Sakazakii Infection Research Articles

Resistance ofEnterobacter sakazakii(Cronobacterspp.) to environmental stresses

To gain a better understanding of the survival and persistence of Enterobacter sakazakii in severe environments. We evaluated the resistance of Ent. sakazakii to various environmental stresses, including heating, drying, water activity (a(w)), and pH. The resistance of Ent. sakazakii to heat varies widely among strains. Most tested strains of Ent. sakazakii exhibited unusual resistance to dry stress, which depends on drying media. Growth of most strains occurred within 24 h at 37 degrees C when the initial a(w) of the medium was adjusted to 0.94 with sucrose or sodium chloride. The minimum pH for growth within 24 h at 37 degrees C was 3.9 or 4.1 for most strains tested. Additionally, there did not appear to be any relationship between resistance to stresses and biofilm-forming ability in Ent. sakazakii planktonic cells. These results indicate that Ent. sakazakii is much more resistant than other Enterobacteriaceae to environmental stresses. Moreover, it is likely that Ent. sakazakii has cross-resistance to dry and thermal stresses. The findings of this study will contribute to an improved understanding of the survival and behaviour of Ent. sakazakii, which will lead to improved strategies for preventing outbreaks of Ent. sakazakii infection.

Surveillance of Stool Samples for the Presence of Enterobacter sakazakii among Korean People

PurposeEnterobacter sakazakii (E. sakazakii) infections are an important cause of life-threatening meningitis, septicemia, and necrotizing enterocolitis in infants. Dried infant formula milk is an important vehicle for E. sakazakii infection. E. sakazakii was isolated in Korea from dried infant formula milk. Although E. sakazakii infection of infants may occur in Korea, its prevalence has not yet been documented. Therefore, we determined the prevalence of E. sakazakii and documented symptoms.Materials and MethodsBetween March and October 2006, 1,146 stool samples were collected from patients at Uijeongbu St. Mary's Hospital. Each fecal swab was dissolved in 10 mL of buffered peptone solution, and enriched culture was streaked onto Druggan-Forsythe-Iversen (DFI) agar. Presumptive E. sakazakii colonies that exhibited a blue-green color during culture on DFI medium were selected. The identity of colonies that developed yellow pigment during culture on TSA was determined using the Vitek system and PCR.ResultsWe isolated 4 E. sakazakii strains whose 16S rRNA sequence alignments had a similarity of 99% with those of 3 E. sakazakii ATCC strains.ConclusionThis is the first report on isolation of E. sakazakii from stool samples and to document the symptoms of Korean patients.

Role of Bacterial OmpA and Host Cytoskeleton in the Invasion of Human Intestinal Epithelial Cells by Enterobacter sakazakii

Enterobacter sakazakii is an emerging pathogen in neonates and infants. Interactions of E. sakazakii with intestinal epithelium could be vital in the pathogenesis of enteric infections and in its systemic dissemination. The present study investigated the interaction of E. sakazakii with human intestinal epithelial (INT407) cells and the role of bacterial outer membrane protein A (OmpA) and host cytoskeleton in these interactions. E. sakazakii invaded INT407 cells with moderate efficiency. An ompA mutant of E. sakazakii was significantly attenuated in its invasiveness, and complementation restored the invasive phenotype significantly. Drugs acting on host cell microfilaments (MF) and microtubules (MT) significantly inhibited bacterial invasion. Localization of both microfilaments (MF) and microtubules (MT) was observed in INT407 cells following E. sakazakii infection. The results suggest that E. sakazakii invasion of INT407 cells involves participation of both MF and MT and bacterial OmpA plays a critical role in invasion.

BIOFILM FORMATION BY ENTEROBACTER SAKAZAKII GROWN IN ARTIFICIAL BROTH AND INFANT MILK FORMULA ON PLASTIC SURFACE

ABSTRACT Biofilm‐forming characteristics of 72 different Enterobacter sakazakii strains on plastic surface were tested. And the influence of the artificial growth medium and infant milk formula (IMF) was also determined. Quantification of biofilm formation was performed with medium including brain heart infusion broth, nutrient broth, tryptic soy broth, 1/20 diluted tryptic soy broth (1/20‐TSB) and differently diluted IMF on plastic microtiter plates. According to the result, reconstituted IMF was the more effective medium for biofilm formation than artificial medium. At normal IMF concentration, 56 strains produced biofilm (77.8%) but most suitable artificial medium and 1/20‐TSB; 12 strains produced biofilm (16.7%). These results indicated that the IMF offers more opportunities of biofilm formation on plastic surface. Therefore, all bottles and equipment should be effectively cleaned for preventing biofilm formation by IMF and E. sakazakii.PRACTICAL APPLICATIONS E. sakazakii could produce biofilm and protect the cells from being affected by disinfectants. It is also more difficult to wash such bacteria off from the surface they attached with biofilm and the chance of cross‐contamination could increase. The ability of biofilm formation could be influenced by different strains and also the growing media. By this study, we observed that IMF is the most effective media for biofilm formation among all the other media tested. Since IMF is the most important vehicle and source of E. sakazakii, it is important to surely clean and disinfect all the processing facilities in the factory and all the utensils, bottles, or teats used for IMF preparation. As biofilm would be formed when contact time increased, it is recommended not to sit the feeding bottles with IMF for a prolonged time to prevent biofilm formation and therefore minimize the opportunity of E. sakazakii infection.

