Financial and social costs

CDI is a costly disease in most countries. In the USA, estimates indicate that there are about 500,000 CDIs every year, which result in $1.1–3.2 billion in health care costs every year (O'Brien et al., Reference O'Brien, Lahue, Caro and Davidson2007). On average, each new CDI infection costs $3000–5000, whereas recurrent infections (more difficult to treat) cost $13,000–18,000 (Dubberke and Wertheimer, Reference Dubberke and Wertheimer2009; Ghantoji et al., Reference Ghantoji, Sail, Lairson, DuPont and Garey2010). Similar high treatment costs have been documented in Europe (Wilcox et al., Reference Wilcox, Cunniffe, Trundle and Redpath1996; Ghantoji et al., Reference Ghantoji, Sail, Lairson, DuPont and Garey2010).

With an increase in the number of human infections, there has also been an increase in social concerns. Over time the incidence of severe disease has increased with more patients requiring surgical removal of the inflamed colon (one in every ten CDI cases – 10%), higher mortality rates, and concern about increased liability (Pepin et al., Reference Pepin, Alary, Valiquette, Raiche, Ruel, Fulop, Godin and Bourassa2005; Sailhamer et al., Reference Sailhamer, Carson, Chang, Zacharias, Spaniolas, Tabbara, Alam, DeMoya and Velmahos2009; Marler, Reference Marler2010). The effects of such medical consequences at the individual and family level are difficult to quantify. Infection control and surveillance initiatives have thus been reinforced in hospitals to reduce the incidence of C. difficile (Pepin et al., Reference Pepin, Valiquette, Alary, Villemure, Pelletier, Forget, Pepin and Chouinard2004; Barbut et al., Reference Barbut, Jones and Eckert2011) with variable success.

Given that C. difficile also infects animals, the disease can have a financial impact on companion animals and livestock also. Financial loss estimates associated with CDI in animals are not available. However, in horses and other companion animals where C. difficile causes enteric disease, the costs associated with veterinary medical treatment are high (several thousand dollars in North America) and generally assumed by the owners. In livestock production, no estimates are yet available either, although there is evidence that C. difficile causes disease and possibly growth delays in production animals (Songer, Reference Songer2004; Kiss and Bilkei, Reference Kiss and Bilkei2005).

Early history of C. difficile as a gut pathogen

C. difficile is a spore-forming anaerobic bacterium that was first isolated from stools of healthy infants in 1935 (Bartlett, Reference Bartlett2008). Although these first bacterial isolates were fatal to hamsters, no attention was directed to the health risks of C. difficile in adults until decades later; C. difficile was deemed normal in the gut of children. In 1962, the same bacterium was isolated from localized infections (e.g. wounds and abscesses) in adults (Smith and King, Reference Smith and King1962). Although these isolates were also fatal to hamsters, the authors concluded that C. difficile was not pathogenic for man.

Only in the late-1970s, additional studies in humans and hamsters confirmed C. difficile as the cause of a severe form of colitis in adults known since the 1890s as pseudomembranous colitis (PMC) (Bartlett, Reference Bartlett2008). In PMC, marked inflammation and cellular debris accumulate over the intestinal surface giving the appearance of a pseudomembrane. Microscopically, PMC was characterized by exuberant inflammatory plaques formed on the surface of the colon protruding from the intestinal wall (Price and Davies, Reference Price and Davies1977). In humans, the cause of PMC was unclear for almost 80 years, until the 1970s when the administration of antibiotics, especially clindamycin and lincomycin, was linked to PMC (Tedesco et al., Reference Tedesco, Stanley and Alpers1974). Initially researchers thought that PMC resulted from a viral infection and the concurrent use of antibiotics (Steer, Reference Steer1975), but finally C. difficile was identified as the microbial cause secondarily linked to antibiotic use; which disrupts the gut flora favoring the opportunistic proliferation of C. difficile (Bartlett et al., Reference Bartlett, Moon, Chang, Taylor and Onderdonk1978; George et al., Reference George, Symonds, Dimock, Brown, Arabi, Shinagawa, Keighley, Alexander-Williams and Burdon1978; Larson et al., Reference Larson, Price, Honour and Borriello1978).

Currently, PMC is almost always (>95%) linked to C. difficile (Hurley and Nguyen, Reference Hurley and Nguyen2002), but not all CDIs result in PMC. In animals, a similar form of inflammation has been reported in a small fraction of piglets infected experimentally with C. difficile. In other animals (e.g. mice, hamsters, horses and calves), various forms of colitis have been described, from mild in most species to severe and fulminant in some horses (Colitis X, first case report was linked to an emerging hyper-virulent C. difficile PCR ribotype 027/NAP1 strain known to be highly problematic in humans) (Songer et al., Reference Songer, Trinh, Dial, Brazier and Glock2009).

During the last decade, the severity of the CDI, including PMC, has increased in populations of children who were previously rarely affected. Today the growing epidemic and the more frequent lack of response to conventional therapies have raised the awareness of CDI to the point where it is increasingly recognized as a global public health challenge, often surpassing the importance of methicillin-resistant Staphylococcus aureus infections (Lessa et al., Reference Lessa, Gould and McDonald2012).

C. difficile in hospitals and risk factors

CDIs were first seen as sporadic cases in humans, particularly in hospitals, but in the 1990s, the frequency increased (>2-fold) as highlighted in an article entitled C. difficile: a pathogen of the nineties (Riley, Reference Riley1998). The problem has been especially notorious in developed nations (Pepin et al., Reference Pepin, Valiquette, Alary, Villemure, Pelletier, Forget, Pepin and Chouinard2004), and continues to extend into the 2000s; now it has been documented in community settings (Fig. 1) (Borgmann et al., Reference Borgmann, Kist, Jakobiak, Reil, Scholz, von Eichel-Streiber, Gruber, Brazier and Schulte2008). Controlling for confounding variables, it is known that such increase is not due to reporting bias (Burckhardt et al., Reference Burckhardt, Friedrich, Beier and Eckmanns2008). Other studies have also shown the remarkable parallel between the increased trend of disease in hospitals and the community (Noren et al., Reference Noren, Akerlund, Back, Sjoberg, Persson, Alriksson and Burman2004). However, the incidence of CDI is much lower (1300-fold) in the community, compared to hospitals, due in part to a lower (37-fold) occurrence of antimicrobial consumption (Noren et al., Reference Noren, Akerlund, Back, Sjoberg, Persson, Alriksson and Burman2004). Compared to other drugs, mortality data associated with drug consumption in the USA showed that among diseases with significant drug-related etiologies, C. difficile enterocolitis primarily associated with antimicrobials had the largest percentage increase in total mentions, with a 203% rise between 1999 and 2003 (Wysowski, Reference Wysowski2007). Today, the increased resistance to many antimicrobials, especially fluoroquinolones has become an emerging global health issue (Spigaglia et al., Reference Spigaglia, Barbanti, Mastrantonio, Brazier, Barbut, Delmee, Kuijper and Poxton2008; Ashiru-Oredope et al., Reference Ashiru-Oredope, Sharland, Charani, McNulty and Cooke2012). Among cases with antimicrobial-associated diarrhea, CDIs account for about 25–30% of all cases. For decades, antimicrobial consumption has been the main predisposing factor for CDI.

