SummaryAdherence of intestinal pathogens, including Escherichia coli O157:H7, to human intestinal epithelial cells is a key step in pathogenesis. Probiotic bacteria, including Lactobacillus helveticus R0052 inhibit the adhesion of E. coli O157:H7 to epithelial cells, a process which may be related to specific components of the bacterial surface. Surface-layer proteins (Slps) are located in a paracrystalline layer outside the bacterial cell wall and are thought to play a role in tissue adherence. However, the ability of S-layer protein extract derived from probiotic bacteria to block adherence of enteric pathogens has not been investigated. Human epithelial (HEp-2 and T84) cells were treated with S-layer protein extract alone, infected with E. coli O157:H7, or pretreated with S-layer protein extract prior to infection to determine their importance in the inhibition of pathogen adherence. The effects of S-layer protein extracts were characterized by phasecontrast and immunofluorescence microscopy and measurement of the transepithelial electrical resistance of polarized monolayers. Pre-treatment of host epithelial cells with S-layer protein extracts prior to E. coli O157:H7 infection decreased pathogen adherence and attaching-effacing lesions in addition to preserving the barrier function of monolayers. These in vitro studies indicate that a non-viable constituent derived from a probiotic strain may prove effective in interrupting the infectious process of an intestinal pathogen.

The microflora of the crop was investigated throughout the broiler production period (0 to 42 days) using PCR combined with denaturing gradient gel electrophoresis (PCR-DGGE) and selective bacteriological culture of lactobacilli followed by amplified ribosomal DNA restriction analysis (ARDRA). The birds were raised under conditions similar to those used in commercial broiler production. Lactobacilli predominated and attained populations of 10 8 to 10 9 CFU per gram of crop contents. Many of the lactobacilli present in the crop (61.9% of isolates) belonged to species of the Lactobacillus acidophilus group and could not be differentiated by PCR-DGGE. A rapid and simple ARDRA method was developed to distinguish between the members of the L. acidophilus group. HaeIII-ARDRA was used for preliminary identification of isolates in the L. acidophilus group and to identify Lactobacillus reuteri and Lactobacillus salivarius. MseI-ARDRA generated unique patterns for all species of the L. acidophilus group, identifying Lactobacillus crispatus, Lactobacillus johnsonii, and Lactobacillus gallinarum among crop isolates. The results of our study provide comprehensive knowledge of the Lactobacillus microflora in the crops of birds of different ages using nucleic acid-based methods of detection and identification based on current taxonomic criteria.The digestive tracts of mammals and birds are home to a diverse collection of bacterial species, collectively referred to as the gut microflora (28). From gnotobiotic animal studies, the microflora is known to influence the biochemistry, immunology, physiology, and nonspecific resistance to intestinal infection of the host (9). The impact of the gut microflora on the nutritional status of farm animals is of particular interest, especially where intensive farming practices are used (4).The crop, ileum, cecum, and colon of poultry are known to harbor bacterial populations (16,27). Recent reports have investigated the composition of the ileal (13) and cecal (35) microflora using bacteriological culture and culture-independent methods. Lactobacilli are numerous in the ileum of broilers, whereas the cecal microflora is dominated by obligately anaerobic bacteria and bacteria yet to be cultivated. From the results of culture-based studies, it has been determined that the microflora of the crop has a simple composition and is dominated by lactobacilli (16,27). Colonization of the surface of the stratified, squamous epithelium of the crop by lactobacilli has been reported by Fuller (6) and Morishita et al. (18). Lactobacillus salivarius, Lactobacillus fermentum or Lactobacillus reuteri, and Lactobacillus acidophilus were the species most commonly detected (16,27). These studies were conducted prior to the reclassification of L. acidophilus, which has been divided into two DNA homology groups containing six related species (5,11,15). DNA homology group A consists of L. acidophilus (A1), Lactobacillus crispatus (A2), Lactobacillus amylovorus (A3), and Lactobacillus gallinarum (A4); DNA homology gr...

