Growth of Enterobacter sakazakii in reconstituted infant formula as affected by composition and temperature

The ability of Enterobacter sakazakii to cause infections in infants, coupled with its documented presence in some lots of commercially manufactured powdered infant formula, raises a concern about the potential for its growth in reconstituted formula, with consequent increased safety risk. A study was done to determine these characteristics in four commercial milk-based powdered infant formulas and two soy-based formulas reconstituted with water and inoculated with a 10-strain mixture of E. sakazakii at populations of 0.02 and 0.53 CFU/ml (ca. 13 CFU/100 g and ca. 409 CFU/100 g of powdered formula, respectively). Reconstituted formulas were stored at 4, 12, 21, and 30 degrees C, and populations were monitored up to 72 h. E. sakazakii did not grow in formulas stored at 4 degrees C, although it was detected by enrichment of all formulas 72 h after reconstitution. Initially at a population of 0.02 CFU/ml, E. sakazakii grew to populations > or = 1 log CFU/ml of reconstituted formulas held at 12, 21, and 30 degrees C for 48, 12, and 8 h, respectively. At an initial population of 0.53 CFU/ml, the pathogen grew to populations > or = 1 log CFU/ml in reconstituted infant formula held at 12 and 21 degrees C for 24 and 8 h, respectively, and to populations 2.55 to 3.14 log CFU/ml when held at 30 degrees C for 8 h. Populations initially at 0.02 and 0.53 CFU/ml of reconstituted formula increased to < or = 0.25 and 0.4 log CFU/ml, respectively, when formulas were held at 30 degrees C for 4 h. Growth was not greatly influenced by the composition of formulas. Results show that the hang time for reconstituted infant formula held at temperatures in neonatal intensive care units should be no longer than 4 h. Portions of reconstituted infant formula not fed to infants should be stored at < or = 4 degrees C, a temperature at which E. sakazakii will not grow.     

Inhibition of growth of Enterobacter sakazakii in reconstituted infant formula by the lactoperoxidase system

Neonatal bacteremia and meningitis caused by the opportunistic pathogen Enterobacter sakazakii have been associated with the consumption of reconstituted powdered infant formula. Lactoperoxidase (LPO), present in mammalian milk, is known to inhibit the growth of enteric pathogens. We undertook a study to determine if the lactoperoxidase system (LPOS) will inhibit the growth of E. sakazakii in a milk-based powdered infant formula reconstituted with water. Initially at 0.04 CFU/ml, E. sakazakii grew to 2.40 to 2.74 log CFU/ml in reconstituted infant formula held at 30 or 37 degrees C for 8 h and to 0.6 log CFU/ ml in formula held for 12 h at 21 degrees C. The pathogen was not detected (less than 1 CFU/227 ml) by enrichment of formula treated with 10 to 30 microg/ml LPO and stored for 24 h at 37 degrees C or 30 microg/ml LPO and stored for 24 h at 30 degrees C. Populations of E. sakazakii, initially at 4.40 log CFU/ml of reconstituted infant formula containing 5 microg/ml LPO, did not increase significantly (P > 0.05) for up to 12 h at 21 and 30 degrees C. Populations either decreased significantly or were unchanged in formula supplemented with 10 microg/ml LPO and stored at 21, 30, or 37 degrees C for up to 24, 8, and 8 h, respectively. Results indicate that LPOS can be used to control the growth of E. sakazakii in reconstituted infant formula, thereby potentially reducing the risk of neonatal infections resulting from consumption of formula that may be contaminated with the pathogen.     

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.     

Inactivation of Enterobacter sakazakii in reconstituted infant formula by monocaprylin

Enterobacter sakazakii is an emerging pathogen that causes meningitis, bacteremia, sepsis, and necrotizing enterocolitis in neonates and children, with a mortality rate of 14%. Epidemiological studies have implicated dried infant formula as the principal source of the pathogen. Caprylic acid is a natural eight-carbon fatty acid present in breast milk and bovine milk and is approved as generally recognizable as safe by the U.S. Food and Drug Administration. The objective of this study was to determine the antibacterial effect of monocaprylin (monoglyceride ester of caprylic acid) on E. sakazakii in reconstituted infant formula. A five-strain mixture of E. sakazakii was inoculated into 10-ml samples of reconstituted infant formula (at 6.0 log CFU/ml) followed by 0, 25, or 50 mM (1%) monocaprylin. The samples were incubated at 37 or 23 degrees C for 0, 1, 6, and 24 h and at 8 or 4 degrees C for 0, 6, 24, and 48 h, and the surviving populations of E. sakazakii at each sampling time were counted. The treatments containing monocaprylin significantly reduced the population of E. sakazakii (P < 0.05) compared with the controls. Monocaprylin (50 mM) reduced the pathogen by >5 log CFU/ml by 1 h of incubation at 37 or 23 degrees C and by 24 h of incubation at 8 or 4 degrees C. Results indicate that monocaprylin could potentially be used to inactivate E. sakazakii in reconstituted infant formula; however, sensory studies are warranted before its use can be recommended.     

