Cronobacter Support
02-19-2009, 07:43 PM
Growth of E. sakazakii
Two different morphological colony types occur when fresh isolates of E. sakazakii are streaked for purity. One type is dry or mucoid with scalloped edges and also rubbery when touched with a loop, whereas the other is typically smooth and nondistinct. The latter type predominates when the isolate is subcultured. Differences in virulence or phenotypic traits between these two colony types is unknown.
E. sakazakii will grow on nonselective media [e.g. tryptic soy agar (TSA)] commonly used in enteric bacteriology. On TSA, colonies 2-3 mm in diameter form within 24 h at 36°C, and if grown at 25°, the colonies will typically be 1-1.5 mm in diameter. Growth is also rapid in tryptic soy broth (TSB) with an increase of 105 to 109 cfu/ml occurring overnight.
When grown in the presence of D-glucose and citrate either aerobically or anaerobically, there are no obvious requirements by E. sakazakii for vitamins, amino acids, or other organic growth factors. The pH ranges for growth can vary considerably, although good growth occurs between pH 5 and 9. Minimum growth temperatures of 5.5 – 8°C have been reported for both clinical and food isolates in reconstituted infant formulas, whereas E. sakazakii cell numbers at 4°C decreased or remained constant during storage. The maximum temperature at which visible growth of E. sakazakii in TSB occurred was at 47°C. While lag and generation times were generally less for food isolates compared to clinical strains, the differences were not statistically significant. Average generation times were 40 min at 23°C and 4.98 h at 10°C, which are less than for most other bacteria typically found in milk or milk products. Hence, E. sakazakii would be expected to predominate if reconstituted formula is stored at nonrefrigerated temperatures. Determination of the competitiveness of E. sakazakii has only been reported against E. cloacae and in that case, E. sakazakii grew to large numbers, whereas there was no growth of E. cloacae. The production of a colicin(s) by E. sakazakii may enable its predominance. Growth of E. sakazakii on surfaces and in biofilms has not been evaluated, although an extracellular polysaccharide which is produced by multiple strains of E. sakazakii, would likely serve to enable attachment of the organism to surfaces and provide protection from disinfectants.
Thermal Tolerance of E. sakazakii
Heat resistance of ten strains (5 clinical and 5 food isolates) of E. sakazakii has been determined 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 of these temperatures, respectively. Compared to other Enterobacteriaceae isolates, E. sakazakii is one of the most thermotolerant organisms in this Family. However, extrapolating thermal inactivation data to HTST pasteurization (72°C, 15 sec) conditions reveals that E. sakazakii will not survive pasteurization. Although E. sakazakii has been isolated from UHT milk, it is unknown if post-pasteurization contamination occurred or if the organism survived UHT treatment. When reconstituted infant formula was boiled (85-100 sec depending on milk type) by exposure to microwaves (2450 MHz, 600W), survival of E. sakazakii was dependent on the milk formula. From an initial inoculum of 100 cfu/ml, no E. sakazakii was detected in four commercial brands; however, in another commercial brand, 20 cfu/ml were detected after microwave heat treatment.
Current Regulations Affecting Occurrence of E. sakazakii in Powdered Infant Formulas
Regulations governing the prevalence of E. sakazakii in powdered infant formulas falls under the hygienic requirements for allowable levels of coliforms. For the Codex Alimentarius, these requirements include a minimum of four of five control samples with < 3 coliforms/g and a maximum of one of five control samples with >3 but £ 20 coliforms/g. Based upon these test parameters, dry milk-based infant formula that contains E. sakazakii at levels of < 1 organism per 100 g of formula would not be reliably detected.
Future Research on E. sakazakii
More research is needed to determine virulence factors, genetic diversity, appropriate recovery and subspeciation methods, environmental niches, and methods for control of this emerging pathogen in foods and the environment.
Two different morphological colony types occur when fresh isolates of E. sakazakii are streaked for purity. One type is dry or mucoid with scalloped edges and also rubbery when touched with a loop, whereas the other is typically smooth and nondistinct. The latter type predominates when the isolate is subcultured. Differences in virulence or phenotypic traits between these two colony types is unknown.
E. sakazakii will grow on nonselective media [e.g. tryptic soy agar (TSA)] commonly used in enteric bacteriology. On TSA, colonies 2-3 mm in diameter form within 24 h at 36°C, and if grown at 25°, the colonies will typically be 1-1.5 mm in diameter. Growth is also rapid in tryptic soy broth (TSB) with an increase of 105 to 109 cfu/ml occurring overnight.
When grown in the presence of D-glucose and citrate either aerobically or anaerobically, there are no obvious requirements by E. sakazakii for vitamins, amino acids, or other organic growth factors. The pH ranges for growth can vary considerably, although good growth occurs between pH 5 and 9. Minimum growth temperatures of 5.5 – 8°C have been reported for both clinical and food isolates in reconstituted infant formulas, whereas E. sakazakii cell numbers at 4°C decreased or remained constant during storage. The maximum temperature at which visible growth of E. sakazakii in TSB occurred was at 47°C. While lag and generation times were generally less for food isolates compared to clinical strains, the differences were not statistically significant. Average generation times were 40 min at 23°C and 4.98 h at 10°C, which are less than for most other bacteria typically found in milk or milk products. Hence, E. sakazakii would be expected to predominate if reconstituted formula is stored at nonrefrigerated temperatures. Determination of the competitiveness of E. sakazakii has only been reported against E. cloacae and in that case, E. sakazakii grew to large numbers, whereas there was no growth of E. cloacae. The production of a colicin(s) by E. sakazakii may enable its predominance. Growth of E. sakazakii on surfaces and in biofilms has not been evaluated, although an extracellular polysaccharide which is produced by multiple strains of E. sakazakii, would likely serve to enable attachment of the organism to surfaces and provide protection from disinfectants.
Thermal Tolerance of E. sakazakii
Heat resistance of ten strains (5 clinical and 5 food isolates) of E. sakazakii has been determined 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 of these temperatures, respectively. Compared to other Enterobacteriaceae isolates, E. sakazakii is one of the most thermotolerant organisms in this Family. However, extrapolating thermal inactivation data to HTST pasteurization (72°C, 15 sec) conditions reveals that E. sakazakii will not survive pasteurization. Although E. sakazakii has been isolated from UHT milk, it is unknown if post-pasteurization contamination occurred or if the organism survived UHT treatment. When reconstituted infant formula was boiled (85-100 sec depending on milk type) by exposure to microwaves (2450 MHz, 600W), survival of E. sakazakii was dependent on the milk formula. From an initial inoculum of 100 cfu/ml, no E. sakazakii was detected in four commercial brands; however, in another commercial brand, 20 cfu/ml were detected after microwave heat treatment.
Current Regulations Affecting Occurrence of E. sakazakii in Powdered Infant Formulas
Regulations governing the prevalence of E. sakazakii in powdered infant formulas falls under the hygienic requirements for allowable levels of coliforms. For the Codex Alimentarius, these requirements include a minimum of four of five control samples with < 3 coliforms/g and a maximum of one of five control samples with >3 but £ 20 coliforms/g. Based upon these test parameters, dry milk-based infant formula that contains E. sakazakii at levels of < 1 organism per 100 g of formula would not be reliably detected.
Future Research on E. sakazakii
More research is needed to determine virulence factors, genetic diversity, appropriate recovery and subspeciation methods, environmental niches, and methods for control of this emerging pathogen in foods and the environment.