KPC-producing Klebsiella Pneumoniae Molecular Epidemiology And Plasmid-mediated KPC Enzyme Dissemination Mechanism

In the recent ten years, the carbapenemase-producing Klebsiella pneumoniae has become a crisis events due to spreading rapidly through the global. The NDA-1metalloenzyme reported from India in2009, the class D enzyme of OXA-48reported in Turkey in2004, and the class A of KPC enzyme reported the United States in2001were all isolated from K. pneumoniae, and caused global cloning related spread. Meanwhile, K. pneumoniae was a notorious "collector" of multidrug resistance plasmids, not only increased their own survival ability, and can also carry resistance genes into other species by the plasmids dissemination, resulting in the spread of the resistance genes. Since2007, the KPC-producing K. Pneumoniae was report in China, they have set up the local area epidemic. This thesis mainly is the objective of the study for prevalence of KPC-producing K. pneumoniae in China and plasmid-mediated KPC enzyme dissemination mechanism..In this study,2971non-duplicated K. pneumoniae isolates were colleted from61hospitals of20provinces in2010, and the susceptibility testing of20common antimicrobial agents were determined. Isolates of K. pneumoniae with resistant or intermediate sensitive to carbapenems were selected to detect KPC-producing carbapenemase producing. Improved Hodge test and PCR amplification and sequencing were used to detect KPC gene. The homology of K. pneumoniae were analyzed by multilocus sequence typing (MLST).The results of drug sensitivity test showed that detection rate of ESBL-producing K. pneumoniae was41.9%, and resistant to imipenem, meropenem, and ertapenem were of1.3%,1.9%and2.7%, respectively. Among75isolates of K. Pneumoniae with resistant or intermediate sensitive to carbapenems, of25isolates were positive by Hodge test, and of25isolates were KPC-2-producing by PCR and sequencing. KPC-producing isolates of K. pneumoniae were mainly isolated from zhejiang province (20isolates), and one strain from each of Beijing, Liaoning, Hubei, Shanghai, and Jiangsu province. In China, the detection ratio of KPC-2-producing K. pneumoniae was of0.84%.According to MLST database, of17isolates among25of isolates of KPC-2-producing K. Pneumoniae were ST11(68%). Four isolates were ST494, and other four isolates were unknown type, belong to the new ST type. Four isolates of KPC-2-producing K. pneumoniae with ST494were recovered from a hospital in zhejiang province.Four of strains of carbapenemase-producing K. pneumoniae were collected from a children’s hospital in hubei provincial. The experiments of PCR and amplification sequencing, MLST, PFGE plasmid analysis, plasmid extraction, conjugation and transformation test, and Southern hybridization were uesed to homology analysis, beta-lactamase identification and plasmid positioning.Homology analysis showed that four strains of K. pneumoniae had an identical profile, and MLST of all four strains were ST439, indicating that the four strains were the same clone. Results of plasmid analysis show that four strains carry plasmid with size of30to300kb. KPC-2gene was located on plasmid with size of30kb, IMP-4gene on plasmid with size of300kb, CTX-M-15on plasmid with size of80kb, and DHA-1gene on plasmid with size of80kb and of300kb, respectively. This study confirms that isolate of K. pneumoniae co-produced KPC-2carbapenemase, IMP-4metallo-β-lactamase, CTX-M-15extended-spectrum β-lactamase, and DHA-1plasmid mediated AmpC β-lactamase.In this study, an isolate of Aeromonas hydrophila, two isolates of Escherichia coli and two isolates of Enterobacter aerogenes were recovered from an inpatient with severe acute pancreatitis. PCR mapping, plasmid analysis, and molecular biology technology were used to analyz for positioning KPC gene in different plasmid and carrying KPC gene transposon structure analysis.All five clinical isolates showed high-level resistance to carbapenem with MIC>32mg/L. The cured A. hydrophila strain, which lacked the KPC-encoding plasmid, showed susceptibility to carbapenems.PCR results showed that blaKPC-2, blaTEM-1, blaSHV-12, and blaDHA-I genes were positive in Two E. aerogenes strains, while the transformants of Two E. aerogenes strains were only positive for blaKPC-2and blaTEM-1For two E. coli strains, blaKPC-1and blasHv-12genes were identified. However, the transformant or transconjugant of two E. coli strains were only positive for the blaKPC-2gene. Differently, an isolate of A. hydrophila and its transconjugants were both positive for the blaKPC-2gene only.Plasmid analysis showed that the five clinical isolates all contain three to five with different sizes. The hybridization results showed that the blaKPC-2gene located in plasmid with size of30kb in conjugant or transformants of an isolate of A. hydrophila and two E. coli strains. However, the blaKPC-2gene was located in a ca.200kb and a ca.170kb plasmid in conjugants of two E. aerogenes strains, respectively. By using PCR mapping and sequencing, the nucleotide sequences of a11467bp fragment was obtained from transconjugants of A. hydrophila. The structures of the fragment contained several putative protein genes with order of Tn3-transposase, Tn3-resolvase,ISKpn8, truncated blaTEM, blaKPC-2, the ISKpn6-like, the KorC, the KlcA, and truncated replication initiator protein gene. The remaining4isolates all had the same structures by PCR mapping and sequencing.