Addition of antifungal skin bacteria to salamanders ameliorates the effects of chytridiomycosis.

Chytridiomycosis, caused by the skin fungus Batrachochytrium dendrobatidis (Bd), has caused population declines of many amphibians in remote protected habitats. Progress has been made in understanding the pathogen's life cycle, documenting its devastating effects on individual amphibians and on populations, and understanding how and why disease outbreaks occur. No research has directly addressed the critical question of how to prevent declines and extinctions caused by outbreaks of the disease. We have identified a number of bacterial species of amphibian skin that inhibit Bd in vitro. Here, we demonstrate that a species of anti-Bd skin bacteria can be successfully added to skins of salamanders Plethodon cinereus, and that addition of this bacterium reduced the severity of a disease symptom in experimentally infected individuals. This is the first demonstration that manipulating the natural skin microbiota of an amphibian species can alter the pathogen's negative effects on infected amphibians and appears to be the first demonstration that an epibiotic manipulation of any wildlife species can lessen the effects of an emerging infectious disease. It suggests that probiotic or bio-augmentation manipulations of cutaneous microbiota could have the potential to reduce susceptibility of amphibians to the disease in nature. This is the first approach suggested that could slow or halt epidemic outbreaks and allow successful reintroductions of amphibian species that have become locally or globally extinct in the wild. Our results also suggest a mechanism for the association of climate change and the likelihood of chytridiomycosis outbreaks via the effects of the former on antifungal bacterial communities.

Antifungal skin bacteria, embryonic survival, and communal nesting in four-toed salamanders, Hemidactylium scutatum.

We examined a novel hypothesis for the maintenance of communal nesting in the salamander, Hemidactylium scutatum, namely that communal nests are more likely than solitary nests to be associated with cutaneous antifungal bacteria, which can inhibit fungal infections of embryos. A communal nest contains eggs of two or more females of the same species. The nesting behavior of H. scutatum females and survival of embryos were determined by frequent nest surveys at three ponds. For communal nests, embryonic survival tended to be higher and catastrophic nest failure was lower. Pure bacterial cultures of resident species were obtained from the salamanders' skins by swabbing and tested against a fungal pathogen of embryos (Mariannaea sp.) in laboratory assays. We found that 27% of females had skin bacteria inhibitory to Mariannaea sp. Communal nests were more likely to have at least one female with antifungal bacteria than were solitary nests. Using a culture-independent assay (denaturing gradient gel electrophoresis of 16S rRNA gene fragments), we found that bacterial species on females and embryos were more similar to each other than they were to bacterial species found in soil within the nest, suggesting that females transmitted skin bacteria to embryos. The presence of anti-Mariannaea skin bacteria identified from the laboratory assays did not prevent fungal presence in field nests. However, once a nest was visibly infected with fungi, presence of anti-Mariannaea bacteria was positively correlated with survival of embryos. Microbe transmission is usually thought to be a cost of group living, but communal nesting in H. scutatum may facilitate the transmission of antifungal bacteria to embryos.

Diversity of cutaneous bacteria with antifungal activity isolated from female four-toed salamanders.

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.

Antibiotics in the human food chain: establishing no effect levels of tetracycline, neomycin, and erythromycin using a chemostat model of the human colonic microflora.

A chemostat model of the healthy human large bowel ecosystem was used to establish no effect levels for tetracycline, neomycin, and erythromycin. For each compound, the equivalent to four oral doses (0, 1.5, 15, and 150 mg/60 kg person/d) was studied. Concentrations of the test compounds in the chemostat medium were intended to simulate fecal levels that might be expected following consumption of food containing antibiotic residue and were based on published oral doses and fecal levels. We monitored the following parameters: short chain fatty acids, bile acids, sulfate reduction, azoreductase and nitroreductase activities, beta-glucosidase and beta-glucuronidase activities, a range of bacterial counts and, lastly, the susceptibility among sentinel bacteria to each test compound. Neomycin and erythromycin reduced bile acid metabolism. Neomycin elevated propionate levels and caused a marginal diminution in azoreductase activity. Based on our results, the no observed effect level (NOEL) of both tetracycline and erythromycin was 15 mg/60 kg person/d. The NOEL for neomycin was 1.5 mg/60 kg person/d.

Ciprofloxacin at low levels disrupts colonization resistance of human fecal microflora growing in chemostats.

We studied the in vitro effects of a range of ciprofloxacin (CI) concentrations on the human intestinal flora's colonization resistance (CR) to Salmonella kedougou NCTC 12173. Four steady state microbial communities were established in chemostats using inocula from a single pool of human feces. Three chemostats were exposed to CI (0.1, 0.43 and 5 microg/mL, respectively); one served as a no-drug control. The CR of each community was tested by three successive daily challenges of 10(8) S. kedougou, each delivered in a 1 mL bolus. There was no colonization of the no-drug chemostat. Likewise, after exposure to only 0.1 microg/mL CI there was no loss of CR and S. kedougou did not colonize. Conversely, both the 0.43 and the 5 microg/mL-exposed floras suffered a loss of CR and these chemostats were colonized. S. kedougou overgrew faster and reached higher counts in the presence of 0.43 than it did in the presence of 5 microg/mL. One possible explanation is that CI had a dose-dependent effect on both the challenge strain and CR. Thus, at higher levels, even though CR was disrupted by CI, so too was the growth of the challenge strain. Since exposure to CI elicited a dose-dependent reduction in Escherichia coli counts [Reg. Pharmacol. Toxicol. 33 (2001) 276] our new data suggest that E. coli may contribute to the CR against salmonella. We further conclude that, even at fecal levels below those reached during therapy, CI may impact the human gut flora sufficiently to facilitate colonization by S. kedougou.

Effects of dietary fat source and subtherapeutic levels of antibiotic on the bacterial community in the ileum of broiler chickens at various ages.

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.

Identification, detection, and enumeration of human bifidobacterium species by PCR targeting the transaldolase gene.

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.