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Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature
ARTICLE IN PRESS FOOD MICROBIOLOGY Food Microbiology 24 (2007) 767–777 www.elsevier.com/locate/fm
Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature Li-Chun Lin, Larry R. Beuchat Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA Received 12 December 2006; received in revised form 7 February 2007; accepted 8 February 2007 Available online 25 February 2007
Abstract 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 log CFU/g of infant rice cereal (aw 0.30, 0.45–0.46, and 0.68–0.69). Cereal was stored at 4, 21, and 30 1C and populations were monitored for up to 12 months. Survival of the pathogen in infant rice, barley, oatmeal, and mixed grain cereals (aw 0.63–0.66, 0.76, or 0.82–0.83) initially containing a population of 4.93–5.64 log CFU/g and held at 4, 21, and 30 1C up to 24 weeks was determined. Populations decreased significantly (pp0.05) in all cereals stored at 21 and 30 1C regardless of aw. Increases in aw or storage temperature accelerated the rate of death of E. sakazakii in dry infant cereals. However, at an initial population of 0.31 log CFU/g, E. sakazakii survived in rice cereal (aw 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 aw 0.63–0.83) stored for up to 24 weeks at 4, 21, or 30 1C. This study demonstrated that E. sakazakii can survive for up to 12 months in infant cereals having a wide range of aw when storage is at temperatures simulating those to which they may be exposed during distribution, at retail, and in the home. r 2007 Elsevier Ltd. All rights reserved. Keywords: Enterobacter sakazakii; Infant cereal; Water activity
1. Introduction Enterobacter sakazakii is a gram-negative, motile, nonspore-forming bacterium belonging to the family Enterobacteriaceae (Farmer et al., 1980). It was originally classified as yellow-pigmented Enterobacter cloacae but is now designated as a separate species on the basis of pigment production (Steigerwalt et al., 1976), DNA–DNA hybridization (Farmer et al., 1980), and other characteristics (Muytjens et al., 1984; Postupa and Aldova, 1984). The first documented case of neonatal meningitis implicating E. sakazakii as the causative agent was in 1958 (Urmenyi and Franklin, 1961). At least 76 cases of E. sakazakii infections and 19 deaths, most involving preterm neonates, infants, and children up to 4 years of age, have since been documented (Iversen and Forsythe, Corresponding author. Tel.: +1 770 412 4740; fax: +1 770 229 3216.
E-mail address: [email protected] (L.R. Beuchat). 0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2007.02.001
2003; Lai, 2001). The primary manifestations of E. sakazakii infections are severe bacteremia (Block et al., 2002; Monroe and Tift, 1979; Noriega et al., 1990), meningitis (Burdette and Santos, 2000; Gallagher and Ball, 1991; Kleiman et al., 1981; Kline, 1988; Willis and Robinson, 1988), and necrotizing enterocolitis (Muytjens et al., 1983; van Acker et al., 2001), with case fatality rates of 40–80% (Bowen and Braden, 2006; Lai, 2001; US Food and Drug Administration, Center for Food Safety and Applied Nutrition, 2002). Biological properties and significance of the bacterium in powdered infant milk formula are described in recent reviews (Bowen and Braden, 2006; Drudy et al., 2006; Gurtler et al., 2005). Outbreaks of infections have implicated powdered milk substitute infant formulas as vehicles of E. sakazakii (Biering et al., 1989; FAO/WHO, 2006; Himelright et al., 2002; Simmons et al., 1989). A survey revealed that 20 of 141 (14.2%) commercially manufactured powdered infant formulas originating from 13 countries contained
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E. sakazakii at populations of 0.36–66 CFU/100 g (Muytjens et al., 1988). A survey of powdered infant formulas available in the Canadian market revealed that contents of 8 of 120 (6.7%) cans were positive for E. sakazakii (Nazarowec-White and Farber, 1997b). Studies have shown that E. sakazakii is more thermotolerant than some Enterobacteriaceae in milk products, e.g., in infant milk formula (Edelson-Mammel and Buchanan, 2004; Iversen et al., 2004; Jung and Park, 2006; Nazarowec-White and Farber, 1997a); however, standard pasteurization practices are thought to be effective for inactivation of the bacterium (Nazarowec-White et al., 1999). Post-pasteurization contamination of powdered infant formulas before packaging may occur in some commercial operations, as evidenced by its isolation from previously unopened cans of formula (Destro, 2006; Himelright et al., 2002; Weir, 2002). E. sakazakii has been isolated from various dry foods, dry food processing plants, and the environment (Iversen and Forsythe, 2003, 2004). The pathogen has been detected in rice seed (Cottyn et al., 2001), rice starch and flour (J.L. Kornacki, personal communication as cited by Richards et al., 2005), brown rice (Jung and Park, 2006), and environmental samples from 8 of 9 food factories, including a cereal factory (Kandhai et al., 2004). Restaino et al. (2006) isolated E. sakazakii from 6 of 18 (33.3%) dry infant cereals as well as from other dry foods and food ingredients. E. sakazakii has been shown to have a high tolerance to desiccation (Breeuwer et al., 2003; EdelsonMammel et al., 2005), but factors affecting its survival in infant cereals have not been reported. Cereals are the most common food for weaning infants at the age of 4–6 months (Awolumate, 1983; Ferna´ndezArtigas et al., 2001). Studies have shown that Escherichia coli O157:H7 (Deng et al., 1998) and toxigenic Bacillus cereus (Jaquette and Beuchat, 1998) survive in dry infant rice cereal stored at 5 1C for at least 24 and 48 weeks, respectively. Characterization of the survival and growth of E. sakazakii in foods has focused on powdered and reconstituted infant formulas (Edelson-Mammel and Buchanan, 2004; Edelson-Mammel et al., 2005; Iversen et al., 2004; Kandhai et al., 2006; Lee et al., 2006; Nazarowec-White and Farber, 1997a, b). Survival characteristics of E. sakazakii in infant cereals as affected by the type of grain component, aw, and storage temperatures under which they are held during distribution or in hospital, day-care center, and home settings have not been reported. The objective of this study was to determine the survival characteristics of E. sakazakii in infant cereals as affected by composition, aw, and storage temperature. 2. Materials and methods 2.1. Strains used Ten strains of E. sakazakii were used. Five strains (2855, 3231, 3234, 3290, and 3295) were isolated from clinical
specimens obtained from infected patients, four strains (2871, 3270, 3437, and 3439) were isolated from foods, and one strain (3396) was isolated from an environmental sample. All strains were obtained from Dr. Jeffrey Farber, Health Canada, Ottawa, Ont., Canada. 2.2. Cereal used Three different lots of the same brand of commercially manufactured dry infant rice, barley, mixed grain, and oatmeal cereals were purchased from local retailers in Griffin, GA. The three lots of each type of cereal were used in three replicate trials. Infant rice cereal contained rice flour, barley cereal contained barley flour, mixed grain cereal contained, wheat, rice, and oat flour, and oat cereal contained oat flour. In addition, all cereals contained added soy oil–lecithin, tri- and dicalcium phosphate, tocopherols (vitamin E), electrolytic iron, zinc sulfate, niacinamide, riboflavin (vitamin B2), pyridoxide hydrochloride (vitamin B6), thiamin (vitamin B1), folic acid, and cyanocobalamin (vitamin B12). 2.3. Preparation of inoculum for cereals Cells of the 10 strains of E. sakazakii in aqueous 15% glycerol solution were stored at 31 1C. Each suspension was thawed and streaked with a wire loop on violet red bile glucose (VRBG) agar (pH 7.4; Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37 1C for 24 h. Cells from single colonies of each strain were streaked on tryptic soy agar (TSA, pH 7.3; Difco; Becton, Dickinson and Co., Sparks, MD), incubated at 37 1C for 24 h, and stored at 4 1C until used to prepare inocula. To prepare inocula for cereals, each strain of E. sakazakii was grown in 9 ml of brain heart infusion (BHI) broth (pH 7.4; Becton, Dickinson and Co.) at 37 1C, with two successive loop transfers (ca. 10 ml) at 24-h intervals. A final transfer of 0.1 ml was made from the second 24-h culture to 400 ml of BHI broth and held in an incubator shaker (New Brunswick Scientific, Edison, NJ) set at 62 rpm for 24 h at 37 1C. The 10 400-ml BHI broth cultures were centrifuged for 15 min at 2700 g in a Marathon 12KBR benchtop refrigerated centrifuge (Fisher Scientific, Pittsburgh, PA) and supernates were decanted. Cells were resuspended in 100 ml of sterile deionized water, centrifuged a second time, and resuspended in 10 ml of sterile deionized water. To determine populations, suspensions were serially diluted in sterile 0.1% peptone water, surface plated on TSA supplemented with 0.1% sodium pyruvate (TSAP) (Sigma-Aldrich, Inc., St. Louis, MO) and VRBG supplemented with 0.1% sodium pyruvate (VRBGP), and incubated at 37 1C for 24 h. Sodium pyruvate was added to enhance the recovery of injured cells (Baird-Parker and Davenport, 1965). Colonies were counted and populations of each of the 10 strains in the suspensions were calculated.
