Tracing of Enterobacter sakazakii isolates in infant milk formula processing by BOX-PCR genotyping.

AIMS: Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe neonatal meningitis, bacteraemia and necrotizing enterocolitis. Contaminated powder infant formulae (PIF) have been identified as one of various infection routes. In this study, E. sakazakii was monitored in the processing environment of a PIF factory to identify possible dissemination routes. METHODS AND RESULTS: The BOX-polymerase chain reaction (PCR), a fingerprinting technique which targets the repetitive BOX sequences, was used in routine to identify points of contamination and investigate clonal persistence. Two hundred E. sakazakii isolates were collected and typed. Most (70%) showed the same fingerprint that revealed the persistence of resident E. sakazakii strains in the processing environment. This method allowed to detect contamination of some PIF by dry-blending ingredients. CONCLUSIONS: Environment was the major cause for contamination of PIF and facilities. Some raw materials delivered as powder were also implicated. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine BOX-PCR genotyping was very useful to trace and investigate in real-time dissemination of micro-organisms in the PIF plant and to implement a series of additional control measures to reduce the risk of final product contamination by E. sakazakii.

Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX-PCR and sequencing of the fliC gene.

AIMS: Enterobacter sakazakii is an emerging food-borne pathogen that can cause rare but severe forms of neonatal meningitis, bacteraemia and necrotizing enterocolitis. A rapid typing method at the strain level is needed to determine the monoclonality or polyclonality of the isolates during outbreaks. METHODS AND RESULTS: The BOX-PCR fingerprinting technique, which targets the repetitive BOX sequences, and sequencing of the flagellin gene, fliC, were evaluated against a panel of 27 Ent. sakazakii strains from clinical and environmental sources. The typeability and discriminatory power of the techniques were compared with those of pulsed-field gel electrophoresis (PFGE), the reference genotyping method. BOX-PCR results yielded 92% agreement with PFGE results, whereas fliC gene sequencing was poorly discriminative. CONCLUSIONS: In our study, BOX-PCR and PFGE were similarly discriminatory to type Ent. sakazakii strains. The weak variability of the Ent. sakazakii fliC gene was related to the absence of the variable central domain present in most fliC genes of Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: The BOX-PCR typing provides an accurate discrimination and a rapid answer to identify clonal isolates of Ent. sakazakii.