【摘要】 目的运用变性高效液相色谱(DHPLC)技术调查广州地区产SHV型β-内酰胺酶肺炎克雷伯菌各种亚型的流行分布情况,试图建立一种方便快捷的分子诊断及其流行病学监测的新方法。方法对73份产超广谱β-内酰胺酶(ESBL)肺炎克雷伯菌进行SHV质粒基因扩增,分别采用变性高效液相色谱(DHPLC)和测序法对扩增产物进行分析,以明确基因类型,并建立各个已知SHV基因亚型的特征性DHPLC图谱库。结果73株产ESBL的肺炎克雷伯菌中68株确定为SHV基因型,其中62株为已知基因型,分别为SHV-1225株,SHV-1a 14株,SHV-17株,SHV-2a 8株,SHV-28 5株,SHV-2 2株,SHV-26和SHV-33各1株；6株为新的SHV基因型,其中5株获得命名；非SHV型菌株5株,分别为LEN-4型1株,OKP型4株。经过DHPLC分析,全部样本均表现为异常的洗脱峰,各种亚型的异常洗脱峰的形态迥异,其敏感性达100％(68/68),特异性为93．2％(68/73)。SHV型质粒基因的突变集中在nt92、nt324～nt402及nt703～nt786这3个区段。结论SHV-12是广州地区产SHV型β-内酰胺酶肺炎克雷伯菌主要的基因亚型,高比例基因变异的检出预示本地区即将或已经面临肺炎克雷伯菌新耐药机制的抵抗和流行,因此必须加强对产ESBL肺炎克雷伯菌的分子流行病学监测,及时调整抗菌策略；DHPLC可作为一种快速敏感的筛查方法用于产ESBL肺炎克雷伯菌的分子流行病学监测。更多还原

【Abstract】 Objective To investigate the molecular epidemiology of SHV-type beta-lactamases producing Klebsiella pneumoniae in Guangzhou,and try to establish a quick and convenient method for their molecular diag- nosis and epidemiological monitoring.Methods SHV plasmid of 73 ESBL-producing Klebsiella pneumoniae strains were amplified by polymerase chain reaction,and the PCR products were analyzed by DHPLC and sequenced.Re- sults Within 73 ESBL-producing Klebsiella pneumoniae strains,68 strains were determined to be SHV-types,in- cluding 25 strains of SHV-12,14 strains of SHV-1a,7 strains of SHV-1,8 strains of SHV-2a,5 strains of SHV- 28,2 strains of SHV-2,1 strain of SHV-26,1 strain of SHV-33 and 6 strains of new SHV-types.Five non-SHV type strains were found in this research,including 1 strain LEN and 4 strains OKP.The patterns of chromatograms for all strains harbored mutations confirmed by sequencing and were different from that of wild type.These chroma- tngrams were also different among all kinds of SHV-genotypes.Sensitivity and specificity of DHPLC were 100% (68/68)and 93.2%(68/73),respectively,when compared to sequencing.Gene mutations of SHV plasmid mainly focused on the three fragments,namely nt92,from nt324 to nt402 and from nt703 to nt786.Conclusion SHV-12 is the predominant genotype of SHV-type beta-lactamnses producing Klebsiella pneumoniae in Guangzhou area.DHPLC method is rapid,sensitive and efficient for mutation detection and epidemiological monitoring of ESBL producing Klebsiella pneumoniae.更多还原