Antibiotic Susceptibility Of ST37 Clostridium Difficile And The Establishment Of Rapid Identification Method Of ST37 Clostridium Difficile By MALDI-TOF Mass Spectrometry

Objective: Clostridium difficile(CD)is a kind of spore-forming,gram-positive,anaerobic bacillus and it is the main pathogen causing antibiotic-associated diarrhea(AAD)and nosocomial infections.The clinical symptoms caused by C.difficile ranging from mild diarrhea to life-threatening pseudomembranous colitis(PMC).The pathogenicity of C.difficile is mainly due to the production of two toxins,enterotoxin A(TcdA)and cytotoxin B(TcdB).The highly virulent type strains,PCR ribotype(RT)027 strains,even produce the third toxin,binary toxin(CDT).The emergence of RT027 strains was responsible for multiple outbreaks in North America and Europe.On the Contrast,only sporadic reports for cases of C.difficile infection(CDI)caused by RT027 in Asia.The most prevalent ribotype seems to be 017 in these regions,which type of strains produces toxin B only.Our previous study have shown that,in China,multilocus sequence type(ST)37 C.difficile,identified by PCR ribotyping as 017,was the main cause of adult patient CDI.Whereas intestinal carrier rate of ST37 strains in healthy adults was not high.In addition,ST37 isolates had significantly higher resistance to fluoroquinolone,macrolide and lincomycins antibiotics than that of other ST isolates.Serious clinical symptoms and outbreaks in the hospital caused by ST37 C.difficile had been reported,which is a major risk of public health security.Therefore,fast-tracking of ST37 isolates has great significance for C.difficile epidemiological monitoring.The commonly used typing methods,such as PCR-ribotyping,MLST and pulsed-field gel electrophoresis typing(PFGE),have the drawbacks of being time-consuming and expensive,and require the use of specialized equipment,making it difficult to use in clinical laboratories.In this study,a rapid method for the detection of C.difficile ST37 based onmatrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF MS)has been established,which will play an important role in the prevention and control of C.difficile infection.Methods: All the strains used in the experiment were collected from Hebei Center for Medical Culture Collection.Among them,64 strains were isolated from diarrhea patients,88 strains were isolated from healthy adults or children,and also included 7 standard strains of C.difficile(ATCC BAA-1870,ATCC BAA-1382,ATCC BAA-1812,ATCC BAA-1804,ATCC 9689,ATCC43255 and ATCC 43598).All 159 strains were identified as C.difficile by MALDI-TOF MS and/or 16 s rDNA sequencing.All the experimental strains were grown on Columbia blood agar plates and passaged twice for subsequent tests.The C.difficile genome was extracted for toxin gene detection and MLST typing.The minimum inhibitory concentrations(MICs)for vancomycin,metronidazole,fidaxomicin,rifaximin,levofloxacin,ciprofloxacin,meropenem,tetracycline,erythromycin,ceftriaxone,ceftazidime,clindamycin and chloramphenicol were determined using the agar dilution method recommended by the Clinical and Laboratory Standards Institute(CLSI).An ethanol/formic acid method was used for extraction of bacterial proteins.The protein mass spectra were collected by Microflex LT and VITEK MS.The rapid identification model of ST37 C.difficile was established by analyzing the mass spectrometry data.44 strains of C.difficile from different regions were used to validate the rapid identification model.Results:1 Among the 159 strains of C.difficile,104 strains were tcdA+tcdB+cdtA-cdtB-,4 strains were tcdA+tcdB+cdtA+cdtB+,21 strains were tcdA-tcdB+cdtA-cdtB-,and 30 strains were non-toxigenic strains tcdA-tcdB-cdtA-cdtB-.All strains were divided into 46 ST types by MLST typing.The most important type was ST3,followed by ST37,ST54,ST2 and ST35,which represented the most prevalent sequence types in the local area.Twenty strains of tcdA-tcdB+cdtA-cdt B-belonged to ST37(20/21).And there was also one strain of tcdA-tcd B+cdt A-cdtB-belonging to ST81 type,whichwas single locus variant(SLV)of ST37.In addition,two new STs were submitted to C.difficile MLST database curated by David Griffiths,which was ST348 and ST349.2 Compared with the resistance rate of the major epidemic STs in Hebei province,we found that ST37 C.difficile showed significantly higher resistance rate to rifaximin,levofloxacin,meropenem,tetracycline,erythromycin,ceftriaxone and chloramphenicol than other STs(P<0.05).There was no statistically significant difference in the resistance rate of all kinds of STs to ciprofloxacin.All isolates were resistant to ceftazidime,and sensitive to the metronidazole,vancomycin and fidaxomicin.3 Comparing the protein profiling of ST37 C.difficile with other STs,the protein mass spectra of ST37 C.difficile was found to have a specific combination of two peaks m/z 3240 and m/z 3285.This set of characteristic peaks can be used to rapidly identify ST37 C.difficile.The sensitivity of this method to identify ST37 C.difficile was 95%,specificity 99%,positive predictive value 95%,and negative predictive value 99%.Two types of mass spectrometer,Microflex LT and VITEK MS,got a consistent result.The results of reproducibility validation showed that m/z 3240 and m/z 3285 peaks were found in 27 spectra of ST37 strains,and without found in 27 spectra of non-ST37 strains,the combination of m/z 3240 and m/z 3285 peaks had a good stability and reproducibility with ST37 C.difficile.In addition,38 C.difficile collected from different regions,including 22 ST37 isolates and 16non-ST37 isolates,were used to validate the ST37 C.difficile rapid detection method.21 strains(95.5%)of ST37 C.difficile were correctly identified out.Conclusions:1 Compared with other STs,the strains of ST37 have a significantly higher resistance rate to a variety of antibiotics.We should be alert to outbreak in the hospital caused by ST37 strains under the pressure of antibiotics.2 A combination of two peaks(m/z 3240 and 3285)has a good correlation with the strains of ST37 and can be used as biomarkers to identify ST37 C.difficile isolates.3 This is the first study to identify C.difficile of ST37 by MALDI-TOF MS used for routine microorganism identification.This technology has the characteristics of accurate,quick,high-throughput and low-cost,and is suitable for the prevention and control of CDI.