Chronic shedding of a SARS-CoV-2 Alpha variant in wastewater.

BACKGROUND: Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program since 2021. Wastewater samples were collected from on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR and results were reported to the health department. RESULTS: One rural, off-campus site consistently produced higher concentrations of SARS-CoV-2 genome copies. Samples from this site were sequenced and contained predominately a derivative of Alpha variant lineage B.1.1.7, detected from fall 2021 through summer 2023. Mutational analysis of reconstructed genes revealed divergence from the Alpha variant lineage sequence over time, including numerous mutations  in the Spike RBD and NTD. CONCLUSIONS: We discuss the possibility that a chronic SARS-CoV-2 infection accumulated adaptive mutations that promoted long-term infection. This study reveals that small wastewater treatment plants can enhance resolution of rare events and facilitate reconstruction of viral genomes due to the relative lack of contaminating sequences.

SARS-CoV-2 wastewater monitoring in rural and small metropolitan communities in Central Michigan.

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program throughout the 2021-2022 academic year. Wastewater samples were collected weekly from ten on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR. Case data reported by Central Michigan District Health Department and CMU were collected and compared with wastewater data. During the delta wave, wastewater detection and on-campus case reports increased rapidly with the start of the academic semester and peaked quickly, compared with a more gradual and prolonged increase in detection and case reports off-campus. During the omicron wave, transmission dynamics were similar on-campus and off-campus. Normalization of on-campus and off-campus wastewater data with pepper mild mottle virus gene expression suggested lower SARS-CoV-2 shedding per person in on-campus compared to off-campus samples during the delta wave, but no difference in virus shedding during the omicron wave. We discuss the possibility that a higher on-campus vaccination rate may have reduced virus shedding per person during the delta wave, but that this effect was lost with the omicron variant. This study suggests that wastewater monitoring is effective in rural and small metropolitan communities when used in conjunction with case reports to understand regional transmission dynamics and the impact of public health policies at a public university on virus shedding in the community.

Genetic Determinants of Escherichia coli Survival in Beach Sand.

Escherichia coli contain a high level of genetic diversity and are generally associated with the guts of warm-blooded animals but have also been isolated from secondary habitats outside hosts. We used E. coli isolates from previous in situ microcosm experiments conducted under actual beach conditions and performed population-level genomic analysis to identify accessory genes associated with survival within the beach sand environment. E. coli strains capable of surviving had been selected for by seeding isolates originating from sand, sewage, and gull waste (n = 528; 176 from each source) into sand, which was sealed in microcosm chambers and buried for 45 days in the backshore beach of Lake Michigan. In the current work, survival-associated genes were identified by comparing the pangenome of viable E. coli populations at the end of the microcosm experiment with the original isolate collection and identifying loci enriched in the out put samples. We found that environmental survival was associated with a wide variety of genetic factors, with the majority corresponding to metabolism enzymes and transport proteins. Of the 414 unique functions identified, most were present across E. coli phylogroups, except B2 which is often associated with human pathogens. Gene modules that were enriched in surviving populations included a betaine biosynthesis pathway, which produces an osmoprotectant, and the GABA (gamma-aminobutyrate) biosynthesis pathway, which aids in pH homeostasis and nutrient use versatility. Overall, these results demonstrate that the genetic flexibility within this species allows for survival in the environment for extended periods. IMPORTANCE Escherichia coli is commonly used as an indicator of recent fecal pollution in recreational water despite its known ability to survive in secondary environments, such as beach sand. These long-term survivors from sand reservoirs can be introduced into the water column through wave action or runoff during precipitation events, thereby impacting the perception of local water quality. Current beach monitoring methods cannot differentiate long-term environmental survivors from E. coli derived from recent fecal input, resulting in inaccurate monitoring results and unnecessary beach closures. This work identified the genetic factors that are associated with long-term survivors, providing insight into the mechanistic basis for E. coli accumulation in beach sand. A greater understanding of the intrinsic ability of E. coli to survive long-term and conditions that promote such survival will provide evidence of the limitations of beach water quality assessments using this indicator.

Comparative genomics of 16 Microbacterium spp. that tolerate multiple heavy metals and antibiotics.

