Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica

Microbiology ◽  
2021 ◽  
Author(s):  
Juan G. Abarca ◽  
Steven M. Whitfield ◽  
Ibrahim Zuniga-Chaves ◽  
Gilbert Alvarado ◽  
Jacob Kerby ◽  
...  
Keyword(s):  
Costa Rica ◽  
Inhibitory Activity ◽  
Batrachochytrium Dendrobatidis ◽  
Mitigation Strategies ◽  
Chytrid Fungus ◽  
Fungal Isolation ◽  
Content Type ◽  
Bacterial Inhibition ◽  
Link Type ◽  
Skin Bacteria

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized – genetically and morphologically – several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas , Streptomyces , Enterobacter , Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

scholarly journals Metabolites Involved in Immune Evasion byBatrachochytrium dendrobatidisInclude the Polyamine Spermidine

2019 ◽  
Vol 87 (5) ◽  
Author(s):  
Louise A. Rollins-Smith ◽  
Antonio C. Ruzzini ◽  
J. Scott Fites ◽  
Laura K. Reinert ◽  
Emily M. Hall ◽  
...  
Keyword(s):  
Small Molecules ◽  
Immune Evasion ◽  
Lymphocyte Proliferation ◽  
Batrachochytrium Dendrobatidis ◽  
Alternative Pathway ◽  
Chytrid Fungus ◽  
Isolation And Identification ◽  
Major Pathway ◽  
Content Type ◽  
The World

ABSTRACTAmphibians have been declining around the world for more than four decades. One recognized driver of these declines is the chytrid fungusBatrachochytrium dendrobatidis, which causes the disease chytridiomycosis. Amphibians have complex and varied immune defenses againstB. dendrobatidis, but the fungus also has a number of counterdefenses. Previously, we identified two small molecules produced by the fungus that inhibit frog lymphocyte proliferation, methylthioadenosine (MTA) and kynurenine (KYN). Here, we report on the isolation and identification of the polyamine spermidine (SPD) as another significant immunomodulatory molecule produced byB. dendrobatidis. SPD and its precursor, putrescine (PUT), are the major polyamines detected, and SPD is required for growth. The major pathway of biosynthesis is from ornithine through putrescine to spermidine. An alternative pathway from arginine to agmatine to putrescine appears to be absent. SPD is inhibitory at concentrations of ≥10 μM and is found at concentrations between 1 and 10 μM in active fungal supernatants. Although PUT is detected in the fungal supernatants, it is not inhibitory to lymphocytes even at concentrations as high as 100 μM. Two other related polyamines, norspermidine (NSP) and spermine (SPM), also inhibit amphibian lymphocyte proliferation, but a third polyamine, cadaverine (CAD), does not. A suboptimal (noninhibitory) concentration of MTA (10 μM), a by-product of spermidine synthesis, enhances the inhibition of SPD at 1 and 10 μM. We interpret these results to suggest thatB. dendrobatidisproduces an “armamentarium” of small molecules that, alone or in concert, may help it to evade clearance by the amphibian immune system.

scholarly journals Antifungal Bacteria on Woodland Salamander Skin Exhibit High Taxonomic Diversity and Geographic Variability

2017 ◽  
Vol 83 (9) ◽  
Author(s):  
Carly R. Muletz-Wolz ◽  
Graziella V. DiRenzo ◽  
Stephanie A. Yarwood ◽  
Evan H. Campbell Grant ◽  
Robert C. Fleischer ◽  
...  
Keyword(s):  
Fungal Pathogen ◽  
Batrachochytrium Dendrobatidis ◽  
Bacterial Species ◽  
Rrna Gene ◽  
Amphibian Skin ◽  
Amphibian Species ◽  
Protective Function ◽  
Content Type ◽  
Skin Bacteria ◽  
Salamander Species

