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The exchange of microbes between humans and the built environment is a dynamic process that has significant impact on health. Most studies exploring the microbiome of the built environment have been predicated on improving our understanding of pathogen emergence, persistence, and transmission. Previous studies have demonstrated that SARS-CoV-2 presence significantly correlates with the proportional abundance of specific bacteria on surfaces in the built environment. However, in these studies, SARS-CoV-2 originated from infected patients. Here, we perform a similar assessment for a clinical microbiology lab while staff were handling SARS-CoV-2 infected samples. The goal of this study was to understand the distribution and dynamics of microbial population on various surfaces within different sections of a clinical microbiology lab during a short period of 2020 Coronavirus disease (COVID-19) pandemic. We sampled floors, benches, and sinks in 3 sections (bacteriology, molecular microbiology, and COVID) of an active clinical microbiology lab over a 3-month period. Although floor samples harbored SARS-CoV-2, it was rarely identified on other surfaces, and bacterial diversity was significantly greater on floors than sinks and benches. The floors were primarily colonized by bacteria common to natural environments (e.g., soils), and benchtops harbored a greater proportion of human-associated microbes, including Staphylococcus and Streptococcus. Finally, we show that the microbial composition of these surfaces did not change over time and remained stable. Despite finding viruses on the floors, no lab-acquired infections were reported during the study period, which suggests that lab safety protocols and sanitation practices were sufficient to prevent pathogen exposures. IMPORTANCE For decades, diagnostic clinical laboratories have been an integral part of the health care systems that perform diagnostic tests on patient's specimens in bulk on a regular basis. Understanding their microbiota should assist in designing and implementing disinfection, and cleaning regime in more effective way. To our knowledge, there is a lack of information on the composition and dynamics of microbiota in the clinical laboratory environments, and, through this study, we have tried to fill that gap. This study has wider implications as understanding the makeup of microbes on various surfaces within clinical laboratories could help identify any pathogenic bacterial taxa that could have colonized these surfaces, and might act as a potential source of laboratory-acquired infections. Mapping the microbial community within these built environments may also be critical in assessing the reliability of laboratory safety and sanitation practices to lower any potential risk of exposures to health care workers.

Objective The aim of this study was to describe effects of vaginal estrogen (VE) on the urogenital microbiome in postmenopausal women with recurrent urinary tract infections (rUTIs). Methods This is a secondary analysis of 17 participants enrolled in a randomized controlled trial of VE versus placebo on urinary tract infection recurrence in postmenopausal women with rUTIs. Paired clean-catch urine samples were collected at baseline and after 6 months of VE and sequenced using 16S rRNA gene sequencing. Sequence reads were analyzed using Quantitative Insights Into Microbial Ecology 2. Changes in α diversity, β diversity, and differentially abundant genera were measured between paired baseline and 6-month samples and between those with a urinary tract infection at 6 months (failures) and those without (successes). Results Of the 17 women, 11 were successes and 6 were failures after 6 months of VE treatment. There was a significant change in α diversity from baseline to month 6 in samples overall (Kruskal-Wallis χ2 = 3.47, P = 0.037) and in the treatment success group (Yuen T = -2.53, P = 0.035). The increase in relative abundance of Lactobacillus crispatus, Lactobacillus gasseri, and Lactobacillus iners AB-1 was correlated with month 6. A relative bloom of L. crispatus compared with L. gasseri was associated with treatment success (Kruskal-Wallis χ2 = 4.9, P = 0.0014). Conclusions Lactobacillus increases in the urogenital microbiome of postmenopausal women with rUTI after 6 months of VE. However, only the relative increase in L. crispatus specifically may be associated with treatment success.

