Phenotypic And Molecular Characterization Of Clinical Carbapenem-Resistant Providencia Rettgeri Strains

Objective To investigate the phenotype,genotype,clonal relateness,as well as the surrounding environment of drug-resistant genes of carbapenem-resistant Providencia rettgeri clinical strains isolated from two hospitals so as to provide reference information for clinical treatment and prevention and control of drug-resistant strains.Methods Twenty strains of carbapenem-resistant Providencia rettgeri were isolated from hospitalized patients in two hospitals.The strains were initially identified by Vitek-2Compact,and the confirmed identification were based on ANI values,the antibiotic susceptibility testing was carried out using Vitek-2 Compact.Common carbapenemase resistance genes were detected using polymerase chain reaction（PCR）.Pulsed field gel electrophoresis（PFGE）and Bio Numerics 7.6 software were used to analyze the clonal relateness of the strains.The plasmids carried by the strains were detected by S1 pulsed field gel electrophoresis（S1-PFGE）and plasmid liquid phase conjugation assay was objected to detect the horizontal transfer characteristics of the plasmids.The sequences of the strains were obtained using Illumina and Nanopore sequencing platforms,and Plasmid Finder,MGE and Res Finder were then used to analyze mobile elements and resistance genes.The plasmid gene structures were draw by Inkscape 0.48.1 software.Results（1）All 20 strains of Providencia rettgeri carried bla _NDM-1 gene,and the resistance rate to ampicillin,ampicillin/sulbactam,cefzolim,ceftriaxone,ceftaxime,ceftazobactam,piperacillin/tazobactam,ceftazobactam/avibactam,meropenem,ertapenem,imipenem and ciprofloxacin was 100%,while the resistance rate to amikacin,tobramycin and gentamicin was 10%.（2）Cluster analysis of PFGE electrophoresis showed that the similarity of PH1～PH6 strains was 97%～100%,while the similarity of PH7～PH12,and QD51～QD56,were 100%,suggesting an nosocomial outbreak in the two hospitals.（3）S1-PFGE electrophoresis showed that 20 strains of Providencia rettgeri carried 1～2 plasmids with the size about 55 kb～350 kb.（4）Bioinformatics analysis showed that the 20 strains carried multiple resistance genes,includingβ-lactamase resistance gene bla _NDM-1（100%）,bla _OXA-1（60%）,bla _OXA-10（10%）,bla _PER-1（5%）,and bla _PER-4（5%）,the quinolone resistance gene qnr D1,the aminoglycoside resistance genes aac（6’）-Ib-cr and aad A16,and other resistance genes,and that strains PH1～PH13 and QD57 carried Col3M plasmids,while QD51～QD56carried Inc C plasmids.In addition,PH1～PH13 contained 4～5 mobile elements,including IS26,Tn6260,cn＿4926＿IS26 and ISEhe3,while QD51～QD57 contained 3～7 mobile elements,including Tn6196,ISEc9 and ISKpn14.（5）In the liquid-phase conjugation experiments,six Providencia rettgeri strains（QD51～QD56）transferred bla _NDM-1 harboring plasmid to Escherichia coli J53Azi^R,and their conjugants were named QD51C～QD56C,respectively.（6）The whole genome and plasmid sequences of PH6,PH12 and QD51 strains were obtained by Illumina and Nanopore sequencing platform.Bioinformatics analysis showed that both PH12 and PH6 strains had Col3M plasmid,which carriedg bla _NDM-1 and bla _OXA-1 genes,and were named as p NDM-PH12（61403 bp）and p NDM-PH6（119003 bp）,respectively.By comparison analysis,it was found that the bla _NDM-1 gene region on p NDM-PH6 plasmid had 9 more copies than that of p NDM-PH12,that is,there was the homologous recombination of the"cat B3-ARR-3-qac E-sul1-tnp A-trp F-ble _MBL-bla _NDM-1"region.In addition,the bla _NDM-1,bla _CMY-6,sul1,aac（6’）-Ib-cr and aac（6’）-Ib3 genes were located on an Inc C plasmid in QD51 strain（named p NDM-QD51,134802 bp）,which could be transferred horizontally through liquid phase conjugation experiments.Conclusion（1）The results indicate that the molecular mechanism of carbapenem resistance in the clinical strains of Providencia rettgeri is due to the bla _NDM-1 gene carried by these strains,which presents a multidrug-resistant phenotype.（2）There were nosocomial outbreaks of caused by the bla _NDM-1-harboring Providencia rettgeri strains in the two hospitals.To the best our knowledge,the nosocomial outbreak caused by co-harboring bla _NDM-1,bla _CMY-6,bla _CTX-M-3,aac（6’）-Ib-cr and aac（6’）-Ib3 Providencia rettgeri strains was firstly reported in the worldwide.（3）The clinical strains of Providencia rettgeri can not only horizontally transfer the bla _NDM-1-harboring plasmid to Escherichia coli J53Azi^Rthrough liquid phase conjugation,but also make the plasmid show diversity through homologous recombination of bla _NDM-1gene region,which has reference significance for the study of the evolution of plasmids carrying carbapenemase genes in Enterobacterales.