Cronobacter Support
05-18-2009, 03:09 PM
1st International Conference on Cronobacter Poster Abstract 38
Phenotypic and genotypic characterization of clinical, environmental and food isolates of Cronobacter species
The purpose of this study was to perform phenotypic and genotypic characterization on over 250 strains of Cronobacter species from around the world. Traditional methods of identification include observation of yellow colony pigmentation on tryptic soy agar (TSA), testing for positive E-glucosidase activity, and a variety of biochemical assays. Traditional phenotypic and genotypic [ribotyping, pulsed-field gel electrophoresis (PFGE), 16S rDNA sequencing, BAX® PCR] methods were performed. The use of 16S rDNA analysis showed 82% identity amongst 260 strains tested, grouping them in 96 clusters, with 50 strains in the largest one (containing C. sakazakii and C. malonaticus). C. muytjensii were able to be separated from other strains, including C. dublinensis. Molecular typing using XbaI-based PFGE was shown to be more discriminatory than automated ribotyping. PFGE analyses of 240 strains (14 eliminated by 16S rDNA as being non-Cronobacter, 6 strains having degradation during PFGE) generated 188 clusters, the largest containing 7 strains. Ribotyping, using the restriction endonuclease EcoRI, grouped the same strains in 158 clusters. From the 260 strains tested, 16 were negative for E. sakazakii by BAX® PCR; among these, 7 were identified as E. sakazakii by the 16S rDNA sequencing. Furthermore, amongst the 14 outliers from the 16S rDNA sequences, 4 were identified as E. sakazakii by API 20E®, 4 produced yellow pigment on TSA and 6 had E-glucosidase activity on DFI and/or ESPM. Of the 260 isolates tested, only 66% were identified as being positive for E. sakazakii by API 20E® and 77% by ID 32E®. While 80% (n=209) of the strains tested presented yellow pigment on TSA, 11% (n=28) formed white colonies, with 2- and 7% showing a strong and pale yellow colonies, respectively. Amongst those white colonies on TSA, 14 were green/blue on DFI/ESPM media. Combining molecular characterization to phenotypic assays will increase the confidence in the development of rapid diagnostic tools for identifying and characterizing this emerging foodborne pathogen.
Franco Pagotto, Karine Hébert, Kevin Tyler and Jeffrey M. Farber
Health Canada, Bureau of Microbial Hazards, Food Directorate, Ottawa, Ontario, Canada.
Phenotypic and genotypic characterization of clinical, environmental and food isolates of Cronobacter species
The purpose of this study was to perform phenotypic and genotypic characterization on over 250 strains of Cronobacter species from around the world. Traditional methods of identification include observation of yellow colony pigmentation on tryptic soy agar (TSA), testing for positive E-glucosidase activity, and a variety of biochemical assays. Traditional phenotypic and genotypic [ribotyping, pulsed-field gel electrophoresis (PFGE), 16S rDNA sequencing, BAX® PCR] methods were performed. The use of 16S rDNA analysis showed 82% identity amongst 260 strains tested, grouping them in 96 clusters, with 50 strains in the largest one (containing C. sakazakii and C. malonaticus). C. muytjensii were able to be separated from other strains, including C. dublinensis. Molecular typing using XbaI-based PFGE was shown to be more discriminatory than automated ribotyping. PFGE analyses of 240 strains (14 eliminated by 16S rDNA as being non-Cronobacter, 6 strains having degradation during PFGE) generated 188 clusters, the largest containing 7 strains. Ribotyping, using the restriction endonuclease EcoRI, grouped the same strains in 158 clusters. From the 260 strains tested, 16 were negative for E. sakazakii by BAX® PCR; among these, 7 were identified as E. sakazakii by the 16S rDNA sequencing. Furthermore, amongst the 14 outliers from the 16S rDNA sequences, 4 were identified as E. sakazakii by API 20E®, 4 produced yellow pigment on TSA and 6 had E-glucosidase activity on DFI and/or ESPM. Of the 260 isolates tested, only 66% were identified as being positive for E. sakazakii by API 20E® and 77% by ID 32E®. While 80% (n=209) of the strains tested presented yellow pigment on TSA, 11% (n=28) formed white colonies, with 2- and 7% showing a strong and pale yellow colonies, respectively. Amongst those white colonies on TSA, 14 were green/blue on DFI/ESPM media. Combining molecular characterization to phenotypic assays will increase the confidence in the development of rapid diagnostic tools for identifying and characterizing this emerging foodborne pathogen.
Franco Pagotto, Karine Hébert, Kevin Tyler and Jeffrey M. Farber
Health Canada, Bureau of Microbial Hazards, Food Directorate, Ottawa, Ontario, Canada.