Thermal Inactivation of Enterobacter sakazakii in ...

60 Journal of Food Protection, Vol. 67, No. 1, 2004, Pages 60–63

Thermal Inactivation of Enterobacter sakazakii in Rehydrated Infant Formula SHARON G. EDELSON-MAMMEL

AND

ROBERT L. BUCHANAN*

Department of Health and Human Services, Food and Drug Administration, Center for Food Safety and Applied Nutrition, 5100 Paint Branch Parkway, College Park, Maryland 20740, USA MS 03-46: Received 4 February 2003/Accepted 1 August 2003

ABSTRACT The presence of low levels of Enterobacter sakazakii in dried infant formula have been linked to outbreaks of meningitis, septicemia, and necrotizing enterocolitis in neonates, particularly those who are premature or immunocompromised. In the current study, the ability of 12 strains of E. sakazakii to survive heating in rehydrated infant formula was determined at 588C with a submerged coil apparatus. The observed D58-values ranged from 30.5 to 591.9 s, with the strains appearing to fall into two distinct heat resistance phenotypes. The z-value of the most heat-resistant strain was 5.68C. When dried infant formula containing this strain was rehydrated with water preequilibrated to various temperatures, a more than 4-log reduction in E. sakazakii levels was achieved by preparing the formula with water at 708C or greater.

Enterobacter sakazakii is a gram-negative non–sporeforming bacterium. It was originally classi ed as ‘‘yellow pigmented’’ Enterobacter cloacae but was subsequently designated a separate species on the basis of pigment production, DNA-DNA hybridization, and other phenotypic characteristics (6, 7). Izard et al. (11), using DNA-DNA hybridization, found that E. sakazakii is a well-de ned species that is distinct from other Enterobacter species. The microorganism is encountered commonly in the environment and in foods, being among the broad group of species collectively referred to as coliforms based on lactose fermentation and resistance to bile salts. On rare occasions, E. sakazakii is associated with severe infections in humans, particularly neonates and immunocompromised infants (1, 2, 8–10, 14–16, 19, 27). Although rare, the primary manifestations of these infections are severe bacteremia and meningitis, with case fatality rates as high as between 40 and 80% (18, 27). E. sakazakii has also been linked to cases of necrotizing enterocolitis (18, 26). Although the vehicle for E. sakazakii has not been identi ed in all cases, dried infant formula has been epidemiologically identi ed as the source of E. sakazakii in at least three outbreaks of neonatal meningitis and one outbreak of necrotizing enterocolitis (2, 3, 5, 10, 17, 24–26). An international survey of dry infant formula from 35 countries found that approximately 14% of the 141 cans examined had detectable levels of E. sakazakii (18). Nazarowec-White and Farber (20) reported that the prevalence of E. sakazakii in dried infant formula available in Canadian retail markets varied between 0 and 12% among the ve manufacturers examined. Thermal treatment of foods just prior to consumption has long been used as a primary means of reducing the risks associated with foodborne pathogens. It has been iden* Author for correspondence. Tel: 301-436-2369; Fax: 301-436-2642; E-mail: [email protected].

ti ed as a practical means of reducing the risk of E. sakazakii in rehydrated infant formulas (12, 13, 17, 22). The effective use of thermal treatments requires accurate information on the heat resistance of the target microorganism; the thermal treatment should be suf cient to inactivate the microorganism of concern while minimizing the loss of nutrients. Nazarowec-White and Farber (21) used a submerged vessel method to evaluate the thermal resistance of pooled isolates of E. sakazakii in reconstituted infant formula; they reported that the microorganism was among the most thermotolerant members of the Enterobacteriaceae encountered in diary products. However, their report was limited to the thermal resistance of the most resistant member of the pooled strains. The objectives of the current study were threefold: (i) determine the thermal resistance with the use of a submerged coil apparatus, (ii) evaluate the diversity in thermal resistance among a variety of E. sakazakii isolates, and (iii) evaluate the effect of rehydrating dried infant formula with water at different temperatures on the survival of the microorganism. MATERIAL AND METHODS E. sakazakii isolates. The E. sakazakii strains used in the study were obtained from the Food and Drug Administration/Center for Food Safety and Applied Nutrition stock culture collection. The original sources of these isolates are provided in Table 1. All cultures were stored in brain heart infusion broth (BHI) with 20% glycerol at 2708C. Four frozen cultures were made of each isolate. Two are used as working cultures and the other two are used for long-term storage of the strains. Infant formula. Cans of commercial dehydrated infant formula forti ed with iron were purchased from local supermarkets. For all experiments, the formula was rehydrated according to the manufacturers’ instructions found on the label, i.e., 60 ml of room temperature water was added for every 8.5 g of dried formula. In all cases, sterile deionized water was used to rehydrate the formula. The only modi cation made was for the ‘‘baby bottle’’ ex-

