Differential survival of potentially pathogenic, septicemia- and meningitis-causing *E. coli* across the wastewater treatment train

The study addresses whether extraintestinal pathogenic E. coli (ExPEC) can survive municipal wastewater treatment and thus pose public health risks via water exposure. Safe water is critical for health; inadequate wastewater treatment correlates with higher disease mortality. Microbes such as E. coli possess stress response systems and genetic elements (e.g., heat shock proteins, locus of heat resistance) that can confer tolerance to disinfection stressors (chlorination, oxidative stress, UV). Evidence suggests selection for treatment resistance within wastewater plants, including naturalized wastewater E. coli strains enriched across treatment and resistant to chlorine, oxidants, and heat, with enhanced biofilm formation. Prior work indicates uropathogenic E. coli (UPEC) survive treatment, with 41.7–59.5% of post-treatment E. coli carrying UPEC virulence genes and some clustering in clinical lineages (e.g., ST131, ST95) with high genomic similarity to clinical isolates and ESBL phenotypes. Other ExPEC pathotypes, including bloodborne E. coli (BBEC) and neonatal meningitic E. coli (NMEC), have been associated with ST95 and ST131 lineages and detected in effluents; about 14.8% of effluent isolates were potential NMEC in one study. The purpose of this study is to comprehensively compare genomes of wastewater-derived ExPEC with clinical BBEC and NMEC to evaluate their similarity and infer potential for waterborne transmission of extraintestinal disease.

Background literature shows: (1) Naturalized wastewater E. coli subpopulations are enriched across treatment and exhibit resistance to chlorine, oxidants, and heat, likely adapted to the WWTP niche. (2) Multiple studies report high prevalence of UPEC-associated virulence determinants in treated effluents, with estimates of 41.7–59.5% of effluent E. coli being potential UPEC. (3) Comparative genomics (Zhi et al., 2020) found wastewater E. coli isolates in clinically relevant sequence types (ST131, ST95) with 96.00–99.49% whole-genome similarity and as few as 2 SNPs in ~0.4 Mb core regions to clinical UPEC; several belonged to O25b-ST131 ESBL-producing clonal group. (4) NMEC and BBEC have been linked to ST95 and ST131 lineages; 14.8% of wastewater effluent E. coli carried NMEC-associated ibeA, suggesting presence of NMEC-like strains post-treatment. These studies motivate examining whether BBEC/NMEC-like ExPEC also differentially survive wastewater treatment and assessing accessory genome contributions to survival and pathogenicity.

