Detergent and sanitizer stresses decrease the thermal resistance of Enterobacter sakazakii in infant milk formula

Infant milk formula has been identified as a potential source of Enterobacter sakazakii. This bacterium can cause a severe form of neonatal meningitis and necrotizing entercolitis. This study determined the effect of acid, alkaline, chlorine and ethanol stresses on the thermal inactivation of E. sakazakii in infant milk formula. Stressed cells were mixed with reconstituted powdered infant milk formula (PIMF) at temperatures between 52 and 58°C for various time periods or mixed with PFMF prior to reconstitution with water at temperatures between 50 and 100°C. The D- and z-values of the cells were determined using linear regression analysis. Detergent and sanitizer stresses decreased the thermal resistance of E. sakazakii in powdered and reconstituted infant milk formula. The values for Z)- acid, alkaline, chlorine and ethanol stressed E. sakazakii at 52-58°C were 14.57-0.54, 12.07-0.37, 10.08-0.40 and 11.61-0.50 min, respectively. The values of alkaline, chlorine and ethanol stressed cells were significantly lower than those of unstressed cells. Only the z-value (4.4°C) of ethanol stressed E. sakazakii was significantly different than that of unstressed cells (4.12°C). Reconstitution at 60°C did not significantly reduce the number of pre-stressed E. sakazakii cells compared with unstressed control cells, whereas significant decreases were obtained at 70°C. Using water at 70°C during the preparation of reconstituted PIMF before feeding infants, may be a suitable and applicable means of reducing the risk of E. sakazakii in the formula. The results of this study may be of use to regulatory agencies, infant milk producers and infant caregivers to design heating processes to eliminate E. sakazakii that may be present in infant milk formula

Cronobacter sakazakii Infection Induced Fatal Clinical Sequels Including Meningitis in Neonatal ICR Mice

Cronobacter sakazakii (C. sakazakii), formerly Enterobacter sakazakii, is an emerging pathogen associated with the ingestion of contaminated reconstituted formula that causes serious illnesses such as bacteremia, septicemia, necrotizing enterocolitis, meningitis and death in low-birth-weight preterm neonatal infants. The objective of this study was to develop an animal model for human neonatal C. sakazakii infections. We acquired timed-pregnant ICR mice and allowed them to give birth naturally. On postnatal day 3.5, each pup was administered orally a total dose of approximately 107 CFU C. sakazakii strain 3439. Mice were observed twice daily for morbidity and mortality. At postnatal day 10.5, the remaining pups were euthanized, and brain, liver, and cecum were excised and analyzed for the presence of C. sakazakii. C. sakazakii was isolated from cecum and other tissues in inoculated mice. In the tissues of C. sakazakii infected mice, meningitis and gliosis were detected in brain. In this study, we confirmed the neonatal ICR mice may be used a very effective animal model for human neonatal C. sakazakii infections

Effect of desiccation, starvation, heat, and cold stresses on the thermal resistance of Enterobacter sakazakii in rehydrated infant milk formula

Enterobacter sakazakii has been implicated in outbreaks of meningitis, septicemia, and necrotizing enterocolitis in immunocompromised and premature neonates. In this study, the effect of desiccation stress, starvation stress, heat shock and cold shock on thermal inactivation of E. sakazakii in rehydrated infant milk formula was evaluated. Stressed cells were mixed with rehydrated infant milk formula at 52, 54, 56, and 58°C for various time periods. The D- and z-values were determined by using linear regression analysis. Z)-values for unstressed E. sakazakii at 52, 54, 56 and 58°C were 15.33, 4.53, 2.00 and 0.53 min, respectively. Desiccation and heat stress, but not starvation or cold stress, caused significant reduction in Z)-values. For example, D52 was 15.33 min for unstressed cells compared with 8.72 and 7.36 after desiccation and heat stress. Z-values of 53 desiccated, starved, heat shocked and cold shocked E. sakazakii were not significantly different from unstressed cells (4.22°C). The results of this study may be of use to regulatory agencies, infant milk producers and infant caregivers to design heating processes to eliminate E. sakazakii that may be present in infant milk formula

Comparison of methods for the microbiological identification and profiling of cronobacter species from ingredients used in the preparation of infant formula

