High Prevalence of Clostridium difficile in Home Gardens in Western Australia

Recently, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant problem, accounting for ∼50% of all CDI cases and reported to affect a younger population without traditional risk factors. Possible sources of CA-CDI are soil, food, and water contaminated by animal feces, and recent reports show overlapping ribotypes of C. difficile in animals, humans, and the environment; however, the epidemiology of CA-CDI and related risk factors need to be better understood. Our research aimed to determine the prevalence of C. difficile in home gardens and on the shoe soles of homeowners in Perth, Western Australia. There were high rates of contamination with C. difficile in gardens, and some of the ribotypes identified had been isolated from human cases of CDI in Western Australia. This study shows that home gardens and shoes may be a source of C. difficile in CA-CDI.

ABSTRACT

In recent years, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant health problem, accounting for ∼50% of all CDI cases. We hypothesized that the home garden environment could contribute to the dissemination of C. difficile spores in the community and investigated 23 homes in 22 suburbs of Perth, Western Australia. We identified a high prevalence of toxigenic C. difficile in this environment. In total, 97 samples consisting of soil (n = 48), compost (n = 15), manure (n = 12), and shoe sole swabs (n = 22) were collected. All samples were cultured anaerobically on C. difficile ChromID agar and enriched in brain heart infusion broth, and isolates were characterized by toxin gene PCR and PCR ribotyping. Two-thirds (67%; 95% confidence interval [CI], 57 to 76%) of home garden samples, including 79% (95% CI, 68 to 91%) of soil, 67% (95% CI, 43 to 90%) of compost, 83% (95% CI, 62% to 100%) of manure, and 32% (95% CI, 12 to 51%) of shoe sole samples, contained C. difficile. Of 87 isolates, 38% (95% CI, 28 to 48%) were toxigenic, and 26 PCR ribotypes (RTs), 5 of which were novel, were identified. The toxigenic C. difficile strain RT014/020 was the most prevalent RT. Interestingly, 19 esculin hydrolysis-negative strains giving white colonies were identified on C. difficile ChromID agar, 5 of which were novel toxigenic RTs that produced only toxin A. Clearly, there is the potential for transmission of C. difficile in the community due to the contamination of home gardens. Our findings highlight the importance of a “One Health” approach to dealing with CDI.

IMPORTANCE Recently, community-associated Clostridium difficile infection (CA-CDI) has emerged as a significant problem, accounting for ∼50% of all CDI cases and reported to affect a younger population without traditional risk factors. Possible sources of CA-CDI are soil, food, and water contaminated by animal feces, and recent reports show overlapping ribotypes of C. difficile in animals, humans, and the environment; however, the epidemiology of CA-CDI and related risk factors need to be better understood. Our research aimed to determine the prevalence of C. difficile in home gardens and on the shoe soles of homeowners in Perth, Western Australia. There were high rates of contamination with C. difficile in gardens, and some of the ribotypes identified had been isolated from human cases of CDI in Western Australia. This study shows that home gardens and shoes may be a source of C. difficile in CA-CDI.

INTRODUCTION

Clostridium (Clostridioides) difficile is a Gram-positive, spore-forming, rod-shaped anaerobic bacterium that resides in the gastrointestinal tracts of humans and animals and extensively contaminates the environment. It is the causative agent of pseudomembranous colitis (PMC) and a predominant cause of life-threatening antimicrobial-associated diarrhea (1). For many years, C. difficile infection (CDI) has been thought of as a health care-associated infection, and increasing incidence, severity, and rates of recurrence make it a major health care threat (2) and a cause of substantial economic losses to health systems (3). According to the Centers for Disease Control and Prevention (CDC), C. difficile ranks in the top five antimicrobial-resistant urgent threats to public health in the United States, with an estimated 223,900 cases and at least 12,800 deaths in 2017, costing around $1 billion in the United States (4).

There have been significant changes in the global epidemiology of CDI recently, and the prevalence of community-associated CDI (CA-CDI) has increased significantly (1). Cases without recent antimicrobial use (5), prolonged inpatient health care, old age (>65 years), and other traditional risk factors accounted for ∼40% of all CDI cases in the United States (2). In Australian communities, ∼26% of cases of CDI were CA-CDI that occurred in younger patients (6). Despite C. difficile being well-studied in the health care environment, information pertaining to routes of transmission and sources of infection in the community, and any association with risk factors, is scarce.

