[Plasmids carried by carbapenems-resistant Klebsiella pneumoniae in burn patients and its correlation with strain transmission].

Objective: To explore the carrier status of carbapenems-resistant Klebsiella pneumoniae (CRKP) plasmids in burn patients and analyze the correlation of these plasmids with the transmission of CRKP. Methods: A retrospective observational study was conducted. A total of 26 CRKP strains, which were isolated from the clinic-related samples of 22 burn patients (with 20 males and 2 females, aged (42±16) years) admitted to the First Affiliated Hospital of Army Medical University (the Third Military Medical University) from January to December 2017, were collected and individually numbered. The plasmids of the strains were extracted by alkali lysis. After determination of the plasmid concentration by a nucleic acid concentration detector, the agarose gel electrophoresis was used to visualize the bands, and rough plasmids typing was performed. The plasmid of the smallest numbered CRKP in each plasmid type was transformed into competent Escherichia coli (E. coli) strain Top10 (hereinafter referred to as TOP10 strain). The growth of each transformed strains and a Top10 strain cultivated in ampicillin containing Luria-Bertani (LB) agar medium overnight was observed, and the proportion of successful transformation was calculated. The plasmids from the smallest numbered plasmid carrying CRKP strain of successfully transformed Top10 strains (hereinafter referred to as the smallest successfully transformed strain) and correspondingly numbered CRKP were extracted, and then, the agarose gel electrophoresis was used to visualize the bands. Aforementioned successfully transformed strains and a TOP10 strain were used for the antimicrobial susceptibility testing with 17 antibiotics commonly used in clinic. The plasmid from the smallest successfully transformed strain was sequenced using the next-generation sequencing technology. Bioinformatics analyses such as protein-coding gene prediction and protein sequence alignment were performed successively. The sequence was subsequently named pKP03-NDM1 according to the carrying of drug resistance gene. According to the whole genome sequence of the plasmid carried by the smallest successfully transformed strain, the polymerase chain reaction, agarose gel electrophoresis, and gene sequencing were used to detect the New Delhi metallo-beta lactamase-1 (blaNDM-1) of plasmids in the remaining 25 strains of CRKP. The ST typing in multilocus sequence typing of 26 strains of CRKP was analyzed based on the literature. Results: Plasmids were successfully extracted from 26 CRKP, with mass concentrations ranging from 19.3 to 189.8 ng/μL. Each of the 26 CRKP carrying plasmids showed at least one band longer than 2 500 bp in the agarose gel electrophoresis, which were roughly divided into 6 patterns of A, B, C, D, E, and F. After overnight cultivation, no growth of strains was observed in LB agar medium containing ampicillin inoculated with the TOP10 strain or TOP10 strains transformed by the plasmid of CRKP patterning A, B, D, or E. In contrast, TOP10 strains transformed by the pattern C plasmid from NO.3 CRKP and the pattern F plasmid from NO.15 CRKP resulted in numerous colony growths, and those transformed strains were named as TOP10-pKP03 and TOP10-pKP15, respectively. The proportion of successful transformation was 1/3. The plasmid carried by TOP10-pKP03 showed a single band in the agarose gel electrophoresis, which was the same size as the largest band of the plasmid from NO.3 CRKP. The TOP10 strain was sensitive to the 17 antibiotics commonly used in clinic. TOP10-pKP03 and TOP10-pKP15 were resistant to penicillins, cephalosporins, and carbapenems but remained sensitive to monocyclic β-lactam, aminoglycosides, quinolones and tigecycline. The full length of the plasmid carried by TOP10-pKP03 was 41 190 bp. In addition to blaNDM-1, this plasmid carried bleMBL, T4SS, bleomycin resistance gene, conjugation transfer elements, and relaxase, etc. The plasmid showed 99% nucleotide identity similarity and the same length to the plasmid pJN24NDM1 extracted from an E. coli isolate JN24. Totally 16 (61.5%) CRKP were confirmed to carrying blaNDM-1 gene, among the ST typing of the 16 strains, 11 strains were ST11, while ST215, ST260, ST395, ST2230, and new ST had 1 strain each. Among the ST typing of 10 blaNDM-1-negative CRKP, 8 strains were ST11, while ST395 and ST2230 had 1 strain each. Conclusions: A blaNDM-1 gene carrying plasmid pKP03-NDM1 was extracted and sequenced from CRKP isolated from burn patients, with a high plasmid carrying rate. Meanwhile, this plasmid may mediate inter-CRKP and CRKP-E. coli horizontal transfer of blaNDM-1, leading to transmission of antimicrobial resistance.