Survival and growth of Enterobacter sakazakii in infant cereal as affected by composition, reconstitution liquid, and storage temperature

Invasive infections caused by Enterobacter sakazakii have occurred predominantly in low-birth-weight neonates and infants younger than 2 months of age. However, infections have also occurred in healthy infants up to 8 months of age and in immunocompromised children up to 4 years of age. The ability of E. sakazakii to survive and grow in infant cereals as affected by composition of the cereal, composition of the reconstitution liquid, and temperature is unknown. A study was done to determine the survival and growth characteristics of E. sakazakii initially at populations of 0.005 and 0.52 CFU/ml of infant rice cereal, oatmeal cereal, or rice with mixed fruit cereal reconstituted with water, milk, or apple juice. Reconstituted cereals were stored at 4, 12, 21, and 30 degrees C, and populations were monitored for up to 72 h. Growth did not occur in reconstituted cereals stored at 4 degrees C or in cereals reconstituted with apple juice and stored at 12 degrees C. Populations (> or =1 CFU/ml) were detected in cereals reconstituted with water or milk and stored at 12, 21, and 30 degres C for 24, 8, and 4 h, respectively. The composition of infant cereals did not markedly affect the survival or growth of E. sakazakii in reconstituted cereals. Populations of E. sakazakii in reconstituted cereal decreased with increases in populations of mesophilic aerobic microflora up to 8 to 9 log CFU/ml, which was concurrent with decreases in pH. E. sakazakii, initially at 2.62 log CFU/ml of rice cereal reconstituted with apple juice (pH 4.32), survived at 40C for at least 14 days. The pathogen grew at 21 and 30 degrees C within 2 days and then decreased to undetectable levels (<1 CFU/10 ml) in cereal stored at 21 degrees C for 5 days or 30'C for 4 days. Initially, at 7.32 log CFU/ml, E. sakazakii was detected in rice cereal stored at 4 degrees C for 50 days. It is recommended that reconstituted infant cereals stored at 21 or 30 degrees C be discarded within 4 h after preparation or stored at -40C, temperatures at which E. sakazakii will not grow.     

Gamma radiation sensitivity of Enterobacter sakazakii in dehydrated powdered infant formula

The observed Enterobacter sakazakii D10-values for tryptic soy broth and dehydrated powdered infant formula were 0.27 +/- 0.05 and 0.76 +/- 0.08 kGy, respectively. A decrease of approximately 3 log in the dehydrated powdered infant formula was obtained by irradiation with 3.0 kGy or rehydration with hot water at 80 degrees C. No recoverable bacteria were found in the powdered infant formula irradiated at 5.0 kGy and stored, either before or after rehydration. A radiation dose of up to 5.0 kGy had no marked effect on the sensory properties of the dehydrated powdered infant formula after rehydration and heating. Gamma radiation could potentially be used to inactivate E. sakazakii in dehydrated powdered infant formula; however, nutritional studies need to be conducted before the use of radiation can be recommended.     

Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature

Enterobacter sakazakii infections in preterm neonates and infants have been epidemiologically associated with consumption of reconstituted powdered infant formula. The bacterium has been isolated from grain, infant cereals, and cereal factory environments. A study was done to determine the survival characteristics of E. sakazakii initially at populations of 0.31 and 5.03 logCFU/g of infant rice cereal (a(w) 0.30, 0.45-0.46, and 0.68-0.69). Cereal was stored at 4, 21, and 30 degrees C and populations were monitored for up to 12 months. Survival of the pathogen in infant rice, barley, oatmeal, and mixed grain cereals (a(w) 0.63-0.66, 0.76, or 0.82-0.83) initially containing a population of 4.93-5.64 logCFU/g and held at 4, 21, and 30 degrees C up to 24 weeks was determined. Populations decreased significantly (p < or = 0.05) in all cereals stored at 21 and 30 degrees C regardless of a(w). Increases in a(w) or storage temperature accelerated the rate of death of E. sakazakii in dry infant cereals. However, at an initial population of 0.31 logCFU/g, E. sakazakii survived in rice cereal (a(w) 0.30-0.69) for up to 12 months at all storage temperatures. Survival of E. sakazakii was not affected by the composition of dry infant rice, barley, mixed grain, and oatmeal cereals (initial a(w) 0.63-0.83) stored for up to 24 weeks at 4, 21, or 30 degrees C. This study demonstrated that E. sakazakii can survive for up to 12 months in infant cereals having a wide range of a(w) when storage is at temperatures simulating those to which they may be exposed during distribution, at retail, and in the home.     

