ORIGINAL RESEARCH
published: 28 February 2017
doi: 10.3389/fmicb.2017.00290
Characterization of
Batrachochytrium dendrobatidis
Inhibiting Bacteria from Amphibian
Populations in Costa Rica
Joseph D. Madison 1*, Elizabeth A. Berg 1, Juan G. Abarca 2, Steven M. Whitfield 3,
Oxana Gorbatenko 4, Adrian Pinto 2, 5 and Jacob L. Kerby 1
1 Department of Biology, University of South Dakota, Vermillion, SD, USA, 2 Centro de Investigación en Estructuras
Microscópicas, Universidad de Costa Rica, San Pedro de Montes de Oca, Costa Rica, 3 Conservation and Research
Department, Zoo Miami, Miami, FL, USA, 4 Life Science Laboratory, Westcore DNA Sequencing Facility, Black Hills State
University, Spearfish, SD, USA, 5 Departamento de Bioquímica, Escuela de Medicina, Centro de Investigación en Biología
Celular y Molecular, Universidad de Costa Rica, San Pedro de Montes de Oca, Costa Rica
Global amphibian declines and extinction events are occurring at an unprecedented rate.
While several factors are responsible for declines and extinction, the fungal pathogen
Batrachochytrium dendrobatidis (Bd) has been cited as a major constituent in these
events. While the effects of this chytrid fungus have been shown to cause broad
scale population declines and extinctions, certain individuals and relict populations have
Edited by:
Reid Harris, shown resistance. This resistance has been attributed in part to the cutaneous bacterial
James Madison University, USA microbiome. Here, we present the first study characterizing anti-Bd bacterial isolates
Reviewed by: from amphibian populations in Costa Rica, including the characterization of two strains
Amy R. Ellison, of Serratia marcescens presenting strong anti-Bd activity. Transcriptome sequencing was
Cardiff University
Jenifer Banning Walke, utilized for delineation of shifts in gene expression of the two previously uncharacterized
Virginia Tech, USA strains of S. marcescens grown in three different treatments comprising Bd, heat-killed
*Correspondence: Bd, and a no Bd control. These results revealed up- and down-regulation of key genes
Joseph D. Madison
joseph.madison@coyotes.usd.edu associated with different metabolic and regulatory pathways. This information will be
valuable in continued efforts to develop a bacterial-based approach for amphibian
Specialty section: protection as well as providing direction for continued mechanistic inquiries of the
This article was submitted to
bacterial anti-Bd response.
Microbial Symbioses,
a section of the journal
Keywords: amphibian, microbiome, Serratia marcescens, RNA-sequencing, Batrachochytrium dendrobatidis
Frontiers in Microbiology
Received: 01 December 2016
Accepted: 13 February 2017 INTRODUCTION
Published: 28 February 2017
Citation: The role of the bacterial microbiome in conferring disease resistance has been the subject of
Madison JD, Berg EA, Abarca JG, intensive study in a number of disease systems. This area has critical importance in elucidating
Whitfield SM, Gorbatenko O, Pinto A the mechanisms of disease dynamics in the context of important host relationships with bacteria.
and Kerby JL (2017) Characterization Inquiries into such systems is an important frontier in many taxa undergoing biodiversity loss.
of Batrachochytrium dendrobatidis
Examples include diseases such as white-nose syndrome caused by Pseudogymnoascus destructans
Inhibiting Bacteria from Amphibian
Populations in Costa Rica. in bats (Hoyt et al., 2015) and chytridiomycosis caused by Batrachochytrium dendrobatidis
Front. Microbiol. 8:290. (hereafter Bd) and Batrachochytrium salamandrivorans (hereafter Bsal) in amphibians (Longcore
doi: 10.3389/fmicb.2017.00290 et al., 1999; Martel et al., 2013). There are also several anthropocentric reasons to decipher these
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Madison et al. Bacteria from Costa Rican Anurans
relationships. Indeed, the role of the bacterial microbiome been suggested in recent communications (Woodhams et al.,
in agriculturally important plant diseases (Berendsen et al., 2016).
2012) and medical applications in human disease (Schwabe and To explore the mechanisms by which individual bacteria may
Jobin, 2013; Kostic et al., 2014) necessitates the need for basic be deterring Bd growth and thus allowing amphibian persistence
research in this field of study. Specifically, the underlying genetic in a disease outbreak, our group has sampled various relict,
mechanisms that bacteria utilize in mitigating disease risk is an recovering, and unaffected amphibian populations in Costa Rica.
understudied area in disease dynamics. While previous work has Bacterial species were isolated in pure culture and assayed for Bd
examined bacterial gene expression in response to disease in inhibition. Of those bacteria that were determined to be strong
humans (Duran-Pinedo et al., 2014), this is generally lacking in anti-Bd candidates, two strains of S. marcescens were selected
many of the other important disease systems of animals where for further characterization. Previous studies have examined
conservation priorities are a concern. other Serratia spp. that have known anti-Bd activity in vitro
This study seeks to characterize amphibian cutaneous bacteria (Woodhams et al., 2014; Becker et al., 2015). It was hypothesized
and their role in ameliorating amphibian population declines. that there would be significant gene up- and down-regulation in
These declines have been attributed to a variety of factors S. marcescens gene expression as a response to Bd which could
including habitat loss, global climate change, disease, and highlight transcriptomic shifts associated with specific bacterial
environmental contaminants (Collins and Storfer, 2003). In response mechanisms. Also examined was the expression of
conjunction with other factors, disease has been directly genes that are involved in canonical S. marcescens antifungal
attributed to extinction of various amphibian species (Crawford pathways. Known antifungal pathways include the production of
et al., 2010). Specifically, the Ranavirus of the Iridoviridae and the broadly antifungal enzymes such as extracellular chitinases and
fungal pathogens Bd and the now emerging Bsal are of concern glucanases, as well as the production of secondary metabolites
vis-à-vis the health of at-risk amphibian populations (Martel such as prodigiosin (Duzhak et al., 2012; Gutiérrez-Román et al.,
et al., 2013). 2015; Tan et al., 2015). Elucidations from this data will allow for
In the wake of devastation that often follows these epizootic more in-depth studies to occur on the mechanisms by which S.
