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Genotypic relatedness of bison and bovine fecal isolates of enterobacter sakazakii to environmental, clinical, and food isolates by pulsed-field gel electrophoresis.

Enterobacter sakazakii continues to be implicated in neonatal infections such as meningitis and necrotizing enterocolitis. Powdered infant formula has been reported to be the most common source of contamination for infected neonates, but the natural habitat of this opportunistic pathogen has been elusive. In addition, the epidemiology and pathogenicity are currently not well understood (1, 2). Research in our laboratory at North Dakota State University has determined that E.sakazakii is present in bison and bovine feces; however, it has not been proved that this is the primary source of E.sakazakii contamination in powdered infant formula. The primary objective of this study was to determine the relatedness of E. sakazakii from various sources, including food, clinical settings, the environment, or from bovine or bison feces using Pulsed-field gel electrophoresis.

Methods. Enterobacter sakazakii isolates obtained from the American Type Culture Collection, North Dakota bison and bovine feces, and one clinical isolate from a neonatal meningitis case were used from our laboratory. Several food, environmental, and clinical isolates were obtained from Cornell University. All isolates were digested with the restriction enzyme XbaI and subjected to pulsed-field gel electrophoresis (PFGE). Analysis was performed using methods from the Fingerprinting II Informatix manual, and combined dendrograms were obtained and compared.

Results. The 40 isolates tested contained anywhere from 9 to 30 DNA fragments indicating a high degree of diversity among the isolates. The Cornell isolates F6-049 and F6-051 had the highest degree of similarity; both of these isolates were from a clinical source. The isolates from bison and bovine feces, designated 52 and N72, respectively, had only 25% similarity to each other. The bison isolate (52) displayed 65% similarity to an isolate from a food source. The bovine isolate (N72) had a 55% similarity to an isolate from a clinical source.

Discussion. Previous studies have found that PFGE is a good measure of genotypic relatedness between Enterobacter sakazakii isolates (3). The results of this study show that there is a high degree of diversity among Enterobacter sakazakii from multiple sources. This also indicates that there is a great deal of genetic diversity among species of Enterobacter sakazakii. However, this does not definitively prove that the isolates from bovine and bison feces are definite pathogens of neonates or the cause of powdered infant formula contamination. Comparison of additional isolates will aid in determining whether the bovine and bison isolates are a potential source of powdered infant formula contamination.


(1.) Restaino L., E. Frampton, W. Linoberg and J. Becker. 2005. A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients and environmental sources. J. Food Prot. 69, 315-322.

(2.) Mutjens, H., and L. Kollee, 1990. E. sakazakii meningitis in neonates: causative role of formula. Pediatr. Infect. Dis. J. 9, 372-372.

(3.) Proudy, I., D. Bougle, E. Coton, M. Coton, R. Leclercq, and M. Vergnaud. 2008. Genotypic characterization of Enterobacter sakazakii isolates by PFGE, BOX-PCR, and sequencing of the fliC gene. J. Appl. Micro. 104:26-34.

Tracy A. Solseng *, Tara Johnson, Liilian M. Nangoh, Heather Vinson,

Margaret Khaitsa and Penelope S. Gibbs

Department of Veterinary and Microbiological Sciences, Great Plains Institute of Food Safety, School of

Food Systems, North Dakota State University, Fargo, ND 58105
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Author:Solseng, Tracy A.; Johnson, Tara; Nangoh, Lillian M.; Vinson, Heather; Khaitsa, Margaret; Gibbs, Pen
Publication:Proceedings of the North Dakota Academy of Science
Article Type:Report
Geographic Code:1USA
Date:Apr 1, 2008
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