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The occurrence of outbreaks of Cronobacter strains causing necrotizing meningitis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 43 Cronobacter isolates and five Enterobacteriaceae strains were used in this study. The strains were characterized using two molecular typing methods, including enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR) and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these two molecular typing tests clearly showed that there was no close relationship between Cronobacter spp. and Enterobacteriaceae strains. Discriminatory index of ERIC-PCR typing for the 43 Cronobacter isolates and five Enterobacteriaceae strains was high (D = 0.984) by Simpson's index of diversity based on the similarity value of more than 80% using BioNumerics version 5.0. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains. These results demonstrate that ERIC-PCR combined with sequence analysis of the gyrB gene may be a reliable, rapid typing strategy for Cronobacter strains. Practical Applications Molecular typing of foodborne pathogenic bacteria has been shown as a useful tool for tracking the source of infection and detection of virulent strains, as well as for determining the geographical and host distribution of possible variants. ERIC-PCR provides rapid clustered results when a large number of Cronobacter spp. isolates need to be subtyped rapidly. Furthermore, partial sequence analysis of the gyrB gene was utilized to differentiate among the closely related isolates. The results of the current study suggest that ERIC-PCR in conjunction with the gyrB sequence analysis would provide a reliable and accurate typing strategy for Cronobacter spp. strains. This combination of techniques can be used as a rapid means of comparing Cronobacter spp. isolates for epidemiological investigations.

The occurrence of outbreaks of Vibrio parahaemolyticus gastroenteritis in China highlights the need for strain characterization and subtyping of this pathogenic species. A total of 56 epidemiologically-unrelated strains of V. parahaemolyticus were isolated from clinical samples, seafood and various environmental sites in the middle-east coastline of China from 2006 to 2008. The isolates were characterized using four molecular typing methods, including ribotyping, enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), pulsed-field gel electrophoresis (PFGE), and sequence analysis of the gyrB gene. Genetic profiles of cluster analysis from these molecular typing tests clearly showed that there were differences in potential pathogenicity among isolates from seafood and its environments. Genetic characterization of two isolates (F13 and QS2) that originated from seafood demonstrated that they were potentially pathogenic. Discriminatory indices of four typing methods for the 56 V. parahaemolyticus isolates were differentiated by Simpson's Index of Diversity. The discriminatory index of ERIC-PCR typing was maximal (D=0.942), while that of sequence analysis of the gyrB gene was minimal (D=0.702). The discriminatory ability was greatly enhanced (D=0.966) when ERIC-PCR was coupled with sequence analysis of the gyrB gene. These results suggest that ERIC-PCR combined with sequence analysis of gyrB gene may be a reliable, rapid typing strategy for V. parahaemolyticus strains.

Cronobacter spp. has caused life-threatening neonatal infections mainly resulted from consumption of contaminated powdered infant formula. A total of 102 vegetable samples from retail markets were evaluated for the presence of Cronobacter spp. Thirty-five presumptive Cronobacter isolates were isolated and identified using API 20E and 16S rDNA sequencing analyses. All isolates and type strains were characterized using enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), and genetic profiles of cluster analysis from this molecular typing test clearly showed that there were differences among isolates from different vegetables. A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) based on the amplification of the gyrB gene (1258 bp) was developed to differentiate among Cronobacter species. A new PCR-RFLP assay based on the amplification of the gyrB gene using Alu I and Hinf I endonuclease combination is established and it has been confirmed an accurate and rapid subtyping method to differentiate Cronobacter species. Sequence analysis of the gyrB gene was proven to be suitable for the phylogenetic analysis of the Cronobacter strains, which has much better resolution based on SNPs in the identification of Cronobacter species specificity than PCR-RFLP and ERIC-PCR. Our study further confirmed that vegetables are one of the most common habitats or sources of Cronobacter spp. contamination in the middle-east coastline of China.

The occurrence of outbreaks of necrotizing meningitis caused by Cronobacter spp. in neonates highlights the need for rapid detection and accurate identification of this pathogenic species. The gold standard for isolation and identification of Cronobacter spp. from powdered infant formula is time consuming and labor intensive. The gyrB gene that encodes the B subunit of DNA gyrase (topoisomerase type II) was found to be suitable for the identification of Cronobacter spp. A region of the gyrB gene of 38 Cronobacter spp. strains and 5 Enterobacter spp. strains was amplified and sequenced, and a pair of primers was designed and synthesized based on the sequence of the gyrB gene. A polymerase chain reaction (PCR) system was developed and optimized to detect Cronobacter spp. The PCR assay amplified a 438 bp DNA product from all 38 Cronobacter spp. strains tested but not from 34 other bacteria. The detection limit was 1.41 pg/PCR (equivalent 282 genomic copies) when the genomic DNA of Cronobacter sakazakii ATCC 29544 was 10-fold diluted. Infant formula powders from 3 different commercial brands were inoculated with strains ATCC 29544 at a level of 56 colony-forming units, and the target fragment were produced after samples were enriched for 6 h at 37 °C. Twenty-five food samples were evaluated by the PCR assay and the conventional method. A PCR product of the expected size was obtained from 3 samples; however, Cronobacter spp. strains were isolated from only 2 samples by the conventional method. This method is a useful tool for rapid identification of Cronobacter spp. in food and potentially environmental samples.