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Objective To investigate the carrying status and characteristics of Clostridium difficile isolated from infants.Methods Two hundred and thirty-eight stool specimens were collected from infant younger than 1 year old,that were hospitalized or outpatient from August to November 2015.Immunochromatography targeted GDH and toxin A&B of C.difficile was used for C.difficile screening,and those positive specimens were inoculated in CDIF and anaerobic culture.C.difficile isolates were genotyped by using slpA sequence typing (slpA ST),and tcdA,tcdB,cdtA and cdtB of C.difficile isolates were detected by PCR.Results Fifty C.difficile strains were isolated from 238 stool samples,and the isolated rates of C.difficile from <3 months,3 months to <6 months,and 6 months to 1 years old groups were 9.3%,17.6% and 27.3%(χ2=6.940,P=0.031<0.05),respectively.52.0%(26/50) of the C.difficile isolates were toxigenic,and 69.2% (18/26) toxigenic isolates harbored tcdA+tcdB+cdtA-cdtB-.Fifty C.difficile isolates were genotyped as 11 slpA STs,slpA ST fr-02 and kr-02 were the commonest genotypes in toxigenic C.difficile isolates;however,that was slpA ST xr-03 in non-toxigenic isolates.Conclusion High C.difficile carriage is found in infants younger than 1 year old,and more than half of C.difficile isolates are toxigenic.Most of toxigenic isolates harbored toxin A and B.The genotype of C.difficile isolates is different between toxigenic isolates and non-toxigenic isolates.
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Objective To develop a digital polymerase chain reaction (PCR) ribotyping method and database for Clostridium difficile genotyping.Methods Sequencer based fluorescence capillary gel electrophoresis was used,instead of agarose gel electrophoresis,to establish the digital PCR-ribotyping of Clostridium difficile.Forty Clostridium difficile reference strains,consisting of 10 PCR-ribotypes (RT),were genotyped by the new digital PCR-ribotyping method to set-up the database.Results The sequencer based fluorescence capillary gel electrophoresis correctly detected PCR-ribotyping products of the 40 reference strains,and showed as digital figure;significant differences of these digital figures were found between the 10 RT.High similar digital figures were shown in twenty-one RT027 strains,three RT002 strains and two RT014 strains.However,seven RT001 strains were typed as four subtypes,and two RT014 strains as two subtypes,respectively.Conclusion A digital PCR-ribotyping,and a reference database consisting of 10 RT are successfully established.
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Objective To develop a multiplex polymerase chain reaction (PCR )method for detecting and genotyping moxifloxacin-resistant Clostridium difficile (C.difficile)isolates.Methods Specific PCR primers of slpA genotypes gr,hr,fr,gc08 and 078 were designed according to the differences of slpA nucleotide sequences in different C.difficile genotypes,and the house-keeping gene tpi specific PCR primers were also added for the construction of multiplex PCR method.Nine common intestinal normal and pathogenic strains were used to verify the specificity of slpA multiplex PCR for the detection of C.difficile.Forty-six C.difficile reference strains,belonging to 11 slpA genotypes,were used to verify the ability of the multiplex PCR method for dectecting and genotyping.Thirty-nine moxifloxacin-resistant clinical isolates were genotyped by the multiplex PCR,and its clinical value was evaluated by comparing with slpA sequence typing (slpA ST)method.Results All the 9 intestinal normal and pathogenic strains were negative when detected by the multiplex PCR.And tpi of 46 C. difficile reference strains were positive,and 36 strains belonging to slpA genotypes gr,hr,fr,gc08 and 078 were genotyped correctly.Other 10 strains which belonged to other 6 genotypes were non-typeable. Among 39 moxifloxacin-resistant clinical isolates,all were positive of tpi,and 32 isolates were typed correctly by the multiplex PCR method,including 22 slpA genotypes gc08,6 genotypes hr,2 genotypes fr,and 2 genotypes 078,which were consistent with slpA ST.However,7 isolates could not be typed by multiplex PCR,which were identified as other genotypes not included in the multiplex PCR by slpA ST. Conclusions A convenient and rapid multiplex PCR method for the detection of C.difficile is established successfully,which can distinguish among five slpA genotypes.slpA genotype gc08 is the common genotype of moxifloxacin-resistant clinical isolates.
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Objective To investigate the genotype and variance of toxin associated genes of moxifloxacin‐resistant Clostridium difficile clinical isolates in Sydney .Methods Twenty‐two moxifloxacin‐resistant Clostridium difficile clinical isolates were collected from Sydney ,which were genotyped by using sequencer capillary gel electrophoresis based PCR‐ribotyping ,and toxin A and B cod‐ing gene tcdA and tcdB ,and binary toxin coding gene cdtA and cdtB were detected by using PCR method .Toxin regulator gene tc‐dC was analyzed by using PCR‐sequencing ,and was aligned with reference sequence of VPI 10463 (Genbank accession number :X92982) ,and the tcdC sequence types of all 22 isolates were identified by using blast tool in NCBI .Results Twenty‐one isolates were genotyped as hypervirulent PCR‐ribotypes 027 (RT027) ,and one isolate as RT078 ;all 22 isolates contained tcdA and tcdB for toxin A and B and cdtA and cdtB for binary toxin (tcdA+ tcdB+ cdtA+ cdtB+ ) .The tcdC sequence types of the 21 RT027 i‐solates belong to sc1 ,and that of the one RT078 isolate belongs to WA39 .Compared with tcdC reference sequence of VPI 10463 ,a consecutive 18 bp deletion (nt341 to 379) and one nucleotide deletion at position 117 were found in the 21 RT027 isolates ,and a consecutive 39 bp deletion (nt330 to 368) and one nucleotide mutation at position 184(C> T) were found in the one RT078 isolate . Conclusion Clostridium difficile hypervirulent RT027 was the common moxifloxacin resistant genotype ;Clostridium difficile hy‐pervirulent RT027 and RT078 clinical isolates contained genes for toxin A and B and binary toxin ,and contained gene sequence mu‐tation in toxin regulator gene tcdC .
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Objective To investigate the genotype and production of toxin A and B of C .difficile clinical isolates collected from Sydney ,Australia .Methods Sixty‐eight C .difficile clinical isolates were collected from Westmead Hospital ,the University of Sydney ,which were genotyped by using PCR‐ribotyping ,and toxin A ,B coding gene tcdA ,tcdB were detected by using PCR meth‐od .Results Thirty‐one PCR‐ribotypes (RTs) were confirmed in the 68 C .difficile clinical isolates ,RT014 (19 .1% ) and RT002 (11 .8% ) were the common genotypes .Sixty‐four of 68 (94 .1% ) isolates contained tcdA and tcdB for toxin A and B .Conclusion The common prevalent PCR‐ribotypes of C .difficile were RT014 and RT002 in Sydney ,most of the C .difficile clinical isolates contained toxin A and B .