Infrequent intra-household transmission of Clostridioides difficile between pet owners and their pets

Summary

Companion animals have been shown to carry Clostridioides difficile strains that are similar or identical to strains found in people, and a small number of studies have shown that pets carry genetically identical C. difficile isolates as their owners, suggesting interspecies transmission. However, the directionality of transmission is ultimately unknown, and the frequency with which animals acquire C. difficile following their owners’ infection is unclear. The goal of this study was to assess how often pets belonging to people with C. difficile infection carry genetically related C. difficile isolates. We enrolled pet owners from two medical institutions (University of Pennsylvania Health System (UPHS) and The Ohio State University Wexner Medical Center (OSUWMC)) who had diarrhea with or without positive C. difficile assays and tested their feces and their pets’ feces for C. difficile using both anaerobic culture and PCR assays. When microorganisms were obtained from both the owner and pet and had the same toxin profile or ribotype, isolates underwent genomic sequencing. Fecal samples were obtained from a total of 59 humans, 72 dogs, and 9 cats, representing 47 complete households (i.e., where a sample was available from the owner and at least one pet). Of these, C. difficile was detected in 30 humans, 10 dogs, and 0 cats. There were only two households where C. difficile was detected in both the owner and pet. In one of these households, the C. difficile isolates were of different toxin profiles/ribotypes (A+/B+ / RT 499 from the owner, A−/B− / RT PR22386 from the dog). In the other household, the isolates were genetically identical (1 SNP difference). Interestingly, the dog from this household had recently received a course of antibiotics (cefpodoxime and metronidazole). Our findings suggest that interspecies transmission of C. difficile occurs infrequently in households with human C. difficile infections.

Introduction

Clostridioides difficile is a spore-forming anaerobic, Gram-positive bacillus that is the leading cause of antibiotic-associated and healthcare-associated diarrhea in humans (Lessa, Winston, McDonald, & Emerging Infections Program, 2015). In recent decades, there has been a marked increase in the incidence and severity of community-acquired C. difficile infections (CDI) (Gould, File, & McDonald, 2015). The source of these community-acquired infections has not been clearly established. People asymptomatically colonized or infected with C. difficile are potential reservoirs (Galdys, Curry, & Harrison, 2014; Miller et al., 2022), but zoonotic, environmental and food-borne transmission of C. difficile to people has also been suggested (Knetsch et al., 2014; Knetsch et al., 2018; Varshney et al., 2014).

Genomic studies have documented intra- and interspecies clonal transmission events for several ribotypes of C. difficile in livestock (Knetsch et al., 2014; Knight et al., 2019), but little is known about the potential for transmission between companion animals and their owners. Pet owners frequently engage in activities that could facilitate transfer of infectious pathogens between pets and owners (e.g., allowing pets to lick their hands and face, picking up fecal matter, sleeping with their pet), and the transmission of bacterial enteropathogens such as Salmonella (Hoelzer, Moreno Switt, & Wiedmann, 2011) and Campylobacter (Montgomery, Robertson, Koski, & al., 2018) between pets and their owners has been demonstrated. However, pets’ roles in the transmission of C. difficile is unknown (Marks, Rankin, Byrne, & Weese, 2011).

