Most of the Dominant Members of Amphibian Skin Bacterial Communities Can Be Readily Cultured

ABSTRACTCurrently, it is estimated that only 0.001% to 15% of bacteria in any given system can be cultured by use of commonly used techniques and media, yet culturing is critically important for investigations of bacterial function. Despite this situation, few studies have attempted to link culture-dependent and culture-independent data for a single system to better understand which members of the microbial community are readily cultured. In amphibians, some cutaneous bacterial symbionts can inhibit establishment and growth of the fungal pathogenBatrachochytrium dendrobatidis, and thus there is great interest in using these symbionts as probiotics for the conservation of amphibians threatened byB. dendrobatidis. The present study examined the portion of the culture-independent bacterial community (based on Illumina amplicon sequencing of the 16S rRNA gene) that was cultured with R2A low-nutrient agar and whether the cultured bacteria represented rare or dominant members of the community in the following four amphibian species: bullfrogs (Lithobates catesbeianus), eastern newts (Notophthalmus viridescens), spring peepers (Pseudacris crucifer), and American toads (Anaxyrus americanus). To determine which percentage of the community was cultured, we clustered Illumina sequences at 97% similarity, using the culture sequences as a reference database. For each amphibian species, we cultured, on average, 0.59% to 1.12% of each individual's bacterial community. However, the average percentage of bacteria that were culturable for each amphibian species was higher, with averages ranging from 2.81% to 7.47%. Furthermore, most of the dominant operational taxonomic units (OTUs), families, and phyla were represented in our cultures. These results open up new research avenues for understanding the functional roles of these dominant bacteria in host health.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00186-17 ◽

2017 ◽

Vol 83 (9) ◽

Cited By ~ 12

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.

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Bacteria from the Genus Arcobacter Are Abundant in Effluent from Wastewater Treatment Plants

Applied and Environmental Microbiology ◽

10.1128/aem.03044-19 ◽

2020 ◽

Vol 86 (9) ◽

Cited By ~ 5

Author(s):

Jannie Munk Kristensen ◽

Marta Nierychlo ◽

Mads Albertsen ◽

Per Halkjær Nielsen

Keyword(s):

Wastewater Treatment ◽

Pathogenic Bacteria ◽

Municipal Wastewater ◽

Wastewater Treatment Plants ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Content Type ◽

Effluent Quality ◽

Culture Independent ◽

Influent Wastewater

ABSTRACT Pathogenic bacteria in wastewater are generally considered to be efficiently removed in biological wastewater treatment plants. This understanding is almost solely based on culture-based control measures, and here we show, by applying culture-independent methods, that the removal of species in the genus Arcobacter was less effective than for many other abundant genera in the influent wastewater. Arcobacter was one of the most abundant genera in influent wastewater at 14 municipal wastewater treatment plants and was also abundant in the “clean” effluent from all the plants, reaching up to 30% of all bacteria as analyzed by 16S rRNA gene amplicon sequencing. Metagenomic analyses, culturing, genome sequencing of Arcobacter isolates, and visualization by fluorescent in situ hybridization (FISH) confirmed the presence of the human-pathogenic Arcobacter cryaerophilus and A. butzleri in both influent and effluent. The main reason for the high relative abundance in the effluent was probably that Arcobacter cells, compared to those of other abundant genera in the influent, did not flocculate and attach well to the activated sludge flocs, leaving a relatively large fraction dispersed in the water phase. The study shows there is an urgent need for new standardized culture-independent measurements of pathogens in effluent wastewaters, e.g., amplicon sequencing, and an investigation of the problem on a global scale to quantify the risk for humans and livestock. IMPORTANCE The genus Arcobacter was unexpectedly abundant in the effluent from 14 Danish wastewater treatment plants treating municipal wastewater, and the species included the human-pathogenic A. cryaerophilus and A. butzleri. Recent studies have shown that Arcobacter is common in wastewater worldwide, so the study indicates that discharge of members of the genus Arcobacter may be a global problem, and further studies are needed to quantify the risk and potentially minimize the discharge. The study also shows that culture-based analyses are insufficient for proper effluent quality control, and new standardized culture-independent measurements of effluent quality encompassing most pathogens should be considered.

