Characterization of Extended-Spectrum β-Lactamase-Producing Escherichia coli Isolates That Cause Diarrhea in Sheep in Northwest China

Abstract

Development of extended-spectrum-β-lactamase (ESBL)-producing Escherichia coli is one the greatest threats faced by mankind. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants (i.e., sheep and goats) in China. The aim of this study was to identify and characterize the resistance profiles, resistomes, and sequence features of 67 ESBL-producing E. coli isolates from sheep in northwest China. The findings showed that bla_CTX-M and bla_TEM were the most prevalent. Interestingly, we found that the resistance gene mcr-1 was widespread in sheep merely from Shaanxi areas, accounting for 19.2% (5/26). The highly prevalent serotypes and FumC-FimH (CH) typing isolates were O8 and C₄H₃₂, respectively. High-risk E. coli clones, such as sequence type 10 (ST10), ST23, ST44, and ST58, were also found in China's sheep population. A total of 67 ESBL-producing isolates were divided into five phylogenetic groups, namely, B1 (n = 47, 70.1%), B2 (n = 1, 1.5%), C (n = 14, 20.9%), E (n = 1, 1.5%), and F (n = 1, 1.5%), with the phylogenetic groups for 3 isolates (4.5%) remaining unknown. Moreover, ESBL-producing E. coli isolates were also characterized by the abundance and diversity of biocide/metal resistance genes and insert sequences. We found that in ESBL-producing E. coli isolates, there were two different types of isolates, those containing ESBL genes or not, which led to large discrepancies between resistance phenotypes and resistomes. In summary, our study provides a comprehensive overview of resistance profiles and genome sequence features in ESBL-producing E. coli and highlights the possible role of sheep as antibiotic resistance gene disseminators into humans. IMPORTANCE Antimicrobial resistance (AMR), especially the simultaneous resistance to several antibiotics (multidrug resistance [MDR]), is one of the greatest threats to global public health in the 21st century. Among animals, chickens, pigs, and cattle are reservoirs of these pathogens worldwide. Nevertheless, there is a knowledge gap on ESBL-producing E. coli from small ruminants in China. This study is the largest and most comprehensive analysis of ESBL-producing E. coli isolates from sheep, including antibiotic resistance profiles, phylogenetic groups, serotypes, multilocus sequence types (MLST), insert sequences (IS), antibiotic resistance genes, disinfectant resistance genes, and heavy metal resistance genes. We recommend extending the surveillance of AMR of sheep-origin E. coli to prevent future public health risks.

Keywords: ESBL-producing E. coli; biocide resistance; insert sequence; metal resistance; resistomes; sheep.

FIG 1

Antimicrobial resistance pattern and frequency of 67 MDR E. coli isolates.

FIG 2

Identification of the molecular epidemiology 67 ESBL-producing E. coli isolates of sheep origin. The minimum spanning tree was obtained using BioNumerics v7.6 software. Each circle represents a special sequence type (ST), and the size of the circle indicates the number of isolates in that ST. The distance labels correspond to the number of discriminating alleles.

FIG 3

Evolutionary tree analysis of the 67 ESBL-producing Escherichia coli strains sequenced in this study. Core genome alignments were obtained using the Roary program, and a maximum likelihood tree was assembled using the FastTree program. Visualization and annotation were carried out through iTOL version 6.52 (https://itol.embl.de; accessed 6 March 2022). Phylogenetic groups, MLST, O and H serotypes, fumC-fimH typing, IS, and the resistance genes of antibiotics, disinfectants, and heavy metals were plotted. Phylogenetic analysis showed a relatively well-supported monophyletic distribution of STs. A dash in the serotypes, fimH, and sequence type (ST) columns indicates an unrecognized serotype, fimH type, and ST in the corresponding database.

FIG 4

MDR rate between isolates containing targeted ESBL genes (ESBLs+) and not containing ESBL genes (ESBLs−).