MATERIALS AND METHODS

Strains used. Ten strains of E. sakazakii were used. Five strains (2855, 3231, 3234, 3290, and 3295) were isolated from clinical samples, four strains (2871, 3270, 3437, and 3439) were isolated from food, and one strain (3396) was isolated from an environmental sample. All strains were obtained from Dr. Jeffrey Farber, Health Canada, Ottawa, Ont., Canada.

Cereals used. Three different lots of the same brand of commercially manufactured dry infant rice, rice with mixed fruit, and oatmeal cereals were purchased from local retailers in

Griffin, Ga. Infant rice, oatmeal, and rice with mixed fruit cereals contained, respectively, rice flour, oat flour, and rice flour, fully riped bananas, pineapple juice concentrate, orange juice concentrate, and pineapple (pineapple, pineapple juice, ascorbic acid). All three cereals contained soy oil-lecithin, tri- and dicalcium phosphate, tocopherols (vitamin E), electrolytic iron, zinc sulfate, niacinamide, riboflavin (vitamin B2), pyridoxine hydrochloride (vitamin B6), thiamin (vitamin B1), folic acid, and cyanocobalamin (vitamin B12).

Liquids used for reconstituting cereals. Three different lots of the same brand of pasteurized whole fat (ca. 3.5%) milk and the same brand of commercially sterilized apple juice supplemented with ascorbic acid (vitamin C) were purchased from local retailers in Griffin, Ga.

Sterile deionized water, milk, and apple juice were adjusted to 21°C for 1.5 h before using to reconstitute dry infant cereals.

Preparation of inoculum. Stock cultures of the ten strains of E. sakazakii in aqueous 15% glycerol solution were stored at -31°C. Each culture was thawed and streaked with a wire loop onto violet red bile glucose agar (VRBG agar, pH 7.4; Oxoid Ltd., Basingstoke, Hampshire, England) and incubated at 37°C for 24 h. Cells from single colonies of each strain were streaked on tryptic soy agar (TSA, pH 7.3; Becton, Dickinson and Co., Sparks, Md.), incubated at 37ºC for 24 h, and stored at 4°C until used to prepare inocula.

To prepare inoculum for cereals, each strain of E. sakazakii was grown in 9 ml of brain heart infusion broth (BHI, pH 7.4; Becton, Dickinson and Co.) at 37°C, with two successive loop transfers (ca.10 µl) of 24-h cultures. A final transfer of 0.1 ml was made from the second 24-h culture to 400 ml of BHI broth and incubated on a platform shaker (New Brunswick Scientific, Edison, N.J.) at 37°C for 24 h at 62 rpm. The ten 400-ml BHI broth cultures were centrifuged

for 15 min at 2,700 x g in a Marathon 12KBR Benchtop Refrigerated Centrifuge (Fisher Scientific, Pittsburgh, Pa.) and the supernatant was decanted. Cells were resuspended in sterile deionized water, centrifuged a second time, and resuspended in 10 ml of sterile deionized water.

To determine populations, suspensions of each strain were serially diluted in sterile 0.1%

peptone water, surface plated (0.1 ml, in duplicate) on TSA supplemented with 0.1% sodium pyruvate (TSAP) (Sigma-Aldrich, Inc., St. Louis, Mo.) and VRBG agar supplemented with 0.1%

sodium pyruvate (VRBGP), and incubated at 25ºC for 48 h and 37ºC for 24 h, respectively.

Sodium pyruvate was added to aid in recovery of injured cells (Baird-Parker and Davenport 1965). Colonies were counted and populations of each of the ten strains in the suspensions were calculated.

Appropriate volumes of suspensions of each strain were combined to give a ten-strain mixture containing approximately equal populations of each strain. The ten-strain mixture was serially diluted in sterile 0.1% peptone water to give populations of 2.42 - 2.65 log CFU/ml and 0.42 - 0.65 log CFU/ml, which served as the high inoculum and low inoculum, respectively, for infant cereals reconstituted with water, milk, or apple juice.

A second series of experiments was done in which the ten-strain mixture was used to inoculate infant rice cereal reconstituted only with apple juice. The purpose of this study was to determine if E. sakazakii would survive in reconstituted cereal at reduced pH (initially at pH 4.32 - 4.33) for up to 50 days at 4 - 30°C. A ten-strain mixture (ca. 10.0 log CFU/ml) prepared as described above served as the high inoculum. The ten-strain mixture was serially diluted in sterile 0.1% peptone water to give ca. 5.0 log CFU/ml which was used as a low inoculum.