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The effect of captivity on the skin microbial symbionts in three Atelopus species from the lowlands of Colombia and Ecuador

PeerJ ◽

10.7717/peerj.3594 ◽

2017 ◽

Vol 5 ◽

pp. e3594 ◽

Cited By ~ 5

Author(s):

Sandra V. Flechas ◽

Ailin Blasco-Zúñiga ◽

Andrés Merino-Viteri ◽

Valeria Ramírez-Castañeda ◽

Miryan Rivera ◽

...

Keyword(s):

Bacterial Community ◽

Batrachochytrium Dendrobatidis ◽

Ex Situ ◽

Rrna Gene ◽

Natural Habitats ◽

Amphibian Species ◽

Skin Bacteria ◽

In Captivity ◽

In The Wild ◽

Microbial Symbionts

Many amphibian species are at risk of extinction in their natural habitats due to the presence of the fungal pathogen Batrachochytrium dendrobatidis (Bd). For the most highly endangered species, captive assurance colonies have been established as an emergency measure to avoid extinction. Experimental research has suggested that symbiotic microorganisms in the skin of amphibians play a key role against Bd. While previous studies have addressed the effects of captivity on the cutaneous bacterial community, it remains poorly studied whether and how captive conditions affect the proportion of beneficial bacteria or their anti-Bd performance on amphibian hosts. In this study we sampled three amphibian species of the highly threatened genus, Atelopus, that remain in the wild but are also part of ex situ breeding programs in Colombia and Ecuador. Our goals were to (1) estimate the diversity of culturable bacterial assemblages in these three species of Atelopus, (2) describe the effect of captivity on the composition of skin microbiota, and (3) examine how captivity affects the bacterial ability to inhibit Bd growth. Using challenge assays we tested each bacterial isolate against Bd, and through sequencing of the 16S rRNA gene, we identified species from thirteen genera of bacteria that inhibited Bd growth. Surprisingly, we did not detect a reduction in skin bacteria diversity in captive frogs. Moreover, we found that frogs in captivity still harbor bacteria with anti-Bd activity. Although the scope of our study is limited to a few species and to the culturable portion of the bacterial community, our results indicate that captive programs do not necessarily change bacterial communities of the toad skins in a way that impedes the control of Bd in case of an eventual reintroduction.

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Culture-Independent and Culture-Dependent Characterization of the Black Soldier Fly Gut Microbiome Reveals a Large Proportion of Culturable Bacteria with Potential for Industrial Applications

Microorganisms ◽

10.3390/microorganisms9081642 ◽

2021 ◽

Vol 9 (8) ◽

pp. 1642

Author(s):

Dorothee Tegtmeier ◽

Sabine Hurka ◽

Sanja Mihajlovic ◽

Maren Bodenschatz ◽

Stephanie Schlimbach ◽

...

Keyword(s):

Gut Microbiome ◽

Amplicon Sequencing ◽

Industrial Applications ◽

Culture Collection ◽

Rrna Gene ◽

Organic Substrates ◽

Culturable Bacteria ◽

Black Soldier Fly ◽

Culture Independent ◽

Culture Dependent

Black soldier fly larvae (BSFL) are fast-growing, resilient insects that can break down a variety of organic substrates and convert them into valuable proteins and lipids for applications in the feed industry. Decomposition is mediated by an abundant and versatile gut microbiome, which has been studied for more than a decade. However, little is known about the phylogeny, properties and functions of bacterial isolates from the BSFL gut. We therefore characterized the BSFL gut microbiome in detail, evaluating bacterial diversity by culture-dependent methods and amplicon sequencing of the 16S rRNA gene. Redundant strains were identified by genomic fingerprinting and 105 non-redundant isolates were then tested for their ability to inhibit pathogens. We cultivated representatives of 26 genera, covering 47% of the families and 33% of the genera detected by amplicon sequencing. Among these isolates, we found several representatives of the most abundant genera: Morganella, Enterococcus, Proteus and Providencia. We also isolated diverse members of the less-abundant phylum Actinobacteria, and a novel genus of the order Clostridiales. We found that 15 of the isolates inhibited at least one of the tested pathogens, suggesting a role in helping to prevent colonization by pathogens in the gut. The resulting culture collection of unique BSFL gut bacteria provides a promising resource for multiple industrial applications.