Cronobacter spp. (formerly Enterobacter sakazakii) can be isolated from a wide range of foods and environments, and its association with neonatal infections has drawn considerable attention from regulatory authorities. The principle route of neonatal infection has been identified as the ingestion of contaminated infant formula. A number of methods have been developed to identify Cronobacter spp., however these were before the most recent (2012 ) taxonomic revision of the genus into seven species. In this study, phenotyping, protein profiling and molecular methods were used to identify Cronobacter strains which had been recently isolated from ingredients used in the preparation of infant formula. Pulsed field gel electrophoresis revealed that different Cronobacter strains had been recovered from the same food products. All isolates were identified as C sakazakii according to four genus specific PCR-probes and protein profiling using MALDI-TOF analysis. However, 16S rDNA sequence analyses and fusA allele sequencing gave more accurate identification: four strains were C sakazakii, one strain was C malonaticus and the remaining strain was C universalis. Multilocus sequence typing showed the strains were different sequence types. These results demonstrate the presence of different Cronobacter species in food ingredients used in the preparation of infant formula, and also the need for molecular identification and profiling methods to be revised according to taxonomic revisions

The Cronobacter genus: ubiquity and diversity

Members of the Cronobacter genus (formerly Enterobacter sakazakii) have become associated with neonatal infections and in particular contaminated reconstituted infant formula. However this is only one perspective of the organism since the majority of infections are in the adult population, and the organism has been isolated from the enteral feeding tubes of neonates on non-formula diets. In recent years methods of detection from food and environmental sources have improved, though accurate identification has been problematic. The need for robust identification is essential in order to implement recent Codex Alimentarius Commission (2008) and related microbiological criteria for powdered infant formula (PIF; intended target age 0-6 months). Genomic analysis of emergent pathogens is of considerable advantage in both improving detection methods, and understanding the evolution of virulence. One ecosystem for Cronobacter is on plant material which may enable the organism to resist desiccation, adhere to surfaces, and resist some antimicrobial agents. These traits may also confer survival mechanisms of relevance in food manufacturing and also virulence mechanisms

Cronobacter spp. as emerging causes of healthcare-associated infection

Background: Until recently, members of the Cronobacter genus (formerly known as Enterobacter sakazakii) were a relatively unknown cause of nosocomial infections. However, their association with infant infections, particularly through the consumption of contaminated reconstituted infant formula in neonatal intensive care units, has resulted in international efforts to improve neonatal health care. Aim: To investigate current understanding of this emergent group of bacterial pathogens and the steps taken to reduce neonatal infection. Methods: A literature review was undertaken to determine current knowledge of the Cronobacter genus with respect to recent taxonomic revisions, sources and clinical relevance. Findings: The majority of severe neonatal meningitis infections are associated with one of the 10 Cronobacter spp., the clonal complex known as C. sakazakii sequence type 4. International efforts by the Food and Agriculture Organization–World Health Organization (WHO) to reduce the risk of neonatal infection by this organism have resulted in improved microbiological safety of powdered infant formula (PIF), but revised guidelines for feeding practices have been problematic. In addition, the majority of infections occur in the adult population and the sources are unknown. Conclusion: International improvements in the microbiological safety of PIF and advice on feeding practices have focused on improving neonatal health care following the heightened awareness of Cronobacter infections in this particular age group. These measures are also likely to reduce neonatal exposure to other opportunistic bacterial pathogens, but a number of unresolved issues remain with respect to the practicalities of feeding premature neonates safely while following WHO advice

A next generation, pilot-scale continuous sterilization system for fermentation media

A new continuous sterilization system was designed, constructed, started up, and qualified for media sterilization for secondary metabolite cultivations, bioconversions, and enzyme production. An existing Honeywell Total Distributed Control 3000-based control system was extended using redundant High performance Process Manager controllers for 98 I/O (input/output) points. This new equipment was retrofitted into an industrial research fermentation pilot plant, designed and constructed in the early 1980s. Design strategies of this new continuous sterilizer system and the expanded control system are described and compared with the literature (including dairy and bio-waste inactivation applications) and the weaknesses of the prior installation for expected effectiveness. In addition, the reasoning behind selection of some of these improved features has been incorporated. Examples of enhancements adopted include sanitary heat exchanger (HEX) design, incorporation of a “flash” cooling HEX, on-line calculation of F(o) and R(o), and use of field I/O modules located near the vessel to permit low-cost addition of new instrumentation. Sterilizer performance also was characterized over the expected range of operating conditions. Differences between design and observed temperature, pressure, and other profiles were quantified and investigated