Genetic Analysis of the Cronobacter sakazakii O4 to O7 O-Antigen Gene Clusters and Development of a PCR Assay for Identification of All C. sakazakii O Serotypes

Abstract

<jats:title>ABSTRACT</jats:title><jats:p>The Gram-negative bacterium<jats:named-content content-type="genus-species">Cronobacter sakazakii</jats:named-content>is an emerging food-borne pathogen that causes severe invasive infections in neonates. Variation in the O-antigen lipopolysaccharide in the outer membrane provides the basis for Gram-negative bacteria serotyping. The O-antigen serotyping scheme for<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>, which includes seven serotypes (O1 to O7), has been recently established, and the O-antigen gene clusters and specific primers for three<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>serotypes (O1, O2, and O3) have been characterized. In this study, the<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O4, O5, O6, and O7 O-antigen gene clusters were sequenced, and gene functions were predicted on the basis of homology.<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O4 shared a similar O-antigen gene cluster with<jats:named-content content-type="genus-species">Escherichia coli</jats:named-content>O103. The general features and anomalies of all seven<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O-antigen gene clusters were evaluated and the relationship between O-antigen structures and their gene clusters were investigated. Serotype-specific genes for O4 to O7 were identified, and a molecular serotyping method for all<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O serotypes, a multiplex PCR assay, was developed by screening against 136 strains of<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>and closely related species. The sensitivity of PCR-based serotyping method was determined to be 0.01 ng of genomic DNA and 10<jats:sup>3</jats:sup>CFU of each strain/ml. This study completes the elucidation of<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O-antigen genetics and provides a molecular method suitable for the identification of<jats:named-content content-type="genus-species">C. sakazakii</jats:named-content>O1 to O7 strains.</jats:p>

Development of a DNA Microarray Method for Detection and Identification of All 15 Distinct O-Antigen Forms of Legionella pneumophila

“…The O antigen exhibits variation in terms of the types of sugars, the arrangement of the sugars within the O unit, and different types of linkages within and between O units (26). This makes LPS one of the most variable cell constituents and provides a basis for the serotyping of many Gram-negative bacteria (31,32,33). So far, only L. pneumophila serogroup O1 LPS gene clusters have been determined.…”

Simultaneous Rapid Detection and Serotyping of Cronobacter sakazakii Serotypes O1, O2, and O3 by Using Specific Monoclonal Antibodies

“…Only strains that were confirmed to be C. sakazakii were then subjected to multiplex PCR to identify the serotype. The PCR conditions and primers were applied as described previously by Sun et al (20). For serotyping of the other Cronobacter species, the conditions and primers were used as described by Jarvis et al (18).…”

“…Although C. sakazakii has been isolated from a variety of different plant-and animal-based food products (14,15), the presence in powdered infant formula (PIF) seems crucial in the infection of neonates (9,12,16). According to an established O-antigen serotyping scheme based on rabbit antisera and a PCRbased serotyping method (17)(18)(19)(20)(21), seven serotypes (O1 to O7) have been identified for C. sakazakii. Serotypes O1 and O2 seem to be most prevalent in PIF samples and in clinical cases, whereas serotype O3 has been isolated quite frequently from PIF but not as often from clinical cases (19,(22)(23)(24).…”

A Cronobacter turicensis O1 Antigen-Specific Monoclonal Antibody Inhibits Bacterial Motility and Entry into Epithelial Cells

“…Within the genus Cronobacter, 17 serogroups have been defined on the basis of the O-antigen-encoding gene clusters (27)(28)(29)(30)(31)(32). For C. turicensis, three O serotypes (serotypes O1 to O3) have been identified (27,28,30), and the chemical structure of the O antigen of the Cronobacter turicensis HPB3287 strain has been determined (33).…”

“…PCR-based identification of the three serotypes described so far (serotypes O1 to O3) was performed using the primers and following the protocols described in the respective publications (27,28,32). The DNA templates were isolated using a Qiagen DNeasy blood and tissue kit (Hilden, Germany).…”