Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that cause illnesses in humans ranging from mild to hemorrhagic enteritis with complications of hemolytic uremic syndrome and even death. Cattle are a major reservoir of STEC, which reside in the hindgut and are shed in the feces, a major source of food and water contaminations. Seven serogroups, O26, O45, O103, O111, O121, O145 and O157, called ‘top-7’, are responsible for the majority of human STEC infections in North America. Additionally, 151 serogroups of E. coli are known to carry Shiga toxin genes (stx). Not much is known about fecal shedding and prevalence and virulence potential of STEC other than the top-7. Our primary objectives were to identify serogroups of STEC strains, other than the top-7, isolated from cattle feces and subtype stx genes to assess their virulence potential. Additional objective was to develop and validate a novel multiplex PCR assay to detect and determine prevalence of six serogroups, O2, O74, O109, O131, O168, and O171, in cattle feces. A total of 351 strains, positive for stx gene and negative for the top-7 serogroups, isolated from feedlot cattle feces were used in the study. Of the 351 strains, 291 belonged to 16 serogroups and 60 could not be serogrouped. Among the 351 strains, 63 (17.9%) carried stx1 gene and 300 (82.1%) carried stx2, including 12 strains positive for both. The majority of the stx1 and stx2 were of stx1a (47/63; 74.6%) and stx2a subtypes (234/300; 78%), respectively, which are often associated with human infections. A novel multiplex PCR assay developed and validated to detect six serogroups, O2, O74, O109, O131, O168, and O171, which accounted for 86.9% of the STEC strains identified, was utilized to determine their prevalence in fecal samples (n = 576) collected from a commercial feedlot. Four serogroups, O2, O109, O168, and O171 were identified as the dominant serogroups prevalent in cattle feces. In conclusion, cattle shed in the feces a number of STEC serogroups, other than the top-7, and the majority of the strains isolated possessed stx2, particularly of the subtype 2a, suggesting their potential risk to cause human infections.
Escherichia coli carrying prophage with genes that encode for Shiga toxins are categorized as Shiga toxin-producing E. coli (STEC) pathotype. Illnesses caused by STEC in humans, which are often foodborne, range from mild to bloody diarrhea with life-threatening complications of renal failure and hemolytic uremic syndrome and even death, particularly in children. As many as 158 of the total 187 serogroups of E. coli are known to carry Shiga toxin genes, which makes STEC a major pathotype of E. coli. Seven STEC serogroups, called top-7, which include O26, O45, O103, O111, O121, O145, and O157, are responsible for the majority of the STEC-associated human illnesses. The STEC serogroups, other than the top-7, called "non-top-7" have also been associated with human illnesses, more often as sporadic infections. Ruminants, particularly cattle, are principal reservoirs of STEC and harbor the organisms in the hindgut and shed in the feces, which serves as a major source of food and water contaminations. A number of studies have reported on the fecal prevalence of top-7 STEC in cattle feces. However, there is paucity of data on the prevalence of non-top-7 STEC serogroups in cattle feces, generally because of lack of validated detection methods. The objective of our study was to develop and validate 14 sets of multiplex PCR (mPCR) assays targeting serogroup-specific genes to detect 137 non-top-7 STEC serogroups previously reported to be present in cattle feces. Each assay included 7-12 serogroups and primers were designed to amplify the target genes with distinct amplicon sizes for each serogroup that can be readily identified within each assay. The assays were validated with 460 strains of known serogroups. The multiplex PCR assays designed in our study can be readily adapted by most laboratories for rapid identification of strains belonging to the non-top-7 STEC serogroups associated with cattle.