Objective To determine whether a single-use stethoscope diaphragm barrier surface remains aseptic when placed on pathogen-contaminated stethoscopes. Methods From May 31 to August 5, 2019, we tested 2 separate barriers using 3 different strains of 7 human pathogens, including extended-spectrum β-lactamase–producing Escherichia coli, methicillin-resistant Staphylococcus aureus, and vancomycin resistant Enterococcus faecium. Results For all diaphragms with either of the 2 barriers tested, no growth was recorded for any of the pathogens. Stethoscopes with aseptic barriers remained sterile for up to 24 hours. These single-use barriers also provided aseptic surfaces when stethoscope diaphragms were inoculated with human specimens, including saliva, stool, urine, and sputum. Conclusion Disposable aseptic diaphragm barriers may provide robust and efficient solutions to reduce transmission of pathogens via stethoscopes.

BackgroundIncreasingly, researchers use protein-coding genes from targeted PCR amplification or direct metagenomic sequencing in community and population ecology. Analysis of protein-coding genes presents different challenges from those encountered in traditional SSU rRNA studies. Most protein-coding sequences are annotated based on homology to other computationally-annotated sequences, which can lead to inaccurate annotations. Therefore, the results of sensitive homology searches must be validated to remove false-positives and assess functionality. Multiple lines of in silico evidence can be gathered by examining conserved domains and residues identified through biochemical investigations. However, manually validating sequences in this way can be time consuming and error prone, especially in large environmental studies.ResultsAn automated pipeline for protein active site validation (PASV) was developed to improve validation and partitioning accuracy for protein-coding sequences, combining multiple sequence alignment with expert domain knowledge. PASV was tested using commonly misannotated proteins: ribonucleotide reductase (RNR), alternative oxidase (AOX), and plastid terminal oxidase (PTOX). PASV partitioned 9,906 putative Class I alpha and Class II RNR sequences from bycatch in a global viral metagenomic investigation with >99% true positive and true negative rates. PASV predicted the class of 2,579 RNR sequences in >98% agreement with manual annotations. PASV correctly partitioned all 336 tested AOX and PTOX sequences.ConclusionsPASV provides an automated and accurate way to address post-homology search validation and partitioning of protein-coding marker genes. Source code is released under the MIT license and is found with documentation and usage examples on GitHub at https://github.com/mooreryan/pasv.

Prior studies have shown that high-fat diet (HFD) can have profound effects on the gastrointestinal (GI) tract microbiome and also demonstrate that bacteria in the GI tract can affect metabolism and lean/obese phenotypes. We investigated whether the composition of viral communities that also inhabit the GI tract are affected by shifts from normal to HFD. We found significant and reproducible shifts in the content of GI tract viromes after the transition to HFD. The differences observed in virome community membership and their associated gene content suggest that these altered viral communities are populated by viruses that are more virulent toward their host bacteria. Because HFD also are associated with significant shifts in GI tract bacterial communities, we believe that the shifts in the viral community may serve to drive the changes that occur in associated bacterial communities., Alterations in diet can have significant impact on the host, with high-fat diet (HFD) leading to obesity, diabetes, and inflammation of the gut. Although membership and abundances in gut bacterial communities are strongly influenced by diet, substantially less is known about how viral communities respond to dietary changes. Examining fecal contents of mice as the mice were transitioned from normal chow to HFD, we found significant changes in the relative abundances and the diversity in the gut of bacteria and their viruses. Alpha diversity of the bacterial community was significantly diminished in response to the diet change but did not change significantly in the viral community. However, the diet shift significantly impacted the beta diversity in both the bacterial and viral communities. There was a significant shift away from the relatively abundant Siphoviridae accompanied by increases in bacteriophages from the Microviridae family. The proportion of identified bacteriophage structural genes significantly decreased after the transition to HFD, with a conserved loss of integrase genes in all four experimental groups. In total, this study provides evidence for substantial changes in the intestinal virome disproportionate to bacterial changes, and with alterations in putative viral lifestyles related to chromosomal integration as a result of shift to HFD. IMPORTANCE Prior studies have shown that high-fat diet (HFD) can have profound effects on the gastrointestinal (GI) tract microbiome and also demonstrate that bacteria in the GI tract can affect metabolism and lean/obese phenotypes. We investigated whether the composition of viral communities that also inhabit the GI tract are affected by shifts from normal to HFD. We found significant and reproducible shifts in the content of GI tract viromes after the transition to HFD. The differences observed in virome community membership and their associated gene content suggest that these altered viral communities are populated by viruses that are more virulent toward their host bacteria. Because HFD also are associated with significant shifts in GI tract bacterial communities, we believe that the shifts in the viral community may serve to drive the changes that occur in associated bacterial communities.

