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. 2015 Mar 1;12(3):177–182. doi: 10.1089/fpd.2014.1842

A Possible Route for Foodborne Transmission of Clostridium difficile?

Barbara M Lund 1,✉, Michael W Peck 1
PMCID: PMC4367519  PMID: 25599421

Abstract

Spores of toxigenic Clostridium difficile and spores of food-poisoning strains of Clostridium perfringens show a similar prevalence in meats. Spores of both species are heat resistant and can survive cooking of foods. C. perfringens is a major cause of foodborne illness; studies are needed to determine whether C. difficile transmission by a similar route is a cause of infection.

Introduction

Clostridium difficile is a major cause of illness in hospitals and other healthcare settings and its incidence is recognized increasingly in the community (Hensgens et al., 2012). In a study in the United States, 32% of cases were classified as community associated (Lessa, 2013). Asymptomatic colonization of healthy individuals by C. difficile has been reported by several groups of workers. Colonization by toxigenic strains was found in 4.0% of 478 healthy individuals. Colonization by nontoxigenic strains was found in a further 3.1% of individuals (Ozaki et al., 2004). In many cases, colonization by a particular strain was transient, but in some cases it was persistent. Studies in healthcare settings and in the community have shown that a high proportion of cases of C. difficile infection (CDI) cannot be matched to previous CDI cases (Eyre et al., 2013; Wilcox, 2013). Thus, sources of C. difficile other than symptomatic patients may play an important role in transmission.

C. difficile and C. perfringens type A occur in foods, particularly meats, but also in vegetables and shellfish, and produce heat-resistant spores. The potential for C. difficile to be transmitted in food has been discussed by several workers including Weese (2009), Rupnik and Songer (2010), Hoover and Rodriguez-Palacios (2013), and Rodriguez-Palacios et al. (2013). C. perfringens is ranked as an important cause of foodborne illness in the United Kingdom (Tam et al., 2012, 2014) and in several countries (Grass et al., 2013) and was estimated as responsible for 10% of cases in the United States (Scallan et al., 2011). Cooked meat or poultry dishes are the predominant foods associated with C. perfringens food poisoning (Grass et al., 2013). Spores of C. perfringens survive cooking; if cooked food is allowed to remain at temperatures between ∼12°C and 50°C, surviving spores can germinate and the vegetative bacteria can multiply. On consumption of the food containing high numbers of C. perfringens, some of the vegetative cells survive passage through the stomach and reach the intestine, where they can multiply and form toxin, causing gastroenteritis. The purpose of this article is to consider the common features of C. difficile and C. perfringens that may be relevant to possible foodborne transmission of C. difficile, and to identify further information that is needed.

Optimum Conditions for Detection and Growth of C. difficile

A major consideration in studying the transmission of C. difficile, and environmental sources, is the efficiency of methods for optimum germination of spores and growth of vegetative bacteria. Various culture media have been used to investigate the prevalence of the organism. Unheated spores of C. difficile or spores heated at 80°C for 10 min showed very low germination on a medium without lysozyme, but this was increased by a thousandfold in the presence of lysozyme, 5 μg/mL, and by a further 100-fold (giving 100% germination) following treatment with sodium thioglycolate and inoculation onto a medium containing lysozyme (Ionesco, 1978). In a similar manner, full recovery of spores of the majority of 108 strains of C. difficile (69 toxigenic, 39 nontoxigenic) heated at 80°C for 10 min required treatment with sodium thioglycolate and inoculation into a medium containing lysozyme (Nakamura et al., 1985). The addition of the bile salt sodium taurocholate to a selective medium increased recovery of spores of C. difficile isolated from clinical samples and heat shocked at 56°C for 10 min, to inactivate vegetative bacteria (Wilson et al., 1982). Taurocholate (and other cholates) and glycine were reported to act as co-germinants for C. difficile spores (Sorg and Sonensheim, 2008), but spores of different clinical isolates differ in response to bile salts (Heeg et al., 2012). In work by Paredes-Sabja et al. (2008), germination occurred in brain–heart infusion medium and was not increased by the inclusion of taurocholate.