Evaluation of multiple strains of Enterobacter sakazakii using fatty acid profiles

Fatty acid profiles are useful for identifying Gram-negative Enterobacter sakazakii strains within the family Enterobacteriaceae. The majority of cases of E. sakazakii infection have involved sepsis, meningitis, or enteritis, especially in neonates and infants. Gas chromatography with flame ionization detection (GC-FID) was utilized for the analysis of cellular fatty acid methyl esters (FAMEs). Thirty E. sakazakii strains isolated from food and environmental sources were cultured for 24 h on brain heart infusion (BHI) agar on three different days at 37 °C. Whole cell FAMEs were obtained by saponification, methylation and extraction into hexane:methyl tert-butyl ether. The day to day variations for the 30 E. sakazakii strains for each fatty acid were determined. Gram-negative bacteria commonly contain combinations of straight-chain, unsaturated, hydroxyl, and cyclo fatty acids. Major fatty acids of E. sakazakii strains evaluated in this study were straight chain 12:0, 14:0, 16:0 and unsaturated 16:1, 18:1, and 17:0 ω cyclo 7–8. Analysis of FAMEs from E. sakazakii strains grown on BHI agar by this rapid GC-FID method is highly reproducible and provides a sensitive procedure for identification of this organism. The fatty acid profile assay could be used to rapidly screen infant formula samples for E. sakazakii and reduce the time required for the current assay by up to 5 days.

Enterobacter sakazakii bacteriophages can prevent bacterial growth in reconstituted infant formula

Reconstituted infant formula has been implicated in outbreaks of Enterobacter sakazakii infections, causing high mortality and serious sequelae. Current prevention methods appear to be insufficient to ensure that such foods are free of E. sakazakii. In this study, the usefulness of bacteriophages for biocontrol of E. sakazakii was investigated. Of a total of six new E. sakazakii phages isolated from sewage and UV irradiated cultures, two were selected for further study by electron microscopy, DNA restriction analysis and SDS-PAGE of structural proteins. Purified phages were used to control bacterial growth in broth medium and reconstituted infant formula. Both phages effectively prevented development of E. sakazakii in formula at various temperatures (12, 24 and 37 °C), the efficiency of which was dependent upon intrinsic lysis properties and the applied phage concentration. We conclude that application of specific bacteriophages may provide a means for efficient prevention of E. sakazakii infection through reconstituted infant formula.

Adhesive properties of Enterobacter sakazakii to human epithelial and brain microvascular endothelial cells

BackgroundEnterobacter sakazakii is an opportunistic pathogen that has been associated with sporadic cases and outbreaks causing meningitis, necrotizing enterocolitis and sepsis especially in neonates. However, up to now little is known about the mechanisms of pathogenicity in E. sakazakii. A necessary state in the successful colonization, establishment and ultimately production of disease by microbial pathogens is the ability to adhere to host surfaces such as mucous membranes, gastric and intestinal epithelial or endothelial tissue.This study examined for the first time the adherence ability of 50 E. sakazakii strains to the two epithelial cell lines HEp-2 and Caco-2, as well as the brain microvascular endothelial cell line HBMEC. Furthermore, the effects of bacterial culture conditions on the adherence behaviour were investigated. An attempt was made to characterize the factors involved in adherence.ResultsTwo distinctive adherence patterns, a diffuse adhesion and the formation of localized clusters of bacteria on the cell surface could be distinguished on all three cell lines. In some strains, a mixture of both patterns was observed. Adherence was maximal during late exponential phase, and increased with higher MOI. The adhesion capacity of E. sakazakii to HBMEC cells was affected by the addition of blood to the bacteria growth medium. Mannose, hemagglutination, trypsin digestion experiments and transmission electron microscopy suggested that the adhesion of E. sakazakii to the epithelial and endothelial cells is mainly non-fimbrial based.ConclusionAdherence experiments show heterogeneity within different E. sakazakii strains. In agreement with studies on E. cloacae, we found no relationship between the adhesive capacities in E. sakazakii and the eventual production of specific fimbriae. Further studies will have to be carried out in order to determine the adhesin(s) involved in the interaction of E. sakazakii with cells and to enhance knowledge of the pathogenesis of E. sakazakii infection.