Fig. 1. Paralell increase in hospitals and the community. C. difficile toxins in fecal samples from patients visiting 40 hospitals and over 2000 physicians in southern Germany. (Reproduced with permission from Borgmann et al. Reference Borgmann, Kist, Jakobiak, Reil, Scholz, von Eichel-Streiber, Gruber, Brazier and Schulte2008; Copyright Eurosurveillance).

Elderly over 65 years old have been always more susceptible to infections (Pepin et al., Reference Pepin, Valiquette, Alary, Villemure, Pelletier, Forget, Pepin and Chouinard2004). Regarding the source of infection, by the end of the 1990s, humans were considered to be the sole reservoirs for infection to other humans (Kaatz et al., Reference Kaatz, Gitlin, Schaberg, Wilson, Kauffman, Seo and Fekety1988). However, studies from the 1980–1990s outside hospitals in the UK demonstrated that C. difficile was present in water bodies in connection with urban settings, soils, root vegetables, and household pets (Borriello et al., Reference Borriello, Honour, Turner and Barclay1983b; al Saif and Brazier, Reference al Saif and Brazier1996). The potential for animal–human and foodborne transmission was then highlighted. Although genetic testing of recovered strains determined that most isolates were capable of producing toxins (necessary for intestinal disease), no molecular typing was reported to determine if they were the same strains affecting humans.

The increasing number of cases inside hospitals maintained the attention on human-to-human transmission, mediated by environmental contamination of the hospital wards and health care personnel (Kaatz et al., Reference Kaatz, Gitlin, Schaberg, Wilson, Kauffman, Seo and Fekety1988). Infections originating in the community, where patients acquire CDI outside hospitals, were considered infrequent and received no attention for disease prevention. No connection or differentiation was acknowledged between community- and hospital-acquired CDI until the last decade. Currently, there are more defined criteria to classify new CDIs as community- or hospital-associated cases based on the site of acquisition or onset of clinical signs (Kuijper and van Dissel, Reference Kuijper and van Dissel2008). A similar differentiation has importantly been used in epidemiological studies in veterinary hospitals since the mid-2000s, especially in Canada (Weese et al., Reference Weese, Toxopeus and Arroyo2006).

Despite the rapid recognition of increased virulence and ecological aspects of C. difficile, medical textbooks continue treating C. difficile as the traditional medical condition acquired only in people exposed to hospitals or long-term health care settings with little attention focused on ecology and prevention. In veterinary medicine, C. difficile is still invariably reported in reference medicine books as an organism associated with clinical disease in animals, with limited emphasis on public health. Most veterinary literature largely remains as review articles.

Although several risk factors for CDI have been identified for humans over the past decades (see Table 1), it is noteworthy to highlight that the ages at which people get CDI (and possible exposure to other unknown risk factors) are parallel but significantly different in hospitals compared to the community. Younger individuals (although less likely to suffer CDI than the elderly) are comparatively more often affected in the community (Hirshon et al., Reference Hirshon, Thompson, Limbago, McDonald, Bonkosky, Heimer, Meek, Mai and Braden2011). The distribution of ages of CDI patients depicted in Fig. 2 highlights that in hospitals most inpatients are significantly older when compared to the age of outpatients treated by the same center during the same period. Regarding the traditionally known risk factors (Table 1), the following excerpt illustrates that disease trends are changing: 36% of patients had no history of antibiotic use within 3 months before symptom onset, and 25% had no underlying medical condition or recent hospital admission and, moreover, were younger than 45 (Kuijper and van Dissel, 2008; Hirshon et al., Reference Hirshon, Thompson, Limbago, McDonald, Bonkosky, Heimer, Meek, Mai and Braden2011). CDI can no longer be considered a disease exclusively acquired in hospitals.

Fig. 2. Affected people in the community are younger than affected people in hospitals. Percentage of humans with CDI in hospitals and the community, Germany 2006. Total number of patients, n = 714. Mov. Avg. = moving average. Horizontal bars represent average (oval) ± S.D., and the medians (vertical ticks). (Data courtesy of Dr S. Borgmann et al.)

Table 1. Risk factors for CDIs

Age is a very important risk factor for disease. Although cases can occur in children elderly are more prone to CDI. Recent studies in long-term care facilities showed that over 50% of patients develop CDI beyond the fourth week after hospital discharge, much longer than for acute-health care settings, highlighting the importance of long-term disease prevention (Pawar et al., Reference Pawar, Tsay, Nelson, Elumalai, Lessa, Clifford McDonald and Dumyati2012). In the community, cancer patients, Crohn's and ulcerative colitis patients, and other individuals receiving immunosuppressants and antibiotics are at risk for CDI. In humans, between 20 and 27% of CDIs that require hospital-level medical treatment are acquired in the community. Based on risk factors known (Table 1), prevention measures could be focused on susceptible individuals.

Theory of person-to-person transmission disproved

The concept of hospital clonality, long suspected to be caused by a single highly infectious strain with clonal dissemination within hospital wards based on fingerprinting qualitative typing techniques, has been increasingly questioned for CDI. Using the latest portable sequencing technology, early in 2012, a whole-genome sequencing study of C. difficile isolates from cases assigned to three hospital outbreaks in UK demonstrated that most consecutive CDIs were due to different strains and so (in their own words) refuted the theory of person-to-person transmission to explain the increase incidence of CDI within hospital wards (Eyre et al., Reference Eyre, Golubchik, Gordon, Bowden, Piazza, Batty, Ip, Wilson, Didelot, O'Connor, Lay, Buck, Kearns, Shaw, Paul, Wilcox, Donnelly, Peto, Walker and Crook2012). For one cluster of CDI involving three people, over 4 days in the same ward the authors concluded that next generation sequencing refutes transmission between suspected linked cases and that isolates of the same strain type are not necessarily linked by person-to-person transmission. Data from this and two other clusters demonstrated that person-to-person transmission within hospitals is not as exclusively high as previously thought. Clostridium difficile strains appear to be introduced to hospitals by incoming patients (and possibly foods/visitation animals) more commonly than earlier suspected.

CDIs and toxin types

Numerous reviews describing the biology and epidemiological changes of CDI in humans are available. In animals, similar papers have been published since the first review describing C. difficile as an emerging pathogen in food animals in 2004 (Songer, Reference Songer2004, Reference Songer2010; Gould and Limbago 2010; Weese, Reference Weese2010; Hensgens et al., Reference Hensgens, Keessen, Squire, Riley, Koene, de Boer, Lipman and Kuijper2012; Rodriguez-Palacios et al., Reference Rodriguez-Palacios, LeJeune and Hoover2012). All reviews indicate that animals and foods are reservoirs of C. difficile strains that produce toxins. From experimental studies in animals, it is possible to say that CDI occurs only when C. difficile opportunistically proliferates in the intestinal tract of its host (animal or human) and produces its toxins that are deleterious to the intestinal wall. In this context, several factors are needed including: (1) the ingestion of C. difficile spores and the persistence of C. difficile in the intestinal tract, (2) the proliferation of C. difficile and the production of toxins in the gut, and (3) an immunologically susceptible host with disarranged gut flora.