Studies on environmental contamination with C. difficile have shown a number of potential sources including soil (7, 8), water (9, 10), animals (11–13), households (14), and food (15–20), in addition to patients colonized with C. difficile (21), with cases acquiring infection through the orofecal route with ingestion of spores. This has been suspected due to the overlapping of ribotypes (RTs) of C. difficile found in the environment, animals, and humans (22, 23). A high positivity rate (40%) of toxigenic C. difficile was observed on shoes in the community (14), suggesting that shoe soles may be a possible vector (24, 25), and, in a very recent study, C. difficile was isolated from 12/25 contaminated outdoor sites (48%) in the domestic environment in Slovenia (26). However, to our knowledge, no comprehensive study has been undertaken to determine the presence of C. difficile in home gardens used for the cultivation of flowers, shrubs, or vegetables. Therefore, we aimed to assess the prevalence of C. difficile in such gardens in Perth, Western Australia (WA). Given that transmission of C. difficile from the gardening environment into the house or elsewhere could occur because of contamination of the soles of shoes, sampling of shoe soles was undertaken also.

RESULTS AND DISCUSSION

In the present study, 97 samples consisting of soil (n = 48), compost (n = 15), manure (n = 12), and shoe sole swabs (n = 22) were collected from 23 homes in 22 different suburbs of Perth, WA, between May and July 2018 (Fig. 1). Of the 23 homes, C. difficile was present by enrichment culture in 22 (96%; confidence interval [CI], 87 to 100%) (Table 1). Two-thirds (67%, 65/97; CI, 58 to 76%) of the garden samples, including 79% (38/48; CI, 68 to 91%) of soils, 67% (10/15; CI, 43 to 91%) of compost, and 83% (10/12; CI, 62 to 100%) of manure, were positive for C. difficile. A total of 87 isolates of C. difficile were recovered from the 65 positive samples as follows: soil (n = 54), manure (n = 11), compost (n = 13), and shoes (n = 9). While home gardens do not appear to have been studied previously in Australia, studies of homes per se recovered C. difficile from 31 to 38% of sites, predominantly household domestic items, pets, various surfaces, dust from vacuum cleaners, bathrooms and toilets, and shoe soles (14, 27, 28). The overall prevalence of C. difficile in our garden samples was higher than other contemporaneous reports (6.5 to 48%) on environmental samples (21, 26, 29–31). The extent of contamination in home gardens in Perth is likely to be due to the practice of adding compost and manure to the poor-quality soils in WA. The soils in the Perth region are very sandy, the depth of topsoil is shallow, and the absence of subsoil results in less water-holding capacity (32) in a relatively arid region.

FIG 1.

Locations and numbers of home gardens in Perth, Western Australia, sampled for C. difficile, by postcode. (Map created using maptools and ggplot2 packages in R Studio.)

TABLE 1.

Overview of sampling locations, types, months, C. difficile-positive samples, and differences between direct culture and enrichment