Alcoholic hepatitis is a severe manifestation of alcohol-associated liver disease with high mortality. Although gut bacteria and fungi modulate disease severity, little is known about the effects of the viral microbiome (virome) in patients with alcohol-associated liver disease. We extracted virus-like particles from 89 patients with alcoholic hepatitis who were enrolled in a multicenter observational study, 36 with alcohol use disorder, and 17 individuals without alcohol use disorder (controls). Virus-like particles from fecal samples were fractionated using differential filtration techniques and metagenomic sequencing was performed to characterize intestinal viromes. We observed an increased viral diversity in fecal samples from patients with alcohol-associated liver disease, with the most significant changes in samples from patients with alcoholic hepatitis. Escherichia-, Enterobacteria-, and Enterococcus phages were overrepresented in fecal samples from patients with alcoholic hepatitis, along with significant increases in mammalian viruses such as Parvoviridae and Herpesviridae. Antibiotic treatment was associated with higher viral diversity. Specific viral taxa, such as Staphylococcus phages and Herpesviridae, were associated with increased disease severity, indicated by a higher median model for end-stage liver disease (MELD) score, and associated with increased 90-day mortality. In conclusion, intestinal viral taxa are altered in fecal samples from patients with alcoholic hepatitis and associated with disease severity and mortality. Our study, for the first time, described an intestinal virome signature associated with alcoholic hepatitis.

Despite their potential importance with regard to tobacco-related health outcomes, as well as their hypothesized role in the production of tobacco-specific N-nitrosamines, bacterial constituents of tobacco products lack characterization. Specifically, to our knowledge, there has been no comprehensive characterization of the effects of storage conditions on the bacterial communities associated with little cigars and cigarillos. To address this knowledge gap, we characterized the bacterial community composition of the tobacco and wrapper components of the following four products: Swisher Sweets Original; Swisher Sweets, Sweet Cherry; Cheyenne Cigars Full Flavor 100’s; and Cheyenne Menthol Box. Each product was stored under three different conditions of temperature and relative humidity to mimic different user storage conditions: room (20°C 50% RH), refrigerator (5°C 18% RH) and pocket (25°C 30% RH). On days 0, 5, 9 and 14, subsamples were collected, the wrapper and tobacco were separated, and their total DNA was extracted separately and purified. Resulting DNA was then used in PCR assays targeting the V3 V4 region of the bacterial 16S rRNA gene, followed by sequencing using Illumina HiSeq 300bp PE. Resulting sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package, followed by analyses in R using the Phyloseq and Vegan packages. A single bacterial phylum, Firmicutes, dominated in the wrapper subsamples whereas the tobacco subsamples were dominated by Proteobacteria. Cheyenne Menthol Box (CMB) samples were characterized by significant differential abundances for 23 bacterial operational taxonomic units (OTUs) in tobacco subsamples and 27 OTUs in the wrapper subsamples between day 0 and day 14 under all conditions. OTUs from the genera Acinetobacter and Bacillus significantly increased in the CMB tobacco subsamples, and OTUs from Bacillus, Streptococcus, Lactobacillus, and Enterococcus significantly increased in the CMB wrapper subsamples over time. These initial results suggest that the bacterial communities of little cigars and cigarillos are dynamic over time and varying storage conditions.

