Antimicrobial Resistance Profile of mcr-1 Positive Clinical Isolates of Escherichia coli in China From 2013 to 2016

Abstract

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

Keywords: Escherichia coli; clinical isolates; mcr-1; multidrug-resistant; plasmid.

FIGURE 1

Frequency and distribution of resistance to antimicrobial agents among 123 E. coli isolates obtained from clinical specimens in China. (A) Frequency of resistance to each antimicrobial agent among 123 E. coli isolates. The number on top of each column represents the percentage of resistant isolates to each antimicrobial agent. PB, polymyxin B; IMP, imipenem; AMP, ampicillin; CFX, cefoxitin; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; CIP, ciprofloxacin; GEN, gentamycin; SXT, sulfamethoxazole-trimethoprim; CM, chloramphenicol; TET, tetracycline. (B) Occurrence of multidrug resistance among 123 E. coli isolates. The x-axis indicates the number of antimicrobial agents. The number on top of each column represents the percentage of resistant isolates to different numbers of antimicrobial agents.

FIGURE 2

Sequence alignment of the eight mcr-1 carrying plasmids and pMCR_1511, pHNSHP45, or pColR598_1. Arrows show the directions of putative open reading frames (ORFs), and the length of the arrow is proportional to the size of each ORF. The gene mcr-1 is marked in red. ISApl1, ISKpn26, and pap2 gene are marked in blue gray, brown, and green. Nickel transport system permease gene (nikA), replication initiation protein (repA), and partition protein (parA) are marked in pink, purple, and black. Conjugal transfer genes are marked in yellow. Regions of homology between sequences (>66%) are indicated by the graded shading.

FIGURE 3

Distribution of the antimicrobial resistance gene is indicated by pentagon (chromosome) or circle (plasmid) in the seven mcr-1 positive strains. The eight mcr-1 carrying plasmids are indicated by red circles and are numbered as 1–8 for p14EC001a, p14EC007a, p14EC017a, p14EC020a, p14EC029a, p14EC033a, p14EC033b, and p14EC047a, respectively.