Rapid, specific detection of Enterobacter sakazakii in infant formula using a real-time PCR assay

Enterobacter sakazakii is a rare cause of invasive infection with high mortality rates in neonates. Powdered milk-based infant formulas have been associated with the E. sakazakii-related outbreaks in premature or other immunocompromised infants. In this study, an assay was developed for the specific detection of E. sakazakii in infant formula using an application of the fluorogenic 5' nuclease assay (TaqMan). A set of primers and probe was designed using the E. sakazakii partial macromolecular synthesis operon: the rpsU gene 3' end and the primase (dnaG) gene 5' end. The specificity of the assay was evaluated using 68 Enterobacter and 55 non-Enterobacter strains. The newly developed assay enables us to detect 100 CFU/ ml in pure culture and in reconstituted infant formula in 50 cycles of PCR without enrichment. The assay was specific enough to discriminate E. sakazakii from all other Enterobacter and non-Enterobacter strains tested. The developed real-time PCR assay could save up to 5 days and eliminate the need for plating samples on selective or diagnostic agars and for biochemical confirmation steps. The real-time PCR assay could be used to rapidly screen infant formula samples for E. sakazakii and would be a boon to food industries and regulatory agencies.     

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

Enterobacter sakazakii is an opportunistic foodborne pathogen that has been isolated from powdered infant milk formula. This study determined the effect of desiccation, starvation, heat and cold stresses on the thermal inactivation of E. sakazakii in rehydrated infant milk formula (RIMF). Stressed cells were mixed with RIMF at 52, 54, 56, and 58 degrees C for various time periods. The D- and z-values were determined by using linear regression analysis. D-values for unstressed E. sakazakii at 52, 54, 56, and 58 degrees C were 15.33, 4.53, 2, and 0.53 min, respectively. Desiccation and heat stresses, but not starvation or cold stress, caused significant (P < 0.05) reduction in D-values. The z-values of desiccated, starved, heat stressed, and cold stressed E. sakazakii were not significantly different from unstressed cells (4.22 degrees C). Thermal resistance of E. sakazakii in RIMF is affected by the environmental stresses; that is, desiccation and heat stresses that may surround the bacterium prior to the contamination of infant 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.     

Effects of extended dry storage of powdered infant milk formula on susceptibility of Enterobacter sakazakii to hot water and ionizing radiation

Infant milk formula has been identified as a potential source of Enterobacter sakazakii, which has been implicated in neonatal meningitis and necrotizing enterocolitis. This study was undertaken to determine whether the length of E. sakazakii storage in powdered infant milk formula (PIMF) affected the ability of the pathogen to survive subsequent reconstitution of the powder with hot water or treatment with gamma radiation. Five E. sakazakii strains were mixed individually with PIMF and kept for up to 12 months at 25 degrees C. After storage PIMF was reconstituted with water at 60 to 100 degrees C or was exposed to < or = 5 kGy of gamma radiation. Without any treatment secondary to drying, E. sakazakii counts decreased < 1 log/g after 1 month but decreased about 4 log/g during storage for 8 to 12 months. Dry storage decreased thermal resistance but increased resistance of E. sakazakii to ionizing radiation in PIMF. Reconstitution of contaminated powder with water at 70 degrees C after 1 month of dry storage reduced E. sakazakii viability slightly, > 2 log/g, and after powder was stored for 12 months all E. sakazakii strains were eliminated. In contrast, desiccation substantially increased the resistance of E. sakazakii strains to ionizing radiation. Although the D-value for E. sakazakii IMF1 following overnight storage in PIMF was 0.98 kGy, > 4 kGy was required to kill 1.5 log/g of the same strain that had survived 12 months in dry PIMF. Results suggested that low-dose irradiation will more effectively eliminate E. sakazakii from PIMF if the treatment is applied shortly after PIMF manufacture.     