Soon after its identification as a pathogen it was determined that the pathogenicity of C. difficile, was mediated via two similar, but structurally and immunologically distinct, virulence factors: Toxins A and B (Bongaerts and Lyerly, Reference Bongaerts and Lyerly1997). Once in the cell, these toxins affect glycosylate Rho GTPase, a key enzyme in signaling pathways regulating actin polymerization. The net effect is a disruption of normal cytoskeletal architecture leading to cell death followed by local and systemic inflammatory reactions (Mazuski et al., Reference Mazuski, Panesar, Tolman and Longo1998; Hamm et al., Reference Hamm, Voth and Ballard2006; Sun et al., Reference Sun, Savidge and Feng2010; Modi et al., Reference Modi, Gulati, Solomon, Monaghan, Robins, Sewell and Mahida2011). Another toxin, called binary toxin, present in a fraction of C. difficile strains may also contribute to disease (Geric et al., Reference Geric, Rupnik, Gerding, Grabnar and Johnson2004, Reference Geric, Carman, Rupnik, Genheimer, Sambol, Lyerly, Gerding and Johnson2006; Terhes et al., Reference Terhes, Urban, Soki, Hamid and Nagy2004; Stare et al., Reference Stare, Delmee and Rupnik2007; Schwan et al., Reference Schwan, Stecher, Tzivelekidis, van Ham, Rohde, Hardt, Wehland and Aktories2009; Sun et al., Reference Sun, Savidge and Feng2010).

Almost always, strains capable of causing disease carry both the toxins A and B, denoted A⁺B⁺. However, since 1999, naturally occurring C. difficile mutant strains, lackng toxin A (A⁻B⁺), have caused major outbreaks in hospitals internationally (al-Barrak et al., Reference al-Barrak, Embil, Dyck, Olekson, Nicoll, Alfa and Kabani1999; Loo et al., Reference Loo, Poirier, Miller, Oughton, Libman, Michaud, Bourgault, Nguyen, Frenette, Kelly, Vibien, Brassard, Fenn, Dewar, Hudson, Horn, Rene, Monczak and Dascal2005; Lyras et al., Reference Lyras, O'Connor, Howarth, Sambol, Carter, Phumoonna, Poon, Adams, Vedantam, Johnson, Gerding and Rood2009; Kuehne et al., Reference Kuehne, Cartman, Heap, Kelly, Cockayne and Minton2010; Sun et al., Reference Sun, Savidge and Feng2010). In the past, A⁻B⁺ strains were uncommon (<5%); nowadays, those strains are problematic and predominant (>30%) in certain regions (Kim et al., Reference Kim, Riley, Kim, Kim, Yong, Lee, Chong and Park2008; Shin et al., Reference Shin, Kuak, Yoo, Kim, Lee, Kang, Whang and Shin2008a, Reference Shin, Kuak, Yoo, Shin and Yoob). Since the strains that do not produce either toxin A or toxin B (A⁻B⁻) are non-pathogenic (Bongaerts and Lyerly 1997; Rupnik et al., Reference Rupnik, Dupuy, Fairweather, Gerding, Johnson, Just, Lyerly, Popoff, Rood, Sonenshein, Thelestam, Wren, Wilkins and von Eichel-Streiber2005), there has been interest in therapeutic microbiology using these strains as probiotics to prevent colonization in susceptible hospitalized people. Experiments in various animal species support the potential benefit. Noteworthy, non-toxigenic strains (A⁻B⁻) are comparatively more common in mature food animals than in people and foods, but A⁻B+ in foods appear to be less common (Bakri et al., Reference Bakri, Brown, Butcher and Sutherland2009; Hensgens et al., Reference Hensgens, Keessen, Squire, Riley, Koene, de Boer, Lipman and Kuijper2012).

In addition to toxin-based studies, the advent of genomics and systems biology have spurred the increasing documentation of other possible virulence factors since the early 2000s. Therapeutically, such knowledge still has not yet resulted in new effective measures to treat CDI in hospitals. Immunologically, most high risk or severely ill patients have low levels of antibodies against toxins A and B, while healthy individuals appear to have higher titers (Kelly and Kyne, Reference Kelly and Kyne2011). Therapeutic interest is now in the use of antibody supplementation to currently approved therapies against CDI, largely based on antimicrobials against C. difficile, i.e. metronidazole and vancomycin, and DNA based vaccines against toxins A and B (Jin et al., Reference Jin, Wang, Zhang, Xiao, Lu and Huang2013). However, increased resistance to such antimicrobials is also increasing (Sinh et al., Reference Sinh, Barrett and Yun2011). Patented human monoclonal antibody technology is in phase clinical trials. Thus far, the therapeutic benefit seems to be present when antibodies are supplemented in mild-moderate cases; but the response is poor in severe CDIs. Epidemiological and experimental data indicate that immune susceptibility and bacterial flora disarrangements are major factors for CDI. Aside from reinforcing hand washing, little has been done on actively involving the communities at risk to prevent exposure to C. difficile.

Increased antimicrobial resistance and ability to produce toxins

Compared to isolates from before 2000, current C. difficile isolates affecting people are more resistant to antibiotics (Warny et al., Reference Warny, Pepin, Fang, Killgore, Thompson, Brazier, Frost and McDonald2005; Sinh et al., Reference Sinh, Barrett and Yun2011). Further, some strains arguably can produce up to 16-to-20 times more toxins (A or B) in vitro compared to regular strains (Loo et al., Reference Loo, Poirier, Miller, Oughton, Libman, Michaud, Bourgault, Nguyen, Frenette, Kelly, Vibien, Brassard, Fenn, Dewar, Hudson, Horn, Rene, Monczak and Dascal2005; Warny et al., Reference Warny, Pepin, Fang, Killgore, Thompson, Brazier, Frost and McDonald2005). Therein, those strains increasingly isolated in current times are often referred to as ‘hyper-virulent’ strains (Mulvey et al., Reference Mulvey, Boyd, Gravel, Hutchinson, Kelly, McGeer, Moore, Simor, Suh, Taylor, Weese and Miller2010). The most widely factor associated with the increased ability to produce toxins in vitro is the presence of a genetic mutation in a gene (tcdC) that normally down-regulates the genes responsible for the production of toxins A and B. In vivo, the association of tcdC polymorphisms with disease severity is less clear. Other virulence factors such as antibiotic-induced adherence to intestinal cells (Deneve et al., Reference Deneve, Delomenie, Barc, Collignon and Janoir2008) and strain-dependent systemic toxin pathogenicity are possibly contributing features (Lanis et al., Reference Lanis, Hightower, Shen and Ballard2012). Of public health relevance, hyper-virulent strains, associated with severe disease in humans, have been increasingly isolated from food animals and foods since 2006 (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Staempfli, Duffield and Weese2007a; Hensgens et al., Reference Hensgens, Keessen, Squire, Riley, Koene, de Boer, Lipman and Kuijper2012).

C. difficile recurrences are increasingly common

Following recovery from a CDI, reinfections in the same individual and treatment failure are occurring with more frequency. Recurrences after treatment of CDI with metronidazole (first drug of choice) have increased from 7% before the year 2000 to 29% thereafter (Kelly and LaMont, Reference Kelly and LaMont2008; Sinh et al., Reference Sinh, Barrett and Yun2011). Although not everyone suffers reinfections, some individuals are overly sensitive. The reasons for such susceptibility are currently under investigation. Low antibody titers effective against the C. difficile toxins (Wilcox, Reference Wilcox2004), and disrupted intestinal flora due to antimicrobials (Rupnik et al., Reference Rupnik, Wilcox and Gerding2009) are among the factors that enhance susceptibility to reinfections. Increasingly, the administration of proton pump inhibitors (widely prescribed antacid) confers more risk for recurrence compared to other classes of antacids (Linsky et al., Reference Linsky, Gupta, Lawler, Fonda and Hermos2010). More common since the year 2000, reported rates of reinfections have varied between 15 and 30%, with recurrences commonly seen among elderly (Kelly and LaMont, 2008).