Inclusion of lysozyme, 5 μg/mL, in a selective medium containing sodium cholate (a less expensive alternative to taurocholate), increased recovery of C. difficile from hospital ward environments, whereas pre-exposure of swabs to alkaline thioglycolate did not further increase recovery (Wilcox et al., 2000). Pre-enrichment in cooked meat broth before plating on a selective medium improved recovery; however, some samples were positive on direct culture but not after enrichment.

According to Limbago et al. (2012), the best recovery of a strain of C. difficile from spiked meat samples was obtained by enrichment in brain–heart infusion broth followed by plating on blood agar or on selective agar media with taurocholate. In an evaluation of culture methods for recovery of the toxigenic C. difficile type strain ATCC 9689 from stool and swab samples, the most sensitive method was heat shock (80°C for 10 min) followed by enrichment in a medium containing cycloserine–cefoxitin mannitol broth with taurocholate, lysozyme (5 μg/mL), and cysteine, followed by plating on a nutrient medium with 5% sheep blood (Hink et al., 2013). Tyrrell et al. (2013) reported that alcohol treatment followed by plating on a selective medium containing horse blood and taurocholate gave the highest recovery from toxin-positive fecal samples; a selective enrichment medium containing lysozyme and taurocholate, without alcohol pretreatment, followed by plating on selective medium containing horse blood and taurocholate enabled recovery of C. difficile from 9% of toxin-positive samples that were negative on direct plating.

A requirement for lysozyme for recovery of spores may result from damage to the spore coat and associated enzymes (Permpoonpattana et al., 2011, 2013). Removal of the exosporium from spores of C. difficile resulted in an increase of colony formation on medium containing taurocholate (Escobar-Cortés et al., 2013). The use of both lysozyme and taurocholate in a culture medium may give maximum recovery of C. difficile spores. The addition of blood to culture media may be a source of lysozyme (Flanagan and Lionetti, 1955).

Consideration is needed of the anaerobic conditions used for culture of C. difficile. Spores of many Clostridium spp. can survive and germinate in aerobic conditions, but the vegetative bacteria will be inactivated in such conditions. The initiation of growth of clostridia can be prevented by the presence of low levels of oxygen. C. perfringens is one of the most easily grown anaerobes, and is less sensitive to oxygen than some other species of anaerobic bacteria (Fredette et al., 1967). C. difficile is reported to require strictly anaerobic conditions (Songer, 2010). The effect of oxygen on initiation of growth of some clostridia is illustrated by its effect on nonproteolytic C. botulinum type E. In an atmosphere of N2:H2:CO2, 84:10:5 by volume, the presence of 0.79% oxygen and a redox potential of +250 mV at pH 7, an inoculum of >104 spores was required to give growth in 5 days at 37°C; in the absence of detectable oxygen (<0.21%), at a redox potential of −400 mV, growth occurred from a single spore in 2 days (Lund et al., 1984). Thus, the most sensitive detection of low numbers of viable spores of some Clostridium spp. by culture media requires strictly anaerobic conditions. A recent article reported successful isolation of C. difficile in culture medium without incubation in an anaerobic chamber (Cadnum et al., 2014). The medium contained cysteine as a reducing agent and was boiled to eliminate oxygen before use; there was no demonstration that very low numbers of C. difficile could be isolated, and the sensitivity of the medium was not determined.

De-aerated liquid media containing reducing agents are used commonly for culture of Clostridium spp. using large inocula, but for epidemiological purposes there is a need to ensure that anaerobic conditions used are sufficient to ensure the most sensitive recovery of C. difficile from samples.

