| Objective:Countries all over the world such as Canada,the United States and European countries have seen outbreaks of hospital-acquired diarrhoea causing high rates of mortality and recurrence since 21 th century.And Clostridium difficile is to blame.Reports said that about 15-25% antibiotic-associated diarrhoea and over 95% pseudomembranous colitis are associated with C.difficile.Toxin A(TcdA)and toxin B(TcdB)are the two main pathogenic factors of this bacterium.Widely-used antibiotics in clinic such as cephalosporins and fluoroquinolones which disrupt the balance of intestinal flora and prompt excessive propagation of toxigenic C.difficile are the most related risk factor.Other risk factors include advanced age,chemotherapeutics,inflammatory bowel disease,organ transplantation,chronic renal disease,immunosuppression and underlying sever complications.Spores,which are resistant to many physical and chemical factors,even the alcoholic hand antiseptics used in hospitals,are vactor of transmission.Data on epidemic of C.difficile in diarrhoea inpatient in Shijiazhuang is limited.So we collected unformed faeces from diarrhoea inpatients to culture C.difficile in order to identify its epidemic and resistance profile to drugs,providing baseline data for prevention and control Clostridium difficile infection in hospitals.In addition,mechanism of resistance to rifamycin in C.difficile will also be involved in this reseach.Method:1 A total of 1013 unformed faeces were collected from diarrhoea adult inpatients in the Second Hospital of Hebei Medical University,the Forth Hospital of Hebei Medical University,the First Hospital of Hebei Medical University and Hbbei general hospital from July,2010 to December,2011,from August,2012 to December 2013 and from January to September,2015.2 Anaerobic enrichment Spore Culture Method were used to culture C.difficile specially.Morphology on the agar plates and under the microscope,special smell and biochemical reaction were measured to confirm the presence of C.difficile.3 Genome were extracted.Toxin gene amplified by multiple PCR were analysed by electrophoresis to determine their toxin types.Primers complementary to the 3' end of the 16 S rDNA gene and the 5' end of the 23 S rDNA gene were used to amplify the variable-length intergenic spacer region.PCR products were separated by electrophoresis to identify the PCR-ribotype of the strains according to the profiles.4 Antimicrobial susceptibility testing was performed using the agar dilution method recommended by the Clinical and Laboratory Standards Institute(CLSI).The antibiotics involved were levofloxacin,ciprofloxacin,ceftazidime,ceftriaxone,meropenem,clindamycin,erythromycin,tetracycline,chloramphenicol,rifampin,rifaximin,metronidazole,vancomycin and fidaxomicin.5 A total of 101 C.difficile were selected from our bacteria library including 69 rifampin-resistant strains,4 rifampin-intermediate strains and 28 rifampin-susceptible strains.Part of the rpoB gene of the strains were amplified and sequenced.Mutations were identified by sequence alignment using rpoB gene of C.difficile 630.Result: 1 172 toxigenic C.difficile were isolated from 1013 faeces and positive rate was 17.0%.Among the 172 C.difficile,168 were tcdA+tcdB+cdtA-cdt B-while 4 were positive for tcdA,tcd B,cdtA and cdtB.No tcdA-tcdB+ was found.34 different PCR-ribotypes were detected,of which HB6 was the most prevalent type and the number was 43,followed by 30 isolates in HB5 and 28 isolates in HB1.In brief,HB6,HB5 and HB1 were the most prevalent types in our region,counting for 58.7%(101/172)of the strains;2 Minimal inhibitory concentration(MIC)of 14 antibiotics to 172 toxigenic C.difficile showed that all the isolates were highly susceptible to metronidazole,vancomycin and fidaxomicin while large portion of the strains were resistant to ciprofloxacin,clindamycin,erythromycin,levofloxacin,tetracycline and the resistance rate were 98.8%,94.2%,77.3%,75%,35.5%,respectively.We also noticed that C.difficile were more sensitive to levofloxacin compared with ciprofloxacin although both of them are the third generation of quinolones.Similarly,all the isolates were resistant to ceftazidime whereas only 26.7% were resistant to ceftriaxone.Low percentage of resistant to rifampin,rifaximin,chloramphenicol,meropenem were observed and the corresponding rates were 14.0%,14.0%,7.0%,2.3%,respectively;3 Moreover,analysis of PCR-ribotype combined with resistance profile revealed that resistance rates were more higher in HB5,HB6 and HB1 than other types.The differences were statistically significant considering erythromycin.HB5 exhibited the highest rates of resistance to all the tested antibiotics among other PCR-ribotypes except ceftazidime,metronidazole,vancomycin and fidaxomicin.And the differences were statistically significant considering ceftriaxone,tetracycline,rifampin and rifaximin.In addition,all the 4 meropenem-resistant C.difficile belonged to HB5.Among 24 rifampin-resistant isolates,22 belonged to HB5 while only 2 were other types(P<0.001);4 Sequencing of part of rpoB gene of 101 C.difficile indicated that mutations were detected in rifampin-resistant strains while didn't in rifampin-susceptible ones.D492 Y,H502N were found in two rifampin-intermediate strains respectively.However,there were still two rifampin-intermediate isolates had no nucleotide change.H502N&R505K,R505K&I548M were the most common mutations observed in rifampin-resistant strains.H502D&D590V,D492 Y,present in rifampin-resistant and rifampin-intermediate strains respectively,were novel mutations related to rifamycin-resistance in C.difficile.Conclusion:1 Of the 172 toxigenic Clostridium difficile,the majority are tcdA+tcdB+ cdtA-cdtB-,counting for 97.7%,while only 4 isolates were positive for tcdA,tcdB,cdtA and cdtB.HB6,HB5 and HB1 were the main prevalent types.The use of fluoroquinolones,cephalosporins and clindamycin in clinic may trigger CDI for the isolated strains were highly resisitant to them.Metronidazole,vancomycin and fidaxomicin can be used as therapeutic drugs since all of the isolates were susceptible.Low resistance rate of C.difficile to rifampin and rifaximin were observed,so they can still serve as alternative therapy for recurrent CDI.When using rifamycin in treatment with CDI,physicians should beware of infections caused by HB5 and drug resistance during the therapy.HB5 exhibited the highest rates of resistance to all the tested antibiotics among other PCR-ribotypes,which make it the multidrug-resistant PCR-ribotype.In particular,HB5 was associated with the prevalence of resistance to rifampin in our region.2 Mutations in rpoB are associated with resistance to rifamycin in C.difficile.3 D492 Y,H502D&D590V of RpoB are novel mutations identified in rifamycin resistant strains. |
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