Sakazakii Cells Research Articles

Specific probiotic strains and their combinations counteract adhesion ofEnterobacter sakazakiito intestinal mucus

Enterobacter sakazakii is an opportunistic pathogen and an occasional contaminant in powdered infant formula. Interaction between specific probiotics and E. sakazakii may reduce the risk of infection. The aim of this study was to characterize in vitro the ability of probiotics (alone and in combinations) to inhibit, compete with and displace the adhesion of E. sakazakii to immobilized human mucus and to assess their capacity to aggregate with pathogen. Specific probiotic strains have proved to aggregate E. sakazakii cells and, through competitive exclusion, inhibition and displacement of the adhered pathogen, were able to inhibit E. sakazakii action on intestinal mucus. The ability to inhibit and to displace adhered pathogen depended on both the probiotic and the pathogen, suggesting that several complementary mechanisms are involved in the processes. We suggest that the selection of specific probiotic strains and their combinations may be a useful means of counteracting E. sakazakii contamination in infant formula and thus to reduce the risk of emerging infection. This approach may also allow the development of new probiotic combinations to counteract the risks associated with other pathogens by improving the intestinal barrier against pathogens.

A comparative study between overlay method and selective-differential media for recovery of stressed Enterobacter sakazakii cells from infant formula

This study compares the performance of different selective-differential media with the overlay method for recovery of stressed cells of Enterobacter sakazakii from infant formula milk (IFM). Five different selective-differential media were used in this study: OK medium, violet red bile agar (VRBA), Druggan–Forsythe–Iversen agar (DFI), Enterobacteriaceae enrichment (EE) agar, and fecal coliform agar (FCA). Tryptic soy agar supplemented with 0.1% sodium pyruvate (TSAP) was used as a control. The overlay method involved applying a thin layer (8 ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1×10 7 CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 °C for 10 min), a freeze–thaw cycle (−20 °C for 24 h, thawed at room temperature, frozen again at −20 °C, and thawed), acidic pH (pH 3.56 for 15 min), alkaline pH (pH 11.04 for 15 min), and desiccation ( E. sakazakii was inoculated onto powdered IFM at a level of ca 1×10 6 CFU/g, held at 21 °C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30 d). No major differences were noticed between the control (TSAP) and the overlay methods. However, the overlay method recovered significantly higher numbers of stressed E. sakazakii cells compared to selective-differential media. Also, the selective-differential media exhibited some variability in terms of their capabilities to recover stressed cells of E. sakazakii. Among all the examined selective-differential media, DFI performed better for recovering stressed E. sakazakii cells. This study suggests that the overlay method may serve as a potential alternative to direct selective plating for best recovery of E. sakazakii from IFM.

Survival of Enterobacter sakazakii in Powdered Infant Formula as Affected by Composition, Water Activity, and Temperature

A study was done to determine survival characteristics of Enterobacter sakazakii in four milk-based and two soybean-based powdered infant formulas. A 10-strain mixture of E. sakazakii was inoculated into the six infant formulas at water activity (aw) 0.25 to 0.30, 0.31 to 0.33, and 0.43 to 0.50 to give low (0.80 log CFU/g) and high (4.66 to 4.86 log CFU/g) populations. At an initial population of 0.80 log CFU/g, E. sakazakii was detected by enrichment in six of six, four of six, and one of six formulas stored for 12 months at 4, 21, and 30°C, respectively. In four of six formulas at aw values of 0.25 to 0.30, initially high populations decreased significantly (P ≤ 0.05), although by less than 1 log CFU/g, within 6 months at 4°C. Populations decreased significantly in all formulas in the aw range of 0.25 to 0.50 during storage for 1 month at 21 or 30°C and again between 1 and 6 months in most formulas. Significant reductions occurred between 6 and 12 months in some formulas. At all storage temperatures, reductions in populations tended to be greater in formulas at aw 0.43 to 0.50 than in formulas at aw 0.25 to 0.30. The rate of inactivation of E. sakazakii in formulas was not markedly influenced by formula composition. Cells from mucoid and nonmucoid colonies formed by two strains on violet red bile glucose agar supplemented with pyruvate were inoculated into a milk-based powdered infant formula and a soybean-based powdered infant formula having a high aw range of 0.43 to 0.86 and stored at 4, 21, and 30°C for up to 36 weeks. With few exceptions, populations of both strains decreased significantly in both formulas within 2 weeks at all temperatures; rates of death increased with increased aw and storage temperature. The presence of mucoidal extracellular materials on the surface of E. sakazakii cells was not associated with protection against death. This study shows that the retention of viability of E. sakazakii in powdered infant formula is affected by aw and temperature. Increases in both parameters cause an increase in the rate of death.