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2.4. Preparation of rice cereal inoculum Ten 100-g portions of a commercially manufactured infant rice cereal (aw 0.35–0.41) were distributed in layers (ca. 2.5 cm deep) in 10 sterile stainless steel bowls (40 cm diameter). Each of the 10 suspensions of E. sakazakii cells was separately spray-inoculated onto the surface of separate 100-g portions. Cell suspension was applied using a chromatography reagent sprayer (Model 422530-0050, Kontes Glass Company, Vineland, NJ). The sprayer was held 45 cm above the surface of the infant rice cereal and suspension was sprayed at ca. 2 psi using nitrogen gas as a carrier. Infant rice cereal was thoroughly stirred using a sterile spoon at 15-s intervals during application of spray inoculum over a 150-s period during which a total of ca. 1.0 ml of suspension per 100 g of infant rice cereal was applied. The inoculated infant rice cereal was deposited in a Ziplocs (Johnson and Son, Inc., Racine, WI) plastic bags, sealed, and vigorously mixed for 2 min. Each 100-g portion of cereal inoculated with a single strain of E. sakazakii was stored for 7 days at 21 1C in sterile polystyrene trays (22 cm long 15 cm wide 5 cm high; Newell Rubbermaid, Inc., Atlanta, GA) placed on wire platforms above the surface of ca. 6 l of saturated potassium acetate in a hermetically sealed plastic tub (81 cm long 33 cm wide 30 cm high). The depth of infant rice cereal inoculum in each tray was ca. 1.5 cm. Two brushless cooling fans (6 cm long 6 cm wide 2.5 cm high; RadioShack, Fort Worth, TX) were attached on opposite internal sides of the tub to facilitate air circulation. Upon equilibration within 14 days, the atmospheric relative humidity inside the tub was ca. 23%. 2.4.1. Determination of populations of E. sakazakii in rice cereal inoculum Within 1 h after inoculation of infant rice cereal and after storage at 21 1C for 7 days, triplicate 10-g samples of each of the 10 portions of inoculated infant rice cereal were analyzed to determine populations of E. sakazakii. Each sample was reconstituted in 90 ml of sterile deionized water at 45 1C, serially diluted in sterile 0.1% peptone water, surface plated (0.1 ml in duplicate) on TSAP and VRBGP agar, and incubated at 25 1C for 48 h and 37 1C for 24 h, respectively, before counting colonies and calculating log CFU/g. Portions of each of the 10 infant rice cereal inocula stored at 21 1C for 7 days were combined and thoroughly mixed for 2 min to give a 10-strain mixture containing approximately equal populations of each strain of E. sakazakii. To determine the population of E. sakazakii in the rice cereal inoculum (aw 0.23) containing a 10-strain mixture, samples (10 g) were immediately combined with 90 ml of sterile deionized water at 45 1C, serially diluted, and surface plated on VRBGP and TSAP agar. Plates were inoculated at 37 1C for 24 h and 25 1C for 48 h, respectively, before colonies were counted. The inoculated rice cereal, containing 7.3–7.5 log CFU/g, served as the high inoculum for rice
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cereal to be stored for up to 12 months. To prepare an inoculum containing a low population of E. sakazakii, the high-population inoculum was serially diluted in infant rice cereal (aw 0.23) to give a population of ca. 2.3 log CFU/g. High-population inoculum or low-population inoculum (2 g) were added to 200 g of uninoculated low-aw infant rice cereal to give desired populations and stored for up to 9 months before analyzing the number of E. sakazakii that survived. Rice, barley, mixed grain, and oatmeal cereals (200 g) at high aw were combined with 2 g of highpopulation inoculum and stored for up to 24 weeks before analysis. 2.5. Preparation of cereals for inoculation and storage 2.5.1. Rice cereal at low aw In the first experiment, survival of E. sakazakii in infant rice cereal at target aw values of 0.23, 0.43, and 0.68 was studied. Three different lots of the same brand of commercially manufactured infant rice cereal were used in three replicate trials. Infant rice cereal from each lot was divided into six of 200-g quantities and placed in polystyrene trays (22 cm long 15 cm wide 5 cm high) at a depth of ca. 2.5 cm. Two of the six trays of each lot were placed on wire platforms inside each of three sterile plastic tubs containing ca. 6 l of saturated solutions of potassium acetate, potassium carbonate, or lithium acetate, which created equilibrium atmospheric relative humidities of ca. 23%, 43%, and 68%, respectively, for the purpose of adjusting the infant rice cereal to target aw values of ca. 0.23, 0.43, and 0.68. Two brushless cooling fans were attached to opposite internal sides of each tub to facilitate air movement. Cereals were stored, with intermittent mixing, in tubs for 14 days at 21 1C, then inoculated with E. sakazakii at high or low population, as described below. 2.5.2. Cereals at high aw A second series of experiments were done using three different lots of the same brand of infant rice, barley, mixed grain, and oatmeal cereals. This study was focused on determining the ability of E. sakazakii to survive at target aw values of ca. 0.68, 0.79, and 0.88. Each lot of each type of cereal was divided into three 200-g quantities and placed in polystyrene trays at a depth of ca. 2.5 cm. Three of the nine trays of each lot and each type of infant cereal were placed on elevated wire platforms inside each of three sterile plastic tubs containing ca. 6 l of saturated solutions of lithium acetate, ammonium sulfate, or sodium benzoate, which created equilibrium atmospheric relative humidities of ca. 68%, 79%, and 88%, respectively, for the purpose of adjusting cereals to aw values of ca. 0.68, 0.79, and 0.88. Two brushless cooling fans were installed inside each tub to facilitate air movement and equilibrium of aw with relative humidity as described above. After storage, with intermittent mixing, for 14 days at 21 1C, cereals at each aw were separately inoculated with high-population inoculum as described below.
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2.6. Inoculation of cereals for storage Infant rice cereal (200 g) initially at aw 0.30, 0.45–0.46, and 0.68–0.69 was inoculated with 2 g of the 10-strain mixture of high- or low-population inoculum, placed in a Ziploc plastic bag (17.7 cm 20.3 cm), sealed, and vigorously mixed for 2 min to give populations of 5.0370.09 log and 0.31 log CFU/g, respectively. Ten grams of inoculated cereal at each aw and containing high or low numbers of E. sakazakii were deposited in 30-ml glass vials and sealed with polyseal liner phenolic caps (Fisher Scientific). Vials were double bagged in Ziploc plastic bags (17.7 cm 20.3 cm), hermetically sealed in plastic tubs, and stored at 4, 21, or 30 1C for up to 12 months before analyzing cereal for the presence (by enrichment) and populations of E. sakazakii. In the second series of experiments, cereals (200 g) adjusted to aw values of 0.63–0.66, 0.76, and 0.82–0.83 were inoculated with 2 g of the 10-strain mixture of highpopulation inoculum, sealed in a Ziploc plastic bag, and vigorously mixed for 2 min to give a population of 5.3970.36 log CFU/g. Sixty grams of each type of inoculated cereal at each aw were deposited in 250-ml glass bottles and hermetically sealed. Cereals were stored at 4, 21, or 30 1C for up to 24 weeks before analyzing to determine the presence (by enrichment) and populations of E. sakazakii. 2.7. Storage of inocula Rice cereal inocula (initial aw, 0.23) containing high (7.31–7.50 log CFU/g) or low (2.31 log CFU/g) numbers of E. sakazakii were each divided into three equal portions, placed in plastic bags, sealed in glass jars, and stored at 4, 21, and 30 1C. Inocula were analyzed for initial populations of E. sakazakii on day 0 (within 1 h after inoculation) and after storage for up to 24 weeks (high-aw cereals) or 9 months (low-aw rice cereal). 2.8. Microbiological analysis of inoculated cereal Each inoculated infant cereal (10 g) initially containing high or low numbers of E. sakazakii was combined with 90 ml of sterile deionized water at 45 1C and vigorously shaken for 1 min. Undiluted mixtures of cereal and water (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions serially diluted in 0.1% sterile peptone (0.1 ml, in duplicate) containing high inoculum (7.3–7.5 log CFU/g) were surface plated on TSAP and VRBGP agar. The VRBGP plates were incubated at 37 1C for 24 h and TSAP plates were incubated at 25 1C for 48 h before positive– presumptive E. sakazakii colonies were counted. Mixtures of cereal initially containing high- or low-population inoculum (10 g) and water (90 ml) were incubated at 37 1C for 24 h. Colonies formed on TSAP were examined for yellow pigmentation. If presumptive E. sakazakii colonies were not detected on TSAP or VRBGP agar,