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TABLE 1. Enterobacter sakazakii strains examined Strain

607 ATCC 29544 ATCC 51329 LCDC 674 CDC A3 (10) SK 90 LCDC 648 EWFAKRC11NNV1493 NQ1-Environ NQ2-Environ NQ3-Environ 4.01C

Source

Clinical isolate, F. Khambaty, FDA ATCC (type strain) ATCC J. M. Farber, Health Canada J. M. Farber, Health Canada J. M. Farber, Health Canada J. M. Farber, Health Canada J. M. Farber, Health Canada Environmental—food manufacturing, M. Kotewicz, FDA Environmental—food manufacturing, M. Kotewicz, FDA Environmental—food manufacturing, M. Kotewicz, FDA Dried infant formula, S. EdelsonMammel, FDA

periments (see below), in which the sterile water was heated to different temperatures before being added to the formula. Determination of thermal resistance with submerged coil apparatus. Thermal trials with the submerged coil apparatus were conducted with the use of modi cations of the techniques described by Buchanan and Edelson (4). Approximately 18 h before the start of a thermal resistance trial, 10 ml of BHI was inoculated with a 0.1-ml aliquot of the frozen working stock culture of the E. sakazakii strain being studied. The culture was incubated at 368C for 18 h. Prior to the start of a heating trial, 15 ml of rehydrated infant formula was inoculated with 1.5 ml of the overnight culture. A 0.4-ml portion was transferred to a precooled 3.6-ml dilution blank and served as the zero-time sample. The initial level of E. sakazakii in the zero-time sample was approximately 108 CFU/ml (Fig. 1). The remainder of the inoculated formula was then injected into the heating coil apparatus (Model 2, Sherwood Instruments, Lynnewood, Mass.) that was preequilibrated to 588C and preprogrammed to deliver appropriately sized aliquots at designated times. After being ejected by the submerged coil, the samples were immediately placed on ice to halt any further thermal inactivation. The samples were then transferred to precooled dilution blanks. For z-value determinations, the heating coil was equilibrated to four additional temperatures: 56, 60, 65, and 708C. Each thermal inactivation trial was repeated at least twice. Survival of E. sakazakii during the rehydration of dried infant formula in baby bottles. Five days before the start of the study, 1,350 g of dried formula was divided evenly between two sterile 4,000-ml beakers. Working under a biological safety hood, approximately 3.5 3 109 CFU in 1.5 ml of a concentrated culture of E. sakazakii strain 607 was added dropwise to the dry infant formula with thorough mixing. The concentrated culture was prepared by centrifuging a 50-ml overnight culture at 4,300 3 g for 15 min and resuspending the pellet in 10 ml of 0.1% buffered peptone water to get an initial concentration of 1.5 to 3.3 3 109 CFU/ml. After the inoculum was added, the formula was dry mixed for an additional 15 min. Preliminary studies with the addition of dye and the consistent counts achieved among the experimental replicates demonstrated that the above protocol uniformly distributed the E. sakazakii throughout the dried infant

