艰难梭菌感染主要ST型菌株生物学特征的比较

Comparison of biological characteristics of major epidemic strains of Clostridium difficile from nosocomial infectious diarrhea

目的探究致医院感染性腹泻艰难梭菌主要序列型别(ST81、ST8和ST42)之间的毒力特征、芽孢形成能力及耐药机制等方面的差异。方法收集2017年9月至2019年9月首都医科大学附属北京友谊医院临床住院腹泻患者送检艰难梭菌培养的稀便标本816份进行艰难梭菌分离和培养。以该院主要序列型别的艰难梭菌ST81(26株)、ST8(15株)和ST42(14株)为实验菌株。应用聚合酶链反应(PCR)和酶联免疫荧光法分别检测艰难梭菌的毒素基因和毒素A/B蛋白的表达;采用平板涂布法检测艰难梭菌的芽孢形成能力;采用琼脂稀释法检测艰难梭菌对11种抗菌药物的耐药性;采用PCR方法扩增耐药基因、测序及氨基酸突变分析艰难梭菌携带的耐药基因和氨基酸的突变位点。计量资料的组间比较采用Kruskal Wallis秩和检验,率的比较采用Fisher精确检验。结果ST81型菌株为tcdA-tcdB+/cdtA-cdtB-毒素型别,ST8和ST42型菌株均为tcdA+tcdB+/cdtA-cdtB-毒素型别,ST42、ST8和ST81型别菌株产生毒素A/B蛋白的产量分别为41.9、2.4和0.83,ST42和ST8组毒素A/B蛋白的产量高于ST81组,差异有统计学意义(均P<0.001),ST42菌株毒素A/B蛋白的产量高于ST8组(P=0.045)。ST81、ST8和ST42型菌株的芽孢形成数量分别为494×10^(5) CFU/ml、160×10^(5) CFU/ml和166×10^(5) CFU/ml,ST81型菌株的芽孢形成数量明显高于ST8和ST42型菌株,差异有统计学意义(均P<0.001)。ST81型菌株为多重耐药菌,对头孢曲松、莫西沙星、红霉素和克林霉素等抗菌药物均耐药,耐药率均为100%(26/26),ST8型菌株对莫西沙星的耐药率为9/15,对红霉素和克林霉素的耐药率均为11/15。ST81型别菌株对莫西沙星的耐药率高于ST8型,差异有统计学意义(P=0.001),ST81型别对红霉素和克林霉素的耐药率高于ST8型,差异均有统计学意义(均P=0.005)。ST42型对头孢曲松和莫西沙星的耐药率分别为0和3/14。ST81型菌株对头孢曲松和莫西沙星的耐药率均高于ST42型,差异有统计学意义(均P<0.001)。ST81型菌株携带红霉素和克林霉素的耐药相关基因ermB阳性率[100%(26/26)]高于ST8组(11/15),差异有统计学意义(P=0.005)。喹诺酮类的耐药基因gyrA和gyrB编码的氨基酸突变分析显示,ST81和ST8型别菌株gyrA和gyrB基因编码的氨基酸同时发生突变,但gyrB基因编码的氨基酸突变位点不同。ST81型菌株为82位点苏氨酸突变为异亮氨酸和426位天冬氨酸突变为缬氨酸,ST8型菌株为82位点苏氨酸突变为异亮氨酸和426位天冬氨酸突变为天冬酰胺,ST42型别菌株gyrA基因编码的氨基酸为单突变,82位点苏氨酸突变为异亮氨酸。结论ST81型和ST42均为多重耐药菌,ST81型芽孢形成能力较强,ST42型别毒力较强。ST81和大部分ST8型对喹诺酮类药物的耐药性较强,需警惕多重耐药性ST81、较强毒力高耐药性ST42和ST8型菌株在医院的播散流行。

Objective This work aims to investigate the virulence features,spore formation and the resistance mechanisms of major sequence types(STs)of clinical Clostridium difficile isolates from nosocomial infectious diarrhea.Methods Clostridium difficile isolates were prospectively collected from 816 loose stool samples of in patients with antibiotic associated diarrhea at the Beijing Friendship Hospital of Capital Medical University from September 2017 to September 2019.The main ST types ST81(26 strains),ST8(15 strains)and ST42(14 strains)of C.difficile were used as experimental strains.The polymerase chain reaction(PCR)and enzyme-linked immunoassay(ELISA)were performed to detect toxin genes and toxin production of different C.difficile ST types,respectively.The count of the colony forming units(CFU)of the strains as conducted by using the brain-heart infusion(BHI)agar plates.The antimicrobial resistance patterns of the strains to eleven kinds of antibiotics were determined by agar dilution method.The antimicrobial resistance genes:gyrA,gyrB and ermB were amplified and sequenced from the stains.Mutations in the resistance genes were analyzed by sequencing.Measure data was compared by Kruskal Wallis Test,differences in the resistance rates in three group were compared using Fisher exact test.Results ST81 strains were identified as the tcdA-tcdB+/cdtA-cdtB-toxin type,ST8 and ST42 strains belonged to tcdA+tcdB+/cdtA-cdtB-toxin type.The toxin production of ST42 strains(41.9)were higher than ST8(2.4)and ST81 groups(0.83)(all P<0.001).The number of spore quantities of ST81,ST8 and ST42 strains were 494×10^(5)CFU/ml,160×10^(5)CFU/ml and 166×10^(5)CFU/ml,respectively.The spore quantities of ST81 strains were much higher than that of ST81 and ST42 strains(all P<0.001).From the in vitro susceptibility test,100%(26/26)ST81 strains were featured as multi-drug resistant(MDR),and they were resistant to moxifloxacin,ceftriaxone,erythromycin and clindamycin.The resistance rates of ST8 strain to moxifloxacin,erythromycin and clindamycin were 9/15,11/15 and 11/15,respectively.ST81 strains had higher resistance rates to moxifloxacin,clindamycin and erythromycin,compared to ST8 strains(P=0.001,P=0.005 and P=0.005).All ST42 strains were susceptible to ceftriaxone and 3/14 ST42 strains were resistant to moxifloxacin.ST81 strains had higher resistance rates to ceftriaxone and moxifloxacin than the ST42 strains(both P<0.001).The positive rate of ermB in ST81 strains(100%,26/26)were higher the ST8 strains(11/15)(P<0.005).Amino acid mutation analysis showed that ST81and ST8 stains had one amino acid substitution in both GyrA and GyrB,but the amino acid substitutions were different in GyrB between two ST types.ST81 strains had two point-mutations:Thr82 replaced by Ile in GyrA,and Asp426 replaced by Val in GyrB.ST8 strains had point-mutation:Thr82 replaced by Ile in GyrA;Asp426 replaced by Asn in GyrB.For ST42 strains,Thr82 was replaced by Ile in GyrA.Conclusions ST81 and ST42 strains were MDR.ST81 had higher spore ability,whereas ST42 strains had more virulence.ST81 strains and most of ST8 strains had high level of fluoroquinolones resistance.It is important to supervise persistently these three ST genotypes to prevent further dissemination.