A total of 16 different strains of Microbacterium spp. were isolated from contaminated soil and enriched on the carcinogen, hexavalent chromium [Cr(VI)]. The majority of the isolates (11 of the 16) were able to tolerate concentrations (0.1 mM) of cobalt, cadmium, and nickel, in addition to Cr(VI) (0.5-20 mM). Interestingly, these bacteria were also able to tolerate three different antibiotics (ranges: ampicillin 0-16 µg ml-1, chloramphenicol 0-24 µg ml-1, and vancomycin 0-24 µg ml-1). To gain genetic insight into these tolerance pathways, the genomes of these isolates were assembled and annotated. The genomes of these isolates not only have some shared genes (core genome) but also have a large amount of variability. The genomes also contained an annotated Cr(VI) reductase (chrR) that could be related to Cr(VI) reduction. Further, various heavy metal tolerance (e.g., Co/Zn/Cd efflux system) and antibiotic resistance genes were identified, which provide insight into the isolates' ability to tolerate metals and antibiotics. Overall, these isolates showed a wide range of tolerances to heavy metals and antibiotics and genetic diversity, which was likely required of this population to thrive in a contaminated environment.

Detection of Influenza A viruses at migratory bird stopover sites in Michigan, USA.

Introduction: Influenza A viruses have the potential to cause devastating illness in humans and domestic poultry. Wild birds are the natural reservoirs of Influenza A viruses and migratory birds are implicated in their global dissemination. High concentrations of this virus are excreted in the faeces of infected birds and faecal contamination of shared aquatic habitats can lead to indirect transmission among birds via the faecal-oral route. The role of migratory birds in the spread of avian influenza has led to large-scale surveillance efforts of circulating avian influenza viruses through direct sampling of live and dead wild birds. Environmental monitoring of bird habitats using molecular detection methods may provide additional information on the persistence of influenza virus at migratory stopover sites distributed across large spatial scales. Materials and methods: In the current study, faecal and water samples were collected at migratory stopover sites and evaluated for Influenza A by real-time quantitative reverse transcriptase PCR. Results and Discussion: This study found that Influenza A was detected at 53% of the evaluated stopover sites, and 7% and 4.8% of the faecal and water samples, respectively, tested positive for Influenza A virus. Conclusion: Environmental monitoring detected Influenza A at stopover sites used by migratory birds.

Potential for gulls to transport bacteria from human waste sites to beaches.

Contamination of recreational beaches due to fecal waste from gulls complicates beach monitoring and may pose a risk to public health. Gulls that feed at human waste sites may ingest human fecal microorganisms associated with that waste. If these gulls also visit beaches, they may serve as vectors, transporting fecal microorganisms to the beach where they may subsequently contaminate sand and water. In this study, samples collected from landfills, treated wastewater storage lagoons, and public beaches demonstrated a spatial and temporal overlap of markers for gull and human-associated microorganisms. In addition, markers for gull, fecal indicator bacteria, and the human-associated marker, HF183, were detected in gull feces and cloacae samples. Further, HF183 was detected in cloacae samples from gulls that were documented by radio-telemetry traveling between human waste sites and public beaches. This study highlights the potential for gulls that visit human waste sites to disperse human-associated microorganisms in the beach landscape.

Influence of Land Use, Nutrients, and Geography on Microbial Communities and Fecal Indicator Abundance at Lake Michigan Beaches.

Microbial communities within beach sand play a key role in nutrient cycling and are important to the nearshore ecosystem function. Escherichia coli and enterococci, two common indicators of fecal pollution, have been shown to persist in the beach sand, but little is known about how microbial community assemblages are related to these fecal indicator bacteria (FIB) reservoirs. We examined eight beaches across a geographic gradient and range of land use types and characterized the indigenous community structure in the water and the backshore, berm, and submerged sands. FIB were found at similar levels in sand at beaches adjacent to urban, forested, and agricultural land and in both the berm and backshore. However, there were striking differences in the berm and backshore microbial communities, even within the same beach, reflecting the very different environmental conditions in these beach zones in which FIB can survive. In contrast, the microbial communities in a particular beach zone were similar among beaches, including at beaches on opposite shores of Lake Michigan. The differences in the microbial communities that did exist within a beach zone correlated to nutrient levels, which varied among geographic locations. Total organic carbon and total phosphorus were higher in Wisconsin beach sand than in beach sand from Michigan. Within predominate genera, fine-scale sequence differences could be found that distinguished the populations from the two states, suggesting a biogeographic effect. This work demonstrates that microbial communities are reflective of environmental conditions at freshwater beaches and are able to provide useful information regarding long-term anthropogenic stress.