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The bacterial community of the lone star tick (Amblyomma americanum)

Parasites & Vectors ◽

10.1186/s13071-020-04550-z ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

L. Paulina Maldonado-Ruiz ◽

Saraswoti Neupane ◽

Yoonseong Park ◽

Ludek Zurek

Keyword(s):

Bacterial Community ◽

Amblyomma Americanum ◽

Rrna Gene ◽

Culturable Bacteria ◽

Core Microbiome ◽

Lone Star Tick ◽

Animal Pathogens ◽

Wide Range ◽

Culture Independent ◽

Culture Dependent

Abstract Background The lone star tick (Amblyomma americanum), an important vector of a wide range of human and animal pathogens, is very common throughout the East and Midwest of the USA. Ticks are known to carry non-pathogenic bacteria that may play a role in their vector competence for pathogens. Several previous studies using the high throughput sequencing (HTS) technologies reported the commensal bacteria in a tick midgut as abundant and diverse. In contrast, in our preliminary survey of the field collected adult lone star ticks, we found the number of culturable/viable bacteria very low. Methods We aimed to analyze the bacterial community of A. americanum by a parallel culture-dependent and a culture-independent approach applied to individual ticks. Results We analyzed 94 adult females collected in eastern Kansas and found that 60.8% of ticks had no culturable bacteria and the remaining ticks carried only 67.7 ± 42.8 colony-forming units (CFUs)/tick representing 26 genera. HTS of the 16S rRNA gene resulted in a total of 32 operational taxonomic units (OTUs) with the dominant endosymbiotic genera Coxiella and Rickettsia (> 95%). Remaining OTUs with very low abundance were typical soil bacterial taxa indicating their environmental origin. Conclusions No correlation was found between the CFU abundance and the relative abundance from the culture-independent approach. This suggests that many culturable taxa detected by HTS but not by culture-dependent method were not viable or were not in their culturable state. Overall, our HTS results show that the midgut bacterial community of A. americanum is very poor without a core microbiome and the majority of bacteria are endosymbiotic.

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Establishement of a national monitoring program based on environmental DNA for amphibians and the chytrid fungus Batrachochytrium dendrobatidis

ARPHA Conference Abstracts ◽

10.3897/aca.4.e65362 ◽

2021 ◽

Vol 4 ◽

Author(s):

Omneya Osman ◽

Johan Andersson ◽

Tomas Larsson ◽

Mats Töpel ◽

Alexander Eiler

Keyword(s):

Batrachochytrium Dendrobatidis ◽

National Level ◽

Monitoring Program ◽

12S Rrna ◽

Rrna Gene ◽

Chytrid Fungus ◽

Rana Arvalis ◽

Amphibian Species ◽

National Monitoring ◽

Wide Range

National monitoring programs provide the basis for evaluating the integrity of ecosystems, their responses to disturbances, and the success of actions taken to conserve or recover biodiversity. In this study, we successfully established a national program for the invasive chytrid fungus Batrachochytrium dendrobatidis (Bd) based on dual TaqMan assays. Amphibian diversity based on metabarcoding of the mitochondrial 12S rRNA gene was also performed. Assays were optimized for sensitive detection of target species from a wide range of amphibian ponds with variable potential of inhibitions for eDNA based detection. An amphibian mock community of 5 species was used to validate the metabarcoding approach while internal standards were used in the case of the dual TaqMan assays. First sampling of over 170 ponds in Norway resulted in Bd detection in 12 environmental samples and one swab sample taken over multiple years indicating the establishment of Bd in Norway. Five amphibian species Bufo bufo, Lissotriton vulgaris, Triturus cristatus, Rana arvalis and Rana temporaria as predicted from data in long-term citizen science reporting systems were widely detected in the collected eDNA samples. Our large scale-monitoring program indicates a low risk of a Bd outbreak and amphibian decline caused by chytridiomycosis but continued monitoring is recommended in the future. These findings indicate that eDNA is an effective method to detect invasive species, and to monitor endangered amphibian species. Still, several shortcomings (such as PCR inhibitors and sample volume) were identified that need to be addressed to improve eDNA-based monitoring at the national level.