events, there are sometimes small relict populations that seem marcescens inhibits Bd growth. Such mechanistic determinations
to have an innate resistance to avoid disease (Chaves-Cordero could be utilized in work developing probiotic bioaugmentation
et al., 2014). This survival has been attributed to a variety tools for use on both captive and wild amphibian populations.
of factors including the cutaneous bacterial microbiome and
host-produced skin peptides (Woodhams et al., 2007a,b,c). An MATERIALS AND METHODS
adaptive immune response has also been investigated with
results that vary by amphibian species and stage of development Bacterial Isolation
(Rollins-Smith, 1998; Ramsey et al., 2010; Poorten et al., 2015). Field sampling for cutaneous bacteria was conducted between
While all of these aspects are important, the possibility of March and November 2012 by searching for amphibians in relict
developing probiotic based protection through exploitation and populations at sites where they had been previously reported
manipulation of the amphibian cutaneous bacterial microbiome (Puschendorf-Fahrenkrug et al., 2005; Hoffmann, 2006; Abarca
has gained recognition as a major research objective. It has et al., 2010; Chaves-Cordero et al., 2014). Bacterial isolates were
been shown that while whole bacterial community composition obtained by swabbing 191 frogs belonging to 12 amphibian
is important (Rebollar et al., 2016a), there are often specific species in neotropical montane regions of Costa Rica (Figure 1;
bacterial species with strong anti-Bd properties (Harris et al., species collected given in Supplementary Table 4). Frogs were
2009). While previous work has examined the transcriptomic captured with plastic bags and handled using fresh disposable
response of Anurans to Bd (Rosenblum et al., 2012a; Ellison nitrile gloves. The capture of transient surface bacteria was
et al., 2014; Price et al., 2015) and also the transcriptome reduced by washing the entire body of individuals for seven
of Bd (Rosenblum et al., 2008, 2012b), there have been no seconds with sterile distilled water. Individuals were swabbed
studies delineating the transcriptomic response of bacteria to over the entire body using a sterile cotton swab. First, swabs were
Bd. However, there have been studies examining the bacterial streaked onto the surface of Reasoner’s 2A (R2A) agar in a petri
metabolites produced in known anti-Bd bacteria (Brucker dish to obtain bacteria; subsequently the washing was continued
et al., 2008a,b; Belden et al., 2014; Loudon et al., 2014). One with sterile distilled water, and a second swab from the frog
important bacteria that has been the subject of various papers was streaked onto chitin agar media for obtaining actinomycetes.
is the anti-Bd bacteria Janthinobacterium lividum (Brucker Plates were cultured at 25–28◦C and colony formation was
et al., 2008a). This bacteria produces a secondary metabolite, observed daily. In the case of R2A media, the formation of
violacein, which has important similarities to the secondary bacterial colonies was observed at 2 or 3 days of isolation,
metabolite prodigiosin produced in another known anti-Bd and one to 2 weeks for actinomycetes. The different colonies
bacteria, Serratia marcescens. J lividum is also known to produce were then purified on a new medium: Luria-Bertani (LB) for
extracellular chitinases similar to those of S. marcescens (Gleave bacteria from R2A media and yeast malt extract agar (YMEA)
et al., 1995). These similarities could present an important for actinomycetes. Each bacterial isolate was cryopreserved with
pathway for general anti-Bd inhbition. These studies also liquid LB media with 40% glycerol and YMEA liquid media with
provide an important overall context for interpreting results 20% glycerol, respectively. All work with amphibians was carried
presented herein. The need for work addressing these issues has out in accordance with the recommendations of the Institutional
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Madison et al. Bacteria from Costa Rican Anurans
kept at room temperature in the dark between absorbance value
recordings. Ninety bacterial isolates were examined using this
method. The mean daily absorbance values of all replicate and
control wells were calculated daily using a spectrophotometer.
A generalized linear model (GLM) was used to compare each
isolate with replicated controls. Increasing absorbance indicates
zoospore reproduction and growth, while decreasing absorbance
indicates zoospore death.
Agar Plate Challenge Assay
For the agar plate challenge assay, plates with TGhL agar were
inoculated with a lawn of Bd (strain JEL 731, isolated from
Craugastor bransfordii at La Selva Biological Station, Costa Rica).
This lawn was then streaked with one of the bacterial isolates
with each isolate being examined in triplicate. Each bacteria was
incubated for 48–72 h at 25◦C and then flooded with 3 mL of
Bd-inoculated broth (3.15 × 106 zoospores per mL) and left for
1–2 h until agar had absorbed most of the Bd-inoculated broth.
