Establishment of digital polymerase chain reaction-ribotyping and database for Clostridium difficile genotyping / 中华传染病杂志

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.

Detection of genotype and toxin associated gene of hypervirulent Clostridium difficile clinical isolates / 国际检验医学杂志

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 .

Antimicrobial resistance and PCR-ribotyping of Shigella responsible for foodborne outbreaks occurred in southern Brazil

Little information about Shigella responsible for foodborne shigellosis is available in Brazil. The present study aimed to investigate the antimicrobial resistance and PCR-ribotyping patterns of Shigella isolates responsible for foodborne outbreaks occurred in Rio Grande do Sul State (RS), Southern Brazil in the period between 2003 and 2007. Shigella strains (n=152) were isolated from foods and fecal samples of victims of shigellosis outbreaks investigated by the Surveillance Service. Identification of the strains at specie level indicated that 71.1 percent of them were S. flexneri, 21.5 percent S. sonnei, and 0.7 percent S. dysenteriae. Ten strains (6.7 percent) were identified only as Shigella spp. An increasing occurrence of S. sonnei was observed after 2004. Most of the strains were resistant to streptomycin (88.6 percent), followed by ampicillin (84.6 percent), and sulfamethoxazole/trimethoprim (80.5 percent). Resistant strains belonged to 73 patterns, and pattern A (resistance to ampicillin, sulfamethoxazole/trimethoprim, tetracycline, streptomycin, chloramphenicol, and intermediate resistance to kanamycin) grouped the largest number of isolates (n=36). PCR-ribotyping identified three banding patterns (SH1, SH2, and SH3). SH1 grouped all S. flexneri and SH2 grouped all S. sonnei. The S. dysenteriae strain belonged to group SH3. According to the results, several Shigella isolates shared the same PCR-rybotyping banding pattern and the same resistance profile, suggesting that closely related strains were responsible for the outbreaks. However, other molecular typing methods need to be applied to confirm the clonal relationship of these isolates.

Post-operative endophthalmitis: Antibiogram & genetic relatedness between Pseudomonas aeruginosa isolates from patients & phacoemulsifiers.

Background & objectives: Though not frequently but there are reports showing phacoemulsifiers as a potent source of infection in post-operative cases of endophthalmitis. This study was carried out to find antibiogram and genetic relatedness between Pseudomonas aeruginosa isolates from a post-cataract surgery endophthalmitis outbreak (3 patients) and internal tubings of 5 phacoemulsifiers. Methods: In vitro antimicrobial sensitivity patterns of the 8 bacterial isolates were observed. Genetic analysis of the bacterial isolates was done using random amplification of polymorphic DNA (RAPD) assay and PCR ribotyping. The resulting DNA band patterns were examined visually and by computer assisted analysis using unweighted pair group method. Results: The three P. aeruginosa patient isolates were found to be different from the five phacoemulsifier isolates in sensitivity towards 3 antibiotics and by genetic analysis (33 and 44% homology by RAPD assay and PCR ribotyping). Two of the patient isolates shared 100 per cent genetic homology by RAPD assay and another pair shared 100 per cent homology by PCR ribotyping. The five isolates from phacoemulsifiers did not share significant genetic homology. There was significant genetic variation between bacterial isolates from patients and phaco emulsifiers. Interpretation & conclusion: Though the three P. aeruginosa isolates obtained from the patients were phenotypically similar and genetically close, they differed from the phaco-machine isolates both genetically, and in their antibiogram profile. However, the five phacoemulsifier isolates were genetically diverse though they shared the same antibiogram profile. Therefore the Ringer’s lactate from phacomachines could not be conclusively proven to be the source of infection.

Application of PCR ribotyping and tDNA-PCR for Klebsiella pneumoniae identification

