 2003). Partial 16s rRNA gene


395



sequence analysis (BSF8 ⁄ 20, AGA GTT TGA TCC TGG CTC AG; BSR534 ⁄ 18, ATT ACC GCG GCT GCT GGC) was performed as previously described for definitive identification (Cai et al. 2003). Sequences were compared with GeneBank strains; >97% of similarity was the cutoff for speciation. Statistical analysis Correlation coefficient analyses (LAB growth, E. coli inhibition assays) were performed using Spearman correlation and adjusted Bonferroni significance tests. Comparisons between the DPM and SBM culture methods (pH 4Æ0 and 0Æ3% bile MRSb average; TSBT tests) were performed using Mann–Whitney statistics. The overall effect of LAB on E. coli growth inhibition on the four strains tested was assessed using Kruskal–Wallis statistics. Analysis was performed using Stata 9Æ.0 and Minitab 14 (Corp LP, College Station, TX, USA). Significance was held at P < 0Æ05.

to the acidic incubation broth. Approximately 50% of isolates identified using the DPM were recovered when the incubated broth was streaked onto MRSa. Conversely, all LAB identified with the SBM method were recovered on MRSa. The overall growth of LAB in acid- and bile MRSb is summarized in Table 1. Overall, 13Æ2% (14 ⁄ 106) and 39Æ6% (42 ⁄ 106) of bovine LAB tested grew at >80% in pH 4Æ0 and 0Æ3% bile MRSb, respectively, compared with the control growth. LAB grew better in 0Æ3% bile MRSb than in pH 4Æ0 MRSb, and the growth rates correlated poorly (r2 = 0Æ27, P = 0Æ07). Most LAB had low or absent growth under aerobic incubation; however, optimal tolerance was observed in 8Æ5% (9 ⁄ 156) of the isolates. Comparing the two isolation methods, bovine LAB recovered with the serial selective broth method grew better in pH 4Æ0 and 0Æ3% bile MRSb than the LAB isolated with the DPM (P < 0Æ000; Table 1). Inhibition of Escherichia coli O157 and F5

Results Isolation of LAB In total, 106 LAB were isolated from 104 specimens (59, 15, 12, 10, 5, and 3, from faeces, cecum, ileum, rumen, rectum, and jejunum, respectively) representing 54 animals. Growth of LAB under acid, bile, and aerobic conditions Although LAB did not grow at pH 2Æ0 MRSb (80% growth rate). a pH 2Æ0 prevented LAB growth; bpH 4Æ0 was more inhibitory than 0Æ3% bile; c0Æ3% bile was more inhibitory than 0Æ15% bile; dLAB isolates identified with serial selective broth had better growth rates than LAB identified with direct plating (P < 0Æ001).

396




% Escherichia coli growth

250 200

%

150 100 a

a

a

50

assay it was unable to identify stimulatory LAB supernatants (Fig. 3). Comparing the two isolation methods, LAB recovered with the DPM had a lesser inhibitory effect on the average E. coli growth rate (79Æ9%) compared with the SPM (60Æ4%; P < 0Æ001); see Fig. 4. Considering the origin of the 10% of isolates that were most resistant to bile and acid, adequate LAB isolates originated most commonly from the ileum (33Æ3%; 4 ⁄ 12) than from faecal (6Æ8%; 4 ⁄ 59), and other gastrointestinal samples (6Æ1%; 2 ⁄ 33) collectively (P = 0Æ03). Three of the

0 Strain: Median:

F5

O157-A

O157-B

O157-C

78·4%

85·0%

70·2%

68·5%

Figure 1 Effect of lactic acid bacteria (LAB) supernatants on the growth rate (%) of four Escherichia coli monoculture assays in tryptose soya broth compared with a paired LAB-free control growth. Observations of >100% and 100% of growth; Fig. 2). Overall, results from the well diffusion assay were moderately comparable with the turbidity test (r2 = 0Æ68; P < 0Æ05), but because of the nature of the

200 150

% 100

50 0

Direct plating method

Serial broths method

Figure 4 Effect of lactic acid bacteria (LAB) supernatants on the average growth rate (% on Y-axis) of four Escherichia coli strains in tryptose soya broth compared with a paired control growth. Observations of >100% and 97% similarity) in the GenBank

