β-内酰胺酶合并OmpK36膜孔蛋白缺失引起肺炎克雷伯菌对碳青霉烯类抗生素敏感性的降低

Reduced carbapenem susceptibility in Klebsiella pneumoniae clinical isolates is due to combination of β-lactamases and the loss of OmpK36 porin

目的研究肺炎克雷伯菌（Klebsiella pneumoniae，Kp）对碳青霉烯类抗生素敏感性降低的分子机制。方法临床分离到2株碳青霉烯敏感性降低的Kp Z2554和Z2110，经脉冲场凝胶电泳（PFGE）证实非同一克隆株。对2株细菌进行药物最低抑菌浓度测定（MIC）、接合试验、质粒图谱分析、等电聚焦电泳（IEF）、特异性PCR扩增和DNA序列分析、外膜蛋白分析以及羰酰氰氯苯腙（CCCP）抑制试验等。结果Kp Z2554和Z2110对亚胺培南和美罗培南的MIC为1～8μg／ml，前者对青霉素类、头孢菌素类和氨曲南等抗生素高度耐药，后者对上述抗生素的耐药程度稍弱于前者，但对头孢西丁的MIC大于512μg／ml。IEF、PCR扩增和DNA序列分析证实Kp Z2554产TEM-1（等电点5．4）和CTX-M-14（等电点7．9）2种β-内酰胺酶，Kp Z2110产TEM-1、CTX-M-14和DHA-1（等电点7．8）3种酶。SDS-PAGE分析显示2株细菌均有相对分子质量（Mr）为39×10^3蛋白条带（OmpK36）的缺失。外膜蛋白基因（ompK35和ompK36）序列分析发现，Kp Z2554和Z2110的ompK35基因的个别位点存在点突变，但氨基酸序列不变。CCCP抑制试验显示CCCP不能提高Kp Z2554和Z2110对碳青霉烯类抗生素的敏感性。结论β-内酰胺酶的产生合并OmpK36膜孔蛋白的缺失可引起蜘对碳青霉烯类抗生素敏感性的降低。

Objective To investigate the molecular mechanism of reduced carbapenem susceptibility in KlebsieUa pneumoniae （ Kp ）. Methods Two clinical isolates of carbapenem-non-susceptible Kp （ strain Z2554 and Z2110 ） were investigated. Pulsed-field gel electrophoresis （ PFGE ） patterns indicated that the two isolates were genetically unrelated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments were carried out in mixed broth cultures. Plasmid DNA preparations were obtained by using an alkalinelysis technique. The crude β-1actamase extracts of Kp isolates and E. coli transconjugant were subjected to analytical isoelectric focusing （IEF）. Specific PCR and DNA sequence analysis were preformed to confirm the β-lactamase type. Outer membrane proteins （OMP） were isolated and examined by SDS-PAGE. Inhibition assays were performed in the presence of an energy uncoupler, carbonyl cyanide m-chlorophenylhydrazone （CCCP）. Results Kp Z2554 and Z2110 showed reduced susceptibility to carbapenems with MICs of 1 to 8 μg/ml. Kp Z2554 was resistant strongly to penicillins, cephalosporins, and aztreonam. Though the resistance level of above-mentioned antibiotics in Kp Z2110 was not so high as strain Z2554, the MIC of cefoxitin was above 512 μg/ml. IEF, PCR and DNA sequence analysis confirmed that Kp Z2554 produced two β-lactamases of TEM-1 （pI of 5.4） and CTX-M-14 （pI of 7.9）, and Kp Z2110 produced three β-1actamases of TEM-1, CTX-M-14, and DHA-1 （ pI of 7.8 ）. SDS-PAGE analysis of OMP revealed that both Kp Z2554 and Z2110 lacked a major OMP of Mr approximately 39 × 10^3（OmpK36） that was present in Kp ATCC13883. The ompK35 and ompK36 genes were amplified by using PCR and were sequenced. The ompK35 gene sequence of Kp Z2554 and Z2110 contained a few silent mutations. In ompK36, several insertions and deletions of short DNA fragment （ 1-6 bp ） and considerable point mutations were found, which resulted in the alteration of open reading frame（ORF） and early termination of translation. The MIC of carbapenems was not change in the presence of CCCP. Conclusion Reduced carbapenem susceptibility in Kp Z2554 and Z2110 isolates is attributable to production of β-1actamases combined with the loss of OmpK36 porin.