Growth and survival of Enterobacter sakazakii in human breast milk with and without fortifiers as compared to powdered infant formula

Enterobacter sakazakii infections often involve debilitated neonates consuming contaminated reconstituted powdered infant formula. There is the possibility that expressed human breast milk can become contaminated with E. sakazakii in the hospital or home setting and through the use of contaminated breast milk fortifiers. In addition, although breast milk has been shown to have some antimicrobial effects, this has not been extensively researched in regards to E. sakazakii. Thus, we examined the survival and growth of 9 strains of E. sakazakii in breast milk, human breast milk with fortifiers and powdered infant formula at 10, 23 and 37 degrees C. The average generation times for clinical, food and environmental isolates in breast milk were 0.94+/-0.04, 0.75+/-0.04 and 0.84+/-0.04 h at 23 degrees C; and 0.51+/-0.03, 0.33+/-0.03 and 0.42+/-0.03 h at 37 degrees C, respectively. E. sakazakii was able to survive up to 12 days in breast milk with fortifiers at 10 degrees C. However, its average generation times among replicates and isolate sources ranged from 11.97+/-3.82 to 27.08+/-4.54 h in breast milk at 10 degrees C. Interestingly, average generation times in breast milk with fortifiers at 23 degrees C (0.83+/-0.05, 0.93+/-0.06 and 0.96+/-0.06 h) and 37 degrees C (0.41+/-0.04, 0.51+/-0.05 and 0.54+/-0.05 h) were longer than in powdered infant formula and breast milk at the same temperatures, indicating that human breast milk fortifiers may have an inhibitory effect on the growth of E. sakazakii. However, the intrinsically ascribed antimicrobial properties of breast milk do not appear to inhibit the growth of this foodborne pathogen in-vitro.     

Survival and growth of Enterobacter sakazakii on fresh-cut fruits and vegetables and in unpasteurized juices as affected by storage temperature

Enterobacter sakazakii is an emerging foodborne pathogen that has caused illnesses and deaths in infants and elderly immunocompromised adults. Outbreaks of E. sakazakii infection have been associated with infant formulas, but the documented presence of this pathogen in a wide variety of ready-to-eat foods, including lettuce and other raw vegetables, makes it important to learn more about its behavior in these environments. We investigated the survival and growth characteristics of E. sakazakii on fresh-cut apple, cantaloupe, strawberry, watermelon, cabbage, carrot, cucumber, lettuce, and tomato and in juices prepared from these fruits and vegetables. Produce and juices were inoculated with E. sakazakii at 2 to 3 log CFU/g and 1 to 2 log CFU/ml, respectively, and stored at 4, 12, or 25 degrees C. Populations either did not change or gradually decreased in fresh-cut produce and juices stored at 4 degrees C but grew at 12 degrees C on fresh-cut apple, cantaloupe, watermelon, cucumber, and tomato and in all juices except apple, strawberry, cabbage, and tomato juices. All fresh-cut fruits and vegetables except strawberry supported growth of E. sakazakii at 25 degrees C. Growth occurred in all juices except apple, strawberry, and cabbage juices, followed by decreases in population to < 1 CFU/ml after 48 to 72 h, which coincided with decreases in pH and an increase in the population of lactic acid bacteria. Increases in total counts occurred in all juices except strawberry juice stored at 25 degrees C and apple and strawberry juices stored at 12 degrees C. Total counts increased in cantaloupe, carrot, cucumber, and lettuce juices stored at 4 degrees C. Populations of molds and yeasts increased in apple and tomato juices stored at 25 degrees C but decreased to <1 CFU/ml in cabbage, lettuce, and cucumber juices. Further characterization of the behavior of E. sakazakii on fresh produce and in unpasteurized juice as affected by commercial packaging and handling practices is needed.     

Effect of high-pressure processing on strains of Enterobacter sakazakii

Four strains of Enterobacter sakazakii were inoculated into tryptic soy broth and reconstituted powdered infant formula and exposed to high-pressure processing. Pressures of 200, 400, and 600 MPa were used for each medium for 1 min. E. sakazakii was reduced by more than 6 log (strains A and B) in both media at 600 MPa. Strain B was significantly (P < or = 0.05) more pressure resistant than the other strains, with just more than a 3-log reduction at 600 MPa in both media. The reconstituted infant formula has a significant (P < or = 0.05) protective effect for certain strains and pressures (strain B at 400 MPa and strain D at 400 and 600 MPa). Differences in log reductions between media (milk and broth) were also observed for certain strains and specific pressures (strain B at 400 MPa and strain D at 400 and 600 MPa; P < or = 0.05). This research showed that E. sakazakii, when present in reconstituted powdered infant formula, can be submitted to high-pressure processing (600 MPa for 1 min) and achieve log reductions ranging from 3 to 6.84, depending on the strain.     

