Population declines of amphibian species in many parts of the world are associated with a lethal fungal pathogen, Batrachochytrium dendrobatidis. Using laboratory challenge assays, we describe the inhibition of B. dendrobatidis by members of eight genera of bacteria isolated from the skin of two amphibian species that exhibit parental care behavior (Plethodon cinereus and Hemidactylium scutatum). We found that members of three genera of bacteria isolated from the skins of the salamander P. cinereus and members of seven genera isolated from the salamander H. scutatum inhibited the growth of B. dendrobatidis. Understanding how B.dendrobatidis interacts with an ecological community of cutaneous flora may be important in explaining and preventing amphibian population declines.

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The effect of dietary fat source (soy oil or a mixture of lard and tallow) and dietary supplementation with antibiotics (a combination of avilamycin at 10 mg kg of feed ؊1 and salinomycin at 40 mg kg of feed ؊1 ) on the bacterial community in the ileum of broiler chickens at different ages (7, 14, 21, and 35 days) was studied using PCR with denaturing gradient gel electrophoresis (DGGE) analysis and bacteriological culture. The bacterial origin of fragments in DGGE profiles was identified by sequencing. Bacterial enumeration results, together with PCR-DGGE profiles, showed that the composition of the microflora was age dependent and influenced by dietary fat source and antibiotic supplementation. An increased incidence of streptococci, enterobacteria, and Clostridium perfringens with age of the chickens was demonstrated. Lactobacilli and C. perfringens were the bacterial groups most strongly affected by the dietary treatments. Moreover, different strains (clonal variants of the alpha-toxin gene) of C. perfringens type A were detected in response to age, dietary fat source, and dietary supplementation with antibiotics.

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Among the microbiota of amphibian skin are bacteria that produce antifungal compounds. We isolated cutaneous bacteria from the skins of three populations of the nest-attending plethodontid salamander Hemidactylium scutatum and subsequently tested the bacterial isolates against two different fungi (related to Mariannaea elegans and Rhizomucor variabilis) that were obtained from dead salamander eggs. The culturable antifungal bacteria were phylogenetically characterized based on 16S rRNA phylogeny, and belonged to four phyla, comprising 14 bacterial families, 16 genera and 48 species. We found that about half of the antifungal bacterial genera and families were shared with a related salamander species, but there was virtually no overlap at the species level. The proportion of culturable antifungal bacterial taxa shared between two large populations of H. scutatum was the same as the proportion of taxa shared between H. scutatum and Plethodon cinereus, suggesting that populations within a species have unique antifungal bacterial species. Approximately 30% of individuals from both salamander species carried anti-M. elegans cutaneous bacteria and almost 90% of P. cinereus and 100% of H. scutatum salamanders carried anti-R. variabilis cutaneous bacteria. A culture independent method (PCR/DGGE) revealed a shared resident bacterial community of about 25% of the entire resident bacterial community within and among populations of H. scutatum. Thus, the culturable antifungal microbiota was far more variable on salamander skins than was the bacterial microbiota detected by PCR/DGGE. The resident cutaneous antifungal bacteria may play an important role in amphibians' innate defense against pathogens, including the lethal chytrid fungus Batrachochytrium dendrobatidis.

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Methods that enabled the identification, detection, and enumeration of Bifidobacterium species by PCR targeting the transaldolase gene were tested. Bifidobacterial species isolated from the feces of human adults and babies were identified by PCR amplification of a 301-bp transaldolase gene sequence and comparison of the relative migrations of the DNA fragments in denaturing gradient gel electrophoresis (DGGE). Two subtypes of Bifidobacterium longum, five subtypes of Bifidobacterium adolescentis, and two subtypes of Bifidobacterium pseudocatenulatum could be differentiated using PCR-DGGE. Bifidobacterium angulatum and B. catenulatum type cultures could not be differentiated from each other. Bifidobacterial species were also detected directly in fecal samples by this combination of PCR and DGGE. The number of species detected was less than that detected by PCR using species-specific primers targeting 16S ribosomal DNA (rDNA). Real-time quantitative PCR targeting a 110-bp transaldolase gene sequence was used to enumerate bifidobacteria in fecal samples. Real-time quantitative PCR measurements of bifidobacteria in fecal samples from adults correlated well with results obtained by culture when either a 16S rDNA sequence or the transaldolase gene sequence was targeted. In the case of samples from infants, 16S rDNA-targeted PCR was superior to PCR targeting the transaldolase gene for the quantification of bifidobacterial populations.

Human subjects consumed biscuits containing either galacto-oligosaccharides or fructo-oligosaccharides in a double-blinded, crossover study. The impact of supplementing the diet with three biscuits per day on the fecal microbiota was evaluated by selective culture of particular bacterial groups, measurement of ␤-galactosidase activity, and nucleic acid-based analytical methods (PCR-denaturing gradient gel electrophoresis [PCR-DGGE] and fluorescent in situ hybridization). The composition of the bifidobacterial populations was monitored at the level of species (PCR-DGGE) and strains (pulsed-field gel electrophoresis of DNA digests), and representative cultures were tested quantitatively for their ability to use galacto-oligosaccharides. Technical improvements to DGGE analysis of the microbiota were made by the use of an internal standard that allowed valid comparisons of fragment staining intensities to be made between profiles, the use of S1 nuclease digestion to remove single-stranded DNA to facilitate cloning of DNA sequences cut from gels, and the extraction of RNA to be used as the template in reverse transcription-PCR-DGGE. RNA-DGGE profiles were markedly different (Dice's similarity coefficient, 58.5%) from those generated by DNA-DGGE. Neither the sizes of the bacterial populations nor the DNA-DGGE profiles of the microbiota were altered by the consumption of the biscuits, but the RNA-DGGE profiles were altered by the detection or increased staining intensity of 16S rRNA gene sequences originating from Bifidobacterium adolescentis and/or Colinsella aerofaciens in the feces of 11 of 15 subjects. ␤-Galactosidase activity was elevated in the feces of some subjects as a result of biscuit consumption. Subjects differed in the ability of the bifidobacterial strains harbored in their feces to use galacto-oligosaccharides. Our observations suggest that a phylogenetic approach to analysis of the gut ecosystem may not always be optimal and that a more physiological (biochemical) method might be more informative.The distal intestinal tract of humans harbors a complex bacterial community, referred to as the microbiota, the composition of which is represented in the feces (24). The intestinal microbiota impacts markedly on the immunology, biochemistry, physiology, and nonspecific disease resistance of the host (14). Additionally, in genetically susceptible hosts, the microbiota is an essential factor in the pathogenesis of inflammatory diseases of the bowel (29). These observations have promoted the view that modification of the composition of the intestinal microbiota by means of dietary supplements might promote health (12,13,27).Probiotics (products containing living bacteria) transiently alter the composition of Lactobacillus or Bifidobacterium populations during the period of administration (8,30,32,33), whereas the consumption of prebiotics (potential substrates for bacterial inhabitants of the intestine) has been reported to selectively increase the number of bifidobacteria (4,6,11,16,20,25,34). Inulin and fructo...