Susceptibility of Clostridium difficile to the food preservatives sodium nitrite, sodium nitrate and sodium metabisulphite Su-Chen Lim a , Niki F. Foster a, b , Thomas V. Riley a, b, * a Microbiology & Immunology, School of Pathology and Laboratory Medicine, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands 6009, Western Australia, Australia b Department of Microbiology, PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Nedlands 6009, Western Australia, Australia article info Article history: Received 30 July 2015 Received in revised form 30 November 2015 Accepted 12 December 2015 Available online 15 December 2015 Keywords: Clostridium difficile Ready-to-eat meat Sodium nitrite Sodium nitrate Sodium metabisulphite abstract Clostridium difficile is an important enteric pathogen of humans and food animals. Recently it has been isolated from retail foods with prevalences up to 42%, prompting concern that contaminated foods may be one of the reasons for increased community-acquired C. difficile infection (CA-CDI). A number of studies have examined the prevalence of C. difficile in raw meats and fresh vegetables; however, fewer studies have examined the prevalence of C. difficile in ready-to-eat meat. The aim of this study was to investigate the in vitro susceptibility of 11 C. difficile isolates of food animal and retail food origins to food preservatives commonly used in ready-to-eat meats. The broth microdilution method was used to determine the minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for sodium nitrite, sodium nitrate and sodium metabisulphite against C. difficile. Checkerboard assays were used to investigate the combined effect of sodium nitrite and sodium nitrate, commonly used in combination in meats. Modal MIC values for sodium nitrite, sodium nitrate and sodium meta- bisulphite were 250 mg/ml, >4000 mg/ml and 1000 mg/ml, respectively. No bactericidal activity was observed for all three food preservatives. The checkerboard assays showed indifferent interaction be- tween sodium nitrite and sodium nitrate. This study demonstrated that C. difficile can survive in the presence of food preservatives at concentrations higher than the current maximum permitted levels allowed in ready-to-eat meats. The possibility of retail ready-to-eat meats contaminated with C. difficile acting as a source of CDI needs to be investigated. © 2015 Elsevier Ltd. All rights reserved. 1. Introduction Clostridium difficile is an anaerobic, spore-forming, Gram-posi- tive bacillus widely found in soil, water and the gastrointestinal tracts of food animals and humans. It causes mild to severe diarrhea and, occasionally, the more serious pseudomembranous colitis and toxic megacolon in humans. Since 2000, there has been a global increase in C. difficile infection (CDI) with heightened severity and mortality, and a rise in community-acquired infection (CA-CDI) in individuals without traditional risk factors of old age or antibiotic usage [1e5]. This has been mainly due to the emergence of so- called hypervirulent strains of C. difficile, particularly PCR- ribotypes 027 and 078, that produce binary toxin (CDT) in addi- tion to toxins A and B. Recently, C. difficile has been found in retail meats, seafoods and vegetables with prevalences up to 42% [6e12]. C. difficile of the same ribotype has been found in foods, food animals and humans [13]. In Canada, Weese et al. (2009) found C. difficile ribotype 078, common in food animals and a cause of disease in humans, in ground meats and poultry [14,15]. In Scotland, ready-to-eat salads were contaminated with C. difficile ribotypes 017 and 001; both are common clinical isolates in Scotland and Europe [16e18]. These findings have led to growing concern that retail foods contami- nated with C. difficile may be one of the reasons for the increased incidence of CDI, particularly in the community. Despite the po- tential for foodborne transmission of C. difficile, there are only a small number of studies that have looked at the prevalence of C. difficile in foods, most of which focused on raw meats and fresh vegetables. To our knowledge, only two studies have investigated the prevalence of C. difficile in raw sausages and only one study has * Corresponding author. Microbiology & Immunology, School of Pathology and Laboratory Medicine, The University of Western Australia, Queen Elizabeth II Medical Centre, Nedlands 6009, Western Australia, Australia. E-mail address: thomas.riley@uwa.edu.au (T.V. Riley). Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe http://dx.doi.org/10.1016/j.anaerobe.2015.12.004 1075-9964/© 2015 Elsevier Ltd. All rights reserved. Anaerobe 37 (2016) 67e71