Companion animals were shown to harbor C. difficile as early as 1983 (Borriello, Honour, Turner, & Barclay, 1983), and the prevalence of C. difficile in healthy adult dogs and cats has been found to range from 3-13% (Rabold et al., 2018; Stone et al., 2016). While the direct transmission of C. difficile between pets and their owners has not been definitively demonstrated, one study found that 2/9 (22%) cats and 2/5 (40%) dogs belonging to owners who had experienced CDI carried C. difficile with identical pulsed field gel electrophoresis profiles as their owners (Loo, Brassard, & Miller, 2016). Similarly, a case report identified genetically identical C. difficile isolates in a 10-month old child and her dog, both of whom had diarrhea (Rodríguez-Pallares et al., 2022). The concurrent colonization or infection of pets and their owners with genetically related strains of C. difficile suggests the possibility of interspecies transmission, but the directionality of transmission (e.g., from pet to owner or from owner to pet) is unclear, and the acquisition of C. difficile from a common environmental source cannot be ruled out. Lefebvre et al. reported that hospital visitation therapy dogs that licked patients or accepted treats from patients were more likely to become colonized with C. difficile than pets that did not lick patients or accept treats (Lefebvre, Arroyo, & Weese, 2006; Lefebvre & Weese, 2009), while another study found that pets living with immunocompromised owners, who are at higher risk of CDI and recurrent CDI (El-Matary et al., 2019; Revolinski & Munoz-Price, 2019), were more likely to be colonized with C. difficile (Weese, Finley, Reid-Smith, Janecko, & Rousseau, 2010). Taken together, these findings suggest that transmission occurred from people to animals. Because patients with CDI can persistently shed C. difficile even following resolution of diarrhea (Jury et al., 2013; Sethi, Al-Nassir, Nerandzic, Bobulsky, & Donskey, 2010) and infect human household contacts (Miller et al., 2022), they might be sources of C. difficile for their pets. Pets that become colonized with C. difficile could then become reservoirs of C. difficile themselves and potentially perpetuate a recurring pet-owner transmission cycle within the household or allow for interhousehold transmission if pets interact with pets or people of other households. However, data are lacking on whether and how frequently pets become colonized with C. difficile following infection of their owners. The objective of this study was thus to assess whether and how often pets residing in the household of people with C. difficile-positive diarrhea carry genetically related isolates of C. difficile.

Materials and Methods:

Patient recruitment:

Patients were recruited from the University of Pennsylvania Health System (UPHS) and The Ohio State University Wexner Medical Center (OSUWMC). At both UPHS and OSUWMC, C. difficile is detected in diarrheic patients with the Xpert C. difficile/Epi, a fecal PCR toxin B gene assay that is considered to be highly sensitive and specific for the presence of toxin-producing C. difficile microorganisms (Pancholi, Kelly, Raczkowski, & Balada-Llasat, 2012). At UPHS, PCR-positive samples are further tested with enzyme immunoassay to distinguish colonization from infection (Polage et al., 2015). All patients at UPHS with a positive C. difficile test in outpatient or inpatient clinical sites were identified as potentially eligible study subjects. With permission from the patient’s physician, UPHS patients with available contact information were contacted by email and invited to participate in the study. UPHS patients who agreed to participate and provided informed consent completed a short online survey asking them about 1) their pets, 2) their household, and 3) the degree of contact they have with their pets. UPHS patients were recruited from May 2019-May 2021, though recruitment was significantly interrupted in 2020 by the COVID pandemic.

OSUWMC patients with a concurrently submitted C. difficile test (both C. difficile-positive and negative individuals were eligible) were approached in person by the study team during their hospitalization. OSUWMC patients who agreed to participate were provided a study kit including an introductory letter describing the study, a consent form, survey, incentivizing gift card form, collection materials for dog feces, and postage-paid return envelope. Surveys gathered data on participants’ 1) health status, 2) their dog’s health and medical history, and 3) their dog’s husbandry and environmental factors. OSUWMC patients were enrolled from May 2016–June 2017.

These studies were approved by the Institutional Review Boards of the University of Pennsylvania and The Ohio State University. Institute for Animal Care and Use Committee approval was not deemed necessary, as owners collected freshly voided fecal samples from their pets themselves.

Patient stool collection:

UPHS and OSUWMC Clinical Microbiology Laboratories store all tested fecal samples from all patients for 7 days as part of routine laboratory protocol before discarding the stool in case additional stool tests are ordered or tests need to be repeated. If a patient consented to participate in the study before their stool sample was discarded, his/her stool sample was transferred from the Clinical Microbiology Laboratory to the OSU or University of Pennsylvania veterinary school microbiology laboratory for anaerobic culture.

Pet stool collection

Pet owners from the UPHS cohort were mailed stool collection kits with instructions to collect a fecal sample from their pet(s) (cats or dogs only) within two weeks of their positive C. difficile test and ship it back overnight to the laboratory in an insulated package with an ice pack. These methods have been used successfully by our team to obtain C. difficile-positive stool samples from people and dogs (Anis, Barnart, Barnard, Kelly, & Redding, 2021; L. Redding et al., 2021).

