Cronobacter Support
04-23-2009, 05:16 PM
1st International Conference on Cronobacter Poster Abstract 4
The taxonomy and identification of Enterobacter sakazakii: a proposed new genus Cronobacter
The organism described as Enterobacter sakazakii is an opportunistic pathogen that can cause infections in neonates. When the species was defined in 1980 it was suggested that it might consist of multiple species. DNA-DNA hybridizations gave no clear generic assignment with 53-54% relatedness to species in two different genera, Enterobacter and Citrobacter. In this study, independent molecular methods were employed to clarify the taxonomic relationship of 210 strains and amendments to the classification of these organisms are proposed. Full length 16S rRNA sequence analysis, automated ribotyping, f-AFLP analysis and DNA-DNA hybridization were performed as previously described. PCR amplification assays targeting the dnaG and gluA genes, biochemical galleries (API20E and ID32E), and E-glucosidase assays were also performed as described. Strains identified as E. sakazakii were divided into separate groups on the basis of their 16S rDNA sequences, ribopatterns and f-AFLP fingerprints. DNA-DNA hybridizations revealed six genomospecies. The phenotypic profiles of the species were determined and biochemical markers identified. All Cronobacter strains expressed E-glucosidase activity and were positively identified by dnaG RT-PCR and E-glucosidase PCR, with no false positive strains. The API20E test system identified 70% of the strains as E. sakazakii however 7/102 strains were mis-identified, as they appeared to belong to other Enterobacter species using 16S rDNA sequence analysis and ribotyping. Using the ID32E gallery with version 3.0 of the apiweb™ database, 189/210 Cronobacter strains (90%) were identified to the species level (as E. sakazakii) with ‘good-excellent’ identification. The nearest significant taxon for all but one of the remaining strains (20/21) was E. sakazakii and none of the other Enterobacteriaceae were misidentified as E. sakazakii with this gallery. This study clarifies the taxonomy and identification of E. sakazakii and proposes a novel genus, Cronobacter.
Carol Iversen1,2,3, Niall Mullane1, Barbara McCardell4, Ben D. Tall4, Angelika Lehner2, Eva Bidlas3, John Marugg3, Ilse Cleenwerk5, Séamus Fanning1, Roger Stephan2 and Han Joosten3
1Centre for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland. 2Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zürich, CH-8057, Zürich, Switzerland. 3Quality and Safety Department, Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland. 4 Centre for Food Safety & Applied Nutrition, US Food and Drug Administration, Laurel, MD 20708, USA.
5BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie – Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.
The taxonomy and identification of Enterobacter sakazakii: a proposed new genus Cronobacter
The organism described as Enterobacter sakazakii is an opportunistic pathogen that can cause infections in neonates. When the species was defined in 1980 it was suggested that it might consist of multiple species. DNA-DNA hybridizations gave no clear generic assignment with 53-54% relatedness to species in two different genera, Enterobacter and Citrobacter. In this study, independent molecular methods were employed to clarify the taxonomic relationship of 210 strains and amendments to the classification of these organisms are proposed. Full length 16S rRNA sequence analysis, automated ribotyping, f-AFLP analysis and DNA-DNA hybridization were performed as previously described. PCR amplification assays targeting the dnaG and gluA genes, biochemical galleries (API20E and ID32E), and E-glucosidase assays were also performed as described. Strains identified as E. sakazakii were divided into separate groups on the basis of their 16S rDNA sequences, ribopatterns and f-AFLP fingerprints. DNA-DNA hybridizations revealed six genomospecies. The phenotypic profiles of the species were determined and biochemical markers identified. All Cronobacter strains expressed E-glucosidase activity and were positively identified by dnaG RT-PCR and E-glucosidase PCR, with no false positive strains. The API20E test system identified 70% of the strains as E. sakazakii however 7/102 strains were mis-identified, as they appeared to belong to other Enterobacter species using 16S rDNA sequence analysis and ribotyping. Using the ID32E gallery with version 3.0 of the apiweb™ database, 189/210 Cronobacter strains (90%) were identified to the species level (as E. sakazakii) with ‘good-excellent’ identification. The nearest significant taxon for all but one of the remaining strains (20/21) was E. sakazakii and none of the other Enterobacteriaceae were misidentified as E. sakazakii with this gallery. This study clarifies the taxonomy and identification of E. sakazakii and proposes a novel genus, Cronobacter.
Carol Iversen1,2,3, Niall Mullane1, Barbara McCardell4, Ben D. Tall4, Angelika Lehner2, Eva Bidlas3, John Marugg3, Ilse Cleenwerk5, Séamus Fanning1, Roger Stephan2 and Han Joosten3
1Centre for Food Safety and Food-borne Zoonomics, UCD Veterinary Sciences Centre, University College Dublin, Belfield, Dublin 4, Ireland. 2Institute for Food Safety and Hygiene, Vetsuisse Faculty, University of Zürich, CH-8057, Zürich, Switzerland. 3Quality and Safety Department, Nestlé Research Centre, Vers-chez-les-Blanc, CH-1000 Lausanne, Switzerland. 4 Centre for Food Safety & Applied Nutrition, US Food and Drug Administration, Laurel, MD 20708, USA.
5BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie – Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium.