肺炎克雷伯菌生物膜形成的相关因素研究

Study on factors related to biofilm formation of Klebsiella pneumonia

目的探讨毒力荚膜血清型、常见毒力基因、耐药基因、碳青霉烯类耐药以及生物膜培养时间等因素与肺炎克雷伯菌生物膜形成的关系。方法比较结晶紫染色定量检测法检测耐碳青霉烯肺炎克雷伯菌组(CRKP组)和肺炎克雷伯菌组(KPN组)的吸光度值(A值),并在生物膜培养的不同时间进行A值比较。PCR扩增6种常见高毒力荚膜血清型基因(K2、K5、K16、K20、K54、K57)、5种常见毒力基因[magA(K1)、rmpA、mrkD、wabG、allS]以及8种耐药基因(blaTEM、blaSHV、blaCTX-M、blaKPC、blaIMP、blaNDM、blaVIM、blaOXA-M)。结果两组肺炎克雷伯菌菌株生物膜形成能力A值为0.454～0.936,在培养12h时,两组生物膜形成能力差异无统计学意义(P>0.05)。在培养18h时,CRKP组形成生物膜的能力较KPN组强(P<0.05)。比较培养12h和18h两个时间段生物膜的A值,CRKP组培养18h的生物膜形成能力较12h更强(P<0.05);KPN组结果相同。24份标本均未检出毒力荚膜血清型基因(K2、K5、K16、K20、K54、K57),5种常见毒力基因[magA(K1),rmpA、mrkD、allS、wabG]仅检出1例(4%)。耐药基因blaTEM占54%、blaSHV占42%、blaKPC占46%、blaIMP占4%。结论培养时间影响结晶紫染色法在临床观察生物膜形成试验中的结果,影响生物膜形成的其他因素以及深层次的原因有待进一步研究和更多实验结果的支持。

Objective To investigate the relationship between the capsular serotype,common virulence genes,drug resistance genes,carbapenem resistance and biofilm culture time with the biofilm formation of Klebsiella pneumoniae.Methods The results of optical density(A)detected by the crystal violet staining quantitative method were compared between the carbapenem resistance Klebsiella pneumoniae(CRKP)group and control group,the A value comparison was conducted at different times of biofilm culture.The six highly virulent capsule serotype genes(K2,K5,K16,K20,K54,K57)were amplified by PCR.At the same time,the five virulence genes:magA(K1),rmpA,mrkD,wabG,allS,and the eight drug-resistant genes(blaTEM,blaSHV,blaCTX-M,blaKPC,blaIMP,blaNDM,blaVIM,blaOXA-M)were amplified too.Results The A value range of biofilm formation capacity in the two groups of Klebsiella pneumoniae was 0.454-0.936,and there was no statistical difference in biofilm formation ability at 12 h between the two groups(P>0.05).The ability of biofilm formation at 18 h culture in the CRKP group was stronger than that in the KPN group(P<0.05).Comparing the A values of biofilm culture between 12 h and 18 h,the biofilm formation ability of the CRKP group at 18 h was stronger than that at 12 h(P<0.05).The results of the control group were the same as that of the CRKP group.The capsule serotype genes(K2,K5,K16,K20,K54,K57)were not detected out in 24 samples,and among the five type of common virulence genes[magA(K1),rmpA,mrkD,allS,wabG],only 1 case(4%)was detected out.The drug resistance gene blaTEM accounted for 54%,blaSHV 42%,blaKPC 46%and blaIMP 4%.Conclusion The culture time affects the results of biofilm formation experiment in clinical observation by crystal violet staining and other aspects of affecting biofilm formation as well as the underlying reasons need to be further studied and supported by more experimental results.