Inhibition of Fungal Pathogens across Genotypes and Temperatures by Amphibian Skin Bacteria

Abstract

Symbiotic bacteria may dampen the impacts of infectious diseases on hosts by inhibiting pathogen growth. However, our understanding of the generality of pathogen inhibition by different bacterial taxa across pathogen genotypes and environmental conditions is limited. Bacterial inhibitory properties are of particular interest for the amphibian-killing fungal pathogens (Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans), for which probiotic applications as conservation strategies have been proposed. We quantified the inhibition strength of five putatively B. dendrobatidis-inhibitory bacteria isolated from woodland salamander skin against six Batrachochytrium genotypes at two temperatures (12 and 18°C). We selected six genotypes from across the Batrachochytrium phylogeny: B. salamandrivorans, B. dendrobatidis-Brazil and four genotypes of the B. dendrobatidis Global Panzootic Lineage (GPL1: JEL647, JEL404; GPL2: SRS810, JEL423). We performed 96-well plate challenge assays in a full factorial design. We detected a Batrachochytrium genotype by temperature interaction on bacterial inhibition score for all bacteria, indicating that bacteria vary in ability to inhibit Batrachochytrium depending on pathogen genotype and temperature. Acinetobacter rhizosphaerae moderately inhibited B. salamandrivorans at both temperatures (μ = 46–53%), but not any B. dendrobatidis genotypes. Chryseobacterium sp. inhibited three Batrachochytrium genotypes at both temperatures (μ = 5–71%). Pseudomonas sp. strain 1 inhibited all Batrachochytrium genotypes at 12°C and four Batrachochytrium genotypes at 18°C (μ = 5–100%). Pseudomonas sp. strain 2 and Stenotrophomonas sp. moderately to strongly inhibited all six Batrachochytrium genotypes at both temperatures (μ = 57–100%). All bacteria consistently inhibited B. salamandrivorans. Using cluster analysis of inhibition scores, we found that more closely related Batrachochytrium genotypes grouped together, suggesting that bacterial inhibition strength may be predictable based on Batrachochytrium relatedness. We conclude that bacterial inhibition capabilities change among bacterial strains, Batrachochytrium genotypes and temperatures. A comprehensive understanding of bacterial inhibitory function, across pathogen genotypes and temperatures, is needed to better predict the role of bacterial symbionts in amphibian disease ecology. For targeted conservation applications, we recommend using bacterial strains identified as strongly inhibitory as they are most likely to produce broad-spectrum antimicrobial agents at a range of temperatures.

Probiotic consortia are not uniformly effective against different amphibian chytrid pathogen isolates

“…Thus, facilitation of B. dendrobatidis growth is not simply a rare phenomenon arising from specific Bd GPL/bacterial combinations, and different Bd GPL isolates may differ systematically in their growth rates when exposed to bacterial filtrates (see also Muletz‐Wolz et al. ). It is unclear why some bacterial strains facilitate B. dendrobatidis growth, but one likely explanation is that certain bacterial metabolites can act as growth substrates or facilitators for fungi (Garbaye, ; Hardoim et al., ).…”

“…These patterns are informative with respect to potential strategies for the application of bacterial probiotics to mitigate B. dendrobatidis and other wildlife pathogens. Conservationists might not always know which particular B. dendrobatidis variant is infecting a local population, preventing targeted application of known strong inhibitors for that variant (Muletz-Wolz et al 2017), and both time and expense may prevent the establishment of such a database de novo if a probiotic intervention is required rapidly. Therefore, we must employ strategies that maximize the chance of successful inhibition in the absence of perfect knowledge of the pathogen.…”

“…The phenomenon of BdGPL growth facilitation has been described previously for single pathogen isolates Bell et al, 2013), but crucially, our results suggest that a bacterial strain's ability to facilitate the growth of B. dendrobatidis extends across a broad suite of pathogen isolates. Thus, facilitation of B. dendrobatidis growth is not simply a rare phenomenon arising from specific BdGPL/bacterial combinations, and different BdGPL isolates may differ systematically in their growth rates when exposed to bacterial filtrates (see also Muletz-Wolz et al 2017). It is unclear why some bacterial strains facilitate B. dendrobatidis growth, but one likely explanation is that certain bacterial metabolites can act as growth substrates or facilitators for fungi (Garbaye, 1994;Hardoim et al, 2015).…”

“…Laboratory and field studies have shown host‐associated bacterial communities protect amphibians from B. dendrobatidis infection and that it is possible to artificially augment the microbiota with probiotic bacteria to improve survivorship in response to the pathogen (Becker et al., ; Bletz et al., ; Harris, Lauer, Simon, Banning, & Alford, ; Jani & Briggs, ; Kueneman et al., ; Muletz, Myers, Domangue, Herrick, & Harris, ; Walke et al., ). However, inhibitory capabilities of individual bacteria are not uniform across the variation presented by B. dendrobatidis (Antwis, Preziosi, Harrison, & Garner, ; Bletz, Myers, et al, ; Muletz‐Wolz et al., ). In addition, previous work has found either no (Becker et al., ) or weak evidence (Bletz, Myers, et al, ) of a phylogenetic signal in the ability of bacterial genera to inhibit a singular B. dendrobatidis isolate.…”

Reduced skin bacterial diversity correlates with increased pathogen infection intensity in an endangered amphibian host

“…Bd infects the skin of amphibians and causes mortality by disrupting the skin electrolyte transport system (Voyles et al, ). However, interactions between Bd and other skin microbes, particularly bacteria, can inhibit Bd colonization and pathogenicity (Harris et al, ; Muletz‐wolz et al, ).…”