Escherichia coli pathotypes (i.e., enteropathogenic and enterotoxigenic) have been identified among the pathogens most responsible for moderate-to-severe diarrhea in low- and middle-income countries (LMICs). Pathogenic E. coli are transmitted from infected human or animal feces to new susceptible hosts via environmental reservoirs such as hands, water, and soil. Commensal E. coli, which includes nonpathogenic E. coli strains, are widely used as fecal bacteria indicator, with their presence associated with increased likelihood of enteric pathogens and/or diarrheal disease. In this study, we investigated E. coli contamination in environmental reservoirs within households (N = 142) in high-population density communities of Harare, Zimbabwe. We further assessed the interconnectedness of the environmental compartments by investigating associations between, and household-level risk factors for, E. coli contamination. From the data we collected, the source and risk factors for E. coli contamination are not readily apparent. One notable exception is the presence of running tap water on the household plot, which is associated with significantly less E. coli contamination of drinking water, handwashing water, and hands after handwashing. In addition, E. coli levels on hands after washing are significantly associated with handwashing water contamination, hand contamination before washing, and diarrhea incidence. Finally, we observed that animal ownership increases E. coli contamination in soil, and E. coli in soil are correlated with contamination on hands before washing. This study highlights the complexity of E. coli contamination in household environments within LMICs. More, larger, studies are needed to better identify sources and exposure pathways of E. coli—and enteric pathogens generally—to identify effective interventions.

• Wastewater irrigation did not impact soil antibiotic resistance bacterial levels. • Biosolids soil amendment increased antibiotic-resistant E. coli and enterococci. • Antibiotic-resistant bacteria persisted in soil with presence on carrots at harvest. • Antibiotic-resistant bacteria decayed faster than total counterparts.

Biosolids land application introduces antibiotic resistance genes (ARGs) and clinically relevant pathogens into agricultural soils, raising concerns about long-term environmental and public health impacts. Despite growing interest in biosolids reuse, there remains a critical need for replicated, longitudinal studies to assess how biosolids amendments shape soil microbiomes and resistomes during crop cultivation. In this replicated longitudinal greenhouse study, we used shotgun metagenomics to characterize the impact of biosolids amendment on the soil microbiome, resistome, virulence factors, and ESKAPE pathogens during carrot cultivation. Biosolids-amended soils exhibited increased richness of microbial genera (e.g., Rhodanobacter, Dyella, and Thermomonas), ARG subtypes (resistance to sulfonamide, tetracycline, fosmidomycin, and macrolides), and virulence factors compared to pristine controls. Notably, all six ESKAPE pathogens, including Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., were detected at elevated relative abundances (1.4- and 3.4-fold) in biosolids-amended soils and remained detectable throughout the 11-week cultivation period. Network analysis revealed statistically supported co-occurrences between microbial taxa and ARGs (with resistance to tetracyclines, beta-lactams, chloramphenicol, and multidrugs), suggesting possible host associations. These findings underscore the ecological and clinical relevance of biosolids amendment and highlight the need for integrated surveillance frameworks to mitigate antimicrobial resistance dissemination in agricultural environments.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli pose a serious threat to human health because of their resistance to the most commonly prescribed antibiotics: penicillins and cephalosporins. In this study, we provide a genomic and metagenomic context for the determinant beta-lactam resistance genes of ESBL-positive E. coli isolated from various wastewater treatment utilities in Oregon, USA. Class A beta-lactamase genes on chromosomes ( bla CTX-M, bla TEM) were clustered with antibiotic resistance genes associated with other classes of antibiotics (sulfonamides and aminoglycosides) along with insertional elements. ESBL genes such as bla CTX-M, bla TEM, and bla SHV were also detected on conjugable plasmids of IncF and IncI incompatibility types. One novel IncF plasmid (pSHV2A_ESBLF) was identified, which carried a multidrug resistance genotype ( bla SHV-2A, aadA22 , aac 3, aph 6, tet A, and sul 1) in addition to a mer (mercury resistance) operon, colicin, and aerobactin genes. Shotgun metagenomic analysis of the ESBL-producing E. coli -originating wastewater samples showed the presence of class A beta-lactamases; however, the ESBL genes identified in the E. coli genomes were below the detection limits. Other ESBL-associated genes (i.e., bla OXA.11, bla FOX.7, and bla GES.17) were identified in the wastewater samples, and their occurrences were correlated with the core microbial genera (e.g., Paraprevotella ). In the E. coli genomes and wastewater samples, tetracycline, aminoglycoside, and beta-lactam resistance determinants frequently co-occurred. The combination of whole-genome and metagenomic analysis provides a holistic description of ESBL-producing organisms and genes in wastewater systems. IMPORTANCE: Using a hybrid sequencing and assembly strategy (short- and long-read sequencing), we identified the distribution of ARGs and virulence factors harbored on plasmids and chromosomes. We further characterized plasmids’ incompatibility types and the co-occurrences of ARGs and virulence factors on plasmids and chromosomes. We investigated the transferability of plasmid-mediated beta-lactams via conjugation. Finally, using shotgun metagenomic analysis of the ESBL-producing Escherichia coli -originated wastewater samples, we described the microbial community, the resistome composition, and the potential associations with plasmid-mediated beta-lactam genes and other ARGs.