Fecal indicator organisms are measured to indicate the presence of fecal pollution, yet the association between indicators and pathogens varies by context. The goal of this study was to empirically evaluate the relationships between indicator Escherichia coli, microbial source tracking markers, select enteric pathogen genes, and potential sources of enteric pathogens in 600 rural Bangladeshi households. We measured indicators and pathogen genes in stored drinking water, soil, and on mother and child hands. Additionally, survey and observational data on sanitation and domestic hygiene practices were collected. Log₁₀ concentrations of indicator E. coli were positively associated with the prevalence of pathogenic E. coli genes in all sample types. Given the current need to rely on indicators to assess fecal contamination in the field, it is significant that in this study context indicator E. coli concentrations, measured by IDEXX Colilert-18, provided quantitative information on the presence of pathogenic E. coli in different sample types. There were no significant associations between the human fecal marker (HumM2) and human-specific pathogens in any environmental sample type. There was an increase in the prevalence of Giardia lamblia genes, any E. coli virulence gene, and the specific E. coli virulence genes stx1/2 with every log₁₀ increase in the concentration of the animal fecal marker (BacCow) on mothers’ hands. Thus, domestic animals were important contributors to enteric pathogens in these households.

Diarrheal illnesses from enteric pathogens are a leading cause of death in children under five in low-and-middle income countries (LMICs). Sanitation is one way to reduce the spread of enteric pathogens in the environment; however, few studies have investigated the effectiveness of sanitation in rural LMICs in reducing pathogens in the environment. In this study, we measured the impact of a sanitation intervention (dual pit latrines, sani-scoop, child potties delivered as part of a randomized control trial, WASH Benefits) in rural Bangladeshi household compounds by assessing prevalence ratios, differences, and changes in concentration of pathogen genes and host-specific fecal markers. We found no difference in prevalence of pathogenic E. coli, norovirus, or Giardia genes in the domestic environment in the sanitation and control arms. The prevalence of the human fecal marker was lower on child hands and the concentration of animal fecal marker was lower on mother hands in the sanitation arm in adjusted models, but these associations were not significant after correcting for multiple comparisons. In the subset of households with ≥ 10 individuals per compound, the prevalence of enterotoxigenic E. coli genes on child hands was lower in the sanitation arm. Incomplete removal of child and animal feces or the compound (versus community-wide) scale of intervention could explain the limited impacts of improved sanitation.

Effective methods for simultaneously measuring viral, bacterial, protozoan, and fungal pathogens in wastewater are needed. Here, we investigate how the sample type and nucleic acid extraction protocols affect broad-range pathogen detection. We compared methods for analyzing wastewater solids and whole influent by dPCR detection of spiked and endogenous targets including DNA and RNA viruses (mpox, norovirus, SARS-CoV-2), bacteria (Clostridium difficile, Campylobacter jejuni), protozoa (Cryptosporidium spp.), fungi (Candida auris), and antibiotic resistance genes. Using selected methods, we then analyzed date-matched (1) solid, (2) centrifuged influent, and (3) whole influent samples collected from 11 facilities at three time points. We demonstrate that one workflow can be used to simultaneously detect all of the targets. Comparing normalization of targets by concentration of PMMoV, carjivirus, and 16S rRNA, we show that using different controls together can complicate the interpretation of concentrations across targets. We observed similar detection rates and nucleic acid concentrations for the targets examined in this study across all three sample types with slightly higher concentrations in centrifuged influent. These results suggest that centrifuged influent is a viable option and can circumvent the limitation of varying residence times for primary settled solids.

Environmental surveillance and clinical diagnostics heavily rely on the polymerase chain reaction (PCR) for target detection. A growing list of microbial threats warrants new PCR-based detection methods that are highly sensitive, specific, and multiplexable. Here, we introduce a PCR-based icosaplex (20-plex) assay for detecting 18 enteropathogen and two antimicrobial resistance genes. This multiplexed PCR assay leverages the self-avoiding molecular recognition system (SAMRS) to avoid primer dimer formation, the artificially expanded genetic information system (AEGIS) for amplification specificity, and next-generation sequencing for amplicon identification. Using parallelized multitarget TaqMan Array Cards (TAC) to benchmark performance of the 20-plex assay on wastewater, soil, and human stool samples, we found 90% agreement on positive calls and 89% agreement on negative calls. Additionally, we show how long-read and short-read sequencing information from the 20-plex can be used to further classify allelic variants of genes and distinguish subspecies. The strategy presented offers sensitive, affordable, and robust multiplex detection that can be used to support efforts in wastewater-based epidemiology, environmental monitoring, and human/animal diagnostics.

Inadequate water, sanitation, and hygiene (WASH) infrastructure may increase exposure to antimicrobial resistance (AMR). In addition, close human-animal interactions and unregulated antibiotic use in livestock facilitate the spread of resistant bacteria. We use metagenomic sequence data and multivariate models to assess how animal exposure and WASH conditions affect the gut resistome and microbiome in 53 pregnant women and 84 children in Ecuador. Here we show improving WASH infrastructure and managing animal exposure may be important in reducing AMR but could also reduce taxonomic diversity in the gut. Escherichia coli , Klebsiella pneumoniae , and clinically relevant antimicrobial resistance genes (ARGs) are detected across all age groups, but the highest abundance is found in children compared to mothers. In mothers, higher animal exposure trends towards a higher number of unique ARGs compared to low animal exposure and is significantly associated with greater taxonomic diversity. In addition, mothers with sewer systems or septic tanks and piped drinking water have fewer unique ARGs compared to those without, and mothers with longer duration of drinking water access have lower total ARG abundance. In contrast, few associations are observed in children, likely due to the dynamic nature of the gut microbiome during early childhood.