Isolation and Characterization of NDM-Positive Escherichia coli from Municipal Wastewater in Jeddah, Saudi Arabia

Abstract

The emergence of resistance to last-resort antibiotics is a public health concern of global scale. Besides direct person-to-person propagation, environmental pathways might contribute to the dissemination of antibiotic-resistant bacteria and antibiotic resistance genes (ARGs). Here, we describe the incidence of blaNDM-1, a gene conferring resistance to carbapenems, in the wastewater of the city of Jeddah, Saudi Arabia, over a 1-year period. blaNDM-1 was detected at concentrations ranging from 10(4) to 10(5) copies/m(3) of untreated wastewater during the entire monitoring period. These results indicate the ubiquity and high incidence of blaNDM-1 in the local wastewater. To track the bacteria carrying blaNDM-1, we isolated Escherichia coli PI7, a strain of sequence type 101 (ST101), from wastewater around the Hajj event in October 2013. Genome sequencing of this strain revealed an extensive repertoire of ARGs as well as virulence and invasive traits. These traits were further confirmed by antibiotic resistance profiling and in vitro cell internalization in HeLa cell cultures. Given that this strain remains viable even after a certain duration in the sewerage, and that Jeddah lacks a robust sanitary infrastructure to fully capture all generated sewage, the presence of this bacterium in the untreated wastewater represents a potential hazard to the local public health. To the best of our knowledge, this is the first report of a blaNDM-1-positive E. coli strain isolated from a nonnosocomial environment in Saudi Arabia and may set a priority concern for the need to establish improved surveillance for carbapenem-resistant E. coli in the country and nearby regions.

FIG 1

Incidence of bla_NDM-1 in the raw wastewater (WW) of a wastewater treatment plant in Jeddah, Saudi Arabia, over a 1-year period.

FIG 2

Genetic map of the plasmid carrying bla_NDM-1 in E. coli PI7 (pKOX_NDM1) and contigs mapping against pKOX_R1 of Klebsiella oxytoca. Relevant characteristics related to antimicrobial resistance (orange), metal resistance (light blue), mobilization elements (purple), and plasmid maintenance and stability (green) are highlighted in the map. (a) pKOX_NDM1 was fully sequenced in this study and shares 99% sequence identity with the reported sequence for K. oxytoca (18). (b) pKOX_R1 was drawn based on the reported sequence for K. oxytoca (18). The inner green circle in the pKOX_R1 map represents the coverage of the contigs in this study mapping against pKOX_R1. Genes highlighted in red type represent those encoding relevant traits mapping outside the contigs found in this study.

FIG 3

(a) Mapping of genomic islands, pathogenicity factors, and antibiotic resistance loci in the E. coli PI7 genome. Yellow and blue lines represent genomic islands predicted by SIGI-HMM and IslandPath/DIMOB, respectively. Red lines represent the genomic locations of all predicted islands. Asterisks correspond to locations of antibiotic efflux pumps. (b) T3SS gene cluster in E. coli PI7. Green arrows represent structural T3SS genes, pink arrows represent genetic elements associated with gene mobility, and gray arrows represent genes encoding hypothetical proteins (HPs) predicted by Secretome 1.1 for noncanonical secretion signals.

FIG 4

In vitro internalization of E. coli PI7, DSM 1103, and DH5α in HeLa cell cultures. (a) Cell internalization of E. coli PI7 versus that of E. coli DSM 1103; (b) confocal microscopy of HeLa cell cultures infected with GFP-tagged E. coli PI7 and GFP-tagged E. coli DH5α (magnification, ×20); (c) z-stack of highlighted box in panel b corresponding to HeLa cells infected with E. coli PI7 (magnification, ×60). Asterisks highlight green fluorescent clusters.