Investigation Of Transmission Mechanisms And Sequence Type Of Carbapenem-resistant KPC-producing Klebsiella Pneumoniae

Clinically-significant Gram-negative species remain mostly Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii. Carbapenem molecules are often the last resort for treating infections due to multidrug resistant isolates. In Enterobacteriaceae, resistance to carbapenems may result from combined mechanisms of resistance associating β-lactamases with weak (if any) intrinsic carbapenemase activity and decreased outer membrane permeability, or from true carbapenemases. Reports of carbapenemases have been increasing over the last few years. This phenotypic grouping of enzymes is a heterogeneous mixture of β-lactamases belonging to molecular Ambler class A (penicillinases), class B (metalloenzymes) and class D (oxacillinases).Carbapenem-hydrolyzing Klebsiella pneumoniae carbapenemase (KPC)-type enzymes (partially inhibited by clavulanic acid) have been identified mostly in Klebsiella pneumoniae. KPC are a group of carbapenemases which belong to Bush group2f, molecular class A. They are capable of hydrolyzing carbapenems, penicillin, cephalosporin, and aztreonam. Threrfore, treatment of infection caused by these pathogens is thus a considerable challenge for clinicians. KPC enzymes differ from the other2f enzymes by two specific characteristics:they are found on transferable plasmids and they are able to hydrolyse the aminothiazoleoxime cephalosporins such as cefotaxime. Plasmid-encoded class A enzyme KPC-1from a K. pneumoniae isolate that confers high-level resistance to carbapenems by itself.KPC was first identified in a K. pneumoniae isolate from North Carolina, and since then the enzyme has been found most frequently in K. pneumoniae. Recently however, it has been detected in multiple genera and species of the Enterobacteriaceae, including Salmonella enterica, K. oxytoca, Enterobacter spp., Citrobacter freundii, E. coli, Serratia marcescens, Proteus mirabilis and Morganella morganii, even in the non-lactosefermenting bacteria isolates of Pseudomonas aeruginosa, Pseudomonas putida and Acinetobacter baumannii. To date, eleven different variants (KPC-2-KPC-12) of the KPC enzyme have been reported, with KPC-2and KPC-3reported the most frequently. Of note, re-sequencing of the blaKPc1gene revealed it to be identical to blaKPc-2-Reports are now appearing that KPC-producing isolates have disseminated worldwide including France, Israel, Greece, Colombia, Argentina, China, Brazil, the U.K., Canada and Poland. Clonal dissemination of KPC-producing K. pneumoniae isolates is often observed, indicating that there are major international clones circulating. Transposons and IS elements are likely associated with the dissemination and spread of bla^pc among different bacterial species. Meanwhile, the horizontal transmission of KPC-harboring plasmid was also observed in a Michigan hospital in the United States and2nd Affiliated Hospital of Zhejiang University in China, respectively. The rapidly spread of this enzyme in the world partial because of clinical detection of carbapenemase producers remains difficult based on a simple phenotypic analysis of antibiotic susceptibility testing. A PCR-based technique may be used for detection of known carbapenemases.KPC has been emerging increasingly, and it makes the resistance mechanism a significant public health concern for their broad-spectrum activity and mobility. Reports appear that KPC-producing isolates have disseminated worldwide including more than ten countries across Asia, America and Europe. Recently, it has been proved ST258is the most frequent clone contributing the worldwide spread of KPC-producing K. pneumoniae, which has been identified in Poland, Norway, Sweden, Israel, Finland, Italy, Germany, Denmark, Hungary, Korea and especially the United States. In2007, KPC-producing K. pneumoniae was firstly reported in Zhejiang province, China. Recently, KPC-producing isolates are on the emerging in many places in China. However, little is known about the sequence type of KPC-producing K. pneumoniae spread in China. The aim of this study was to analyze the molecular epidemiology of KPC-producing K. pneumoniae circulating in China on the basis of sequence typing. In the study, ninety-five carbapenem-resistant KPC-2-producing K. pneumoniae isolates from13hospitals of nine cities covering five provinces in China were analyzed.1. Antimicrobial susceptibility