Pet owners from the OSUWMC cohort were provided stool collection kits with instructions to collect two consecutive fecal samples per dog from up to two household dogs and return them by mail to the study team. Participants were requested to send dog fecal samples within 3 days from study enrollment. Dog-owning patients also had the option of electing to participate in a follow-up component of the study, involving collecting the same samples approximately one month after they returned their original kit.

Culture and isolation of C. difficile:

The following protocols were used at UPHS and at OSU for culturing C. difficile.

UPHS protocol:

Fecal samples first underwent selective enrichment by mixing with cooked meat broth and incubated anaerobically for 48 hours. A 1-ml aliquot of the broth underwent alcohol shock with 0.5 ml of 100% ethanol, sat for 60 minutes at room temperature, then was inoculated onto CCFA/ChromID C. difficile agar and Columbia CNA agar. Inoculated plates were incubated at 36°C under anaerobic growth conditions for seven days and checked for growth every other day. Suspect colonies, identified by colony morphology and characteristic odor, were isolated and confirmed to be C. difficile by Maldi-TOF identification and/or RapID^™ ANA II System.

To confirm culture results for toxigenic isolates, all samples underwent qPCR using an in-house multiplex PCR that detects the tcdA and tcdB genes and has been validated for use in canine stool (Anis et al., 2021).

OSU protocol:

Fecal samples (100-300g) were placed in C. difficile moxalactam norfloxacin (CDMN) selective enrichment broth with 0.1% sodium taurocholate, as previously described and incubated within sealed tubes at 37°C for 7 days (36-38). Following enrichment, 2 ml of the sample underwent an alcohol shock, was centrifuged for 10 min at 3,800 g, and the resulting pellet was transferred onto CDMN agar with 7% horse blood. These plates were incubated anaerobically at 37°C for 48–72 hr. A single colony with typical morphology was subcultured onto Columbia blood agar (CBA) with 5% horse blood and incubated anaerobically at 37°C for 48 hr. Resulting colonies were evaluated by Gram stain, colony morphology, characteristic odor and L-proline aminopeptidase activity; colonies that were suspected to be C. difficile underwent confirmatory testing. DNA was extracted from fresh colonies grown on blood agar plates, using a Qiagen DNeasy Blood & Tissue Kit (Qiagen). Isolates were confirmed as C. difficile through PCR amplification of the triose phosphate isomerase (tpi) gene, as previously described (Fry, Thakur, Abley, & Gebreyes, 2012). Isolates confirmed as C. difficile were tested for the presence of tcdA, tcdB, and cdtB to characterize toxigenic potential. TcdA- and tcdB-specific primers were used in a multiplex PCR, as described previously (Lemee et al., 2004). CdtB-specific primers were used to identify the presence of the binary toxin gene as previously described (O’Shaughnessy et al., 2019). Isolates also underwent capillary electrophoresis PCR - ribotyping as previously described (Fawley et al., 2015). International designations (i.e., ribotype 078) were used for strains where reference strains were available; otherwise, new ribotype designation numbers were assigned. Samples that were negative for the toxins or had a toxin profile inconsistent with the identified ribotype were rechecked using the multiplex method described by Persson, Torpdahl, and Olsen (2008). All reactions and agarose gels were run alongside a reaction with DNA from strain ATCC 9689 as a positive control, and a reaction without DNA as a negative control.

Whole genome sequencing and analysis:

When C. difficile was isolated from both the patient and pet sample at UPHS, cultured isolates were sent to the PennCHOP Microbiome Sequencing Core for nucleic acid extraction, high-throughput sequencing, sequence-data management and data analysis. Briefly, DNA was extracted from pure cultures using the Qiagen DNeasy PowerSoil kit. Genomic libraries for C. difficile isolates were generated from 1 ng of DNA using the NexteraXT kit (Illumina, San Diego, CA, USA). Libraries were sequenced on the Illumina HiSeq using 2x125 bp chemistry in High Output mode. Whole genome shotgun sequencing data was processed for assembly through the Sunbeam pipeline (https://github.com/sunbeam-labs/sunbeam) using Cutadapt (Martin, 2011), Trimmomatic (Bolger, Lohse, & Usadel, 2014), and Komplexity (Clarke et al., 2019) for low quality and low complexity filtering, and BWA (Li & Durbin, 2009) for alignment to host DNA sequence for removal. C. difficile genomes were assembled using SPADES (v3.14.0) (Prjibelski, Antipov, Meleshko, Lapidus, & Korobeynikov, 2020) and genome quality was assessed with CheckM (v1.1.3) (Parks, Imelfort, Skennerton, Hugenholtz, & Tyson, 2015). Sequence typing was performed for each isolate using MLST 160 v.2.19.0 by analyzing housekeeping genes from assembled contigs and comparing the sequence variation against characterized C. difficile sequence types in the PubMLST database as previously described (L. E. Redding et al., 2022). Single-copy core gene sequences (Lee, 2019) from assembled and reference genomes were used to infer a phylogenetic tree to determine relatedness of isolates from patients and their pets. Single-nucleotide variants (SNVs) within the core genes were calculated by performing a pairwise alignment using the Biostrings library (v2.54.0) in R. Finally, to identify antibiotic resistance genes, a diamond BLASTx screen for each isolate was performed using the isolate’s DNA sequences aligned with the Comprehensive Antibiotic Resistance Database (CARD) protein homology database.

