Genetic Diversity of Cronobacter sakazakii Isolates Collected from a Swiss Infant Formula Production Facility

Abstract

In this study, 141 Cronobacter isolates that were collected based on a hygienic monitoring program performed in a powdered infant formula production facility in Switzerland between September 2011 and October 2012 were further characterized. Isolates were identified to the species level by molecular methods, and strains of Cronobacter sakazakii were further subtyped by applying PCR-based O-antigen serotyping, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). C. sakazakii was the most prevalent species identified (93.6%). Among this collection of isolates, representatives of all but one O-antigen serotype (serotype O5) were recognized. MLST analysis of 19 selected isolates revealed that most of the typeable isolates belonged to sequence type (ST) 4. Correlations between ST4 and serotype O2 and between ST83 and serotype O7 were observed. PFGE analysis revealed clusters with multiple isolates, including strains from samples collected at different time points and sampling sources. Generally, the observed heterogeneity among strains collected over the 13 months of the monitoring program was high, suggesting a constant flux among strains rather than a selection for persisting organisms.

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain

“…The predominant Cronobacter species cultured from the four PIF production environments studied was C. sakazakii. This finding is in agreement with others, including Müller et al (32), who reported on the microbial ecology of a Swiss PIF facility, Mozrová et al (43), who studied a dairy farm and its environment in the Czech Republic, and Pan et al (20), who reported on Cronobacter species found in commercially available PIF products.…”

“…The same ST has also been identified among nonclinical isolates, including those cultured from PIF and follow-up formula (20,30,32). An example of the latter is C. sakazakii SP291, which was included in this study but did not cluster with any of the 14 pulsotypes.…”

“…This protocol has been used to describe some of the diversity related to the genus (6,(29)(30)(31). Both PFGE and MLST have been widely applied to study the genomic diversity of Cronobacter isolated from manufacturing facilities, commercial PIF, and follow-up formula, as well as clinical isolates (20,32,33). These reports highlight the dominance of C. sakazakii and the importance of the ST-4 clonal complex as the etiological agent in meningitis cases.…”

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

“…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).…”

“…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.…”