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USGS2020

Repetitive sampling and control threshold improve 16S rRNA results from produced waters associated with hydraulically fractured shales

Sequencing microbial DNA from deep subsurface environments is complicated by a number of issues ranging from contamination to non-reproducible results. Many samples obtained from these environments - which are of great interest due to the potential to stimulate microbial methane generation - contain low biomass. Therefore, samples from these environments are difficult to study as sequencing results can be easily impacted by contamination. In this case, the low amount of sample biomass may be effectively swamped by the contaminating DNA and generate misleading results. Additionally, performing field work in these environments can be difficult, as researchers generally have limited access to and time on site. Therefore, optimizing a sampling plan to produce the best results while collecting the greatest number of samples over a short period of time is ideal. This study aimed to recommend an adequate sampling plan for field researchers obtaining microbial biomass for 16S rRNA gene sequencing, applicable specifically to oil and gas-producing environments. Forty-nine different samples were collected by filtering specific volumes of produced water from a hydraulically fractured well producing from the Niobrara Shale. Water was collected in two different sampling events 24 hours apart. Four to five samples were collected from 11 specific volumes. These samples along with eight different blanks were submitted for analysis. DNA was extracted from each sample, and quantitative polymerase chain reaction (qPCR) and 16S rRNA Illumina MiSeq gene sequencing were performed to determine relative concentrations of biomass and microbial community composition, respectively. The qPCR results varied across sampled volumes, while no discernible trend correlated contamination to volume of water filtered. This suggests that collecting a larger volume of sample may not result in larger biomass concentrations or better representation of a sampled environment. Researchers could prioritize collecting many low volume samples over few high-volume samples. Our results suggest that there also may be variability in the concentration of microbial communities present in produced waters over short (i.e., hours) time scales, which warrants further investigation. Submission of multiple blanks is also vital to determining how contamination or low biomass effects may influence a sample set collected from an unknown environment.

Montana, Wyoming, Colorado, New Mexico, Texas, Okl↗

USGS2021

Comparative genomics analyses support the reclassification of Bisgaard taxon 40 as Mergibacter gen. nov., with Mergibacter septicus sp. nov. as type species: Novel insights into the phylogeny and virulence factors of a Pasteurellaceae family member associated with mortality events in seabirds

The Pasteurellaceae family has been associated with fatal diseases in numerous avian species. Several new taxa within this family, including Bisgaard taxon 40, have been recently described in wild birds, but their genomic characteristics and pathogenicity are not well understood. We isolated Bisgaard taxon 40 from four species of seabirds, including one sampled during a mass, multi-species mortality event in Florida, United States. Here, we present a comprehensive phenotypic and genetic characterization of Bisgaard taxon 40 and comparative genomic analysis with reference strains from the Pasteurellaceae family, aiming at determining its phylogenetic position, antimicrobial susceptibility profile, and identifying putative virulence factors. In silico multilocus sequence-based and whole-genome-based phylogenetic analysis clustered all Bisgaard taxon 40 strains together on a distinct branch separated from the other members of the Pasteurellaceae family, indicating that Bisgaard taxon 40 could represent a new genus. These findings were further supported by protein similarity analyses using the concatenation of 31 conserved proteins and other taxonomic approaches such as the percentage of conserved protein test. Additionally, several putative virulence factors were identified, including those associated with adhesion (capsule, ompA , ompH ) and colonization ( exbD , fur , galU , galE , lpxA , lpxC , and kdsA ) of the host and a cytolethal distending toxin ( cdt ), which may have played a role in disease development leading to the mortality event. Considerably low minimum inhibitory concentrations (MICs) were found for all the drugs tested, in concordance with the absence of antimicrobial resistance genes in these genomes. The novel findings of this study highlight genomic and phenotypic characteristics of this bacterium, providing insights into genome evolution and pathogenicity. We propose a reclassification of these organisms within the Pasteurellaceae family, designated as Mergibacter gen. nov., with Mergibacter septicus sp. nov. as the type species. The type strain is Mergibacter septicus A25201 T (=DSM 112696).

Frontiers in Microbiology↗

USGS2005

Genotyping of the fish rhabdovirus, viral haemorrhagic septicaemia virus, by restriction fragment length polymorphisms

The aim of this study was to develop a standardized molecular assay that used limited resources and equipment for routine genotyping of isolates of the fish rhabdovirus, viral haemorrhagic septicaemia virus (VHSV). Computer generated restriction maps, based on 62 unique full-length (1524 nt) sequences of the VHSV glycoprotein ( G ) gene, were used to predict restriction fragment length polymorphism (RFLP) patterns that were subsequently grouped and compared with a phylogenetic analysis of the G -gene sequences of the same set of isolates. Digestion of PCR amplicons from the full-length G -gene by a set of three restriction enzymes was predicted to accurately enable the assignment of the VHSV isolates into the four major genotypes discovered to date. Further sub-typing of the isolates into the recently described sub-lineages of genotype I was possible by applying three additional enzymes. Experimental evaluation of the method consisted of three steps: (i) RT-PCR amplification of the G -gene of VHSV isolates using purified viral RNA as template, (ii) digestion of the PCR products with a panel of restriction endonucleases and (iii) interpretation of the resulting RFLP profiles. The RFLP analysis was shown to approximate the level of genetic discrimination obtained by other, more labour-intensive, molecular techniques such as the ribonuclease protection assay or sequence analysis. In addition, 37 previously uncharacterised isolates from diverse sources were assigned to specific genotypes. While the assay was able to distinguish between marine and continental isolates of VHSV, the differences did not correlate with the pathogenicity of the isolates.