ABSTRACT Diverse bacteria inhabit amphibian skin; some of those bacteria inhibit growth of the fungal pathogen Batrachochytrium dendrobatidis. Yet there has been no systematic survey of anti-B. dendrobatidis bacteria across localities, species, and elevations. This is important given geographic and taxonomic variations in amphibian susceptibility to B. dendrobatidis. Our collection sites were at locations within the Appalachian Mountains where previous sampling had indicated low B. dendrobatidis prevalence. We determined the numbers and identities of anti-B. dendrobatidis bacteria on 61 Plethodon salamanders (37 P. cinereus, 15 P. glutinosus, 9 P. cylindraceus) via culturing methods and 16S rRNA gene sequencing. We sampled co-occurring species at three localities and sampled P. cinereus along an elevational gradient (700 to 1,000 meters above sea level [masl]) at one locality. We identified 50 anti-B. dendrobatidis bacterial operational taxonomic units (OTUs) and found that the degree of B. dendrobatidis inhibition was not correlated with relatedness. Five anti-B. dendrobatidis bacterial strains occurred on multiple amphibian species at multiple localities, but none were shared among all species and localities. The prevalence of anti-B. dendrobatidis bacteria was higher at Shenandoah National Park (NP), VA, with 96% (25/26) of salamanders hosting at least one anti-B. dendrobatidis bacterial species compared to 50% (7/14) at Catoctin Mountain Park (MP), MD, and 38% (8/21) at Mt. Rogers National Recreation Area (NRA), VA. At the individual level, salamanders at Shenandoah NP had more anti-B. dendrobatidis bacteria per individual (μ = 3.3) than those at Catoctin MP (μ = 0.8) and at Mt. Rogers NRA (μ = 0.4). All salamanders tested negative for B. dendrobatidis. Anti-B. dendrobatidis bacterial species are diverse in central Appalachian Plethodon salamanders, and their distribution varied geographically. The antifungal bacterial species that we identified may play a protective role for these salamanders. IMPORTANCE Amphibians harbor skin bacteria that can kill an amphibian fungal pathogen, Batrachochytrium dendrobatidis. Some amphibians die from B. dendrobatidis infection, whereas others do not. The bacteria that can kill B. dendrobatidis, called anti-B. dendrobatidis bacteria, are thought to influence the B. dendrobatidis infection outcome for the amphibian. Yet how anti-B. dendrobatidis bacterial species vary among amphibian species and populations is unknown. We determined the distribution of anti-B. dendrobatidis bacterial species among three salamander species (n = 61) sampled at three localities. We identified 50 unique anti-B. dendrobatidis bacterial species and found that all of the tested salamanders were negative for B. dendrobatidis. Five anti-B. dendrobatidis bacterial species were commonly detected, suggesting a stable, functional association with these salamanders. The number of anti-B. dendrobatidis bacteria per individual varied among localities but not among co-occurring salamander species, demonstrating that environment is more influential than host factors in structuring the anti-B. dendrobatidis bacterial community. These anti-B. dendrobatidis bacteria may serve a protective function for their salamander hosts.

scholarly journals Inhibition of Local Immune Responses by the Frog-Killing Fungus Batrachochytrium dendrobatidis

2014 ◽  
Vol 82 (11) ◽  
pp. 4698-4706 ◽  
Author(s):  
J. Scott Fites ◽  
Laura K. Reinert ◽  
Timothy M. Chappell ◽  
Louise A. Rollins-Smith
Keyword(s):  
Immune Responses ◽  
Batrachochytrium Dendrobatidis ◽  
Single Injection ◽  
Delayed Type Hypersensitivity ◽  
Chytrid Fungus ◽  
Content Type ◽  
Adaptive Immune ◽  
Secondary Injection

ABSTRACTAmphibians are suffering unprecedented global declines. A leading cause is the infectious disease chytridiomycosis caused by the chytrid fungusBatrachochytrium dendrobatidis. Chytridiomycosis is a skin disease which disrupts transport of essential ions leading to death. Soluble factors produced byB. dendrobatidisimpair amphibian and mammalian lymphocytesin vitro, but previous studies have not shown the effects of these inhibitory factorsin vivo. To demonstratein vivoinhibition of immunity byB. dendrobatidis, a modified delayed-type-hypersensitivity (DTH) protocol was developed to induce innate and adaptive inflammatory swelling in the feet ofXenopus laevisby injection of killed bacteria or phytohemagglutinin (PHA). Compared to previous protocols for PHA injection in amphibians, this method induced up to 20-fold greater inflammatory swelling. Using this new protocol, we measured DTH responses induced by killed bacteria or PHA in the presence ofB. dendrobatidissupernatants. Swelling induced by single injection of PHA or killed bacteria was not significantly affected byB. dendrobatidissupernatants. However, swelling caused by a secondary injection of PHA, was significantly reduced byB. dendrobatidissupernatants. As previously describedin vitro, factors fromB. dendrobatidisappear to inhibit lymphocyte-mediated inflammatory swelling but not swelling caused by an inducer of innate leukocytes. This suggests thatB. dendrobatidisis capable of inhibiting lymphocytes in a localized response to prevent adaptive immune responses in the skin. The modified protocol used to induce inflammatory swelling in the present study may be more effective than previous methods to investigate amphibian immune competence, particularly in nonmodel species.

