OBJECTIVETo analyze the ribotyping fingerprint of Enterobacter sakazakii (E. sakazakii) isolated from food and its typing power.

METHODSTwo standard strains and twenty-eight isolates of E.sakazakii were analyzed by the DuPont Riboprinter(TM) microbial characterization system. The relevant database was established and the fingerprint patterns were analyzed with BioNumerics software.

RESULTSThis system grouped two standard strains and twenty-eight E.sakazakii isolates into 26 ribotypes, and four ribotypes included two strains respectively, the other twenty-two strains showed different ribotypes. The lowest similarity was 31.58%. The number of bands by ribotyping was approximately ten and the molecular weight of these bands ranged from 1 to 50 kb. By the clustering program in BioNumerics, these isolates could be grouped into four clusters.

CONCLUSIONThe automatic ribotyping method is convenient and fast in E.sakazakii typing.

Cronobacter sakazakii ; classification ; genetics ; isolation & purification ; DNA, Bacterial ; DNA, Ribosomal ; Food Microbiology ; Ribotyping ; methods

OBJECTIVETo establish and compare the pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA) and automated ribotyping for subtyping of Citrobacter strains.

METHODSPFGE protocol was optimized in terms of plug preparation procedure, restriction enzymes and configuration of electrophoretic parameters. MLVA method was evaluated by finding variable number tandem repeats in two genomes of Citrobacter strains. The ribotyping was performed by using the automated RiboPrinter system.

RESULTSWe optimized the plug preparation procedure, focused on the cell suspension concentration (turbidity of 2.5 to 3.5), SDS addition (no SDS needed) and lysis time (1 h), and selected the appropriate restriction enzyme (XbaI) and the electrophoretic parameters (1.0 s-20.0 s for 19 h) of PFGE. There was nearly no discriminatory power of MLVA between Citrobacter strains. For 51 Citrobacter strains, automated ribotyping gave a D-value of 0.9945, while PFGE gave a D-value of 0.9969. Both PFGE and automated ribotyping clustered strains from the same sources (with the same species from the same place at the same time identified as the same source) and divided strains from different sources (from different years, places and hosts) into different subtypes.

CONCLUSIONPFGE protocol established in this paper and automated ribotyping are suitable for application in Citrobacter subtyping.

Automation ; Citrobacter ; classification ; genetics ; Electrophoresis, Gel, Pulsed-Field ; methods ; Minisatellite Repeats ; genetics ; Multilocus Sequence Typing ; methods ; Phylogeny ; Ribotyping ; methods

We describe a riboprinting scheme for identification of unknown Acanthamoeba isolates at the species level. It involves use of the PCR-RFLP of small subunit ribosomal RNA gene (riboprint) of 24 reference strains by 4 kinds of restriction enzymes. Seven strains in morphological group I and III were identified at species level with their unique sizes of PCR product and riboprint type by Rsa I. Unique restriction fragment length patterns of 17 strains in group II by Dde I, Taq I and Hae III were classified into: (1) four taxa that were identifiable to the species level, (2) a subgroup of 4 taxa and a pair of 2 taxa that were identical to each other, and (3) a species complex of 7 taxa assigned to A. castellanii complex that were closely related. These results were consistent with that of 18s rDNA sequence analysis. This approach provides an alternative to the rDNA sequencing for rapid identification of a new clinical isolate or large number of environmental isolates of Acanthamoeba.
Acanthamoeba/classification/genetics/*isolation & purification ; Animals ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; RNA, Protozoan ; RNA, Ribosomal ; Ribotyping/*methods

BACKGROUND/AIMS: The incidence and severity of Clostridium difficile infection (CDI) have increased worldwide, resulting in a need for rapid and accurate diagnostic methods. METHODS: A retrospective study was conducted to compare CDI diagnosis methods between January 2014 and December 2014. The stool samples, which were obtained in presumptive CDI patients, were compared for their diagnostic accuracy and rapidity, including real-time polymerase chain reaction (PCR) of toxin genes, C. difficile toxin assay, and culture for C. difficile. RESULTS: A total of 207 cases from 116 patients were enrolled in this study and 117 cases (56.5%) were diagnosed as having CDI. Among the 117 cases, the sensitivities of real-time PCR, C. difficile toxin assay, and culture for C. difficile were 87.2% (102 cases; 95% CI, 80.7%–92.8%), 48.7% (57 cases; 95% CI, 41.0%–59.8%), and 65.0% (76 cases; 95% CI, 60.2%–78.5%), respectively (P < 0.005). Notably, 34 cases (29.0%) were diagnosed with CDI by real-time PCR only. The time required to obtain results was 2.27 hours (136.62±82.51 minutes) for real-time PCR, 83.67 hours (5,020.66±3,816.38 minutes) for toxin assay, and 105.79 hours (6,347.68±3,331.46 minutes) for culture (P < 0.005), respectively. CONCLUSIONS: We confirmed that real-time PCR of toxin genes is the most effective diagnostic method for accurate and early diagnosis of CDI. It also helps to diagnose hypervirulent CDI, such as ribotype 027 infection.
Clostridium difficile ; Clostridium ; Diagnosis ; Early Diagnosis ; Humans ; Incidence ; Methods ; Polymerase Chain Reaction ; Real-Time Polymerase Chain Reaction ; Retrospective Studies ; Ribotyping