Reported Prevalence of C. difficile in Food Animals and in Food

C. difficile has been found in the intestinal tract of many types of food animals, including cattle, pigs, sheep, and poultry, as well as dogs and cats (Hensgens et al., 2012; Koene et al., 2012). Ribotypes in cattle, pigs, and poultry included those causing disease in humans. Reports of the prevalence of toxigenic C. difficile in retail samples of meat and, less frequently, in other foods have been summarized by Hensgens et al. (2012). In North America relatively high prevalence rates have been reported in uncooked meat products, in up to 42% of beef, 41% of pork and 44% of turkey samples, whereas lower prevalence rates, of up to 4.3% and 2.7% in ground beef/pork and chicken meat, respectively, have been reported in Europe. In contrast to many North American reports, a survey of 1755 retail meat samples, including ground beef, ground poultry, chicken breast, and pork chops, tested by U.S. state public health laboratories and 60 samples tested by Centers for Disease Control and Prevention, failed to detect C. difficile (Limbago et al., 2012). Differences in reported prevalence may be due in part to the use of different methodology. Toxigenic C. difficile was found in 23–50% of uncooked meats in the United States, in 14% of ready-to-eat summer sausage, and in 63% of ready-to-eat braunschweiger (Songer et al., 2009). Ribotypes 078 and 027, which are found commonly in human infection, were detected. Most strains isolated from foods have a genotype identical to those of human and animal isolates from the same geographic area or other parts of the world (Rupnik and Songer, 2010).

In a Canadian study, toxigenic C. difficile was isolated from 28 of 230 (12%) of samples of retail ground beef and ground pork (Weese et al., 2009). Of 28 positive samples, 20 were positive by enrichment but not by direct plating. The detection threshold of the enrichment method was stated as ≤10 spores/g. In four ground beef samples that were positive by direct culture, 20–240 spores/g were found and in four ground pork samples that were positive by direct culture 20–60 spores/g were detected. Toxigenic C. difficile was detected in 12.8% of chicken meat samples but could only be detected by enrichment, indicating that the numbers present were ≤10 spores/g (Weese et al., 2010).

These reports indicate that toxigenic C. difficile may be present in meat products, usually at a low concentration.

Reported Prevalence of Food-Poisoning Strains of C. perfringens Type A in Food

C. perfringens is often found in retail samples of raw meat and poultry. In order to determine the incidence of type A strains carrying the cpe gene, which is essential for causing food poisoning, Wen and McClane (2004) surveyed 887 non-outbreak, retail samples of meats, poultry, and seafood. Of these samples, 31% were contaminated with C. perfringens with a most probable number (MPN) up to 32/g, 24% were contaminated with C. perfringens type A, and only 13 samples (∼1.4%) were contaminated with cpe-positive strains of type A. In the majority of samples, C. perfringens was present as vegetative cells rather than as spores. In representative cpe-positive strains, the cpe gene was located on the chromosome, rather than on a plasmid; strains with a chromosomal cpe gene are reported to have a much higher heat resistance than strains with a plasmid cpe gene (Sarker et al., 2000). In 395 samples of cooked beef (kidney and flesh) sold in the streets in the Ivory Coast, the prevalence of C. difficile spores (12.4%) was reported greater than that of C. perfringens type A spores (5.1%); the numbers present were not determined (Kouassi et al., 2014).

Heat Resistance of Spores of C. difficile and C. perfringens

Studies in which heated spores of C. difficile were treated with alkaline thioglycolate and recovered on a medium with added lysozyme showed the highest heat resistance (Table 1). Kamiya et al. (1989) showed that when spores of four clinical strains were heated at 70°C for 10 min and plated on brain–heart infusion medium supplemented with glucose, soluble starch, and cysteine-HCl (GS-BHI), recovery rates were >10% for 2 strains and <0.01% for 2 strains. With all these strains, after heating at 80°C for 10 min, there was a marked decline in relative recovery rates of spores. After heating at 60°–75°C for 10 min, recovery of spores was increased to ∼100% by inoculation onto GS-BSI plus taurocholate, but after heating at 80°C for 10 min the relative recovery rate was reduced to 2.9–0.05% of spores. When spores heated at 85°C for 10 min were recovered by treatment with thioglycolate and inoculated onto GS-BHI plus lysozyme, recovery rates approaching 100% were obtained; after heating at 90°C for 10 min, relative recovery rates were 47.2–10.0% and heating at 100°C for 10 min reduced the relative recovery rates to 2.1–-0.20%. Studies by some workers involved recovery of survivors on media containing blood, which may be a source of lysozyme. Studies shown in Table 1, using multiple strains from animal and food sources and including genotypes of public health relevance, showed that spores were liable to survive heating at 71°C and higher temperatures.