The more recurrences a person has, the more likely he/she is to have a recurrence again. The risk of recurrence goes from about 20% after the initial CDI episode to about 40 and 60% after the first and two-or-more recurrences, respectively (McFarland, Reference McFarland2008). In at least 10% of cases, subsequent infections are caused by a new C. difficile strain that is molecularly different from that of the first CDI episode (Wilcox et al., Reference Wilcox, Fawley, Settle and Davidson1998; Noren et al., Reference Noren, Akerlund, Back, Sjoberg, Persson, Alriksson and Burman2004; Hell et al., Reference Hell, Permoser, Chmelizek, Kern, Maass, Huhulescu, Indra and Allerberger2011). As more discriminatory typing methods become available (e.g., multiple-locus variable number tandem repeat analysis, MLVA; or next generation sequencing) (Marsh et al., 2011; Eyre et al., Reference Eyre, Golubchik, Gordon, Bowden, Piazza, Batty, Ip, Wilson, Didelot, O'Connor, Lay, Buck, Kearns, Shaw, Paul, Wilcox, Donnelly, Peto, Walker and Crook2012), it is likely that more recurrences will be recognized to be indeed due to different strains, and not due to persistent infections. Currently, reinfection with different strains indicates that there are unrecognized sources of C. difficile in the community that serve as the source of infection for convalescent people following hospital discharge. Numerous studies have shown that animals, foods, and recreational environments can be sources of C. difficile strains similar or identical to those causing diseases in humans (Janezic et al., Reference Janezic, Ocepek, Zidaric and Rupnik2012). Table 2 summarizes reported sources of C. difficile in the community.

Table 2. Reported sources of C. difficile outside hospitals (in the community)

Companion animals – household pets

In modern times, especially in urban areas, pets are an integral part of the family, sharing human lifestyles, bedrooms, and beds. Recent estimates indicate that between 14 and 62% of pet owners allow dogs and cats on their beds (Chomel and Sun, Reference Chomel and Sun2011; Montgomery et al., Reference Montgomery, Xiao and Cama2011). Although dogs and cats have been shown to carry toxigenic strains of C. difficile in their feces since the 1980s (Borriello et al., Reference Borriello, Honour, Turner and Barclay1983b), the current striking genetic similarity between isolates from animals and humans indicate that zoonosis may be occurring (Lefebvre et al., Reference Lefebvre, Arroyo and Weese2006). Most companion animals that harbor C. difficile do so asymptomatically (Weese et al., Reference Weese, Finley, Reid-Smith, Janecko and Rousseau2010a). However, if risk factors are prevalent, that parallel those of humans (antimicrobial administration), dogs and cats may develop diarrhea (Weese et al., Reference Weese, Weese, Bourdeau and Staempfli2001). No PMC or C. difficile bacteremia has being documented in dogs or cats.

Screening studies indicated that up to 10% of household pets may carry C. difficile, representing a risk for owners (Weese et al., Reference Weese, Finley, Reid-Smith, Janecko and Rousseau2010a). Although no direct transmissibility from pets to humans has been documented, the presence of virulent strains of C. difficile (including PCR ribotype 027) in ‘therapy’ dogs indicate that in hospitals, visitation animals might carry strains within and outside health-care facilities (Lefebvre et al., Reference Lefebvre, Arroyo and Weese2006). Pets owned by an immune-compromised person are more likely to be colonized by C. difficile (Weese et al., Reference Weese, Finley, Reid-Smith, Janecko and Rousseau2010a). However, in one study that examined the zoonotic risk, the strains isolated from dogs and households were different (Weese et al., Reference Weese, Finley, Reid-Smith, Janecko and Rousseau2010a) another indication against direct-contact transmission. Although the authors concluded that dogs were not a significant source of household C. difficile contamination, all isolates from dogs were indistinguishable from historical isolates recovered from ill humans in the same geographical region, including emerging PCR ribotype 027. Therefore, it is advisable to prevent close contact between susceptible people and pets with diarrhea. It is also important to highlight that inadvertent infections with C. difficile (or other enteric human pathogens) in healthy-looking pets could occur in association with the consumption of raw pet foods (Weese et al., Reference Weese, Rousseau and Arroyo2005; Finley et al., Reference Finley, Reid-Smith and Weese2006). Avoiding the inclusion of raw meats in pet diets is always a good practice to reduce the risk of transmission of C. difficile and other zoonotic pathogens, especially if high-risk individuals are in the household.

In veterinary hospitals, outbreaks of severe diarrhea associated with C. difficile have been reported in small animal clinics (Weese and Armstrong, Reference Weese and Armstrong2003). Therefore, pets may become inadvertent carriers of C. difficile spores following routine veterinary visits or hospitalization. To date no studies have assessed the potential of dogs and cats to be vehicles of C. difficile strains out of veterinary hospitals and within food or livestock production systems.

Companion animals – horses

C. difficile has also been studied in horses since the mid-1980s (Ehrich et al., Reference Ehrich, Perry, Troutt, Dellers and Magnusson1984). Today, it is known that in the community up to 7% of healthy horses can carry C. difficile (Medina-Torres et al., Reference Medina-Torres, Weese and Staempfli2011), but the proportion of animals shedding the pathogen varies across studies as it depends on culture methods, the animals’ age, and management conditions. Adult horses are less likely to carry the bacterium compared to neonatal foals. Overall, between 2 and 30% of horses were found to be carrying spores at any given time without showing signs of disease (Baverud et al., Reference Baverud, Gustafsson, Franklin, Aspan and Gunnarsson2003). However, like other species, horses can also develop diarrhea and forms of serious colitis (Weese et al., Reference Weese, Toxopeus and Arroyo2006; Songer et al., Reference Songer, Trinh, Dial, Brazier and Glock2009). As in humans, antimicrobials increase the risk of horses being affected with CDI (Weese et al., Reference Weese, Toxopeus and Arroyo2006). In young foals, antimicrobials are also a predisposing factor (Arroyo et al., Reference Arroyo, Weese and Staempfli2004). Co-infection with C. perfringens may explain enterocolitis in some foals (Uzal et al., Reference Uzal, Diab, Blanchard, Moore, Anthenill, Shahriar, Garcia and Songer2011). In equine hospitals, strict isolation and infection control measures are thus widely recommended to avoid outbreaks (Baverud, Reference Baverud2004).

Some ethnic societies by tradition still rely on horse power to work agricultural lands to produce foods, especially fresh produce (Lengacher et al., Reference Lengacher, Kline, Harpster, Williams and Lejeune2011). In many regions, regulated production and slaughter of horse meat is allowed to sustain, at least partially, local economies and traditions (USDA, 1997). Since horse manure can contain C. difficile spores for years (Baverud et al., Reference Baverud, Gustafsson, Franklin, Aspan and Gunnarsson2003) its use as traditional organic fertilizer highlights the risk for fresh produce contamination (Pell, Reference Pell1997). To date, there are no studies addressing the role of horses in food and environmental health and safety associated with C. difficile. Nevertheless, farmers and animal handlers should be aware of the risk of finding C. difficile on horse manure and the potential for dissemination to susceptible members of the family or the community.