Each plate had two bacterial strains, with pairs randomized across
four replicates. Pictures were taken of the bacteria every 24 h for
FIGURE 1 | Map showing amphibian collection sites in Costa Rica 3 days. ImageJ software was used to standardize measurements.
where amphibians were swabbed for bacterial isolates. Species Zone of inhibition (ZOI) was measured by calculating the area
collected at each site are given. of each zone [Area = (ZOI length–bacteria length) × (ZOI
width–bacteria width)]. A GLM was used to compare each
isolate to a theoretical control where the ZOI = 0. All of the
Commission on Biodiversity at the University of Costa Rica. isolates but one exhibited a ZOI, but only 38 of the 86 had
Amphibian sampling resulting in the bacteria used in this study a significant p-value (p < 0.05). After 2 days of growth at
was done in conjunction with Adrian Pinto at the University of 20◦C, the resulting zone of inhibition was quantified. Of those
Costa Rica, with research permits approved by the Institutional bacteria that were determined to be strong anti-Bd candidates
Commission on Biodiversity at the University of Costa Rica and identified using 16S rDNA sequencing, two strains of S.
(Resolutions 014). marcescens were selected for further characterization. The two
Cell-Free Supernatant Challenge Assay strains had 91% 16S rDNA partial sequence homology (BLAST
Needleman-Wunsch alignment). Both strains of S. marcescens
A cell-free supernatant assay was initially used to examine anti-
used in this study were isolated from a single captured and
Bd potential of bacterial isolate extracts. This assay followed
released specimen of Agalychnis annae from a successfully
closely to the protocol outlined by Bell et al. (2013). Briefly,
translocated population established from a relict population in
bacterial isolates were first inoculated into 2 mL of sterile
Costa Rica.
tryptone gelatin hydrolysate lactose (TGhL) medium and
incubated at 25◦C for 48 h. They were then centrifuged at
5,796 g for 10min to pellet cells. The supernatant was transferred 16S rDNA Sequencing
to 1.5 mL filter microtubes and centrifuged again at 5,796 g DNA was extracted from 36 bacterial isolates with the greatest
for 10min until only peptides remained suspended in the Bd-inhibitory properties. DNA extraction utilized a Qiagen
broth. Experimental and control wells were divided on 96-well DNeasy Blood and Tissue kit (Qiagen-Hilden, Germany). DNA
microplates. The experimental wells contained 50 µL of Bd- preparation for sequencing was done according to manufacturer
inoculated TGhL broth (3.15 × 106 zoospores per mL), 45µL directions. Briefly, extracted DNA was amplified using the
TGhL medium, and 5µL of the bacterial isolate. Three controls eubacterial primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′)
were used. The positive control wells contained 50µL of Bd, and 1492r (5′-ACCTTGTTACGACTT-3′). This amplified
45µL TGhL medium, and 5µL deionized water. The negative product was then sequenced with the original primers as well as
control wells contained 50µL of heat killed Bd (raised to 60◦C the internal primers 907r (5′-CCGTCAATTCMTTTRAGTTT-
for 30–60 min), 45µL of TGhL medium, and 5 µL deionized 3′) and 704f (5′-GTAGCGGTGAAATGCGTAGA-3′). The
water. The final wells were blanks and contained 95µL TGhL BigDye Terminator v3.1 cycle sequencing kit was utilized in
medium and 5µL deionized water. Complete plates contained sequencing. Sequencing was carried out using an Applied
four replicates of each bacterial isolate and eight replicates of Biosystems 3500 Genetic Analyzer (Life Technologies-
the positive, negative, and medium-only controls. Absorbance Carlsbad, California, USA). The output sequencing data
values were recorded after plate setup was completed (day 0) was assembled with Geneious (version 7.0) and subjected
and then every 24 h on a spectrophotometer (BioTek Take 3) to taxonomic identification using BLAST in the NCBI
at 492 nm until maximum Bd growth (day 14). Plates were database.
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Madison et al. Bacteria from Costa Rican Anurans
Bacterial and Fungal Growth Analysis
All fungal growth was done in lactose TGhL broth as
per previous studies (Bell et al., 2013). Growth curves
of S. marcescens were also acquired under the varying
treatments (control, Bd, heat-killed Bd). These growth
curves were acquired using serial dilutions followed
by plating and counting of colony forming units to
avoid the non-distinguishing reading of zoospores from
bacterial cells by the spectrophotometer which would
have resulted in skewed bacterial cell counts between
treatments.
RNA-Sequencing
Bd strain JEL731 was used in this experiment. S. marcescens
(described above) were isolated from the cutaneous layer of A.
annae. Both S. marcescens strains were subsequently shipped
from Costa Rica to the University of South Dakota and
grown in culture on LB agar. TGhL broth was inoculated
with Bd, mixed, and spiked with S. marcescens according to
our experimental conditions. Each experimental condition was
done in triplicate with bacterial populations of both strains FIGURE 2 | Growth curve of bacteria in all treatments obtained using
(at 20◦C). Experimental conditions included a no-Bd zoospore serial dilution plating. RNA was extracted at T = 12 h (indicated by vertical
control, heat-killed Bd zoospore control (killed by exposure to line). Bd1 and Bd2 indicate S. marcescens strains one and two grown with
20 min at 50◦C; Johnson et al., 2003), and live-Bd zoospores. Bd, respectively. Heat-killed Bd1 and heat-killed Bd2 indicates S. marcescens
strains one and two grown with heat killed Bd, respectively. No Bd1 and no
The concentration of Bd zoospores used for both heat-killed
Bd2 indicates S. marcescens strains one and two grown without any Bd,
and live-Bd zoospore experimental conditions were 25 104× respectively.
