fmicb-09-01604 July 16, 2018 Time: 16:25 # 1 ORIGINAL RESEARCH published: 18 July 2018 doi: 10.3389/fmicb.2018.01604 Increased Numbers of Culturable Inhibitory Bacterial Taxa May Mitigate the Effects of Batrachochytrium dendrobatidis in Australian Wet Tropics Frogs Sara C. Bell1*†, Stephen Garland2 and Ross A. Alford1 1 College of Science and Engineering, James Cook University, Townsville, QLD, Australia, 2 College of Public Health, Medical and Veterinary Sciences, James Cook University, Townsville, QLD, Australia Symbiotic bacterial communities resident on amphibian skin can benefit their hosts. Edited by: For example, antibiotic production by community members can control the pathogen Eria Alaide Rebollar, Batrachochytrium dendrobatidis (Bd) and it is possible for these community members to National Autonomous University of Mexico (Morelos), Mexico be used as probiotics to reduce infection levels. In the early 1990s, the emergence of Bd Reviewed by: caused declines and disappearances of frogs in the Australian Wet Tropics; the severity Molly Bletz, of its effects varied among species and sites. Some species have since recolonized Technische Universitat Braunschweig, despite enzootic Bd within their populations. This variation in history among species and Germany Matthew Henry Becker, sites provided an opportunity to investigate the role of anti-fungal cutaneous bacteria in Smithsonian Institution, United States protecting frogs against Bd infection. We collected cutaneous swab samples from three *Correspondence: species of frogs at two upland and two lowland sites in the Wet Tropics, and used Sara C. Bell [email protected] in vitro challenge assays to identify culturable Bd-inhibitory bacterial isolates for further †Present address: analysis. We sequenced DNA from cultured inhibitory isolates to identify taxa, resulting Sara C. Bell, in the classification of 16 Bd-inhibitory OTUs, and determined whether inhibitory taxa Australian Institute of Marine Science, were associated with frog species, site, or intensity of infection. We present preliminary Townsville, QLD, Australia results showing that the upper limit of Bd infection intensity was negatively correlated Specialty section: with number of inhibitory OTUs present per frog indicating that increased numbers of This article was submitted to Bd-inhibiting taxa may play a role in reducing the intensity of Bd infections, facilitating Microbial Symbioses, a section of the journal frog coexistence with enzootic Bd. One upland site had a significantly lower prevalence Frontiers in Microbiology of Bd infection, a significantly higher proportion of frogs with one or more culturable Bd- Received: 31 October 2017 inhibitory OTUs, a greater number of inhibitory bacterial genera present per frog, and Accepted: 27 June 2018 Published: 18 July 2018 statistically significant clustering of individual frogs with similar Bd-inhibitory signatures Citation: when compared to all other sites. This suggests that Bd-inhibitory taxa are likely to be Bell SC, Garland S and Alford RA particularly important to frogs at this site and may have played a role in their ability to (2018) Increased Numbers recolonize following population declines. Our findings suggest that the use of multi-taxon of Culturable Inhibitory Bacterial Taxa May Mitigate the Effects Bd-inhibitory probiotics to support at-risk amphibian populations may be more effective of Batrachochytrium dendrobatidis than single-taxon alternatives. in Australian Wet Tropics Frogs. Front. Microbiol. 9:1604. Keywords: amphibian, cutaneous bacteria, Batrachochytrium dendrobatidis, microbiota, cell-free supernatant, doi: 10.3389/fmicb.2018.01604 inhibitory bacteria, chytridiomycosis, disease mitigation Frontiers in Microbiology| www.frontiersin.org 1 July 2018| Volume 9| Article 1604 fmicb-09-01604 July 16, 2018 Time: 16:25 # 2 Bell et al. Bacterial Assemblages Correlate With Infection INTRODUCTION (Woodhams et al., 2007; Lam et al., 2010; Walke et al., 2011; Flechas et al., 2012; Bell et al., 2013; Daskin et al., 2014; Becker Antibiotic-producing bacterial symbionts can protect their hosts et al., 2015; Holden et al., 2015; Madison et al., 2017). from disease (Currie et al., 1999; Haas and Défago, 2005; Scott Patterns of frog decline and recovery in the Australian Wet et al., 2008; Mao-Jones et al., 2010; Mattoso et al., 2012). For Tropics following the arrival of the pathogen Bd in the late example, a high proportion of bacteria isolated from healthy coral 1980s (Laurance et al., 1996; Berger et al., 1999; McDonald and can produce antibiotic compounds active against coral pathogens Alford, 1999) differed among species and sites. Frog populations (Ritchie, 2006; Mao-Jones et al., 2010; Zhang et al., 2013). of all species at lowland sites (below 400 m) did not appear However, the best-studied mutualisms exist between insects to experience declines, while several species, including Litoria and their bacterial symbionts. Both fungus-farming ants and nannotis (waterfall frog) and Litoria rheocola (common mist pine beetles house antibiotic-producing bacteria in specialized frog), suffered either declines or local extirpation at all upland cuticular compartments to control pathogens that threaten their rainforest sites (above 400 m; Richards et al., 1993; McDonald food supply (Currie et al., 1999, 2006; Scott et al., 2008). In and Alford, 1999). Some populations of these species have addition, solitary digger wasps use antibiotic-producing bacteria subsequently reappeared at some but not all sites from which to protect their cocooned larvae against pathogens (Kroiss they disappeared (McDonald et al., 2005; Woodhams and Alford, et al., 2010), and fungus-farming ants maintain a bacterial 2005; Skerratt et al., 2010; McKnight et al., 2017). This suggests biofilm on their cuticles to control a fungal pathogen (Mattoso multiple, potentially independent appearances of resistance to et al., 2012). Variation in the bacterial strain present can cause Bd, or loss of virulence by the pathogen (McKnight et al., 2017). differential morbidity in fungus-farming ants (Poulsen et al., Another species, Litoria serrata (green-eyed tree frog) appeared 2010), demonstrating that antibiotic production by symbionts is to suffer temporary population declines at upland sites, followed important for host protection. by recovery within a few years (Richards et al., 1993; McDonald Antibiotic production by symbiotic bacteria is also important and Alford, 1999; Richards and Alford, 2005). in vertebrates. In two bird species, the European Hoopoe and The mechanisms allowing population recovery or the Green Woodhoopoe, nestlings harbor antibiotic-producing recolonization are likely to vary among species and sites bacteria in their uropygial glands to control feather-degrading (McKnight et al., 2017). There is substantial genetic separation pathogens (Soler et al., 2008; Martín-Vivaldi et al., 2010). among Wet Tropics frog populations (Schneider et al., 1998; Symbiotic bacterial communities resident on amphibian skin can Cunningham, 2001; Hoskin et al., 2005; Richards et al., 2010; also produce, antibiotic compounds that contribute to the control Bell et al., 2012); variation among populations in both innate and of Batrachochytrium dendrobatidis (Bd; Becker et al., 2009, 2015; acquired immune defenses due to selection pressures imposed Harris et al., 2009a,b; Lam et al., 2010; Kueneman et al., 2016), a by the pathogen may affect host resistance to Bd and ultimately pathogenic fungus responsible for worldwide amphibian declines survival (Rollins-Smith et al., 2002, 2011; Woodhams et al., (Berger et al., 1998; Stuart et al., 2004; Skerratt et al., 2007; Wake 2010; Savage and Zamudio, 2011; Fites et al., 2012, 2014; Bataille and Vredenburg, 2008). et al., 2015; McKnight et al., 2017; Voyles et al., 2018). Pathogen One aspect of community ecology theory predicts that virulence may also vary among regions (Berger et al., 2005; complex communities, with higher numbers of species and Farrer et al., 2011; Becker et al., 2017). Therefore, resistance to Bd hence more potential interactions, are generally more resistant to infection is likely to have been acquired independently at these invasion than simple communities with fewer species (Robinson sites. and Valentine, 1979). This has been demonstrated in soil bacterial Symbiotic cutaneous bacteria are considered to be an communities (Matos et al., 2005; van Elsas et al., 2012; Hol et al., important component of the amphibian innate immune defense 2015; Hu et al., 2016), and also in the locust gut (Dillon et al., system (Woodhams et al., 2007, 2014; Harris et al., 2009a). It is 2005) where higher numbers of inoculated bacterial isolates have likely that the suite of cutaneous bacteria present on amphibian been associated with greater resistance against pathogens. skin can evolve broad antimicrobial activity against invading Studies of amphibian skin microbiota have produced similar pathogens much faster than adaptation of the host’s innate and results. In vitro studies have demonstrated the potential acquired defenses (Rosenberg et al., 2007; King et al., 2016; importance of multi-species bacterial communities in resistance Rosenberg and Zilber-Rosenberg, 2016). Therefore it is possible to Bd (Loudon et al., 2014; Piovia-Scott et al., 2017). Co-cultured that at least some of the recolonized frogs in the Wet Tropics synthetic multi-species communities were more effective against uplands have developed resistance to Bd through acquisition or Bd than single