Table 1.

Heat Resistance of Spores of Clostridium difficile and C. perfringens

Heating medium Recovery of spores Strains tested D value at specified temperature Reference
C. difficile
 Phosphate buffer Alkaline thioglycolate treatment. Medium plus lysozyme (10 μg/mL) 108 strains D100°C=2.5–33 min Nakamura et al. (1985)
 Distilled water Alkaline thioglycolate treatment. Medium plus lysozyme (10 μg/mL) 4 strains D100°C=∼4–6 min Kamiya et al. (1989)
 Phosphate-buffered saline Medium, 5% sheep blood agar 20 strains D71°C=∼30 min Rodriguez-Palacios et al. (2010)
 Phosphate –buffered saline Blood agar 22 strains D85°C=6.0–8.5 min Rodriguez-Palacios and Lejeune (2011)
 Gravy, 0% fat; lean ground beef, 3% fat; ground beef 30% fat. Blood agar 4 strains D96°C=0.59–1.19 min Rodriguez-Palacios and Lejeune (2011)
 Gravy, 0% fat; lean ground beef, 3% fat; ground beef 30% fat. Blood agar 4 strains D85°C=2.5–3.3 min Rodriguez-Palacios and Lejeune (2011)
 Gravy, 0% fat; lean ground beef, 3% fat; ground beef 30% fat. Blood agar 4 strains D71°C=47–71 min Rodriguez-Palacios and Lejeune (2011)
C. perfringens
 Culture medium Brain–heart infusion agar 5 strains; chromosomal cpe gene D100°C=30–124 min Sarker et al. (2000)
 Culture medium Brain–heart infusion agar 7 strains; plasmid cpe gene D100°C=0.5–1.9 min Sarker et al. (2000)
 Culture medium Brain–heart infusion agar 14 strains; chromosomal cpe gene D100°C=30–170 min Wen and McClane (2004)
 Phosphate buffer Brain–heart infusion agar 10 strains; chromosomal (plus one plasmid) cpe gene D95°C=>7.5 min Grant et al. (2008)
 Phosphate buffer Brain–heart infusion agar 5 strains; plasmid cpe gene D95°C=<7.5 min Grant et al. (2008)
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D value, time at specified temperature for 10-fold reduction in viable numbers.

Treatment of heated spores with alkaline thioglycolate probably ruptures disulphide bonds in the outer layers of the spore, increasing the spore permeability (Gould and Hitchins, 1963).

Enzymes with lysozyme activity have been reported in many types of raw food and in animal tissue (Lund and Peck, 1994). Hen egg-white lysozyme is stable to heat treatment, and some activity remained after heating 5–50 μg/mL at 90°C for 20 min in meat slurry pH 6.5–6.6 (Peck and Fernandez, 1995). The heat stability may be reduced by the presence of other proteins or increased in the presence of components such as some sugars and polysaccharides. The measured heat-resistance of spores of C. difficile was higher when heated spores were treated with alkaline thioglycolate and recovered on medium containing lysozyme (Table 1). This has also been reported for spores of nonproteolytic C. botulinum and C. perfringens (Peck et al., 1993; Barach et al., 1974).

Spores of C. perfringens can be sublethally injured by heat treatment, and spores heated at 105°C for up to 20 min or at 120°C for up to 25 s showed increased recovery on a medium containing lysozyme (Barach et al., 1974). Removal of exchangeable cations Ca++ and Na+ from C. perfringens spores by treatment with acid or alkali decreased survival of spores after heating at 95°C and plating on a medium without lysozyme; addition of lysozyme to the culture medium increased the number of survivors, and this was increased more dramatically by treatment of the heated spores with alkali before plating on medium containing lysozyme (Ando and Tsuzuki, 1983). Recovery on a medium without added lysozyme showed that spores had a very high heat-resistance, and spores of strains with a chromosomal cpe gene showed much higher heat-resistance than that of strains with a plasmid-borne cpe gene (Table 1). The majority of strains of C. perfringens associated with food poisoning carry a chromosomal cpe gene (Wen and McClane, 2004).

