Mechanism Of Plasmid-mediated Multidrug Resistance In Citrobacter Freundii

Carbapenem anbiotics possess broadest spectrum of activity and greatest potencyagainst Gram-positive and Gram-negative bacteria. Their activity is not affected byβ-lactamases, AmpC (AmpC-type enzymes) and ESBLs (extended-spectrumβ-lactamases, ESBLs). Unfortunately, recent emergence of carbapenem-resistantpathogens seriously challenges use of this class of lifesaving drugs. Carbapenemasesare specific β-lactamases with ability to hydrolyze carbapenems. Three majormolecular classes of carbapenemases, namely A, B and D, are recognized. Classes Aand D are serine-beta-lactamases, whereas class B is metallo-beta-lactamases. Themost important mechanism of carbapenem resistance is productions ofcarbapenemases.The aim of this study is to characterize the detailed molecular mechanisms ofcoproduction of plasmid-encoding carbapenemases KPC-2and NDM-1in Citrobacterfreundii strain112298, a multidrug-resistance isolate from a Chinese hospital.In this study, we collected70carbapenem-resistance isolates from a singlehospital in Guangzhou city, from which strain112298was selected for further indeepth study due coproduction of KPC and NDM. Strain112298was isolated from ahuman case of septic shock in a Chinese teaching hospital. Bacterial speciesidentification was performed by16S rRNA gene sequencing. The majorcarbapenemase and ESBL genes were detected by PCR followd by ampliconsequencing. A modified CarbaNP test was conducted to detectjavascript:void(0);javascript:void(0);carbapenemase, respectively. The minimum inhibitoryconcentration values were determined by using VITEK2, and antimicrobialsusceptibility was judged by Clinical and Laboratory Standards Institute standard. Theresistance plasmid was transfer into Escherichia coli though conjugal transfer,followed by plasmid DNA isolation from transconjugant, and then fully sequencedthrough high throughput sequencing and compared with closely related plasmids. Strain112298produced two carbapenemases KPC and NDM, which wereencoded by two different plasmids p112298-KPC (accession number KP987215) andp112298-NDM (accession number: KP987216), and contributed to carbapenemresistance of112298. p112298-NDM, but not p112298-KPC, could be transferred intoE. coli through conjugation experiments, generating a transconjugant112298-NDM-EC600. Both p112298-NDM and p112298-KPC could be transferredinto E. coli through electroporation, yielding two corresponding electroporants112298-NDM-TOP10and112298-KPC-TOP10, respectively.112298harboredblaNDM-1, blaSHV, blaKPC-2, blaOXA-1group and blaCTX-M-9group;112298-NDM-EC600and112298-NDM-TOP10contained blaNDM-1and blaSHV, while112298-KPC-TOP10harbored blaKPC-2, blaOXA-1group and blaCTX-M-9group. Class A/B, B, B, and Acarbapenemase activity was detected in112298,112298-NDM-EC600,112298-NDM-TOP10, and112298-KPC-TOP10, respectively. As determined by theantibiotic susceptibility test,112298was resistant to all the penicillin, β-lactamaseinhibitor, monobactam, cephalosporin, carbapenem, fluoroquinolone, furane, andsulfanilamide drugs tested but remained to be susceptible to aminoglycosides, while112298-NDM-EC600,112298-NDM-TOP10, and112298-KPC-TOP10werepenicillins, β-lactamase inhibitors, monobactam, cephalosporins, carbapenems testedbut remained to be susceptible to fluoroquinolones, furanes, aminoglycosides, andsulfanilamide tested.p112298-KPC was a novel untypeable plasmid while p112298-NDM was anIncX3type one. In p112298-KPC, a Tn1721-based blaKPC-2-carrying transposon isassociated with several additional resistance modules with various mobile elements,constituting a single multidrug resistance region. Assembly of these resistancemodules is likely mediated by homologous recombination between five copies ofintact or truncated IS26elements at different sites within the MDR region.p112298-NDM is a very close derivate of pNDM-HN380. blaNDM-1in p112298-NDMis carried by a Tn125variant, which differs from the prototype Tn125as observedpNDM-BJ01by disruption of upstream copy of ISAba125by IS5and absence ofdownstream copy of ISAba125. Production of KPC-2and NDM-1by p112298-KPC and p112298-NDM,respectively, makes C. freundii112298highly resistant to carbapenems. This fact isworrisome to a higher degree because the blaKPC-2gene is associated with a lot ofresistance genes (accounting for resistance to cephalosporin, chloramphenicol,chromate, fosfomycin, quaternary ammonium, rifampicin and sulphonamide) andmobile elements (such as insertion sequences, transposons and class1integron),constituting a complex mosaic structure of MDR in p112298-KPC. Coexistence of alarge amount of resistance genes is a single isolate denotes its rapid evolution and itspotent ability to acquire and keep different resistance genes, which will facilitate itsdissemination and persistence under different antimicrobial selection pressures.Future studies are needed to evaluate prevalence of the above plasmids amongenvironmental, animal and human isolates in China and other countries.