White-nose syndrome is an emerging infectious disease that has led to a dramatic decline in cave-... more White-nose syndrome is an emerging infectious disease that has led to a dramatic decline in cave-hibernating bat species. White-nose syndrome is caused by the newly described fungal pathogen Geomyces destructans, which infects the ear, muzzle, and wing membranes of bats. Although the exact mechanism by which the fungus causes death is not yet understood, G. destructans leads to a high mortality rate in infected animals. While the primary mechanism of infection appears to be bat-to-bat transfer, it is still unclear what role human activity may play in the spread of this pathogen. Here we evaluate the effectiveness of decontamination protocols that can be utilized by speleologists to reduce the likelihood of spreading this dangerous pathogen to naïve bats or uninfected hibernacula. Our results show that pre-cleaning to remove muds and/ or sediments followed by the use of commercially available disinfectants can effectively remove G. destructans from caving fabrics. Alternatively, immersion in water above 50 uC for at least 20 minutes effectively destroys the fungal spores. These results have allowed the development of a decontamination protocol (http://www.fws.gov/ WhiteNoseSyndrome/cavers.html) that, when appropriately followed, can greatly reduce the likelihood of the human mediated transfer of G. destructans from an infected to uninfected site.
The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has been responsible for over ... more The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has been responsible for over 650,000 deaths worldwide. Transmission of SARS-CoV-2 occurs primarily through airborne transmission or direct human contact, demonstrating the importance of social distancing measures and the use of face masks to prevent infection. Nonetheless, the persistence of coronavirus on surfaces means that disinfection is important to limit the possibility of contact transmission. In this paper, the potential for various surfaces in show caves to serve as sources for SARS-CoV-2 infection is examined. Given the isoelectric potential (pI) of SARS and SARS-like coronaviruses, it is likely that they are adsorbed via electrochemical interactions to (limestone) rock surfaces, where the high humidity, pH and presence of biocarbonate ions will quickly lead to inactivation. Nonetheless, show caves contain infrastructure made of other non-porous surfaces that are more permissive for maintaining coronavirus...
The study of cave microbiology deals with the microscopic life that resides in caves. Microscopic... more The study of cave microbiology deals with the microscopic life that resides in caves. Microscopic life has historically been grouped into the Monera, one of the five kingdoms of life, and a pretty broad group that includes any microscopic organism that lacks a nucleus; also historically known as the prokaryotes (Whittaker, 1969). The protozoa, which are also microscopic and include such organisms as the amoeba, do contain a nucleus and are classed within the eukaryotes (Whittaker, 1969). While viruses are also of microscopic, they are not technically alive and, although fascinating, will not be discussed within the scope of this review. Animals, plants and fungi all constitute the forms of life that can be seen with the naked eye (macroscopic) and are nucleus-containing eukaryotes.
Barite (BaSO 4) speleothems have been reported from caves around the globe and interpreted to hav... more Barite (BaSO 4) speleothems have been reported from caves around the globe and interpreted to have chiefly formed in phreatic, hypogene, hydrothermal settings. Here we report two contrasting types of barite speleothems (bluish tabular crystals in a shallow pool and actively dripping greenish stalactites), which today form at lower temperatures in the non-hydrothermal and vadose environment of Lechuguilla Cave, New Mexico, USA. Scanning electron microscopy analysis, along with energy-and wavelength-dispersive X-ray spectroscopy (EDS, WDS), as well as X-ray diffraction (XRD), characterize the habit and chemical composition as barite. Fractionation of the minor element calcium is related to growth along different crystal faces whereas variations in strontium concentration are mirrored in blue color zoning of the pool crystals. Two possible modes of non-hydrothermal barite precipitation are discussed: (1) intense evaporation driven by thermal atmospheric convection cells or (2) mixing of bariumrich, sulfate-poor water with water rich in sulfate. Both processes, in isolation or in combination, lead to supersaturation and could explain formation of the investigated barite speleothems. Observations of three types of microbes on the pool barite crystals showing evidence of incrustation raises the question whether there is a potential involvement of microbial activity in the temperate barite precipitation in Lechuguilla Cave.
Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the g... more Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the genomes of five proteobacteria isolated from Lechuguilla Cave in New Mexico, USA. Genome-based phylogeny indicates that each strain belongs to a distinct genus. Two Rhizobiaceae isolates possess the genomic potential for the biosynthesis of acyl-homoserine lactone.
Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly... more Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly associated with soils, animal feeds and water. Some members of the Acinetobacter have been implicated in hospital-acquired infections, with broad-spectrum antibiotic resistance. Here we report the whole genome sequence of LC510, an Acinetobacter species isolated from deep within a pristine location of the Lechuguilla Cave. Pairwise nucleotide comparison to three type strains within Acinetobacter assigned LC510 as an Acinetobacter pittii isolate. Scanning of the LC510 genome identified two genes coding for beta-lactamase resistance, despite the fact that LC510 was isolated from a portion of the cave not previously visited by humans and protected from anthropogenic input. The ability to produce acyl-homoserine lactone (AHL) signal in culture medium, an observation that is consistent with the identification of the luxI and luxR homologs in its genome, suggests that cell-to-cell communicati...
Despite the importance of karst aquifers as a source of drinking water, little is known about the... more Despite the importance of karst aquifers as a source of drinking water, little is known about the role of microorganisms in maintaining the quality of this water. One of the limitations in exploring the microbiology of these environments is access, which is usually limited to wells and surface springs. In this study, we compared the microbiology of the Madison karst aquifer sampled via the potentiometric lakes of Wind Cave with surface sampling wells and a spring. Our data indicated that only the Streeter Well (STR), which is drilled into the same hydrogeologic domain as the Wind Cave Lakes (WCL), allowed access to water with the same low biomass (1.56-9.25 × 10 3 cells mL −1). Filtration of ∼300 L of water from both of these sites through a 0.2 µm filter allowed the collection of sufficient cells for DNA extraction, PCR amplification of 16S rRNA gene sequences, and identification through pyrosequencing. The results indicated that bacteria (with limited archaea and no detectable eukaryotic organisms) dominated both water samples; however, there were significant taxonomic differences in the bacterial populations of the samples. The STR sample was dominated by a single phylotype within the Gammaproteobacteria (Order Acidithiobacillales), which dramatically reduced the overall diversity and species richness of the population. In WCL, despite less organic carbon, the bacterial population was significantly more diverse, including significant contributions from the Gammaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Planctomycetes, Fusobacter, and Omnitrophica phyla. Comparisons with similar oligotrophic environments suggest that karst aquifers have a greater species richness than comparable surface environs. These data also demonstrate that Wind Cave provides a unique opportunity to sample a deep, subterranean aquifer directly, and that the microbiology of such aquifers may be more complex than previously anticipated.
Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing ... more Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothems appealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave "Grotte des Collemboles" via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units (OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with (4 OTUs, 17%) and (9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinked to the culturable strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three stud...
The U.S. Culture Collection Network held a meeting to share information about how culture collect... more The U.S. Culture Collection Network held a meeting to share information about how culture collections are responding to the requirements of the recently enacted Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (CBD). The meeting included representatives of many culture collections and other biological collections, the U.S. Department of State, U.S. Department of Agriculture, Secretariat of the CBD, interested scientific societies, and collection groups, including Scientific Collections International and the Global Genome Biodiversity Network. The participants learned about the policies of the United States and other countries regarding access to genetic resources, the definition of genetic resources, and the status of historical materials and genetic sequence information. Key topics included what constitutes access and how the CBD Access and Benefit-Sharing Clearing-...
Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with ... more Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that p...
A total of eight Ensifer sp. strains were isolated from two pristine cave environments. One strai... more A total of eight Ensifer sp. strains were isolated from two pristine cave environments. One strain was isolated from a cave water pool located in the Wind Cave National Park, South Dakota, USA and the remaining seven strains were isolated from Lechuguilla Cave of Carlsbad Caverns National Park, New Mexico, USA. Whole genome sequencing and comparative genomic analyses of the eight isolates compared to various type strains from the genera Ensifer and Sinorhizobium demonstrates that although members in these genera can be phylogenetically separated into two distinct clades, the percentage of conserved proteins (POCP) between various type strains from Ensifer and Sinorhizobium are consistently higher than 50%, providing strong genomic evidence to support the classification of the genera Ensifer and Sinorhizobium into a single genus.
Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore re... more Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore reservoirs of resistance elements and reflective of the natural history of antibiotics and resistance. In a previous study, we discovered that multi-drug resistance is common in bacteria isolated from Lechuguilla Cave, an underground ecosystem that has been isolated from the surface for over 4 Myr. Here we use whole-genome sequencing, functional genomics and biochemical assays to reveal the intrinsic resistome of Paenibacillus sp. LC231, a cave bacterial isolate that is resistant to most clinically used antibiotics. We systematically link resistance phenotype to genotype and in doing so, identify 18 chromosomal resistance elements, including five determinants without characterized homologues and three mechanisms not previously shown to be involved in antibiotic resistance. A resistome comparison across related surface Paenibacillus affirms the conservation of resistance over millions of years and establishes the longevity of these genes in this genus.
