高毒力肺炎克雷伯菌新型变种感染病例分析及耐药机制研究

Molecular identification and antimicrobial resistance analysis of a new hypervirulent variant of Klebsiella pneumoniae

目的：研究高毒力肺炎克雷伯菌新型变种（ hvKP）临床感染分布及对常用抗菌药物的耐药情况,分析相关耐药机制,并对毒力表型进行探讨。方法回顾性研究。收集2011年1月至2013年12月南昌大学第一附属医院分离的210株非重复肺炎克雷伯菌及其药敏结果,筛选出12株hvKP菌；多位点序列分型（ MLST）及肠杆菌科基因间重复一致序列（ ERIC）-PCR对hvKP菌株进行分子流行病学研究；产超广谱β内酰胺酶（ ESBL）菌株和碳青霉烯酶菌株检测分别应用双纸片协同及确认试验和改良Hodge试验；PCR及测序检测耐药基因型、血清荚膜分型及毒力基因；黏液丝试验确定菌株的高黏液型（HM）表型。结果 hvKP菌对15种抗生素的耐药情况严重,ESBL检出率高达8/12,1株改良Hodge试验阳性。12株hvKP中共发现5种 ERIC-MLST分子克隆分型,A/ST23和 B/ST263为主要型别,分别占5/12和4/12。 PCR及测序结果显示,blaKPC-21株,qnrA19株,qnrB47株, qnrS17株,blaCTX-M-36株,blaCTX-M-147株,blaTEM-110株,blaSHV-124株。 K1血清型11株,K2血清型1株,未检出其他血清型。毒力基因magA、rmpA和产气菌素检出率分别为11/12、9/12和9/12。此外,kfu基因、mrkD基因、wabG基因及allS基因的携带率分别为9/12、10/12、4/12和2/12。 HM阳性率为8/12。结论分离出多重耐药hvKP菌株,耐药机制复杂多样,由多种主要分子克隆引起,且存在强毒性血清型和多种毒力基因,应引起临床高度重视,必须采取有效措施控制其传播。（中华检验医学杂志,2015,38：392-396）

Objective To investigate the infection case and drug resistance patterns of new hypervirulent （ hypermucoviscous） clinical variant of Klebsiella pneumoniae（ hvKP） strains, and the virulence andresistancemechanisms.Methods Retrospectivestudy.TwohundredandtenKlebsiellapneumoniae isolates were collected in the First Affiliated Hospital of Nanchang University from January 2011 to December 2013, and 12 hvKP were identified. Multilocus sequence typing （ MLST） and enterobacterial repetitive intergenic consensus （ ERIC）-PCR were used to type the hvKP isolates. The extended spectrumβ-lactamase and carbapenemase producing Klebsiella pneumoniae were screened by the double disc synergy test and the modified Hodge test, respectively. PCR and DNA sequencing were carried out to detect their resistance related genes, serotype and virulence genes. Hypermucoviscosity phenotype （ HM ） of all strains was determinedbystringtest.Results HvKPwereresistantto15antibioticsandtheextended-spectrumbeta-lactamase producing rate was 8/12. In addition, one strain was detected positive by the modified Hodge test. Among the 12 hvKP strains, five ERIC-MLST molecular types were identified, of which A/ST23 and B/ST263 were the most predominant types, accounting for 5/12 and 4/12, respectively. The results of PCR and DNA secquencing showed that 2 were positive for blaKPC-2 （ 2/12 ） , 9 positive for qnrA1 （ 9/12 ） , 6＆amp;nbsp;positive for qnrB4 （6/12）, 8 positive for qnrS1 （8/12）, 6 positive for blaCTX-M-3 （6/12）, 7 positive for blaCTX-M-14（7/12）, 10 positive for blaTEM-1（10/12）, and 4 positive for blaSHV-12（4/12）. Eleven hvKP strains were serotype K1, while only one strain was serotype K2. Serotype K3, K5, K20, K54 and K57 were not detected. The positive rate of virulence genes magA, rmpA and aerobactin were 11/12,9/12 and 9/12, respectively. In addition, the positive rate of kfu, mrkD, wabG and allS were 9/12,10/12,4/12 and 2/12, respectively.ThepositiverateofHMwas8/12.Conclusions TheresistancemechanismofhvKPstrains was complex and diverse, in which strong toxicity serotype and multiple virulence genes exist, which need attention from clinic. Effective infection control measure should be conducted in order to control the outbreak of resistant hvKP strains.