Diversity of O Antigens within the Genus Cronobacter: from Disorder to Order

Abstract

bCronobacter species are Gram-negative opportunistic pathogens that can cause serious infections in neonates. The lipopolysaccharides (LPSs) that form part of the outer membrane of such bacteria are possibly related to the virulence of particular bacterial strains. However, currently there is no clear overview of O-antigen diversity within the various Cronobacter strains and links with virulence. In this study, we tested a total of 82 strains, covering each of the Cronobacter species. The nucleotide variability of the O-antigen gene cluster was determined by restriction fragment length polymorphism (RFLP) analysis. As a result, the 82 strains were distributed into 11 previously published serotypes and 6 new serotypes, each defined by its characteristic restriction profile. These new serotypes were confirmed using genomic analysis of strains available in public databases: GenBank and Pub-MLST Cronobacter. Laboratory strains were then tested using the current serotype-specific PCR probes. The results show that the current PCR probes did not always correspond to genomic O-antigen gene cluster variation. In addition, we analyzed the LPS phenotype of the reference strains of all distinguishable serotypes. The identified serotypes were compared with data from the literature and the MLST database (www.pubmlst.org/cronobacter/). Based on the findings, we systematically classified a total of 24 serotypes for the Cronobacter genus. Moreover, we evaluated the clinical history of these strains and show that Cronobacter sakazakii O2, O1, and O4, C. turicensis O1, and C. malonaticus O2 serotypes are particularly predominant in clinical cases.

CRISPR– cas Loci Profiling of Cronobacter Sakazakii Pathovars

“…Initially this was achieved by applying random fragment length polymorphism profiling across the O-antigen region and PCR assays targeting the presumed conserved serotype-specific genes wzx and wzy [14]. These methods were able to distinguish a total of seven serotypes in C. sakazakii, thus considerably less than the 189 defined sequence types for this species [6,15]. The O-antigen analysis has been supported in part by chemical composition determination of the frequently isolated serotypes C. sakazakii O:1 and O:2 [16,17].…”

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

“…Despite their rapidity, some molecular methods lack specificity, depending on the chosen primers (22,29). The recent reassignment of C. sakazakii serotypes O5 and O6 to C. malonaticus highlights the unreliability of PCR-based serotyping for the detection of all sequence-based variations of the O antigen of C. sakazakii (21,22,30). Additionally, PCR methods may be not convenient for smaller laboratories, since they require expensive equipment and highly trained employees.…”

“…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).…”

“…According to an established O-antigen serotyping scheme based on rabbit antisera and a PCRbased serotyping method (17-21), 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).Today, the contamination of PIF by C. sakazakii is being detected using conventional microbiological methods. Optimized procedures for the isolation and identification of Cronobacter spp.…”

“…In light of the need for a reliable and inexpensive rapid detection method, several PCRbased protocols for the identification of C. sakazakii at the genus, species, and serotype levels have been established (28). Despite their rapidity, some molecular methods lack specificity, depending on the chosen primers (22,29). The recent reassignment of C. sakazakii serotypes O5 and O6 to C. malonaticus highlights the unreliability of PCR-based serotyping for the detection of all sequence-based variations of the O antigen of C. sakazakii (21,22,30).…”