致肝脓肿高毒力肺炎克雷伯菌的表型及分子特征

Molecular characteristics of liver abscess-causing Klebsiella pneumoniae

目的探究导致肝脓肿高毒力肺炎克雷伯菌的分子特征。方法用VITEK-2细菌鉴定仪鉴定2014年1月至2016年1月丽水市中心医院肝脓肿患者脓肿穿刺液中分离的细菌。应用拉丝试验鉴定菌株的高黏性,用多位点序列分型(MLST)和血清型分型(K分型)对菌株进行分子分型,并用S1核酸酶脉冲场凝胶电泳(S1-PFGE)对菌株质粒谱进行分析。结果 57例肝脓肿患者接受肝脏脓肿穿刺引流并做脓液培养。44例患者的脓液培养到不同的致病菌,培养阳性率为77.2%。在培养到的44株病原菌中,其中2株为大肠埃希菌,产酸克雷伯菌、金黄色葡萄球菌各1株,而肺炎克雷伯菌为40株,占肝脓肿致病菌的90.9%。40株肺炎克雷伯菌中,拉丝阳性率为67.5%(27/40),K1为主要血清分型,占62.5%(25/40),其次为K2型,占17.5%(7/40)。ST23为主要ST分型,占47.5%(19/40),其次为ST86和ST65,各占7.5%(3/40)。同时发现一些未报道过的致肝脓肿肺炎克雷伯菌新ST分型,如ST218、ST1941、ST76、ST2159、ST660和ST485。40株致肝脓肿肺炎克雷伯菌中总共检测到12种质粒谱,包括带有一个质粒、多个质粒或不带质粒的谱型。其中带有一个近220 kb的质粒谱为主要谱型,共涉及19株菌,占47.5%,12株菌带有一个质粒,大小为140~250 kb。4株菌带有2个或3个质粒,5株菌不含有质粒。结论拉丝试验和血清学分型不能鉴定所有的高毒力肺炎克雷伯菌;高毒力肺炎克雷伯菌很多为ST23型,但其进化整体上较为分散;高毒力肺炎克雷伯菌菌株可以不携带质粒。

Objective To analyze the molecular characteristics of liver abscess-causing Klebsiella pneumoniae(LA-Kp). Methods CT-guided liver abscess subcutaneous drainage and sample collection were performed on patients with liver abscess in our hospital during January 2014 to January 2016. The isolates were identified using VITEK-2. String test was used to define the hypermucoviscous phenotype. Multilocus Sequence Typing(MLST) and serotyping were applied to identify the molecular typings. S1-PFGE was carried out for detecting and determining the sizes of the endogenous plasmids in the strains. Results A total of 57 patients received percutaneous drainage therapy were enrolled. Pus samples were collected and overall rate of positive bacterial culture was 77.2%(44/57), of which 90.9%(40/44) were identified as K. pneumoniae. Other pathogens were Escherichia. coli(2/44), Klebsiella oxytoca(1/44) and Staphylococcus aureus(1/44). The positive rate of string test of LA-Kp was 67.5%(27/40). K1 serotype was the dominant serotype 62.5%(25/40), followed by K2 17.5%(7/40). MLST analysis revealed a total of 16 sequence types. ST23 was the major sequence type, accounting for 47.5%(19/40). ST86(n=3) and ST65(n=3) were also identified. Six STs including ST218, ST1941, ST76, ST2159, ST660 and ST485 that had not been reported previously in LA-Kp were identified. A total of 12 different plasmid profiles were observed among 40 LA-Kp strains, including strains harboring a single pK20144-like plasmid and strains harboring no or multiple plasmids. Among them, 19 strains(47.5%) contained a plasmid with similar size as pK2044(around 220 kb), and 12 strains harbored a plasmid ranging from 140 to 250 kb. Interestingly, 4 strains harbored two or three plasmids, which had not been reported previously. Moreover, 5 LA-Kp strains contained no plasmid. Conclusion String test, MLST and serotyping can not identify all hypervirulent K. pneumoniae. The presence of a replicating plasmid is not essential for hypervirulent K. pneumoniae.