Risk factor analysis

In the OSUWMC cohort, animal-level health and environmental factors were investigated as risk factors for pets’ fecal shedding of C. difficile. The difference in distribution of each factor among colonized and non-colonized animals was investigated using Fisher’s exact test or the Wilcoxon rank-sum test, as appropriate. A p-value ≤ 0.05 was considered statistically significant in all analyses.

Results:

At OSUWMC, a total of 32 human patients and their 45 dogs were enrolled (i.e., all completed a survey and returned at least one dog fecal sample) (Figure 1). Most (29/30; 97%) enrolled individuals reported their dogs lived indoor/outdoor, with close contact between themselves and the dog being tested (lick owner’s face several times a week or more frequently: 16/42 (38%)); lick owner’s hands several times a week or more frequently: 23/42 (55%)). Within two weeks of patient enrollment, two fecal samples had been obtained from 44/45 (98%) and only one fecal sample was obtained from 1/45 dogs. Additional samples from 23 of the 45 dogs (51%) were provided at a later time point, a median of 53 days (range: 41-295) after submission of the prior dog sample(s) (Table 1). Overall, C. difficile was detected in 17 of 32 OSUWMC households (53.1%) from either the pet or the human, with one or more toxigenic isolate in 13 of these households (76.5%); in 8/17 (47.1%) of these households, only the pet was C. difficile-positive (Table 1). The proportion of dogs that were positive for C. difficile was similar at patient enrollment (7/45; 15.6%) and follow-up testing (2/23; 8.7%). In only one of the 32 sampled households (3.1%) was the pet and owner C. difficile-positive. The isolates obtained concurrently from the pet and owner were of different toxin profiles and ribotypes (A+/B+ / RT 499 for the owner, A−/B− / RT PR22386 for the dog; Table 1), suggesting genetically distinct isolates, and were not sequenced. Ribotypes varied across animals and patients (Table 1). No demographic or environmental variables were significantly associated with pets’ fecal shedding of C. difficile (Table 2).

Figure 1:

Participation scheme and results obtained in two cohorts of pet owners from the University of Pennsylvania Health System (UPHS) and the Ohio State University Wexner Medical Center (OSUWMC).

Table 1:

Distribution of samples and PCR results obtained for C. difficile from patients/pets at The Ohio State University Wexner Medical Center (OSUWMC). H indicates human samples, D indicates dog samples. Red cells denote a positive result for C. difficile, while green cells denote a negative result. Brown cells indicate a sample was not available for that time point. T0 refers to the time of enrollment (due to sampling methodology, human samples were obtained up to two weeks prior to household paired dog samples). T1 refers to the second timepoint (median of 53 days (range: 41-295) after first dog sample(s) submitted; T=0). Toxin profiles and ribotypes, when available, of the C. difficile isolates are indicated in the relevant cells. All isolates were CDT-.

