CHARACTERIZATION, TYPING, AND DETECTION OF PATHOGENIC ESCHERICHIA COLI

Authors: Fratamico, Pina; Uhlich, Gaylen; Liu, Yanhong

Submitted to: UJNR Food & Agricultural Panel Proceedings
Publication Type: Proceedings
Publication Acceptance Date: 8/20/2005
Publication Date: 10/23/2005
Citation: Fratamico, P.M., Uhlich, G.A., Liu, Y. 2005. Characterization, typing, and detection of pathogenic escherichia coli. UJNR Food & Agricultural Panel Proceedings.

Interpretive Summary:

Technical Abstract: A clearer understanding of the ability of Escherichia coli O157:H7 to survive and persist in food and the environment and the availability of reliable methods for detection and identification of E. coli O157:H7 and other pathogenic E. coli will facilitate the development of more effective control strategies. To address these areas of research, Congo red-binding and -nonbinding phase variants of Escherichia coli O157:H7 strain ATCC 43895 were identified. The Congo red-binding variant, strain 43895OR, produced a dry, aggregative colony on Congo red plates that was similar to the red, dry and rough (rdar) phenotype characteristic of multicellular behavior in certain strains of Salmonella. In contrast, variant 43895OW produced a smooth and white (saw) colony morphology. In the current study, it was demonstrated that, like the rdar strains of S. Typhimurium, strain 43895OR formed large aggregates in broth cultures, firm pellicles on glass at the air-medium interface, and biofilm formation on both glass and polystyrene. However, unlike the rdar strains of Salmonella, strain 43895OR did not stain with calcofluor, an indicator of the beta-1,4 glucose linkages of cellulose. Scanning electron microscopy (SEM) of strain 43895OR fixed on agar showed large amounts of extracellular matrix (ECM) while strain 43895OW was totally devoid of ECM. However, SEM of biofilms indicated that strain 43895OW expressed ECM with curli fibers during attachment to glass. Strain 43895OR produced greater amounts of biofilm than strain 43895OW on polystyrene, glass, stainless steel, and Teflon, and biofilm formation was greater on polystyrene in rich rather than nutrient-limited medium. Biofilm cells of both strains showed increased resistance to hydrogen peroxide and a quaternary ammonium sanitizer when compared to their respective planktonic cells. This study showed that the rdar phenotype of E. coli O157:H7 strain 43895OR is important in multicellular growth, biofilm formation, and resistance to sanitizers. In a second study, DNA microarrays were developed for rapid identification of different serogroups of E. coli on a single platform. Oligonucleotides, as well as PCR products from genes in the O-antigen gene clusters of E. coli serogroups O7, O104, O111, and O157, were spotted onto glass slides. This was followed by hybridization with labeled Long PCR products of the entire O antigen gene clusters of these serogroups. Results demonstrated that microarrays consisting of either oligonucleotides or PCR products generated specific signals for each serogroup. Thirdly, a real-time multiplex PCR assay was developed and used for detection of E. coli O157:H7 in food. The multiplex PCR, employing TaqMan probes and targeting the E. coli O157:H7 fliC or eae, stx1, stx2, and wzy genes, was used to detect E. coli O157:H7 inoculated at ca. 2 CFU/ 25g or 25 ml in apple cider, ground beef, lettuce, and raw milk after 8 h of enrichment in RapidChek Enrichment broth. This research furthers the understanding of the mechanism of biofilm formation in E. coli O157:H7 and provides the tools for rapid detection and identification of E. coli O157:H7 and other pathogenic E. coli in foods.