zoospores/mL (measured with a hemocytometer). Cultures were
incubated at 20◦C with RNA being extracted in bacterial
exponential growth phase at 12 h (Figure 2). RNA extraction qPCR Verification
was done by initially stabilizing bacterial RNA with RNAprotect A subset of differentially expressed genes (DEGs) identified
bacteria reagent (Qiagen-Hilden, Germany). Enzymatic lysis was through RNA-sequencing were verified using RT-qPCR. RNA
subsequently used using an EDTA (ethylenediaminetetraacetic was from the same samples utilized in the RNA sequencing
acid)/lysozyme solution buffered with Tris. RNA purification and were reverse transcribed to cDNA using the Quantitect
was then carried out using an RNeasy kit (Qiagen-Hilden, Reverse Transcription Kit (Qiagen-Hilden, Germany). Real-time
Germany) following standard protocols. Purified RNA was PCR was done on the StepOnePlus Real-Time qPCR-system
then transported from the University of South Dakota to the (Applied Biosystems-Foster City, California) using SYBR Green
WestCore DNA core facility for sequencing preparation and chemistry as provided by the SYBR Green PCR Kit (Qiagen-
sequencing. Subsequent rRNA depletion of total-RNA for both Hilden, Germany). All qPCR was carried out with no-RT and
bacteria and fungi was accomplished using a modified protocol no-template negative controls. The narG (nitrate reductase)
for a Ribo-Zero rRNA Removal Kit (Illumina, San Diego, CA). gene and fadE (acyl-conezyme A dehydrogenase) gene were
The modified protocol included probes from both the yeast examined due to their significant upregulation observed in RNA-
and bacterial rRNA Removal Kits which were combined in a seq analysis. Other genes examined included chiA (chitinase A)
1:1 ratio to the recommended concentration for a one probe and pigM (key-regulatory enzyme in the prodigiosin production
kit. Clean-up of the rRNA reduced RNA was done using pathway) to confirm lack of differential expression as seen in
a RNA-Clean and Concentrator kit (Zymo Research-Irvine, RNA-seq analysis. The reference genes dnaE and rplU were
CA) following manufacturer instructions. RNA quality (RIN utilized for statistical comparison. Every gene analyzed had
score) was obtained on a LabChip GX (Caliper, a PerkinElmer expression determined under every experimental condition (Bd
company, Hopkin, MA) using the PicoRNA assay. RNA quantity treatment, HK-Bd treatment, and no-Bd control). All primer
was measured on a Qubit 2.0 Fluorometer (Thermo Fisher sequences used were either from the literature or designed in
Scientific, Waltham, MA, USA). Subsequent library preparation NCBI using the Primer BLAST tool followed by verification in
for sequencing utilized the Illumina ScriptSeq RNA-Seq Library our laboratory (primer sequences, Supplementary Table 1).
Kit. Library quantification was done with a LabChip GX using the
DNA High Sensitivity assay and Qubit 2.0 Fluorometer. Indices Statistical Analyses
were added for a six sample multiplex on a one lane flowcell. De-multiplexing of raw data was done on Illumina BaseSpace
The Illumina MiSeq platform was used for the sequencing run as part of the BaseSpace MiSeq Reporter workflow. Analysis
using the version 3 reagents kit to obtain 2 × 76 bp reads of raw RNA-seq data was done using the Rockhopper 2
(paired-end). software platform (McClure et al., 2013; Tjaden, 2015). Raw
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Madison et al. Bacteria from Costa Rican Anurans
g-zipped fastq files were uploaded directly to the Rockhopper
platform with paired-end reads being combined. Assembly
and mapping of the raw data was done using the default
settings. Reads were mapped to the S. marcescens WW4
reference genome (NCBI Reference Sequence NC_020211.1).
Significant differential gene expression analysis was also done
using Rockhopper 2. Briefly, assembled/mapped files were
subjected to upper-quartile normalization. Expression data was
then subjected to the Rockhopper algorithm assuming a negative
binomial distribution for estimation of p-values. P-values were
then used to obtain q-values in which false discovery rate is taken
into account using the Benjamini-Hochberg procedure. Analysis
of qPCR data for differential gene expression was completed
using the11Ct method (Livak and Schmittgen, 2001).
RESULTS
Hosts and Isolates
Amphibian species A. annae, Agalychnis lemur, C. bransfordii,
and Oophaga pumilio had the greatest number of anti-Bd
bacterial isolates (Supplementary Table 3). A. lemur hosted two
bacterial species that were shown to enhance Bd growth. None of
the tested isolates from species Craugastor ranoides, Craugastor
taurus, or Hyalinobatrachium colymbiphyllum exhibited anti-Bd
properties. However, we note that not all of the strains isolated
were tested, and bacterial strains are known to exist from H.
colymbiphyllum that are not culturable or have culturing bias FIGURE 3 | Box plot estimates of the cell-free supernatant challenge
(Walke et al., 2011). A. annae exhibited the highest diversity of assay for each of the bacteria strains. The box represents the interquartile
range (IQR) and the top and bottom whiskers represent Q3—(1.5 IQR) and
anti-Bd bacteria, with 7 different genera represented in anti-Bd
Q1—(1.5 IQR), respectively. Bacteria IDs on the x-axis are referenced in
isolates. Supplementary Table 3. Lower values signify greater inhibition of Bd growth.
Identified anti-Bd bacteria were in the phyla Proteobacteria, B0 corresponds to a Bd-only control.
Bacteroidetes, Actinobacteria, and Firmicutes. Proteobacteria
was the most common phyla being found on 5 of the 12
amphibian species. The following genera were also identified: that exhibited anti-Bd properties (p < 0.05) in the cell-free
Alcaligenes, Bacillus, Chyrseobacterium, Enterobacteriaceae, supernatant assay but not in the agar-based assay.
