| Objective1.To identify the molecular epidemiological characteristics of C.difficile isolated from clinical diarrhea patients in Zhejiang Province.2.To explore the global evolution and phylogeny of C.difficile ST2,which was one of the most prevalent strains.3.To reveal the antimicrobial susceptibility patterns and biological phenotypes of 40 ST2strains and explore its correlations with genomic characteristics.Methods1.Clinical stool specimens were collected from different wards of hospitals in several cities of Zhejiang Province from 2016 to 2018.Colonies of C.difficile were cultured and MLST were determined for comparing epidemic characteristics.2.A total of 40 ST2 strains collected from mainland China(18 in Zhejiang,10 in Hebei,2 in Hunan),overseas(2 in Hong Kong)and Asian-Pacific regions(8 in South Korea,Japan,Singapore,and Australia)over the years were selected for whole genome sequencing.Together with 142global ST2 genome sequences obtained from NCBI.Single nucleotide polymorphism(SNPs),homologous recombinant,insertion and deletion sites(Indel),transposons(Tn),antimicrobial resistance genes(AMR)and virulence factors(VF)were aligned to reference genome W0022a.All these genomic characteristics were analyzed for molecular evolution and phylogeny of C.difficile ST2.3.Antimicrobial susceptibility patterns and biological phenotypes of 40 ST2 strains were tested to explore their correlations with antimicrobial resistance genes and genomic features.Results1.Totally,5406 stool specimens were collected,806 C.difficile strains were cultivated,and75 MLST types were obtained from 19 wards of 15 hospitals in 10 cities of Zhejiang Province.The predominant genotype in Zhejiang was ST39,followed by ST54,ST3,ST37,ST35 and ST2.The positive rate of C.difficile was relatively high in clinical diarrhea patients in Zhejiang Province,especially in Huzhou city,Jinhua city and Lishui city(c~2=108.73,P<0.001)and in patients under 18 years and adults over 51 years old(c~2=13.43,P=0.004).2.According to the whole genome analysis,the total length of the genome of C.difficile ST2was about 4.19 Mb.14795 SNPs,1352 homologous recombination and 4613 Indel were detected,and the ratio of recombination/mutation(r/m)within the deep-branching phylogeny was 2.31.3.Two distinct lineages(L1 and L2)of global ST2 strains were discriminated in global phylogeny by the maximum likelihood tree.Notably,one sub-lineage(L2b)of L2 primarily comprised of strains from China(75.8%),accounting for 73.5%of L2b.L1 and L2a substantially consisted of strains from Europe and North America,accounting for 91.6%and 82.9%,respectively.The r/m ratio of three lineages were:L1:1.26,L2a:2.32,L2b:2.60,respectively.Besides,a specific nonsynonymous SNP was found at the 5923 position of tcd B gene in L2b strains,thymidine was mutated into guanine,which altered the glycosylation function of the tcd B.4.According to the sequence alignment of AMR genes,cde A and van XYG genes existed in all strains,and cde A gene was related to quinolones resistance.However,the other AMR genes only existed in a few strains.Neither did polymorphism of tcd A and tcd B genes was found in the comparison to virulence factors.5.The antimicrobial susceptibility patterns showed that the resistance rates varied for antimicrobials of ST2 strains.The highly resistance rate to quinolones(levofloxacin,ciprofloxacin)and clindamycin were observed,accounting for 82.5%,92.5%and 85.0%,respectively.The resistance rate to fusidic acid and erythromycin was 62.5%and 25.0%.And the resistance rates to rifampicin,moxifloxacin,gatifloxacin,tetracycline and pairacillin tazobactam were 5.0%,10.0%,15.0%,7.5%and 2.5%,respectively.All strains were susceptible to metronidazole,vancomycin.6.Real-time cytotoxin expression experiments showed that L2b strains expressed lower toxin B concentrations(5.11±3.20 ng/μL)than those in L1(10.49±15.82 ng/μL)and L2a(13.92±2.39 ng/μL)(c~2=12.10,P=0.002).Conclusions1.The risk of nosocomial infections of C.difficile was relatively high in clinical diarrhea patients in Zhejiang Province.We should strengthen the monitor and control the nosocomial infections of C.difficile due to the diverse distribution of dominant ST types among different cities and population.2.Global C.difficile ST2 was evolved into two lineages,and the L2b Chinese sub-lineage was reported for the first time.A specific nonsynonymous SNP was found in L2b,which could provide scientific guidance for other genome evolution and phylogeny studies.3.The discoveries of AMR gene cde A and high resistance rates to quinolones provided scientific basis for further studies on resistance mechanism of C.difficile and supported for antimicrobial selection in clinical patients.4.The strains in L2b Chinese sub-lineage presented a low hypovirulence characteristic,which could lead mild clinical diarrhea symptoms or asymptomatic colonization among patients.Therefore,the prevention and control measures of nosocomial infection of C.difficile should be reinforced. |
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