Survival and Growth of Enterobacter sakazakii on Fresh-Cut Fruits and Vegetables and in Unpasteurized Juices as Affected by Storage Temperature

Enterobacter sakazakii is an emerging foodborne pathogen that has caused illnesses and deaths in infants and elderly immunocompromised adults. Outbreaks of E. sakazakii infection have been associated with infant formulas, but the documented presence of this pathogen in a wide variety of ready-to-eat foods, including lettuce and other raw vegetables, makes it important to learn more about its behavior in these environments. We investigated the survival and growth characteristics of E. sakazakii on fresh-cut apple, cantaloupe, strawberry, watermelon, cabbage, carrot, cucumber, lettuce, and tomato and in juices prepared from these fruits and vegetables. Produce and juices were inoculated with E. sakazakii at 2 to 3 log CFU/g and 1 to 2 log CFU/ml, respectively, and stored at 4, 12, or 25°C. Populations either did not change or gradually decreased in fresh-cut produce and juices stored at 4°C but grew at 12°C on fresh-cut apple, cantaloupe, watermelon, cucumber, and tomato and in all juices except apple, strawberry, cabbage, and tomato juices. All fresh-cut fruits and vegetables except strawberry supported growth of E. sakazakii at 25°C. Growth occurred in all juices except apple, strawberry, and cabbage juices, followed by decreases in population to <1 CFU/ml after 48 to 72 h, which coincided with decreases in pH and an increase in the population of lactic acid bacteria. Increases in total counts occurred in all juices except strawberry juice stored at 25°C and apple and strawberry juices stored at 12°C. Total counts increased in cantaloupe, carrot, cucumber, and lettuce juices stored at 4°C. Populations of molds and yeasts increased in apple and tomato juices stored at 25°C but decreased to <1 CFU/ml in cabbage, lettuce, and cucumber juices. Further characterization of the behavior of E. sakazakii on fresh produce and in unpasteurized juice as affected by commercial packaging and handling practices is needed.

New Investigator Award, Blue Ribbon Abstract Award: Fatal Enterobacter sakazakii infection associated with powdered infant formula in a neonatal intensive care unit in New Zealand

BACKGROUND: Enterobacter sakazakii (E. sakazakii), a gram-negative bacillus of the Enterobacteriacae family, is a rare cause of meningitis, bacteremia, and necrotizing enterocolitis in infants. The population at particular risk is infants born prematurely, of low birthweight, or with compromised immunity. In 2004, an outbreak investigation was initiated in our neonatal intensive care unit (NICU) when a 5-day-old premature baby was found to be growing E. sakazakii in blood culture. The index case (Baby A) was born at 33 weeks gestation and admitted to NICU for observation. She received few medications and was fed with a combination of expressed breastmilk and infant formula. Five days after birth, Baby A became unwell. Despite treatment, she suffered progressive neurological damage and died on day 18. METHODS: The organism identification was confirmed and background microbiology isolate rates ascertained: 0-2 isolates per year were found, but no isolate had originated from NICU. A review of international literature revealed an association between E. sakazakii infection in neonates and the use of powdered infant formula. The cohort of neonates present in NICU had feces samples collected for culture along with demographic and risk factor/exposure data. A case patient was any neonate diagnosed with E. sakazakii infection (confirmed or suspected) or colonization who had been in NICU during the 5-day study period. Samples of medications, water, powdered infant formula, and breastmilk and samples from surfaces and equipment in formula preparation areas were among environmental specimens cultured. All aspects of infant formula preparation and use were reviewed, along with patient care practices within NICU. Use of sterile ready-to feed formula was implemented. RESULTS: Of 24 babies screened, four babies were identified as being colonized with E. sakazakii. None became clinically unwell. Two types of powdered infant formula were positive for E. sakazakii. Pulsed-field gel electrophoresis (PFGE) analysis found that E. sakazakii isolated from the index case, all four colonized cases, and one batch of powdered infant formula were all indistinguishable. The index case and two colonized babies had received this type of formula. CONCLUSIONS: Powdered infant formula is not a sterile product and may be contaminated with E. sakazakii. International recommendations advocating the use of sterile liquid formula for NICU babies should be widely publicized.

Enterobacter sakazakii Infections in Neonates Associated with Intrinsic Contamination of a Powdered Infant Formula

AbstractWe report an outbreak of Enterobacter sakazakii infection and colonization in neonates related to an infant formula contaminated during the manufacturing process. The outbreak occurred in a 2O-bed neonatal intensive care unit during a six-week period in 1988, and involved a total of four infants. Three infants had sepsis and three had bloody diarrhea; all patients responded to intravenous antibiotics and recovered without complications. The E sakazakii isolated from the formula had the same plasmid and multilocus enzyme profile as those isolated from patients. This outbreak demonstrates the significance of commercially contaminated formulas and emphasizes the need to limit contamination and multiplication of bacteria in enteral formulas.