*Strain from Argentinean regional cheese; primers reported: PLB16, 5¢-AGA GTT TGA TCC TGG CTC AG-3¢; MLB16, 5¢-GGC TGC TGG CAC GTA GTT AG-3¢ (Vasek et al. 2001). Strain from Malaysian Chili Bo (Mora et al. 2000).

six further characterized isolates (95th percentile) were Lactobacillus plantarum recovered from the ileum of young calves (Table 2). 16S rRNA gene sequence analysis confirmed the selected strains as L. plantarum and Pediococcus acidilactici (Table 3). Discussion Selection of organisms based on objective in vitro testing is an important and often overlooked step in the development of effective veterinary probiotics. This study has demonstrated that relatively simple and logical in vitro testing can identify species-specific micro-organisms that could presumably have a greater chance of being effective in vivo. Results from this study indicate that the stepwise series of broth method may yield a more suitable (i.e. resistant and inhibitory) selection of LAB from bovine samples compared with simple direct plating. To date, various methodologies have been attempted but no formal comparative studies are available for cattle. This is the first available screening study identifying L. plantarum as an intestinal micro-organism from bovine 398

samples with potential for probiotic use against E. coli. Previous studies have failed identifying bovine L. plantarum as a promising species for prophylactic use (Mayra-Makinen et al. 1983; Brashears et al. 2003; Rodriguez-Palacios et al. 2004; Timmerman et al. 2005). The resistance to extended exposure to acid pH and high bile concentrations in the selected LAB strains, namely L. plantarum and P. acidilactici, might confer on them the ability to better survive and colonize during intestinal transit by enhancing their intestinal growth advantages in vivo as it has been suggested for other LAB (Gilliland et al. 1984; Chou and Weimer 1999; Haller et al. 2001). However, to test these potential advantages, further in vivo colonization studies are required as other digestive and enzymatic factors may render LAB nonviable. By definition, LAB intended for probiotic use should exert beneficial health effects following administration (Heczko et al. 2006). This study aimed to select bovine LAB with the potential ability to inhibit E. coli of public health importance (i.e. O157 types) and of relevance for the bovine industry (i.e. F5 and other O-pathogenic



serotypes; Lee et al. 2008; Sargeant et al. 2007). However, 6Æ6% of LAB isolated promoted the growth of the pathogenic E. coli tested, which highlights the importance of systematic screening and in vitro testing of potential probiotics prior to clinical use. Similar findings have not been reported in screening studies in cattle. Thus, safety studies and randomized controlled clinical trials are needed to characterize LAB when intended for probiotic purposes. The reasons for the LAB stimulatory effect on E. coli remain unclear but warrant further investigation. Previous pathogenesis studies have shown that LAB may inhibit E. coli due in part to a dose-dependant bactericidal effect of organic acids (Ogawa et al. 2001) Although it is unclear if such effect is biologically possible in vivo, diffusion down a concentration gradient of undissociated organic acids is necessary to penetrate into E. coli cells and induce damage independently from their intracellular effect on pH (Presser et al. 1997). Correlation analysis suggested a pH-dependant inhibitory effect, however the precise mechanism by which the LAB supernatants modified the growth of E. coli was not presently elucidated and requires further evaluation. Extrapolation of in vitro inhibitory activity to the living intestinal environment should be made cautiously, as other inhibitory mechanisms independent from organic acid synthesis might exist as it has been demonstrated for other L. plantarum strains (Brink et al. 2006; Collado et al. 2007; Fuentes et al. 2008). Likewise, potential adverse effects of excessive organic acid production (including d-lactic acid) should be considered. A strong additive antibacterial effect has also been documented when LAB antimicrobials interact with low concentrations of lactic acid (NikuPaavola et al. 1999). Hence, a more local effect influencing the intestinal environment might also occur but needs to be studied in cattle. The identification of L. plantarum from the ileum and cecum suggests that this species might be a normal intestinal inhabitant in cattle from early ages. No similar findings have been reported in cattle; however, the ability of these L. plantarum strains to remain viable in the intestinal tract may indicate adaptation and a desirable ecological feature to sustain potential probiotic benefits (Heczko et al. 2006). Further studies are needed to determine whether these isolates are permanent inhabitants of the bovine gastrointestinal tract or transient after the ingestion of environmental strains. In either case, the identification of these naturally occurring L. plantarum strains from bovine intestinal segments seems promising. In a recent selection study, a bovine P. acidilactici strain C135, phenotypically identified with API 50 CH, showed adequate bile and acid resistance but variable inhibitory activity against E. coli O157 in vitro (Brashears et al. 2003). Owing to the lack of an accurate genetic


