Amphibians are experiencing global population declines and are the most threatened vertebrate class (Stuart et al., 2004; Hoffmann et al., 2010). Indeed, 41 percent of amphibian species are threatened. Among the many factors that contribute to amphibian declines, the fungal disease chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis (Bd), is the largest disease threat to the biodiversity of any vertebrate group (Wake and Vredenburg, 2008; Hoffmann et al., 2010). Alarmingly, disease-induced declines occur in protected, pristine environments, such as national parks, where up to 92.5 percent of critically endangered amphibian species are affected by Bd (Kilpatrick et al., 2010).

Amphibians have several defenses against Bd, including symbiotic bacteria living on their skins (Harris et al., 2006). Numerous anti-fungal bacterial species have been cultured from amphibians (Lauer et al., 2007; Woodhams et al., 2007), and their presence is associated with disease resistance (Becker and Harris, 2010). Additionally, using culture-independent techniques, diverse host-specific bacterial communities have been observed on amphibians (McKenzie et al., 2011; Kueneman et al., 2013; Loudon et al., 2013; Walke et al., 2014). One promising mitigation strategy against Bd is to augment the bacterial communities on amphibians' skin to increase disease resistance (Harris et al., 2009b; Bletz et al., 2013). This approach of using bacterial probiotics has been successful in laboratory experiments and one field trial (Becker et al., 2009; Vredenburg et al., 2011).

Amphibian-associated bacteria can inhibit Bd by producing anti-fungal metabolites (Brucker et al., 2008a,b). Several anti-Bd metabolites are produced from amphibian skin bacteria, such as violacein and indole-3-carboxaldehyde (I3C) from Janthinobacterium lividum and 2,4-diacetylphloroglucinol is produced Lysobacter gummosus (Brucker et al., 2008a,b). Additionally, evidence from free-living salamanders in nature suggests that the concentration of bacterially produced metabolites on their skins can be high enough to kill pathogenic fungi, such as Bd (Brucker et al., 2008b). Moreover, a positive correlation was found between the abundance of the amphibian symbiont J. lividum and violacein on red-backed salamanders; salamanders with more J. lividum and violacein had less morbidity due to chytridiomycosis (Becker et al., 2009).

A recent model on the assembly of beneficial microbiomes suggests that the microbiota's defensive function is a by-product of interspecific competition among microbial species (Scheuring and Yu, 2012). If microbial population densities reach the point where competition occurs and enough energy is available, defensive chemical production can be triggered that may defend the host. Without conditions of interspecific competition (pure culture), a weaker defensive function may occur. For example, bacteria cultured separately have been found to not produce anti-fungal compounds, but do produce anti-fungal compounds when cultured together (De Boer et al., 2007). In this study, we tested the hypothesis that conditions that favor interspecific competition will result in greater inhibition of Bd than under conditions of no interspecific competition. We hypothesized that greater inhibition of Bd would be caused by the production of emergent metabolites (i.e., metabolites produced in co-culture but not produced in mono-culture) that are only found with interspecific competition.

Combinations of bacterial isolate's CFSs exhibited several different relationships in the inhibition of Bd, demonstrating that the isolates, and the concentrations of an isolate's CFSs, are important factors in Bd inhibition. In some cases, the inhibition caused by two isolates CFSs had an additive effect on Bd growth inhibition. Other combinations inhibited Bd growth synergistically. Some combinations did not demonstrate any effect on Bd growth.

The in vitro model of combining CFSs can represent spatially separated isolates on amphibian skin whose secreted metabolites combine in the interstices between them. The different relationships among the pairwise combinations between the four isolates in high, medium and low concentrations demonstrate the variability of bacterial interactions that are likely found in nature. It is unknown how isolates are distributed on amphibian skins, much less their relative population densities, but it is likely to be variable even on a single host individual. In an additive relationship, the metabolites of both bacterial isolates are contributing equally to the inhibition of Bd growth. Synergistic relationships may be the result of anti-fungals with different modes of action (Ghannoum and Rice, 1999). As a hypothetical example, in our system a metabolite from the first bacterial isolate may puncture the cell wall of Bd, whereas the metabolites from the second bacterial isolate may inhibit translation.

Emergent metabolites were identified that were present in bacterial co-cultures, but not present in bacterial mono-cultures. CFSs from co-cultures were often more inhibitory of Bd than combining CFSs from mono-cultures. Co-culturing is a model of isolates on the skins that are in the same or close spatial location and are competing with each other by starting to produce additional inhibitory metabolites, and these new metabolites are one reason for greater inhibition of Bd. Furthermore, emergent metabolites may be synergistically interacting with the metabolites produced in mono-culture and other emergent metabolites to enhance their collective killing power. We determined that these emergent metabolites are anti-Bd and determined the identity and IC₅₀ of the most inhibitory metabolite, tryptophol. Although it is inhibitory, it is much less potent than previously identified anti-Bd metabolites such as violacein (Brucker et al., 2008b). Tryptophol has been found to be produced by fungi such as Ceratocystis adipose (Guzmán-López et al., 2007), Candida albicans (Lingappa et al., 1969), Pythium ultimum (Rey et al., 2001), Drechslera nodulosum (Sugawara and Strobel, 1987), and Zygosaccharomyces priorianus (Rosazza et al., 1973). The mechanism that tryptophol uses to inhibit Bd is unknown. We hypothesize that these emergent metabolites are interacting to cause the greater Bd inhibition found in the co-culture assays. The strong inhibition of Bd caused by the co-culture CFSs supports the hypothesis that interspecific competition led to greater Bd inhibition than intraspecific competition alone (De Boer et al., 2007; Scheuring and Yu, 2012). Furthermore, it suggests that competing microbial species in amphibian skins would enhance disease protection.