Salmonella contamination of poultry is a global public health problem. The objective of this study was to map the distribution of Salmonella on the young chicken carcass, to improve poultry inspection and food safety. Young chickens (n = 70) in the Cornish game hen class were obtained at retail over a 3-year period. Carcasses were aseptically sectioned into 12 parts, and then Salmonella was isolated from whole-part incubations by conventional culture methods. Isolates were characterized for serotype and antibiotic resistance, and by pulsed-field gel electrophoresis (PFGE). Salmonella incidence was 21.5% (181 of 840) for parts and 57.1% (40 of 70) for carcasses. The number of contaminated parts per carcass ranged from 0 to 12, with a mean of 4.5 among contaminated carcasses. Chi-square analysis indicated that Salmonella incidence differed (P < 0.05) among parts, with rib back (38.6%) and sacral back (34.3%) being the most contaminated. Among the 40 contaminated carcasses, there were 37 different patterns of contamination among parts. Of the 33 carcasses with more than one contaminated part, 12.1% contained two serotypes, 33.3% contained two or more antibiotic resistance profiles, and 100% contained two or more PFGE patterns. The most common serotype was Typhimurium (94.5%), and most (97.2%) isolates were resistant to multiple antibiotics. These results indicated a diverse pattern of Salmonella contamination among carcasses and that multiple subtypes of Salmonella were often present on contaminated carcasses. Thus, whole-carcass incubation succeeded by characterization of multiple isolates per carcass is needed to properly assess and manage this risk to public health.
Escherichia coli O103, harbored in the hindgut and shed in the feces of cattle, can be enterohemorrhagic (EHEC), enteropathogenic (EPEC), or putative non-pathotype. The genetic diversity particularly that of virulence gene profiles within O103 serogroup is likely to be broad, considering the wide range in severity of illness. However, virulence descriptions of the E. coli O103 strains isolated from cattle feces have been primarily limited to major genes, such as Shiga toxin and intimin genes. Less is known about the frequency at which other virulence genes exist or about genes associated with the mobile genetic elements of E. coli O103 pathotypes. Our objective was to utilize whole genome sequencing (WGS) to identify and compare major and putative virulence genes of EHEC O103 (positive for Shiga toxin gene, stx1, and intimin gene, eae; n = 43), EPEC O103 (negative for stx1 and positive for eae; n = 13) and putative non-pathotype O103 strains (negative for stx and eae; n = 13) isolated from cattle feces. Six strains of EHEC O103 from human clinical cases were also included. All bovine EHEC strains (43/43) and a majority of EPEC (12/13) and putative non-pathotype strains (12/13) were O103:H2 serotype. Both bovine and human EHEC strains had significantly larger average genome sizes (P < 0.0001) and were positive for a higher number of adherence and toxin-based virulence genes and genes on mobile elements (prophages, transposable elements, and plasmids) than EPEC or putative non-pathotype strains. The genome size of the three pathotypes positively correlated (R2 = 0.7) with the number of genes carried on mobile genetic elements. Bovine strains clustered phylogenetically by pathotypes, which differed in several key virulence genes. The diversity of E. coli O103 pathotypes shed in cattle feces is likely reflective of the acquisition or loss of virulence genes carried on mobile genetic elements.
The current study was undertaken to evaluate chlorine resistance among strains of Salmonella Kentucky isolated from chicken carcasses. Selected strains (n = 8) were exposed to 30 ppm of chlorine in 10% buffered peptone water (pH 7.4) for 0 to 10 min at 4°C and 150 rpm. The initial level (mean ± SD) of Salmonella Kentucky was 6.18 ± 0.09 log CFU/ml and did not differ (P > 0.05) among strains. A two-way analysis of variance indicated that the level of Salmonella Kentucky in chlorinated water was affected (P < 0.05) by a time by strain interaction. Differences among strains increased as a function of chlorine exposure time. After 10 min of chlorine exposure, the most resistant strain (SK145) was 5.63 ± 0.54 log CFU/ml, whereas the least resistant strain (SK275) was 3.07 ± 0.29 log CFU/ml. Significant differences in chlorine resistance were observed for most strain comparisons. Death of Salmonella Kentucky was nonlinear over time and fitted well to a power law model with a shape parameter of 0.34 (concave upward). Time (minutes) for a 1-log reduction of Salmonella Kentucky differed (P < 0.05) among strains: >10 min for SK145, 6.0 min for SK254, 1.5 min for SK179, and 0.3 to 0.65 min for other strains. Results of this study indicate that strain is an important variable to include in models that predict changes in levels of Salmonella Kentucky in chlorinated water.
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