Genomic insights into virulence, antimicrobial resistance, and adaptation acumen of Escherichia coli isolated from an urban environment

Abstract

Populations of common commensal bacteria such as Escherichia coli undergo genetic changes by the acquisition of certain virulence and antimicrobial resistance (AMR) encoding genetic elements leading to the emergence of pathogenic strains capable of surviving in the previously uninhabited or protected niches. These bacteria are also reported to be prevalent in the environment where they survive by adopting various recombination strategies to counter microflora of the soil and water, under constant selection pressure(s). In this study, we performed molecular characterization, phenotypic AMR analysis, and whole genome sequencing (WGS) of E. coli (n = 37) isolated from soil and surface water representing the urban and peri-urban areas. The primary aim of this study was to understand the genetic architecture and pathogenic acumen exhibited by environmental E. coli. WGS-based analysis entailing resistome and virulome profiling indicated the presence of various virulence (adherence, iron uptake, and toxins) and AMR encoding genes, including bla_NDM-5 in the environmental isolates. A majority of our isolates belonged to phylogroup B1 (73%). A few isolates in our collection were of sequence type(s) (ST) 58 and 224 that could have emerged recently as clonal lineages and might pose risk of infection/transmission. Mobile genetic elements (MGEs) such as plasmids (predominantly) of the IncF family, prophages, pipolins, and insertion elements such as IS1 and IS5 were also observed to exist, which may presumably aid in the propagation of genes encoding resistance against antimicrobial drugs. The observed high prevalence of MGEs associated with multidrug resistance in pathogenic E. coli isolates belonging to the phylogroup B1 underscores the need for extended surveillance to keep track of and prevent the transmission of the bacterium to certain vulnerable human and animal populations.

Importance: Evolutionary patterns of E. coli bacteria convey that they evolve into highly pathogenic forms by acquiring fitness advantages, such as AMR, and various virulence factors through the horizontal gene transfer (HGT)-mediated acquisition of MGEs. However, limited research on the genetic profiles of environmental E. coli, particularly from India, hinders our understanding of their transition to pathogenic forms and impedes the adoption of a comprehensive approach to address the connection between environmentally dwelling E. coli populations and human and veterinary public health. This study focuses on high-resolution genomic analysis of the environmental E. coli isolates aiming to understand the genetic similarities and differences among isolates from different environmental niches and uncover the survival strategies employed by these bacteria to thrive in their surroundings. Our approach involved molecular characterization of environmental samples using PCR-based DNA fingerprinting and subsequent WGS analysis. This multidisciplinary approach is likely to provide valuable insights into the understanding of any potential spill-over to human and animal populations and locales. Investigating these environmental isolates has significant potential for developing epidemiological strategies against transmission and understanding niche-specific evolutionary patterns.

Keywords: Escherichia coli; antimicrobial resistance; environment; genome analysis; virulence.

Fig 1

Heatmap depicting resistance profiles of the isolates obtained by experimental analysis using the Kirby–Bauer disc diffusion assay. The isolates are defined as resistant, intermediate, and susceptible for each antimicrobial drug according to the Clinical & Laboratory Standards Institute (CLSI, 2021) guidelines. Yellow color indicates sensitive, and pink color indicates intermediate while red color indicates resistance to respective antibiotics.

Fig 2

Dendrogram based on phylogenetic analysis of 37 environmental E. coli isolates using (A) ERIC-PCR, (B) REP-PCR, and (C) RAPD-PCR banding analysis by GelJ (29). Dotted boxes represent clusters in which most of them are segregated indicating clonality.

Fig 3

Graphical representation of specific biofilm formation value of each of the isolates. NA097 served as a positive control while DH5α was used as a negative control. While the isolates showing an SBF value of 0.5 or more were considered as strong biofilm formers, those with SBF value less than 0.1 were graded weak biofilm formers; those having SBF value between 0.1 and 0.5 were moderate biofilm formers.

Fig 4

Whole genome comparative analysis of 29 environmental E. coli isolates using BRIG, with E. coli str. K-12 substr. MG1655 as the reference. Each ring represents a genome, and the rings were color-coded based on their ST, and the genome names were labeled.

Fig 5

Principal coordinate analysis plot based on the presence/absence binary matrix of the pan genome reflecting the clonality among the isolates belonging to the same phylogroup, irrespective of their source. The isolates have been color-coded according to their respective phylogroups.

Fig 6

Whole genome-based prediction of (A) antimicrobial resistance and (B and C) virulence determinants of 29 environmental isolates using the Comprehensive Antibiotic Resistance Database (32) and Virulence factor database (33), respectively.

Fig 7

Graphical representation of the occurrence and frequency of mobile genetic elements among the isolates: (A) plasmids obtained using BacAnt (34); (B) insertion elements obtained using ISEScan (35). IS1 (252 out of total 1,407 IS copies) was found to be maximally present while ISKRA4 (3 out of 1,407 IS copies) was found to be the least frequent.