Results in Table 1 indicate that spores of C. difficile may be less heat resistant than those of C. perfringens, although variations in methodology may account for some of the reported differences. Importantly, however, spores of C. difficile as well as those of C. perfringens are likely to survive many processes for cooking foods, which involve heating to an internal temperature of 70° for up to 2 min or up to 74°C (WHO, 2006; ACMSF, 2007; FSA, 2010, 2013; FDA, 2013; Lund, 2014).

Spores of C. perfringens that survive cooking of foods can germinate and multiply if cooked foods are allowed to remain at temperatures between 12°C and 50°C. Guidelines in the United States and in the United Kingdom aim to ensure that exposure of cooked foods to this range of temperature is minimized, so that no more than a 10-fold increase in numbers of C. perfringens occurs during cooling of cooked food (Le Marc et al., 2008; FDA, 2013). A computer program is available that enables prediction of growth from surviving C. perfringens spores during any specified cooling curve (Le Marc et al., 2008).

Discussion

It is clear that spores of C. difficile, like those of enterotoxigenic C. perfringens, are liable to survive cooking of meat and other foods to a core temperature of 74°C or to 70°C for up to 2 min and therefore, like those of C. perfringens, they may germinate and allow growth of vegetative bacteria if cooked food is maintained at permissive temperatures, or they may remain as resistant spores. Although there have been no reported cases of food-associated CDI in humans, consumption of beef has been reported as a risk factor for C. difficile infection in patients in the community (Søes et al., 2014). For C. perfringens, there is information on the temperatures allowing germination and growth and on rates of growth, enabling determination of the rate of growth during cooling of cooked foods; this allows specification of temperature controls and a cooling regimen to prevent growth of this bacterium.

The optimum temperature for growth of C. difficile is stated as 30°–37°C, and the organism grows at 25°C and 45°C (Rainey et al., 2009), but there appears to be a lack of information on the minimum and maximum temperatures allowing growth, and on rates of growth. Thus, conditions required to prevent growth of the bacterium during cooling of cooked foods are not known.

In the case of C. perfringens, growth of bacteria to give >105/g in the food consumed is considered, in general, to result in food poisoning (Heredia and Labbé, 2013). In the case of C. difficile, the infectious dose of spores or of vegetative bacteria that results in colonization, and possibly disease in vulnerable people, is unknown. In order to assess the risk of transmission of C. difficile in cooked foods and, if necessary, to devise conditions to prevent this transmission, the following information is needed, bearing in mind the wide genetic diversity of strains (Knetsch et al., 2012):

  • • the anaerobic conditions required for the most sensitive detection and isolation of the bacterium

  • • the optimum culture medium for maximum recovery of heated and unheated spores and vegetative bacteria

  • • further data on spore heat-resistance in foods

  • • the range of temperatures that allow growth

  • • the effect of temperature on rate of growth

  • • the effect of combinations of factors (e.g., temperature, pH, and NaCl) on growth

  • • an estimate of the number of vegetative bacteria or spores needed to result in colonization of healthy adults and of vulnerable members of the population

Conclusions

Research is needed to establish whether infection with C. difficile can be caused by transmission on food. Spores of C. difficile, like those of C. perfringens, can occur in meat and survive temperatures and times recommended for cooking. Germination of spores of C. perfringens and vegetative growth can occur in cooked meats if they are maintained at temperatures of 12°–52°C, and following consumption gastroenteritis can result. Information is needed on conditions in which surviving spores of C. difficile would germinate and vegetative bacteria would multiply in cooked meat dishes, or whether the spores would persist, and whether the spores or vegetative bacteria would result in asymptomatic or symptomatic infection after consumption of the meat.

Acknowledgments

This work was supported by the BBSRC Institute Strategic Programme on Gut Health and Food Safety (grant number BB/J004529/1).

Disclosure Statement

No competing financial interests exist.

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