Denaturing gradient gel electrophoresis (DGGE) of DNA fragments generated by PCR with 16S ribosomal DNA-targeted group-specific primers was used to detect lactic acid bacteria (LAB) of the genera Lactobacillus, Pediococcus, Leuconostoc, and Weissella in human feces. Analysis of fecal samples of four subjects revealed individual profiles of DNA fragments originating not only from species that have been described as intestinal inhabitants but also from characteristically food-associated bacteria such as Lactobacillus sakei, Lactobacillus curvatus, Leuconostoc mesenteroides, and Pediococcus pentosaceus. Comparison of PCR-DGGE results with those of bacteriological culture showed that the food-associated species could not be cultured from the fecal samples by plating on Rogosa agar. On the other hand, all of the LAB species cultured from feces were detected in the DGGE profile. We also detected changes in the types of LAB present in human feces during consumption of a milk product containing the probiotic strain Lactobacillus rhamnosus DR20. The analysis of fecal samples from two subjects taken before, during, and after administration of the probiotic revealed that L. rhamnosus was detectable by PCR-DGGE during the test period in the feces of both subjects, whereas it was detectable by culture in only one of the subjects.

Four sourdoughs (A to D) were produced under practical conditions by using a starter mixture of three commercially available sourdough starters and a baker's yeast constitutively containing various species of lactic acid bacteria (LAB). The sourdoughs were continuously propagated until the composition of the LAB flora remained stable. Two LAB-specific PCR-denaturing gradient gel electrophoresis (DGGE) systems were established and used to monitor the development of the microflora. Depending on the prevailing ecological conditions in the different sourdough fermentations, only a few Lactobacillus species were found to be competitive and became dominant. In sourdough A (traditional process with rye flour), Lactobacillus sanfranciscensis and a new species, L. mindensis, were detected. In rye flour sourdoughs B and C, which differed in the process temperature, exclusively L. crispatus and L. pontis became the predominant species in sourdough B and L. crispatus, L. panis, and L. frumenti became the predominant species in sourdough C. On the other hand, in sourdough D (corresponding to sourdough C but produced with rye bran), L. johnsonii and L. reuteri were found. The results of PCR-DGGE were consistent with those obtained by culturing, except for sourdough B, in which L. fermentum was also detected. Isolates of the species L. sanfranciscensis and L. fermentum were shown by randomly amplified polymorphic DNA-PCR analysis to originate from the commercial starters and the baker's yeast, respectively.The production of sourdough bread can be traced back to ancient times (24). The products are characterized by their unique flavor, enhanced shelf life and nutritional value, and favorable technological properties (16,25). Sourdough is an intermediate product and contains metabolically active microorganisms. The microbial ecology of the sourdough fermentation is determined by ecological factors described by Hammes and Gänzle (16). Endogenous factors are determined by the chemical and microbiological components of the dough, and exogenous factors are determined by temperature and atmosphere. In practice, strong effects are exerted by process parameters such as dough yield, amount and composition of the starter, number of propagation steps, and fermentation time. The impact of these parameters during continuous propagation of sourdough causes the selection of a characteristic microflora consisting of lactic acid bacteria (LAB) and usually yeasts. Microbiological studies have revealed that 43 species of LAB, mostly species of the genus Lactobacillus, and more than 23 species of yeasts, especially species of the genera Saccharomyces and Candida (6, 23), occur in this ecological niche. As shown for certain industrial sourdough processes (4), such microbial associations may endure for years, although the fermentation process runs under nonaseptic conditions.Based on common principles used in artisanal and industrial processes, Böcker et al. (5) defined three types of sourdough. Type I sourdoughs are produced with traditional techni...