Lysinibacillus,Microbacterium, Pseudomonas, Sphingobacterium,
Staphylococcus, and Stenotrophomonas. The Serratia genus Transcriptomic Analysis
was the most common identified within the selected anti-Bd All extracted RNA had RNA integrity numbers (RINs) >9.0
bacteria isolates; S. marcescens was the most common species indicating that all extracted RNA samples were within tolerance
(Supplementary Table 5) and was found on three of the for use in sequencing. Raw sequencing data of two S. marcescens
amphibian species. strains from A. annae were referenced against the S. marcescens
WW4 genome with >80% mapping (Table 1). Additionally, 47–
Bacterial Challenge Assays 74% of reads were mapped to protein coding genes (Table 1).
Of the 90 bacterial isolates examined in the cell-free supernatant All DEGs identified in each treatment were compared to a no-
challenge assay, 10 (11.1%) exhibited Bd inhibition where p < Bd control. In the live Bd treatment this experiment found 111
0.05 and 2 (2.2%) enhanced Bd growth (Figure 3; Supplementary differentially expressed genes (DEGs) in S. marcescens strain one
Table 2). Of the 86 isolates examined in the agar-based (Supplementary Table 6) and 100 DEGs in S. marcescens strain
challenge assay (Figure 4; Supplementary Table 3), 38 (44.2%) two (Supplementary Table 7). For the heat-killed Bd treatment,
of isolates were characterized as anti-Bd (p < 0.05). All but S. marcescens strain one had 96 DEGs (Supplementary Table
one isolate produced a zone-of-inhibition during the experiment 8) and S. marcescens strain two had 73 DEGs (Supplementary
(Supplementary Table 3). Table 9). Pooling results from both strains of S. marcescens used
We also compared the 68 isolates which were examined in this study, only three significant DEGs were identified in
in both assays; overall 30 exhibited anti-Bd properties (44.1%; the Bd treatment while five significant DEGs were identified in
Supplementary Table 4). Of these 30 isolates, 28 isolates (93.3%) the heat-killed Bd treatment. We were able to identify the top
were shown to inhibit Bd using the agar-based method but not 15 up- and down-regulated genes in each treatment in each
the cell-free supernatant method, and four isolates (13.3%) were strain as well as the significant DEGs that were shared with
shown to inhibit Bd using both methods. There were 2 isolates both strains pooled (Figure 5). Genes that were significantly
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Madison et al. Bacteria from Costa Rican Anurans
TABLE 1 | Sequencing results including total reads for each multiplexed
sample, reads mapped to reference genome, and mapped genes aligning
to protein coding genes.
*Sequenced Total Mapped Aligning to protein
sample reads reads coding genes
SMWW4 Control (1) 3239079 2664279 (82%) 1491996 (56%)
SMWW4 Control (2) 3814486 3258485 (85%) 2378694 (73%)
SMWW4 Bd (1) 3938104 3201797 (81%) 2145204 (67%)
SMWW4 Bd (2) 3912551 3348231 (86%) 2477691 (74%)
SMWW4 HKBd (1) 4097349 3361055 (82%) 1579696 (47%)
SMWW4 HKBd (2) 3139545 2623075 (84%) 1914845 (73%)
*Both strains were mapped to the S.marcescens strain WW4 (SMWW4) with correlating
experimental growth condition (Control, B. dendrobatidis present (Bd), heat-killed B.
dendrobatidis present (HKBd) and biological replicate number.
were isolated from amphibian species in recovering and
relict populations from a major epizootic event. These strains
represent important data for understanding the role the bacterial
microbiome has in conferring Bd resistance. Understanding
which bacterial species are anti-Bd and the underlying gene
expression is meaningful for proposing mechanisms of anti-
Bd action and subsequent verification studies using gene
knockout and complementation. Such work will be invaluable
to understanding disease resistance and the role of the bacterial
microbiome. Additionally, this work is critical for the future
FIGURE 4 | Box plot estimates of the agar based challenge assay for development of novel in situ microbiome engineering tools for
each of the bacteria strains. The box represents the interquartile range conservation.
(IQR) and the top and bottom whiskers represent Q3—(1.5 IQR) and Q1—(1.5 In general, the bacterial genera that were identified in this
IQR), respectively. Bacteria IDs on the x-axis are referenced in Supplementary study as having anti-Bd activity were similar to those found in
Table 4. Larger values signify greater inhibition of Bd growth.
other studies both in the tropics and in temperate zones (Lam
et al., 2010; Flechas et al., 2012). The bacterial isolates most
common in this study weremembers of the genus Serratia, part of
upregulated in both strains included those associated with the the phylumProteobacteria. Serratiawere the strongest candidates
nitrate reductases and the associated transport machinery (narG, for bioaugmentation efforts identified in this study owing to their
narH, and nirC). Also of interest and shared between both strains rapid growth in vitro, apparent anti-Bd properties, and common
was a putatative oxalate-formate antiporter coding gene (yhjX) distribution across various Costa Rican amphibian species. They
and the acyl-conezyme A dehydrogenase gene (fadE). We also have also been shown to exhibit anti-Bd properties in previous
used a Venn diagram to visualize the differentially expressed work examining amphibian microbiomes (Woodhams et al.,
genes of S. marcescens in strains one and two as a response to 2007b; Antwis et al., 2015). While the S. marcescens used in
live-Bd and heat-killed Bd (Figure 6). this study have excellent potential for bioaugmentation efforts
RNA-sequencing expression results were validated by and mechanistic inquiries, certain strains of S. marcescens are
examination of six genes in the presence of the two experimental known opportunistic human pathogens (Hejazi and Falkiner,
treatments and control (Figure 7). The narG gene showed 1997). Caution should be taken in working with uncharacterized
upregulation in Bd and heat-killed Bd treatments with much wild-type strains of this bacteria including safety education of
stronger upregulation in response to the live Bd treatment. The personnel and BSL-2 standards in all laboratory work.