Household		T0	T1
1	H	A+/B+ RT AI-78
	D1
	D2
2	H
	D1	A+/B+
	D2
3	H
	D1	A−/B−
	D2
4	H	A+/B+ RT 499
	D1	A−/B− RT PR22386
		A−/B− RT PR22386
5	H	A−/B
	D1
	D2
6	H
	D1		A+/B+ RT 014/4
			A+/B+ RT 014/4
7	H
	D1
	D2	A+/B+ RT 009
8	H
	D1	A−/B−
9	H	A+/B+ RT 009
	D1
10	H	A+/B− RT 241
	D1
11	H	A+/B− RT AI-37
	D1
	D2
12	H
	D1	A+/B+ RT 106
13	H
	D1	A−/B− RT 076
		A+/B+ RT PR22401
14	H	A−/B−
	D1
15	H
	D1		A+/B+ RT 002/2
16	H	A+/B− RT 412
	D1
	D2
17	H	A+/B+ RT PR22405
	D1

Table 2:

Owner, household and dog factors for dogs with positive and negative stool samples for Clostridioides difficile from OSUWMC cohort, Ohio, USA.

Variable	Dog C. difficile-positive3		Dog C. difficile negative		P-value1
	N	Num (%)	N	Num (%)
Household demographics
Num people in household (median; range)	8	2.5 (1-9)	34	2 (1-9)	0.92
One or more higher risk person in household	9	3 (33%)	35	8 (23%)	0.7
Owner C. difficile-positive3	9	1 (11%)	36	12 (33%)	0.2
Owner ever told by their doctor they have C. difficile	6	3 (50%)	32	11 (34%)	0.7
Dog demographics
Age in yrs (median; range)	8	7.5 (1.5-11)	35	6 (0.3-13)	0.72
Male sex	8	6 (75%)	35	21 (60%)	0.7
Spayed/neutered	8	6 (75%)	35	30 (86%)	0.6
Size	7		35		0.9
Small (< 20 lbs)		3 (43%))		10 (35%
Medium (20-55 lbs)		2 (29%)		14 (40%)
Large (>55 lbs)		2 (29%)		11 (31%)
Dog medical history (within past 3 months)
Dog received an antimicrobial	8	1 (13%)	35	1 (3%)	0.3
Dog received a drug that may reduce immune function	8	0	35	2 (6%)	1.0
Dog medical history (within past 2 weeks)
Had diarrhea4	8	0	35	3 (9%)	1.0
Had vomiting5	8	0	35	2 (6%)	1.0
Dog diet and habits
Any household dogs have at least weekly contact with livestock	8	1 (13%)	35	2 (6%)	0.5
Any household dogs have at least weekly contact with wildlife	8	1 (13%)	35	8 (23%)	1.0
Any household dogs in the past 2 weeks fed raw animal product treats	8	0	35	5 (14%)	0.6

¹Fisher’s exact test, unless otherwise stated

²Two-sample Wilcoxon rank-sum (Mann–Whitney) test

³Clostridioides difficile isolated from one or more stool sample

⁴Defined as 3 or more loose or liquid stools per day

⁵Defined as 3 or more episodes per day

At UPHS, a total of 1,054 C. difficile-positive patients were sent an email inviting them to participate in the study. Of these, a total of 49 (4.6%) pet owners agreed to participate (Figure 1). Fecal samples could only be obtained from a total of 26 human patients due to delays in response of some of the participants (i.e., their fecal sample had already been discarded by the hospital by the time they responded). Fecal samples were also obtained from 9 cats, and 27 dogs from 24 distinct households (Table 3). There were 15 complete household pairs/groups, where a fecal sample was available from both the patient and at least one pet, and 9 incomplete household pairs/groups, where fecal samples were available from pets only (Table 3). Only 1 of 36 (2.8%) pets from the UPHS cohort was C. difficile-positive.

Table 3:

Distribution of samples and culture/PCR results obtained for Clostridioides difficile from C. difficile-positive patients and their pets at the University of Pennsylvania Healthy System (UPHS). H indicates samples from human patients, D indicates samples from dogs, C indicates samples from cats. Red cells denote a positive result for C. difficile on anaerobic culture, while green cells denote a negative result. Note that all human samples were positive by PCR performed at the hospital, but C. difficile isolates could not be cultured from all human samples. Brown cells indicate a sample was not available. Toxin profiles and sequence types, when available, of the C. difficile isolates are indicated in the relevant cells.