Post code	Sampling date (mo-day)	Sample type	No. of samples	Enrichment broth		Direct culture		Ribotype(s)a
				House positive	% positive	House positive	% positive
6006	Jun-18	Soil, soil, manure, shoe	4	3	75	1	25	QX 597, 010, 014/020, 103
6009	Jun-18	Soil, soil, manure, shoe	4	3	75	1	25	QX 638, QX 601, QX 639, 010
6010	May-18	Soil, compost, manure, soil, shoe	5	2	40	0	0	125, QX 639, 039
6014	Jun-18	Soil, compost, manure, shoe	4	4	100	4	100	056, 125, 076, QX 189
6018	Jun-18	Soil, compost, manure, shoe	4	4	100	3	75	054, Unique, 125, 056
6019	May-18	Soil, compost, soil, shoe	4	1	25	0	0	QX 077
6020	Jun-18	Soil, soil, soil, shoe	4	3	75	1	25	125, 014/020, 051
6024	Jun-18	Soil, compost, manure, shoe	4	2	50	2	50	125, 014/020, QX 597, 125
6026	Jun-18	Soil, soil, soil, soil, soil, compost, compost, manure	8	6	75	4	50	010, 081, 014/020, 051
6054	Jun-18	Soil, soil, soil, shoe	4	4	100	3	75	QX 597, 014/020, 286, 125
6059	Jun-18	Soil, compost, soil, shoe	4	3	75	1	25	125, Unique, 286, 010, 014/020
6062	Jun-18	Soil, soil, soil, shoe	4	4	100	0	0	010, 287, 014/020
6066	Jun-18	Soil, compost, manure, shoe	4	3	75	3	75	QX 141, 125014/020
6066	Jun-18	Soil, compost, manure, shoe	4	3	75	2	50	106, 125, 014/020
6076	Jun-18	Soil, compost, manure, shoe	4	4	100	4	100	Unique, 054, 014/020
6101	Jul-18	Soil, soil, manure, shoe	4	3	75	2	50	051, 125
6112	Jun-18	Soil, soil, soil, shoe	4	2	50	1	25	014/020
6150	Jun-18	Soil, compost, soil, shoe	4	2	50	2	50	010, 287
6151	Jun-18	Soil, compost, soil, shoe	4	3	75	2	50	014/020, 125
6162	Jun-18	Soil, soil, soil, shoe	4	0	0	0	0	0
6152	Jun-18	Soil, soil, soil, shoe	4	3	75	2	50	QX 639, QX 400, QX 637, Unique, 054
6163	Jun-18	Soil, compost, manure, shoe	4	1	25	1	25	QX 077, 125, QX 189
6163	Jul-18	Soil, compost, soil, shoe	4	2	50	1	25	51
		Total	97	65	67	40	41

^aQX, internally assigned ribotype; Unique, novel ribotype pattern isolated for the first time in the laboratory.

Of the 87 isolates of C. difficile, 33 (38%; CI, 28 to 48%) were toxigenic, comprising A⁺ B⁺ CDT⁺ (n = 1), A⁺ B⁺ CDT⁻ (n = 27), A⁻ B⁻ CDT⁺ (n = 1), and A⁺ B⁻ CDT⁻ (n = 4) isolates, and 54 (62%; CI 52 to 72%) were nontoxigenic isolates (A⁻ B⁻ CDT⁻). Of note, 37% (CI, 24 to 50%) of the soil isolates harbored toxigenic C. difficile strains, comparable to soil studies elsewhere (44% in Slovenia, 73% in Belgium, 67% and 99.5% in Zimbabwe) (21, 29, 31, 33).

Animal manure and manure-based composts contain C. difficile spores that eventually get disseminated into the environment (34). Livestock manure including piglet feces, biosolids, or compost is used as fertilizers directly in agricultural practice, and proper storage of manure, including anaerobic digestion or aeration of sludge or composting, significantly reduces the bacterial load by the generation of metabolic heat from microorganisms in the contaminated manure (35). However, C. difficile spores are highly recalcitrant to extreme temperatures, oxygen, chemical disinfectants, and UV light (36–38). Hence, C. difficile spores can easily survive in compost and manure even after chemical and/or composting treatments.

The soles of shoes provide an obvious mechanism to contaminate households from the immediate outside environment. This has been studied and reported on earlier (14, 24). In the present study, C. difficile was found on 7 of 22 (32%; CI, 12 to 51%) shoe sole samples, and most (60%; CI, 30 to 90%) were toxigenic strains. The prevalence of positive shoe soles was slightly lower but of a similar magnitude to previous studies (14, 24). However, these figures are higher than a previous study in hospital settings where C. difficile was found on 7 of 41 shoe sole samples (17.1%) collected from health professionals in an acute care hospital in the United States (39), suggesting that sources/reservoirs of C. difficile, such as animal manure and compost, are important for the transmission of C. difficile in home gardens.

A total of 26 different RTs were identified, including 5 novel RTs. Soil samples showed the greatest diversity (24 different RTs; Shannon’s index, 2.939; Simpson’s, 0.9335), followed by shoe sole samples (6 RTs; Shannon’s index, 1.677; Simpson’s, 0.7901), manure (5 RTs; Shannon’s index, 1.414; Simpson’s, 0.7107), and compost (6 RTs; Shannon’s index, 1.411; Simpson’s, 0.6627). C. difficile RT 014/020 was the only RT found in all samples (soil, compost, manure, and shoe soles) and was the most prevalent (20.7%, 18/87; CI, 12 to 29%) followed by RTs 054, 056, 103, 106, and QX 076 (all toxigenic) and nontoxigenic RTs 125 (17.2%, 15/87; CI, 9 to 25%), 010 (9.2%, 8/87; CI, 3 to 15%), 051, and 287 (Fig. 2). Interestingly, a number of esculin hydrolysis-negative strains (19/87, 21.8%; CI, 13 to 31%) were found that produced white colonies on ChromID C. difficile agar plates (see below).