FIGURE 1. Examples of thermal inactivation curves observed with E. sakazakii. Each point represents the average of two separate thermal inactivation trials. m, strain 607; v, strain 51329. formula. The inoculated formula was then stored under refrigeration in a sterile, screw-cap, wide-mouth Nalgene bottle until needed. The concentration of microorganisms in the formula when hydrated according to the manufacturers’ directions was approximately 106 CFU/ml. To determine bacterial survival, 25.5-g portions of inoculated formula were weighed out and transferred to prelabeled, sterilized baby bottles. The formula was rehydrated by the addition of 180 ml of sterile deionized water that had been preequilibrated to the desired temperature. After addition of the water, the caps were screwed back onto the bottles. The bottles were then gently agitated by hand at room temperature for 10 min and analyzed for E. sakazakii. Each water temperature was evaluated three times. The cooling rate of the formula after addition of the heated water to the bottle was measured with the use of a second set of trials, in which a thermometer was inserted through the nipple so that it was appropriately positioned in the formula when the cap was secured. The temperature was then read and recorded at speci c intervals over the course of the 10-min holding time. The cooling rate determination for each rehydration temperature was performed three times. Plating and enumeration. All samples and appropriate dilutions were surface plated with the use of a spiral plater (Spiral Biotech, Bethesda, Md.) onto tryptic soy agar (Difco, Detroit, Mich.) containing 1% sodium pyruvate. The sodium pyruvate was added to aid in the recovery of the thermally injured cells. The plates were incubated overnight and then enumerated with an automatic plate counter (Spiral Biotech). D-value and z-value determinations. Standard regression analysis was performed by log-linear models in Excel (Microsoft) and the D-value determined by taking the negative reciprocal of the slope. The z-value was found using linear regression of the log D-values of ve temperatures: 56, 58, 60, 65, and 708C.

RESULTS Substantial variation in thermal resistance was observed among the 12 isolates (Table 2), with almost a 20fold differential existing between the most (607) and least (51329) heat-resistant strains. The inactivation curves were log-linear, indicating rst-order inactivation kinetics (Fig. 1). The distribution of heat resistance among the various isolates was bimodal, with half of the strains having D-

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EDELSON-MAMMEL AND BUCHANAN


TABLE 2. D-values (s) of 12 Enterobacter sakazakii strains heated at 588C in infant formula with a submerged coil apparatus Strain

na

Range

51329 NQ2-Environ NQ3-Environ LCDC 674 CDC A3(1) NQ1-Environ EWFAKRC11NNV1493 29544 SK 90 LCDC 648 4.01C 607

2 2 2 2 2 2 2 6 2 2 2 3

30.4–30.5 30.2–32.8 31.6–37.3 33.7–40.2 31.7–43.3 47.2–48.5 303.0–312.5 333.3–400.0 454.6–476.2 526.3–555.6 555.6–588.2 526.3–625.0

a b

Mean 6 SDb

30.5 31.5 34.4 36.9 37.5 47.9 307.8 367.1 465.4 540.9 571.9 591.9

6 6 6 6 6 6 6 6 6 6 6 6

0.1 1.8 4.1 4.6 8.2 1.0 6.7 23.4 15.3 20.7 23.1 49.9

Number of replicate thermal inactivation trials. Standard deviations calculated for strains where n 5 2 are for the purposes of comparison only and should be interpreted in relation to the usual restriction of this statistic to n $ 3.

values of less than 50 s and the other half having D-values of more than 300 s. The heat resistance of strain 607, the most heat-resistant strain at 588C, was determined at ve temperatures to calculate the microorganism’s z-value (Table 3). The relationship between D-value and heating temperature was loglinear, with an R2 value of 0.998. The calculated z-value for strain 607 was 5.68C. The thermal resistance of E. sakazakii strain 607, as determined with the submerged coil apparatus, was such that exposure to temperatures at or above 708C should provide virtually instantaneous inactivation of the microorganism. A simple but practical test of this hypothesis was performed by rehydrating dried infant formula containing strain 607 at a level of approximately 106 CFU/ml of formula when rehydrated in individual baby bottles (Table 4). Essentially no inactivation occurred with 508C water, 1-D of inactivation was observed with 608C, and 4-D or more inactivation was observed with water temperatures $708C.