Household		Sample results
1	H
	D1
	D2
2	H
	C1
	C2
	C3
	C4
3	H	A+/B+
	D1
4	H	A−/B+
	D1
5	H	A+/B+
	D1
	D2
	D3
	D4
	D5
6	H
	D1
7	H
	C1
8	H
	D1
9	H
	C1
10	H	A+/B+
	D1
11	H	A+/B+
	D1
	D2
	D3
	D4
12	H
	C1
	C2
13	H	A+/B+
	D1
	D2
14	H	A+/B+
	D1
15	H
	D1
	D2
16	H	A+/B+ ST 8
	D1	A+/B+ ST 8
17	H	A+/B+
	D1
	C1
18	H	A−/B+
	D1
19	H	A+/B+
	D1
20	H
	C1
21	H
	D1
22	H
	D1
23	H
	D1
24	H
	D1

The UPHS household in which the dog and owner had C. difficile-positive stool samples consisted of a 55-year-old woman and her adult dog. The owner, whose stool was positive for C. difficile on both PCR and EIA (i.e., confirmed infected), reported being the primary caretaker of the dog, but she did not report allowing the dog to lick her face or hands or share sleeping quarters with it. The owner also reported that her dog had recently been on a course of antimicrobials (including cefpodoxime and metronidazole). The isolates from this pet/owner pair were both from Clade 1, sequence type 8, which is one of the most common strains of clinical importance in human CDI (Knight, Elliott, Chang, Perkins, & Riley, 2015; O’Grady, Riley, & Knight, 2021). The human- and dog-derived strains differed by only 1 SNV among the single copy core genes. Using the negative binomial distribution of SNVs from the assembly of Vibrio campbellii technical replicates as reference (Gu et al., 2020), the binomial test of 1 SNV between genomes results in a p-value >0.91, implying that these two isolates are identical. These microorganisms were genetically distinct (>50 SNPs difference) from C. difficile isolates from cows, dogs, and pediatric patients obtained from a different study (L. E. Redding et al., 2022) (Figure 2). Seventeen antibiotic resistance genes were identified, including 13 vancomycin resistance markers, two beta-lactamase genes conferring resistance to carbapenems, one C. difficile-associated efflux pump, and one clindamycin resistance methyltransferase (Supplementary document). The genome sequences of the two isolates from the pet-positive/owner-positive household were deposited in GenBank, with accession numbers SAMN25977879 (human isolate) and SAMN25977878 (dog isolate).

Figure 2:

Dendogram showing the number of single nucleotide polymorphisms between C. difficile isolates from different sources, including a patient with C. difficile infection and their pet (“S1_Patient” and “S1_Pet”), a neonatal dairy calf (“S2_B”), a puppy from an unrelated home as the patient-pet pair (“S2_C”), a human pediatric patient (“S2_H”), and a high-quality reference genome downloaded from NCBI RefSeq (“RefSeq”).

Discussion

In this study, we found that pets of patients with C. difficile infection infrequently shed C. difficile, and co-occurrence of isolates with identical genomic features/ribotypes occurred in only a single household. Together, these findings suggest that interspecies transmission of C. difficile is rare within a household, in contrast to human-to-human transmission (Miller et al., 2022). Results from other epidemiological studies corroborate our findings. In one study, healthy community-dwelling pet owners were less likely to be colonized with C. difficile than non-pet owners (Galdys et al., 2014), and in a large-scale study of healthy pet owners and their pets, colonization with C. difficile never occurred contemporaneously in both the pet and owner (Rabold et al., 2018). In another study, none of eight mammalian pets belonging to patients with recurrent CDI were found to carry C. difficile (Shaughnessy et al., 2016). In yet another study, pet ownership was found to be protective against the recurrence of CDI, with increased contact between pet owners and pets associated with greater degrees of protection (Laurel E. Redding et al., 2020). It is unclear why pets appear to only infrequently acquire C. difficile from their owners. It may be that the canine gut microbiota is better able to provide colonization resistance against C. difficile, even when in an environment where C. difficile is present, because of canine behaviors that result in frequent acquisition of diverse microflora (e.g., coprophagia, being close to the ground, licking paws and anuses).

