Early Detection of Severe Acute Respiratory Syndrome Coronavirus 2 Variants Using Traveler-based Genomic Surveillance at 4 US Airports, September 2021-January 2022.

We enrolled arriving international air travelers in a severe acute respiratory syndrome coronavirus 2 genomic surveillance program. We used molecular testing of pooled nasal swabs and sequenced positive samples for sublineage. Traveler-based surveillance provided early-warning variant detection, reporting the first US Omicron BA.2 and BA.3 in North America.

Long-read metagenomic sequencing reveals shifts in associations of antibiotic resistance genes with mobile genetic elements from sewage to activated sludge.

BACKGROUND: There is concern that the microbially rich activated sludge environment of wastewater treatment plants (WWTPs) may contribute to the dissemination of antibiotic resistance genes (ARGs). We applied long-read (nanopore) sequencing to profile ARGs and their neighboring genes to illuminate their fate in the activated sludge treatment by comparing their abundance, genetic locations, mobility potential, and bacterial hosts within activated sludge relative to those in influent sewage across five WWTPs from three continents. RESULTS: The abundances (gene copies per Gb of reads, aka gc/Gb) of all ARGs and those carried by putative pathogens decreased 75-90% from influent sewage (192-605 gc/Gb) to activated sludge (31-62 gc/Gb) at all five WWTPs. Long reads enabled quantification of the percent abundance of ARGs with mobility potential (i.e., located on plasmids or co-located with other mobile genetic elements (MGEs)). The abundance of plasmid-associated ARGs decreased at four of five WWTPs (from 40-73 to 31-68%), and ARGs co-located with transposable, integrative, and conjugative element hallmark genes showed similar trends. Most ARG-associated elements decreased 0.35-13.52% while integrative and transposable elements displayed slight increases at two WWTPs (1.4-2.4%). While resistome and taxonomic compositions both shifted significantly, host phyla for chromosomal ARG classes remained relatively consistent, indicating vertical gene transfer via active biomass growth in activated sludge as the key pathway of chromosomal ARG dissemination. CONCLUSIONS: Overall, our results suggest that the activated sludge process acted as a barrier against the proliferation of most ARGs, while those that persisted or increased warrant further attention. Video abstract.

Critical evaluation of short, long, and hybrid assembly for contextual analysis of antibiotic resistance genes in complex environmental metagenomes.

In the fight to limit the global spread of antibiotic resistance, the assembly of environmental metagenomes has the potential to provide rich contextual information (e.g., taxonomic hosts, carriage on mobile genetic elements) about antibiotic resistance genes (ARG) in the environment. However, computational challenges associated with assembly can impact the accuracy of downstream analyses. This work critically evaluates the impact of assembly leveraging short reads, nanopore MinION long-reads, and a combination of the two (hybrid) on ARG contextualization for ten environmental metagenomes using seven prominent assemblers (IDBA-UD, MEGAHIT, Canu, Flye, Opera-MS, metaSpades and HybridSpades). While short-read and hybrid assemblies produced similar patterns of ARG contextualization, raw or assembled long nanopore reads produced distinct patterns. Based on an in-silico spike-in experiment using real and simulated reads, we show that low to intermediate coverage species are more likely to be incorporated into chimeric contigs across all assemblers and sequencing technologies, while more abundant species produce assemblies with a greater frequency of inversions and insertion/deletions (indels). In sum, our analyses support hybrid assembly as a valuable technique for boosting the reliability and accuracy of assembly-based analyses of ARGs and neighboring genes at environmentally-relevant coverages, provided that sufficient short-read sequencing depth is achieved.

Evaluation of Preparedness and Recovery Needs of Private Well Users After the Great Louisiana Flood of 2016.

CONTEXT: The August 2016 Louisiana flood marked the second 500-year flood in the state in 1 year. OBJECTIVE: The aim of this study was to identify private well user needs in the aftermath of the flood and to develop disaster planning and recovery recommendations for flood-prone well-reliant communities. DESIGN: A descriptive cross-sectional study was conducted to collect information from a convenience sample of flood-impacted well users via surveys and water sampling kits, which were distributed to well users 9 to 11 weeks after floodwaters receded (n = 106). SETTING: Surveys and kits were distributed at roadside flood response and recovery stations set up by local churches in French Settlement, Livingston Parish, Louisiana, an area at the epicenter of the flood-impacted area. PARTICIPANTS: Subjects were included if they self-reported having a flood-impacted well. MAIN OUTCOME MEASURES: Surveys collected information to characterize knowledge gaps, risk perceptions, flood impacts, resource accessibility, and well maintenance barriers. Well water tests evaluated total coliform and Escherichia coli. RESULTS: Among those in low-risk flood zones (n = 22), 27% were in areas designated as having flooded. Among flood-impacted wells that were shock chlorinated after the flood (n = 16), 31.3% tested positive for total coliform and 12.5% for E coli. Only 26% of respondents received well-related information after the disaster. CONCLUSIONS: Results highlight critical needs for disaster planning and well user education in flood-prone areas, changes to flood risk maps, and concerns with the efficacy of disinfection strategies. Information and resources needs for flood-impacted well users are presented and recommendations on how to improve flood preparedness and recovery are made.