Accurate predictions across multiple fields of microbiome research have far-reaching benefits to society, but there are few widely accepted quantitative tools to make accurate predictions about microbial communities and their functions. More discussion is needed about the current state of microbiome analysis and the tools required to overcome the hurdles preventing development and implementation of predictive analyses., Accurate predictions across multiple fields of microbiome research have far-reaching benefits to society, but there are few widely accepted quantitative tools to make accurate predictions about microbial communities and their functions. More discussion is needed about the current state of microbiome analysis and the tools required to overcome the hurdles preventing development and implementation of predictive analyses. We summarize the ideas generated by participants of the Mid-Atlantic Microbiome Meet-up in January 2019. While it was clear from the presentations that most fields have advanced beyond simple associative and descriptive analyses, most fields lack essential elements needed for the development and application of accurate microbiome predictions. Participants stressed the need for standardization, reproducibility, and accessibility of quantitative tools as key to advancing predictions in microbiome analysis. We highlight hurdles that participants identified and propose directions for future efforts that will advance the use of prediction in microbiome research.

Standardized conditions for collection, preservation and storage of urine for microbiome research have not been established. We aimed to identify the effects of the use of preservative AssayAssure® (AA), and the effects of storage time and temperatures on reproducibility of urine microbiome results. We sequenced the V3–4 segment of the 16S rRNA gene to characterize the bacterial community in the urine of a cohort of women. Each woman provided a single voided urine sample, which was divided into aliquots and stored with and without AA, at three different temperatures (room temperature [RT], 4 °C, or −20 °C), and for various time periods up to 4 days. There were significant microbiome differences in urine specimens stored with and without AA at all temperatures, but the most significant differences were observed in alpha diversity (estimated number of taxa) at RT. Specimens preserved at 4 °C and −20 °C for up to 4 days with or without AA had no significant alpha diversity differences. However, significant alpha diversity differences were observed in samples stored without AA at RT. Generally, there was greater microbiome preservation with AA than without AA at all time points and temperatures, although not all results were statistically significant. Addition of AA preservative, shorter storage times, and colder temperatures are most favorable for urinary microbiome reproducibility.

Lotic surface water sites (e.g. creeks) are important resources for localized agricultural irrigation. However, there is concern that microbial contaminants within untreated surface water may be transferred onto irrigated soil and crops. To evaluate this issue, water samples were collected between January 2017 and August 2018 from a freshwater creek used to irrigate kale and radish plants on a small farm in the Mid-Atlantic, United States. In addition, on one sampling date, a field survey was conducted in which additional water (creek source and point-of-use) and soil samples were collected to assess the viral and bacterial communities pre- and post- irrigation. All samples were processed for DNA extracts and shotgun sequenced on the Illumina HiSeq platform. The resulting metagenomic libraries were assembled de novo and taxonomic and functional features were assigned at the contig and peptide level. From these data, we observed that Betaproteobacteria (e.g. Variovorax) dominated the water, both at the source and point-of-use, and Alphaproteobacteria (e.g. Streptomyces) dominated both pre- and post-irrigated soil. Additionally, in the creek source water there were variations in the abundance of the dominant bacterial genera and functional annotations associated with seasonal characteristics (e.g. water temperature). Antibiotic resistance genes and virulence factors were also identified in the creek water and soil, with the majority specific to their respective habitat. Moreover, an analysis of clustered regularly interspaced short palindromic repeat (CRISPR) arrays showed the persistence of certain spacers through time in the creek water, as well as specific interactions between creek bacteriophages and their hosts. Overall, these findings provide a more holistic picture of bacterial and viral composition, dynamics, and interactions within a freshwater creek that can be utilized to further our knowledge on its suitability and safety for irrigation.