filaments, two regions of potential motor engagement were created on the vesicle surface. Geometr... more filaments, two regions of potential motor engagement were created on the vesicle surface. Geometric constraints limited the engaged motor number at each contact point to no more than 3, as confirmed by laser trap stall force measurements. These points of vesicle-actin filament contact resulted in an effective tug of war between two motor teams that were physically separated on the vesicle surface. The vesicle's directional outcome at an intersection was visualized using high spatial and temporal 3D super resolution imaging. For vesicles that physically contacted both actin filaments at the intersection, 55% of vesicles continued straight through the intersection, 35% switched filaments, while only 10% terminated. These outcomes were successfully simulated by a model in which the directional outcome was dependent on the interfilament gap, the vesicle's azimuthal approach angle to the intersection, the stochastic attachment and detachment of motors within a team (i.e. a team's force generation), and the Brownian motion of the vesicle while attached to the actin filaments. All of these physical factors contributed to which motor ensemble won the tug of war at the intersection and thus the vesicle's directional outcome. This simplified biomimetic system and resultant data provide an experimental framework for understanding how MyoVa motor teams navigate their cargo through evermore complex 3D actin networks that exist within cells.
Helicobacter presence and viability in waters is not well characterized. The identification of na... more Helicobacter presence and viability in waters is not well characterized. The identification of natural reservoirs and infection sources may provide novel insights into its waterborne transmission. The goal of this study was to investigated the occurrence of Helicobacter spp. in natural freshwaters from Roraima Tepui, a little studied and unique ecosystem of the Guayana Shield. Freshwaters collected from two localities at Roraima Tepui were cultured in HP selective broth and agar for Helicobacter pylori and analysed by fluorescent in situ hybridization (FISH), specific PCR assays, 16S rRNA gene sequencing and phylogenetic analysis. The presence of other bacteria in freshwater enrichments was determined using clone library sequencing of the 16S rRNA gene and phylogenetic inferences. Helicobacter spp. were detected by semi-nested PCR and FISH in freshwater enrichments from both sites. Coccoid viable but nonculturable (VBNC) cells were evidenced using 16S rRNA gene Helicobacter species and H. pylorispecific probes. Partial 16S rRNA gene sequences of two HP enrichments showed high similarity to H. pylori and Helicobacter nemestrinae (99-100 %). Other bacteria such as Serratia, Aquitalea, Chromobacterium, Mycobacterium, Acinetobacter, Curvibacter and Dysgonomonas were also detected using complete 16S rRNA gene sequences, with Serratia, Aquitalea and Chromobacterium the most common genera (40.9, 18.2 and 15.2 %, respectively). This is the first time that Helicobacter spp. have been reported in freshwaters of a tepui ecosystem. Our results contribute to the current knowledge of these bacteria in the aquatic environment and expand their known/potential sites outside the human host.
monitoring are possible, a framework for deciding how to prioritize inventory and monitoring acti... more monitoring are possible, a framework for deciding how to prioritize inventory and monitoring activities, and references to specific protocols that are already in place at NPS cave parks.
Despite extremely starved conditions, caves contain surprisingly diverse microbial communities. O... more Despite extremely starved conditions, caves contain surprisingly diverse microbial communities. Our research is geared toward understanding what ecosystems drivers are responsible for this high diversity. To asses the effect of rock fabric and mineralogy, we carried out a comparative geomicrobiology study within Carlsbad Cavern, New Mexico, USA. Samples were collected from two different geologic locations within the cave: WF1 in the Massive Member of the Capitan Formation and sF88 in the calcareous siltstones of the Yates Formation. We examined the organic content at each location using liquid chromatography mass spectroscopy and analyzed microbial community structure using molecular phylogenetic analyses. In order to assess whether microbial activity was leading to changes in the bedrock at each location, the samples were also examined by petrology, X-ray diffraction (XRD) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Our results suggest that on the chemically complex Yates Formation (sF88), the microbial community was significantly more diverse than on the limestone surfaces of the Capitan (WF1), despite a higher total number of cells on the latter. Further, the broader diversity of bacterial species at sF88 reflected a larger range of potential metabolic capabilities, presumably due to opportunities to use ions within the rock as nutrients and for chemolithotrophic energy production. The use of these ions at sF88 is supported by the formation of a corrosion residue, presumably through microbial scavenging activities. Our results suggest that rock fabric and mineralogy may be an important driver of ecosystem function and should be carefully reviewed when carrying out microbial community analysis in cave environments.