FIG 2.

Summary of prevalence, ribotype, and toxin gene profile for 87 C. difficile isolates from home gardens in Perth, Western Australia. PCR ribotype pattern analysis was performed by creating a neighbor-joining tree using the Pearson correlation (optimization, 5%; curve smoothing, 1%). *, reference strain; NA, not applicable.

PCR ribotyping of all 87 isolates demonstrated 26 distinctive RTs including clinically important toxigenic RTs, such as RTs 014/020, 056, 054, 103, 081, and 106, that have been identified previously in humans with CDI in WA. C. difficile RT 014/020 remains the most prevalent strain of C. difficile in Australia, found in both hospital-acquired infection and CA-CDI (40–43). RT 014/020 is also a major strain found in pigs in Australia (44), as well as lawn and soils (8, 45). Therefore, it is not surprising that a high prevalence in soils and manure was found in our study, suggesting that pig manure may be a source of environmental contamination. The relationship between pig and human strains of RT 014/020 in Australia, as demonstrated by single nucleotide variant (SNV) analysis following whole-genome sequencing (WGS), is suggestive of zoonotic transmission (46).

Viable counts were conducted on all soil, compost, and manure samples. The overall recovery of C. difficile from all of the samples was 41% (40/97; CI, 31 to 51% [median, 2; range, 0 to 4]) and 67% (65/97; CI, 58 to 76% [median, 3; range, 0 to 6]) by direct culture and enrichment, respectively, which is a significant difference (P = 0.0003, chi-square). C. difficile recovery was highest from manure (67%, 8/12; CI, 40 to 93% [median, 0; range, 0 to 1]) followed by soil (44%, 21/48; CI, 30 to 58% [median, 1; range, 0 to 3]), compost (33%, 5/15; CI, 9 to 57% [median, 0; range, 0 to 1]), and shoe soles (27%, 6/22; CI, 8 to 46% [median, 0; range, 0 to 1]) by direct culture. For enrichment, shoe sole sponge samples (32%, 7/22; CI, 12 to 51% [median, 0; range, 0 to 1]) also had the lowest rate, with the highest from manure at 83% (10/12; CI, 62 to 100% [median, 0; range, 0 to 1]), followed by soil at 79% (38/48; CI, 68 to 91% [median, 2; range, 1 to 5]), and compost at 67% (10/15; CI, 42 to 91% [median, 0; range, 0 to 2]) (P = 0.0007, chi-square). The average maximum C. difficile viable counts were in compost (2,028 CFU/g) followed by manure (619 CFU/g) and soil (554 CFU/g).

C. difficile-infected animals shed up to 10,000 spores per gram of feces (47). The concentration of viable C. difficile in cattle feces has been reported as 2.5 × 10⁴ CFU/ml (11). In an earlier study of lawns in WA, the concentration of viable C. difficile was 1,200 CFU/g (8). The infectious dose of C. difficile for humans remains unclear; however, it is likely that the concentration of spores in home gardens is sufficient to cause human CDI. Shoe soles could serve as a vector for the dissemination of spores between the environment, households, and the community.

Of the 19 esculin hydrolysis-negative strains of C. difficile that failed to produce black colonies on ChromID C. difficile agar, 5 were RT 125, and 5 were toxigenic but produced only toxin A (A⁺ B⁻ CDT⁻). These latter five and the remaining novel isolates were assigned internal “QX” RTs. All five tcdA⁺ only isolates were QX 597. Such strains have been reported in France (48) where the pathogenicity locus (PaLoc) for the toxin A only positive strain was located at a site in the genome that differed from the usual site. For these strains, a novel model of PaLoc evolution was proposed that merged two “Mono-Toxin PaLoc” into the classical “Bi-Toxin PaLoc” proposed (48). Further work is required to investigate these variant strains of C. difficile isolated from environmental samples.