Shotgun metagenomics, which allows for broad sampling of viral diversity, has uncovered genes that are widely distributed among virioplankton populations and show linkages to important biological features of unknown viruses. Over 25% of known dsDNA phage carry the DNA polymerase I (polA) gene, making it one of the most widely distributed phage genes. Because of its pivotal role in DNA replication, this enzyme is linked to phage lifecycle characteristics. Previous research has suggested that a single amino acid substitution might be predictive of viral lifestyle. In this study Chesapeake Bay virioplankton were sampled by shotgun metagenomic sequencing (using long and short read technologies). More polA sequences were predicted from this single viral metagenome (virome) than from 86 globally distributed virome libraries (ca. 2,100, and 1,200, respectively). The PolA peptides predicted from the Chesapeake Bay virome clustered with 69% of PolA peptides from global viromes; thus, remarkably the Chesapeake Bay virome captured the majority of known PolA peptide diversity in viruses. This deeply sequenced virome also expanded the diversity of PolA sequences, increasing the number of PolA clusters by 44%. Contigs containing polA sequences were also used to examine relationships between phylogenetic clades of PolA and other genes within unknown viral populations. Phylogenic analysis revealed five distinct groups of phages distinguished by the amino acids at their 762 (Escherichia coli IAI39 numbering) positions and replication genes. DNA polymerase I sequences from Tyr762 and Phe762 groups were most often neighbored by ring-shaped superfamily IV helicases and ribonucleotide reductases (RNRs). The Leu762 groups had non-ring shaped helicases from superfamily II and were further distinguished by an additional helicase gene from superfamily I and the lack of any identifiable RNR genes. Moreover, we found that the inclusion of ribonucleotide reductase associated with PolA helped to further differentiate phage diversity, chiefly within lytic podovirus populations. Altogether, these data show that DNA Polymerase I is a useful marker for observing the diversity and composition of the virioplankton and may be a driving factor in the divergence of phage replication components.

The use of irrigation water sourced from reclamation facilities and untreated surface water bodies may be a practical solution to attenuate the burden on diminishing groundwater aquifers. However, comprehensive microbial characterizations of these water sources are generally lacking, especially with regard to variations through time and across multiple water types. To address this knowledge gap we used a shotgun metagenomic approach to characterize the taxonomic and functional variations of microbial communities within two agricultural ponds, two freshwater creeks, two brackish rivers, and three water reclamation facilities located in the Mid-Atlantic, United States. Water samples (n = 24) were collected from all sites between October and November 2016, and filtered onto 0.2 μm membrane filters. Filters were then subjected to total DNA extraction and shotgun sequencing on the Illumina HiSeq platform. From these data, we found that Betaproteobacteria dominated the majority of freshwater sites, while Alphaproteobacteria were abundant at times in the brackish waters. One of these brackish sites was also host to a greater abundance of the bacterial genera Gimesia and Microcystis. Furthermore, predicted microbial features (e.g. antibiotic resistance genes (ARGs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) arrays) varied based on specific site and sampling date. ARGs were found across samples, with the diversity and abundance highest in those from a reclamation facility and a wastewater-impacted freshwater creek. Additionally, we identified over 600 CRISPR arrays, containing ∼2600 unique spacers, suggestive of a diverse and often site-specific phage community. Overall, these results provide a better understanding of the complex microbial community in untreated surface and reclaimed waters, while highlighting possible environmental and human health impacts associated with their use in agriculture.