Sampling and isolate recovery: Chlorine-stressed E. coli were obtained by treating raw sewage (from 10 Alberta WWTPs) with 3% sodium hypochlorite to 0.3–0.5 ppm free chlorine, achieving ~4 log10 reduction (Colilert QuantiTray). Residual chlorine was quenched with sodium thiosulfate; selective culture used ColiTag or lauryl trypticase broth/BCG, then X-Gluc plates at 44.5 °C, followed by confirmation on blood agar and Vitek 2 identification. Wastewater treatment-resistant isolates were collected from secondary-treated and finished effluents at three Calgary WWTPs (grit removal, primary clarification, activated sludge, secondary clarification, UV 25–30 mJ/cm2). Effluents were filtered (100 mL) onto X-Gluc plates; blue colonies were picked, grown in LB, and confirmed as E. coli by Vitek 2. In total, 1212 effluent isolates were collected; 261 random were confirmed as E. coli. Combined with chlorine-stressed isolates, 376 presumed chlorine-tolerant and 261 treatment-resistant isolates were available for screening. Screening for ExPEC markers: PCR assays were used to exclude naturalized wastewater strains (uspC-1530-flhDC marker) and confirm E. coli (uidA). Remaining isolates were screened for ExPEC-associated virulence genes (papC, sfa/foc, iroN, ibeA, fyuA, chuA, kpsM) and the ST131 and O25b-ST131 markers using specified cycling conditions. PCR results were later confirmed in silico via whole-genome sequencing. Whole-genome sequencing and assembly: Presumptive wastewater ExPEC (W-ExPEC) were defined as isolates with ≥3 ExPEC virulence genes, or any positive for ibeA or ST131 lineage markers. Eighty-six W-ExPEC were sequenced (Illumina HiSeq X, PE150). Reads were quality-trimmed with Trimmomatic (SLIDINGWINDOW=4:15, LEADING=3, TRAILING=3, MINLEN=36) and assembled with SPAdes (v3.11.1; -careful; -k 21,33,55,77). Contigs <1000 bp were excluded. Comparative genomics: A local repository of 320 clinical ExPEC (BBEC and NMEC) genomes was built from NCBI. Core-genome SNP analysis across W-ExPEC (n=86) and clinical (n=320) was performed using REALPHY v1.1 with one assembly as reference, followed by MEGA-X to calculate pairwise SNP differences across a ~417 kbp core backbone. W-ExPEC with <250 core SNPs to any clinical strain were retained (n=37) for deeper analyses. Pairwise whole-genome similarity was computed with REALPHY (percentage of C-ExPEC genome mapping to each W-ExPEC), using 96.03% as a lower threshold for shared pathogenic phenotype. Each W-ExPEC was also compared to 46 intestinal pathogenic and naturalized wastewater E. coli to contextualize similarities. Phylogeny, phylogroups, and MLST: Maximum-likelihood core-genome trees were built with RAxML (v8.2.12) using REALPHY alignments, including reference strains of known phylogroups. Phylogroups were assigned by ClermonTyper, and MLST was performed (mlst 2.19.0, E. coli #1 scheme). Visualization used ggplot2, ape, and ggtree in R. Pan-genome and accessory genome analyses: Roary (v3.13.0) was used to compute a pan-genome for the 86 W-ExPEC, their closest clinical matches (n=38), reference UPEC (n=9), naturalized wastewater strains (n=5), and lab reference strains (n=4). Accessory gene presence/absence clustering generated an accessory genome tree. Virulence and antibiotic resistance gene screening: ABRicate (v1.0.1) screened genomes against VFDB and Ecoli_vf (virulence) and CARD (resistance) databases (≥90% coverage, ≥80% identity). Counts of virulence genes (VGs) and antibiotic resistance genes (ARGs) were appended to the accessory gene tree.

• From 637 wastewater E. coli isolates, 247 carried at least one ExPEC-associated virulence gene; 7 carried all seven screened genes. The most prevalent were fyuA and chuA; sfa/foc and ibeA were least common. Twenty-two isolates were positive for the ST131 lineage marker. Eighty-six isolates met criteria as presumptive wastewater ExPEC (W-ExPEC) and were sequenced.
• Thirty-seven W-ExPEC had close core-genome similarity to clinical BBEC/NMEC (≤250 SNPs across ~417 kbp). Examples: wastewater strains 2F5 and 2F6 differed from BBEC_156 by only 5 SNPs; 3G9 differed from BBEC_211 by 3 SNPs; 1G6 differed from NMEC_9 by 7 SNPs.
• Whole-genome similarity between these 37 W-ExPEC and clinical counterparts ranged 96.04–99.74%. Notable pairs: 1G6 with NMEC_9 (99.72%); 389 and 4G1 with BBEC_38 (99.62% and 99.58%) and with NMEC_4 (99.59% and 99.55%). Some W-ExPEC matched up to 48 clinical strains above threshold. In contrast, similarity to intestinal pathogenic or naturalized wastewater E. coli was 65.9–95.3% (mean 84.9%); none exceeded 96.03%.
• Phylogeny/MLST: Of 37 W-ExPEC, 34 clustered in phylogroup B2 and 3 in phylogroup A alongside clinical ExPEC. Sequence types among W-ExPEC: ST131 (n=18), ST95 (n=8), ST73 (n=2), ST10 (n=2), ST127 (n=2), and one each of ST357, ST538, ST44; two unknown STs. Clinical matches mirrored these lineages.
• Pan-genome: 26,865 genes total with 2,133 core genes (~8% of pan-genome). W-ExPEC averaged 4,729 genes; core comprised ~45% of W-ExPEC genomes. Accessory genome clustering grouped strains into three clusters: Cluster 1 likely enteric E. coli; Cluster 2 included naturalized wastewater strains (e.g., ST635) and some minor ExPEC lineages; Cluster 3 contained most W-ExPEC and clinical ExPEC (major lineages ST131, ST95, ST73, ST127, plus ST538, ST357).
• Virulence genes: W-ExPEC with clinical matches carried extensive virulence repertoires (195–278 VGs). ST95, ST127, and ST73 tended to have higher VG counts. None of the 37 W-ExPEC carried intestinal pathotype markers (eaeA, stx1, stx2, rfbE), supporting extraintestinal pathogenic potential. Pairwise VG profiles between W-ExPEC and clinical counterparts were highly similar (e.g., 3G11 vs BBEC_211 shared 214 VGs; 3B9/4G1 vs NMEC_4 shared 276 VGs).
• Antibiotic resistance genes: W-ExPEC carried 43–56 ARGs; clinical counterparts generally had more. ST131 W-ExPEC had the highest ARG counts, followed by ST44 and ST73. Clinically significant ARGs included aminoglycoside-modifying enzymes (e.g., AAC(3)-IId; AAC(3)-Ile and AAC(6')-Ib-cr in some ST131), APH(3")-Ib and APH(6)-Id across multiple lineages, and beta-lactamases (ampC, ampH, blaCTX-M-15, blaOXA-1, blaTEM-181, blaCTX-M-14), indicating ESBL phenotypes among several isolates. Some W-ExPEC and clinical pairs had identical ARG profiles (e.g., 3E4 with BBEC_267; 3G9 with BBEC_211; 3B9/4G1 with NMEC_4).
• Accessory overlap with naturalized strains: While naturalized wastewater strains had few VGs and are likely non-pathogenic, W-ExPEC shared some accessory genes with them, suggesting common genetic capacities aiding survival through treatment.

