Evidence of Naturalized Stress-Tolerant Strains of Escherichia coli in Municipal Wastewater Treatment Plants

Abstract

Escherichia coli has been proposed to have two habitats—the intestines of mammals/birds and the nonhost environment. Our goal was to assess whether certain strains of E. coli have evolved toward adaptation and survival in wastewater. Raw sewage samples from different treatment plants were subjected to chlorine stress, and ∼59% of the surviving E. coli strains were found to contain a genetic insertion element (IS30) located within the uspC-flhDC intergenic region. The positional location of the IS30 element was not observed across a library of 845 E. coli isolates collected from various animal hosts or within GenBank or whole-genome reference databases for human and animal E. coli isolates (n = 1,177). Phylogenetics clustered the IS30 element-containing wastewater E. coli isolates into a distinct clade, and biomarker analysis revealed that these wastewater isolates contained a single nucleotide polymorphism (SNP) biomarker pattern that was specific for wastewater. These isolates belonged to phylogroup A, possessed generalized stress response (RpoS) activity, and carried the locus of heat resistance, features likely relevant to nonhost environmental survival. Isolates were screened for 28 virulence genes but carried only the fimH marker. Our data suggest that wastewater contains a naturalized resident population of E. coli. We developed an endpoint PCR targeting the IS30 element within the uspC-flhDC intergenic region, and all raw sewage samples (n = 21) were positive for this marker. Conversely, the prevalence of this marker in E. coli-positive surface and groundwater samples was low (≤5%). This simple PCR assay may represent a convenient microbial source-tracking tool for identification of water samples affected by municipal wastewater.IMPORTANCE The results of this study demonstrate that some strains of E. coli appear to have evolved to become naturalized populations in the wastewater environment and possess a number of stress-related genetic elements likely important for survival in this nonhost environment. The presence of non-host-adapted strains in wastewater challenges our understanding of using E. coli as a microbial indicator of wastewater treatment performance, suggesting that the E. coli strains present in human and animal feces may be very different from those found in treated wastewater.

Heat and Pressure Resistance in Escherichia coli Relates to Protein Folding and Aggregation

“…A high frequency of heat resistant Enterobacteriaceae was reported in beef (Dlusskaya et al, 2011;Mercer et al, 2015;), in raw milk cheese after thermization at 60 • C, and in Daqu, a solid state cereal fermentation that reaches temperatures of up to 60 • C (Marti et al, 2016;Boll et al, 2017;Wang et al, 2018). The high frequency of LHR-positive strains of E. coli in chlorine-treated waste-water (Zhi et al, 2016) relates to the contribution of the LHR to chlorine resistance (Wang et al, 2020). The presence of the transmissible LHR in pathogenic organisms associated with the food and water supply may present significant challenges to public health.…”

Determination of Survival of Wildtype and Mutant Escherichia coli in Soil

“…However, several studies have isolated E. coli from various natural environments such as municipal wastewater, freshwater, beach water, beach sand and soils (Jiménez et al, 1989;Brennan et al, 2010;Chiang et al, 2011;Byappanahalli et al, 2012;Zhi et al, 2016). The capacity of these E. coli strains to survive for long periods of time and grow in the external environment raises questions about the validity of its continued use as indicator of water quality (Brennan et al, 2010).…”

Daqu Fermentation Selects for Heat-Resistant Enterobacteriaceae and Bacilli

“…Even when considering that multiple copies of the LHR may be present in one strain, this study demonstrates that an increase of the fermentation temperature exerts a strong selective pressure for LHR-positive Enterobacteriaceae. A high frequency of LHR-positive Escherichia coli organisms has been previously found for meat (13), cheese (31), and wastewater (17); however, the LHR has not been identified in spontaneous solid-state fermentation or in K. cowanii. The temperature of Daqu ranged from 55 to 60°C for 6 days; at this temperature range, even LHR-positive Kosakonia and Enterobacter spp.…”

“…The reservoir and selective pressure for LHR-positive or spoVA 2mob -positive microorganisms remain unknown; to date, conclusions on environmental conditions selecting for LHR-positive E. coli were based solely on the frequency of LHR-positive isolates in specific ecosystems (12,17,18). Ecosystems that maintain a temperature of 60°C for an extended period may select for heat-resistant bacteria.…”