The impact of microbially contaminated irrigation water on risks to produce safety and public health is a complex issue that is not well understood. This study tracked fecal indicators, pathogenic bacteria, and total bacterial communities from a creek water irrigation source to irrigated produce to assess the impact of irrigation events on soil and produce-associated microbiota. Kale and radishes were drip-irrigated using Mid-Atlantic creek water in October 2017. Plant and soil samples were collected immediately before and after irrigation, and for 3 consecutive days thereafter. All samples (n = 134), including irrigation water, were tested for generic Escherichia coli and total coliforms (TC) using standard membrane filtration or direct plating, and for Salmonella enterica and Listeria monocytogenes by selective enrichment. DNA extracted from all samples was PCR-amplified for the V3-V4 region of the 16S rRNA gene for bacterial community profiling. In soil, TC levels were significantly higher immediately and 3 days post-irrigation compared to pre-irrigation (p 0.01). E. coli levels in soil increased after irrigation, but the difference was not significant (p = 0.31), and die-off was not observed. No E. coli were detected on kale leaves. TC increased over the study period on radish roots (p 0.01) but not kale leaves (p = 0.43). Although target pathogens were detected in irrigation water, S. enterica was detected from only one post-irrigation kale sample and L. monocytogenes was not detected in the field. The 16S rRNA gene sequencing data revealed differences in bacterial community structure and composition across sample types and showed that radish soil and root surface bacterial communities were more strongly influenced by irrigation compared to kale samples. This study provides insights into the impact of irrigation water on fresh produce microbiota, revealing that, although irrigation did influence crop-associated microbiota (especially below ground) in the field, bacterial pathogens were not likely transferred to the crop.

The Mid-Atlantic Microbiome Meet-up (M3) organization brings together academic, government, and industry groups to share ideas and develop best practices for microbiome research. In January of 2018, M3 held its fourth meeting, which focused on recent advances in biodefense, specifically those relating to infectious disease, and the use of metagenomic methods for pathogen detection. Presentations highlighted the utility of next-generation sequencing technologies for identifying and tracking microbial community members across space and time. However, they also stressed the current limitations of genomic approaches for biodefense, including insufficient sensitivity to detect low-abundance pathogens and the inability to quantify viable organisms. Participants discussed ways in which the community can improve software usability and shared new computational tools for metagenomic processing, assembly, annotation, and visualization. Looking to the future, they identified the need for better bioinformatics toolkits for longitudinal analyses, improved sample processing approaches for characterizing viruses and fungi, and more consistent maintenance of database resources. Finally, they addressed the necessity of improving data standards to incentivize data sharing. Here, we summarize the presentations and discussions from the meeting, identifying the areas where microbiome analyses have improved our ability to detect and manage biological threats and infectious disease, as well as gaps of knowledge in the field that require future funding and focus. Electronic supplementary material The online version of this article (10.1186/s40168-018-0582-5) contains supplementary material, which is available to authorized users.

Objective Zero-valent iron sand filtration can remove multiple contaminants, including some types of pathogenic bacteria, from contaminated water. However, its efficacy at removing complex viral populations, such as those found in reclaimed water used for agricultural irrigation, has not been fully evaluated. Therefore, this study utilized metagenomic sequencing and epifluorescent microscopy to enumerate and characterize viral populations found in reclaimed water and zero-valent iron-sand filtered reclaimed water sampled three times during a larger greenhouse study. Results Zero-valent iron-sand filtered reclaimed water samples had significantly less virus-like particles than reclaimed water samples at all collection dates, with the reclaimed water averaging between 108 and 109 and the zero-valent iron-sand filtered reclaimed water averaging between 106 and 107 virus-like particles per mL. In addition, for both sample types, viral metagenomes (viromes) were dominated by bacteriophages of the order Caudovirales, largely Siphoviridae, and genes related to DNA metabolism. However, the proportion of sequences homologous to bacteria, as well as the abundance of genes possibly originating from a bacterial host, was higher in the viromes of zero-valent iron-sand filtered reclaimed water samples. Overall, zero-valent iron-sand filtered reclaimed water had a lower total concentration of virus-like particles and a different virome community composition compared to unfiltered reclaimed water. Electronic supplementary material The online version of this article (10.1186/s13104-019-4251-y) contains supplementary material, which is available to authorized users.

