Clostridioides difficile is a spore-forming, anaerobic bacterium that can colonise the gastrointestinal tract of both humans and animals. In humans, C. difficile can cause infections (C. difficile infection, CDI), with symptoms ranging from diarrhoea to severe pseudomembranous colitis. Traditionally, CDI was regarded as a primarily nosocomial disease, but it is now increasingly found in persons outside the healthcare setting [Reference Hensgens1]. In community-acquired CDI, ribotype 078 (RT078) is emerging as a cause of infection [Reference Goorhuis2]. This type is predominant among pigs and cattle, animals that are frequently found positive for C. difficile [Reference Weese3]. Previous research into RT078 has shown that pig farmers and their pigs shared identical C. difficile strains and that transmission occurred either via direct contact or via the environment [Reference Knetsch4, Reference Keessen5]. In a study among persons living near livestock farms in the Netherlands, the prevalence of C. difficile carriage was low (1.2%) and 0.2% carried RT 078 [Reference Zomer6].

C. difficile has also been found in a wide range of animals other than pigs and cattle, including horses, dogs and cats, and the most common strains found in human CDI also occur in cats and dogs [Reference Koene7]. This suggests that household pets could serve as a potential source of C. difficile for humans (and vice versa), or that there is a common source of exposure. Indeed Loo et al. found that transmission may occur between CDI patients and their household members and domestic pets [Reference Loo, Brassard and Miller8]. However, other studies on C. difficile isolates from households have revealed no overlap in ribotypes between dogs or cats and their owners, or between dogs and the household environment [Reference Weese9, Reference Rabold10].

If zoonotic transmission of C. difficile occurs, veterinary healthcare workers who are in close contact with diseased and possibly diarrhoeic animals might be at an increased risk of acquiring C. difficile and potentially contribute to spreading C. difficile in the community. Therefore, the aim of this study is to investigate the prevalence of C. difficile carriage and risk factors including occupational contact with different types of animals in veterinary healthcare workers.

The medical ethical committee of the University Medical Center Utrecht reviewed this study and granted it an official exemption for approval under the medical research involving human subjects act (WMO) (number 18-389/C). This study is part of the Antibiotic-Resistant Bacteria in Dutch Veterinary healthcare workers study (Dutch acronym: AREND), in which the presence of ESBL-producing Escherichia coli and Klebsiella pneumoniae, colistin-resistant E. coli and K. pneumoniae, and C. difficile was determined in persons working in veterinary healthcare. Veterinary personnel (aged 18 years or older) was recruited between August 2018 and March 2019, through flyers sent to veterinary clinics, articles and recruitment at a veterinary conference (KNMvD voorjaarsdagen 2018). All participants signed an informed consent form. Participants sent in a faecal sample collected at home and completed a web-based questionnaire covering potential risk factors for C. difficile carriage (Supplementary material). To avoid clustering, participants working in the same clinic were assigned to participate in different months.

Faecal samples were sent to the laboratory by regular mail and upon arrival were either processed the same day or stored at 4 °C for up to 2 days. C. difficile was cultured by suspending approximately 1 g of faeces in 9 ml of C. difficile enrichment modified broth (Mediaproducts) with C.D.M.N. Selective Supplement (Oxoid) and incubated at 37 °C for 10–15 days under anaerobic conditions. The suspension was inoculated onto ChromID C. difficile agar (bioMérieux) directly, as well as following ethanol shock and incubated for 2–5 days under anaerobic conditions. A maximum of three suspected colonies per person were selected for further testing. Bacterial species were confirmed using Matrix-Assisted Laser Desorption/Ionisation Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) (Bruker). Subsequently, C. difficile positive isolates were genetically identified as C. difficile by polymerase chain reaction (PCR) for the presence of the gluD gene [Reference Paltansing11]. Further C. difficile characterisation was performed by PCR ribotyping and by determining the presence of toxin A (tcdA), toxin B (tcdB) and the binary toxin (cdtA/cdtB) genes [Reference Persson, Torpdahl and Olsen12, Reference Fawley13].