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Ecological Succession of Bacterial Communities during Conventionalization of Germ-Free Mice

Applied and Environmental Microbiology ◽

10.1128/aem.05239-11 ◽

2012 ◽

Vol 78 (7) ◽

pp. 2359-2366 ◽

Cited By ~ 39

Author(s):

Merritt G. Gillilland ◽

John R. Erb-Downward ◽

Christine M. Bassis ◽

Michael C. Shen ◽

Galen B. Toews ◽

...

Keyword(s):

Bacterial Community ◽

16S Rrna ◽

Bacterial Communities ◽

Structural Heterogeneity ◽

Ecological Succession ◽

Rrna Gene ◽

Gi Tract ◽

Content Type ◽

Germ Free ◽

Over Time

ABSTRACTLittle is known about the dynamics of early ecological succession during experimental conventionalization of the gastrointestinal (GI) tract; thus, we measured changes in bacterial communities over time, at two different mucosal sites (cecum and jejunum), with germfree C57BL/6 mice as the recipients of cecal contents (input community) from a C57BL/6 donor mouse. Bacterial communities were monitored using pyrosequencing of 16S rRNA gene amplicon libraries from the cecum and jejunum and analyzed by a variety of ecological metrics. Bacterial communities, at day 1 postconventionalization, in the cecum and jejunum had lower diversity and were distinct from the input community (dominated by eitherEscherichiaorBacteroides). However, by days 7 and 21, the recipient communities had become significantly diverse and the cecal communities resembled those of the donor and donor littermates, confirming that transfer of cecal contents results in reassembly of the community in the cecum 7 to 21 days later. However, bacterial communities in the recipient jejunum displayed significant structural heterogeneity compared to each other or the donor inoculum or the donor littermates, suggesting that the bacterial community of the jejunum is more dynamic during the first 21 days of conventionalization. This report demonstrates that (i) mature input communities do not simply reassemble at mucosal sites during conventionalization (they first transform into a “pioneering” community and over time take on the appearance, in membership and structure, of the original input community) and (ii) the specific mucosal environment plays a role in shaping the community.

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Rice SST Variation Shapes the Rhizosphere Bacterial Community, Conferring Tolerance to Salt Stress through Regulating Soil Metabolites

mSystems ◽

10.1128/msystems.00721-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Tengxiang Lian ◽

Yingyong Huang ◽

Xianan Xie ◽

Xing Huo ◽

Muhammad Qasim Shahid ◽

...

Keyword(s):

Salt Stress ◽

Bacterial Community ◽

Management Practices ◽

High Throughput Sequencing ◽

Crop Productivity ◽

Soil Salinization ◽

Rrna Gene ◽

Content Type ◽

Rice Mutant ◽

Rhizosphere Bacterial Community

ABSTRACT Some plant-specific resistance genes could affect rhizosphere microorganisms by regulating the release of root exudates. In a previous study, the SST (seedling salt tolerant) gene in rice (Oryza sativa) was identified, and loss of SST function resulted in better plant adaptation to salt stress. However, whether the rice SST variation could alleviate salt stress via regulating soil metabolites and microbiota in the rhizosphere is still unknown. Here, we used transgenic plants with SST edited in the Huanghuazhan (HHZ) and Zhonghua 11 (ZH11) cultivars by the CRISPR/Cas9 system and found that loss of SST function increased the accumulation of potassium and reduced the accumulation of sodium ions in rice plants. Using 16S rRNA gene amplicon high-throughput sequencing, we found that the mutant material shifted the rhizobacterial assembly under salt-free stress. Importantly, under salt stress, the sst, HHZcas, and ZH11cas plants significantly changed the assembly of the rhizobacteria. Furthermore, the rice SST gene also affected the soil metabolites, which were closely related to the dynamics of rhizosphere microbial communities, and we further determined the relationship between the rhizosphere microbiota and soil metabolites. Overall, our results show the effects of the rice SST gene on the response to salt stress associated with the soil microbiota and metabolites in the rhizosphere. This study reveals a helpful linkage among the rice SST gene, soil metabolites, and rhizobacterial community assembly and also provides a theoretical basis for improving crop adaptation through soil microbial management practices. IMPORTANCE Soil salinization is one of the major environmental stresses limiting crop productivity. Crops in agricultural ecosystems have developed various strategies to adapt to salt stress. We used rice mutant and CRISPR-edited lines to investigate the relationships among the Squamosa promoter Binding Protein box (SBP box) family gene (SST/OsSPL10), soil metabolites, and the rhizosphere bacterial community. We found that during salt stress, there are significant differences in the rhizosphere bacterial community and soil metabolites between the plants with the SST gene and those without it. Our findings provide a useful paradigm for revealing the roles of key genes of plants in shaping rhizosphere microbiomes and their relationships with soil metabolites and offer new insights into strategies to enhance rice tolerance to high salt levels from microbial and ecological perspectives.