A novel actinobacterium, designated MM109 T , was isolated from a moonmilk deposit collected from... more A novel actinobacterium, designated MM109 T , was isolated from a moonmilk deposit collected from the cave 'Grotte des Collemboles' located in Comblain-au-Pont, Belgium. Based on a polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological, and physiological characterization, the isolate has been affiliated to the genus Streptomyces. Multilocus sequence analysis based on the 16S rRNA gene and five other housekeeping genes (atpD, gyrB, rpoB, recA and trpB) showed that the MM109 T isolate is sufficiently distinct from its closest relative, Streptomyces peucetius strain AS 4.1799 T , as to represent a novel species. The phylogenetic distinctiveness of the taxon represented by isolate MM109 T was supported by the isolation and identification of additional twelve moonmilk-derived isolates, which according to multilocus sequence analysis were clustered along with MM109 T. Scanning electron microscopy observations revealed complex and diversified structures within a MM109 T colony, made from branching vegetative mycelia. The spore chains of the MM109 T isolate undergo complete septation at the late stages of the morphological differentiation process, leading to the formation of packs of smooth cylindrical-shaped spores. Isolate MM109 T produces several intracellular and diffusible pigments, particularly an intracellular green-pigmented secondary metabolite, which was identified through UPLC-ESI-MS analysis as ferroverdin A, an iron-chelating molecule formerly extracted and characterized from Streptomyces sp. strain WK-5344. The isolate MM109 T is thus considered to represent a novel species of Streptomyces, for which the name Streptomyces lunaelactis sp. nov. is proposed with the type strain MM109 T (=DSM 42149 T = BCCM/LMG 28326 T).
Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to... more Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366-385 nm) elicits a distinct orange-yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange-yellow wing fluorescence (n580) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n588) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n522) and 100% of nonfluorescent (n540) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of longwave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.
Here, we report the whole-genome sequences and annotation of five oligotrophic bacteria from two ... more Here, we report the whole-genome sequences and annotation of five oligotrophic bacteria from two sites within the Lechuguilla Cave in the Carlsbad Caverns National Park, NM. Three of the five genomes contain an acyl-homoserine lactone signal synthase ortholog (luxI) that is involved in cell-to-cell communication via quorum sensing.
Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. T... more Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.
White-nose syndrome is an emerging infectious disease that has led to a dramatic decline in cave-... more White-nose syndrome is an emerging infectious disease that has led to a dramatic decline in cave-hibernating bat species. White-nose syndrome is caused by the newly described fungal pathogen Geomyces destructans, which infects the ear, muzzle, and wing membranes of bats. Although the exact mechanism by which the fungus causes death is not yet understood, G. destructans leads to a high mortality rate in infected animals. While the primary mechanism of infection appears to be bat-to-bat transfer, it is still unclear what role human activity may play in the spread of this pathogen. Here we evaluate the effectiveness of decontamination protocols that can be utilized by speleologists to reduce the likelihood of spreading this dangerous pathogen to naïve bats or uninfected hibernacula. Our results show that pre-cleaning to remove muds and/ or sediments followed by the use of commercially available disinfectants can effectively remove G. destructans from caving fabrics. Alternatively, immersion in water above 50 uC for at least 20 minutes effectively destroys the fungal spores. These results have allowed the development of a decontamination protocol (http://www.fws.gov/ WhiteNoseSyndrome/cavers.html) that, when appropriately followed, can greatly reduce the likelihood of the human mediated transfer of G. destructans from an infected to uninfected site.
The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has been responsible for over ... more The COVID-19 pandemic, caused by the novel coronavirus SARS-CoV-2, has been responsible for over 650,000 deaths worldwide. Transmission of SARS-CoV-2 occurs primarily through airborne transmission or direct human contact, demonstrating the importance of social distancing measures and the use of face masks to prevent infection. Nonetheless, the persistence of coronavirus on surfaces means that disinfection is important to limit the possibility of contact transmission. In this paper, the potential for various surfaces in show caves to serve as sources for SARS-CoV-2 infection is examined. Given the isoelectric potential (pI) of SARS and SARS-like coronaviruses, it is likely that they are adsorbed via electrochemical interactions to (limestone) rock surfaces, where the high humidity, pH and presence of biocarbonate ions will quickly lead to inactivation. Nonetheless, show caves contain infrastructure made of other non-porous surfaces that are more permissive for maintaining coronavirus...
The study of cave microbiology deals with the microscopic life that resides in caves. Microscopic... more The study of cave microbiology deals with the microscopic life that resides in caves. Microscopic life has historically been grouped into the Monera, one of the five kingdoms of life, and a pretty broad group that includes any microscopic organism that lacks a nucleus; also historically known as the prokaryotes (Whittaker, 1969). The protozoa, which are also microscopic and include such organisms as the amoeba, do contain a nucleus and are classed within the eukaryotes (Whittaker, 1969). While viruses are also of microscopic, they are not technically alive and, although fascinating, will not be discussed within the scope of this review. Animals, plants and fungi all constitute the forms of life that can be seen with the naked eye (macroscopic) and are nucleus-containing eukaryotes.