Fate of Enterobacter sakazakii attached to or in biofilms on stainless steel upon exposure to various temperatures or relative humidities

Survival of Enterobacter sakazakii dried on the surface of stainless steel and exposed to 43% relative humidity, as affected by temperature, was studied. Populations of E. sakazakii (7.4 to 8.6 log CFU per coupon) on coupons dried for 2 h at 22 degrees C decreased significantly (P < or = 0.05) at 4, 25, and 37 degrees C within 10, 3, and 1 day(s), respectively, but the pathogen remained viable for up to 60 days. At a given storage temperature and time, reductions were significantly greater when cells had been suspended in water rather than in infant formula before drying. Formation of biofilm by E. sakazakii on stainless steel immersed in M9 medium, which contains minimal concentrations of nutrients, and infant formula at 25 degrees C and subsequent survival of cells at 25 degrees C as affected by exposure to 23, 43, 68, 85, and 100% relative humidity were investigated. Some of the cells in these biofilms survived under all test relative humidities for up to 42 days. The overall order of survival as affected by relative humidity was 100 > 23 = 43 = 68 > 85% relative humidity, regardless of the medium in which the biofilm was formed. Reduction in viability of cells was significantly greater in biofilm that had formed in M9 medium than in biofilm formed in infant formula. Results indicate that infant formula provides protection for attached cells, as well as cells in biofilm, against lethality on exposure to desiccation. These results are useful when predicting the survival characteristics of E. sakazakii on stainless steel surfaces in processing and preparation kitchen environments.     

Detection of Enterobacter sakazakii strains by real-time PCR

A precise 5' nuclease (TaqMan) real-time PCR was developed and validated in house for the specific detection of Enterobacter sakazakii isolates. Specifically designed nonpatented primers and a hydrolysis (TaqMan) probe were used to target the 16S rRNA gene. All 27 E. sakazakii and 141 non-E. sakazakii strains tested with the real-time PCR were identified correctly. To monitor false-negative results, an internal amplification control was coamplified with the same primers used for the E. sakazakii DNA. The detection probability of the assay was 56% when an E. sakazakii cell suspension containing 10(2) CFU/ml was used as template in the PCR (0.5 CFU per reaction) and 100% with a 10(3) CFU/ml suspension. This PCR assay should be very useful for the diagnostic detection of E. sakazakii in foods, especially powdered infant formula, after cultural enrichment.     

Survival of Enterobacter sakazakii on fresh produce as affected by temperature, and effectiveness of sanitizers for its elimination

A study was done to determine the survival characteristics of Enterobacter sakazakii on the surface of apples, cantaloupes, strawberries, lettuce, and tomatoes stored at 4, 12, and 25 degrees C for 8-28 days. Populations significantly decreased (por=50 microg/ml, were equivalent in killing E. sakazakii on apples. Populations of E. sakazakii on apples treated with 10 microg/ml chlorine dioxide for 1 or 5 min were significantly reduced (por=4.00 log CFU/apple. Reductions of >or=3.70 log CFU/tomato were achieved by treatment with 10 microg/ml chlorine or chlorine dioxide or 40 microg/ml Tsunami 200 for 5 min. Reductions in populations of E. sakazakii on lettuce treated with chlorine at 10, 50, and 100 microg/ml for 1 min ranged from 1.61 to 2.50 log CFU/sample (26+/-4 g), compared to populations remaining on lettuce washed with water. Chlorine was less effective in killing E. sakazakii on lettuce than on apples or tomatoes. Treatment of lettuce with Tsunami 200 (40 and 80 microg/ml) for 5 min caused a reduction of >or=5.31 log CFU/sample. Results provide insights to predicting survival characteristics of E. sakazakii on produce and the efficacy of sanitizers in killing the bacterium.     

