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

Access to clean water is crucial for public health, and effective wastewater treatment is a vital public health intervention. Inadequate wastewater treatment correlates with increased disease mortality globally. The ability of pathogens to evolve resistance to water treatment is a significant concern. *Escherichia coli*, a model bacterium, possesses multiple mechanisms to tolerate various stressors, including those associated with water treatment like chlorination, osmotic stress, and oxidative stress. Growing evidence shows that water treatment may select for the evolution of resistance to disinfection processes. Studies have identified subpopulations of *E. coli*, including naturalized wastewater strains and extraintestinal pathogenic *E. coli* (ExPEC), demonstrating enhanced resistance to water treatment. Previous research has shown that ExPEC strains, including those causing urinary tract infections (UPEC), survive wastewater treatment. More recently, concern has risen regarding the survival of other ExPEC pathotypes, such as bloodborne *E. coli* (BBEC) and neonatal meningitic *E. coli* (NMEC), which share sequence types with UPEC strains found in treated wastewater. This study aimed to comprehensively analyze the survival and genetic characteristics of NMEC and BBEC strains in treated wastewater effluents and chlorinated sewage, exploring the potential for waterborne transmission of diseases like septicemia and meningitis.

A significant body of research highlights the importance of safe water for public health, linking inadequate water, sanitation, and hygiene to millions of deaths and disability-adjusted life years lost annually. The impact of wastewater treatment on public health is well-established, with studies showing a strong correlation between inadequate treatment and increased disease mortality. The adaptability of microbes and their ability to develop resistance to water treatment stressors, such as chlorination and oxidative stress, are documented. *E. coli*, in particular, has demonstrated multiple mechanisms for tolerating these stressors, including stress-specific transcription factors and cross-resistance strategies. Previous research has focused on the survival of UPEC strains in wastewater treatment, showing that a substantial proportion of surviving *E. coli* possess UPEC-associated virulence genes and pathogenicity islands. The prevalence of ExPEC strains in wastewater, including those belonging to clinically relevant sequence types, raises concerns about potential transmission.

This study involved the collection of *E. coli* isolates from chlorinated sewage and treated wastewater effluents from multiple wastewater treatment plants in Alberta, Canada. Chlorine-stressed isolates were obtained from chlorinated sewage samples, while wastewater treatment-resistant isolates were collected from partially treated and finished effluents. Isolates were screened for ExPEC-associated virulence genes and molecular markers using PCR. Presumptive ExPEC isolates (86 in total) were selected for whole-genome sequencing using an Illumina HiSeq X platform. Bioinformatic analyses were performed to assess core genome similarity, pairwise whole-genome similarity, core genome phylogenetic relationships, phylogrouping, multilocus sequence typing (MLST), pan-genomic analysis, accessory genome clustering, and virulence and antibiotic resistance gene composition. Specific tools and databases used included Trimmomatic, SPAdes, REALPHY, MEGA-X, Roary, ABRicate, VFDB, Ecoli_vf, and CARD. The study also utilized previously established thresholds for whole-genome similarity to define the pathogenic potential of wastewater isolates.

Of 637 wastewater *E. coli* isolates, 86 were identified as presumptive ExPEC based on virulence gene screening. These isolates clustered within major ExPEC lineages (ST131, ST95, ST73, and others). Core genome SNP analysis revealed that 37 W-ExPEC strains shared close core genome similarity (≤250 SNPs in a ~417 kbp backbone) with clinical BBEC and NMEC strains. Pairwise whole-genome comparisons showed that these 37 W-ExPEC strains exhibited >96.03% whole-genome similarity with at least one clinical BBEC or NMEC strain, indicating a high degree of genetic relatedness. Phylogenetic analysis placed the majority of W-ExPEC strains within phylogroup B2, a major phylogroup associated with ExPEC pathotypes. Pan-genomic analysis revealed that W-ExPEC strains shared a similar accessory gene profile with their clinical counterparts, suggesting similar pathogenic potential. Comparison of virulence and antibiotic resistance genes showed that W-ExPEC strains possessed extensive virulence gene repertoires and harbored several clinically important antibiotic resistance genes (e.g., aminoglycoside modification enzymes, beta-lactamases). Some W-ExPEC strains even shared identical ARG profiles with their clinical counterparts.

The high degree of similarity between W-ExPEC and C-ExPEC strains across the core, whole, and accessory genomes strongly suggests that many ExPEC strains surviving wastewater treatment are highly pathogenic, with the potential to cause septicemia and meningitis. The study supports previous findings demonstrating the survival of UPEC strains in wastewater treatment and extends this observation to BBEC and NMEC pathotypes. The shared accessory genes between ExPEC and naturalized wastewater strains may explain the differential survival of ExPEC during water treatment. The significant presence of antibiotic resistance genes in these wastewater strains further compounds the public health concern. Potential transmission routes for ExPEC from wastewater-contaminated environments include recreational water exposure and potentially drinking water, considering the significant volumes of wastewater effluents impacting drinking water sources and surface waters used for various purposes.

This study provides strong evidence that clinically relevant BBEC and NMEC strains survive municipal wastewater treatment processes. The high genomic similarity between wastewater ExPEC and clinical ExPEC strains, coupled with their resistance to water treatment and antibiotic resistance, raises significant public health concerns. Further research is needed to fully understand the environmental persistence and transmission of these pathogens and the associated risks.

The initial identification of presumptive ExPEC strains relied on virulence gene screening, which may not capture all potential ExPEC strains due to the lack of a single definitive marker. The comparative genomic analyses were limited by the size of the local genome database of clinical BBEC and NMEC strains, potentially underestimating the true prevalence of ExPEC in wastewater. The study did not investigate the specific mechanisms of water treatment resistance in these strains, nor did it directly assess the epidemiological link between waterborne exposure and disease incidence.