While infrequent, interspecies transmission events do appear possible, as shown in our study by the close identity between one household’s human and pet C. difficile isolates. However, it is ultimately very difficult to demonstrate the directionality of transmission or whether both the person and animal were infected from the same source. In one study where pets from four households carried C. difficile microorganisms with identical PFGE profiles as their owners (Loo et al., 2016), one of the cats tested positive for C. difficile only on the second home visit (i.e., not at baseline), suggesting delayed transmission from human-to-animal. Because CDI is often a hospital-acquired infection, it would be reasonable to assume that transmission mostly occurs in the human-to-animal direction. Data on hospital therapy animals seem to support this, as dogs participating in animal assistance interventions in human healthcare facilities were more likely to shed C. difficile than dogs participating in non-healthcare facilities, and dogs that licked patients’ hands or accepted treats from them were even more likely to acquire C. difficile (Lefebvre, Reid-Smith, Waltner-Toews, & Weese, 2009). In this study, the isolates from both the dog and the owner contained antibiotic resistance genes to vancomycin, which is used to treat CDI at UPHS, further suggesting that this isolate originated with the human patient.

Animal-to-human transmission appears less likely, though not impossible. A case report (Rodríguez-Pallares et al., 2022) appears to describe such a transmission event, where a 10-month old infant who lived with a dog with chronic diarrhea (that was later tested and found to be carrying C. difficile) became ill with a genetically identical strain of C. difficile as her dog. However, even in this scenario, it was uncertain whether the dog carried C. difficile prior to the child, and it is unclear in general whether C. difficile is a cause of canine diarrhea or an incidental finding in dogs with diarrhea (Marks et al., 2011). Interestingly, as in the above-described case, the only dog in our cohort that carried an identical C. difficile isolate as its owner had recently been on a course of antimicrobials itself, which could have led to dysbiosis of the dog’s gut microbiome and enhanced susceptibility to C. difficile colonization or infection.

A limitation of this study, as in others examining this topic, is that we were not able to assess whether pets were colonized prior to the patient developing C. difficile infection. To do so, we would have needed to test pets before their owner became infected. However, because C. difficile infection occurs at a rate of 14.9-71 community-acquired cases per 100,000 person-years, it is highly impractical to follow sufficient numbers of patients prospectively in hopes of identifying those that develop CDI. Further, sampling methodology prohibited simultaneous sample collection from people and their pets, with pets sampled up to two weeks following C. difficile sampling in people. Additionally, we did not document C. difficile shedding among humans after discharge from the hospital, and the magnitude of household contamination or degree of pet exposure is unknown. Nonetheless, it is known that human shedding persists for weeks after the resolution of clinical signs (Sethi et al., 2010). Another limitation was the low participation rate. Other studies investigating this topic have also reported low participation rates (Loo et al., 2016),(Laurel E. Redding et al., 2020). In a previous case-control study investigating the effect of pet ownership on the recurrence of CDI (Laurel E. Redding et al., 2020), we found that responders to our survey were significantly more likely to have experienced prior episodes of CDI, recurrence of CDI, and gastric acid suppression than non-responders, and thus may have been more invested in contributing to research aimed at better understanding the epidemiology of their condition. Our results may therefore not be completely generalizable. However, the fact that this study was carried out at two different institutions provides some mitigation against this possibility. Finally, the sensitivity of the detection methods we used might have underestimated the prevalence of C. difficile and prevented us from obtaining paired isolates from pets and owners. Anaerobic culture has been shown to have reduced sensitivity compared to molecular methods (Blanco, Alvarez-Perez, & Garcia, 2013), and we found a small number of human fecal samples that tested positive by PCR at the hospital but yielded no isolates on anaerobic culture. To mitigate this possibility, we used both anaerobic culture and PCR on all fecal samples; however, non-toxigenic isolates, which are arguably of less clinical relevance than toxigenic isolates, may not have been detected.

In conclusion, the contemporaneous colonization of pets and their owners with C. difficile was infrequent, suggesting infrequent interspecies transmission. While pets did sometimes carry C. difficile, there was only one of 47 households where the pet and owner harbored genetically identical isolates. Moreover, the dog in question in that household had recently received antimicrobial therapy itself, which may have resulted in gut dysbiosis and predisposition to C. difficile colonization/infection.

Impacts:

• We tested the pets of people with C. difficile infection to determine whether pets and their owners carry genetically related strains of C. difficile

• Pets and owners contemporaneously carried C. difficile in only two of 47 households, and genetically related isolates in the pet and owner occurred in only one household

• Interspecies transmission of C. difficile within a household appeared to be rare