Decontamination of poultry carcasses using steam or hot water in combination with rapid cooling, chilling or freezing of carcass surfaces

Reconstituted infant formula has been implicated in outbreaks of Enterobacter sakazakii infections, causing high mortality and serious sequelae. Current prevention methods appear to be insufficient to ensure that such foods are free of E. sakazakii. In this study, the usefulness of bacteriophages for biocontrol of E. sakazakii was investigated. Of a total of six new E. sakazakii phages isolated from sewage and UV irradiated cultures, two were selected for further study by electron microscopy, DNA restriction analysis and SDS-PAGE of structural proteins. Purified phages were used to control bacterial growth in broth medium and reconstituted infant formula. Both phages effectively prevented development of E. sakazakii in formula at various temperatures (12, 24 and 37 degrees C), the efficiency of which was dependent upon intrinsic lysis properties and the applied phage concentration. We conclude that application of specific bacteriophages may provide a means for efficient prevention of E. sakazakii infection through reconstituted infant formula.     

THERMAL RESISTANCE, SURVIVAL AND INACTIVATION OF ENTEROBACTER SAKAZAKII (CRONOBACTER SPP.) IN POWDERED AND RECONSTITUTED INFANT FORMULA

Enterobacter sakazakii has recently been recognized as an opportunistic foodborne pathogen, and dry infant formula serves as the mode of transmission. The objectives of this study were to investigate the heat resistance, survival and inactivation under room and refrigeration temperatures storage of dry and reconstituted infant formula milk (IFM). E. sakazakii strains (eight strains) showed a wide variability in heat resistance at different temperatures (55, 60 and 63C). The D-values at 55C ranged from 1.51 to 14.83 min, at 60C from 0.17 to 2.71 min and at 63C from 0.05 to 0.88 min. The calculated z values for the studied E. sakazakii strains ranged from 3.76–10.11C. Microwave oven heating of 60-mL portions of reconstituted IFM for 40–50 s was effective in eradicating inoculated E. sakazakii. Storing powdered IFM for 15 days at 4C resulted in at least a 1-log reduction in E. sakazakii strains, whereas storing reconstituted IFM at 4C for 2 weeks resulted in more than a 2-log reduction in E. sakazakii.

PRACTICAL APPLICATIONS

This study shows that E. sakazakii strains differ widely in their heat resistance. No differences were observed between biofilm formers and nonformers in terms of heat-resistance in thermal inactivation kinetics experiments. Conventional high temperature short-time pasteurization processes are considered sufficient to inactivate all E. sakazakii strains, and a household microwave oven (40–50 s for 60-mL portions) can be used to inactivate E. sakazakii if present in reconstituted infant formula milk (IFM). Growth of E. sakazakii can be inhibited in powdered and reconstituted IFM by refrigeration. Also, it is recommended that reconstituted IFM be discarded or refrigerated if not immediately consumed. The probiotic L. acidophilus ATCC 4356 was not effective in inhibiting E. sakazakii in powdered or reconstituted IFM.     

Inactivation of Enterobacter sakazakii in infant milk formula by gamma irradiation: determination of D10-value

ABSTRACT:  Enterobacter sakazakii is an emerging foodborne pathogen that has caused several cases of meningitis and necrotizing entercolitis in infants and has been associated with infant formulas. Five strains of E. sakazakii were inoculated individually into brain heart infusion broth and rehydrated or dehydrated infant milk formula and exposed to ionizing radiation. E. sakazakii strains in brain heart infusion broth and rehydrated infant milk formula (RIMF) were exposed to irradiation dose of up to 1 kGy while strains in dehydrated infant milk formula (DIMF) were exposed to irradiation dose of up to 9 kGy. The D₁₀-values were determined by using a linear regression model. Average calculated D₁₀-values ranged from 0.21 to 0.29 kGy, 0.24 to 0.37 kGy, and 1.06 to 1.71 kGy in brain heart infusion broth, RIMF, and DIMF, respectively. The results obtained from this study will be useful for powdered infant milk formula industries to reduce the risk associated with E. sakazakii .     

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 (8ml) of each of the selective media onto TSAP after spreading a sample onto TSAP. Reconstituted IFM was inoculated by ca 1x10(7)CFU/ml of a mixture of four strains of E. sakazakii and subjected to different stress conditions: heat (55 degrees C for 10min), a freeze-thaw cycle (-20 degrees C for 24h, thawed at room temperature, frozen again at -20 degrees C, and thawed), acidic pH (pH 3.56 for 15min), alkaline pH (pH 11.04 for 15min), and desiccation (E. sakazakii was inoculated onto powdered IFM at a level of ca 1x10(6)CFU/g, held at 21 degrees C, water activity of the inoculated product was 0.29 and examined at 0, 15, and 30d). 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.