Shotgun Metagenomics Reveals Taxonomic and Functional Shifts in Hot Water Microbiome Due to Temperature Setting and Stagnation.

Hot water premise plumbing has emerged as a critical nexus of energy, water, and public health. The composition of hot water microbiomes is of special interest given daily human exposure to resident flora, especially opportunistic pathogens (OPs), which rely on complex microbial ecological interactions for their proliferation. Here, we applied shotgun metagenomic sequencing to characterize taxonomic and functional shifts in microbiomes as a function of water heater temperature setting, stagnation in distal pipes, and associated shifts in water chemistry. A cross-section of samples from controlled, replicated, pilot-scale hot water plumbing rigs representing different temperature settings (39, 42, and 51°C), stagnation periods (8 h vs. 7 days), and time-points, were analyzed. Temperature setting exhibited an overarching impact on taxonomic and functional gene composition. Further, distinct taxa were selectively enriched by specific temperature settings (e.g., Legionella at 39°C vs. Deinococcus at 51°C), while relative abundances of genes encoding corresponding cellular functions were highly consistent with expectations based on the taxa driving these shifts. Stagnation in distal taps diminished taxonomic and functional differences induced by heating the cold influent water to hot water in recirculating line. In distal taps relative to recirculating hot water, reads annotated as being involved in metabolism and growth decreased, while annotations corresponding to stress response (e.g., virulence disease and defense, and specifically antibiotic resistance) increased. Reads corresponding to OPs were readily identified by metagenomic analysis, with L. pneumophila reads in particular correlating remarkably well with gene copy numbers measured by quantitative polymerase chain reaction. Positive correlations between L. pneumophila reads and those of known protozoan hosts were also identified. Elevated proportions of genes encoding metal resistance and hydrogen metabolism were noted, which was consistent with elevated corrosion-induced metal concentrations and hydrogen generation. This study provided new insights into real-world factors influencing taxonomic and functional compositions of hot water microbiomes. Here metagenomics is demonstrated as an effective tool for screening for potential presence, and even quantities, of pathogens, while also providing diagnostic capabilities for assessing functional responses of microbiomes to various operational conditions. These findings can aid in informing future monitoring and intentional control of hot water microbiomes.

Interactive effects of temperature, organic carbon, and pipe material on microbiota composition and Legionella pneumophila in hot water plumbing systems.

BACKGROUND: Several biotic and abiotic factors have been reported to influence the proliferation of microbes, including Legionella pneumophila, in hot water premise plumbing systems, but their combined effects have not been systematically evaluated. Here, we utilize simulated household water heaters to examine the effects of stepwise increases in temperature (32-53 °C), pipe material (copper vs. cross-linked polyethylene (PEX)), and influent assimilable organic carbon (0-700 µg/L) on opportunistic pathogen gene copy numbers and the microbiota composition, as determined by quantitative polymerase chain reaction and 16S rRNA gene amplicon sequencing. RESULTS: Temperature had an overarching influence on both the microbiota composition and L. pneumophila numbers. L. pneumophila peaked at 41 °C in the presence of PEX (1.58 × 105 gene copies/mL). At 53 °C, L. pneumophila was not detected. Several operational taxonomic units (OTUs) persisted across all conditions, accounting for 50% of the microbiota composition from 32 to 49 °C and 20% at 53 °C. Pipe material most strongly influenced microbiota composition at lower temperatures, driven by five to six OTUs enriched with each material. Copper pipes supported less L. pneumophila than PEX pipes (mean 2.5 log10 lower) at temperatures ≤ 41 °C, but showed no difference in total bacterial numbers. Differences between pipe materials diminished with elevated temperature, probably resulting from decreased release of copper ions. At temperatures ≤ 45 °C, influent assimilable organic carbon correlated well with total bacterial numbers, but not with L. pneumophila numbers. At 53 °C, PEX pipes leached organic carbon, reducing the importance of dosed organic carbon. L. pneumophila numbers correlated with a Legionella OTU and a Methylophilus OTU identified by amplicon sequencing. CONCLUSIONS: Temperature was the most effective factor for the control of L. pneumophila, while microbiota composition shifted with each stepwise temperature increase. While copper pipe may also help shape the microbiota composition and limit L. pneumophila proliferation, its benefits might be constrained at higher temperatures. Influent assimilable organic carbon affected total bacterial numbers, but had minimal influence on opportunistic pathogen gene numbers or microbiota composition. These findings provide guidance among multiple control measures for the growth of opportunistic pathogens in hot water plumbing and insight into the mediating role of microbial ecological factors.