identification of such strain no objective comparisons can be made to the P. acidolactici strain of the present study. To date, no scientific reports exist to consider this species a probiotic strain with clinical benefits in cattle. However, of interest, intestinal P. acidolactici strains have been recently suggested to be promising in humans (Millette et al. 2008). Further studies are therefore recommended. This study identified bovine intestinal L. plantarum and P. acidilactici as promising LAB for potential probiotic purposes against E. coli in cattle. These LAB species can be isolated from plants, and certain strains have shown consistent fermentative benefits while improving silage quality and decreasing the load of pathogenic bacteria (Cai et al. 1999; Whiter and Kung 2001; Klocke et al. 2006). Of interest, certain L. plantarum and P. acidilactici strains have showed synergistic fermentative benefits in grass silage (Fitzsimons et al. 1992). The biological features and the optimal aerotolerance of these species confer the selected strains a special status for further testing as potential oral probiotics in cattle. Natural ingestion of LAB in the diet would be preferable over the transient administration of commercial products, which are often prone to deterioration during storage and manipulation (Weese 2002). The complex nature of the intestinal flora and the partial modification of E. coli O157 faecal shedding in cattle by L. acidophilus NP51 (Sargeant et al. 2007) indicate the need for new reinforcing strains. Lactobacillus plantarum and P. acidilactici are alternatives to be evaluated. Acknowledgements This study was supported by the Ontario Ministry of Agriculture and Food, Canada. The E. coli strains were kindly provided by Dr Roger Johnson, Laboratory of Foodborne Zoonoses, Health Canada, Guelph, Canada. The authors are grateful to Dr Hugh Cai, Animal Health Laboratory, University of Guelph, for sequence analysis. References Alvarez-Olmos, M.I. and Oberhelman, R.A. (2001) Probiotic agents and infectious diseases: a modern perspective on a traditional therapy. Clin Infect Dis 32, 1567–1576. Brashears, M.M., Jaroni, D. and Trimble, J. (2003) Isolation, selection, and characterization of lactic acid bacteria for a competitive exclusion product to reduce shedding of Escherichia coli O157:H7 in cattle. J Food Protect 66, 355– 363. Brink, M., Todorov, S.D., Martin, J.H., Senekal, M. and Dicks, L.M. (2006) The effect of prebiotics on production of antimicrobial compounds, resistance to growth at low pH and