10 ml of pre-enrichment mixture was combined with 90 ml of Enterobacteriaceae enrichment broth (Becton, Dickinson and Co.) supplemented with 0.1% sodium pyruvate (EEP) and incubated at 37 1C for 24 h. These enrichments were streaked onto TSAP and VRBGP agar and incubated at 25 1C for 48 h and 37 1C for 24 h, respectively. Presumptive colonies of E. sakazakii formed on TSAP agar were subjected to confirmation assays using the API 20E Identification System (bioMe´rieux, Hazelwood, MO) and the Microbact 12A/B Identification kit (Oxoid) according to manufacturers’ instructions. 2.9. Measurement of aw Measurement of aw of infant cereals was done immediately after opening boxes in which they were commercially packaged, after equilibration in tubs with various atmospheric relative humidities for 14 days at 21 1C, and after inoculation and storage for up to 12 months using an aw meter (Aqua Lab Model CX2, Decagon Devices, Inc., Pullman, WA). 2.10. Statistical analysis All experiments were performed in triplicate. Data were analyzed using the general linear model on SAS software (Statistical Analysis Systems Institute, Cary, NC). The least significant difference test was used to determine significant differences (pp0.05) in populations of E. sakazakii detected in infant cereals as affected by aw, composition, storage temperature, and storage time. 3. Results and discussion 3.1. Changes in the aw of inoculated infant rice cereal (initial aw 0.30–0.69) The aw of infant cereals purchased at a local supermarket ranged from 0.35 to 0.41. The purpose of exposing infant rice cereal to atmospheric relative humidities of 23%, 43%, and 68% was to mimic conditions to which open containers of cereal might be exposed in hospital, day-care center, and home settings. Changes in the aw of infant rice cereal during subsequent storage for 12 months at 4, 21, and 30 1C are shown in Fig. 1. During storage, the cereal initially at aw 0.30 and 0.46 increased or decreased slightly upon equilibration with fluctuating relative humidities in the storage chambers. 3.2. Survival in infant rice cereal (initial aw 0.30–0.69) containing low inoculum E. sakazakii survived in infant rice cereal (aw 0.30–0.69) inoculated at a population (0.31 log CFU/g) and stored for up to 12 months at 4, 21, and 30 1C (Table 1). With the exception of cereal at aw 0.46 stored at 30 1C, the pathogen was detected by enrichment of cereal stored for 12 months,
ARTICLE IN PRESS L.-C. Lin, L.R. Beuchat / Food Microbiology 24 (2007) 767–777
1.0
771
High inoculum
Low inoculum
Water activity
0.8 0.6 0.4 0.2 0.0 0 1
3
6
9
12 0 1 3 Storage time (months)
6
9
12
Fig. 1. Water activity of infant rice cereal (initially at aw 0.30–0.69) inoculated with E. sakazakii at 0.31 log CFU/g (low inoculum) and 5.03 log CFU/g (high inoculum) and stored at 4 (J), 21 (&), and 30 1C (W) for up to 12 months.
Table 1 Survival of E. sakazakii in infant cereal (initial population was 0.31 log CFU/g) as affected by low aw (0.30–0.69) and storage temperaturea Initial aw
0.30
Storage temp. (1C)
4 21 30
0.46
4 21 30
0.69
4 21 30
Detection medium
Storage time (months) 0
1
3
6
9
12
TSAP VRBGP TSAP VRBGP TSAP VRBGP
2 2 2 2 2 2
1 1 1 1 2 2
2 2 2 2 1 1
2 2 2 2 3 3
2 2 0 0 2 2
1 1 1 1 2 2
TSAP VRBGP TSAP VRBGP TSAP VRBGP
2 2 2 2 2 2
0 0 0 0 2 2
1 1 2 2 2 2
2 2 2 2 2 2
0 0 0 0 0 0
2 2 2 2 0 0
TSAP VRBGP TSAP VRBGP TSAP VRBGP
1 1 1 1 1 1
2 2 2 2 1 1
2 2 1 1 0 0
2 2 2 2 0 0
2 2 1 1 0 0
2 2 2 2 1 1
a Numbers indicate positive samples out of three tested (three replicate experiments). Detection limit was 1 CFU/10 g.
regardless of the aw or temperature. Breeuwer et al. (2003) reported that stationary phase E. sakazakii cells were more resistant than E. coli, Salmonella, and other Enterobacteriaceae to desiccation stress. Retention of viability was attributed to accumulation of trehalose in cells. Our observations on the exceptional resistance of E. sakazakii to desiccation are in agreement with these findings. 3.3. Survival in infant rice cereal (initial aw 0.30–0.68) containing high inoculum Fig. 2 shows populations of E. sakazakii recovered on TSAP from infant rice cereal (initial aw 0.30–0.68) inoculated with the pathogen at a population of 5.03 log
CFU/g and stored at 4, 21, or 30 1C. Significantly higher populations of E. sakazakii were recovered from most samples on TSAP than on VRBGP agar (not shown), which is in agreement with observations by Gurtler and Beuchat (2005) showing the inability of VRBGP agar to support resuscitation and colony formation by stressed cells of E. sakazakii. Populations decreased significantly (p p 0.05) as storage time progressed, regardless of the aw or temperature. Increased aw and storage temperature caused increased rates of death. At most storage time/ temperature combinations, the population of E. sakazakii in cereal initially at aw 0.68 decreased to a lower level than in cereal initially at aw 0.45, which in turn was lower than in cereal initially at aw 0.30. Populations of E. sakazakii in infant rice cereal (initial aw 0.30–0.68) stored at 4, 21, and 30 1C for 12, 6, and 3 months, respectively, as determined on TSAP, decreased by 0.52–1.51, 1.86–4.04, and 3.25–4.77 log CFU/g. With the exception of rice cereal (aw 0.68) stored at 30 1C for 12 months, E. sakazakii was recovered by direct plating or enrichment of at least one of three replicate samples representing all experimental parameter combination. Edelson-Mammel et al. (2005) reported that an initial E. sakazakii population of ca. 6 log CFU/g of powdered infant formula (aw 0.14–0.27) decreased by 2.4 log CFU/g during storage at 20–22 1C for 150 days (ca. 5 months), followed by an additional 1-log decrease during the next 534 days. In our study, populations in infant rice cereal (initial aw 0.30) stored at 21 1C for 6 months decreased by 1.86 log CFU/g, indicating a similar loss of viability over time in dried rice cereal and infant formula. Results show that E. sakazakii can survive in dry infant rice cereal (initial aw 0.30–0.69) stored at 4–30 1C for at least 12 months. Christian and Stewart (1973) reported that Staphylococcus aureus and Salmonella Newport survived in dry foods (aw 0–0.53) at 25 1C for 27 weeks. In the same study, both microorganisms were observed to survive better at lower aw (0–0.22). In contrast, Deng et al. (1998) reported that survival of E. coli O157:H7 in infant rice cereal (aw 0.35–0.73) was enhanced at higher aw. Populations in rice cereal (aw 0.35) initially containing E. coli O157:H7 at a population of 6.31 log CFU/g
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772
6
4°C
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6
21°C
30°C
5 4 3 2 1 0 0
1
3
6
9
12 0 1
3
6
9
12
Storage time (months) Fig. 2. Population (log CFU/g) of E. sakazakii recovered from infant rice cereal as affected by initial aw 0.30 (J), 0.45 (&), and 0.68 (W) and storage at 4, 21, or 30 1C for up to 12 months. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