PCR analysis of 16S-23S internal transcribed spacer (PCR ribotyping) and tRNA intergenic spacer (tDNA-PCR) were evaluated for their effectiveness in identification of clinical strains of Klebsiella pneumoniae and differentiation with related species. For this purpose both methods were applied to forty-three clinical isolates biochemically identified as K. pneumoniae subsp. pneumoniae isolated from patients clinical specimens attended at five hospitals in three Brazilian cities. References strains of K. pneumoniae subsp. pneumoniae, K. pneumoniae subsp. ozaenae, K. oxytoca, K. planticola and Enterobacter aerogenes were also analyzed. Both PCR methods showed specific patterns for each species. A conserved PCR ribotype pattern was observed for all clinical K. pneumoniae isolates, while differing from other related analyzed species. tDNA-PCR revealed five distinct patterns among the K. pneumoniae clinical isolates studied, demonstrating a predominant group with 90,6 percent of isolates presenting the same pattern of K. pneumoniae type strain. Both PCR-based methods were not able to differentiate K. pneumoniae subspecies. On the basis of the results obtained, both methods were efficient to differentiate the Klebsiella species analyzed, as well as E. aerogenes. Meanwhile tDNA-PCR revealed different tRNA arrangements in K. pneumoniae, suggesting intra-species heterogeneity of their genome organization, the polymorphism of the intergenic spacers between 16S and 23S rRNA genes appears to be highly conserved whithin K. pneumoniae clinical isolates, showing that PCR ribotyping can be an useful tool for identification of K. pneumoniae isolates.

Molecular Analysis of Clostridium difficile Isolates by Arbitrarily Primed-Polymerase Chain Reaction and Polymerase Chain Reaction-Ribotyping / 감염

BACKGROUND: Clostridium difficile is known as the major cause of nosocomially acquired diarrhea. Various phenotypic and genotypic methods have been used to subtype C. difficile strains. The purpose of the present study is to evaluate several typing methods which can be used as tools for subtyping C. difficile isolates for epidemiological studies. METHODS: In two Korean tertiary care hospitals, a total of 81 C. difficile isolates were collected from symptomatic, hospitalized patients in 1998. All isolates were examined for the release of toxin A and toxin B by PCR assay and cell culture assay. Also arbitrarily primed-PCR and PCR-ribotyping profiles were determined for the typing of C. difficile strains on a genetic level. RESULTS: The toxin B gene was detected in 65.4% (54/81) of isolates by both PCR assay and cell cultureassay. Nine types were identified with T-7 primer, and 13 types were identified with PG-05 primer in AP- PCR. Sixteen types were identified in PCR-ribotyping. When two typing methods were compared, reproducibility by PCR-ribotyping was 100%, while it was only 83% and 33% AP-PCR with primer T-7, and PG-05, respectively. The discrimination index was 0.88 for PCR-ribotyping, 0.82 for AP-PCR with primer T-7 and 0.81 with primer PG-05. CONCLUSION: These data suggest that PCR-ribotyping provides a reproducible, discriminatory, and simple alternative to conventional molecular approaches for typing strains of C. difficile.

Biochemical, Serological and Virulence-associated Characteristics of Yersinia enterocolitica Isolated in Korea / 감염

BACKGROUND: Yersinia enterocolitica is an inhabitant in lower intestinal tract of many domestic and wild animals as well as in nature. Of the several forms of diseases by Y. enterocolitica, acute enteritis, especially in young children, is most common. Infection of the bacteria is usually occurred through fecal-oral route by contaminated foods or water, especially mountainspring water. Therefore, we isolated Y. enterocolitica from feces of pigs and mountainspring water and investigated the biochemical, serological, genetical and virulence-associated characteristics of the bacteria for the epidemiological analysis. METHODS: Fifty-one strains of Y. enterocolitica were isolated from the samples and identified by biochemical tests and VITEK system. We performed antimicrobial susceptibility test, in vitro virulence-associated characteristics, biotyping, serotyping, and PCR-ribotyping and compared the methods, such as biotype, serotype and PCR-ribotyping, to differentiate the isolates. RESULTS: Biochemical properties of the isolates were very similar to those of reference strains. They were susceptible to chloramphenicol, tetracycline, norfloxacin, but resistant to bacitracin, ampicillin in antimicrobial susceptibility test. In vitro virulence-associated tests, the positive strains were 27 isolates (52.9%) in CRMOX+, 10 isolates (19.6%) in CV+, 30 isolates (58.8%) in CD+, 20 isolates (39.2%) in esculin+ and 20 isolates in salicin+ (39.2%). Thirty-one strains of 51 isolates (61%) harbored about 70 kb plasmid presumably associated with virulence factor. Biotypes and serotypes of the strains were 3B, 3A, 4 and O:3, O:13, O:16 in order, respectively. Type I having 700 and 800 bps products was the most common type of PCR-ribotypes. CONCLUSION: Biochemical properties, biotypes and serotypes of the isolates were similar with previously reports in Korea. PCR-ribotyping, a genetical method, could be a more effective tool for differentiating isolates of Yersinia enterocolitica than conventional methods such as serotype, biotype.