Food animals – pigs

As in companion animals, C. difficile was also isolated from pigs in the early 1980s (Jones and Hunter, Reference Jones and Hunter1983). Since then, over 60 published studies have served to now recognize C. difficile as an enteric pathogen in this domesticated species. Among pigs, young piglets have the highest risk for disease development (Post et al., Reference Post, Jost and Songer2002). For this reason, pigs have been increasingly used as models to study the pathogenesis of this disease (Keel and Songer, Reference Keel and Songer2007, Reference Keel and Songer2011; Steele et al., Reference Steele, Feng, Parry and Tzipori2010; Scaria et al., Reference Scaria, Janvilisri, Fubini, Gleed, McDonough and Chang2011). Mortality and morbidity rates in pigs are largely uncertain, but some estimates indicate that up to 100% of litters and individual piglets can be affected in infected farrowing facilities (Songer, Reference Songer2004). In non-fatal cases, weaning weights of diseased pigs can be 10% below the expected average weight (Songer, Reference Songer2004). In older animals, there is one report of an association between C. difficile and increased mortality in sows that received antimicrobial treatment (Kiss and Bilkei, 2005).

During processing, the isolation of C. difficile from healthy pigs close to the harvest time, and from processed carcasses (<2.5%) support the potential for food contamination (Norman et al., Reference Norman, Harvey, Scott, Hume, Andrews and Brawley2009; Weese et al., Reference Weese, Rousseau, Deckert, Gow and Reid-Smith2011, Susick et al., Reference Susick, Putnam, Bermudez and Thakur2012). Recent isolation of C. difficile from mesenteric lymph nodes at harvest (<1%) indicates that pathogen dissemination from the gut to muscles tissues via the circulatory and lymphatic system is possible (Susick et al., Reference Susick, Putnam, Bermudez and Thakur2012).

Swine-derived C. difficile isolates have garnered the greatest attention from public health personnel because PCR ribotype 078 – the increasingly documented emerging human strain in the community – is the major strain among porcine isolates. PCR ribotype 078 isolates have accounted for up to 80% of all swine isolates in most studies involving pigs in North America and Europe (Keel et al., Reference Keel, Brazier, Post, Weese and Songer2007; Debast et al., Reference Debast, van Leengoed, Goorhuis, Harmanus, Kuijper and Bergwerff2009; Songer et al., 2009). In humans, the same strain has increased its association with human disease by at least 6-fold from 2000 to 2008 (Goorhuis et al., Reference Goorhuis, Bakker, Corver, Debast, Harmanus, Notermans, Bergwerff, Dekker and Kuijper2008). Regarding the type of production system, no differences have been found between the prevalence of C. difficile in organic and conventional swine operations (Keessen et al., Reference Keessen, van den Berkt, Haasjes, Hermanus, Kuijper and Lipman2011), or between conventional and antibiotic-free operations (Susick et al., Reference Susick, Putnam, Bermudez and Thakur2012).

Food animals – cattle

Until recently little attention was directed to the study of C. difficile in ruminants. The first published report described C. difficile in veal calves with diarrhea in 2002 (Porter et al., Reference Porter, Reggiardo, Glock, Keel and Songer2002). The first published study quantifying the impact of C. difficile in the bovine industry determined in 2004 the role of the pathogen as a cause of diarrhea in young calves, the effect of seasonality, and its implications for public health (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006). In that study, a case-control study of calves <28 days of age – from 102 dairy farms in Canada – showed that significantly more calves with diarrhea were positive for C. difficile toxins compared to the control group, suggesting an association of C. difficile with intestinal disease. In further experimental studies, the same group could not induce disease when calves fed colostrums were given orally high numbers of toxigenic C. difficile (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Staempfli, Duffield and Weese2007a, Reference Rodriguez-Palacios, Stampfli, Stalker, Duffield and Weeseb). Subsequent studies in calf ranches have supported the association of intestinal lesions with C. difficile (Hammitt et al., Reference Hammitt, Bueschel, Keel, Glock, Cuneo, DeYoung, Reggiardo, Trinh and Songer2008). Colonization of neonatal calves infected under natural conditions was detected within 24 h of birth and lasted for at least 6 days after detection, indicating that calves were indeed amplifiers of toxigenic C. difficile (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Staempfli, Duffield and Weese2007a). Histological lesions were mild and restricted to ileum and colon. In veal calves, the rate of C. difficile shedding and associated diarrhea increases as animals are treated with antibiotics upon entry to finishing operations (Costa et al., Reference Costa, Stampfli, Arroyo, Pearl and Weese2011). Strain clonal diversity and shedding prevalence in young farm animals decrease with age (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006; Zidaric et al., Reference Zidaric, Pardon, Dos Vultos, Deprez, Brouwer, Roberts, Henriques and Rupnik2012).

In older cattle, C. difficile shedding decreased over time during the finishing period and was not affected by the administration of antimicrobials (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Pickworth, Loerch and Lejeune2011b). At the time of harvest, C. difficile can be found in healthy feedlot steers and culled dairy cattle, highlighting the risk for carcass and food contamination (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Koohmaraie and LeJeune2011a, Reference Rodriguez-Palacios, Pickworth, Loerch and Lejeuneb; Thitaram et al., Reference Thitaram, Frank, Lyon, Siragusa, Bailey, Lombard, Haley, Wagner, Dargatz and Fedorka-Cray2011). In Belgium, the frequency of shedding at slaughter was about 7% (Rodriguez et al., Reference Rodriguez, Taminiau, Van Broeck, Avesani, Delmee and Daube2012). Younger cattle used for food production, i.e. veal calves, although representing <2% of all meat consumed in the USA, can also have C. difficile strains of relevance for disease in humans (Costa et al., Reference Costa, Stampfli, Arroyo, Pearl and Weese2011; Houser et al., Reference Houser, Soehnlen, Wolfgang, Lysczek, Burns and Jayarao2012). Regardless of its association with enteric disease, C. difficile isolates derived from cattle were the first to draw attention to the potential for foodborne transmissibility involving current epidemic human strains PCR ribotypes 017, 027, 077, 014, and 078 (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006, Reference Rodriguez-Palacios, Reid-Smith, Staempfli, Daignault, Janecko, Avery, Martin, Thomspon, McDonald, Limbago and Weese2009, Reference Rodriguez-Palacios, LeJeune and Hoover2012; Keel et al., Reference Keel, Brazier, Post, Weese and Songer2007; Hammitt et al., Reference Hammitt, Bueschel, Keel, Glock, Cuneo, DeYoung, Reggiardo, Trinh and Songer2008) (Compare Fig. 3 and 4). Antimicrobial resistance against new-class linezolid, but not tigecycline, has been observed in C. difficile from cattle at harvest in the USA (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Koohmaraie and LeJeune2011a).

Fig. 3. The same strains that have been observed in animals and foods (see fig. 4) have been predominant and responsible for a major fraction of severe CDIs in people. Note seasonal peaks and that 5 (of over 300 possible) UK PCR ribotypes have accounted for over half of all human cases despite hospital infection control efforts (see Seasonality section below). A) Reproduced with permission from Hensgens et al., Reference Hensgens, Goorhuis, Notermans, van Benthem and Kuijper2009, Copyright Eurosurveillance. B) Note increase of PCR-027 (Courtesy Dr. EJ Kuijper, personal communication). Compiled from Hensgens et al., Reference Reil, Erhard, Kuijper, Kist, Zaiss, Witte, Gruber and Borgmann2011, Reference Reil, Hensgens, Kuijper, Jakobiak, Gruber, Kist and Borgmann2012).

