First Report of Coexistence of bla SFO-1 and bla NDM-1 β-Lactamase Genes as Well as Colistin Resistance Gene mcr-9 in a Transferrable Plasmid of a Clinical Isolate of Enterobacter hormaechei

Abstract

Many antimicrobial resistance genes usually located on transferable plasmids are responsible for multiple antimicrobial resistance among multidrug-resistant (MDR) Gram-negative bacteria. The aim of this study is to characterize a carbapenemase-producing Enterobacter hormaechei 1575 isolate from the blood sample in a tertiary hospital in Wuhan, Hubei Province, China. Antimicrobial susceptibility test showed that 1575 was an MDR isolate. The whole genome sequencing (WGS) and comparative genomics were used to deeply analyze the molecular information of the 1575 and to explore the location and structure of antibiotic resistance genes. The three key resistance genes (bla _SFO-1, bla _NDM-1, and mcr-9) were verified by PCR, and the amplicons were subsequently sequenced. Moreover, the conjugation assay was also performed to determine the transferability of those resistance genes. Plasmid files were determined by the S1 nuclease pulsed-field gel electrophoresis (S1-PFGE). WGS revealed that p1575-1 plasmid was a conjugative plasmid that possessed the rare coexistence of bla _SFO-1, bla _NDM-1, and mcr-9 genes and complete conjugative systems. And p1575-1 belonged to the plasmid incompatibility group IncHI2 and multilocus sequence typing ST102. Meanwhile, the pMLST type of p1575-1 was IncHI2-ST1. Conjugation assay proved that the MDR p1575-1 plasmid could be transferred to other recipients. S1-PFGE confirmed the location of plasmid with molecular weight of 342,447 bp. All these three resistant genes were flanked by various mobile elements, indicating that the bla _SFO-1, bla _NDM-1, and mcr-9 could be transferred not only by the p1575-1 plasmid but also by these mobile elements. Taken together, we report for the first time the coexistence of bla _SFO-1, bla _NDM-1, and mcr-9 on a transferable plasmid in a MDR clinical isolate E. hormaechei, which indicates the possibility of horizontal transfer of antibiotic resistance genes.

Keywords: Enterobacter hormaechei; IncHI2; WGS; blaNDM–1; blaSFO–1; mcr-9; mobile elements; plasmid.

FIGURE 1

A conjugative plasmid p1575-1. AR (ARGs), acquired antibiotic resistance determinant genes; VF, virulence factors; ORF1-27, bla_NDM–1; ORF1-39, qnrS1; ORF1-174, Relaxase; ORF1-222, tetD; ORF1-226, bla_SFO–1.

FIGURE 2

(A) Ring diagram representation of plasmid p1575-1. From the inside to the outside, the first circle represents the scale; the second circle represents GC content; the third circle represents the GC skew; the fourth and sixth circles represent the COG to which each CDS belongs; the fifth circle represents the backbone; the seventh to 10th circles represent p1575-1, pNIHE14-1904-mcr9, pECL-90-2, and pIHI2-323, respectively. GC, guanine + cytosine; bla_SFO–1, extended-spectrum β-lactamases (ESBLs); bla_NDM–1, New Delhi metallo-β-lactamase-1 gene; mcr-9, colistin resistance gene; qnrs1, fluoroquinolones gene. (B) Comparative analysis of the mcr-9-harboring plasmid characterized in this study with closely related plasmid pNIHE14-1904-mcr9. Open reading frames (ORFs) are portrayed by arrows and are depicted in different colors based on their predicted gene functions. The genes associated with the T4SS are indicated by dark blue arrows, while the genes involved in replication are indicated by red arrows. Resistance genes are indicated by yellow arrows, and accessory genes are indicated by light blue and purple arrows. Orange arrows represent the skeletal gene of the plasmid, and blue shading denotes shared regions of homology among different plasmids.

FIGURE 3

bla_SFO–1, bla_NDM–1, and mcr-9 gene contigs. Genetic environments surrounding the bla_NDM–1, bla_SFO–1 and mcr-9 genes in plasmid p1575-1. (A) The DNA fragments flanking the bla_SFO–1 gene in plasmid p1575-1. (B) The DNA fragments flanking the bla_NDM–1 gene in plasmid p1575-1. (C) The DNA fragments flanking the mcr-9 gene in plasmid p1575-1. (D) The IS26–bla_LAP–2–qnrS1–IS26 module. Colored arrows indicate open reading frames, with dark green, dark yellow, dark blue, purple, and red arrows representing other genes, mobile and accessory elements, the individual conjugation-related genes, hypothetical proteins and transposases, and antibiotic resistance genes, respectively.

FIGURE 4

S1-nuclease pulsed-field gel electrophoresis profiles. M, Salmonella enterica serotype Braenderup strain H9812; 1,1575; 2, p1575-1-EC600; 3, EC600.