Factors Shaping the Human Exposome in the Built Environment: Opportunities for Engineering Control.

The "exposome" is a term describing the summation of one's lifetime exposure to microbes and chemicals. Such exposures are now recognized as major drivers of human health and disease. Because humans spend ∼90% of their time indoors, the built environment exposome merits particular attention. Herein we utilize an engineering perspective to advance understanding of the factors that shape the built environment exposome and its influence on human wellness and disease, while simultaneously informing development of a framework for intentionally controlling the exposome to protect public health. Historically, engineers have been focused on controlling chemical and physical contaminants and on eradicating microbes; however, there is a growing awareness of the role of "beneficial" microbes. Here we consider the potential to selectively control the materials and chemistry of the built environment to positively influence the microbial and chemical components of the indoor exposome. Finally, we discuss research gaps that must be addressed to enable intentional engineering design, including the need to define a "healthy" built environment exposome and how to control it.

A dynamic and complex monochloramine stress response in Escherichia coli revealed by transcriptome analysis.

Despite the widespread use of monochloramine in drinking water treatment, there is surprisingly little information about its mode of action. In this study, DNA microarrays were used to investigate the global gene expression of Escherichia coli cells exposed to sub-lethal concentrations of monochloramine, with a focus on temporal dynamics. Genes induced by monochloramine were associated with several stress response functions, including oxidative stress, DNA repair, multidrug efflux, biofilm formation, antibiotic resistance, and cell wall repair. The diversity of functional associations supports a model of monochloramine action involving multiple cellular targets including cell membranes, nucleic acids, and proteins. These data suggest that E. coli responds to monochloramine exposure by activating diverse defense responses rather than a single antioxidant system and the exposure may also induce biofilm formation. The induction of multidrug efflux pumps and specific antibiotic resistance genes further suggests that exposure to monochloramine may contribute to reduced susceptibility to some antibiotics.

Multiphasic analysis of the temporal development of the distal gut microbiota in patients following ileal pouch anal anastomosis.

BACKGROUND: The indigenous gut microbiota are thought to play a crucial role in the development and maintenance of the abnormal inflammatory responses that are the hallmark of inflammatory bowel disease. Direct tests of the role of the gut microbiome in these disorders are typically limited by the fact that sampling of the microbiota generally occurs once disease has become manifest. This limitation could potentially be circumvented by studying patients who undergo total proctocolectomy with ileal pouch anal anastomosis (IPAA) for the definitive treatment of ulcerative colitis. A subset of patients who undergo IPAA develops an inflammatory condition known as pouchitis, which is thought to mirror the pathogenesis of ulcerative colitis. Following the development of the microbiome of the pouch would allow characterization of the microbial community that predates the development of overt disease. RESULTS: We monitored the development of the pouch microbiota in four patients who underwent IPAA. Mucosal and luminal samples were obtained prior to takedown of the diverting ileostomy and compared to samples obtained 2, 4 and 8 weeks after intestinal continuity had been restored. Through the combined analysis of 16S rRNA-encoding gene amplicons, targeted 16S amplification and microbial cultivation, we observed major changes in structure and function of the pouch microbiota following ileostomy. There is a relative increase in anaerobic microorganisms with the capacity for fermentation of complex carbohydrates, which corresponds to the physical stasis of intestinal contents in the ileal pouch. Compared to the microbiome structure encountered in the colonic mucosa of healthy individuals, the pouch microbial community in three of the four individuals was quite distinct. In the fourth patient, a community that was much like that seen in a healthy colon was established, and this patient also had the most benign clinical course of the four patients, without the development of pouchitis 2 years after IPAA. CONCLUSIONS: The microbiota that inhabit the ileal-anal pouch of patients who undergo IPAA for treatment of ulcerative colitis demonstrate significant structural and functional changes related to the restoration of fecal flow. Our preliminary results suggest once the pouch has assumed the physiologic role previously played by the intact colon, the precise structure and function of the pouch microbiome, relative to a normal colonic microbiota, will determine if there is establishment of a stable, healthy mucosal environment or the reinitiation of the pathogenic cascade that results in intestinal inflammation.