One case of facilitation occurred in co-culture between Pseudomonas sp. and Janthinobacterium sp. This result is intriguing since when the bacteria were grown separately and then combined, they did not result in facilitation. Previous work using a similar protocol to determine bacteria's effect on Bd found facilitation of Bd growth by some isolate's CFSs (Bell et al., 2013). Pseudomonas can produce a number of antifungal metabolites and Janthinobacterium has been widely studied as an anti-Bd bacterium, so facilitation of Bd was surprising. In our study, the co-culture lacked metabolites that were found in the Janthinobacterium mono-culture. This shift in metabolites may have led to the facilitation of Bd.

When the “separate then combined” assays were repeated, the results differed to some extent between trials. In particular, synergy between certain pairs of isolates in the first experiment was not always found in the second experiment. A possible reason for this is that isolates were under somewhat different densities in culture due to stochastic variation. As a result, isolates produced different concentrations of metabolites that then interacted differently when combined. An additional reason may be the age of the media used or the use of different batches of media that had slight chemical differences. Also, it is possible that mutation in laboratory stocks led to different outcomes in the assays. However, all of the results demonstrate that additive and synergistic interactions occur among amphibians' skin bacteria, which depend on a variety of factors, including bacterial species identify and chemical concentration.

The inhibitory interactions between different bacterial species are not the only chemical interactions that inhibit Bd within the amphibian skin system. Myers et al. (2012) found synergistic interactions between the antimicrobial peptides (AMPs) from the skin of the frog, Rana muscosa, and the known anti-Bd metabolite 2,4-diacetylphloroglucinol (2,4-DAPG), which is produced by the bacterium Pseudomonas fluorescens. It is likely that AMPs interact with other bacterially-produced metabolites to inhibit Bd, especially since we found that bacteria produce a high richness of metabolites. Bacteria also produce AMPs that may inhibit Bd, although we focused on smaller metabolites that are produced by bacteria.

Our results support the hypothesis that competition between different bacterial species causes greater Bd inhibition. Bacterial species compete through the production of defensive compounds, which leads to their competitive dominance. Therefore, the host obtains a defensive benefit as a by-product of microbial competition. Results from theory suggest that beneficial, antibiotic-producing bacteria are favored over bacteria that do not produce such compounds as long as the host supplies adequate resources for antibiotic production (Scheuring and Yu, 2012). Although we expect that competition has driven the production of emergent metabolites and an overall greater inhibitory function, competition was not measured, and it is possible that other interactions, such as cooperation, led to our results.

This work reveals the complexity of microbial interactions and emphasizes the importance of considering such interactions when developing a bacterial probiotic as a conservation strategy. Furthermore, our results suggest that a bacterial probiotic with multiple bacterial species is more promising than a bacteria probiotic with a single bacterial species. Although a single trial to test bacterial probiotics with multiple bacterial species was unsuccessful on amphibians (Bletz et al., 2013), more work is needed to fully assess the potential of such therapy. Probiotic trials with a single isolate have been successful in some cases (Harris et al., 2009a,b), but not in others (Becker et al., 2011; Bletz et al., 2013). This variation could be explained by the probiotic interacting synergistically with the resident microbiota to inhibit Bd in the successful cases, but not synergistically inhibiting Bd or even facilitating Bd the unsuccessful cases. Bacterial probiotics with multiple bacterial species have been successful in mouse intestines (Lawley et al., 2012). In this study, the successful combination of bacteria used in the probiotic was selected by trial and error. This strategy was successful, however not efficient. It is likely important to choose bacteria that will compete interspecifically and produce more inhibitory metabolites. We propose choosing bacteria that display additive or synergistic relationships when in combination with other isolates over a range of concentrations and be highly inhibitory when co-cultured. If a bacterial isolate does not have an effect on Bd growth and does not produce any effect when combined with a second bacterial isolate, then it should be excluded from further consideration as part of a probiotic solution. To test whether the addition of multiple probiotics on amphibians worked in vivo, the defensive function of the mucosome could be measured, which would determine if the chemicals on an amphibian are anti-fungal prior to infection by a pathogen (Woodhams et al., 2014). Our results provide support for the use of a co-culture method in probiotic development that may lead to a more effective probiotic solution.

In conclusion, we found that metabolites produced by amphibians' skin bacteria in monoculture can be combined, and they can interact synergistically with each other to inhibit Bd. In addition, we found that when these bacteria were co-cultured they were often even more inhibitory to Bd, and they produced emergent metabolites that were not made by the bacteria when in monoculture. Furthermore, we identified the emergent metabolite tryptophol, which was the most active emergent metabolite against Bd. These results will aid in developing combination of probiotics to mitigate the amphibian disease chytridiomycosis, in addition to contributing to our knowledge of bacterial and chemical interactions.