decreased to 3.19 and 1.22 log CFU/g within 24 weeks at 5 1C and 11 weeks at 25 1C, respectively. The rate of death of E. coli O157:H7 was more rapid than that of E. sakazakii in infant rice cereal held at similar aw and temperature conditions. Jaquette and Beuchat (1998) reported that the rate of death of vegetative cells of B. cereus in infant rice cereal (aw 0.27–0.78) stored for 36 weeks at 5, 25, 35, or 45 1C was not influenced by aw. Death was more rapid at the higher storage temperatures, however, regardless of the aw, which is in agreement with observations on E. sakazakii and E. coli O157:H7. 3.4. Survival in infant rice, barley, mixed grain, and oatmeal cereals (initial aw 0.63–0.83) containing high inoculum Changes in aw in infant rice, barley, mixed grain, and oatmeal cereals (initially at aw 0.63–0.66, 0.76, and 0.82–0.83) inoculated with E. sakazakii (4.93–5.64 log CFU/g) and stored at 4, 21, or 30 1C for 24 weeks are shown in Fig. 3. The aw of infant cereals stored at 30 1C was measured only up to 14 weeks, beyond which samples were not available for analysis. The aw of infant cereals changed more when cereals were stored at 30 1C than when stored at 4 or 21 1C, a phenomenon attributed to fluctuation in relative humidity within the storage chamber. Survival of E. sakazakii in infant rice, barley, mixed grain, and oatmeal cereals (initial aw 0.63–0.83) stored at 4, 21, and 30 1C are shown in Figs. 4–6, respectively. Initial populations were 4.93–5.64 log CFU/g. As in experiments with rice cereal at low aw (0.30–0.68), significantly higher numbers of E. sakazakii were recovered when cereals at high aw were plated on TSAP than on VRBGP agar (not shown). Populations decreased at the slowest rate in cereals
stored at 4 1C, e.g., reductions of 0.49–0.84, 0.38–0.94, and 1.13–1.53 log CFU/g occurred during storage of cereals at aw 0.63–0.66, 0.76, and 0.82–0.83, respectively, for up to 24 weeks (Fig. 4); this compares to reductions of at least 3.93 log CFU/g within 8 weeks at 30 1C (Fig. 6). Populations of E. sakazakii in infant rice, barley, mixed grain, and oatmeal cereals decreased significantly (pp0.05) as storage time at 21 1C (Fig. 5) and 30 1C (Fig. 6) progressed. Storage of cereals initially at aw 0.82–0.83 for 24 weeks at 4 1C resulted in decreases in populations of E. sakazakii that were significantly (pp0.05) lower in cereals than in cereals initially at aw 0.63–0.76 (Fig. 4). Populations of E. sakazakii in infant rice, barley, and oatmeal cereals stored at 21 1C for 14, 14, and 6 weeks, respectively, were significantly reduced when the initial aw was 0.82–0.83 compared to populations at aw 0.76, and at aw 0.76 compared to populations at aw 0.63–0.66 (Fig. 5). However, survival of E. sakazakii in infant cereals stored at 30 1C for 14 weeks was not affected by aw (Fig. 6). At a given initial aw (0.63–0.83), populations of E. sakazakii significantly decreased in infant rice, barley, mixed grain, and oatmeal cereals as storage temperature increased. With few exceptions, the composition of the four types of infant cereals did not markedly affect the survival of E. sakazakii, regardless of aw or storage temperature. Shown in Table 2 are the longest storage times at which E. sakazakii was detected by enrichment (X1 CFU/10 g) in cereals (aw 0.63–0.83) stored at 21 and 30 1C. Cereals initially containing 4.93–5.64 log CFU/g were stored at 21 1C for 2, 4, 6, 8, 14, and 24 weeks and at 30 1C for 2, 4, 6, 8, 10 and 14 weeks. With the exceptions of barley and oatmeal cereals at aw 0.82–0.83, the pathogen was detected in all cereals stored at 21 1C for 24 weeks. E. sakazakii was
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0.9
773
Mixed grain
Rice
0.8
0.7
Water activity
0.6
0.5 0.9
Barley
Oatmeal
0.8
0.7
0.6
0.5 0
4
8
12 14
24 0 4 Storage time (weeks)
8
12 14
24
Fig. 3. Water activity of infant rice, barley, mixed grain, and oatmeal cereals inoculated with E. sakazakii at 5.39 log CFU/g and stored at 4 (J), 21 (&), and 30 1C (W) for up to 24 weeks.
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0
2
4
6
8
14
24 0 2 4 6 Storage time (weeks)
8
14
24
Fig. 4. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 4 1C for up to 24 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g).
detected in all cereals (aw 0.63–0.66) stored at 30 1C for 10 weeks but not 14 weeks. Maximum storage times at which the pathogen was detected in cereals were decreased with increased aw and temperature. Higher numbers of E.
sakazakii survived in cereals (aw 0.63–0.83) stored at 4 1C (Fig. 4) than at 21 or 31 1C. With few exceptions, the composition of cereals did not significantly affect the survival of E. sakazakii, regardless of aw or storage temperature.
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774
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0 2
4
6
8
24 0
14
2
4
6 8
14
24
Storage time (weeks) Fig. 5. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 21 1C for up to 24 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0
2
4
6
8
10
14 0
2
4
6
8
10
14
Storage time (weeks) Fig. 6. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 30 1C for up to 14 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
3.5. Survival in infant rice cereal inoculum (initial aw 0.23) Survival of E. sakazakii in the rice cereal inoculum stored for up to 9 months at 4, 21, and 30 1C was determined. Infant rice cereal inoculum (initial aw 0.23) from which a portion had been used to inoculate infant rice cereal (initial aw 0.30–0.69) (Fig. 7) or infant rice, barley, mixed grain, and oatmeal cereals (initial aw
0.63–0.83) (Fig. 8) was monitored for populations of E. sakazakii. The pathogen was detected by direct plating or enrichment of high-population (7.31 log CFU/g) rice inoculum stored for 9 months at 4, 21, and 30 1C but not in low-population (2.31 log CFU/g) inoculum stored at 21 or 30 1C. The trend showing decreases in populations as affected by storage temperature is similar to that observed for inoculated infant rice cereal (initial aw 0.30).
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8
Table 2 Survival of E. sakazakii (initial populations were 4.93–5.64 log CFU/g) in infant rice, barley, mixed grain, and oatmeal cereals as affected by high aw (0.63–0.83) at 21 and 30 1Ca Storage temp. (1C)
Longest storage time (weeks) at which E. sakazakii was detected Rice
Barley
Mixed grain
Oatmeal
0.63–0.66
21 30
24 10
24 10
24 10
24 10
0.76
21 30
24 8
24 10
24 10
24 8
0.82–0.83
21 30
24 10
14 8
24 6
14 8
4 3 2
0
7 E.sakazakii (log CFU/g)
5
0
8
6 5 4 3 2 1 0 3 6 Storage time (months)
6
1
a Cereals were stored at 21 and 30 1C for up to 24 and 14 weeks, respectively. Samples (10 g) were pre-enriched in 90 ml of deionized water at 37 1C for 24 h, enriched in 90-ml EEP broth at 37 1C for 24 h, and streaked on TSAP, followed by incubation at 25 1C for 48 h. Numbers indicate the longest storage time (weeks) at which E. sakazakii was detected in at least one of three samples tested (three replicate experiments). Detection limit was 1 CFU/10 g.
0 0.5 1
7 E.sakazakii (log CFU/g)
Initial aw
775
9
Fig. 7. Populations (log CFU/g) of E. sakazakii recovered from infant rice cereal (initial aw 0.23) used as high inoculum (solid symbols) and low inoculum (open symbols) to inoculate infant rice cereal (initial aw 0.30–0.69) as affected by storage temperature at 4 (, J), 21 (’, &), and 30 1C (m, W) up to 9 months. Detection limit was 1 log CFU/g (10 CFU/g).
2
4
6
8 14 Storage time (weeks)
24
Fig. 8. Populations (log CFU/g) of E. sakazakii recovered from infant rice cereal (initial aw 0.23) used to inoculate infant rice, barley, mixed grain, and oatmeal cereals (aw 0.63–0.83) as affected by storage temperature at 4 (J), 21 (&), and 30 1C (W) up to 24 weeks. Detection limit was 1 log CFU/g (10 CFU/g).
the probability of its survival in cereals at the time of reconstitution and feeding to infants. Kandhai et al. (2006) reported that powdered infant formula inoculated with lag, exponential, and stationary phase of E. sakazakii and stored for up to 4 weeks prior to reconstitution in sterile water did not show a longer lag time or different specific growth rate compared to cells in other phases of growth. Richards et al. (2005) reported that E. sakazakii can grow in infant rice cereal reconstituted with water, milk, and liquid infant formula at 12, 21, and 30 1C. Further investigations are needed to determine the survival and growth characteristics of E. sakazakii in reconstituted infant cereal as affected by the composition of the liquid used for reconstitution and the storage temperature after reconstitution. Acknowledgment This work was supported, in part, by a grant from the International Life Sciences Institute—North America (ILSI N.A.). The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI N.A.
