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Low- and middle-income countries (LMICs) bear the largest mortality burden of antibiotic-resistant infections. Small-scale animal production and free-roaming domestic animals are common in many LMICs, yet data on zoonotic exchange of gut bacteria and antibiotic resistance genes (ARGs) in low-income communities are sparse. Differences between rural and urban communities with regard to population density, antibiotic use, and cohabitation with animals likely influence the frequency of transmission of gut bacterial communities and ARGs between humans and animals. Here, we determined the similarity in gut microbiomes, using 16S rRNA gene amplicon sequencing, and resistomes, using long-read metagenomics, between humans, chickens, and goats in a rural community compared to an urban community in Bangladesh. Gut microbiomes were more similar between humans and chickens in the rural (where cohabitation is more common) than the urban community, but there was no difference for humans and goats in the rural versus the urban community. Human and goat resistomes were more similar in the urban community, and ARG abundance was higher in urban animals than rural animals. We identified substantial overlap of ARG alleles in humans and animals in both settings. Humans and chickens had more overlapping ARG alleles than humans and goats. All fecal hosts from the urban community and rural humans carried ARGs on chromosomal contigs classified as potentially pathogenic bacteria, including Escherichia coli, Campylobacter jejuni, Clostridioides difficile, and Klebsiella pneumoniae. These findings provide insight into the breadth of ARGs circulating within human and animal populations in a rural compared to urban community in Bangladesh. IMPORTANCE While the development of antibiotic resistance in animal gut microbiomes and subsequent transmission to humans has been demonstrated in intensive farming environments and high-income countries, evidence of zoonotic exchange of antibiotic resistance in LMIC communities is lacking. This research provides genomic evidence of overlap of antibiotic resistance genes between humans and animals, especially in urban communities, and highlights chickens as important reservoirs of antibiotic resistance. Chicken and human gut microbiomes were more similar in rural Bangladesh, where cohabitation is more common. Incorporation of long-read metagenomics enabled characterization of bacterial hosts of resistance genes, which has not been possible in previous culture-independent studies using only short-read sequencing. These findings highlight the importance of developing strategies for combatting antibiotic resistance that account for chickens being reservoirs of ARGs in community environments, especially in urban areas.

Indirect exposure to waterborne viruses increases the risk of infection, especially among children with frequent hand-to-mouth contacts. Here, we quantified the transfer of one bacteriophage (MS2) and two enteric viruses (adenovirus and coxsackievirus) from liquid to skin. MS2, a commonly used enteric virus surrogate, was used to compare virus transfer rates in a volunteer trial to those obtained using human cadaver skin and synthetic skin. MS2 transfer to volunteer skin was similar to transfer to cadaver skin but significantly different from transfer to synthetic skin. The transfer of MS2, adenovirus, and coxsackievirus to cadaver skin was modeled using measurements for viruses attaching to the skin (adsorbed) and viruses in liquid residual on skin (unadsorbed). We find virus transfer per surface area is a function of the concentration of virus in the liquid and the film thickness of liquid retained on the skin and is estimable using a linear model. Notably, the amount of MS2 adsorbed on the skin was on average 5 times higher than the amount of adenovirus and 4 times higher than the amount of coxsackievirus. Quantification of pathogenic virus retention to skin would thus be overestimated using MS2 adsorption data. This study provides models of virus transfer useful for risk assessments of water-related activities, demonstrates significant differences in the transfer of pathogenic virus and MS2, and suggests cadaver skin as an alternative testing system for studying interactions between viruses and skin.IMPORTANCE Enteric viruses (viruses that infect the gastrointestinal tract) are responsible for most water-transmitted diseases. They are shed in high concentrations in the feces of infected individuals, persist for an extended period of time in water, and are highly infective. Exposure to contaminated water directly (through ingestion) or indirectly (for example, through hand-water contacts followed by hand-to-mouth contacts) increases the risk of virus transmission. The work described herein provides a quantitative model for estimating human-pathogenic virus retention on skin following contact with contaminated water. The work will be important in refining the contribution of indirect transmission of virus to risks associated with water-related activities.

