摘要
目的探究临床分离铜绿假单胞菌常见群体感应(QS)系统调控基因和毒力基因的携带和表达情况及与耐药的相关性。方法收集检验科各类临床标本中分离出的铜绿假单胞菌97株,应用vitek2-compact仪器法及标准纸片扩散法检测抗菌药物的耐药性,运用聚合酶链式反应(PCR)方法检测4种QS系统调控基因及7种毒力基因的携带情况,对结果做统计学分析;荧光定量PCR(qRT-PCR)检测调控基因LasI、Rh1R和毒力基因exoS、PCN的表达情况,对两类基因的相对表达量进行线性分析。结果97株分离株对11种抗菌药物的耐药率:妥布霉素、庆大霉素、阿米卡星的耐药率小于10%且阿米卡星最低为2.06%,对其余药物耐药率均在11%以上,最高的为亚胺培南25.77%,43株为多重耐药菌株,且呼吸系统来源的菌株对抗菌药物耐药性更高。PCR基因检测结果显示:4种QS系统调控基因LasI、LasR、Rh1I、Rh1R检出率均为100%;7种毒力基因检出率最高的为exoT 98.97%(96/97),最低的为exoU 13.40%(13/97),PCN 43.30%(42/97),其余毒力基因检出率皆在86%以上。未携带毒力基因的菌株对头孢他啶等6种抗菌药物耐药,携带有7种毒力基因的菌株对头孢他啶耐药。PCN、exoS、exoU阳性菌株中,非多重耐药铜绿假单胞菌(NMDR-PA)所占比例更高;PCN+菌株对哌拉西林/他唑巴坦耐药率更高且差异有统计学意义;与exoS+/exoU-菌株相比,exoS-/exoU+菌株耐药的抗菌药物种类明显下降。NMDR-PA中调控基因rh1R、LasI的相对表达量更高且LasI的差异有统计学意义;调控基因rh1R与毒力基因exoS、调控基因LasI与毒力基因PCN之间的表达呈正向线性关系。结论铜绿假单胞菌耐药现象仍较严重且表现多重耐药,调控基因稳定存在于菌株中,除exoU、PCN外,其余毒力基因exoS、exoT、exoY、ToxA、LasB携带率均较高,调控基因rh1R、LasI能正向调节毒力基因的表达,QS系统调控基因与毒力基因均会影响铜绿假单胞菌的耐药性。
Objective To investigate the correlation of carriage and expression of quorum sensing(QS)system regulatory genes and virulence genes with drug resistance in Pseudomonas aeruginosa strains isolated from clinical sources.Methods A total of 97 strains of Pseudomonas aeruginosa isolated from various clinical specimens in the laboratory were collected.Firstly,Drug-resistance were detected by the vitek2-compact instrument method and the standard disc diffusion method,4 QS system-regulated genes and 7 virulence genes were detected by polymerase chain reaction(PCR).After that,the expression of regulatory genes LasI,Rh1R and virulence genes exoS and PCN were detected by real-time fluorescence quantitative PCR(qRT-PCR),and moreover linearly analysis was used to analyze the correlation of the two types of genes.Results The resistance rates of 97 strains to 11 antibacterial drugs.The resistance rate of tobramycin,gentamicin,and amikacin was less than 10%,and the minimum amikacin was 2.06%.The resistance rate to the remaining drugs was all above 11%,and the highest was imipenem 25.77%,and 43 strains were multi-drug resistant bacteria,and the respiratory-derived strains were more resistant to antimicrobials.The results of PCR gene detection showed:the detection rates of the four QS system-regulated genes LasI,LasR,Rh1I and Rh1R were all 100%;the highest detection rate of 7 virulence genes was exoT 98.97%(96/97),and the lowest was exoU 13.40%(13/97),PCN 43.30%(42/97),and the rest were above 86%.The strains without virulence genes were resistant to 6 antibiotics including ceftazidime,and the strains with 7 virulence genes were only resistant to ceftazidime;among the PCN,exoS,and exoU positive strains,the proportion of non-multidrug resistant Pseudomonas aeruginosa(NMDR-PA)was higher;PCN+strains were more resistant to piperacillin/tazobactam and the difference was statistically significant;compared with exoS+/exoU-strains,the types of antibiotics resistant to exoS-/exoU+strains decreased significantly.The relative expression levels of regulatory genes rh1R and LasI in NMDR-PA were higher and the difference of LasI was statistically significant;the expression of the regulatory gene rh1R and the virulence gene exoS and the regulatory gene LasI and the virulence gene PCN showed a positive linear relationship.Conclusion The drug-resistance of Pseudomonas aeruginosa is still serious and multi-drug resistance.QS system regulatory genes exist in the strain stably,except for exoU and PCN,the other virulence genes exoS,exoT,exoY,ToxA and LasB have higher carrying rates,the regulatory genes rh1R and LasI positively regulate the expression of virulence genes,both QS system regulatory genes and virulence genes affect drug resistance in Pseudomonas aeruginosa.
作者
周凯
时翠销
宋国滨
黄颖
徐元宏
Zhou Kai;Shi Cuixiao;Song Guobin;Huang Ying;Xu Yuanhong(Dept of Clinical Laboratory, The First Affiliated Hospital of Anhui Medical University, Hefei 230022;Dept of Clinical Laboratory, East Hospital Affiliated to Tongji University, Shanghai 200123)
出处
《安徽医科大学学报》
CAS
北大核心
2022年第5期748-753,共6页
Acta Universitatis Medicinalis Anhui
基金
国家自然科学基金(编号:81171606)。
关键词
铜绿假单胞菌
耐药性
群体感应系统
毒力基因
Pseudomonas aeruginosa
drug resistance
quorum sensing system
virulence genes
