T11-08 Quantification of Six Major Non-O157 Escherichia coli Serogroups in Feces of Feedlot Cattle by Spiral Plating and Quantitative Real-time PCR Methods

Tuesday, July 28, 2015: 10:30 AM
C124 (Oregon Convention Center)
Pragathi Belagola Shridhar, Kansas State University, Manhattan, KS
Lance Noll, Kansas State University, Manhattan, KS
Ellen Kim, Kansas State University, Manhattan, KS
Charley Cull, Kansas State University, Manhattan, KS
Diana Dewsbury, Kansas State University, Manhattan, KS
Xiaorong Shi, Kansas State University, Manhattan, KS
Natalia Cernicchiaro, Kansas State University, Manhattan, KS
David Renter, Kansas State University, Manhattan, KS
Jianfa Bai, Kansas State University, Manhattan, KS
TG Nagaraja, Kansas State University, Manhattan, KS
Introduction: Shiga Toxin-producing non-O157 E. coli of serogroups O26, O45, O103, O111, O121 and O145 are major foodborne pathogens.  Cattle are a major reservoir and shed the organisms in the feces.  Data on fecal concentration of non-O157 E. coli in cattle is limited because of lack of validated quantification methods.  

Purpose: To evaluate the applicability of spiral plating method and multiplex real-time quantitative PCR (qPCR) to quantify six non-O157 E. coli serogroups in cattle feces.  

Methods: Cattle fecal samples (n = 1,152) collected from eight commercial feedlots were suspended in E. coli broth.  Diluted fecal suspensions were spiral plated onto selective chromogenic medium.  Concentration (CFU/g) was determined by counting chromogenic colonies using a counting grid and testing ten randomly picked colonies individually by a multiplex conventional PCR targeting six serogroups and four virulence genes.  Concentration of each serogroup was determined based on the proportion of colonies positive for the serogroup.  DNA extracted from fecal suspensions was subjected to two qPCR assays: Assay 1 - O26, O103, and O111; Assay 2 - O45, O121, and O145.  Fecal suspensions incubated at 40°C for 6 h were subjected to a culture method for the detection of six non-O157 E. coli serogroups.

Results: A total of 787 (68.3%) samples were positive by culture method for at least one of the six non-O157 E. coli serogroups, and of those 139 (17.7%) were quantifiable (> log 2.9) by spiral plating method and 493 (42.8%; > log 4) by qPCR assays.  E. coli O103 was the predominant serogroup quantified by spiral plating method (8.7%) and qPCR (25.5%).  

Significance: Spiral plating method can be used to determine the concentration of non-O157 E. coli serogroups and those that carry Shiga Toxin genes in cattle feces.  The qPCR assays are more sensitive but allow quantification only at the serogroup level.