Identification of proteins involved in osmotic stress response in Enterobacter sakazakii by proteomics

Enterobacter sakazakii is considered an opportunistic food-borne pathogen, causing rare but significant illness especially in neonates. It has been proposed that the organism is relatively resistant to osmotic and dry stress compared to other species of the Enterobacteriaceae group. To understand the mechanisms involved in osmotic stress response, 2-DE protein analysis coupled to MALDI-TOF MS was employed to investigate changes in the protein profiles of E. sakazakii cells in response to two different types of osmotic stress (physical desiccation and growth in hyperosmotic media). In total, 80 differentially expressed protein spots corresponding to 53 different protein species were identified. Affiliation of proteins to functional categories revealed that a considerable number of the differentially expressed proteins from desiccated and hyperosmotic grown samples belonged to the same functional category but were regulated in opposite directions. Our data show that the protein pattern of NaCl-grown cultures reflect more or less a general down-regulation of central metabolic pathways, whereas adaptation of (non-growing) cells in a desiccated state represents an accumulation of proteins that serve some structural or protective role. The most striking effects observed for both types of osmotic stress in E. sakazakii were a significant down-regulation of the motility apparatus and the formation of filamentous cells.

Survival of Enterobacter sakazakii in a Dehydrated Powdered Infant Formula

A quantity of dehydrated powdered infant formula was prepared to contain Enterobacter sakazakii strain 607 at approximately 106 CFU/ml when rehydrated according to the manufacturer's instructions. The survival of the microorganism in the dry formula was followed for 2 years, during which samples periodically were rehydrated and analyzed for viable E. sakazakii. During the initial 5 months of storage at room temperature, viable counts declined approximately 2.4 log cycles. During the subsequent 19 months, the concentration of viable E. sakazakii declined an additional 1.0 log cycle. These results indicate that a small percentage of E. sakazakii cells can survive for extended periods in dehydrated powdered infant formula.

Desiccation and heat tolerance of Enterobacter sakazakii.

Enterobacter sakazakii is an opportunistic pathogen which has been isolated at low levels from powdered infant formulas. This study was performed to demonstrate that Ent. sakazakii is not particularly thermotolerant, but can adapt to osmotic and dry stress. We determined the heat, osmotic and dry stress resistance of Ent. sakazakii. The D-value at 58 degrees C ranged from 0.39 to 0.60 min, which is comparable with that of other Enterobacteriaceae, but much lower than reported previously (Nazarowec-White and Farber 1997, Letters in Applied Microbiology 24: 9-13). However, stationary phase Ent. sakazakii cells were found to be more resistant to osmotic and dry stress than Escherichia coli, Salmonella and other strains of Enterobacteriaceae tested. Further analysis indicated that the dry resistance is most likely linked to accumulation of trehalose in the cells. The high tolerance to desiccation provides a competitive advantage for Ent. sakazakii in dry environments, as found in milk powder factories, and thereby increases the risk of postpasteurization contamination of the finished product. Understanding of the physiology and survival strategies of Ent. sakazakii is an important step in the efforts to eliminate this bacterium from the critical food production environments.