Food animals – poultry

This is the food animal species that has been studied the least. The first studies that highlighted the potential relevance of poultry as carriers of toxigenic strains are from Africa (Simango, Reference Simango2006; Simango and Mwakurudza, Reference Simango and Mwakurudza2008). In Zimbawe, Simango and colleagues showed that up to 30% of free-range chickens carried toxigenic C. difficile with antimicrobial resistance patterns of relevance for humans (Simango, Reference Simango2006; Simango and Mwakurudza, Reference Simango and Mwakurudza2008). These results indicate that the risk of transmission via foods/animals to susceptible people in Africa, where the rate of HIV-infected patients in the community at risk for CDI is high (Onwueme et al., Reference Onwueme, Fadairo, Idoko, Onuh, Alao, Agaba, Lawson, Ukomadu and Idoko2011), might be a relevant factor to consider for targeted intervention. The prevalence of C. difficile in free range poultry in Zimbabwe could be extrapolated to comparable societies where free range poultry is common practice including some Asian and Latin American countries where C. difficile has been problematic in humans (Legaria et al., Reference Legaria, Lumelsky and Rosetti2003; Rupnik et al., Reference Rupnik, Kato, Grabnar and Kato2003; Huang et al., Reference Huang, Wu, Wang, Zhang, Fang, Palmgren, Weintraub and Nord2008; Balassiano et al., Reference Balassiano, Yates, Domingues and Ferreira2012).

Recent studies on poultry commercial operations have also documented the relevance of C. difficile in Europe and North America, where free-range production contributes less to the food supply. In Slovenia, one study conducted on an intensive commercial farming system reported that the percentage of birds colonized with C. difficile was higher than that reported in African free-range poultry, with prevalence decreasing with age (Zidaric et al., Reference Zidaric, MZemljic, Janezic, Kocuvan and Rupnik2008), as shown in calves, other animals, and children (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006; Enoch et al., Reference Enoch, Butler, Pai, Aliyu and Karas2011). In that study, over 60% of birds carried C. difficile in early production, but it was significantly less frequent (below 2%) as animals approached harvest time. In Austria, 5% of poultry tested had C. difficile (Indra et al., Reference Indra, Lassnig, Baliko, Much, Fiedler, Huhulescu and Allerberger2009). In agreement, the percentage of poultry and turkey colonized at harvest in the USA approached zero in intensive rearing facilities in Ohio (Rodriguez-Palacios et al., unpublished data). More recently, the prevalence of C. difficile in commercial chickens was also comparable at 2% prior to harvest in Texas in a study conducted by the U.S. Department of Agriculture (Harvey et al., Reference Harvey, Norman, Andrews, Hume, Scanlan, Callaway, Anderson and Nisbet2011). Clearly, much more work needs to be done in this field also, especially because, inexplicably, the prevalence of C. difficile in poultry meats, at least in North America, is significantly higher postharvest, ranging between 6 and 12% of chickens (Weese et al., Reference Weese, Reid-Smith, Avery and Rousseau2010b; Harvey et al., Reference Harvey, Norman, Andrews, Hume, Scanlan, Callaway, Anderson and Nisbet2011). Considering that Campylobacter illnesses in humans are often associated with the consumption of poultry (Moran et al., Reference Moran, Scates and Madden2009; Scallan et al., Reference Scallan, Hoekstra, Angulo, Tauxe, Widdowson, Roy, Jones and Griffin2011), it is possible that the risk of infection with C. difficile via ingestion of contaminated foods is comparable, simply because spores are expected to be more resistant to heat than the vegetative and viable but not culturable forms of Campylobacter spp.

In summary, from the available reports of C. difficile in animals, two general conclusions can be drawn. First, similar to humans, newborn and young animals are more frequently colonized by C. difficile than adult animals; however, unlike humans neonatal animals are at higher risk of being affected with enteric disease. Second, also similar to humans, animals in most studies exhibit a small diversity of C. difficile strains, although the high strain diversity observed in some cattle and poultry studies (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006; Zidaric et al., Reference Zidaric, MZemljic, Janezic, Kocuvan and Rupnik2008; Avbersek et al., Reference Avbersek, Janezic, Pate, Rupnik, Zidaric, Logar, Vengust, Zemljic, Pirs and Ocepek2009) could be a reflection of culture methodology and farm variability. High strain diversity usually indicates successful colonization with minimal selection forces. Environmental and host associated factors are possibly contributing to the selection of a few predominant strains.

Waters and the environment

In general, spore-forming bacteria including clostridia are microorganisms that last a long time in the environment. With very few exceptions, C. difficile produce spores that can survive for months in the environment (Baverud et al., Reference Baverud, Gustafsson, Franklin, Aspan and Gunnarsson2003), but not many publications are available in this regard. A British publication described the presence of toxigenic C. difficile in soils, wells, recreational waters, veterinary clinics, and in households (al Saif and Brazier, 1996). In Africa, soils in rural areas of Zimbawe inhabited by free-range chicken also had toxigenic C. difficile (Simango, Reference Simango2006). Despite these publications, little attention has been placed on the environment as a source of infectious spores and on its role in human and animal infections. C. difficile spores are disseminated via air in indoor environments (Roberts et al., Reference Roberts, Smith, Snelling, Kerr, Banfield, Sleigh and Beggs2008) (see Dissemination below). At the farm level, this area of research remains largely unexplored.

C. difficile in foods

Discovering that a particular microorganism becomes an emerging food safety concern should not be surprising. Foods have been a historic source of exposure for many pathogens. However, despite the expectedness of such an event, skepticism is natural. C. difficile was first recovered from foods and animals in 1980. In 1982, it was suspected as the cause of PMC that occurred after an elderly patient consumed canned salmon (Gurian et al., Reference Gurian, Ward and Katon1982). However, the patient had other health issues (hypochloremia) and the food item was not cultured. During 1981–1983, two studies reported finding no C. difficile in cooked foods from hospital menus; but the studies did not acknowledge that fresh foods or undercooked foods could be a source of exposure. It is important to note that culture methodology for food and environmental samples might have been suboptimal at the time since concurrent sampling of hospital air and walls yielded no C. difficile in the same studies. Further discussion about potential foodborne transmission went on until 1983 (Borriello et al., Reference Borriello, Honour and Barclay1983a).

In retrospect, we now know that thorough cooking (at least 96 °C, 15 min) should eliminate the amount of C. difficile expected to be found in most foods (Rodriguez-Palacios and LeJeune, Reference Rodriguez-Palacios, Koohmaraie and LeJeune2011). During the 20-year period 1982–2002, there was only one publication on C. difficile and foods. It was a report of an incidental finding of C. difficile in packed meats published by Broda et al. (Reference Broda, DeLacy, Bell, Braggins and Cook1996) .

Raw and ready-to-eat foods

Raw ground beef and pork were among the first food products to be found contaminated with C. difficile. In 1994, Broda and her colleagues, studying microorganisms that caused gas ‘blown pack’ spoilage in ready-to-eat meats incidentally found C. difficile (Broda et al., Reference Broda, DeLacy, Bell, Braggins and Cook1996). The next study conducted on raw meat commercial diets for dogs and cats also found C. difficile in a sample of turkey-based diet (Weese et al., Reference Weese, Rousseau and Arroyo2005). Despite the frequent occurrence of C. difficile in foods, its public health significance has generally been under-recognized or viewed with skepticism. A specially designed study base on MLVA have highlighted that the isolation (and prevalence) of C. difficile in the food supply is real and not due to laboratory contamination of the food samples (Curry et al., Reference Curry, Marsh, Schlackman and Harrison2012).

