Contaminated powered infant formula (PIF) has been identified to be linked with a number of clinical outbreaks of rare but life-threatening infections in infants associated with Cronobacter. The overall objective of this thesis is to evaluate the impact of microbiologically active substances in PIF on the growth and detection of Cronobacter spp. Depending on preparation, storage and feeding practices, a potential rapid growth of Cronobacter spp. in reconstituted PIF is possible. The first aim was to investigate the effectiveness of using hot water of 70°C or higher on inactivation of Cronobacter spp. during PIF rehydration. It was showed that PIF reconstituted with water of 80°C in plastic bottle caused a cell reduction of Cronobacter spp. of greater than 3 log and this might thus reduce substantially the risk of infection in infants. Although the effect of fermented infant formula (FIF) on suppression of Cronobacter spp. growth was shown to be partially offset by water of 80°C, this inhibitive effect based on the bioactive substances emerging after infant formula fermentation was not strong enough. It was assumed that a direct reconstitution of FIF with water of 80°C may lead to a greater inactivation of Cronobacter spp. and result in a reduction of potential infection risk in higher magnitude. Incorporation of organic acids in PIF contributes to an alternative in controlling Cronobacter growth in rehydrated PIF. The present study first reported the synergistic effect of combination of infant formula moderately acidified with organic acid to pH 6.0 and physiological infant gastric acidity of pH 5.0: Under the simulated neonatal gastric acid condition, the slightly acidified infant formula which did not show inhibitive effect solely reduced significantly the Cronobacter population. Compared with other acidification interventions reported to date where a low pH of ≤ 5.0 was achieved, the acidified formula presented in this work is expected to improve the taste due to the natural pH value and yet preserve the bacteriostatic activity.Microbiologically active substances present in infant formula product such competitive flora and/or antimicrobial components can inhibit the growth of Cronobacter and further affect the effectiveness of the detection method for Cronobacter. A new protocol for rapid detection of Cronobacter spp. from PIF and raw materials using impedance method combined with rRNA-based lateral flow assay has been evaluated, particularly on products/matrices with high amounts of competing background flora and on inhibitive specialised infant formulae. Compared with the reference method based on ISO/TS 22964, a higher sensitivity and specificity were observed, and the detection time was substantially reduced to 29 hours to obtain a final confirmation for Cronobacterin this new protocol whereas the standard protocol needs 6 days. The present work has also represented a comparative assessment of various systems including chromogenic agars, FISH, real time PCR, 16S rRNA gene sequencing, API and MALDI-TOF MS for the detection and identification of Cronobacter spp.. While the phenotypic systems based on biochemical reactions gave false positive or false negative outcomes for Cronobacter spp., the molecular-based identification systems have shown an accuracy of 100% in differentiating Cronobacter from non-Cronobacter strains. Both 16S rRNA gene sequencing and MALDI-TOFMS assigned isolates to species level, but the identification of MALDI-TOFMS was more discriminating and unambiguous, with results provided more rapidly within a few minutes.
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