fadE gene was also significantly upregulated but only in the Bd In understanding S. marcescens and their anti-Bdmechanisms
treatment. Other genes examined included chiA (chitinase A) in more detail as it pertains to the cutaneous microbiome,
and pigM (key-regulatory enzyme in the prodigiosin production we found that the two S. marcescens strains examined in this
pathway) to confirm the lack of differential expression as seen study had various transcriptomic shifts in response to Bd. The
in RNA-sequencing. Reference genes dnaE and rplU showed no responses seen were varied and implicated various metabolic
significant differential expression. and regulatory processes. The genes validated using RT-qPCR
had complete congruency with the RNA-sequencing data. This
DISCUSSION should be seen as a small but important validation of the
significantly expressed genes identified in the RNA-sequencing
This work is the first to highlight gene expression in anti- portion of the study as well as validation of lack of expression in
Bd bacterial strains. More importantly, many of these bacteria genes coding for enzymes known to have antifungal activity.
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Madison et al. Bacteria from Costa Rican Anurans
FIGURE 5 | Gene expression data presented as a heat map for the 15 most up- and down-regulated genes for Bd and heat-killed Bd treatments
relative to a no-Bd control. Corresponding gene expression values are given for those only in the top 15 in one treatment. Corresponding genes that were not
significantly up or down-regulated relative to a no-Bd control but were in the top 15 in a different treatment are denoted by a value of 0. Gene names are given on the
vertical axis. Treatments are given on the bottom horizontal axis. Sm1 and Sm2 denote S. marcescens strain one and two. Bd indicates live B. dendrobatidis and
HKBd indicates heat-killed B. dendrobatidis. 1.2 indicates pooled expression data for both strains. A gene name of unk indicates an unnamed gene. An asterisk (*) by
a gene name indicates an antisense gene.
The upregulation of nitrate reductases and nitrite transporters upregulation of genes encoding the catalytic nitrate reductase
was one of the few gene sets shown to be upregulated in α-subunit and transitory β-subunit which are both part of the
both strains and in both treatments. Specifically, the significant membrane bound reductase (NAR) complex is of interest. This
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Madison et al. Bacteria from Costa Rican Anurans
biosynthesis. Chitinases have been previously shown to target the
β-1,4 n-acetylglucosamine linkages of the fungal chitin polymer
which constitutes a substantial component of the fungal cell
wall (Chet and Inbar, 1994). Also, bacterial glucanases have
been shown to have a similar effect in the targeting of glucan
linkages critical for fungal cell wall structure (Hong and Meng,
2003). The importance of prodigiosin, a secondary metabolite
shown to have antifungal characteristics, was also examined in
the context of the key regulatory enzymes of the prodigiosin
biosynthesis pathway. These enzymes, which are encoded by
the prodigiosin biosysnthesis gene cluster (pig cluster), were
not shown to be upregulated in this study. The downstream
effects of this lack of expression was also confirmed visually as
S. marcescens in culture exhibited only a faint red color (color
consistent with the production of the red pigment prodigiosin)
under the experimental conditions used in this study. This can
most likely be attributed to the known temperature dependence
of prodigiosin production in S. spp. (Williams, 1973; Woodhams
et al., 2014). The temperatures used for co-culturing in this study
FIGURE 6 | Venn diagram depicting shared significantly expressed (20◦C) are not generally known to be the most advantageous
DEGs from each of the treatment conditions (Bd and heat killed-Bd; for prodigiosin production. The lack of upregulation seen in
both vs. no-Bd control) in both S. marcescens isolates examined.
the chitinase (chi) genes as well as those involved in glucanase
production was also of interest. While not being upregulated
directly, there was still a consistent amount of basal expression
data coupled with upregulation of the inner membrane nitrite which may indicate constitutive production of these products
transporter in both strains seems to indicate a response to which is unaffected by the presence of Bd or heat-killed Bd.
anaerobic conditions in which nitrate reduction is utilized for While differential expression is of interest and often informative
energy conservation (Richardson et al., 2001). The reason for the of an organism’s response (e.g., bacteria to Bd) there may
utilization of the membrane-bound nitrate reductase machinery be products that are basally expressed and indifferent to the
other than a response to anaerobic conditions and available presence of a stimulatory organism such as Bd. The only way in
nitrate is unclear but certainly occurring in both Bd and heat- which to empirically evaluate the importance of such products
killed Bd treatments. being produced in an anti-Bd response would be the generation
Also upregulated in both strains under both conditions was of knockouts or knockdowns of the genes of interest in line with
a putative oxalate-formate antiporter coding gene. The need to molecular Koch’s postulates (Falkow, 1988).
