Characterization of a multidrug resistant C. difficile meat isolate
Introduction
Clostridium difficile is a major cause of enteric disease in humans, with fecal–oral route as the primary mode of transmission. C. difficile disease has been traditionally regarded as a nosocomial infection in humans, especially in those receiving prolonged antimicrobial therapy (Bartlett, 1997, Hookman and Barkin, 2009, McDonald et al., 2006). However, in recent years, an increase in the number and severity of C. difficile infection in humans has been reported, particularly in those involving communities outside the hospital environment and low risk subjects (McDonald et al., 2006, Rupnik et al., 2009). Recent studies conducted by investigators worldwide have indicated the occurrence of C. difficile in a variety of food animals. A rise in isolation rates of the pathogen from animal reservoirs is one potential reason attributed to the increased reports of human C. difficile infections (Rupnik et al., 2009, Thitaram et al., 2011). In addition, C. difficile has been isolated from raw and ready-to-eat meats at retail stores (Harvey et al., 2011a, Songer et al., 2009, Weese et al., 2009, Weese et al., 2010). The detection of genotypically similar and identical C. difficile strains implicated from human infections in foods and food animals (Rodriguez-Palacios et al., 2011, Weese et al., 2011) further strengthens the potential role of food as a source of community associated C. difficile disease (Marsh et al., 2011, Songer et al., 2009). Although a limited number of studies in the U.S. investigated the prevalence of C. difficile in meats, an in-depth characterization of the food isolates has not been performed. The prevalence of C. difficile in foods observed in previous investigations varies from 0 to 40% (Harvey et al., 2011b, Limbago et al., 2012, Songer et al., 2009, Weese et al., 2009). A report by the US Department of Health and Human Services reported the highest rate of C. difficile associated hospitalizations in the New England region in 2010 and 2011 (Steiner et al., 2012). The Centers for Disease Control and Prevention (CDC) in collaboration with the Connecticut Department of Public Health (DPH) has initiated the Emerging Infections Program (EIP) for C. difficile surveillance with the objectives (1) to determine the population-based incidence of community- and healthcare-associated C. difficile infection, and (2) to characterize C. difficile strains that are responsible for infection in the population under surveillance, with a focus on strains from community-associated cases.
Recent studies have suggested the possible emergence of new virulent or antibiotic resistant C. difficile strains by horizontal gene transfer between toxigenic and non-toxigenic strains (Brouwer et al., 2013). In addition, a closely related foodborne pathogen, Clostridium perfringens was found to acquire virulence by way of horizontal gene transfer in the gut environment (Kobayashi et al., 2009, Popoff and Bouvet, 2013). Moreover, the CDC has listed C. difficile as one of the three urgent threats in their recent report on emerging pathogens with antibiotic resistance (Antibiotic Resistance Threats in the United States, 2013). A genome-wide characterization of C. difficile food isolates would provide their virulence and antimicrobial resistance profiles, besides indicating the genetic relatedness and clonality between food and clinical isolates.
In this manuscript, as a part of investigating the prevalence of C. difficile in retail meat sold in Connecticut, we report the results on phenotypic and molecular characterization of a multidrug resistant C. difficile isolate from pork, and present the draft genome of the C. difficile meat isolate with special reference to its drug resistance.
Section snippets
Sample collection and detection of C. difficile in meat
A total of 100 samples each of ground beef, ground pork and chicken wings were procured from 23 geographically distant retail stores in Connecticut. All the procured samples were brought to the laboratory on ice and processed within 24 h of receipt. Qualitative detection of C. difficile was performed as described previously (Weese et al., 2010), with modification for a higher detection limit. Fifty grams each of ground beef, ground pork or a chicken wing was separately added to 50 ml of C.
Results and discussion
In this study, we investigated the prevalence of C. difficile in 100 samples each of ground beef, ground pork and chicken wings purchased from different geographic locations in Connecticut. We followed direct plating and selective enrichment followed by alcohol shock in CDMN agar. In order to increase the detection limit, a larger volume (1 ml) was pour plated instead of plating 100 μl on to CDMN agar. Also, the whole meat–CDMN broth mixture was enriched for further plating followed by an alcohol
Conclusion
In summary, we confirmed that C. difficile occurs at very low levels in raw meat sold in Connecticut. The isolates were non-toxigenic, but found to have an array of antibiotic resistance genes and mobile elements, which can potentially contribute to the generation of multi-drug resistant toxigenic strains. Further studies are warranted to delineate the source of contamination and the effect of cooking and other processing operations on the viability of C. difficile spores in meat.
The following
Nucleotide sequence accession numbers
This Whole Genome Shotgun project has been deposited in DDBJ/EMBL/GenBank under accession number JFAF00000000. The version described in this paper is the first version, JFAF01000000. http://www.ncbi.nlm.nih.gov/nuccore/JFAF00000000.
Acknowledgment
This research was funded by the United States Department of Agriculture—National Institute of Food and Agriculture (USDA-NIFA) Critical and Emerging Food Safety Issues program grant # 2010–03567.
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