Stability of microbiota facilitated by host immune regulation: informing probiotic strategies to manage amphibian disease.

Microbial communities can augment host immune responses and probiotic therapies are under development to prevent or treat diseases of humans, crops, livestock, and wildlife including an emerging fungal disease of amphibians, chytridiomycosis. However, little is known about the stability of host-associated microbiota, or how the microbiota is structured by innate immune factors including antimicrobial peptides (AMPs) abundant in the skin secretions of many amphibians. Thus, conservation medicine including therapies targeting the skin will benefit from investigations of amphibian microbial ecology that provide a model for vertebrate host-symbiont interactions on mucosal surfaces. Here, we tested whether the cutaneous microbiota of Panamanian rocket frogs, Colostethus panamansis, was resistant to colonization or altered by treatment. Under semi-natural outdoor mesocosm conditions in Panama, we exposed frogs to one of three treatments including: (1) probiotic - the potentially beneficial bacterium Lysinibacillus fusiformis, (2) transplant - skin washes from the chytridiomycosis-resistant glass frog Espadarana prosoblepon, and (3) control - sterile water. Microbial assemblages were analyzed by a culture-independent T-RFLP analysis. We found that skin microbiota of C. panamansis was resistant to colonization and did not differ among treatments, but shifted through time in the mesocosms. We describe regulation of host AMPs that may function to maintain microbial community stability. Colonization resistance was metabolically costly and microbe-treated frogs lost 7-12% of body mass. The discovery of strong colonization resistance of skin microbiota suggests a well-regulated, rather than dynamic, host-symbiont relationship, and suggests that probiotic therapies aiming to enhance host immunity may require an approach that circumvents host mechanisms maintaining equilibrium in microbial communities.

Skin penetration enhancement of mefenamic acid by ethanol and 1,8-cineole can be explained by the 'pull' effect.

The simultaneous skin permeation of drug and penetration enhancer have been studied in vitro. Simple formulations of mefenamic acid in PEG400 incorporating various proportions of ethanol or 1,8-cineole were prepared and applied to porcine ear skin in diffusion cells under infinite conditions. Receptor phases were assayed for mefenamic acid by HPLC and ethanol or 1,8-cineole by GC. Concentration-dependent permeation profiles were obtained for both ethanol or 1,8-cineole, in addition to concentration-dependent enhancement of mefenamic acid. When the steady state flux of mefenamic acid was plotted against ethanol or 1,8-cineole, linear relationships were observed with r2 values of 0.988 and 0.999, respectively. The close connection between rates of excipient and solute permeation is generally referred to as the 'pull' (or 'drag') effect, where in this case permeation of the enhancer facilitated permeation of the solute. This appears to be sufficient to account for the enhancing activity of ethanol and 1,8-cineole, notwithstanding initial modulations that may occur within the stratum corneum.