scholarly journals Single-Phase PacBio De Novo Assembly of the Genome of the Chytrid Fungus Batrachochytrium dendrobatidis, a Pathogen of Amphibia

2018 ◽  
Vol 7 (21) ◽  
Author(s):  
Nicholas Sumpter ◽  
Margi Butler ◽  
Russell Poulter
Keyword(s):  
New Zealand ◽  
Genome Assembly ◽  
De Novo Assembly ◽  
Single Phase ◽  
Batrachochytrium Dendrobatidis ◽  
De Novo ◽  
Chytrid Fungus ◽  
Content Type

Here, we present an updated genome assembly of the diploid chytrid fungus Batrachochytrium dendrobatidis strain RTP6. This strain is part of the global panzootic lineage (BdGPL) and was isolated in Dunedin, New Zealand.

scholarly journals The Bacterially Produced Metabolite Violacein Is Associated with Survival of Amphibians Infected with a Lethal Fungus

2009 ◽  
Vol 75 (21) ◽  
pp. 6635-6638 ◽  
Author(s):  
Matthew H. Becker ◽  
Robert M. Brucker ◽  
Christian R. Schwantes ◽  
Reid N. Harris ◽  
Kevin P. C. Minbiole
Keyword(s):  
Batrachochytrium Dendrobatidis ◽  
Plethodon Cinereus ◽  
Chytrid Fungus ◽  
Experimental Exposure ◽  
Amphibian Species ◽  
Mortality And Morbidity ◽  
Antifungal Metabolite ◽  
Microbial Species ◽  
Skin Bacteria ◽  
Janthinobacterium Lividum

ABSTRACT The disease chytridiomycosis, which is caused by the chytrid fungus Batrachochytrium dendrobatidis, is associated with recent declines in amphibian populations. Susceptibility to this disease varies among amphibian populations and species, and resistance appears to be attributable in part to the presence of antifungal microbial species associated with the skin of amphibians. The betaproteobacterium Janthinobacterium lividum has been isolated from the skins of several amphibian species and produces the antifungal metabolite violacein, which inhibits B. dendrobatidis. In this study, we added J. lividum to red-backed salamanders (Plethodon cinereus) to obtain an increased range of violacein concentrations on the skin. Adding J. lividum to the skin of the salamander increased the concentration of violacein on the skin, which was strongly associated with survival after experimental exposure to B. dendrobatidis. As expected from previous work, some individuals that did not receive J. lividum and were exposed to B. dendrobatidis survived. These individuals had concentrations of bacterially produced violacein on their skins that were predicted to kill B. dendrobatidis. Our study suggests that a threshold violacein concentration of about 18 μM on a salamander's skin prevents mortality and morbidity caused by B. dendrobatidis. In addition, we show that over one-half of individuals in nature support antifungal bacteria that produce violacein, which suggests that there is a mutualism between violacein-producing bacteria and P. cinereus and that adding J. lividum is effective for protecting individuals that lack violacein-producing skin bacteria.

scholarly journals Phyllobacterium loti sp. nov. isolated from nodules of Lotus corniculatus

2014 ◽  
Vol 64 (Pt_3) ◽  
pp. 781-786 ◽  
Author(s):  
Maximo Sánchez ◽  
Martha-Helena Ramírez-Bahena ◽  
Alvaro Peix ◽  
María J. Lorite ◽  
Juan Sanjuán ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Sequence Similarity ◽  
Carbon Sources ◽  
Lotus Corniculatus ◽  
Culture Conditions ◽  
Rrna Gene ◽  
Content Type ◽  
Link Type ◽  
The 16S Rrna Gene

Strain S658T was isolated from a Lotus corniculatus nodule in a soil sample obtained in Uruguay. Phylogenetic analysis of the 16S rRNA gene and atpD gene showed that this strain clustered within the genus Phyllobacterium . The closest related species was, in both cases, Phyllobacterium trifolii PETP02T with 99.8 % sequence similarity in the 16S rRNA gene and 96.1 % in the atpD gene. The 16S rRNA gene contains an insert at the beginning of the sequence that has no similarities with other inserts present in the same gene in described rhizobial species. Ubiquinone Q-10 was the only quinone detected. Strain S658T differed from its closest relatives through its growth in diverse culture conditions and in the assimilation of several carbon sources. It was not able to reproduce nodules in Lotus corniculatus. The results of DNA–DNA hybridization, phenotypic tests and fatty acid analyses confirmed that this strain should be classified as a representative of a novel species of the genus Phyllobacterium , for which the name Phyllobacterium loti sp. nov. is proposed. The type strain is S658T( = LMG 27289T = CECT 8230T).

