Title

Enterobacter sakazakii: A study on inactivation, detection and virulence

Date of Completion

January 2006

Keywords

Agriculture, Food Science and Technology|Biology, Microbiology|Health Sciences, Pathology

Degree

Ph.D.

Abstract

Enterobacter sakazakii is an emerging pathogen transmitted through contaminated infant formula, causing meningitis, meningo-encephalitis, necrotizing enterocolitis and sepsis in neonates and infants. In spite of the renewed research interest on E. sakazakii in the recent past, many lacunae exist in our understanding of the pathogen, especially regarding how it brings about disease in the host. Development of effective antimicrobial ingredients to prevent growth of E. sakazakii in infant formula would improve the safety of the formula preparations. The present study revealed that monocaprylin, the monoglyceride ester of caprylic acid (C-8 fatty acid) at 50 mM level reduced E. sakazakii count by >5 log CFU/ml by 1 h of incubation at 37 or 23°C, and by 24 h of incubation at 8 or 4°C in reconstituted infant formula. Rapid tools for E. sakazakii detection in infant formula would promote effective monitoring and early detection of contamination of infant formula. A PCR developed using primers based on the outer membrane protein A ( ompA) gene of E. sakazakii was found to be specific to E. sakazakii strains and could detect 10-1 CFU E. sakazakii of per ml of reconstituted infant formula. Identifying the virulence factors of E. sakazakii and delineating their role in the pathogenic mechanism will help us to identify new targets for therapeutic or prophylactic intervention strategies against this pathogen. Outer membrane protein A (OmpA) was identified as a major fibronectin binding protein of E. sakazakii by ligand immuno blotting assay. An isogenic mutant strain in the ompA of E. sakazakii was found to be significantly (P < 0.05) attenuated in its invasiveness into brain microvascular endothelial cells and human intestinal epithelial (INT 407) cells. Complementation of the mutant with the ompA gene restored the invasive phenotype significantly (P < 0.05). The invasiveness of E. sakazakii into INT 407 cells was inhibited by drugs acting on both microfilament and microtubule components of the cytoskeleton. Further, E. sakazakii was found to induce localization of both microfilaments and microtubules in infected INT 407 cells.^