Veterinary Microbiology↗

USGS2003

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

Aims: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. Methods and Results: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep‐PCR genomic profiles) and E. coli (Vitek ® GNI+) and enterococci (API ® rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at ≥85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API ® profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high‐level resistance to some antibiotics. Conclusions: Gull faeces could be a major contributor of E. coli (10 5 –10 9 CFU g −1 ) and enterococci (10 4 –10 8 CFU g −1 ) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. Significance and Impact of the Study: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.

Journal of Applied Microbiology↗

USGS2022

Natural inactivation of MS2, poliovirus type 1 and Cryptosporidium parvum in an anaerobic and reduced aquifer

Aims The study of microbial inactivation rates in aquifer systems has most often been determined in aerobic and oxidized systems. This study examined the inactivation (i.e. loss of infectivity) of MS2, poliovirus type 1 (PV1) and Cryptosporidium parvum in an anaerobic and reduced groundwater system that has been identified as storage zones for aquifer storage and recovery (ASR) facilities. Methods and Results Anaerobic and reduced (ORP < − 250 mV) groundwater from an artesian well was diverted to an above-ground, flow-through mesocosm that contained diffusion chambers filled with MS2, PV1 or Cryptosporidium parvum . The respective infectivity assays were performed on microorganisms recovered from the diffusion chambers during 30- to 58-day experiments. The net reduction in infectivity was 5.73 log 10 over 30 days for MS2, 5.00 log 10 over 58 days for PV1 and 4.07 log 10 over 37 days for C . parvum . The best fit inactivation model for PV1 was the log-linear model and the Weibull model for MS2 and C . parvum , with respective inactivation rates (95% confidence interval) of 0.19 (0.17–0.21) log 10 day −1 , 0.31 (0.19–0.89) log 10 day −1 and 0.20 (0.14–0.37) log 10 day −1 . Conclusions The groundwater geochemical conditions in this aquifer enhanced the inactivation of MS2, PV1, and C . parvum at rates approximately 2.0–5.3-fold, 1.2–17.0-fold, and 4.5–5.6-fold greater, respectively, than those from published studies that used diffusion chambers in aerobic-to-anoxic groundwater systems, with positive redox potentials. Significance and Impact of the Study Geochemical conditions like those in the aquifer zone in this study can naturally and significantly reduce concentrations of microbial indicators and pathogens of human health concern in injected surface water. Appropriate storage times for injected surface water could complement above-ground engineered processes for microorganism removal and inactivation (e.g. filtration, disinfection) by naturally increasing overall microorganism log-inactivation rates of ASR facilities.

Journal of Applied Microbiology↗

USGS2025

Bacterial community diversity and potential eco-physiological roles in toxigenic blooms composed of Microcystis, Aphanizomenon or Planktothrix

Cyanobacterial toxicity, cyanotoxins, and their impact on aquatic ecosystems and human health are well documented. In comparison, less is known about bloom-associated bacterial communities. Co-occurring bacteria can influence bloom development, physiology and collapse, and may also provide a niche for pathogenic bacteria. Existing research focuses on the cyanosphere of Microcystis -dominated blooms, despite the increasing prevalence of filamentous genera ( Aphanizomenon and Planktothrix ). This pilot study aimed to broaden our understanding of the bacterial consortia attached to morphologically distinct cyanobacteria (coccoid and filamentous) dominating phytoplankton communities and to explore their potential roles in amplifying the impacts of cyanobacterial blooms. We investigated four shallow freshwater bodies across three continents and two climate zones: an urban pond in the USA, a dammed reservoir and a natural lake in Poland, and an urban water body in Singapore. Amplicon sequencing (16S rRNA gene) was used to characterize bacterial communities, while shotgun metagenomics identified nitrogen- and phosphorus-cycling genes to infer potential eco-physiological functions. Cyanobacteria dominated bacterioplankton assemblages at all sites (>35.6%), with bloom composition influencing toxigenic profiles. A mixed bloom of Microcystis , Snowella , and Aphanizomenon had the broadest range of cyanotoxin synthetase genes ( mcy E, cyr J, ana F and sxt A). Microcystis blooms correlated with increased Roseomonas , while Planktothrix co-occurred with Flavobacterium – both bacteria likely contribute to nutrient-cycling within blooms and represent potential opportunistic pathogens for aquatic organisms and humans. The Microcystis cyanosphere exhibited the highest number of significant positive correlations with bacteria (19 relations), compared to Planktothrix and Aphanizomenon (11 and 2 relations, respectively). Non-diazotrophic blooms of Microcystis and Planktothrix showed greater abundances of nitrogen – ( ure B, gln A, nar B, and nar HZ) and phosphorus-cycling genes ( pho BHPR and ppk 1), indicating a strong dependence on associated bacteria for nutrient acquisition compared to diazotrophic Aphanizomenon . These findings suggest that Aphanizomenon -dominated blooms may be sustained by simpler microbiomes. Our results provide preliminary evidence of cyanosphere heterogeneity potentially shaped by the dominance or coexistence of three morphologically and eco-physiologically distinct genera of cyanobacteria. A comprehensive knowledge of the taxonomy and functional roles of bloom-associated microbiomes is therefore essential to understand bloom activity, evaluate the environmental threat, and develop effective strategies for prevention and mitigation.

Frontiers in Microbiology↗