Cryptic lineages of the genus Escherichia.

Extended multilocus sequence typing (MLST) analysis of atypical Escherichia isolates was used to identify five novel phylogenetic clades (CI to CV) among isolates from environmental, human, and animal sources. Analysis of individual housekeeping loci showed that E. coli and its sister clade, CI, remain largely indistinguishable and represent nascent evolutionary lineages. Conversely, clades of similar age (CIII and CIV) were found to be phylogenetically distinct. When all Escherichia lineages (named and unnamed) were evaluated, we found evidence that Escherichia fergusonii has evolved at an accelerated rate compared to E. coli, CI, CIII, CIV, and CV, suggesting that this species is younger than estimated by the molecular clock method. Although the five novel clades were phylogenetically distinct, we were unable to identify a discriminating biochemical marker for all but one of them (CIII) with traditional phenotypic profiling. CIII had a statistically different phenotype from E. coli that resulted from the loss of sucrose and sorbitol fermentation and lysine utilization. The lack of phenotypic distinction has likely hindered the ability to differentiate these clades from typical E. coli, and so their ecological significance and importance for applied and clinical microbiology are yet to be determined. However, our sampling suggests that CIII, CIV, and CV represent environmentally adapted Escherichia lineages that may be more abundant outside the host gastrointestinal tract.

Genetic diversity and population structure of Escherichia coli isolated from freshwater beaches.

Escherichia coli is an important member of the gastrointestinal tract of humans and warm-blooded animals (primary habitat). In the external environment outside the host (secondary habitat), it is often considered to be only a transient member of the microbiota found in water and soil, although recent evidence suggests that some strains can persist in temperate soils and freshwater beaches. Here we quantified the population genetic structure of E. coli from a longitudinal collection of environmental strains isolated from six freshwater beaches along Lake Huron and the St. Clair River in Michigan. Multilocus enzyme electrophoresis (MLEE) and multilocus sequence typing (MLST) revealed extensive genetic diversity among 185 E. coli isolates with an average of 40 alleles per locus. Despite evidence for extensive recombination generating new alleles and genotypic diversity, several genotypes marked by distinct MLEE and MLST profiles were repeatedly recovered from separate sites at different times. A PCR-based phylogrouping technique showed that the persistent, naturalized E. coli belonged to the B1 group. These results support the hypothesis that persistent genotypes have an adaptive advantage in the secondary habitat outside the host.

Spatial and temporal changes in microbial community structure associated with recharge-influenced chemical gradients in a contaminated aquifer.

In a contaminated water-table aquifer, we related microbial community structure on aquifer sediments to gradients in 24 geochemical and contaminant variables at five depths, under three recharge conditions. Community amplified ribsosomal DNA restriction analysis (ARDRA) using universal 16S rDNA primers and denaturing gradient gel electrophoresis (DGGE) using bacterial 16S rDNA primers indicated: (i). communities in the anoxic, contaminated central zone were similar regardless of recharge; (ii). after recharge, communities at greatest depth were similar to those in uncontaminated zones; and (iii). after extended lack of recharge, communities at upper and lower aquifer margins differed from communities at the same depths on other dates. General aquifer geochemistry was as important as contaminant or terminal electron accepting process (TEAP) chemistry in discriminant analysis of community groups. The Shannon index of diversity (H) and the evenness index (E), based on DGGE operational taxonomic units (OTUs), were statistically different across community groups and aquifer depths. Archaea or sulphate-reducing bacteria 16S rRNA abundance was not clearly correlated with TEAP chemistry indicative of methanogenesis or sulphate reduction. Eukarya rRNA abundance varied by depth and date from 0 to 13% of the microbial community. This contaminated aquifer is a dynamic ecosystem, with complex interactions between physical, chemical and biotic components, which should be considered in the interpretation of aquifer geochemistry and in the development of conceptual or predictive models for natural attenuation or remediation.