4. Conclusions Results show that E. sakazakii, initially at populations as low as 0.31 log CFU/g (2 CFU/g), can survive at 4, 21, or 30 1C for up to 12 months in infant cereals in an aw range of 0.30–0.83. Results show that very small numbers of E. sakazakii, if present in infant cereals, can survive for up to 12 months in cereals exposed to conditions under which they may be held during distribution and storage. Tolerance of the pathogen to these conditions enhances
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Survival of Enterobacter sakazakii in infant cereal as affected by composition, water activity, and temperature Li-Chun Lin, Larry R. Beuchat Center for Food Safety and Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223-1797, USA Received 12 December 2006; received in revised form 7 February 2007; accepted 8 February 2007 Available online 25 February 2007
Abstract 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 log CFU/g of infant rice cereal (aw 0.30, 0.45–0.46, and 0.68–0.69). Cereal was stored at 4, 21, and 30 1C and populations were monitored for up to 12 months. Survival of the pathogen in infant rice, barley, oatmeal, and mixed grain cereals (aw 0.63–0.66, 0.76, or 0.82–0.83) initially containing a population of 4.93–5.64 log CFU/g and held at 4, 21, and 30 1C up to 24 weeks was determined. Populations decreased significantly (pp0.05) in all cereals stored at 21 and 30 1C regardless of aw. Increases in aw or storage temperature accelerated the rate of death of E. sakazakii in dry infant cereals. However, at an initial population of 0.31 log CFU/g, E. sakazakii survived in rice cereal (aw 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 aw 0.63–0.83) stored for up to 24 weeks at 4, 21, or 30 1C. This study demonstrated that E. sakazakii can survive for up to 12 months in infant cereals having a wide range of aw when storage is at temperatures simulating those to which they may be exposed during distribution, at retail, and in the home. r 2007 Elsevier Ltd. All rights reserved. Keywords: Enterobacter sakazakii; Infant cereal; Water activity
1. Introduction Enterobacter sakazakii is a gram-negative, motile, nonspore-forming bacterium belonging to the family Enterobacteriaceae (Farmer et al., 1980). It was originally classified as yellow-pigmented Enterobacter cloacae but is now designated as a separate species on the basis of pigment production (Steigerwalt et al., 1976), DNA–DNA hybridization (Farmer et al., 1980), and other characteristics (Muytjens et al., 1984; Postupa and Aldova, 1984). The first documented case of neonatal meningitis implicating E. sakazakii as the causative agent was in 1958 (Urmenyi and Franklin, 1961). At least 76 cases of E. sakazakii infections and 19 deaths, most involving preterm neonates, infants, and children up to 4 years of age, have since been documented (Iversen and Forsythe, Corresponding author. Tel.: +1 770 412 4740; fax: +1 770 229 3216.
E-mail address: [email protected] (L.R. Beuchat). 0740-0020/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2007.02.001
2003; Lai, 2001). The primary manifestations of E. sakazakii infections are severe bacteremia (Block et al., 2002; Monroe and Tift, 1979; Noriega et al., 1990), meningitis (Burdette and Santos, 2000; Gallagher and Ball, 1991; Kleiman et al., 1981; Kline, 1988; Willis and Robinson, 1988), and necrotizing enterocolitis (Muytjens et al., 1983; van Acker et al., 2001), with case fatality rates of 40–80% (Bowen and Braden, 2006; Lai, 2001; US Food and Drug Administration, Center for Food Safety and Applied Nutrition, 2002). Biological properties and significance of the bacterium in powdered infant milk formula are described in recent reviews (Bowen and Braden, 2006; Drudy et al., 2006; Gurtler et al., 2005). Outbreaks of infections have implicated powdered milk substitute infant formulas as vehicles of E. sakazakii (Biering et al., 1989; FAO/WHO, 2006; Himelright et al., 2002; Simmons et al., 1989). A survey revealed that 20 of 141 (14.2%) commercially manufactured powdered infant formulas originating from 13 countries contained
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E. sakazakii at populations of 0.36–66 CFU/100 g (Muytjens et al., 1988). A survey of powdered infant formulas available in the Canadian market revealed that contents of 8 of 120 (6.7%) cans were positive for E. sakazakii (Nazarowec-White and Farber, 1997b). Studies have shown that E. sakazakii is more thermotolerant than some Enterobacteriaceae in milk products, e.g., in infant milk formula (Edelson-Mammel and Buchanan, 2004; Iversen et al., 2004; Jung and Park, 2006; Nazarowec-White and Farber, 1997a); however, standard pasteurization practices are thought to be effective for inactivation of the bacterium (Nazarowec-White et al., 1999). Post-pasteurization contamination of powdered infant formulas before packaging may occur in some commercial operations, as evidenced by its isolation from previously unopened cans of formula (Destro, 2006; Himelright et al., 2002; Weir, 2002). E. sakazakii has been isolated from various dry foods, dry food processing plants, and the environment (Iversen and Forsythe, 2003, 2004). The pathogen has been detected in rice seed (Cottyn et al., 2001), rice starch and flour (J.L. Kornacki, personal communication as cited by Richards et al., 2005), brown rice (Jung and Park, 2006), and environmental samples from 8 of 9 food factories, including a cereal factory (Kandhai et al., 2004). Restaino et al. (2006) isolated E. sakazakii from 6 of 18 (33.3%) dry infant cereals as well as from other dry foods and food ingredients. E. sakazakii has been shown to have a high tolerance to desiccation (Breeuwer et al., 2003; EdelsonMammel et al., 2005), but factors affecting its survival in infant cereals have not been reported. Cereals are the most common food for weaning infants at the age of 4–6 months (Awolumate, 1983; Ferna´ndezArtigas et al., 2001). Studies have shown that Escherichia coli O157:H7 (Deng et al., 1998) and toxigenic Bacillus cereus (Jaquette and Beuchat, 1998) survive in dry infant rice cereal stored at 5 1C for at least 24 and 48 weeks, respectively. Characterization of the survival and growth of E. sakazakii in foods has focused on powdered and reconstituted infant formulas (Edelson-Mammel and Buchanan, 2004; Edelson-Mammel et al., 2005; Iversen et al., 2004; Kandhai et al., 2006; Lee et al., 2006; Nazarowec-White and Farber, 1997a, b). Survival characteristics of E. sakazakii in infant cereals as affected by the type of grain component, aw, and storage temperatures under which they are held during distribution or in hospital, day-care center, and home settings have not been reported. The objective of this study was to determine the survival characteristics of E. sakazakii in infant cereals as affected by composition, aw, and storage temperature. 2. Materials and methods 2.1. Strains used Ten strains of E. sakazakii were used. Five strains (2855, 3231, 3234, 3290, and 3295) were isolated from clinical
specimens obtained from infected patients, four strains (2871, 3270, 3437, and 3439) were isolated from foods, and one strain (3396) was isolated from an environmental sample. All strains were obtained from Dr. Jeffrey Farber, Health Canada, Ottawa, Ont., Canada. 2.2. Cereal used Three different lots of the same brand of commercially manufactured dry infant rice, barley, mixed grain, and oatmeal cereals were purchased from local retailers in Griffin, GA. The three lots of each type of cereal were used in three replicate trials. Infant rice cereal contained rice flour, barley cereal contained barley flour, mixed grain cereal contained, wheat, rice, and oat flour, and oat cereal contained oat flour. In addition, all cereals contained added soy oil–lecithin, tri- and dicalcium phosphate, tocopherols (vitamin E), electrolytic iron, zinc sulfate, niacinamide, riboflavin (vitamin B2), pyridoxide hydrochloride (vitamin B6), thiamin (vitamin B1), folic acid, and cyanocobalamin (vitamin B12). 2.3. Preparation of inoculum for cereals Cells of the 10 strains of E. sakazakii in aqueous 15% glycerol solution were stored at 31 1C. Each suspension was thawed and streaked with a wire loop on violet red bile glucose (VRBG) agar (pH 7.4; Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37 1C for 24 h. Cells from single colonies of each strain were streaked on tryptic soy agar (TSA, pH 7.3; Difco; Becton, Dickinson and Co., Sparks, MD), incubated at 37 1C for 24 h, and stored at 4 1C until used to prepare inocula. To prepare inocula for cereals, each strain of E. sakazakii was grown in 9 ml of brain heart infusion (BHI) broth (pH 7.4; Becton, Dickinson and Co.) at 37 1C, with two successive loop transfers (ca. 10 ml) at 24-h intervals. A final transfer of 0.1 ml was made from the second 24-h culture to 400 ml of BHI broth and held in an incubator shaker (New Brunswick Scientific, Edison, NJ) set at 62 rpm for 24 h at 37 1C. The 10 400-ml BHI broth cultures were centrifuged for 15 min at 2700 g in a Marathon 12KBR benchtop refrigerated centrifuge (Fisher Scientific, Pittsburgh, PA) and supernates were decanted. Cells were resuspended in 100 ml of sterile deionized water, centrifuged a second time, and resuspended in 10 ml of sterile deionized water. To determine populations, suspensions were serially diluted in sterile 0.1% peptone water, surface plated on TSA supplemented with 0.1% sodium pyruvate (TSAP) (Sigma-Aldrich, Inc., St. Louis, MO) and VRBG supplemented with 0.1% sodium pyruvate (VRBGP), and incubated at 37 1C for 24 h. Sodium pyruvate was added to enhance the recovery of injured cells (Baird-Parker and Davenport, 1965). Colonies were counted and populations of each of the 10 strains in the suspensions were calculated.