Prevalence of C. difficile carriage with 95% confidence intervals (CIs) was determined with the Wilson method [Reference Wilson14]. Using univariable logistic regression analysis, crude odds ratios (ORs) with 95% CIs were calculated to study potential risk factors for C. difficile carriage. A P-value < 0.05 was used to determine significance. Analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC, USA).

Of 515 veterinary healthcare workers that signed the informed consent form, 482 (93.6%) returned the faecal sample and completed the questionnaire. The median age of participants was 38 years (range 20–70 years), and 84.9% were female. The participants worked in veterinary clinics located in 310 different postal code areas. The prevalence of C. difficile carriage was 2.3% (11/482; 95% CI 1.3–4.0). Three persons carried C. difficile RT078 (prevalence 0.6%; 95% CI 0.2–1.8), see Table 1. Other ribotypes with toxin genes tcdA and tcdB were found in five participants (006, 046, 351 and two unidentified ribotypes that did not match any isolate in the established database). Three persons carried ribotypes without toxin genes (009, 039 and one unidentified ribotype). The three persons carrying RT078 all worked in different postal code areas. Two were veterinarians frequently working with companion animals, and one also worked with horses. The third person was a veterinary assistant who indicated not to have frequent animal contact at work but had non-occupational contact with pigs in the last 4 weeks, and had a partner who was a pig farmer. All three held animals at home, including dogs, cats and horses. Potential non-work-related risk factors that were present in these persons were having a young child going to day care (n = 1), use of proton pump inhibitors (PPI) or antacids due to acid reflux (n = 2) and use of antibiotics in the past 6 months (n = 1). More characteristics, including those of persons carrying other C. difficile strains, are shown in Table 1.

Table 1. Characteristics of veterinary healthcare workers who were carrier of Clostridioides difficile

tcdA, toxin A gene; tcdB, toxin B gene; cdtA/cdtB, binary toxin genes; PPI, proton pump inhibitor; proc., procedures; UNK, unknown.

^a Weekly or more often.

^b Rabbit, Guinea pig, hamster, rat and/or mouse.

^c Including: ADHD medication, oral contraceptives, medication for depression, sleeping pills/tranquilizers, antidiabetic agents, antihypertensive agents, chemotherapy, statins, laxatives.

^d Including: gastric mucosal irritation, acid reflux, gastric cancer, colon polyps, colon cancer, irritable bowel syndrome, Crohn's disease, ulcerative colitis, coeliac disease.

^e Including: vomiting, nausea, abdominal pain or cramps, mucus or blood in the stool, pale stool, diarrhoea (≥3 times a day).

The results of the univariate risk factor analysis for C. difficile carriage are shown in Supplementary material, Table S1. Pig contact (not work related) in the past 4 weeks was the only statistically significant animal-related risk factor (OR 6.8; 95% CI 1.3–34.0). Several hygiene-related factors were associated with an increased risk, including almost never washing hands after patient contact (OR 12.7; 95% CI 1.2–129.2) and poor hygiene practices at home: regularly/sometimes washing hands before food preparation (OR 5.4; 95% CI 1.1–25.6); almost never washing hands after toilet use (OR 7.3; 95% CI 1.3–40.8); and not changing the kitchen dishcloth on a daily basis (OR 8.3; 95% CI 1.1–65.0). Other risk factors were health and medication-related: having acid reflux (OR 4.2; 95% CI 1.1–16.3) and using medication for depression (such as venlafaxine, lithium and monoamine oxidase inhibitors) (OR 10.0; 95% CI 2.4–41.0).

The prevalence of C. difficile carriage of 2.3% (95% CI 1.3–4.0) in veterinary healthcare workers was not significantly higher compared to the prevalence of 1.2% (95% CI 0.9–1.7; n = 30/2432) that was found in a large Dutch population study among persons living in a rural area with a high density of livestock farms in 2014–2015 [Reference Zomer6]. It was lower than the prevalence of 5.1% (95% CI 3.8–6.9) in 765 stool samples of a population of asymptomatic patients with significant comorbidity and medication use on admission to Dutch hospitals [Reference Terveer15]. All carriers were female, which was most likely caused by an overrepresentation (85%) of female participants. The majority of C. difficile positive isolates (72.7%; n = 8/11) contained a toxigenic variant. This is comparable to the distribution of toxigenic/non-toxigenic variants in the paper by Zomer et al. (70.0%; n = 21/30) [Reference Zomer6]. RT078 was the most prevalent ribotype (n = 3; 27.3%), while it was the second most prevalent type in the aforementioned study, after RT014. RT014 was not detected in the present study. In the Dutch sentinel surveillance of CDI in 2019–2020 RT014 was the most frequently isolated ribotype (18.1%), whereas RT078 accounted for 8.7% of CDI [Reference Vendrik16].

