Emergence of Neonatal Sepsis Caused by MCR-9- and NDM-1-Co-Producing Enterobacter hormaechei in China

Abstract

Mobile colistin resistance (mcr) genes represent an emerging threat to public health. Reports on the prevalence, antimicrobial profiles, and clonality of MCR-9-producing Enterobacter cloacae complex (ECC) isolates on a national scale in China are limited. We screened 3,373 samples from humans, animals, and the environment and identified eleven MCR-9-positive ECC isolates. We further investigated their susceptibility, epidemiology, plasmid profiles, genetic features, and virulence potential. Ten strains were isolated from severe bloodstream infection cases, especially three of them were recovered from neonatal sepsis. Enterobacter hormaechei was the most predominant species among the MCR-9-producing ECC population. Moreover, the co-existence of MCR-9, CTX-M, and SHV-12 encoding genes in MCR-9-positive isolates was globally observed. Notably, mcr-9 was mainly carried by IncHI2 plasmids, and we found a novel ~187 kb IncFII plasmid harboring mcr-9, with low similarity with known plasmids. In summary, our study presented genomic insights into genetic characteristics of MCR-9-producing ECC isolates retrieved from human, animal, and environment samples with one health perspective. This study is the first to reveal NDM-1- and MCR-9-co-producing ECC from neonatal sepsis in China. Our data highlights the risk for the hidden spread of the mcr-9 colistin resistance gene.

Keywords: Enterobacter cloacae complex; IncHI2; MCR-9; neonatal; sepsis.

Figure 1

Schematic procedure of the study. BRICS, Blood Bacterial Resistant Investigation Collaborative Systems; WGS, whole-genome sequencing.

Figure 2

Characteristics of MCR-9-producing ECC-associated cases. (A) Study sites and geographical distribution of MCR-9-producing ECC isolates. Seven provinces shown in color were included in the surveillance for MCR-9. (B) Clinical characteristics and epidemiological characteristics of MCR-9-producing ECC-associated isolates.

Figure 3

Core-genome phylogeny and resistome of 70 MCR-9-positive ECC isolates from NCBI genome database and the eleven isolates from this work. E. hormaechei, E. cloacae, E. kobei, and E. arburiae isolates are indicated in blue, green, yellow, and pink. Scale bars represent the number of substitutions per site. The ARGs distribution of MCR-9-positive ECC isolates is presented on the right. The ARGs groups are indicated with different colors. Isolates identified in this study are shown in red.

Figure 4

(A) S1-PFGE and Southern blot of eleven MCR-positive isolates. S1-PFGE patterns of Salmonella isolates and their relevant conjugants. Southern blot-hybridization of S1-nuclease digested DNA using a specific probe (mcr-9). M: XbaI digested total DNA of Salmonella enterica serotype Braenderup H9812 as a size marker. (B) Genetic contexts of mcr-9 genes in eleven ECC isolates. Blue rectangles highlight identical regions.

Figure 5

Circular representation of the studied mcr-9-carrying plasmids. (A) Analysis of the IncFII plasmid p53287-MCR-9 carried by the bacteremia associated E hormaechei strain 53287. (B) Sequence comparison of the mcr-9-encoding IncHI2 plasmids p57244-MCR-9, p51118-MCR-9, and p61363-MCR-9. GC content and GC Skew were represented on the inner map’s distance scale (kb). Each plasmid was compared to its most closely-related plasmid. The red arc around the map indicated ORFs. Certain important genes were also indicated on the ring.

Figure 6

Virulence profiles of MCR-9-positive ECC isolates. (A) Serum-killing assay. Survival in a serum-killing assay of ECC strains. The survival is denoted in percentage. The bars denote means and standard errors of the mean. (B) Galleria mellonella infection assay. Survival at 144 h in a G mellonella assay of ECC strains. The survival is denoted in percentage. The bars denote means and standard errors of the mean.