The Detection,Genotyping And Antimicrobial Resistance Study Of Clostridium Difficile

Purpose:The widespread use of antibiotics makes the incidence of antibiotic-associated diarrhea(AAD)increased.Clostridium difficile(CD)is one of the most important pathogens for AAD.In recent years,the incidence and severity of Clostridium difficile infection(CDI)rised highly as the result of the prevalence of high-virulent C.difficile strains,which brought about severe challenges for clinical treatment.This study investigated the molecular epidemiology and antibiotic resistance of C.difficile isolated from two tertiary care hospitals in Shandong Province,to aim of providing the suitable and efficient laboratory test method and strategy for the effective prevention and control of C.difficile infection.Methods:This study collected 504 stool specimens from patients with suspected antibiotic-associated diarrhea in the Affiliated Hospital of Qingdao University and the Zibo Centeral Hospital from March 2016 to May 2017.Glutamate dehydrogenase(GDH)and Clostridium difficile toxin A & B(CDAB)were detected by enzyme-linked immunoassay(EIA),and anaerobic culture and toxin gene testing were conducted meanwhile,for evaluating the sensitivity and specificity of GDH and CDAB for the diagnosis of CDI.Multilocus sequence typing and PCR-ribotyping were performed on 85 C.difficile isolates,to analyze the molecular epidemiological characteristics of clinical C.difficile isolates from Shandong Province.The clinical severity scores were assessed according to the severity of infectious symptoms of patients with CDI,to observe the relationship between different genotype C.difficile isolates and their clinical severity.The resistance of C.difficile strains to six commonly used antimicrobial drugs were detected by agar dilution methods.The amino acid variations of GyrA and GyrB were detected to observe the relationship between the genotype and the resistant phenotype of ciprofloxacin and moxifloxacin,and to study the role of the amino acid variations of GyrA and GyrB in the resistance mechanism of C.difficile strains to quinolone antibiotics.Results:504 fecal samples were conducted by EIA methods and anaerobic cultured.It was found that 115 samples were GDH-EIA positive(22.8%,115/504),39 samples were CDAD-EIA positive(7.7%,39/504),85 samples were anaerobic culture positive(16.9%,85/504).Further toxin gene detections were performed on the C.difficile strains isolated in the study,and found that 72 strains were toxin gene positive,with the toxin carrying rate of 84.7%(72/85).Compared with the anaerobic culture results,the GDH-EIA test had high sensitivity(98.8%)and specificity(92.6%),but the positive results only indicated that C.difficile were present in the samples,no matter if they were the toxingenic strains.Compared with toxigenic culture(including anaerobic culture and toxin gene detection),CDAB-EIA test had high specificity(100.0%)and low sensitivity(54.2%).Therefor,it was not recommended for CDI diagnosis in laboratory alone.The sensitivity of the scheme combining GDH and CDAB for the diagnosis of CDI was only 54.2%(39/72).And further addition of toxigenic culture to the scheme,the sensitivity for the diagnosis of CDI could be increased to 100%.Combined with the characteristics of GDH,CDAB and toxigenic culture,we recommended a three-step laboratorial diagnosis arithmetic for the rational application of regional laboratories.The results of GDH negative could exclude the diagnosis of CDI,and the results of CDAB positive could support the diagnosis of CDI.When the GDH and CDAB results were inconsistent,toxigenic culture should be carried out,which was considered an economic and efficient laboratorial diagnosis strategy for CDI.85 C.difficile strains were studied by the toxin gene detection and molecular typings,and it showed that 61 strains were A+B+CDT–(71.8%,61/85),10 strains were A–B+CDT–(11.8%,10/85),one strain was A+B+CDT+(1.2%,1/85),and 13 strains were A–B–CDT–(15.3%,13/85).The most common genotypes of C.difficile in this region were RT046/ST35(11.8%,10/85),RT014/ST2(10.6%,9/85)and RT017/ST37(10.6%,9/85),in which RT046/ST35 was the most common genotype of A+B+CDT– C.difficile strains(16.4%,10/61),and RT017/ST37 was the most usual genotype of A–B+CDT– C.difficile strains.A high-virulence C.difficile strain of which genotype was RT027/ST1 and toxingeic type was A+B+CDT+,was first found in Shandong Province.Five new types of multilocus sequence typing(MLST)were first discovered and named: ST450,ST451,ST452,ST453,and ST454.The clinical severity scores of patients with CDI showed that the severity scores of patients infected with A-B+CDT-C.difficile strains(3.50±0.85)were higher than those of patients infected with A+B+CDT-C.difficile strains(2.59±0.93,P<0.05),and the severity scores of patients infected with RT046/ST35 C.difficile strains(3.80±0.92)were higher than those of patients infected with RT001/ST3(2.63±0.74)and RT020/ST2(2.57±0.79)C.difficile strains(P<0.05).The antibiotic sensitivity tests in vitro showed that all C.difficile strains were sensitive to vancomycin and metronidazole,and the resistant rates of C.difficile strains to ciprofloxacin,erythromycin and clindamycin were 97.6%(83/85),64.7%(55/85)and 58.8%(50/85),respectively.All the toxigenic C.difficile strains were resistant to ciprofloxacin,and the resistant rates of the toxigenic C.difficile strains to erythromycin(70.8%,51/72),clindamycin(65.3%,47/72)and tetracycline(38.9%,28/72)were higher than that of the non-toxigenic strains(P<0.05).The main amino acid substitutions of Thr82→Ile and Ser366→Ala were discovered to be related to the increase of the minimal inhibit concentration(MIC)to moxifloxacin by the detection of GyrA and GyrB amino acid variation.They were consistent that the resistant phenotypes with the genotypes,which suggesting that GyrA and GyrB amino acid variations might be the main resistant mechanism of C.difficile strains to moxifloxacin.Conclusion:The combination of GDH and CDAB,supplemented by toxigenic culture when necessary,could diagnostic CDI efficiently and accurately.RT046/ST35,RT014/ST2 and RT017/ST37 CD strains were the most common strains in the region,and the high-virulence strain(RT027/ST1)was first isolated in Shandong Province.The resistant rates of C.difficile isolates to ciprofloxacin,erythromycin and clindamycin were high,and vancomycin and metronidazole were still the first-line antibiotics for the therapy of CDI.