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Phylogenetic investigation of skin sloughing rates in frogs: relationships with skin characteristics and disease-driven declines

Proceedings of The Royal Society B Biological Sciences ◽

10.1098/rspb.2018.2378 ◽

2019 ◽

Vol 286 (1896) ◽

pp. 20182378 ◽

Cited By ~ 4

Author(s):

Michel E. B. Ohmer ◽

Rebecca L. Cramp ◽

Craig R. White ◽

Peter S. Harlow ◽

Michael S. McFadden ◽

...

Keyword(s):

Batrachochytrium Dendrobatidis ◽

Phylogenetic Signal ◽

Interspecific Variation ◽

Phylogenetic Position ◽

Turnover Rates ◽

Amphibian Skin ◽

Amphibian Species ◽

Wide Range ◽

Skin Morphology ◽

And Function

Amphibian skin is highly variable in structure and function across anurans, and plays an important role in physiological homeostasis and immune defence. For example, skin sloughing has been shown to reduce pathogen loads on the skin, such as the lethal fungus Batrachochytrium dendrobatidis ( Bd ), but interspecific variation in sloughing frequency is largely unknown. Using phylogenetic linear mixed models, we assessed the relationship between skin turnover rate, skin morphology, ecological traits and overall evidence of Bd -driven declines. We examined skin sloughing rates in 21 frog species from three continents, as well as structural skin characteristics measured from preserved specimens. We found that sloughing rate varies significantly with phylogenetic group, but was not associated with evidence of Bd -driven declines, or other skin characteristics examined. This is the first comparison of sloughing rate across a wide range of amphibian species, and creates the first database of amphibian sloughing behaviour. Given the strong phylogenetic signal observed in sloughing rate, approximate sloughing rates of related species may be predicted based on phylogenetic position. While not related to available evidence of declines, understanding variation in sloughing rate may help explain differences in the severity of infection in genera with relatively slow skin turnover rates (e.g. Atelopus ).

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Age, gut location and diet impact the gut microbiome of a tropical herbivorous surgeonfish

FEMS Microbiology Ecology ◽

10.1093/femsec/fiz179 ◽

2019 ◽

Author(s):

Lara Parata ◽

Shaun Nielsen ◽

Xing Xing ◽

Torsten Thomas ◽

Suhelen Egan ◽

...

Keyword(s):

Coral Reef ◽

Gastrointestinal Tract ◽

Bacterial Community ◽

Gut Microbiota ◽

Bacterial Communities ◽

Food Source ◽

Amplicon Sequencing ◽

Rrna Gene ◽

Bacterial Symbionts ◽

Adult Fish

Abstract Herbivorous fishes play important ecological roles in coral reefs by consuming algae that can otherwise outcompete corals, but we know little about the gut microbiota that facilitates this process. This study focussed on the gut microbiota of an ecologically important coral reef fish, the convict surgeonfish Acanthurus triostegus. We sought to understand how the microbiome of this species varies along its gastrointestinal tract and how it varies between juvenile and adult fish. Further, we examined if the bacteria associated with the diet consumed by juveniles contributes to the gut microbiota. 16S rRNA gene amplicon sequencing showed that bacterial communities associated with the midgut and hindgut regions were distinct between adults and juveniles, however, no significant differences were seen for gut wall samples. The microbiota associated with the epilithic algal food source was similar to that of the juvenile midgut and gut wall but differed from the microbiome of the hindgut. A core bacterial community including members of taxa Epulopiscium and Brevinemataceae was observed across all gastrointestinal and diet samples, suggesting that these bacterial symbionts can be acquired by juvenile convict surgeonfish horizontally via their diet and then are retained into adulthood.

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