The antibiotic resistances of 45 lactic acid bacteria strains belonging to the genera Lactobacillus, Streptococcus, Lactococcus, Pediococcus, and Leuconostoc were investigated. The objective was to determine antibiotic resistances and to verify these at the genetic level, as is currently suggested by the European "qualified presumption of safety" safety evaluation system for industrial starter strains. In addition, we sought to pinpoint possible problems in resistance determinations. Primers were used to PCR amplify genes involved in ␤-lactam antibiotic, chloramphenicol, tetracycline, and erythromycin resistance. The presence of ribosomal protection protein genes and the ermB gene was also determined by using a gene probe. Generally, the incidences of erythromycin, chloramphenicol, tetracycline, or ␤-lactam resistances in this study were low (<7%). In contrast, aminoglycoside (gentamicin and streptomycin) and ciprofloxacin resistances were higher than 70%, indicating that these may constitute intrinsic resistances. The genetic basis for ciprofloxacin resistance could not be verified, since no mutations typical of quinolone resistances were detected in the quinolone determining regions of the parC and gyrA genes. Some starter strains showed low-level ampicillin, penicillin, chloramphenicol, and tetracycline resistances, but no known resistance genes could be detected. Although some strains possessed the cat gene, none of these were phenotypically resistant to chloramphenicol. Using reverse transcription-PCR, these cat genes were shown to be silent under both inducing and noninducing conditions. Only Lactobacillus salivarius BFE 7441 possessed an ermB gene, which was encoded on the chromosome and which could not be transferred in filter-mating experiments. This study clearly demonstrates problems encountered with resistance testing, in that the breakpoint values are often inadequately identified, resistance genes may be present but silent, and the genetic basis and associated resistance mechanisms toward some antibiotics are still unknown.

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Members of the genus Lactobacillus are common inhabitants of the gut, yet little is known about the traits that contribute to their ecological performance in gastrointestinal ecosystems. Lactobacillus reuteri 100-23 persists in the gut of the reconstituted Lactobacillus-free mouse after a single oral inoculation. Recently, three genes of this strain that were specifically induced (in vivo induced) in the murine gut were identified (38). We report here the detection of a gene of L. reuteri 100-23 that encodes a high-molecular-mass surface protein (Lsp) that shows homology to proteins involved in the adherence of other bacteria to epithelial cells and in biofilm formation. The three in vivo-induced genes and lsp of L. reuteri 100-23 were inactivated by insertional mutagenesis in order to study their biological importance in the murine gastrointestinal tract. Competition experiments showed that mutation of lsp and a gene encoding methionine sulfoxide reductase (MsrB) reduced ecological performance. Mutation of lsp impaired the adherence of the bacteria to the epithelium of the mouse forestomach and altered colonization dynamics. Homologues of lsp and msrB are present in the genomes of several strains of Lactobacillus and may play an important role in the maintenance of these bacteria in gut ecosystems.The guts of mammals are colonized by a complex collection of microorganisms (the gut microbiota) that influence biochemical, physiological, immunological, and nonspecific disease resistance characteristics of the host (10). The bacterial composition of this ecosystem has been shown to be remarkably stable over time (43). Members of the gut microbiota have presumably coevolved with their host species and must possess traits that enable them to establish and maintain themselves in a lotic (flowing) and competitive environment. On the other hand, there are microbes present in the gastrointestinal tract that have a transient existence (allochthonous components), and this situation raises questions about what factors distinguish resident members of the microbiota from allochthonous members (27).Lactobacillus reuteri strain 100-23 is a true (autochthonous) resident of the murine gut because it adheres to the nonsecretory epithelium of the forestomach and persists at constant population levels in particular regions of the gut throughout the life of the murine host (13, 39).We maintain a colony of mice whose gut microbiota is devoid of lactobacilli but is otherwise equivalent to that of conventional mice (32). These reconstituted Lactobacillus-free (RLF) mice permit the study of Lactobacillus traits against a standardized bacteriological background. L. reuteri 100-23 and the RLF mouse gut therefore provide an appropriate model system with which to study the molecular basis of Lactobacillus autochthony.While L. reuteri 100-23 inhabits the murine gut, genes encoding methionine sulfoxide reductase (msrB), xylose isomerase (xylA), and a protein with low similarity to methionine synthase II (met) are specifically induced (38). Add...

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