产ESBL肺炎克雷伯菌16SrRNA甲基化酶分布与耐药性研究

The distribution and drug resistances of 16SrRNA methylase of the extended spectrum β-lactamase klebsiella pneumoniae

目的探讨产ESBL肺炎克雷伯菌临床分离株16SrRNA甲基化酶基因分布以及与耐药谱的关系。方法采用PCR法检测苍南县人民医院临床分离出的69株非重复产ESBL肺炎克雷伯菌中的16SrRNA甲基化酶基因(rmt B和arm A),通过DNA测序,确定ESBL基因及整合子基因;质粒接合试验和质粒消除试验确定16SrRNA甲基化酶基因传播途径,ERIC-PCR技术进行基因分型。结果 69株产ESBL肺炎克雷伯菌rmt B阳性菌20株(28.99%),其中2株同时携带有rmt B和arm A。20株检测rmt B阳性株均携带有CTX-M基因,14株为CTX-M-14基因,6株为CTX-M-15基因;14株携带TEM-l基因;8株携带SHV基因中,5株SHV-12基因,3株SHV-11基因;3株携带OXA-10基因;3株携带VBE-1基因。另有12株携带有intl阳性,含有5种不同耐药基因盒,分别携带drf A25、drf A1、drf A12、aad A1、aad A2、sat和blaVEB-1基因。ERIC-PCR法显示20株16SrRNA甲基化酶基因阳性的肺炎克雷伯菌主要分为5型,A型为优势克隆菌株。质粒接合和消除试验发现A型克隆株KP5和KP16 rmt B均位于一质粒上并通过接合传播。结论产ESBL肺炎克雷伯菌临床分离株中普遍存在16SrRNA甲基化酶基因rmt B,导致对多种氨基糖苷类抗生素高水平耐药。rmt B通过水平基因传播和克隆传播两种方式进行播散,并且存在同时产ESBLs、16SrRNA甲基化酶和I类整合子肺炎克雷伯菌传播。

Objective To explore the distribution of 16SrRNA methylase in the extended spectrum β -lactamase klebsiella pneummoniae and its relationship with drug resistant spectrum. Methods Sixty - nine clinical isolated strains of non - repetitive ESBL klebsiella pneumonia were collected in the People＇s Hospital of Cangnan County. 16SrRNA methylation enzyme （rmtB and armA ） was detected by PCR, and the genotype of ESBL and integron were analyzed. Plasmid conjugation test and plasmid elimination method were used to determine dissemination of 16SrRNA methylase. ERIC - PCR genotyping technology was used to establish DNA fingerprinting. Results In 69 samples, 20 isolates were rmtB positive （28. 99% ） , and 2 of which were also armA and rmtB positive. All positive isolates carried the CTX - M gene, including 14 strains carried CTX - M - 14 gene and 6 strains carried CTX - M - 15 gene. Besides, 14 strains carried TEM - 1 gene. A total of 8 strains carried the SHV gene, including 5 strains carried SHV- 12 gene and 3 strains carried SHV -11 gene. 3 strains carried OXA - 10 gene, and 3 strains carried VBE - 1 gene. In addition, the intl gene was found in 12 strains of 20 rmtB positive strains, which were divided into five kinds of gene cassettes, including drfA25, drfA1, drfA12, aadA1, aadA2, sat and blavEB-1 respectively. The strains of 16SrRNA methylase positive were divided into five genotypes by ERIC -PCR, and A genotype was the main strain clones. From conjugative plasmid and elimination test, it is found that both of rmtB from KP 5 and KP 16 were located in a plasmid and they can disseminate by conjugation . Conclusion It iscommonly found that 16SrRNA methylase gene rmtB exists in the extended spectrum β-lactamase klebsiella pnemnmoniae, which could lead to resist to almost all amino glycoside. Both of horizontal gene transfer and colonial spread were responsible for the dissemination of the rmtB. In addition, the ESBLs, 16SrRNA methylation enzymes and class I integron can spread in Klebsiella pneumonia at same time.