Cronobacter Research Laboratory
Research Scientist: Dr. Franco Pagotto
Research Technical Support : Karine Hébert, Kevin Tyler,
Bureau of Microbial Hazards, Health Canada
Enterobacter sakazakii, is an emerging foodborne pathogen that has drawn attention to the scientific community for the last 50 years. This organism was first characterized in 1929 as "yellow-pigmented coliform" and was discovered to be the causative agent of septicemia in infants. In the 1960s the "yellow pigmented coliform" was suspected to also be the cause of two terminal neonatal meningitis. By the 1980s this pathogen was classified as a new species, Enterobacter sakazakii and was found to severely affect infants and neonates by causing sepsis, necrotic enterocolitis and meningitis.
This organism was reclassified into the new genus Cronobacter in 2008. The type species is Cronobacter sakazakii. Other species within this genus now include Cronobacter malonaticus, Cronobacter turicensis, Cronobacter genomospecies 1, Cronobacter muytjensii, Cronobacter dublinensis subsp. dublinensis, Cronobacter dublinensis subsp. lausannensis, and Cronobacter dublinensis subsp. lactaridi.
While the natural reservoir for Cronobacter spp. remains unknown, it has been isolated from a variety of foods (powdered infant formula, fermented bread, raw lettuce, raw rice, minced beef, sausage meat, sous and tamarind drinks, fermented cassava, crab meat, cheese, fresh and dried foods, soybean, herbs, spices) as well as different environment (hospitals, processing plants, homes, dust, vacuum cleaners, even in the intestines of stable flies).
The vast majority of Cronobacter spp. infections happen in hospital neonatal intensive care units. Improper preparation, handling and storage of infant formula are considered the most likely causes of contamination. Powdered infant formulas (PIF) are not commercially sterile products. Cronobacter is able to survive in PIF and so there is a risk of infection when reconstituted formula (powdered formula with water added to it) is kept at room temperature for prolonged periods of time as Cronobacter grows very quickly at room temperature.
Investigations have revealed that infant formula can become contaminated with Cronobacter spp.:
- through the raw material used for producing the formula;
- through contamination of the formula or other dry ingredients after pasteurization;
- through contamination of the formula as it is being reconstituted by the caregiver just prior to feeding.
Cronobacter spp. can cause illness in all age groups, but infants (children less than 1 year old) who are fed reconstituted PIF are at higher risk. Those at greatest risk for Cronobacter spp. infection are neonates (first 28 days), particularly pre-term, low birth weight or immuno-compromised infants. Infants of HIV-positive mothers are also at risk both because they may specifically require infant formula and may be more susceptible to infection.
Cronobacter spp. are considered emerging pathogens for stroke patients who suffer from dysphagia and aspiration pneumonia. It is thought that Cronobacter may be able to colonise the mouths of patients with acute stroke, with possible development of antibiotic resistance to commonly used antibiotics used to treat aspiration pneumonia. Cronobacter has also been considered to be a contaminant of fluid thickeners such as modified maize starch powders, and food supplements that include milk shakes and build-up soups.
The long-term goals of our laboratory include the following:
- Develop a non-primate animal model to assess the virulence and pathogenicity of Cronobacter species;
- Assess whether all strains of Cronobacter are pathogenic, and whether there are differences in virulence amongst strains;
- Develop a dose-response model for Cronobacter using a non-primate animal model;
- Determine the virulence factors of Cronobacter species;
- Determine if other Enterobacteriaceae present in powdered infant formula are involved in causing illness in neonates
Current research activities also include the following:
- Molecular characterization using riboprinting and pulsed-field gel electrophoresis (PFGE) of in-house strains of Cronobacter species;
- Develop a standardized (PFGE) protocol as part of a PulseNet International collaboration effort;
- Compare current methodologies (e.g., FDA, ISO) for isolation of Cronobacter species in powdered infant formula. This is ongoing with method development being done in-house;
- Undertake full genome sequencing in collaboration with McGill University / Genome Quebec
F.J. Pagotto, R.F. Lenati and J.M. Farber. 2008. Enterobacter sakazakii. In M.P. Doyle and L.R. Beuchat (ed.), Food Microbiology: Fundamentals and Frontiers, 3rd edition, ASM Press, pp. 271-291.
J.M. Farber, F.J. Pagotto and J.-L. Cordier. 2008. Regulatory aspects. In J.M. Farber and S.J. Forsythe (ed.), Enterobacter sakazakii, ASM Press, pp. 235-253.
J.M. Farber, F.J. Pagotto and R. Lenati. 2008. Pathogenicity of Enterobacter sakazakii. In J.M. Farber and S.J. Forsythe (ed.), Enterobacter sakazakii, ASM Press, pp. 127-144.
Pagotto, F.J., Nazarowec White, M., Bidawid, S., and Farber, J.M. 2003. Virulence properties of Enterbacter sakazakii: infectivity and enterotoxin production in vitro and in vivo. Journal of Food Protection 66: 370-375.
R.F. Lenati, D.L. O'Connor, K.C. Hébert, J.M. Farber, and F.J. Pagotto. 2008. Growth and survival of Enterobacter sakazakii in human breast milk with and without fortifiers as compared to powdered infant formula. International Journal of Food Microbiology 122: 171-179.
G. Palcich, C. de Moraes Gillio, L.C. Aragon-Alegro, F.J.. Pagotto, J.M. Farber, M. Landgraf., and M.T. Destro. 2009. Enterobacter sakazakii in dried-infant formulas and milk kitchens of maternity wards in Sao Paulo, Brazil. Journal of Food Protection 72: 37-42.
MacLean, L.L., Pagotto, F., Farber, J.M., and Perry, M.B. 2009. The structure of the O-antigen in the endotoxin of the emerging food pathogen Cronobacter (Enterobacter) muytjensii strain 3270. Carbohydrate Research 344: 667-671.
MacLean, L.L., Pagotto, F., Farber, J.M., and Perry, M.B. 2009. Structure of the antigenic repeating pentasaccharide unit of the LPS O-polysaccharide of Cronobacter sakazakii implicated in the Tennessee outbreak. Biochemistry and Cell Biology 87: 459-465.
Pagotto, F.J., and Farber, J.M. 2009. Cronobacter spp. (Enterobacter sakazakii): Advice, policy and research in Canada. International Journal of Food Microbiology 136: 238-245.
MacLean, L.L., Vinogradov, E., Pagotto, F., Farber, J.M., and Perry, M.B. 2009. Characterization of the O-antigen in the lipopolysaccharide of Cronobacter (Enterobacter) malonaticus 3267. Biochemistry and Cell Biology 87: 927-932.
Miled-Bennour, R., Ells, T.C., Pagotto, F.J., Farber, J.M., Kérouanton, A., Meheut, T., Colin, P., Joosten, H., Leclercq, A., and Besse, N.G. 2010. Genotypic and phenotypic characterisation of a collection of Cronobacter (Enterobacter sakazakii) isolates. International Journal of Food Microbiology 139: 116-125.
MacLean, L.L., Vinogradov, E., Pagotto, F., Farber, J.M., and Perry, M.B. 2010. The structure of the O-antigen of Cronobacter sakazakii isolate HPB2855 involved in a neonatal infection. Carbohydrate Research 345:1932-1937.
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