Strain R0052, isolated from a North American dairy starter culture, was initially identified as Lactobacillus acidophilus based on phenotypic analyses. However, upon sequencing the 16S rRNA gene, it became clear that the isolate was very highly related to Lactobacillus suntoryeus, Lactobacillus helveticus and Lactobacillus gallinarum, as similarities ranging from 99·3 to 99·8 % were observed. As an initial screening test to investigate the relatedness of strain R0052 and reference strains of L. suntoryeus, L. helveticus and L. gallinarum, the partial sequences for the genes encoding the alpha subunit of ATP synthase (atpA), RNA polymerase alpha subunit (rpoA), phenylalanyl-tRNA synthase alpha subunit (pheS), the translational elongation factor Tu (tuf), a surface-layer protein (slp) and the Hsp60 chaperonins (groEL) were determined and they revealed high relatedness between all of the strains. The determination of the 16S–23S rRNA internally transcribed spacer (ITS) sequences revealed 98·3–100 % similarity between L. suntoryeus and L. helveticus strains. SDS-PAGE of whole-cell proteins did not distinguish between these species. Fluorescent amplified fragment length polymorphism (FAFLP) could distinguish between these taxa, but they still constituted a single cluster within the L. acidophilus group. Finally, DNA–DNA hybridization experiments between strain R0052 and the type strains of L. helveticus and L. suntoryeus yielded reassociation values above 70 % and confirmed that these names are synonyms.

Thirty-eight isolates of Lactobacillus gallinarum cultured from the crops of broiler chickens were screened for the presence of genes encoding S-layer proteins. All of the isolates had two S-protein genes, which were designated Lactobacillus gallinarum S-protein (lgs) genes. One gene in each isolate was either lgsA or lgsB. The Lactobacillus isolates were further characterized by pulsed-field gel electrophoresis of DNA digests, which grouped the isolates into 17 genotypes (strains). The second gene in each of eight representative strains was sequenced and shown to differ among strains (lgsC, lgsD, lgsE, lgsF, lgsG, lgsH, and lgsI). The genome of each strain thus encoded a common S-protein (encoded by either lgsA or lgsB) and a strain-specific S-protein. The extraction of cell surface proteins from cultures of the eight strains showed that each strain produced a single S-protein that was always encoded by the strain-specific lgs gene. Two of the strains were used to inoculate chickens maintained in a protected environment which were Lactobacillus-free prior to inoculation. DNAs and RNAs extracted from the digesta of the chickens were used for PCR and reverse transcription-PCR, respectively, to demonstrate the presence and transcription of lgs genes in vivo. In both cases, only the strain-specific gene was transcribed. Both of the strains adhered to the crop epithelium, consistent with published data predicting that S-proteins of lactobacilli are adhesins. The results of this study provide a basis for the investigation of gene duplication and sequence variation as mechanisms by which bacterial strains of the same species can share the same habitat.Lactobacilli are commonly detected in gut samples collected from animal species, especially those from rodents, pigs (reviewed in reference 35), and chickens (13,15,20,24,45). In the chicken gut, lactobacilli are present in the crop (15), the ileum (20, 24), and the ceca (45). Lactobacilli dominate the relatively simple microbiota of the chicken crop (13), where at least some strains adhere to the crop epithelial surface (8, 12). Lactobacillus gallinarum, Lactobacillus crispatus, Lactobacillus johnsonii, Lactobacillus salivarius, and Lactobacillus reuteri persist in the crop throughout the life of broilers raised under commercial farming conditions (15). The first three of these species are members of the Lactobacillus acidophilus complex, which contains six closely related species in two subgroups (11,18,22). DNA homology group A contains L. acidophilus (A1), L. crispatus (A2), Lactobacillus amylovorus (A3), and L. gallinarum (A4), whereas DNA homology group B contains Lactobacillus gasseri (B1) and L. johnsonii (B2).Surface-associated crystalline protein layers, termed S-layers, have been detected on cells of strains belonging to group A of the L. acidophilus complex (GAA) (4), with each strain having two different S-protein genes. S-proteins are the individual subunits that comprise S-layers (reviewed in references 30 and 32). It has been speculated that S-layers may ...

Probiotics, known for their prophylactic and therapeutic properties, are routinely used by the medical community in various regions of the world. In some Asian countries, these products are controlled as pharmaceutical substances and must adhere to strict regulatory guidelines. However, outside of Europe where the European Food Safety Authority has recently adopted a Qualified Presumption of Safety approach for probiotics used in food and feed, current safety requirements do not necessitate screening for the presence of virulence and other risk factors, which may result in the inadvertent use of probiotic strains harboring harmful genes. A safety evaluation was conducted on Enterococcus faecium R0026 and Bacillus subtilis R0179 used in several commercial probiotic products marketed in Asia. Molecular techniques were used to verify the identity of each strain and antibiotic resistance profiles were determined towards clinically relevant antibiotics. Strains were subsequently screened for the presence of enterotoxins and virulence factors and were subjected to 28 days of repeated high-dose oral toxicity testing in rats. No risk factors or aberrant activities were identified using such a detailed approach. Thus, both microbes were deemed to pose low risk to the consumer and, therefore, safe for use as probiotics.