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

Abstract

Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.

FIG 1

Dendrogram showing the 14 clusters of the 168 strains, denoted clusters C1 to C14, generated using BioNumerics v7.1. The similarity cutoff value was 90%. Both the optimization and tolerance values were 1.0%.

FIG 1

Dendrogram showing the 14 clusters of the 168 strains, denoted clusters C1 to C14, generated using BioNumerics v7.1. The similarity cutoff value was 90%. Both the optimization and tolerance values were 1.0%.

FIG 2

MLST distributions of 133 PIF and 31 clinical isolates. (a) PIF isolates; (b) all 164 PIF and clinical isolates studied. Distinct sequence types are shown in different colors.

FIG 3

The multigenome microarray identification of Cronobacter species and the comparison with MLST and PFGE subtyping method results.

FIG 4

Swimming ability of Cronobacter isolates compared to that of the reference strain S. Typhimurium DT104 13348. The reference strain showed an average swimming activity of 60 mm after 8 h of incubation at 37°C. (a) E657, C. sakazakii serotype O:1, clinical origin; (b) CQ8, C. sakazakii serotype O:4, PIF origin; (c) ATCC BAA-894, C. sakazakii serotype O:1, PIF origin; (d) 206N, C. sakazakii serotype O:2, clinical origin.

FIG 5

Four types of cell morphology observed with Congo red dye binding. (a) red, dry, and rough (RDAR); (b) brown, dry, and rough (BDAR); (c) red and smooth (RAS); (d) brown and smooth (BAS).

FIG 6

Comparisons among ST-1 and ST-4 isolates, of PIF or clinical origin, for the ability to swim. (a) CQ74, ST-1/PIF; (b) CQ113, ST-1/PIF; (c) E654, ST-1/clinical; (d) E657, ST-1/clinical; (e) CQ14, ST-4/PIF; (f) CQ27, ST-4/PIF; (g) 206N, ST-4/clinical; (h) E788, ST-4/clinical.