A study was conducted to examine the diversity and composition of denitrifying bacteria that vary across environmental gradients in both nitrate and salinity within an unconfined beach aquifer. The findings suggest that diversity in these enzymes results from adaptation to a particular environment rather than just random microheterogeneity alone.

Wastewater-based epidemiology (WBE) expanded rapidly in response to the COVID-19 pandemic. As the public health emergency has ended, researchers and practitioners are looking to shift the focus of existing wastewater surveillance programs to other targets, including bacteria. Bacterial targets may pose some unique challenges for WBE applications. To explore the current state of the field, the National Science Foundation-funded Research Coordination Network (RCN) on Wastewater Based Epidemiology for SARS-CoV-2 and Emerging Public Health Threats held a workshop in April 2023 to discuss the challenges and needs for wastewater bacterial surveillance. The targets and methods used in existing programs were diverse, with twelve different targets and nine different methods listed. Discussions during the workshop highlighted the challenges in adapting existing programs and identified research gaps in four key areas: choosing new targets, relating bacterial wastewater data to human disease incidence and prevalence, developing methods, and normalizing results. To help with these challenges and research gaps, the authors identified steps the larger community can take to improve bacteria wastewater surveillance. This includes developing data reporting standards and method optimization and validation for bacterial programs. Additionally, more work is needed to understand shedding patterns for potential bacterial targets to better relate wastewater data to human infections. Wastewater surveillance for bacteria can help provide insight into the underlying prevalence in communities, but much work is needed to establish these methods.IMPORTANCEWastewater surveillance was a useful tool to elucidate the burden and spread of SARS-CoV-2 during the pandemic. Public health officials and researchers are interested in expanding these surveillance programs to include bacterial targets, but many questions remain. The NSF-funded Research Coordination Network for Wastewater Surveillance of SARS-CoV-2 and Emerging Public Health Threats held a workshop to identify barriers and research gaps to implementing bacterial wastewater surveillance programs., Competing Interests: RUH is managing member of AquaVitas, LLC and founder of the ASU non-profit project OneWaterOneHealth operating in the same intellectual space. LPS is employed by Ceres Nanosciences.

Microbial source tracking (MST) identifies sources of fecal contamination in the environment using host-associated fecal markers. While there are numerous bacterial MST markers that can be used herein, there are few such viral markers. Here, we designed and tested novel viral MST markers based on tomato brown rugose fruit virus (ToBRFV) genomes. We assembled eight nearly complete genomes of ToBRFV from wastewater and stool samples from the San Francisco Bay Area in the United States. Next, we developed two novel probe-based reverse transcription-PCR (RT-PCR) assays based on conserved regions of the ToBRFV genome and tested the markers' sensitivities and specificities using human and non-human animal stool as well as wastewater. The ToBRFV markers are sensitive and specific; in human stool and wastewater, they are more prevalent and abundant than a commonly used viral marker, the pepper mild mottle virus (PMMoV) coat protein (CP) gene. We used the assays to detect fecal contamination in urban stormwater samples and found that the ToBRFV markers matched cross-assembly phage (crAssphage), an established viral MST marker, in prevalence across samples. Taken together, these results indicate that ToBRFV is a promising viral human-associated MST marker. IMPORTANCE Human exposure to fecal contamination in the environment can cause transmission of infectious diseases. Microbial source tracking (MST) can identify sources of fecal contamination so that contamination can be remediated and human exposures can be reduced. MST requires the use of host-associated MST markers. Here, we designed and tested novel MST markers from genomes of tomato brown rugose fruit virus (ToBRFV). The markers are sensitive and specific to human stool and highly abundant in human stool and wastewater samples., Competing Interests: The authors declare a conflict of interest. A.N., B.J.F., M.K.W., and A.B.B., are co-inventors on a U.S. provisional patent application #63/387,657 that has been filed and relates to the methods presented in this manuscript. The other authors declare no competing interests.