Agricultural ponds have a great potential as a means of capture and storage of water for irrigation. However, pond topography (small size, shallow depth) leaves them susceptible to environmental, agricultural, and anthropogenic exposures that may influence microbial dynamics. Therefore, the aim of this project was to characterize the bacterial and viral communities of pond water in the Mid-Atlantic United States with a focus on the late season (October–December), where decreasing temperature and nutrient levels can affect the composition of microbial communities. Ten liters of freshwater from an agricultural pond were sampled monthly, and filtered sequentially through 1 and 0.2 μm filter membranes. Total DNA was then extracted from each filter, and the bacterial communities were characterized using 16S rRNA gene sequencing. The remaining filtrate was chemically concentrated for viruses, DNA-extracted, and shotgun sequenced. Bacterial community profiling showed significant fluctuations over the sampling period, corresponding to changes in the condition of the pond freshwater (e.g., pH, nutrient load). In addition, there were significant differences in the alpha-diversity and core bacterial operational taxonomic units (OTUs) between water fractions filtered through different pore sizes. The viral fraction was dominated by tailed bacteriophage of the order Caudovirales, largely those of the Siphoviridae family. Moreover, while present, genes involved in virulence/antimicrobial resistance were not enriched within the viral fraction during the study period. Instead, the viral functional profile was dominated by phage associated proteins, as well as those related to nucleotide production. Overall, these data suggest that agricultural pond water harbors a diverse core of bacterial and bacteriophage species whose abundance and composition are influenced by environmental variables characteristic of pond topology and the late season.

The discovery of abundant viruses in the oceans and on land has ushered in a quarter century of groundbreaking advancements in our understanding of viruses within ecosystems. Two types of observations from environmental samples--direct counts of viral particles and viral metagenomic sequences--have been critical to these discoveries. Accurate direct counts have established ecosystem-scale trends in the impacts of viral infection on microbial host populations and have shown that viral communities within aquatic and soil environments respond to both short term and seasonal environmental change. Direct counts have been critical for estimating viral production rate, a measurement essential to quantifying the implications of viral infection for the biogeochemical cycling of nutrients within ecosystems. While direct counts have defined the magnitude of viral processes; shotgun sequences of environmental viral DNA--virome sequences--have enabled researchers to estimate the diversity and composition of natural viral communities. Virome-enabled studies have found the virioplankton to contain thousands of viral genotypes in communities where the most dominant viral population accounts for a small fraction of total abundance followed by a long tail of diverse populations. Detailed examination of long virome sequences has led to new understanding of genotype-to-phenotype connections within marine viruses and revealed that viruses carry metabolic genes that are important to maintaining cellular energy during viral replication. Increased access to long virome sequences will undoubtedly reveal more genetic secrets of viruses and enable us to build a genomics rulebook for predicting key biological and ecological features of unknown viruses.

Tobacco products, specifically cigarettes, are home to microbial ecosystems that may play an important role in the generation of carcinogenic tobacco-specific nitrosamines (TSNAs), as well as the onset of multiple adverse human health effects associated with the use of these products. Therefore, we conducted time-series experiments with five commercially-available brands of cigarettes that were either commercially-mentholated, custom-mentholated, user-mentholated or non-mentholated. To mimic user storage conditions, the cigarettes were incubated for 14 days under three different temperatures and relative humidities (i.e., pocket, refrigerator and room). Overall, 360 samples were collected over the course of two weeks and total DNA was extracted, PCR amplified for the V3V4 hypervariable region of the 16S rRNA gene and sequenced using Illumina MiSeq. A subset of samples (n=32) was also analyzed via liquid chromatography with tandem mass spectrometry for two TSNAs: N'- nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Comparative analyses of the five tobacco brands revealed bacterial communities dominated by Pseudomonas, Pantoea, and Bacillus, with Pseudomonas relatively stable in abundance regardless of storage condition. In addition, core bacterial operational taxonomic units (OTUs) were identified in all samples and included Bacillus pumilus, Rhizobium sp., Sphingomonas sp., unknown Enterobacteriaceae, Pantoea sp., Pseudomonas sp., Pseudomonas oryzihabitans, and Pseudomonas putida. Additional OTUs were identified that significantly changed in relative abundance between day 0 and day 14, influenced by brand and storage condition. In addition, small but statistically significant increases in NNN levels were observed in user- and commercially-mentholated brands between day 0 and day 14 at pocket conditions.. These data suggest that manufacturing and user manipulations, such as mentholation, and storage conditions, may directly impact the microbiome of cigarette tobacco as well as the levels of carcinogens.