upregulate the antiport machinery for oxalate decarboxylation to While not showing differential expression, the observed
formate in response to Bd is at this point not fully understood. expression of the proteins associated with the type six secretion
However, the production of oxalate by other fungi and the system (T6SS) was also of interest. The expression of amajority of
necessary genes in the Bd genome for production of oxalate the 13 core genes includingVgrG (TssI) andHcp (TssD) associated
suggest such production by Bd is theoretically possible and with the T6SS could have implications in direct inhibition of
potentially necessary (Benny, 1995; Bindschedler et al., 2016). Bd by S. marcescens. While the use of such a system has been
This coupled with recent work showing amphibian mortality shown to inhibit competing bacteria in other S. marcescens
due to oxalate nephropathy from an unknown oxalate source strains (Murdoch et al., 2011), the existence of effector proteins
may suggest a specific mechanism by which Bd could kill associated with the T6SS that are antifungal in nature are at this
amphibians (Tokiwa et al., 2015). This possible mechanism point unclear. However, direct competition with other bacteria
deserves further consideration, especially owing to the fact that could allow S. marcescens to propagate to higher levels at the
specific mechanisms for Bd induced mortality are not fully bacterial community level allowing other factors discussed above
understood. Evidence for the epidermal dysfunction hypothesis to play a larger role in the system. This preliminary evidence
presented by Voyles et al. (2009) still has no clear biochemical on the role of a T6SS in S. marcescens and other gram negative
mechanism delineated for the disruption of epidermal channels bacteria in the amphibian-Bd system therefore merits further
involved in electrolyte transport. Whether this response and the examination.
formation of nitrite into the periplasmic space has any direct The upregulation of 2-dehydro, 3-deoxy-phosphogluconate
inhibitory effects on Bd growth is also unclear but worth further aldolase gene, coding for a key regulatory enzyme in the
consideration. Entner-Duodoroff (ED) pathway, was seen in both S. marcescens
Differential expression of the commonly associated strains in response to heat-killed Bd. The reason behind
antimicrobial genes of S. marcescens was not seen. These include this upregulation in response to both strains of heat-
the upregulation of extracellular chitinases and glucanases as killed Bd treatments is unclear but could also merit further
well as the key regulatory enzymes involved in prodigiosin consideration.
Frontiers in Microbiology | www.frontiersin.org 8 February 2017 | Volume 8 | Article 290
Madison et al. Bacteria from Costa Rican Anurans
FIGURE 7 | qPCR verification of a select group of genes identified in RNA-sequencing. Error bars represent 95% confidence intervals. All gene expression
was compared to dnaE and rplU reference genes.
The upregulation of various antisense-RNAs was also biological replicates could have been beneficial, both strains came
intriguing and could represent a point of regulation which is from the same individual and were the only two strains collected
part of a response to Bd. All asRNAs were associated with in suburbanHeredia, Costa Rica. It should also be noted that each
ribosomal RNAs representing possible regulation of translation. sample of assumedly clonal bacterial cells should have variation
Curiously, all differentially expressed asRNAs were only observed following an approximate normal distribution due to the high
in strain one with down-regulation as a response to Bd and population (n > 10,000,000) of bacterial cells in each tube.
upregulation as a response to heat-killed Bd. These results seem Setting up multiple growth flasks would be a better indicator of
to indicate clear differences of a bacterial reaction to Bd and population sensitivity (and resulting expression profile) to small
heat-killed Bd in strain one. The generation of asRNAs are and potentially uncontrollable differences in growth conditions.
known to have significant regulatory effects on gene expression We have included results from both the comparisons between
as well as functional roles (Sesto et al., 2013). Specifically, asRNAs individual bacterial populations as well as comparisons with both
have been shown to induce (i.e., agr locus of Staphylococcus populations pooled. The introduction of other S. marcescens
aureus) as well as attenuate (i.e., asRNA dependent rep-mRNA from other amphibian species and individuals would nullify
conformations for attenuation of replication control) various the comparison of bacteria growing in the same environment
bacterial functions (Brantl, 2002). However, the exact role of such although would still be interesting but ultimately outside the
regulation and function in this system remains unclear. scope of this study.
The two strains of S. marcescens used in this study both had The results from the transcriptomic analysis portion of this
high homology to sequenced strain WW4. However, there are study provide important information of a bacterial species
obvious physiological differences in these strains as indicated with strongly inhibitory Bd properties. The use of meta-
by the few (eight) genes that were differentially expressed in omics approaches as suggested by Rebollar et al. (2016b)
both. The strains used were collected at the same time but could also be utilized in further studies delineating whole
remained in culture for different amounts of time before being bacterial microbiome response to Bd. However, we suggest that
cryoarchived representing a possible source of the observed conclusions delineating species-specific bacterial mechanisms
differences. The unstable genomes of wild-type bacteria and of interest for probiotic development from such methods
potential for lateral gene transfer could account for differences should be done with caution. Many bacteria identified through
in the aforementioned timeframe and highlights the difficulty such techniques have no reference genome or at the least
in working with such bacteria. While the inclusion of more have large repeat regions creating challenging areas for de
Frontiers in Microbiology | www.frontiersin.org 9 February 2017 | Volume 8 | Article 290
Madison et al. Bacteria from Costa Rican Anurans
novo assembly in species-specific determinations (NP-hard). All of these approaches though, have seen limitations and
Interpreting data and trusting available workflows is also lack broad applicability. Therefore, we recommend in situ
problematic and fails to recognize these considerations in such microbiome engineering tools as a novel direction for
analyses. Determination of error in workflows can also be continued amelioration efforts. Specifically, addition of
challenging and would entail complex error propagation (e.g., known antifungal genes and the conferring of protective
Taylor series expansion) strategies that are currently limited mechanisms to specific members of a resident bacterial
in implementation. Any meta-omic interpretations should be population with plasmid or phage-based genetic modifications
reserved for the community-level in the interest of excluding may be possible. This is an area our group is currently exploring
erroneous conclusions. Such data would still be of great benefit with great interest for use in the amphibian-disease system
in continued research initiatives. and beyond. Recent interest in the literature (Sheth et al.,
Several of the amphibian species examined had few to no anti- 2016) seems to address this potential use of the bacterial
Bd bacterial isolates. The number of isolates examined from each microbiome.