Barite (BaSO 4) speleothems have been reported from caves around the globe and interpreted to hav... more Barite (BaSO 4) speleothems have been reported from caves around the globe and interpreted to have chiefly formed in phreatic, hypogene, hydrothermal settings. Here we report two contrasting types of barite speleothems (bluish tabular crystals in a shallow pool and actively dripping greenish stalactites), which today form at lower temperatures in the non-hydrothermal and vadose environment of Lechuguilla Cave, New Mexico, USA. Scanning electron microscopy analysis, along with energy-and wavelength-dispersive X-ray spectroscopy (EDS, WDS), as well as X-ray diffraction (XRD), characterize the habit and chemical composition as barite. Fractionation of the minor element calcium is related to growth along different crystal faces whereas variations in strontium concentration are mirrored in blue color zoning of the pool crystals. Two possible modes of non-hydrothermal barite precipitation are discussed: (1) intense evaporation driven by thermal atmospheric convection cells or (2) mixing of bariumrich, sulfate-poor water with water rich in sulfate. Both processes, in isolation or in combination, lead to supersaturation and could explain formation of the investigated barite speleothems. Observations of three types of microbes on the pool barite crystals showing evidence of incrustation raises the question whether there is a potential involvement of microbial activity in the temperate barite precipitation in Lechuguilla Cave.
Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the g... more Genomic resources remain scarce for bacteria isolated from oligotrophic caves. We sequenced the genomes of five proteobacteria isolated from Lechuguilla Cave in New Mexico, USA. Genome-based phylogeny indicates that each strain belongs to a distinct genus. Two Rhizobiaceae isolates possess the genomic potential for the biosynthesis of acyl-homoserine lactone.
Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly... more Acinetobacter are Gram-negative bacteria belonging to the sub-phyla Gammaproteobacteria, commonly associated with soils, animal feeds and water. Some members of the Acinetobacter have been implicated in hospital-acquired infections, with broad-spectrum antibiotic resistance. Here we report the whole genome sequence of LC510, an Acinetobacter species isolated from deep within a pristine location of the Lechuguilla Cave. Pairwise nucleotide comparison to three type strains within Acinetobacter assigned LC510 as an Acinetobacter pittii isolate. Scanning of the LC510 genome identified two genes coding for beta-lactamase resistance, despite the fact that LC510 was isolated from a portion of the cave not previously visited by humans and protected from anthropogenic input. The ability to produce acyl-homoserine lactone (AHL) signal in culture medium, an observation that is consistent with the identification of the luxI and luxR homologs in its genome, suggests that cell-to-cell communicati...
Despite the importance of karst aquifers as a source of drinking water, little is known about the... more Despite the importance of karst aquifers as a source of drinking water, little is known about the role of microorganisms in maintaining the quality of this water. One of the limitations in exploring the microbiology of these environments is access, which is usually limited to wells and surface springs. In this study, we compared the microbiology of the Madison karst aquifer sampled via the potentiometric lakes of Wind Cave with surface sampling wells and a spring. Our data indicated that only the Streeter Well (STR), which is drilled into the same hydrogeologic domain as the Wind Cave Lakes (WCL), allowed access to water with the same low biomass (1.56-9.25 × 10 3 cells mL −1). Filtration of ∼300 L of water from both of these sites through a 0.2 µm filter allowed the collection of sufficient cells for DNA extraction, PCR amplification of 16S rRNA gene sequences, and identification through pyrosequencing. The results indicated that bacteria (with limited archaea and no detectable eukaryotic organisms) dominated both water samples; however, there were significant taxonomic differences in the bacterial populations of the samples. The STR sample was dominated by a single phylotype within the Gammaproteobacteria (Order Acidithiobacillales), which dramatically reduced the overall diversity and species richness of the population. In WCL, despite less organic carbon, the bacterial population was significantly more diverse, including significant contributions from the Gammaproteobacteria, Firmicutes, Chloroflexi, Actinobacteria, Planctomycetes, Fusobacter, and Omnitrophica phyla. Comparisons with similar oligotrophic environments suggest that karst aquifers have a greater species richness than comparable surface environs. These data also demonstrate that Wind Cave provides a unique opportunity to sample a deep, subterranean aquifer directly, and that the microbiology of such aquifers may be more complex than previously anticipated.
Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing ... more Moonmilk are cave carbonate deposits that host a rich microbiome, including antibiotic-producing Actinobacteria, making these speleothems appealing for bioprospecting. Here, we investigated the taxonomic profile of the actinobacterial community of three moonmilk deposits of the cave "Grotte des Collemboles" via high-throughput sequencing of 16S rRNA amplicons. Actinobacteria was the most common phylum after Proteobacteria, ranging from 9% to 23% of the total bacterial population. Next to actinobacterial operational taxonomic units (OTUs) attributed to uncultured organisms at the genus level (~44%), we identified 47 actinobacterial genera with (4 OTUs, 17%) and (9 OTUs, ~16%) as the most abundant in terms of the absolute number of sequences. Streptomycetes presented the highest diversity (19 OTUs, 3%), with most of the OTUs unlinked to the culturable strains that were previously isolated from the same deposits. Furthermore, 43% of the OTUs were shared between the three stud...
The U.S. Culture Collection Network held a meeting to share information about how culture collect... more The U.S. Culture Collection Network held a meeting to share information about how culture collections are responding to the requirements of the recently enacted Nagoya Protocol on Access to Genetic Resources and the Fair and Equitable Sharing of Benefits Arising from their Utilization to the Convention on Biological Diversity (CBD). The meeting included representatives of many culture collections and other biological collections, the U.S. Department of State, U.S. Department of Agriculture, Secretariat of the CBD, interested scientific societies, and collection groups, including Scientific Collections International and the Global Genome Biodiversity Network. The participants learned about the policies of the United States and other countries regarding access to genetic resources, the definition of genetic resources, and the status of historical materials and genetic sequence information. Key topics included what constitutes access and how the CBD Access and Benefit-Sharing Clearing-...
Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with ... more Moonmilk is a karstic speleothem mainly composed of fine calcium carbonate crystals (CaCO3) with different textures ranging from pasty to hard, in which the contribution of biotic rock-building processes is presumed to involve indigenous microorganisms. The real microbial input in the genesis of moonmilk is difficult to assess leading to controversial hypotheses explaining the origins and the mechanisms (biotic vs. abiotic) involved. In this work, we undertook a comprehensive approach in order to assess the potential role of filamentous bacteria, particularly a collection of moonmilk-originating Streptomyces, in the genesis of this speleothem. Scanning electron microscopy (SEM) confirmed that indigenous filamentous bacteria could indeed participate in moonmilk development by serving as nucleation sites for CaCO3 deposition. The metabolic activities involved in CaCO3 transformation were furthermore assessed in vitro among the collection of moonmilk Streptomyces, which revealed that p...
A total of eight Ensifer sp. strains were isolated from two pristine cave environments. One strai... more A total of eight Ensifer sp. strains were isolated from two pristine cave environments. One strain was isolated from a cave water pool located in the Wind Cave National Park, South Dakota, USA and the remaining seven strains were isolated from Lechuguilla Cave of Carlsbad Caverns National Park, New Mexico, USA. Whole genome sequencing and comparative genomic analyses of the eight isolates compared to various type strains from the genera Ensifer and Sinorhizobium demonstrates that although members in these genera can be phylogenetically separated into two distinct clades, the percentage of conserved proteins (POCP) between various type strains from Ensifer and Sinorhizobium are consistently higher than 50%, providing strong genomic evidence to support the classification of the genera Ensifer and Sinorhizobium into a single genus.
Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore re... more Antibiotic resistance is ancient and widespread in environmental bacteria. These are therefore reservoirs of resistance elements and reflective of the natural history of antibiotics and resistance. In a previous study, we discovered that multi-drug resistance is common in bacteria isolated from Lechuguilla Cave, an underground ecosystem that has been isolated from the surface for over 4 Myr. Here we use whole-genome sequencing, functional genomics and biochemical assays to reveal the intrinsic resistome of Paenibacillus sp. LC231, a cave bacterial isolate that is resistant to most clinically used antibiotics. We systematically link resistance phenotype to genotype and in doing so, identify 18 chromosomal resistance elements, including five determinants without characterized homologues and three mechanisms not previously shown to be involved in antibiotic resistance. A resistome comparison across related surface Paenibacillus affirms the conservation of resistance over millions of years and establishes the longevity of these genes in this genus.