399


in the presence of bile, and adhesion of probiotic cells to intestinal mucus. J Appl Microbiol 100, 813–820. Cai, Y., Kumai, S., Ogawa, M., Benno, Y. and Nakase, T. (1999) Characterization and identification of Pediococcus species isolated from forage crops and their application for silage preparation. Appl Environ Microbiol 65, 2901– 2906. Cai, H., Archambault, M. and Prescott, J.F. (2003) 16S ribosomal RNA sequence-based identification of veterinary clinical bacteria. J Vet Diagn Invest 15, 465–469. Chou, L.S. and Weimer, B. (1999) Isolation and characterization of acid- and bile-tolerant isolates from strains of Lactobacillus acidophilus. J Dairy Sci 82, 23–31. Collado, M.C., Surono, I.S., Meriluoto, J. and Salminen, S. (2007) Potential probiotic characteristics of Lactobacillus and Enterococcus strains isolated from traditional dadih fermented milk against pathogen intestinal colonization. J Food Protect 70, 700–705. Elam, N.A., Gleghorn, J.F., Rivera, J.D., Galyean, M.L., Defoor, P.J., Brashears, M.M. and Younts-Dahl, S.M. (2003) Effects of live cultures of Lactobacillus acidophilus (strains NP45 and NP51) and Propionibacterium freudenreichii on performance, carcass, and intestinal characteristics, and Escherichia coli strain O157 shedding of finishing beef steers. J Anim Sci 81, 2686–2698. Ewaschuk, J.B., Naylor, J.M., Chirino-Trejo, M. and Zello, G.A. (2004) Lactobacillus rhamnosus strain GG is a potential probiotic for calves. Can J Vet Res 68, 249–253. Ewaschuk, J.B., Zello, G.A. and Naylor, J.M. (2006) Lactobacillus GG does not affect d-lactic acidosis in diarrheic calves, in a clinical setting. J Vet Intern Med 20, 614–619. Filho-Lima, J.V., Vieira, E.C. and Nicoli, J.R. (2000) Antagonistic effect of Lactobacillus acidophilus, Saccharomyces boulardii and Escherichia coli combinations against experimental infections with Shigella flexneri and Salmonella enteritidis subsp. typhimurium in gnotobiotic mice. J Appl Microbiol 88, 365–370. Fitzsimons, A., Duffner, F., Curtin, D., Brophy, G., O’kiely, P. and O’connell, M. (1992) Assessment of Pediococcus acidilactici as a potential silage inoculant. Appl Environ Microbiol 58, 3047–3052. Fuentes, S., Egert, M., Jimenez-Valera, M., MonteolivaSanchez, M., Ruiz-Bravo, A. and Smidt, H. (2008) A strain of Lactobacillus plantarum affects segmented filamentous bacteria in the intestine of immunosuppressed mice. FEMS Microbiol Ecol 63, 65–72. Gilliland, S.E., Staley, T.E. and Bush, L.J. (1984) Importance of bile tolerance of Lactobacillus acidophilus used as a dietary adjunct. J Dairy Sci 67, 3045–3051. Haller, D., Colbus, H., Ganzle, M.G., Scherenbacher, P., Bode, C. and Hammes, W.P. (2001) Metabolic and functional properties of lactic acid bacteria in the gastro-intestinal ecosystem: a comparative in vitro study between bacteria of intestinal and fermented food origin. Syst Appl Microbiol 24, 218–226.

400


Hawes, S.E., Hillier, S.L., Benedetti, J., Stevens, C.E., Koutsky, L.A., Wolner-Hanssen, P. and Holmes, K.K. (1996) Hydrogen peroxide-producing lactobacilli and acquisition of vaginal infections. J Infect Dis 174, 1058–1063. Heczko, P.B., Strus, M. and Kochan, P. (2006) Critical evaluation of probiotic activity of lactic acid bacteria and their effects. J Physiol Pharmacol 57(Suppl. 9), 5–12. Klocke, M., Mundt, K., Idler, C., McEniry, J., O’Kiely, P. and Barth, S. (2006) Monitoring Lactobacillus plantarum in grass silages with the aid of 16S rDNA-based quantitative real-time PCR assays. Syst Appl Microbiol 29, 49–58. Lee, J.H., Hur, J. and Stein, B.D. (2008) Occurrence and characteristics of enterohemorrhagic Escherichia coli O26 and O111 in calves associated with diarrhea. Vet J 176, 205–209. Ljungh, A. and Wadstrom, T. (2006) Lactic acid bacteria as probiotics. Curr Issues Intest Microbiol 7, 73–89. de Man, J.C., Rogosa, M. and Sharpe, M.E. (1960) A medium for the cultivation of lactobacilli. J Appl Bacteriol 23, 130–135. Mayra-Makinen, A., Manninen, M. and Gyllenberg, H. (1983) The adherence of lactic acid bacteria to the columnar epithelial cells of pigs and calves. J Appl Bacteriol 55, 241–245. Millette, M., Dupont, C., Shareck, F., Ruiz, M.T., Archambault, D. and Lacroix, M. (2008) Purification and identification of the pediocin produced by Pediococcus acidilactici MM33, a new human intestinal strain. J Appl Microbiol 104, 269–275. Mora, D., Fortina, M.G., Parini, C., Daffonchio, D. and Manachini, P.L. (2000) Genomic subpopulations within the species Pediococcus acidilactici detected by multilocus typing analysis: relationships between pediocin AcH ⁄ PA-1 producing and non-producing strains. Microbiology 146, 2027–2038. Niku-Paavola, M.L., Laitila, A., Mattila-Sandholm, T. and Haikara, A. (1999) New types of antimicrobial compounds produced by Lactobacillus plantarum. J Appl Microbiol 86, 29–35. Ogawa, M., Shimizu, K., Nomoto, K., Tanaka, R., Hamabata, T., Yamasaki, S., Takeda, T. and Takeda, Y. (2001) Inhibition of in vitro growth of Shiga toxin-producing Escherichia coli O157:H7 by probiotic Lactobacillus strains due to production of lactic acid. Int J Food Microbiol 68, 135–140. Peterson, R.E., Klopfenstein, T.J., Erickson, G.E., Folmer, J., Hinkley, S., Moxley, R.A. and Smith, D.R. (2007) Effect of Lactobacillus acidophilus strain NP51 on Escherichia coli O157:H7 fecal shedding and finishing performance in beef feedlot cattle. J Food Protect 70, 287–291. Presser, K.A., Ratkowsky, D.A. and Ross, T. (1997) Modelling the growth rate of Escherichia coli as a function of pH and lactic acid concentration. Appl Environ Microbiol 63, 2355– 2360. Rodriguez-Palacios, A., Weese, J.S., Duffield, T. and Staempfli, H.R. (2004) Effect of oral probiotics on calf diarrhea:



