An in vitro methodology which mimics in vivo human upper gastrointestinal transit was developed. The transit tolerance of potentially probiotic Lactobacillus and Bifidobacterium species was determined by exposing washed cell suspensions at 37°C to a simulated gastric juice (pH 2·0), containing pepsin (0·3% w/v) and sodium chloride (0·5% w/v), and a simulated small intestinal juice (pH 8·0), containing pancreatin USP (1 g l−1) and sodium chloride (5 g l−1), and monitoring changes in total viable count periodically. The methodology was also employed to determine the effect of adding milk proteins (1 g l−1), hog gastric mucin (1 g l−1) and soyabean trypsin‐chymotrypsin inhibitor [SBTCI] (1 g l−1) on transit tolerance. The majority (14 of 15) of isolates lost >90% viability during simulated gastric transit. Only one isolate, Lactobacillus fermentum KLD, was considered intrinsically resistant. The addition of milk proteins, singly and in combination, generally improved gastric transit tolerance. In this regard, two isolates, Lact. casei 212.3 and Bifidobacterium infantis 25962, exhibited 100% gastric transit tolerance in the presence of milk proteins. In general, the addition of hog gastric mucin did not influence simulated gastric transit tolerance of lactobacilli but tended to increase that of bifidobacteria. However, it increased that of Lact. casei 242 and Lact. salivarius 43338 but diminished that of B. bifidum 2715 and B. animalis Bo. Selected bile salts‐resistant isolates were intrinsically tolerant to simulated small intestinal transit. Only Lact. casei F19 and B. adolescentis 15703T showed significant reduction in viability after 240 min. In general, the addition of milk proteins and SBTCI did not affect simulated small intestinal transit tolerance. However, they significantly improved the intrinsic resistance of Lact. casei F19 but diminished that of B. breve 15700T. It is concluded that, whereas the majority of bile salts‐resistant lactobacilli and bifidobacteria may be intrinsically sensitive to gastric transit, they are intrinsically resistant to small intestinal transit. In addition, it is postulated that milk proteins and mucin may function as both buffering agents and inhibitors of digestive protease activity in vivo, thereby protecting ingested bacterial strains during upper gastrointestinal transit.

In recent years, the time-honored reputation of lactobacilli as promoters of gastrointestinal and female urogenital health has been qualified. This has occurred due to a rare association with human infection in the presence of certain predisposing factors and their potential to act as a source of undesirable antibiotic resistance determinants to other members of the indigenous microbiota. This necessitates greater caution in their selection for use in microbial adjunct nutrition and disease management (prophylaxis and therapy). It was against this background that 46 Lactobacillus strains from human and dairy sources were assayed for susceptibility to 44 antibiotics. All strains were resistant to a group of 14 antibiotics, which included inhibitors of cell wall synthesis (cefoxitin [30 microg] and aztreonam [30 microg]), protein synthesis (amikacin [30 microg], gentamicin [10 microg], kanamycin [30 microg], and streptomycin [10 microg]), nucleic acid synthesis (norfloxacin [10 microg], nalidixic acid [30 microg], sulphamethoxazole [100 microg], trimethoprim [5 microg], co-trimoxazole [25 microg], and metronidazole [5 microg]), and cytoplasmic membrane function (polymyxin B [300 microg] and colistin sulphate [10 microg]). All strains were susceptible to tetracycline (30 microg), chloramphenicol (30 microg), and rifampicin (5 microg). Four human strains and one dairy strain exhibited atypical resistance to a penicillin, bacitracin (10 microg), and/or nitrofurantoin (300 microg). One human strain was also resistant to erythromycin (15 microg) and clindamycin (2 microg). These resistances may have been acquired due to antibiotic exposure in vivo, but conclusive evidence is lacking in this regard. Seven microorganism-drug combinations were evaluated for beta-lactamase activity using synergy and nitrocefin tests. The absence of activity suggested that cell wall impermeability appeared responsible for beta-lactam resistance. The occurrence of a minority of lactobacilli with undesirable, atypical resistance to certain antibiotics demonstrates that not all strains are suitable for use as probiotics or bacteriotherapeutic agents. The natural resistance of lactobacilli to a wide range of clinically important antibiotics may enable the development of antibiotic/probiotic combination therapies for such conditions as diarrhea, female urogenital tract infection, and infective endocarditis.

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Minimum inhibitory contentrations (MICs) of selected inhibitors of cell wall synthesis (benzylpenicillin, ampicillin, and vancomycin), protein synthesis (gentamicin, streptomycin, tetracycline, chloramphenicol, and erythromycin), and nucleic acid synthesis (co-trimoxazole, rifampicin, and metronidazole) were determined by gradient diffusion (E test; AB Biodisk, Solna, Sweden) on deMan, Rogosa, Sharpe (MRS) agar for Lactobacillus strain GG and 11 closely related, rapidly growing, facultatively anaerobic, potentially probiotic Lactobacillus rhamnosus strains. All strains were resistant to vancomycin (MIC90 > or = 256 microg/ml), co-trimoxazole (MIC90 > or = 32 microg/ml), metronidazole (MIC90 > or = 32 microg/ml), gentamicin (MIC90 > or = 128 microg/ml), and streptomycin (MIC90 > or = 256 microg/ml), and sensitive to pencillin G (MIC90 > 0.375 microg/ml), ampicillin (MIC90 > 0.750 microg/ml), rifampicin (MIC90 > 0.375 microg/ml), tetracycline (MIC90 > 1.5 microg/ml), chloramphenicol (MIC90 > 8 microg/ml), and erythromycin (MIC90 > 2 microg/ml). E test MICs were also determined for L. acidophilus National Collection of Food Bacteria (NCFB) 1748 and L. reuteri Deutsche Sammlung von Mikroorganismen 20016T by the inoculum application method recommended by the manufacturer (swabbing), with and without antibiotic prediffusion for 1 h at room temperature, and by an alternative inoculum application (agar overlay) method, without antibiotic prediffusion. Antibiotic prediffusion increased the MICs for penicillin G, ampicillin, tetracycline, and chloramphenicol by up to 2 log2 MIC dilutions without changing antibiotic susceptibility category. Agar overlay application also increased the MICs for these antibiotics as well as for gentamicin by up to 3 log2 MIC dilutions without changing antibiotic susceptibility category. Exact agreement between MICs determined by swab and agar overlay application without antibiotic prediffusion was strain dependent: 54.5% for strain DSM 20016T and 72.7% for strain NCFB 1748. The swab and agar overlay gradient diffusion methods provide a reliable basis for antibiotic susceptibility testing of rapidly growing, facultatively anaerobic lactobacilli, using MRS agar as test medium and are readily applicable for testing individual isolates as needed.