Findings demonstrate that many E. coli surviving wastewater treatment are genetically extremely similar to clinical BBEC and NMEC across core, whole, and accessory genomes, implying comparable extraintestinal pathogenic potential and differential survival through treatment. The dominance of major ExPEC lineages (ST131, ST95, ST73, ST127) among wastewater isolates supports independent evolution of treatment resistance across multiple lineages. High virulence gene loads without intestinal pathotype markers and substantial ARG burdens, including ESBL-associated genes, underscore public health concerns. The similarity and shared accessory content between W-ExPEC and naturalized wastewater strains suggest overlapping adaptive traits contributing to treatment survival (e.g., stress resistance, iron acquisition). The results support a model wherein ExPEC are repeatedly introduced to WWTPs via community infections, face strong disinfection selection pressures, and persist post-treatment, enabling environmental exposure and potential feedback into human populations. Likely transmission routes include recreational water and de facto reuse in drinking water sources; these may be underrecognized compared with foodborne routes. The study urges comprehensive epidemiological investigations into waterborne transmission of extraintestinal infections (UTIs, septicemia, neonatal meningitis) and surveillance of ARG-laden ExPEC in water systems.

Wastewater-derived E. coli from chlorinated sewage and finished effluents in Alberta, Canada, include numerous isolates that are nearly indistinguishable from clinical BBEC and NMEC at core, whole-genome, and accessory levels, frequently within major ExPEC lineages and carrying abundant virulence and antibiotic resistance determinants (including ESBLs). These observations, together with prior evidence for UPEC persistence, indicate that ExPEC pathotypes may have evolved differential resistance to wastewater disinfection, raising the possibility of waterborne transmission of UTIs, septicemia, and meningitis. Future work should: (1) quantify environmental persistence and prevalence of ExPEC downstream of WWTPs; (2) conduct epidemiological studies linking water exposure to extraintestinal infections; (3) dissect accessory genes and mechanisms underlying treatment resistance; and (4) broaden comparative genomic repositories to better capture ExPEC diversity and transmission dynamics.

• Virulence gene panels cannot uniquely define ExPEC pathotypes; thus initial screening likely missed some ExPEC.
• PCR screening emphasized ST131 markers, potentially biasing selection and under-representing other ExPEC lineages (e.g., ST95, ST73, ST10, ST127).
• The clinical comparison set, though large (n=320), is finite; some wastewater strains lacking >96.03% similarity to a clinical counterpart may still be ExPEC but without a close match in the repository.
• Some sequence types were underrepresented among clinical references, potentially limiting detection of matches for minor ExPEC lineages.
• Genomic similarity infers pathogenic potential but does not directly measure virulence phenotypes or environmental survival mechanisms in situ.