Analyzing Antibiotic Resistance in Bacteria from Wastewater in Pakistan Using Whole-Genome Sequencing

Abstract

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.

Keywords: Pakistan; antibiotic resistance; carbapenem; plasmid; wastewater; whole-genome sequencing.

Figure 1

Summary of ARGs detected in the sequenced isolates, (A) E. coli and (B) rest of the isolates. The species column indicates the predicted species for each isolate. The phylotypes and serotypes for E. coli were determined along with the MLST for each isolate if the scheme was available. * Pasteur MLST scheme for Acinetobacter baumannii 51AC1. “-” in the MLST indicates that the MLST scheme is not available or an unknown MLST. The total ARGs are summarized by bacterial isolate identified in silico, and conferring resistance as summarized by antibiotic category. (A) Summary of ARGs, point mutations and plasmid replicons in E. coli isolates from this study. (B) The ARGs in the sequenced isolates are those belonging to Klebsiella and other individual species sequenced in this study. BTL, β-lactams; QNL, quinolone; SUL, sulfonamide; TMP, trimethoprim; AMG, aminoglycoside; C, chloramphenicol; FOS, Fosfomycin; M, macrolide; T, tetracycline; G, Glycopeptide; EFP; multi-drug efflux RND transporters; Q; quaternary ammonium compound; T; point mutations for tigecycline. FD, fosmidomycin; Plasmids; the plasmids replicons hits for each isolate.

Figure 1

Summary of ARGs detected in the sequenced isolates, (A) E. coli and (B) rest of the isolates. The species column indicates the predicted species for each isolate. The phylotypes and serotypes for E. coli were determined along with the MLST for each isolate if the scheme was available. * Pasteur MLST scheme for Acinetobacter baumannii 51AC1. “-” in the MLST indicates that the MLST scheme is not available or an unknown MLST. The total ARGs are summarized by bacterial isolate identified in silico, and conferring resistance as summarized by antibiotic category. (A) Summary of ARGs, point mutations and plasmid replicons in E. coli isolates from this study. (B) The ARGs in the sequenced isolates are those belonging to Klebsiella and other individual species sequenced in this study. BTL, β-lactams; QNL, quinolone; SUL, sulfonamide; TMP, trimethoprim; AMG, aminoglycoside; C, chloramphenicol; FOS, Fosfomycin; M, macrolide; T, tetracycline; G, Glycopeptide; EFP; multi-drug efflux RND transporters; Q; quaternary ammonium compound; T; point mutations for tigecycline. FD, fosmidomycin; Plasmids; the plasmids replicons hits for each isolate.

Figure 2

Comparison map of p41EC2 with other E. coli plasmids from NCBI.