RT078 has been reported as the predominant type in pigs in the Netherlands [Reference Koene7], but only a minority of the veterinary workers had frequent occupational contact with pigs (n = 19; 3.9%), and only one of the three RT078 C. difficile positives had (non-occupational) contact with pigs. We found an association between C. difficile carriage and non-occupational contact with pigs, although this was based on only two C. difficile positive persons.

To our knowledge, this is the first study that investigated C. difficile carriage in veterinary healthcare workers. Most of the participants (>85%) had occupational contact with dogs and cats, and 69% had occupational contact with companion animals only and not with livestock. There are around 2400 veterinary clinics in the Netherlands of which 60% are companion animal clinics, 15% are livestock clinics, 5% are horse clinics, and 20% are mixed clinics [Reference Bergevoet, Benus and van der Valk17]. The distribution of participants in our study working with companion animals (90%), livestock (23%) and horses (16%) is therefore representative for the country. The exact number of clinics represented in our study is unknown, but personnel from veterinary clinics located in 310 different 4-digit postal code areas were included (from a total of 4070 of these areas in the Netherlands).

C. difficile carriage has been described in healthy and diarrhoeic companion animals [Reference Weese3]. Furthermore, studies in veterinary clinics demonstrated C. difficile being present in companion animals visiting the clinic as well as on the clinic's surfaces, suggesting potential transmission at the clinic [Reference Alvarez-Perez18, Reference Villagomez-Estrada19]. We found an increased risk of C. difficile carriage for poor hand hygiene after patient contact, which could indicate a potential route of exposure via patients. However, since the prevalence in veterinary healthcare workers was low, the risk of transmission was likely very small.

Although clinical and epidemiological risk factors of CDI have been studied frequently [Reference Bloomfield and Riley20], studies on risk factors of C. difficile carriage are still scarce, especially for community-acquired carriage [Reference Crobach21]. Known risk factors of C. difficile carriage in the healthcare setting include recent hospitalisation and the use of specific medication, such as immunosuppressant, antibiotics and PPI or H₂ blockers [Reference Crobach21]. Among predominantly healthy young infants, the risk was increased in infants with a pet dog [Reference Stoesser22], and in the general population antibiotic use was previously identified as a risk factor [Reference Zomer6]. We found a non-significant association between antibiotic use and C. difficile carriage, presumably due to the small number of participants that were C. difficile positive. Furthermore, having acid reflux (but not the use of PPI or antacids) as well as the use of medication for depression was associated with a higher risk of C. difficile carriage. This association that was found with certain types of medication could be explained by the influence that they have on the microbiome [Reference Crobach21, Reference Cussotto23], and both CDI and carriage have been associated with an altered microbiome and a decreased bacterial diversity in the gut [Reference Crobach24].

This study had some limitations. First, due to the small number of C. difficile positive participants, estimates of potential risk factors are weak. To obtain robust insights into general risk factors for C. difficile carriage, large population studies are needed. Second, we did not include a control group of persons without occupational animal contact, since we were mainly interested in specific occupational risk factors in veterinary healthcare. The prevalence in veterinary healthcare workers was compared to the prevalence that was found in a large Dutch population study performed 4 years earlier [Reference Zomer6]. Finally, the risk factors assessed in this study are based on self-reporting, it is possible that some exposures were under- or overreported due to recall bias.

In conclusion, the prevalence of C. difficile carriage in veterinary healthcare workers was low and no indications were found that working in veterinary care increased the risk of C. difficile carriage.