Further development of sample preparation and detection methods for O157 and the top 6 non-O157 STEC serogroups in cattle feces
Introduction
As production of food and agricultural products in large centralized facilities increases, so does the risk of transmission of pathogenic microorganisms. Shiga toxin-producing Escherichia coli (STEC) are food borne pathogens responsible for mild to severe illness in humans, sometimes leading to hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) (Hussein, 2007). For decades, one STEC serotype (O157:H7) was considered to be a major public health concern (Bettelheim, 2007). More recently it has been estimated that 20–50% of STEC infections are caused by non-O157 serogroups, equivalent to 37,000 illnesses annually in the U.S. (Brooks et al., 2005, Johnson et al., 2006). Six non-O157 serogroups (O26, O45, O103, O111, O121, O145) are identified as a major public health concern due to their frequent association with HC and HUS (Brooks et al., 2005, Johnson et al., 2006). The U.S. Department of Agriculture Food Safety and Inspection Service (USDA-FSIS) now includes the “big-6” non-O157 serogroups as adulterants in beef trim (U.S. Department of Agriculture, 2011). Cattle have been identified as a main reservoir for STEC, and contaminated beef products have been implicated as sources of human infection (Bettelheim, 2007, Gill and Gill, 2010, Hussein, 2007). The association between STEC serotypes found in cattle and those causing human illness is not well understood (Norman et al., 2012).
Food safety monitoring systems are vital, but are only as sensitive, reliable, and feasible as their applied methodologies. Determining STEC prevalence in samples of cattle origin is complex. Effective methods are available for detection of O157, but the same methods cannot be applied to the non-O157 serogroups due to the complexity and diversity of these pathogens, which has prohibited the development of a standardized isolation and culturing method (Bettelheim, 2007, Norman et al., 2012). Genes for synthesis of the outer membrane O-antigens (wzx, wzy) can be used to discriminate STEC based on serogroup in molecular assays (Lin et al., 2011). Several PCR assays have been developed for such serogroup-based detection in food and carcass samples (Fratamico et al., 2011, Lin et al., 2011, Monday et al., 2007, Paton and Paton, 1998, Wang et al., 2013), however few studies have thoroughly evaluated PCR for sensitivity and accuracy for detection of STEC serogroups in cattle feces (Bai et al., 2012, Paddock et al., 2012). Cattle feces are a complex matrix, complete with unique challenges such as assay inhibitors (Wang et al., 2013), which necessitates further development of feces-specific detection and isolation procedures.
The abundance and proximity of E. coli and stx-encoding bacteriophage across diverse environments allow for dynamic exchange of mobile genetic elements resulting in ever evolving STEC genomes and pathogenicity (Allison, 2007, Brandt et al., 2011). Due to globalization and industrial centralization of the primary and secondary food production chains, the focus and transfer of food related pathogens and their mobile genetic elements across global distances are more possible than ever (Chase-Topping et al., 2008). The potential for emerging and novel pathogens to enter the food supply is invariably present. Therefore, monitoring systems are imperative to secure food safety and minimize the associated human health risk. Effectively, monitoring systems must be reliable and sensitive. In order to gain a better understanding of the potential impact of non-O157 STEC in cattle and the associated human health risks, accurate and economically feasible serogroup detection methods are required. The main objective of this study was to validate and/or further develop detection methods for O157 and non-O157 STEC in cattle feces. Initially, we evaluated a current PCR assay (Lin et al., 2011) for the detection of potential STEC serogroups in feces, and subsequently enhanced the sensitivity for the detection of serogroups in cattle feces by developing a novel multiplex PCR (mPCR) assay. Both fresh and autoclaved feces (to eliminate potentially competing background microflora) were used to evaluate the effects of enrichment on PCR detection of STEC. Conventional culturing methods were compared to optical density (OD) measurements, and quantitative PCR (qPCR) serogroup gene copy numbers to allow for estimations of cell numbers between the different values.
Section snippets
Pure culture optical density, colony forming units, gene copy numbers
The procedures used for determining the limit of detection and ability of the assay to detect STEC associated serogroups in feces is outlined in Fig. 1. Limit of detection was determined using two serotypes of E. coli, O157:H7 (EDL933; Public Health Agency of Canada) and O26:H11 (source: human; Laboratory for Foodborne Zoonoses, Public Health Agency of Canada, Guelph, Ontario, Canada), and E. coli ATCC 25922 (Public Health Agency of Canada) was used as a control. Cells were streaked on nutrient
Pure culture optical density, colony forming units, gene copy numbers
The OD, CFUs, and serogroup-specific DNA copy numbers were determined for pure cultures of O157:H7 and 26:H11 (Fig. 2). Average OD ranged from 0.01 to 0.78 and 0.01 to 0.97 from 0 to 8 h for O157:H7 and O26:H11 respectively. Average CFU/mL for O157:H7 ranged from 3.6 × 106 at time 0 to 1.5 × 109 at 8 h. Average CFU/mL for O26:H11 ranged from 4.7 × 106 to 2.6 × 109 from 0 to 8 h, respectively. Over 8 h, serogroup DNA copy numbers increased from 9.7 × 102 to 3.4 × 105 for O157 and 1.6 × 103 to 3.3 × 105 for O26 per
Discussion
In current assays, the detection and isolation of STEC most commonly involve enrichment, IMS, plating on selective or non-selective media for phenotypic identification, and latex agglutination with O-serogroup specific antiserum (primarily for O157) (Wang et al., 2013). Molecular assays utilizing the sensitivity of PCR to screen for genes coding: O-antigen synthesis, Shiga toxins, and flagellar gene targets are often utilized (Paddock et al., 2012). Serogroup O157 can be distinguished from
Conclusion
Advances in molecular biology and other technological progress hold the potential to improve the utility of detection assays for potentially pathogenic microorganisms within the food chain. Enhanced detection of STEC can decrease the market circulation of products compromising human health, aid in diagnostics, and support outbreak management. Our multiplex PCR assay to detect seven potentially pathogenic serogroups has been verified for a number of advantages over previous methods including
Acknowledgements
The authors would like to thank Vic Gannon and Roger Johnson (Public Health Agency of Canada, Guelph, Ontario) for donating the STEC strains used in this study. We thank Jenilee Peters, Susanne Trapp, Yidong Han, Ruth Barbieri, and Amanda Harvey for their assistance in the laboratory. For expertise and valuable discussion, we thank Rahat Zaheer, Brandon Gilroyed and Shaun Cook. This research was supported by a grant from Alberta Innovates BioSolutions (2014R036R).
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