scholarly journals Faecalicoccus acidiformans gen. nov., sp. nov., isolated from the chicken caecum, and reclassification of Streptococcus pleomorphus (Barnes et al. 1977), Eubacterium biforme (Eggerth 1935) and Eubacterium cylindroides (Cato et al. 1974) as Faecalicoccus pleomorphus comb. nov., Holdemanella biformis gen. nov., comb. nov. and Faecalitalea cylindroides gen. nov., comb. nov., respectively, within the family Erysipelotrichaceae

2014 ◽  
Vol 64 (Pt_11) ◽  
pp. 3877-3884 ◽  
Author(s):  
Celine De Maesschalck ◽  
Filip Van Immerseel ◽  
Venessa Eeckhaut ◽  
Siegrid De Baere ◽  
Margo Cnockaert ◽  
...  
Keyword(s):  
16S Rrna ◽  
16S Rrna Gene ◽  
Type Species ◽  
Gene Sequence ◽  
Sequence Similarity ◽  
16S Rrna Gene Sequence ◽  
Rrna Gene ◽  
Rrna Gene Sequence ◽  
Content Type ◽  
Link Type

Strains LMG 27428T and LMG 27427 were isolated from the caecal content of a chicken and produced butyric, lactic and formic acids as major metabolic end products. The genomic DNA G+C contents of strains LMG 27428T and LMG 27427 were 40.4 and 38.8 mol%. On the basis of 16S rRNA gene sequence similarity, both strains were most closely related to the generically misclassified Streptococcus pleomorphus ATCC 29734T. Strain LMG 27428T could be distinguished from S. pleomorphus ATCC 29734T based on production of more lactic acid and less formic acid in M2GSC medium, a higher DNA G+C content and the absence of activities of acid phosphatase and leucine, arginine, leucyl glycine, pyroglutamic acid, glycine and histidine arylamidases, while strain LMG 27428 was biochemically indistinguishable from S. pleomorphus ATCC 29734T. The novel genus Faecalicoccus gen. nov. within the family Erysipelotrichaceae is proposed to accommodate strains LMG 27428T and LMG 27427. Strain LMG 27428T ( = DSM 26963T) is the type strain of Faecalicoccus acidiformans sp. nov., and strain LMG 27427 ( = DSM 26962) is a strain of Faecalicoccus pleomorphus comb. nov. (type strain LMG 17756T = ATCC 29734T = DSM 20574T). Furthermore, the nearest phylogenetic neighbours of the genus Faecalicoccus are the generically misclassified Eubacterium cylindroides DSM 3983T (94.4 % 16S rRNA gene sequence similarity to strain LMG 27428T) and Eubacterium biforme DSM 3989T (92.7 % 16S rRNA gene sequence similarity to strain LMG 27428T). We present genotypic and phenotypic data that allow the differentiation of each of these taxa and propose to reclassify these generically misnamed species of the genus Eubacterium formally as Faecalitalea cylindroides gen. nov., comb. nov. and Holdemanella biformis gen. nov., comb. nov., respectively. The type strain of Faecalitalea cylindroides is DSM 3983T = ATCC 27803T = JCM 10261T and that of Holdemanella biformis is DSM 3989T = ATCC 27806T = CCUG 28091T.

scholarly journals EspM Is a Conserved Transcription Factor That Regulates Gene Expression in Response to the ESX-1 System

mBio ◽  
2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Kevin G. Sanchez ◽  
Micah J. Ferrell ◽  
Alexandra E. Chirakos ◽  
Kathleen R. Nicholson ◽  
Robert B. Abramovitch ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Protein Transport ◽  
Transcriptional Response ◽  
Secretion System ◽  
Pathogenic Species ◽  
Macrophage Infection ◽  
Content Type ◽  
Link Type ◽  
Phagosomal Membrane