In 2007, the first study documenting human epidemic strains of C. difficile in foods (specifically, in 20% of retail ground meats), documented the regional and international relevance of the finding (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Staempfli, Duffield and Weese2007a). Subsequent studies have confirmed that this pathogen can be found in other foods tested. Now, scientific reports describe toxigenic C. difficile in meats in several countries. Although the percentages of meat packages that have been contaminated with C. difficile have ranged from 3 to 42%, the overall expected real prevalence of C. difficile contamination under natural conditions at the store level (by sampling 1–2 retail packages of meat per store) has been determined to be at about 6% (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Reid-Smith, Staempfli, Daignault, Janecko, Avery, Martin, Thomspon, McDonald, Limbago and Weese2009). Poultry has been the type of meat least studied. One study found no C. difficile in retail poultry (Indra et al., Reference Indra, Lassnig, Baliko, Much, Fiedler, Huhulescu and Allerberger2009); however recent studies indicate that poultry meats can also carry toxigenic strains (Weese et al., Reference Weese, Reid-Smith, Avery and Rousseau2010b; Harvey et al., Reference Harvey, Norman, Andrews, Hume, Scanlan, Callaway, Anderson and Nisbet2011). In the USA, the frequency of contamination of retail chicken has been documented to be between 9 and 18%, with all the edible animal parts (legs, wings, thighs, etc.) having comparable frequencies of contamination (Weese et al., Reference Weese, Reid-Smith, Avery and Rousseau2010b). Of concern, emerging C. difficile PCR ribotype 078 strains was found in retail chicken in both Canada and the USA (Weese et al., Reference Weese, Reid-Smith, Avery and Rousseau2010b; Harvey et al., Reference Harvey, Norman, Andrews, Hume, Scanlan, Callaway, Anderson and Nisbet2011). This strain is an emerging strain in humans, in hospitals, and the community, in food production environments and in retail foods (Rupnik et al., Reference Rupnik, Widmer, Zimmermann, Eckert and Barbut2008). The earlier identification of C. difficile in animals, with the subsequent increase of incidence of PCR ribotype 078 among people with CDI over the last decade indicates that this pathogen strain is likely moving from animals to humans (Goorhuis et al., Reference Goorhuis, Bakker, Corver, Debast, Harmanus, Notermans, Bergwerff, Dekker and Kuijper2008; Hensgens et al., Reference Hensgens, Keessen, Squire, Riley, Koene, de Boer, Lipman and Kuijper2012). At the processing plant, there is now molecular evidence to suspect persistence and potential cross-contamination of retail food (pork) products with unique MLVA types belonging to the PCR ribotype 078 clone over time (Curry et al., Reference Curry, Marsh, Schlackman and Harrison2012).

Fig. 4. Frequency of isolation of toxigenic C. difficile of distinct PCR ribotypes from humans, young cattle, and various foods. Note the relative importance of food animal derived strains in cases of human disease, and the presence of some ribotypes in Europe and North America (i.e., PCR-078). Data compiled from Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006; Keel et al., Reference Keel, Brazier, Post, Weese and Songer2007; Bauer et al., 2011; Reil et al., Reference Reil, Erhard, Kuijper, Kist, Zaiss, Witte, Gruber and Borgmann2011; Hensgens et al., Reference Hensgens, Keessen, Squire, Riley, Koene, de Boer, Lipman and Kuijper2012.

Convenient ready-to-eat products (deli meats and minimally processed fruits and vegetables) are gaining market share. Unlike other infamous foodborne bacteria, such as Escherichia coli O157:H7, the spores formed by C. difficile that are often found in these products are highly resistant to current recommended cooking food safety guidelines. Molecular studies confirmed in Scotland that ready-to-eat salads were contaminated with C. difficile strains linked to human disease (Bakri et al., Reference Bakri, Brown, Butcher and Sutherland2009). Clostridium difficile was first isolated from root vegetables in 1996 (al Saif and Brazier, Reference al-Barrak, Embil, Dyck, Olekson, Nicoll, Alfa and Kabani1996). More recently, it has been isolated from vegetables in North America; (J. G. Songer, 2007, personal communication; Rodriguez-Palacios and LeJeune (2007), unpublished data; Metcalf et al., Reference Metcalf, Costa, Dew and Weese2010). C. difficile have also been isolated from shellfish and fish, which are often consumed undercooked or raw (Metcalf et al., Reference Metcalf, Avery, Janecko, Matic, Reid-Smith and Weese2011). In Europe, the highest rate of food contamination was reported last year in edible mollusks in Italy, 49% (Pasquale et al., 2012). Several reviews are available summarizing the studies documenting C. difficile in foods (Indra et al., Reference Indra, Lassnig, Baliko, Much, Fiedler, Huhulescu and Allerberger2009; Gould and Limbago, Reference Gould and Limbago2010; Weese, Reference Weese2010). In Latin America, the first report is from Costa Rica, where a molecular clinical genotype was found in 2% of food samples; notoriously, the isolates were susceptible to the antibiotics to which the clinical isolates were highly resistant (Quesada-Gomez et al., 2013). Unless proven otherwise, antimicrobial discrepancy between genetically related strains should not be used to deem two isolates as non-related (Eyre et al., 2012).

Seasonality

As with many other diseases, there could be parallel in the seasonal trends in CDI associated with the prevalence of the causative bacterium in animals, foods and humans (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Reid-Smith, Staempfli, Daignault, Janecko, Avery, Martin, Thomspon, McDonald, Limbago and Weese2009). The number of cases of CDI in humans is higher during winter months, at least in northern latitudes (Burckhardt et al., Reference Burckhardt, Friedrich, Beier and Eckmanns2008; Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Reid-Smith, Staempfli, Daignault, Janecko, Avery, Martin, Thomspon, McDonald, Limbago and Weese2009; Reil et al., Reference Reil, Hensgens, Kuijper, Jakobiak, Gruber, Kist and Borgmann2012). That seasonal increase has been partly attributed to a larger number of cases associated with seasonal respiratory and enteric viral infections that require antimicrobial administration or hospitalization (Polgreen et al., Reference Polgreen, Yang, Lucas, Bohnett and Cavanaugh2010). In foods and food animals, at least three independent studies document the same seasonal pattern in North America (higher prevalence in winter) (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Stampfli, Duffield, Peregrine, Trotz-Williams, Arroyo, Brazier and Weese2006, Reference Rodriguez-Palacios, Reid-Smith, Staempfli, Daignault, Janecko, Avery, Martin, Thomspon, McDonald, Limbago and Weese2009; Norman et al., Reference Norman, Harvey, Scott, Hume, Andrews and Brawley2009; Kho Reference Kho Sugeng2012). Although seasonality patterns could occur independently in parallel as a function of climatic variations (Naumova et al., Reference Naumova, Jagai, Matyas, DeMaria, MacNeill and Griffiths2007), it is also possible that the prevalence of C. difficile at least in food animals, some foods, and people could be epidemiologically connected. It is important to note that earlier studies did not identify seasonal patterns in human disease (Tvede et al., Reference Tvede, Schiotz and Krasilnikoff1990).

Together, the molecular characteristics and virulence markers of food and food animal-derived C. difficile isolates indicate that the presence of emerging strains in vegetables and meats (and possibly the seasonality) might have a direct, yet unproven, connection with the epidemiology of CDI in humans. Although confirming such a connection might take some time, there is enough epidemiological evidence to take action and enhance prevention through education to minimize the risk of inadvertent exposure to C. difficile among individuals at risk.