filaments, two regions of potential motor engagement were created on the vesicle surface. Geometr... more filaments, two regions of potential motor engagement were created on the vesicle surface. Geometric constraints limited the engaged motor number at each contact point to no more than 3, as confirmed by laser trap stall force measurements. These points of vesicle-actin filament contact resulted in an effective tug of war between two motor teams that were physically separated on the vesicle surface. The vesicle's directional outcome at an intersection was visualized using high spatial and temporal 3D super resolution imaging. For vesicles that physically contacted both actin filaments at the intersection, 55% of vesicles continued straight through the intersection, 35% switched filaments, while only 10% terminated. These outcomes were successfully simulated by a model in which the directional outcome was dependent on the interfilament gap, the vesicle's azimuthal approach angle to the intersection, the stochastic attachment and detachment of motors within a team (i.e. a team's force generation), and the Brownian motion of the vesicle while attached to the actin filaments. All of these physical factors contributed to which motor ensemble won the tug of war at the intersection and thus the vesicle's directional outcome. This simplified biomimetic system and resultant data provide an experimental framework for understanding how MyoVa motor teams navigate their cargo through evermore complex 3D actin networks that exist within cells.
Helicobacter presence and viability in waters is not well characterized. The identification of na... more Helicobacter presence and viability in waters is not well characterized. The identification of natural reservoirs and infection sources may provide novel insights into its waterborne transmission. The goal of this study was to investigated the occurrence of Helicobacter spp. in natural freshwaters from Roraima Tepui, a little studied and unique ecosystem of the Guayana Shield. Freshwaters collected from two localities at Roraima Tepui were cultured in HP selective broth and agar for Helicobacter pylori and analysed by fluorescent in situ hybridization (FISH), specific PCR assays, 16S rRNA gene sequencing and phylogenetic analysis. The presence of other bacteria in freshwater enrichments was determined using clone library sequencing of the 16S rRNA gene and phylogenetic inferences. Helicobacter spp. were detected by semi-nested PCR and FISH in freshwater enrichments from both sites. Coccoid viable but nonculturable (VBNC) cells were evidenced using 16S rRNA gene Helicobacter species and H. pylorispecific probes. Partial 16S rRNA gene sequences of two HP enrichments showed high similarity to H. pylori and Helicobacter nemestrinae (99-100 %). Other bacteria such as Serratia, Aquitalea, Chromobacterium, Mycobacterium, Acinetobacter, Curvibacter and Dysgonomonas were also detected using complete 16S rRNA gene sequences, with Serratia, Aquitalea and Chromobacterium the most common genera (40.9, 18.2 and 15.2 %, respectively). This is the first time that Helicobacter spp. have been reported in freshwaters of a tepui ecosystem. Our results contribute to the current knowledge of these bacteria in the aquatic environment and expand their known/potential sites outside the human host.
monitoring are possible, a framework for deciding how to prioritize inventory and monitoring acti... more monitoring are possible, a framework for deciding how to prioritize inventory and monitoring activities, and references to specific protocols that are already in place at NPS cave parks.
Despite extremely starved conditions, caves contain surprisingly diverse microbial communities. O... more Despite extremely starved conditions, caves contain surprisingly diverse microbial communities. Our research is geared toward understanding what ecosystems drivers are responsible for this high diversity. To asses the effect of rock fabric and mineralogy, we carried out a comparative geomicrobiology study within Carlsbad Cavern, New Mexico, USA. Samples were collected from two different geologic locations within the cave: WF1 in the Massive Member of the Capitan Formation and sF88 in the calcareous siltstones of the Yates Formation. We examined the organic content at each location using liquid chromatography mass spectroscopy and analyzed microbial community structure using molecular phylogenetic analyses. In order to assess whether microbial activity was leading to changes in the bedrock at each location, the samples were also examined by petrology, X-ray diffraction (XRD) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Our results suggest that on the chemically complex Yates Formation (sF88), the microbial community was significantly more diverse than on the limestone surfaces of the Capitan (WF1), despite a higher total number of cells on the latter. Further, the broader diversity of bacterial species at sF88 reflected a larger range of potential metabolic capabilities, presumably due to opportunities to use ions within the rock as nutrients and for chemolithotrophic energy production. The use of these ions at sF88 is supported by the formation of a corrosion residue, presumably through microbial scavenging activities. Our results suggest that rock fabric and mineralogy may be an important driver of ecosystem function and should be carefully reviewed when carrying out microbial community analysis in cave environments.
