RESUMO
Objective To investigate the capsular polysaccharide (CPS) serotypes and molecular characteristics of carbapenem resistant hypermucoviscous Klebsiella pneumoniae (CR-HMKP) and study the possible mechanism of carbapenem resistance. Methods A retrospective study was conducted on 18 nonduplicate CR-HMKP strains which were collected from the First Affiliated Hospital of Nanchang University from 2012 to 2016. The clinical data were retrieved from medical records. The capsular serotypes, resistance genes and virulence factors were detected by polymerase chain reaction and DNA sequencing. Antimicrobial susceptibility testing was determined on VITEK 2 compact system. The CR-HMKP strains were characterized molecularly by using PCR, multilocus sequence typing (MLST) and pulsed field gel electrophoresis (PFGE). Modified carbapenem inactivation method was used to screen carbapenemase-producing strains. Plasmid conjugation transfer experiments were carried out to study transmission of carbapenem resistance. Results Eighteen (2.7％) CR-HMKP isolates were identified, which belonged to 4 serotypes, including wzi128-K1 (n=1), wzi206-K57 (n=1), wzi2-K2 (n=2), and wzi64-K14.64 (n=14). PCR and sequencing analysis identified blaNDM-1 gene in 2 CR-HMKP strains, blaKPC-2 gene in 17 strains, qnrS1 gene in 18 strains, blaCTX-M-3 gene in 3 strains, blaCTX-M-14 gene in 18 strains, blaTEM-1 gene in 16 strains, blaSHV-12 gene in 17 strains, and rmtB in 5 strains. All the 18 CR-HMKP strains carried virulence-associated genes, including rmpA (88.9％, 16/18), magA (5.6％, 1/18), iroN (83.3％, 15/18), aerobactin (27.8％, 5/18), rmpA2 (66.7％, 12/18) and mrkD (100％, 18/18). Three sequence types (STs) were identified by MLST, including ST11 (15 strains), ST86 (2 strains), and ST412 (1 strain). PFGE resulted in three major PFGE clusters, of which cluster A corresponds to ST1 isolates, and cluster B corresponds to ST86 isolates, and cluster C corresponds to ST412 isolates. All the blaKPC-2- positive strains belonged to ST11. Plasmid conjugation was successful in 5 (27.8％) of the 18 CR-HMKP isolates. Conclusions wzi64-K14.64 is the predominant capsule serotype of the CR-HMKP strains in this hospital. KPC-2 gene conjugationmay contribute to the emergence of CR-HMKP isolates. In addition, CRHMKP strain may be the highly prevalent ST11, and highly virulent CPS serotypes harboring K1/K2.
RESUMO
Objective To investigate the antimicrobial resistant mechanisms of high level carbapenem resistant Klebsiella pneumoniae infection of burn patients .Methods A retrospective study was conducted on totally 18 non-repetitive high level CR-KP which were isolated from burn patients hospitalized between July 2014 and June 2015.MIC of antibiotics were determined by using the GN 13 cards and agar dilution method.The specific PCR and DNA sequence analysis were performed to confirm the β-lactamase type.Plasmid conjugation transfer experiments and southem hybridization were applied to study the mode of carbapenem resistance transmission .Outer membrane proteins ( Omps) were isolated and examined by PCR and ( sodium dodecyl sulfate polyacrylamide gel electrophores ) SDS-PAGE.Pulsed-field gel electrophoresis( PFGE ) and Multilocus sequence typing ( MLST ) was used to determine the genotypes . Results Susceptibility of antimicrobial agents indicated that all these strains with multiple drug resistance . The resistance rate to piperacillin/tazobactam, ceftriaxone, levofloxacin, ciprofloxacin, gentamicin, tobramycin, cefotetan, ceftazidime, cefepime, aztreonam, imipenem, and meropenem was 100％ (18/18).Moreover, the resistance rate of CR-KP isolates to amikacin was 72.2％ ( 13/18 ) , compound sulfamethoxazole was 61.1％(11/18), tigecycline was 0％(0/18).Conjugation study with Escherictda coli J53 resulted in the transfer of significant reduced carbapenem susceptibility from donors (MICs increased at least 8-fold).By PCR, eighteen strains of Klebsiella pneumoniae carried NDM-1 gene, 5 strains carried KPC-2 gene.The blaNDM-1 was transferable by plasmids.Southern blot hybridization indicated that the blaNDM-1 gene was located on plasmid in size of 46 kb.The plasmid belonged to incompatibility group IncX 3.Seven types of CR-KP were detected by PFGE.In addition, MLST assigned them to sequence type ( ST)11, ST395, ST17, ST37, ST263, ST14 and ST76 types.SDS-PAGE and ompK35/36 genes sequence analysis of Omp indicated that there was absence of outer membrane proteins OmpK 36 in ST11, ST395, ST37 strains.However, the other STs strains expressed lower quantities of OmpK 36.Conclusions High level carbapenem resistance in K.pneumoniae causing infection in burn patients is attributable to production of plasmid-mediated metallo· β-laetamase NDM-1 combined with porin OmpK36 deficiency or low expression .The K.pneumoniae with NDM-1 and KPC-2 carbapenemase were detected .
