| Objectives:Carbapenem-resistant Enterobacter cloacae complex（CRECC）has increasingly emerged as a major cause of healthcare-associated infections,and colistin is one of the last resort antibiotics for treatment.Mobile colistin resistance（mcr）-9 is a member of a growing family of mcr genes,which are reported to be an inducible gene encoding an acquired phosphoethanolamine transferase.We identified the presence of mcr-9 in CRECC strains isolated at our institution.We aimed to determine the clinical features,molecular characteristics,genomic structure,and phenotypic impact of mcr-9carriage in different patients,as well as to determine the pathogenicity of mcr-9 positive strains.Methods:A total of 24 Enterobacter cloacae complex（ECC）strains were collected from our institution from 2018 to 2021,and clinical data from patients were collected.Subsequently,antibiotic resistance genes and the popular characteristics of the strains were determined by PCR,whole-genome sequencing（WGS）,and bioinformatic analysis.A total of six CRECC strains carrying mcr-9 were identified,and the phenotypic impact of mcr-9 was verified by colistin induction assays.We assessed the sequence similarity of mcr-9-harbouring-plasmid from our institution and compared to plasmids for which sequence data is publicly available.Growth curves and in vitro competition assays were used to clarify the fitness cost of mcr-9 on the strains.In addition,biofilm formation assay,serum killing assay,and in vivo virulence assay of the Galleria mellonella infection model were used to clarify the pathogenicity of mcr-9 positive strains.Results:The major sequence type（ST）of the 24 ECC strains were ST177 and ST171.All 23 CRECC strains carried the metallo-β-lactamase gene(bla NDM-1,bla NDM-5,or bla IMP-4,except for the CRECC410 strain carried both bla NDM-5and bla IMP-4),the extended-spectrumβ-lactamase(bla TEM-1),and a quinolone resistance gene（aac（6’）Ib-cr）.mcr-9 was identified in IncHI2/2A or IncHI2/2A+Inc N plasmids.One of the p NDM-068001plasmids carrying mcr-9.1 could be a hybrid plasmid formed by a Tn6360-like bla NDM-1region inserted into an mcr-9.1-positive IncHI2/2A plasmid.Sequence alignments showed that the mcr-9.1 was mainly composed of"IS903B-mcr-9.1-wbu C-IS26",the p ECL414-1plasmid sequence in which IS1B was located downstream of mcr-9.2.The relative expression levels of mcr-9.2,qse C,and qse B genes were found to increase in CRECC414strain under the induction of sub-inhibitory concentrations of colistin,and the minimum inhibitory concentration of colistin was also increased.Based on in vitro competition assays,the strains carrying mcr-9 were found to have a fitness cost and a slowed growth rate.In addition,the CRECC78 strain showed increased biofilm production and high serum resistance.Conclusions:There may have been clonal transmission of ST177 clone between the pediatric intensive care unit,neonatal ward,respiratory medicine and pediatric ward of our hospital between October 21,2018,to June 6,2019.The spread of mcr-9 in the CRECC strain was primarily driven by the IncHI2/2A and IncHI2/2A+Inc N plasmid.The mcr-9.2gene was inducible in the CRECC414 strain.mcr-9 may impose a fitness burden on the strain.The increased biofilm production of the CRECC78 strain may contribute to its high pathogenicity.Furthermore,these findings provide a basis for studying the regulatory mechanisms of mcr-9 expression and highlight the importance of continuous monitoring of strains carrying mcr-9 in the future. |
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