TABLE 4. Survival of Enterobacter sakazakii strain 607 during the rehydration of dried infant formula with water at different temperatures

a

Surviving E. sakazakii (log CFU/ml)

Temperature (8C) of water added to dry infant formula

Replicate A

Replicate B

Replicate C

Mean

100 90 80 70 60 50 Unheated

,2.0a ,2.0 ,2.0 ,2.0 4.7 5.8 5.9

,2.0 ,2.0 ,2.0 ,2.0 4.8 5.6 5.9

,2.0 ,2.0 ,2.0 ,2.0 4.8 5.8 5.9

,2.0 ,2.0 ,2.0 ,2.0 4.7 5.7 5.9

Lower limit of detection; 100 CFU/ml (log CFU/ml 5 2.0).

mined D58-values in rehydrated infant formula for a pooled group of ve clinical isolates and a second pooled group of ve food isolates. The reported D 58-values were 327 and 206 s for the two pooled groups, respectively. Although the D58-values observed in the current study (Table 2) overlapped those reported by Nazarowec-White and Farber (21), by examining individual strains, it became apparent that thermal resistance among E. sakazakii strains varied as much as 20-fold (Table 2). Furthermore, among the isolates examined in the current study, several were substantially more heat resistant than the pooled isolates examined by Nazarowec-White and Farber (21). Strain 607 was consistently the most heat resistant in our laboratory. Interestingly, on the basis of the 12 strains examined, the thermal resistance of E. sakazakii appears to fall into two distinct phenotypes (Table 2), which suggests that this microorganism might have a relatively simple set of genetic determinants for thermal resistance. Because we were interested in establishing the stringency of thermal treatments that might be needed to control E. sakazakii, strain 607 was used in subsequent experimentation. The underlying assumption is that strain 607 is also the most thermally resistant strain at other temperatures.

DISCUSSION The thermal resistance of E. sakazakii was previously studied by Nazarowec-White and Farber (21), who deterTABLE 3. D-values (s) of Enterobacter sakazakii strain 607 heated at ve temperatures in infant formula with a submerged coil apparatus

a b

Temp (8C)

na

Range

56 58 60 65 70

3 3 3 2 2

1,111.1–1,428.6 526.3–625.0 238.1–277.8 34.0–36.4 3.8–3.9

Mean 6 SDb

1,263.2 6 591.9 6 264.6 6 35.2 6 3.9 6

159.1 49.9 22.9 1.7 0.1

Number of replicate thermal inactivation trials. Standard deviations calculated for strains where n 5 2 are for the purposes of comparison only and should be interpreted in relation to the usual restriction of this statistic to n $ 3.

FIGURE 2. Temperature of rehydrated infant formula after hot water of various temperatures was added to baby bottles containing dried infant formula. The temperature at time 5 0 was that of the water just prior to it being added to the bottle.


The effect of temperature on thermal resistance, as measured by a z-value of 5.68C, was consistent with z-values of 5.6 and 6.08C for pooled food and pooled clinical isolates, respectively, reported by Nazarowec-White and Farber (21). These z-values are also consistent with the 4 to 68C z-values observed with most foodborne, non–sporeforming pathogenic bacteria. Although the thermal resistance of the more heat resistant E. sakazakii is greater than some enteric pathogens (21), it has been reported to be less resistant than Listeria monocytogenes (23). Standard pasteurization practices have been reported to be effective for the destruction of E. sakazakii (23). As previously mentioned, various investigators have identi ed the reheating of formula after rehydration as a means for reducing the risk associated with low levels of E. sakazakii (12, 13, 17, 22). Kindle et al. (13) reported that heating 150-ml portions of various infant formula for 85 to 100 s achieved a mean temperature of 82 to 938C and lead to more than 4-D inactivation of E. sakazakii. However, concerns related to the potential for scalding when baby bottles are removed from the microwave has generally prevented the use of the approach. The results of the current study indicate that an alternative approach is to rehydrate the dried infant formula with the use of water with a temperature of 708C or more. The 4-D or more inactivation observed in the current study would be expected to reduce the risk of a viable cell of E. sakazakii surviving by at least four orders of magnitude. Considering that the levels of E. sakazakii observed in dried infant formula have generally been 1 CFU/100 g of dry formula or less, a 4-D treatment would virtually assure that a serving would not contain this enteric bacterium. Although con rmation will be required, the short duration and relatively low temperature of this approach would be expected to have a minimal effect on the nutrient pro le of dried infant formulas. Future research that veri es that this degree of inactivation is achieved with a variety of E. sakazakii strains in a range of dried infant formula formulations would be bene cial.

THERMAL INACTIVATION OF E. SAKAZAKII

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ACKNOWLEDGMENT

21.

We thank Mary Porteous for her technical assistance in the conduct of this study.

22.

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