testing and Mininal Inhibitory Concentrations determinationThe MICs of antimicrobial agents were determined by Etest (bioMerieux, France) according to the manufacturer's instructions and were interpreted as recommended by the Clinical and Laboratory Standards Institute (CLSI) update of June2010guidelines. All isolates exhibited resistance to imipenem, meropenem and/or ertapenem, with MICs ranging from3to>32mg/L. In addition, no susceptible isolates was detected in antimicrobial susceptibility testing to ceftazidime, cefepime, cefoxitin, piperacillin-tazobactam and cefoperazone-sulbactam. These isolates were demonstrated variable susceptibilities to ciprofloxacin and amikacin. Moreover, all isolates were susceptible to colistin (MIC≤2mg/L) and tigecycline (MIC≤2mg/L).2. The relatedness of carbapenem-resistant K. pneumoniae PFGE and MLST analysisPFGE analysis of the95clinical isolates of KPC-producing K. pneumoniae showed nine clones including A,B,C,D,E,F,G,H and I. Among these clones, clone B, C and E were the dominant clones and spread widely among hospitals in Hangzhou, Ningbo, Nanjing and Hefei cities. MLST showed seven STs based on the analysis of the seven housekeeping genes among the95clinical isolates of KPC-producing K. pneumoniae according to the MLST database of the K. pneumoniae. The prevalent clone was ST11(61/95), which was detected in isolates from Hangzhou, Ningbo, Nanjing and Hefei. Furthermore, six types were identified:ST15, ST23, ST349, ST351, ST438, and ST439. To understand the significance of PFGE pattern similarities and differences,14KPC-2-producing K. pneumoniae isolates represented different STs and regions were selected for PFGE and plasmid analysis. The MLST data were in concordance with the results generated by PFGE and the isolates with ST11shared the similarity of>80%in PFGE patterns. Plasmids from14selected transformants had a diversity of HindⅢ restriction maps.3. PCR amplification and DNA sequencing of extended-spectrum P-lactamases (ESBL) and plasmid-mediated AmpC β-lactamase genesAll original isolates were screened by PCR with specific primers for blaCTX-M, blaTEM, blasHV, blaVEB and six families of plasmid-mediated AmpC β-lactamase genes (MOX, CIT, DHA, ACC, EBC, FOX). Sequencing confirmed the blaTEM gene as blaTEM-1in43isolates and the blaVEB gene was not detected in any of the K. pneumoniae isolates. Among the61ST11isolates,38isolates contained ESBL genes(blaCTX-M-3, blaCTX-M-14, and blaSHV-12), five isolates contained AmpC gene(blaDHA-1), and three of them contained both ESBL and AmpC genes. Simultaneously, of the32isolates with other STs (ST15, ST23, ST349, ST351and ST439),9contained ESBL genes (blaCTX-M-3, blaCTX-M-14, blaSHV-12),17contained an AmpC gene (blaDHA-1), and4contained both ESBL and AmpC genes. In the other two isolates with ST438, no ESBL and AmpC genes were detected.4. Genetic environment of blaKPC geneA series of primers were designed at the base of blaKPC-surrounding sequences and PCR experiments were performed according to previously reported conditions. The obtained amplification products were sequenced. As reported previously, the environment surrounding blaKPC-2in plasmid from China is composed of a partial Tn4401structure and a Tn3-based element with the gene order of Tn3-transposase, Tn3-resolvase,ISKpn8, the blaKPC-1gene, and the ISKpn6-like element. In addition, there are another two variants. In our study, it has been observed that all the three structures as reported appeared in the95isolates, including33isolates with the original structure,12isolates with the structure of variant1and50isolates with the structure of variant2.The above results showed:1. Carbapenem-resistant K. pneumoniae are resistant to most antimicrobial agents. However, isolates were susceptible to colistin and tigecycline. Clinicians could select antimicrobial agents in combination according to drug concentrations.2. The dominant clone of KPC-producing K. pneumoniae in China is ST11, which is closely related to ST258that has been reported worldwide.3. The ESBL and/or the plasmid-mediated AmpC genes were detected in six STs besides of ST438. However in KPC-2-producing K. pneumoniae, it is hard to distinguish the difference of the presence of resistant genes between the STs.4. The special environment surrounding blaKPC-2in plasmid from China is composed of a partial Tn4401structure and a Tn3-based element, which may account for the high mobility of this resistance mechanism.