clinical trials published between 1973–2003. J Vet Intern Med 18, 395–396. Salminen, S. and Arvilommi, H. (2001) Probiotics demonstrating efficacy in clinical settings. Clin Infect Dis 32, 1577– 1578. Sargeant, J.M., Amezcua, M.R., Rajic, A. and Waddell, L. (2007) Pre-harvest interventions to reduce the shedding of E. coli O157 in the faeces of weaned domestic ruminants: a systematic review. Zoonoses Public Health 54, 260–277. Song, Y.L., Kato, N., Matsumiya, Y., Liu, C.X., Kato, H. and Watanabe, K. (1999) Identification of Lactobacillus species of human origin by a commercial kit, API50CHL. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi 10, 77–82. Sookkhee, S., Chulasiri, M. and Prachyabrued, W. (2001) Lactic acid bacteria from healthy oral cavity of Thai volunteers: inhibition of oral pathogens. J Appl Microbiol 90, 172–179. Timmerman, H.M., Mulder, L., Everts, H., van Espen, D.C., van der Wal, E., Klaassen, G., Rouwers, S.M., Hartemink, R. et al. (2005) Health and growth of veal calves fed milk


replacers with or without probiotics. J Dairy Sci 88, 2154– 2165. Vasek, O.M., Hebert, E.M., de Giori, S.G., Raya, R. and Fusco, A.J. (2001) Secuencia parcial del gen 16S rRNA de cepas constituyentes de un fermento para la elaboracio´n de Queso Artesanal de Corrientes. Available at: http:// www.unne.edu.ar/Web/cyt/cyt/2001/8-Exactas/E-051.pdf. Accessed 10 January 2008. Weese, J.S. (2002) Microbiologic evaluation of commercial probiotics. J Am Vet Med Assoc 220, 794–797. Weese, J.S. and Rousseau, J. (2005) Evaluation of Lactobacillus pentosus WE7 for prevention of diarrhea in neonatal foals. J Am Vet Med Assoc 226, 2031–2034. Whiter, A.G. and Kung, L. Jr (2001) The effect of a dry or liquid application of Lactobacillus plantarum MTD1 on the fermentation of alfalfa silage. J Dairy Sci 84, 2195–2202. Yoon, I.K. and Stern, M.D. (1995) Influence of direct-fed microbials on ruminal microbial fermentation and performance of ruminants: a review. Asian–Austral J Anim Sci 8, 533–555.


401