Separation of the bacterial species, Escherichia coli, from mixed-species microbial communities for transcriptome analysis.

BACKGROUND: The study of bacterial species interactions in a mixed-species community can be facilitated by transcriptome analysis of one species in the community using cDNA microarray technology. However, current applications of microarrays are mostly limited to single species studies. The purpose of this study is to develop a method to separate one species, Escherichia coli as an example, from mixed-species communities for transcriptome analysis. RESULTS: E. coli cells were separated from a dual-species (E. coli and Stenotrophomonas maltophilia) community using immuno-magnetic separation (IMS). High recovery rates of E. coli were achieved. The purity of E. coli cells was as high as 95.0% separated from suspended mixtures consisting of 1.1 - 71.3% E. coli, and as high as 96.0% separated from biofilms with 8.1% E. coli cells. Biofilms were pre-dispersed into single-cell suspensions. The reagent RNAlater (Ambion, Austin, TX) was used during biofilm dispersion and IMS to preserve the transcriptome of E. coli. A microarray study and quantitative PCR confirmed that very few E. coli genes (only about eight out of 4,289 ORFs) exhibited a significant change in expression during dispersion and separation, indicating that transcriptional profiles of E. coli were well preserved. CONCLUSIONS: A method based on immuno-magnetic separation (IMS) and application of RNAlater was developed to separate a bacterial species, E. coli as an example, from mixed-species communities while preserving its transcriptome. The method combined with cDNA microarray analysis should be very useful to study species interactions in mixed-species communities.

Bayesian network expansion identifies new ROS and biofilm regulators.

Signaling and regulatory pathways that guide gene expression have only been partially defined for most organisms. However, given the increasing number of microarray measurements, it may be possible to reconstruct such pathways and uncover missing connections directly from experimental data. Using a compendium of microarray gene expression data obtained from Escherichia coli, we constructed a series of Bayesian network models for the reactive oxygen species (ROS) pathway as defined by EcoCyc. A consensus Bayesian network model was generated using those networks sharing the top recovered score. This microarray-based network only partially agreed with the known ROS pathway curated from the literature and databases. A top network was then expanded to predict genes that could enhance the Bayesian network model using an algorithm we termed 'BN+1'. This expansion procedure predicted many stress-related genes (e.g., dusB and uspE), and their possible interactions with other ROS pathway genes. A term enrichment method discovered that biofilm-associated microarray data usually contained high expression levels of both uspE and gadX. The predicted involvement of gene uspE in the ROS pathway and interactions between uspE and gadX were confirmed experimentally using E. coli reporter strains. Genes gadX and uspE showed a feedback relationship in regulating each other's expression. Both genes were verified to regulate biofilm formation through gene knockout experiments. These data suggest that the BN+1 expansion method can faithfully uncover hidden or unknown genes for a selected pathway with significant biological roles. The presently reported BN+1 expansion method is a generalized approach applicable to the characterization and expansion of other biological pathways and living systems.

[Effects of acid soils on the biodegradation of hydrocarbons].

Biodegradation efficiency of hydrocarbons and melioration of micro-ecosystem conditions in acid soils should be seriously concerned due to either occurrence of acid polluted soils or acidification during bioremediation process. The influence of acid situation on degrading microbes and the biodegradation rate were figured out by monitoring variations of biomass, microbial activities and petroleum contents with time in acid and alkaline polluted soils in laboratory. Injecting degrading microbes and meliorating micro-ecosystem conditions of acid soils were conducted. The results showed that acid soils (pH = 5.4-5.7) had extreme restraint on local microbe numbers and activities, and biodegradation rate almost reached zero. Injection of degrading microbes could not remarkably reduce the restriction of acid conditions. The microbe numbers quickly went down from 10(6) cells/g dried soil to zero in 14 days and the Fluorescein Diacetate (FDA) activities were only about 0.10 Abs/g dried soil. However, addition of bio-carriers could effectively improve micro-ecosystem conditions in acid soils, thus notably diminish the restraint to some extent. In 19 days, the amount of microorganisms decreased from 2 x 10(6) to 2.2 x 10(2) cells/g dried soil. And up to 49 days, about 13% hydrocarbons were removed from the polluted soils by microbes.