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2.4. Preparation of rice cereal inoculum Ten 100-g portions of a commercially manufactured infant rice cereal (aw 0.35–0.41) were distributed in layers (ca. 2.5 cm deep) in 10 sterile stainless steel bowls (40 cm diameter). Each of the 10 suspensions of E. sakazakii cells was separately spray-inoculated onto the surface of separate 100-g portions. Cell suspension was applied using a chromatography reagent sprayer (Model 422530-0050, Kontes Glass Company, Vineland, NJ). The sprayer was held 45 cm above the surface of the infant rice cereal and suspension was sprayed at ca. 2 psi using nitrogen gas as a carrier. Infant rice cereal was thoroughly stirred using a sterile spoon at 15-s intervals during application of spray inoculum over a 150-s period during which a total of ca. 1.0 ml of suspension per 100 g of infant rice cereal was applied. The inoculated infant rice cereal was deposited in a Ziplocs (Johnson and Son, Inc., Racine, WI) plastic bags, sealed, and vigorously mixed for 2 min. Each 100-g portion of cereal inoculated with a single strain of E. sakazakii was stored for 7 days at 21 1C in sterile polystyrene trays (22 cm long 15 cm wide 5 cm high; Newell Rubbermaid, Inc., Atlanta, GA) placed on wire platforms above the surface of ca. 6 l of saturated potassium acetate in a hermetically sealed plastic tub (81 cm long 33 cm wide 30 cm high). The depth of infant rice cereal inoculum in each tray was ca. 1.5 cm. Two brushless cooling fans (6 cm long 6 cm wide 2.5 cm high; RadioShack, Fort Worth, TX) were attached on opposite internal sides of the tub to facilitate air circulation. Upon equilibration within 14 days, the atmospheric relative humidity inside the tub was ca. 23%. 2.4.1. Determination of populations of E. sakazakii in rice cereal inoculum Within 1 h after inoculation of infant rice cereal and after storage at 21 1C for 7 days, triplicate 10-g samples of each of the 10 portions of inoculated infant rice cereal were analyzed to determine populations of E. sakazakii. Each sample was reconstituted in 90 ml of sterile deionized water at 45 1C, serially diluted in sterile 0.1% peptone water, surface plated (0.1 ml in duplicate) on TSAP and VRBGP agar, and incubated at 25 1C for 48 h and 37 1C for 24 h, respectively, before counting colonies and calculating log CFU/g. Portions of each of the 10 infant rice cereal inocula stored at 21 1C for 7 days were combined and thoroughly mixed for 2 min to give a 10-strain mixture containing approximately equal populations of each strain of E. sakazakii. To determine the population of E. sakazakii in the rice cereal inoculum (aw 0.23) containing a 10-strain mixture, samples (10 g) were immediately combined with 90 ml of sterile deionized water at 45 1C, serially diluted, and surface plated on VRBGP and TSAP agar. Plates were inoculated at 37 1C for 24 h and 25 1C for 48 h, respectively, before colonies were counted. The inoculated rice cereal, containing 7.3–7.5 log CFU/g, served as the high inoculum for rice
769
cereal to be stored for up to 12 months. To prepare an inoculum containing a low population of E. sakazakii, the high-population inoculum was serially diluted in infant rice cereal (aw 0.23) to give a population of ca. 2.3 log CFU/g. High-population inoculum or low-population inoculum (2 g) were added to 200 g of uninoculated low-aw infant rice cereal to give desired populations and stored for up to 9 months before analyzing the number of E. sakazakii that survived. Rice, barley, mixed grain, and oatmeal cereals (200 g) at high aw were combined with 2 g of highpopulation inoculum and stored for up to 24 weeks before analysis. 2.5. Preparation of cereals for inoculation and storage 2.5.1. Rice cereal at low aw In the first experiment, survival of E. sakazakii in infant rice cereal at target aw values of 0.23, 0.43, and 0.68 was studied. Three different lots of the same brand of commercially manufactured infant rice cereal were used in three replicate trials. Infant rice cereal from each lot was divided into six of 200-g quantities and placed in polystyrene trays (22 cm long 15 cm wide 5 cm high) at a depth of ca. 2.5 cm. Two of the six trays of each lot were placed on wire platforms inside each of three sterile plastic tubs containing ca. 6 l of saturated solutions of potassium acetate, potassium carbonate, or lithium acetate, which created equilibrium atmospheric relative humidities of ca. 23%, 43%, and 68%, respectively, for the purpose of adjusting the infant rice cereal to target aw values of ca. 0.23, 0.43, and 0.68. Two brushless cooling fans were attached to opposite internal sides of each tub to facilitate air movement. Cereals were stored, with intermittent mixing, in tubs for 14 days at 21 1C, then inoculated with E. sakazakii at high or low population, as described below. 2.5.2. Cereals at high aw A second series of experiments were done using three different lots of the same brand of infant rice, barley, mixed grain, and oatmeal cereals. This study was focused on determining the ability of E. sakazakii to survive at target aw values of ca. 0.68, 0.79, and 0.88. Each lot of each type of cereal was divided into three 200-g quantities and placed in polystyrene trays at a depth of ca. 2.5 cm. Three of the nine trays of each lot and each type of infant cereal were placed on elevated wire platforms inside each of three sterile plastic tubs containing ca. 6 l of saturated solutions of lithium acetate, ammonium sulfate, or sodium benzoate, which created equilibrium atmospheric relative humidities of ca. 68%, 79%, and 88%, respectively, for the purpose of adjusting cereals to aw values of ca. 0.68, 0.79, and 0.88. Two brushless cooling fans were installed inside each tub to facilitate air movement and equilibrium of aw with relative humidity as described above. After storage, with intermittent mixing, for 14 days at 21 1C, cereals at each aw were separately inoculated with high-population inoculum as described below.
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2.6. Inoculation of cereals for storage Infant rice cereal (200 g) initially at aw 0.30, 0.45–0.46, and 0.68–0.69 was inoculated with 2 g of the 10-strain mixture of high- or low-population inoculum, placed in a Ziploc plastic bag (17.7 cm 20.3 cm), sealed, and vigorously mixed for 2 min to give populations of 5.0370.09 log and 0.31 log CFU/g, respectively. Ten grams of inoculated cereal at each aw and containing high or low numbers of E. sakazakii were deposited in 30-ml glass vials and sealed with polyseal liner phenolic caps (Fisher Scientific). Vials were double bagged in Ziploc plastic bags (17.7 cm 20.3 cm), hermetically sealed in plastic tubs, and stored at 4, 21, or 30 1C for up to 12 months before analyzing cereal for the presence (by enrichment) and populations of E. sakazakii. In the second series of experiments, cereals (200 g) adjusted to aw values of 0.63–0.66, 0.76, and 0.82–0.83 were inoculated with 2 g of the 10-strain mixture of highpopulation inoculum, sealed in a Ziploc plastic bag, and vigorously mixed for 2 min to give a population of 5.3970.36 log CFU/g. Sixty grams of each type of inoculated cereal at each aw were deposited in 250-ml glass bottles and hermetically sealed. Cereals were stored at 4, 21, or 30 1C for up to 24 weeks before analyzing to determine the presence (by enrichment) and populations of E. sakazakii. 2.7. Storage of inocula Rice cereal inocula (initial aw, 0.23) containing high (7.31–7.50 log CFU/g) or low (2.31 log CFU/g) numbers of E. sakazakii were each divided into three equal portions, placed in plastic bags, sealed in glass jars, and stored at 4, 21, and 30 1C. Inocula were analyzed for initial populations of E. sakazakii on day 0 (within 1 h after inoculation) and after storage for up to 24 weeks (high-aw cereals) or 9 months (low-aw rice cereal). 2.8. Microbiological analysis of inoculated cereal Each inoculated infant cereal (10 g) initially containing high or low numbers of E. sakazakii was combined with 90 ml of sterile deionized water at 45 1C and vigorously shaken for 1 min. Undiluted mixtures of cereal and water (0.25 ml in quadruplicate and 0.1 ml in duplicate) and suspensions serially diluted in 0.1% sterile peptone (0.1 ml, in duplicate) containing high inoculum (7.3–7.5 log CFU/g) were surface plated on TSAP and VRBGP agar. The VRBGP plates were incubated at 37 1C for 24 h and TSAP plates were incubated at 25 1C for 48 h before positive– presumptive E. sakazakii colonies were counted. Mixtures of cereal initially containing high- or low-population inoculum (10 g) and water (90 ml) were incubated at 37 1C for 24 h. Colonies formed on TSAP were examined for yellow pigmentation. If presumptive E. sakazakii colonies were not detected on TSAP or VRBGP agar,
10 ml of pre-enrichment mixture was combined with 90 ml of Enterobacteriaceae enrichment broth (Becton, Dickinson and Co.) supplemented with 0.1% sodium pyruvate (EEP) and incubated at 37 1C for 24 h. These enrichments were streaked onto TSAP and VRBGP agar and incubated at 25 1C for 48 h and 37 1C for 24 h, respectively. Presumptive colonies of E. sakazakii formed on TSAP agar were subjected to confirmation assays using the API 20E Identification System (bioMe´rieux, Hazelwood, MO) and the Microbact 12A/B Identification kit (Oxoid) according to manufacturers’ instructions. 2.9. Measurement of aw Measurement of aw of infant cereals was done immediately after opening boxes in which they were commercially packaged, after equilibration in tubs with various atmospheric relative humidities for 14 days at 21 1C, and after inoculation and storage for up to 12 months using an aw meter (Aqua Lab Model CX2, Decagon Devices, Inc., Pullman, WA). 2.10. Statistical analysis All experiments were performed in triplicate. Data were analyzed using the general linear model on SAS software (Statistical Analysis Systems Institute, Cary, NC). The least significant difference test was used to determine significant differences (pp0.05) in populations of E. sakazakii detected in infant cereals as affected by aw, composition, storage temperature, and storage time. 3. Results and discussion 3.1. Changes in the aw of inoculated infant rice cereal (initial aw 0.30–0.69) The aw of infant cereals purchased at a local supermarket ranged from 0.35 to 0.41. The purpose of exposing infant rice cereal to atmospheric relative humidities of 23%, 43%, and 68% was to mimic conditions to which open containers of cereal might be exposed in hospital, day-care center, and home settings. Changes in the aw of infant rice cereal during subsequent storage for 12 months at 4, 21, and 30 1C are shown in Fig. 1. During storage, the cereal initially at aw 0.30 and 0.46 increased or decreased slightly upon equilibration with fluctuating relative humidities in the storage chambers. 3.2. Survival in infant rice cereal (initial aw 0.30–0.69) containing low inoculum E. sakazakii survived in infant rice cereal (aw 0.30–0.69) inoculated at a population (0.31 log CFU/g) and stored for up to 12 months at 4, 21, and 30 1C (Table 1). With the exception of cereal at aw 0.46 stored at 30 1C, the pathogen was detected by enrichment of cereal stored for 12 months,
ARTICLE IN PRESS L.-C. Lin, L.R. Beuchat / Food Microbiology 24 (2007) 767–777
1.0
771
High inoculum
Low inoculum
Water activity
0.8 0.6 0.4 0.2 0.0 0 1
3
6
9
12 0 1 3 Storage time (months)
6
9
12
Fig. 1. Water activity of infant rice cereal (initially at aw 0.30–0.69) inoculated with E. sakazakii at 0.31 log CFU/g (low inoculum) and 5.03 log CFU/g (high inoculum) and stored at 4 (J), 21 (&), and 30 1C (W) for up to 12 months.
