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

Abstract

cCronobacter 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. The epidemiological link between Cronobacter infection in neonates and contaminated powdered infant formula (PIF) has been previously established (4, 5), with C. sakazakii sequence type 4 (ST-4) being linked to cases of meningitis (6). Outbreaks have been associated with contaminated food products and the presence of this bacterium in PIF production environments (5, 7).In order to rapidly and accurately characterize Cronobacter species in PIF and its associated environments, several molecularbased protocols have been developed, which include direct target gene detection and subtyping methods (8-20). Pulsed-field gel electrophoresis (PFGE) is an accepted method for tracking isolates across the food chain, and this approach is generally considered suitable for epidemiological studies (12,(21)(22)(23)(24)(25)(26)(27)(28). A multilocus sequence typing (MLST) scheme for Cronobacter species was developed, which focuses on single nucleotide polymorphisms associated with seven housekeeping genes (including atpD, fusA, glnS, gltB, gyrB, infB, and pps) and identifies their associated alleles (29). This protocol has been used to describe some of the diversity related to the genus (6, 29-31). 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 we...

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

“…Blažková et al (22) reported that only C. sakazakii serotypes O1, O2, and O4 were isolated from clinical cases, while C. sakazakii serotypes O3 and O7 were not found, even though their occurrence in PIF samples has been described (22). In contrast, Yan et al reported in two recently published studies (24,29) that C. sakazakii serotypes O1 to O3 are the most common serotypes in clinical isolates. The distribution of C. sakazakii serotypes in clinical isolates allows the assumption that some C. sakazakii serotypes might be more pathogenic than others.…”

“…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).…”

Survival of Cronobacter in powdered infant formula and their variation in biofilm formation

“…; Yan et al . ). For example, C. sakazakii ST1, ST4, ST8 and ST12 strains are regarded as pathovars that have been linked to human illnesses, with C. sakazakii ST4 CC being predominantly associated with neonatal meningitis (Joseph and Forsythe ; Joseph et al .…”

“…; Yan et al . ). To date, no unique virulence traits have been identified in the CC pathovars (Masood et al .…”

Advancements in Microarray Utility for Detection and Tracking of Foodborne Microbes in the Genomic Era

“…For instance, during the last decade, scientists at the Center of Food Safety and Applied Nutrition (CFSAN), FDA in conjunction with Affymetrix, Inc., have developed several microarray assays [25] for the investigation of genetic diversity and phylogenetic relatedness of Escherichia coli O157:H7 [23,26], STEC [22,27], Salmonella [19], Listeria monocytogenes [21], Cronobacter [28][29][30] and enteric viruses [15,17].…”