A novel actinobacterium, designated MM109 T , was isolated from a moonmilk deposit collected from... more A novel actinobacterium, designated MM109 T , was isolated from a moonmilk deposit collected from the cave 'Grotte des Collemboles' located in Comblain-au-Pont, Belgium. Based on a polyphasic taxonomic approach comprising chemotaxonomic, phylogenetic, morphological, and physiological characterization, the isolate has been affiliated to the genus Streptomyces. Multilocus sequence analysis based on the 16S rRNA gene and five other housekeeping genes (atpD, gyrB, rpoB, recA and trpB) showed that the MM109 T isolate is sufficiently distinct from its closest relative, Streptomyces peucetius strain AS 4.1799 T , as to represent a novel species. The phylogenetic distinctiveness of the taxon represented by isolate MM109 T was supported by the isolation and identification of additional twelve moonmilk-derived isolates, which according to multilocus sequence analysis were clustered along with MM109 T. Scanning electron microscopy observations revealed complex and diversified structures within a MM109 T colony, made from branching vegetative mycelia. The spore chains of the MM109 T isolate undergo complete septation at the late stages of the morphological differentiation process, leading to the formation of packs of smooth cylindrical-shaped spores. Isolate MM109 T produces several intracellular and diffusible pigments, particularly an intracellular green-pigmented secondary metabolite, which was identified through UPLC-ESI-MS analysis as ferroverdin A, an iron-chelating molecule formerly extracted and characterized from Streptomyces sp. strain WK-5344. The isolate MM109 T is thus considered to represent a novel species of Streptomyces, for which the name Streptomyces lunaelactis sp. nov. is proposed with the type strain MM109 T (=DSM 42149 T = BCCM/LMG 28326 T).
Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to... more Definitive diagnosis of the bat disease white-nose syndrome (WNS) requires histologic analysis to identify the cutaneous erosions caused by the fungal pathogen Pseudogymnoascus [formerly Geomyces] destructans (Pd). Gross visual inspection does not distinguish bats with or without WNS, and no nonlethal, on-site, preliminary screening methods are available for WNS in bats. We demonstrate that long-wave ultraviolet (UV) light (wavelength 366-385 nm) elicits a distinct orange-yellow fluorescence in bat-wing membranes (skin) that corresponds directly with the fungal cupping erosions in histologic sections of skin that are the current gold standard for diagnosis of WNS. Between March 2009 and April 2012, wing membranes from 168 North American bat carcasses submitted to the US Geological Survey National Wildlife Health Center were examined with the use of both UV light and histology. Comparison of these techniques showed that 98.8% of the bats with foci of orange-yellow wing fluorescence (n580) were WNS-positive based on histologic diagnosis; bat wings that did not fluoresce under UV light (n588) were all histologically negative for WNS lesions. Punch biopsy samples as small as 3 mm taken from areas of wing with UV fluorescence were effective for identifying lesions diagnostic for WNS by histopathology. In a nonlethal biopsy-based study of 62 bats sampled (4-mm diameter) in hibernacula of the Czech Republic during 2012, 95.5% of fluorescent (n522) and 100% of nonfluorescent (n540) wing samples were confirmed by histopathology to be WNS positive and negative, respectively. This evidence supports use of longwave UV light as a nonlethal and field-applicable method to screen bats for lesions indicative of WNS. Further, UV fluorescence can be used to guide targeted, nonlethal biopsy sampling for follow-up molecular testing, fungal culture analysis, and histologic confirmation of WNS.
Here, we report the whole-genome sequences and annotation of five oligotrophic bacteria from two ... more Here, we report the whole-genome sequences and annotation of five oligotrophic bacteria from two sites within the Lechuguilla Cave in the Carlsbad Caverns National Park, NM. Three of the five genomes contain an acyl-homoserine lactone signal synthase ortholog (luxI) that is involved in cell-to-cell communication via quorum sensing.
Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. T... more Antibiotic resistance is a global challenge that impacts all pharmaceutically used antibiotics. The origin of the genes associated with this resistance is of significant importance to our understanding of the evolution and dissemination of antibiotic resistance in pathogens. A growing body of evidence implicates environmental organisms as reservoirs of these resistance genes; however, the role of anthropogenic use of antibiotics in the emergence of these genes is controversial. We report a screen of a sample of the culturable microbiome of Lechuguilla Cave, New Mexico, in a region of the cave that has been isolated for over 4 million years. We report that, like surface microbes, these bacteria were highly resistant to antibiotics; some strains were resistant to 14 different commercially available antibiotics. Resistance was detected to a wide range of structurally different antibiotics including daptomycin, an antibiotic of last resort in the treatment of drug resistant Gram-positive pathogens. Enzyme-mediated mechanisms of resistance were also discovered for natural and semi-synthetic macrolide antibiotics via glycosylation and through a kinase-mediated phosphorylation mechanism. Sequencing of the genome of one of the resistant bacteria identified a macrolide kinase encoding gene and characterization of its product revealed it to be related to a known family of kinases circulating in modern drug resistant pathogens. The implications of this study are significant to our understanding of the prevalence of resistance, even in microbiomes isolated from human use of antibiotics. This supports a growing understanding that antibiotic resistance is natural, ancient, and hard wired in the microbial pangenome.
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Papers by Hazel Barton