Table 1 Survival of E. sakazakii in infant cereal (initial population was 0.31 log CFU/g) as affected by low aw (0.30–0.69) and storage temperaturea Initial aw
0.30
Storage temp. (1C)
4 21 30
0.46
4 21 30
0.69
4 21 30
Detection medium
Storage time (months) 0
1
3
6
9
12
TSAP VRBGP TSAP VRBGP TSAP VRBGP
2 2 2 2 2 2
1 1 1 1 2 2
2 2 2 2 1 1
2 2 2 2 3 3
2 2 0 0 2 2
1 1 1 1 2 2
TSAP VRBGP TSAP VRBGP TSAP VRBGP
2 2 2 2 2 2
0 0 0 0 2 2
1 1 2 2 2 2
2 2 2 2 2 2
0 0 0 0 0 0
2 2 2 2 0 0
TSAP VRBGP TSAP VRBGP TSAP VRBGP
1 1 1 1 1 1
2 2 2 2 1 1
2 2 1 1 0 0
2 2 2 2 0 0
2 2 1 1 0 0
2 2 2 2 1 1
a Numbers indicate positive samples out of three tested (three replicate experiments). Detection limit was 1 CFU/10 g.
regardless of the aw or temperature. Breeuwer et al. (2003) reported that stationary phase E. sakazakii cells were more resistant than E. coli, Salmonella, and other Enterobacteriaceae to desiccation stress. Retention of viability was attributed to accumulation of trehalose in cells. Our observations on the exceptional resistance of E. sakazakii to desiccation are in agreement with these findings. 3.3. Survival in infant rice cereal (initial aw 0.30–0.68) containing high inoculum Fig. 2 shows populations of E. sakazakii recovered on TSAP from infant rice cereal (initial aw 0.30–0.68) inoculated with the pathogen at a population of 5.03 log
CFU/g and stored at 4, 21, or 30 1C. Significantly higher populations of E. sakazakii were recovered from most samples on TSAP than on VRBGP agar (not shown), which is in agreement with observations by Gurtler and Beuchat (2005) showing the inability of VRBGP agar to support resuscitation and colony formation by stressed cells of E. sakazakii. Populations decreased significantly (p p 0.05) as storage time progressed, regardless of the aw or temperature. Increased aw and storage temperature caused increased rates of death. At most storage time/ temperature combinations, the population of E. sakazakii in cereal initially at aw 0.68 decreased to a lower level than in cereal initially at aw 0.45, which in turn was lower than in cereal initially at aw 0.30. Populations of E. sakazakii in infant rice cereal (initial aw 0.30–0.68) stored at 4, 21, and 30 1C for 12, 6, and 3 months, respectively, as determined on TSAP, decreased by 0.52–1.51, 1.86–4.04, and 3.25–4.77 log CFU/g. With the exception of rice cereal (aw 0.68) stored at 30 1C for 12 months, E. sakazakii was recovered by direct plating or enrichment of at least one of three replicate samples representing all experimental parameter combination. Edelson-Mammel et al. (2005) reported that an initial E. sakazakii population of ca. 6 log CFU/g of powdered infant formula (aw 0.14–0.27) decreased by 2.4 log CFU/g during storage at 20–22 1C for 150 days (ca. 5 months), followed by an additional 1-log decrease during the next 534 days. In our study, populations in infant rice cereal (initial aw 0.30) stored at 21 1C for 6 months decreased by 1.86 log CFU/g, indicating a similar loss of viability over time in dried rice cereal and infant formula. Results show that E. sakazakii can survive in dry infant rice cereal (initial aw 0.30–0.69) stored at 4–30 1C for at least 12 months. Christian and Stewart (1973) reported that Staphylococcus aureus and Salmonella Newport survived in dry foods (aw 0–0.53) at 25 1C for 27 weeks. In the same study, both microorganisms were observed to survive better at lower aw (0–0.22). In contrast, Deng et al. (1998) reported that survival of E. coli O157:H7 in infant rice cereal (aw 0.35–0.73) was enhanced at higher aw. Populations in rice cereal (aw 0.35) initially containing E. coli O157:H7 at a population of 6.31 log CFU/g
ARTICLE IN PRESS L.-C. Lin, L.R. Beuchat / Food Microbiology 24 (2007) 767–777
772
6
4°C
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6
21°C
30°C
5 4 3 2 1 0 0
1
3
6
9
12 0 1
3
6
9
12
Storage time (months) Fig. 2. Population (log CFU/g) of E. sakazakii recovered from infant rice cereal as affected by initial aw 0.30 (J), 0.45 (&), and 0.68 (W) and storage at 4, 21, or 30 1C for up to 12 months. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
decreased to 3.19 and 1.22 log CFU/g within 24 weeks at 5 1C and 11 weeks at 25 1C, respectively. The rate of death of E. coli O157:H7 was more rapid than that of E. sakazakii in infant rice cereal held at similar aw and temperature conditions. Jaquette and Beuchat (1998) reported that the rate of death of vegetative cells of B. cereus in infant rice cereal (aw 0.27–0.78) stored for 36 weeks at 5, 25, 35, or 45 1C was not influenced by aw. Death was more rapid at the higher storage temperatures, however, regardless of the aw, which is in agreement with observations on E. sakazakii and E. coli O157:H7. 3.4. Survival in infant rice, barley, mixed grain, and oatmeal cereals (initial aw 0.63–0.83) containing high inoculum Changes in aw in infant rice, barley, mixed grain, and oatmeal cereals (initially at aw 0.63–0.66, 0.76, and 0.82–0.83) inoculated with E. sakazakii (4.93–5.64 log CFU/g) and stored at 4, 21, or 30 1C for 24 weeks are shown in Fig. 3. The aw of infant cereals stored at 30 1C was measured only up to 14 weeks, beyond which samples were not available for analysis. The aw of infant cereals changed more when cereals were stored at 30 1C than when stored at 4 or 21 1C, a phenomenon attributed to fluctuation in relative humidity within the storage chamber. Survival of E. sakazakii in infant rice, barley, mixed grain, and oatmeal cereals (initial aw 0.63–0.83) stored at 4, 21, and 30 1C are shown in Figs. 4–6, respectively. Initial populations were 4.93–5.64 log CFU/g. As in experiments with rice cereal at low aw (0.30–0.68), significantly higher numbers of E. sakazakii were recovered when cereals at high aw were plated on TSAP than on VRBGP agar (not shown). Populations decreased at the slowest rate in cereals
stored at 4 1C, e.g., reductions of 0.49–0.84, 0.38–0.94, and 1.13–1.53 log CFU/g occurred during storage of cereals at aw 0.63–0.66, 0.76, and 0.82–0.83, respectively, for up to 24 weeks (Fig. 4); this compares to reductions of at least 3.93 log CFU/g within 8 weeks at 30 1C (Fig. 6). Populations of E. sakazakii in infant rice, barley, mixed grain, and oatmeal cereals decreased significantly (pp0.05) as storage time at 21 1C (Fig. 5) and 30 1C (Fig. 6) progressed. Storage of cereals initially at aw 0.82–0.83 for 24 weeks at 4 1C resulted in decreases in populations of E. sakazakii that were significantly (pp0.05) lower in cereals than in cereals initially at aw 0.63–0.76 (Fig. 4). Populations of E. sakazakii in infant rice, barley, and oatmeal cereals stored at 21 1C for 14, 14, and 6 weeks, respectively, were significantly reduced when the initial aw was 0.82–0.83 compared to populations at aw 0.76, and at aw 0.76 compared to populations at aw 0.63–0.66 (Fig. 5). However, survival of E. sakazakii in infant cereals stored at 30 1C for 14 weeks was not affected by aw (Fig. 6). At a given initial aw (0.63–0.83), populations of E. sakazakii significantly decreased in infant rice, barley, mixed grain, and oatmeal cereals as storage temperature increased. With few exceptions, the composition of the four types of infant cereals did not markedly affect the survival of E. sakazakii, regardless of aw or storage temperature. Shown in Table 2 are the longest storage times at which E. sakazakii was detected by enrichment (X1 CFU/10 g) in cereals (aw 0.63–0.83) stored at 21 and 30 1C. Cereals initially containing 4.93–5.64 log CFU/g were stored at 21 1C for 2, 4, 6, 8, 14, and 24 weeks and at 30 1C for 2, 4, 6, 8, 10 and 14 weeks. With the exceptions of barley and oatmeal cereals at aw 0.82–0.83, the pathogen was detected in all cereals stored at 21 1C for 24 weeks. E. sakazakii was