Irrespective of the type of food product tested, the most important and concerning finding is that emerging hyper-virulent strains of C. difficile (PCR ribotypes 027 and 078) are among the most predominant genotypes recovered from foods (Fig. 4). The reasons for the predominance of these ribotypes are unknown, but increased sporulation rates could favor some strains (Akerlund et al., Reference Akerlund, Persson, Unemo, Norén, Svenungsson, Wullt and Burman2008) to become endemic in the environment.

Dissemination of C. difficile

More recently there have been growing concerns regarding biosecurity and further global dissemination (Clements et al., Reference Clements, Magalhaes, Tatem, Paterson and Riley2010). Hyper-virulent strains of C. difficile that were first reported in humans and animals in Eastern North America and Western Europe in the early 2000s (Warny et al., Reference Warny, Pepin, Fang, Killgore, Thompson, Brazier, Frost and McDonald2005; Kuipjer et al., 2008) have been identified in sporadic cases and outbreaks of disease in humans in more distant locations, including Australia, Japan, Korea, and Singapore, since 2007 (Sawabe et al., Reference Sawabe, Kato, Osawa, Chida, Tojo, Arakawa and Okamura2007; Tae et al., Reference Tae, Jung, Song, Kim, Choi, Lee, Hwang, Kim and Lee2009; Clements et al., Reference Clements, Magalhaes, Tatem, Paterson and Riley2010; Lim et al., Reference Lim, Ling, Lee, Koh, Tan, Kuijper, Goh, Low, Ang, Harmanus, Lin, Krishnan, James and Lee2011). Transcontinental commercial flights and the importation of live animals from places where emerging strains are documented have been listed as possibilities for dissemination (Clements et al., Reference Clements, Magalhaes, Tatem, Paterson and Riley2010) of lineages that emerged in North America (He et al., 2012). At the regional level, studies in white-tailed deer (common visitors to livestock grazing areas and abundant in North America, Europe, and New Zealand) and wild birds have been documented to be an important factor for C. difficile dissemination in a suburban agricultural region, with tangible exposure potential to humans and animals in the USA (Rodriguez-Palacios et al., unpublished data; French et al., Reference French, Rodriguez-Palacios and LeJeune2010). C. difficile has been isolated from several other wildlife species since the 1980s, including feral swine populations (Thakur et al., Reference Thakur, Sandfoss, Kennedy-Stoskopf and Deperno2011). Air dissemination studies have increased in recent years. Studies conducted around the vicinity of pig farms indicate that aerial dissemination for short distances is possible with downstream currents. In bathrooms, C. difficile has been found surrounding toilets, presumably due to aerosolization of fecal particles during flushing. Not surprisingly, this is of preventive relevance since seemingly identical C. difficile strains have been isolated from pigs and from toilets used by the farm workers in an integrated swine operation (Norman et al., Reference Norman, Harvey, Scott, Hume, Andrews and Brawley2009).

Genome association studies

Whole-genome, microarray-based studies indicate that food animals might have been the original sources of some emerging epidemic strains of C. difficile, particularly newly emerging PCR ribotype 078 (Stabler et al., Reference Stabler, Gerding, Songer, Drudy, Brazier, Trinh, Witney, Hinds and Wren2006; Goorhuis et al., Reference Goorhuis, Bakker, Corver, Debast, Harmanus, Notermans, Bergwerff, Dekker and Kuijper2008; Bakker et al., Reference Bakker, Corver, Harmanus, Goorhuis, Keessen, Fawley, Wilcox and Kuijper2010). MLVA analysis continues to indicate that hospitalized humans and food animals, and foods are carrying clonally related strains (Marsh et al., Reference Marsh, Tulenko, Shutt, Thompson, Weese, Songer, Limbago and Harrison2011; Koene et al., Reference Koene, Mevius, Wagenaar, Harmanus, Hensgens, Meetsma, Putirulan, van Bergen and Kuijper2012). However, no conclusive studies are available to determine if animal shedding or food contamination are associated with changing patterns of disease in humans. Rather, it is possible that the pathogen constantly moves between humans, animals, and the environment, partly evolving and adapting as it moves across temporal and spatial niches. Given the spore forming nature of C. difficile, it is possible that inter-species transmission occurs from environmental sources and that some level of host adaptation (Janvilisri et al., Reference Janvilisri, Scaria, Thompson, Nicholson, Limbago, Arroyo, Songer, Grohn and Chang2009) and clonality has also ensued in parallel over millions of years (Stabler et al., Reference Stabler, Gerding, Songer, Drudy, Brazier, Trinh, Witney, Hinds and Wren2006; He et al., Reference He, Sebaihia, Lawley, Stabler, Dawson, Martin, Holt, Seth-Smith, Quail, Rance, Brooks, Churcher, Harris, Bentley, Burrows, Clark, Corton, Murray, Rose, Thurston, van Tonder, Walker, Wren, Dougan and Parkhill2010). Horizontal gene transfer and homologous recombination are very frequent genetic events in C. difficile. It is possible that the epidemiology of C. difficile will continue to evolve. Genomic approaches are increasingly used to understand virulence pathways and to provide modern alternatives for rapid diagnosis and treatment (Forgetta et al., Reference Forgetta, Oughton, Marquis, Brukner, Blanchette, Haub, Magrini, Mardis, Gerding, Loo, Miller, Mulvey, Rupnik, Dascal and Dewar2011; Eyre et al., 2012), but prevention strategies remain a challenge mostly due to limited information on disease ecology (outside hospitals) and inherent problems with integration of knowledge across disciplines. Addressing this issue, here we identify areas where recommendations should be expanded (Table 3). We have also proposed a list of simple educational measures for prophylactic use, which is under multidisciplinary consideration.

Recommending thorough cooking, kitchen hygiene, and minimize exposure

To date, most food safety guidelines available to the community instruct people to cook most foods at determined minimum internal temperatures to achieve a significant (6 log units) reduction of major foodborne pathogens to make most meals safe. These ranges vary from 63 °C to 74 or 85 °C (CFIA, 2010; USDA, 2011). Because recent quantitative studies have shown that C. difficile spores can survive extended heating at 71 °C (160°F), the minimum temperature recommended for cooking of meats (Rodriguez-Palacios et al., Reference Rodriguez-Palacios, Reid-Smith, Staempfli and Weese2010), it is necessary to heat foods at higher temperatures to inactivate C. difficile spores. Based on quantitative analysis with C. difficile isolates derived from foods, food animals, and humans (Meisel-Mikolajczyk et al., Reference Meisel-Mikolajczyk, Kaliszuk-Kaminska and Martirosian1995; Rodriguez-Palacios and LeJeune, Reference Rodriguez-Palacios, Koohmaraie and LeJeune2011), heating foods to 85 °C for 10–15 min could be a reasonable strategy to minimize the counts of C. difficile in foods. Alternatively, heating at 96 °C (sub-boiling) could reduce 6 log₁₀ within 2–3 min (Rodriguez-Palacios and LeJeune, Reference Rodriguez-Palacios, Koohmaraie and LeJeune2011). Thorough cooking at boiling temperatures, a common household practice, is ideal, and could be emphasized. As C. difficile could still survive cooking temperatures and multiply in heated foods, it is also recommended that foods be properly chilled and stored as indicated for other clostridial foodborne pathogens.