ABSTRACT Pathogenic mycobacteria encounter multiple environments during macrophage infection. Temporally, the bacteria are engulfed into the phagosome, lyse the phagosomal membrane, and interact with the cytosol before spreading to another cell. Virulence factors secreted by the mycobacterial ESX-1 (ESAT-6-system-1) secretion system mediate the essential transition from the phagosome to the cytosol. It was recently discovered that the ESX-1 system also regulates mycobacterial gene expression in Mycobacterium marinum (R. E. Bosserman, T. T. Nguyen, K. G. Sanchez, A. E. Chirakos, et al., Proc Natl Acad Sci U S A 114:E10772–E10781, 2017, https://doi.org/10.1073/pnas.1710167114), a nontuberculous mycobacterial pathogen, and in the human-pathogenic species M. tuberculosis (A. M. Abdallah, E. M. Weerdenburg, Q. Guan, R. Ummels, et al., PLoS One 14:e0211003, 2019, https://doi.org/10.1371/journal.pone.0211003). It is not known how the ESX-1 system regulates gene expression. Here, we identify the first transcription factor required for the ESX-1-dependent transcriptional response in pathogenic mycobacteria. We demonstrate that the gene divergently transcribed from the whiB6 gene and adjacent to the ESX-1 locus in mycobacterial pathogens encodes a conserved transcription factor (MMAR_5438, Rv3863, now espM). We prove that EspM from both M. marinum and M. tuberculosis directly and specifically binds the whiB6-espM intergenic region. We show that EspM is required for ESX-1-dependent repression of whiB6 expression and for the regulation of ESX-1-associated gene expression. Finally, we demonstrate that EspM functions to fine-tune ESX-1 activity in M. marinum. Taking the data together, this report extends the esx-1 locus, defines a conserved regulator of the ESX-1 virulence pathway, and begins to elucidate how the ESX-1 system regulates gene expression. IMPORTANCE Mycobacterial pathogens use the ESX-1 system to transport protein substrates that mediate essential interactions with the host during infection. We previously demonstrated that in addition to transporting proteins, the ESX-1 secretion system regulates gene expression. Here, we identify a conserved transcription factor that regulates gene expression in response to the ESX-1 system. We demonstrate that this transcription factor is functionally conserved in M. marinum, a pathogen of ectothermic animals; M. tuberculosis, the human-pathogenic species that causes tuberculosis; and M. smegmatis, a nonpathogenic mycobacterial species. These findings provide the first mechanistic insight into how the ESX-1 system elicits a transcriptional response, a function of this protein transport system that was previously unknown.

scholarly journals Amphritea ceti sp. nov., isolated from faeces of Beluga whale (Delphinapterus leucas)

2014 ◽  
Vol 64 (Pt_12) ◽  
pp. 4068-4072 ◽  
Author(s):  
Young-Ok Kim ◽  
Sooyeon Park ◽  
Doo Nam Kim ◽  
Bo-Hye Nam ◽  
Sung-Min Won ◽  
...  
Keyword(s):  
Phylogenetic Trees ◽  
Sequence Similarity ◽  
Beluga Whale ◽  
Delphinapterus Leucas ◽  
Rrna Gene ◽  
Phenotypic Properties ◽  
Content Type ◽  
Link Type ◽  
Beluga Whale Delphinapterus Leucas ◽  
Type Strains

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped or ovoid bacterial strain, designated RA1T, was isolated from faeces collected from Beluga whale (Delphinapterus leucas) in Yeosu aquarium, South Korea. Strain RA1T grew optimally at 25 °C, at pH 7.0–8.0 and in the presence of 2.0 % (w/v) NaCl. Neighbour-joining, maximum-likelihood and maximum-parsimony phylogenetic trees based on 16S rRNA gene sequences revealed that strain RA1T joins the cluster comprising the type strains of three species of the genus Amphritea , with which it exhibited 95.8–96.0 % sequence similarity. Sequence similarities to the type strains of other recognized species were less than 94.3 %. Strain RA1T contained Q-8 as the predominant ubiquinone and summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C18 : 1ω7c and C16 : 0 as the major fatty acids. The major polar lipids of strain RA1T were phosphatidylethanolamine, phosphatidylglycerol, two unidentified lipids and one unidentified aminolipid. The DNA G+C content of strain RA1T was 47.4 mol%. The differential phenotypic properties, together with the phylogenetic distinctiveness, revealed that strain RA1T is separated from other species of the genus Amphritea . On the basis of the data presented, strain RA1T is considered to represent a novel species of the genus Amphritea , for which the name Amphritea ceti sp. nov. is proposed. The type strain is RA1T ( = KCTC 42154T = NBRC 110551T).

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