Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.

Escherichia coli O157:H7 is a major food safety concern through contamination of beef and produce. Non-antibiotic interventions to minimize O157:H7 in food animals and products are highly desired and one strategy to improve food safety is to reduce O157:H7 in cattle, a main asymptomatic reservoir, through probiotic administration. Non-pathogenic E. coli populating the intestine represent a compelling probiotic source, as these strains are already host-adapted. The challenge is to identify non-pathogenic strains capable of competing with O157:H7 for nutrients or those producing compounds to inhibit O157:H7 growth. Here, E. coli isolated from cattle fecal and recto-anal junction swab samples were sequenced and screened for the ability to compete with O157:H7. Fourteen genetically distinct, non-Shiga toxin-encoding, non-O157:H7 E. coli strains were recovered, and individual isolates representative of each strain were assessed. Nearly all strains possessed complete genetic pathways for the utilization of carbon and nitrogen sources required for O157:H7 colonization of the cattle intestine. Growth curve assays were conducted, and growth metrics were compared between bovine non-O157:H7 E. coli isolates and two O157:H7 isolates. While no strain outperformed both O157:H7 strains for all nutrients tested, at least one strain outperformed O157:H7 for each of the carbon sources tested. No strain grew significantly better than O157:H7 in media supplemented with ethanolamine. A “highly competitive” consortium of 4 non-O157:H7 isolates that grew significantly better than O157:H7 reduced O157:H7 counts CFU/mL by 1.53 log₁₀ and >0.72 log₁₀ under anaerobic and aerobic conditions, respectively, in competition assays. A consortium of “low-competitive” strains reduced O157:H7 counts by >0.47 log₁₀ and >0.51 log₁₀ under anaerobic and aerobic conditions. These results suggest that cattle harbor non-O157:H7 E. coli strains capable of limiting O157:H7 growth in vitro. Surveys of commensal non-O157:H7 isolates from cattle using growth-based phenotypic assays may be useful in identifying E. coli strains capable of outcompeting O157:H7 in the bovine intestine for further in vivo testing as probiotics.