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Enterobacter sakazakii has been implicated as the causal organism in a severe form of neonatal meningitis, with reported mortality rates of 40 to 80%. Dried infant formula has been identified as a potential source of the organism in both outbreaks and sporadic cases. In this study, clinical and foodborne isolates of E. sakazakii were evaluated for enterotoxin production by the suckling mouse assay. In addition, suckling mice were challenged both orally and by intraperitoneal injection. Of 18 E. sakazakii strains evaluated, four were found to test positive for enterotoxin production. All strains of E. sakazakii were lethal to suckling mice at 10(8) CFU per mouse by intraperitoneal injection, while two strains caused death by the peroral route. In in vitro assays, CHO, Vero, and Y-1 cells demonstrated both cell lysis and rounding when exposed to E. sakazakii strain LA filtrates. This is the first report describing any putative virulence factors of E. sakazakii.

Enterobacter sakazakii has been implicated in a severe form of neonatal meningitis. Although studies have failed to identify an environmental source for the organism, dried infant formula has been implicated in outbreaks and sporadic cases of E. sakazakii meningitis. The high mortality rate (50 to 75%), the severity of the infection in infants, and the lack of information on the incidence, survival, and growth of E. sakazakii in foods led to this study. Experiments were undertaken to determine the incidence of E. sakazakii in dried infant formula, the temperature range for growth, and the growth characteristics of E. sakazakii in reconstituted dried infant formula. Strains of E. sakazakii were isolated from dried infant formula available on the Canadian retail market. The prevalence varied from 0 to 12% in samples from five different companies. For both clinical and food isolates, minimum growth temperatures of 5.5 to 8.0°C were observed by using a temperature-gradient incubator. The potential growth of E. sakazakii was followed by using a mixture of food and clinical isolates in three different formulas incubated at 4, 10, and 23°C. Average generation times were 40 min at 23°C and 4.98 h at 10°C. E. sakazakii strains did not grow at 4°C and began to die off during storage at this temperature. The results of this study stress the importance of using aseptic methods and proper temperature control in the preparation, use, and storage of dried infant formula.

M . N AZ A RO WE C -W HI T E A ND J .M . F A RB ER . 1997. Enterobacter sakazakii, designated a unique species in 1980, has been implicated in a rare but severe form of neonatal meningitis, with dried-infant formula being implicated as the mode of transmission. The high mortality rate (40-80%) and the lack of information about this organism led to a study of the heat resistance of Ent. sakazakii in reconstituted dried-infant formula. Ten Canadian Ent. sakazakii strains (5 clinical and 5 food isolates) were used to determine the heat resistance of this organism at 52, 54, 56, 58 and 60°C in reconstituted dried-infant formula. D-values of 54·8, 23·7, 10·3, 4·2 and 2·5 min were obtained for each temperature, respectively. The overall calculated zvalue was 5·82°C. In a comparison of the D-values of several members of the Enterobacteriaceae in dairy products, Ent. sakazakii appeared to be one of the most thermotolerant organisms. The importance of process control during manufacture and the use of aseptic procedures during the preparation, use and storage of dried-infant formula is discussed.