amphibian species is small compared to the breadth of the entire The results and insights provided here will serve as a critical
microbial community on the cutaneous layer. Additionally, not foundation for future studies interested in bacterial responses
all isolates from field sampling were used in this study. Microbial to Bd and continued work on bioaugmentation efforts with S.
communities on amphibians are extremely variable between marcescens. Specifically, those genes shown upregulated in both
populations and individuals within the same population across strains of S. marcescens would be of interest for further work
temporal and spatial distributions (Vredenburg et al., 2010). including the construction of bacterial knockouts as well as
Continued sampling and bioassays of the bacteria of these complementary studies in Bd. Lastly, this data will aid in future
relict populations may uncover additional bacteria that exhibit and ongoing work addressing manipulation of the amphibian
anti-Bd properties. Additionally, the number of anti-Bd isolates microbiome as well as other disease systems throughmicrobiome
does not necessarily correlate with the success of an amphibian engineering efforts.
population; few isolates may still provide protection against
chytridiomycosis.
It is also possible that these relict populations have not yet AUTHOR CONTRIBUTIONS
acquired anti-Bd bacteria, but instead persist in the presence
JM was involved in the following: anti-Bd assays, RNA
of Bd because of other immune defenses. While symbiotic
sequencing, RT-qPCR, data analysis, and writing the manuscript.
bacteria are an effective defense against Bd, other innate
EB was involved in the following: anti-Bd assays and related
defenses such as antimicrobial peptides (AMPs) secreted by
data analysis. JA was involved in the following: isolation and
dermal glands are important in conferring anti-Bd properties
initial characterization of the bacterial isolates and writing the
(Rollins-Smith and Conlon, 2005; Conlon, 2011). AMP secretion
manuscript. OG was involved in the following: RNA sequencing
has been shown to be variable between amphibian species
laboratory work. SW was involved in the following: isolation
and families creating many possible dynamics between AMPs
and initial characterization of the bacterial isolates and writing
and the amphibian bacterial microbiome (Conlon, 2011).
the manuscript. AP was involved in the following: isolation
Three of the amphibian species examined in this study
and initial characterization of the bacterial isolates and writing
belong to families known to secrete AMPs with anti-fungal
the manuscript. JK was involved in the following: project
properties and also host isolates identified as anti-Bd by
oversight of all aspects of the study. All authors partook in
this study (Apponyi et al., 2004; Amiche et al., 2008;
commenting/editing of the manuscript and have approved the
Conlon, 2011). However, Incilius holdridgei of the Bufonidae
given manuscript for submission. Additionally, all authors agree
family are known to not secrete any distinguishable AMPs
to be accountable for all aspects of the presented work.
with antifungal properties. Interestingly, this species also
lives in areas with the ideal environmental conditions for
Bd growth (Abarca et al., 2010) thus making it possible FUNDING
that symbiotic bacteria play a strong role in resistance to
chytridiomycosis. In addition to innate immune responses, the This research was done with the support of the United States
adaptive immune system can also provide significant benefit in Fish and Wildlife Service (USFWS Number: 46-6003541) and
anti-Bd activity. The relationship between innate and immune by the Research Center for Cellular and Molecular Biology,
responses may also be important in continuing amelioration University of Costa Rica (CIBCM Project Number: 801-B2-029).
efforts. Sequencing data reported in this publication was supported by
Several control efforts have been suggested to mitigate an Institutional Development Award (IDeA) from the National
amphibian decline due to chytridiomycosis, such as inoculating Institute of General Medicine Sciences of the National Institutes
ponds with sodium chloride or the use of anti-fungal of Health under grant number P20GM103443. Its contents are
drugs (Stockwell et al., 2012). Also proposed is probiotic solely the responsibility of the authors and do not necessarily
bioaugmentation with protective bacteria known to inhibit represent official views of NIGMS or NIH. Support for this
Bd. Bioaugmentation targets amphibian species that are research was also supplemented by the JohnW. Carlson Research
being released into areas where Bd has colonized and where grant through the University of South Dakota College of Arts and
populations are experiencing decline due to chytridiomycosis. Sciences.
Frontiers in Microbiology | www.frontiersin.org 10 February 2017 | Volume 8 | Article 290
Madison et al. Bacteria from Costa Rican Anurans
ACKNOWLEDGMENTS All transcriptome sequencing files are accessible through
the NCBI database with accession number GSE84057 and
All transcriptome sequencing work was performed in WestCore, ID 200084057. Included are raw data files, processed
the SD BRIN DNA Core Facility. WestCore’s research staff data files, and metadata files giving the experimental
contributed valuable technical expertise to this project. The parameters and conditions. All 16S rDNA sequencing
advice and help of Cynthia Anderson was especially appreciated. and metadata files are available through the NCBI
database with accession numbers KX928035-KX928072.
SUPPLEMENTARY MATERIAL All assembled sequences were checked for chimeras
using DECIPHER (Wright et al., 2012). All R scripts
Additional Supplementary Material for this article can be used for the generation of graphs and statistical analyses
found online at: http://journal.frontiersin.org/article/10.3389/ are available through GitHub, (user: kvasir7, repository:
fmicb.2017.00290/full#supplementary-material dissertation_research).
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