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Molecular analysis of the Enterobacter sakazakii O-antigen gene locus.

ABSTRACT: Nucleotide polymorphism associated with the O-antigen-encoding locus, rfb, in Enterobacter sakazakii was determined by PCR-restriction fragment length polymorphism analysis. Based on the analysis of these DNA profiles, 12 unique banding patterns were detected among a collection of 62 strains from diverse origins. Two common profiles were identified and were designated serotypes O:1 and O:2. DNA sequencing of the 12,500-bp region flanked by galF and gnd identified 11 open reading frames, all with the same transcriptional direction. Analysis of the proximal region of both sequences demonstrated remarkable heterogeneity. A PCR assay targeting genes specific for the two prominent serotypes was developed and applied for the identification of these strains recovered from food, environmental, and clinical samples.

SUBMITTER: Mullane N

PROVIDER: S-EPMC2446543 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Molecular analysis of the Enterobacter sakazakii O-antigen gene locus.

Mullane N N O'Gaora P P Nally J E JE Iversen C C Whyte P P Wall P G PG Fanning S S

Applied and environmental microbiology 20080425 12

Nucleotide polymorphism associated with the O-antigen-encoding locus, rfb, in Enterobacter sakazakii was determined by PCR-restriction fragment length polymorphism analysis. Based on the analysis of these DNA profiles, 12 unique banding patterns were detected among a collection of 62 strains from diverse origins. Two common profiles were identified and were designated serotypes O:1 and O:2. DNA sequencing of the 12,500-bp region flanked by galF and gnd identified 11 open reading frames, all with ...[more]

PMID: 18441119

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Project description:BackgroundEnterobacter sakazakii is a foodborne pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. The current FDA detection method includes two enrichment steps, the subculturing of the second enrichment broth on a selective agar (VRBG), a further subculturing of selected grown colonies on TSA and the subsequent biochemical identification of yellow-pigmented colonies by API20E. However, there is a strong need for simplified methods for isolation and identification of E. sakazakii. In this study, two chromogenic media, which allow to indicate presumptive E. sakazakii colonies by the alpha glucosidase activity, as well as a newly developed 1,6-alpha-glucosidase based conventional PCR assay and a rRNA oligonucleotide probe based commercial test system for identification of presumptive E. sakazakii were evaluated on 98 target and non-target strains. The methods were compared with respect to specificity aspects.ResultsA total of 75 presumptive E. sakazakii and 23 non-target strains were analysed by using chromogenic media, alpha-glucosidase based PCR assay, and the VIT assay. For most presumptive E. sakazakii strains on the chromogenic media, the PCR and VIT assay confirmed the identification. However, for a number of presumptive E. sakazakii isolates from fruit powder, the alpha-glucosidase PCR and VIT assay did not correspond to the typical E. sakazakii colonies on DFI and ESIA. Further characterization by API32E identification, phylogenetic analysis of partial 16S rRNA sequences and ribotyping strongly suggested, that these strains did not belong to the species E. sakazakii. The newly developed alpha-glucosidase based PCR assay as well as the commercially available VIT Enterobacter sakazakii identification test showed an excellent correlation with the 16S rRNA data, and are thus well suited for identification of E. sakazakii.ConclusionThe results indicate that presumptive colonies on ESIA and DFI media need further species identification. Both evaluated molecular methods, the alpha-glucosidase PCR and the 16S RNA in situ hybridisation test (VIT), although based on completely different target regions and methodologies performed equally well in terms of specificity.

Project description:BackgroundFollowing the association of Cronobacter spp. to several publicized fatal outbreaks in neonatal intensive care units of meningitis and necrotising enterocolitis, the World Health Organization (WHO) in 2004 requested the establishment of a molecular typing scheme to enable the international control of the organism. This paper presents the application of Next Generation Sequencing (NGS) to Cronobacter which has led to the establishment of the Cronobacter PubMLST genome and sequence definition database (http://pubmlst.org/cronobacter/) containing over 1000 isolates with metadata along with the recognition of specific clonal lineages linked to neonatal meningitis and adult infectionsResultsWhole genome sequencing and multilocus sequence typing (MLST) has supports the formal recognition of the genus Cronobacter composed of seven species to replace the former single species Enterobacter sakazakii. Applying the 7-loci MLST scheme to 1007 strains revealed 298 definable sequence types, yet only C. sakazakii clonal complex 4 (CC4) was principally associated with neonatal meningitis. This clonal lineage has been confirmed using ribosomal-MLST (51-loci) and whole genome-MLST (1865 loci) to analyse 107 whole genomes via the Cronobacter PubMLST database. This database has enabled the retrospective analysis of historic cases and outbreaks following re-identification of those strains.ConclusionsThe Cronobacter PubMLST database offers a central, open access, reliable sequence-based repository for researchers. It has the capacity to create new analysis schemes 'on the fly', and to integrate metadata (source, geographic distribution, clinical presentation). It is also expandable and adaptable to changes in taxonomy, and able to support the development of reliable detection methods of use to industry and regulatory authorities. Therefore it meets the WHO (2004) request for the establishment of a typing scheme for this emergent bacterial pathogen. Whole genome sequencing has additionally shown a range of potential virulence and environmental fitness traits which may account for the association of C. sakazakii CC4 pathogenicity, and propensity for neonatal CNS.

Project description:BACKGROUND: Enterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue. This study examined for the first time the adherence ability of 50 E. sakazakii strains to the two epithelial cell lines HEp-2 and Caco-2, as well as the brain microvascular endothelial cell line HBMEC. Furthermore, the effects of bacterial culture conditions on the adherence behaviour were investigated. An attempt was made to characterize the factors involved in adherence. RESULTS: Two distinctive adherence patterns, a diffuse adhesion and the formation of localized clusters of bacteria on the cell surface could be distinguished on all three cell lines. In some strains, a mixture of both patterns was observed. Adherence was maximal during late exponential phase, and increased with higher MOI. The adhesion capacity of E. sakazakii to HBMEC cells was affected by the addition of blood to the bacteria growth medium. Mannose, hemagglutination, trypsin digestion experiments and transmission electron microscopy suggested that the adhesion of E. sakazakii to the epithelial and endothelial cells is mainly non-fimbrial based. CONCLUSION: Adherence experiments show heterogeneity within different E. sakazakii strains. In agreement with studies on E. cloacae, we found no relationship between the adhesive capacities in E. sakazakii and the eventual production of specific fimbriae. Further studies will have to be carried out in order to determine the adhesin(s) involved in the interaction of E. sakazakii with cells and to enhance knowledge of the pathogenesis of E. sakazakii infection.

Dataset Information

Molecular analysis of the Enterobacter sakazakii O-antigen gene locus.

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Molecular analysis of the Enterobacter sakazakii O-antigen gene locus.

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