ABSTRACT
The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.
Subject(s)
Bacterial Typing Techniques/standards , DNA, Bacterial/genetics , Electrophoresis, Gel, Pulsed-Field/standards , Molecular Epidemiology/standards , Vibrio parahaemolyticus/classification , Vibrio parahaemolyticus/genetics , Bacterial Typing Techniques/methods , Cluster Analysis , Deoxyribonucleases, Type II Site-Specific/metabolism , Genotype , Humans , Molecular Epidemiology/methodsABSTRACT
A rapid pulsed-field gel electrophoresis (PFGE) protocol for subtyping of Streptococcus suis serotype 2 was developed and evaluated using 27 clinical isolates from 22 epidemiologically unrelated patients. Results were matched against antibiogram, virulence genotyping and multi locus sequence typing (MLST). PFGE appeared to be the most discriminatory with numerical index of discrimination (D) equal to 0.87.
Subject(s)
Electrophoresis, Gel, Pulsed-Field/methods , Streptococcus suis/classification , Animals , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Typing Techniques , DNA, Bacterial/analysis , Humans , Microbial Sensitivity Tests , Serotyping , Streptococcus suis/drug effects , Streptococcus suis/genetics , Streptococcus suis/pathogenicity , Time Factors , Virulence/geneticsABSTRACT
A novel polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) of the hsp65 gene was used for the routine identification of mycobacteria in a high throughput clinical laboratory. A total of 2036 clinical isolates were tested by PRA in conjunction with other methods. The PRA identification of M. tuberculosis complex was 100% sensitive and specific, and 74.5% of nontuberculous mycobacteria (NTM) were correctly identified. It gave highly consistent results for Mycobacterium avium complex (MAC) species and for most isolates of M. fortuitum, M. chelonae, and M. kansasii. It had proven to be highly robust and stable despite usage on such a large-scale and is thus particularly suitable for use in high throughput laboratories in areas with a high incidence of tuberculosis.
Subject(s)
Bacterial Proteins , Mycobacterium Infections/diagnosis , Mycobacterium/classification , Polymerase Chain Reaction/economics , Polymerase Chain Reaction/methods , Polymorphism, Restriction Fragment Length , Algorithms , Bacterial Typing Techniques , Chaperonin 60 , Chaperonins/genetics , DNA, Bacterial/analysis , Genotype , Humans , Mycobacterium/genetics , Mycobacterium/isolation & purification , Mycobacterium Infections/microbiology , Quality Control , Reproducibility of ResultsABSTRACT
We used multiple-locus variable-number tandem repeat analysis (MLVA) and pagA sequencing to genotype a Bacillus anthracis isolate from a fatal case of human anthrax in Hong Kong. The isolate has a unique MLVA genotype, is related to the Sterne and Ames strains, and is consistent with genotypes identified in China.