Background There is a paucity of data regarding the microbial constituents of tobacco products and their impacts on public health. Moreover, there has been no comparative characterization performed on the bacterial microbiota associated with the addition of menthol, an additive that has been used by tobacco manufacturers for nearly a century. To address this knowledge gap, we conducted bacterial community profiling on tobacco from user- and custom-mentholated/non-mentholated cigarette pairs, as well as a commercially-mentholated product. Total genomic DNA was extracted using a multi-step enzymatic and mechanical lysis protocol followed by PCR amplification of the V3-V4 hypervariable regions of the 16S rRNA gene from five cigarette products (18 cigarettes per product for a total of 90 samples): Camel Crush, user-mentholated Camel Crush, Camel Kings, custom-mentholated Camel Kings, and Newport Menthols. Sequencing was performed on the Illumina MiSeq platform and sequences were processed using the Quantitative Insights Into Microbial Ecology (QIIME) software package. Results In all products, Pseudomonas was the most abundant genera and included Pseudomonas oryzihabitans and Pseudomonas putida, regardless of mentholation status. However, further comparative analysis of the five products revealed significant differences in the bacterial compositions across products. Bacterial community richness was higher among non-mentholated products compared to those that were mentholated, particularly those that were custom-mentholated. In addition, mentholation appeared to be correlated with a reduction in potential human bacterial pathogens and an increase in bacterial species resistant to harsh environmental conditions. Conclusions Taken together, these data provide preliminary evidence that the mentholation of commercially available cigarettes can impact the bacterial community of these products. Electronic supplementary material The online version of this article (doi:10.1186/s40168-017-0235-0) contains supplementary material, which is available to authorized users.

Background Since 1982, specific serotypes of Shiga toxin-producing Escherichia coli (STEC) have been recognized as significant foodborne pathogens acquired from contaminated beef and, more recently, other food products. Cattle are the major reservoir hosts of these organisms, and while there have been advancements in food safety practices and industry standards, STEC still remains prevalent within beef cattle operations with cattle hides implicated as major sources of carcass contamination. To investigate whether the composition of hide-specific microbial communities are associated with STEC prevalence, 16S ribosomal RNA (rRNA) bacterial community profiles were obtained from hide and fecal samples collected from a large commercial feedlot over a 3-month period. These community data were examined amidst an extensive collection of prevalence data on a subgroup of STEC that cause illness in humans, referred to as enterohemorrhagic E. coli (EHEC). Fecal 16S rRNA gene OTUs (operational taxonomic units) were subtracted from the OTUs found within each hide 16S rRNA amplicon library to identify hide-specific bacterial populations. Results Comparative analysis of alpha diversity revealed a significant correlation between low bacterial diversity and samples positive for the presence of E. coli O157:H7 and/or the non-O157 groups: O26, O111, O103, O121, O45, and O145. This trend occurred regardless of diversity metric or fecal OTU presence. The number of EHEC serogroups present in the samples had a compounding effect on the inverse relationship between pathogen presence and bacterial diversity. Beta diversity data showed differences in bacterial community composition between samples containing O157 and non-O157 populations, with certain OTUs demonstrating significant changes in relative abundance. Conclusions The cumulative prevalence of the targeted EHEC serogroups was correlated with low bacterial community diversity on pre-harvest cattle hides. Understanding the relationship between indigenous hide bacterial communities and populations may provide strategies to limit EHEC in cattle and provide biomarkers for EHEC risk assessment. Electronic supplementary material The online version of this article (doi:10.1186/s40168-016-0155-4) contains supplementary material, which is available to authorized users.