ARTICLE IN PRESS L.-C. Lin, L.R. Beuchat / Food Microbiology 24 (2007) 767–777
0.9
773
Mixed grain
Rice
0.8
0.7
Water activity
0.6
0.5 0.9
Barley
Oatmeal
0.8
0.7
0.6
0.5 0
4
8
12 14
24 0 4 Storage time (weeks)
8
12 14
24
Fig. 3. Water activity of infant rice, barley, mixed grain, and oatmeal cereals inoculated with E. sakazakii at 5.39 log CFU/g and stored at 4 (J), 21 (&), and 30 1C (W) for up to 24 weeks.
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0
2
4
6
8
14
24 0 2 4 6 Storage time (weeks)
8
14
24
Fig. 4. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 4 1C for up to 24 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g).
detected in all cereals (aw 0.63–0.66) stored at 30 1C for 10 weeks but not 14 weeks. Maximum storage times at which the pathogen was detected in cereals were decreased with increased aw and temperature. Higher numbers of E.
sakazakii survived in cereals (aw 0.63–0.83) stored at 4 1C (Fig. 4) than at 21 or 31 1C. With few exceptions, the composition of cereals did not significantly affect the survival of E. sakazakii, regardless of aw or storage temperature.
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774
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0 2
4
6
8
24 0
14
2
4
6 8
14
24
Storage time (weeks) Fig. 5. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 21 1C for up to 24 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
6
Rice
Mixed grain
Barley
Oatmeal
5 4 E.sakazakii (log CFU/g)
3 2 1 0 6 5 4 3 2 1 0 0
2
4
6
8
10
14 0
2
4
6
8
10
14
Storage time (weeks) Fig. 6. Population (log CFU/g) of E. sakazakii recovered from infant rice, barley, mixed grain, and oatmeal cereals as affected by initial aw 0.63–0.66 (J), 0.76 (&), and 0.82–0.83 (W) and storage at 30 1C for up to 14 weeks. Bars indicate standard deviations. Detection limit was 1 log CFU/g (10 CFU/g). Values less than 1 log CFU/g but more than 0 log CFU/g indicate that E. sakazakii was detected in one or more replicate samples.
3.5. Survival in infant rice cereal inoculum (initial aw 0.23) Survival of E. sakazakii in the rice cereal inoculum stored for up to 9 months at 4, 21, and 30 1C was determined. Infant rice cereal inoculum (initial aw 0.23) from which a portion had been used to inoculate infant rice cereal (initial aw 0.30–0.69) (Fig. 7) or infant rice, barley, mixed grain, and oatmeal cereals (initial aw
0.63–0.83) (Fig. 8) was monitored for populations of E. sakazakii. The pathogen was detected by direct plating or enrichment of high-population (7.31 log CFU/g) rice inoculum stored for 9 months at 4, 21, and 30 1C but not in low-population (2.31 log CFU/g) inoculum stored at 21 or 30 1C. The trend showing decreases in populations as affected by storage temperature is similar to that observed for inoculated infant rice cereal (initial aw 0.30).
ARTICLE IN PRESS L.-C. Lin, L.R. Beuchat / Food Microbiology 24 (2007) 767–777
8
Table 2 Survival of E. sakazakii (initial populations were 4.93–5.64 log CFU/g) in infant rice, barley, mixed grain, and oatmeal cereals as affected by high aw (0.63–0.83) at 21 and 30 1Ca Storage temp. (1C)
Longest storage time (weeks) at which E. sakazakii was detected Rice
Barley
Mixed grain
Oatmeal
0.63–0.66
21 30
24 10
24 10
24 10
24 10
0.76
21 30
24 8
24 10
24 10
24 8
0.82–0.83
21 30
24 10
14 8
24 6
14 8
4 3 2
0
7 E.sakazakii (log CFU/g)
5
0
8
6 5 4 3 2 1 0 3 6 Storage time (months)
6
1
a Cereals were stored at 21 and 30 1C for up to 24 and 14 weeks, respectively. Samples (10 g) were pre-enriched in 90 ml of deionized water at 37 1C for 24 h, enriched in 90-ml EEP broth at 37 1C for 24 h, and streaked on TSAP, followed by incubation at 25 1C for 48 h. Numbers indicate the longest storage time (weeks) at which E. sakazakii was detected in at least one of three samples tested (three replicate experiments). Detection limit was 1 CFU/10 g.
0 0.5 1
7 E.sakazakii (log CFU/g)
Initial aw
775
9
Fig. 7. Populations (log CFU/g) of E. sakazakii recovered from infant rice cereal (initial aw 0.23) used as high inoculum (solid symbols) and low inoculum (open symbols) to inoculate infant rice cereal (initial aw 0.30–0.69) as affected by storage temperature at 4 (, J), 21 (’, &), and 30 1C (m, W) up to 9 months. Detection limit was 1 log CFU/g (10 CFU/g).
2
4
6
8 14 Storage time (weeks)
24
Fig. 8. Populations (log CFU/g) of E. sakazakii recovered from infant rice cereal (initial aw 0.23) used to inoculate infant rice, barley, mixed grain, and oatmeal cereals (aw 0.63–0.83) as affected by storage temperature at 4 (J), 21 (&), and 30 1C (W) up to 24 weeks. Detection limit was 1 log CFU/g (10 CFU/g).
the probability of its survival in cereals at the time of reconstitution and feeding to infants. Kandhai et al. (2006) reported that powdered infant formula inoculated with lag, exponential, and stationary phase of E. sakazakii and stored for up to 4 weeks prior to reconstitution in sterile water did not show a longer lag time or different specific growth rate compared to cells in other phases of growth. Richards et al. (2005) reported that E. sakazakii can grow in infant rice cereal reconstituted with water, milk, and liquid infant formula at 12, 21, and 30 1C. Further investigations are needed to determine the survival and growth characteristics of E. sakazakii in reconstituted infant cereal as affected by the composition of the liquid used for reconstitution and the storage temperature after reconstitution. Acknowledgment This work was supported, in part, by a grant from the International Life Sciences Institute—North America (ILSI N.A.). The opinions expressed herein are those of the authors and do not necessarily represent the views of ILSI N.A.
4. Conclusions Results show that E. sakazakii, initially at populations as low as 0.31 log CFU/g (2 CFU/g), can survive at 4, 21, or 30 1C for up to 12 months in infant cereals in an aw range of 0.30–0.83. Results show that very small numbers of E. sakazakii, if present in infant cereals, can survive for up to 12 months in cereals exposed to conditions under which they may be held during distribution and storage. Tolerance of the pathogen to these conditions enhances
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