Reconstitution and inoculation of infant cereals. Thirty grams of infant rice, rice with mixed fruit, or oatmeal cereals were reconstituted with 180 ml of water, milk, or apple juice at a

ratio of 1:6 (g:ml, dry cereal:liquid) in a sterile 500-ml screw-cap glass bottle (Pyrex, Corning Incorporated Life Sciences, Acton, Mass.). The thoroughly mixed, reconstituted cereal (21°C) was inoculated 0.3 ml of low- or high-inoculum to give ca. 3.63 and 363 CFU/100 g of dry cereal, respectively, or ca. 0.005 and 0.52 CFU/ml of reconstituted cereal. Inoculated

reconstituted cereal was thoroughly mixed for 15 s followed by incubation at 4, 12, 21, and 30°C for up to 72 h.

In the second series of experiments, 30 g of infant rice cereal were reconstituted with 180 ml of apple juice at a ratio of 1:6 (g:ml, dry cereal:liquid) in a sterile 500-ml screw-cap glass bottle. The thoroughly mixed, reconstituted cereal was inoculated with 1.0 ml of low- or high-inoculum to give ca. 2.62 and 7.32 log CFU/ml of reconstituted cereal, respectively. Inoculated reconstituted cereal was thoroughly mixed for 15 s followed by incubation at 4, 12, 21, and 30°C for up to 50 days.

Microbiological analysis of inoculated cereal. In the first experiment, inoculated infant rice, rice with mixed fruit, and oatmeal cereals reconstituted with water, milk, and apple juice were analyzed for the presence (by enrichment) of E. sakazakii and populations of E. sakazakii and mesophilic aerobic bacteria within 30 min (0 h) after inoculation and after incubating for 4, 8, 12, 24, 48, and 72 h at 4, 12, 21, or 30°C. In the second series of experiments, infant rice cereal reconstituted with apple juice, inoculated with E. sakazakii, and incubated at 4, 12, 21, and 30°C was analyzed for the presence (by enrichment) and populations of E. sakazakii within 30 min (0 h) after inoculation and at 24-h intervals throughout 14 days and at 50 days.

At each sampling time, bottles containing reconstituted inoculated infant cereals were vigorously shaken by hand for 15 s. Cereals (0.25 ml, in quadruplicate and 0.1 ml, in duplicate) were surface plated on TSAP and VRBGP agar and incubated at 25 ºC for 48 h and 37ºC for 24

h, respectively. Reconstituted inoculated infant cereals serially diluted in sterile 0.1% peptone water (0.1 ml, in duplicate) were also surface plated on TSAP and VRBGP agar. The VRBGP plates were incubated at 37°C for 24 h and TSAP plates were incubated at 25ºC for 48 h.

Presumptive-positive E. sakazakii colonies formed on VRBGP agar were counted. Populations of mesophilic aerobic bacteria (total plate counts) and presumptive E. sakazakii (yellow

pigmented colonies) were determined by counting colonies formed on TSAP.

In addition to surface-plating samples on TSAP and VRBGP agar, 10 ml of inoculated reconstituted infant cereals was combined with 90 ml of Enterobacteriaceae enrichment broth (pH 7.2; Becton, Dickinson and Co.) supplemented with 0.1% sodium pyruvate (EEP) and incubated at 37°C for 24 h. If presumptive E. sakazakii colonies were not detected on TSAP or VRBGP agar, enrichments were streaked on TSAP and VRBGP agar and incubated at 25°C for 48 h and 37°C for 24 h, respectively. Presumptive colonies of E. sakazakii formed on TSAP and VRBGP agar were subjected to confirmation assays using the API 20E identification system (bioMérieux, Hazelwood, Mo.) and the Microbact 12A/B identification kit (Oxoid) according to manufacturers’ instructions.

pH measurement. The pH of inoculated reconstituted infant rice, rice with mixed fruit, and oatmeal cereals was monitored. Five milliliters of inoculated reconstituted cereal was dispensed in a 30-ml medicine cup (Medegen Medical Products, Gallaway, Tenn.) and analyzed within 1 h after inoculation and after incubating for 4, 8, 12, 24, 48, and 72 h at 4, 12, 21, or 30°C using a benchtop pH meter with flat-surface pH electrode (Denver Instrument Company, Denver, Colo.). For the second experiment in which inoculated infant rice cereal was

reconstituted with apple juice, pH values were monitored daily throughout the 14-day storage period and at 50 days.

Statistical analysis. All experiments were replicated three times. Data were analyzed using the general linear model on SAS software (Statistical Analysis Systems Institute, Cary, N.C.). The least significant difference test was used to determine if changes in populations of E.

sakazakii and mesophilic aerobic bacteria and pH in inoculated reconstituted infant cereals were significantly (p ≤ 0.05) affected by the composition of infant cereals, storage temperature after reconstitution, and storage time.