In the present study, multiple strains of C. difficile were often recovered from a single sample of soil, manure, or compost, including both esculin hydrolysis-positive and -negative strains, resulting in black and nonblack colonies, respectively, on ChromID C. difficile agar that differed in RT. ChromID C. difficile agar contains a chromogenic substrate and, after esculin is hydrolyzed, glucose and esculetin react with ferric citrate to give black colonies for presumptive identification of C. difficile (49). There have been reports of esculin hydrolysis-negative strains of C. difficile isolated from human diarrheal stools that produced white colonies on ChromID C. difficile agar (49). These strains were mainly binary toxin-positive C. difficile RT 023 (A⁺ B⁺ CDT⁺), an emerging strain capable of causing severe disease (49, 50).

There are some limitations to this study. First, the research was conducted in 23 homes in suburban Perth, WA. Australia is a big country, indeed a continent, and the landscape consists of a wide range of soils. Much of the land in WA, and other states, is affected by acidity, salinity, and alkalinity, which often limits vegetable or plant growth (32). Furthermore, discrepancies in the environmental conditions, including rainfall fluctuations over an extended period of time, significantly affect soil microbial communities, and microorganisms are not evenly distributed in gardens in some states. Hence, extensive studies involving home garden samples from different states of Australia are advisable before generalizing our results to the rest of Australia. Second, there were no data pertaining to CDI being diagnosed within the households sampled; however, given that the volunteers in this project worked in the pathology industry, it is very unlikely that they failed to mention such an important piece of information. Third, spore quantification was only carried out once. Finally, there were several novel isolates that did not match with international reference RTs, making it challenging to compare with RTs reported from other countries.

In conclusion, this report establishes the potential for transmission of C. difficile in the community because of the contamination of home gardens. We showed that home gardens may be a source for the dissemination of C. difficile in the environment, which subsequently cause CA-CDI. Taken together, retail meat, soil, compost, manure, shoe soles, vegetables, public lawns, hospitals and hospital grounds, household environments, companion animals, and production animals are possible sources or reservoirs for CA-CDI. These findings imply that the epidemiology of CA-CDI is complicated, not simple, and that more sophisticated work is required to unravel these potentially interrelated factors. However, with such a high prevalence of C. difficile in home gardens, home gardens may also be one of these factors, and further investigation is required.

MATERIALS AND METHODS

Sample collection and preparation.

Permission was obtained from PathWest Laboratory Medicine (WA) to contact laboratory staff via email and ask for volunteers to collect samples from their gardens; thus, this was a convenience sample. The volunteers were provided with a sampling kit consisting of sterile powder-free gloves; containers for soil, animal manure, and compost; and sterile Polywipe sponges (Medical Wire and Equipment Co. Ltd, UK) for collecting shoe sole samples. Instructions were provided, and volunteers were asked to return the samples as soon as possible. The 97 garden samples were processed in 5 batches (29 samples processed in batch 1, 40 in batch 2, 20 in batch 3, and 4 samples each in batch 4 and batch 5). The study was conducted from May to July (2018) inclusive (autumn to early winter) in the metropolitan area of Perth, Western Australia (Fig. 1).

Approximately 100 g each of soil, compost, or manure was collected into a sterile 250-ml container at each home using a sterile wooden spatula. Soil samples were collected from about 2.5 cm below the top surface. Manure and compost were collected from bags of product that had been acquired from commercial sources or heaps stored at home.

Shoe soles were wiped with a sterile Polywipe sponge across the whole surface of the bottom of a pair of shoes and the sponge placed in the sterile bag provided. All samples were placed in a biohazard bag and transported at ambient temperature to the laboratory on the same day of sample collection and stored at 4°C for up to 48 h before processing.

Isolation of C. difficile.

Approximately 5 g of soil/manure/compost was enriched in 90-ml volumes of brain heart infusion broth supplemented with 1 g/liter taurocholate, 10 mg/liter cefoxitin, and 200 mg/liter cycloserine (BHIB-S) (PathWest Media, Mount Claremont, Western Australia), which was prereduced for 4 h in an anaerobic chamber (A35; Don Whitley Scientific Ltd, Shipley, West Yorkshire, UK) with an atmosphere of 10% hydrogen, 10% carbon dioxide, and 80% nitrogen at 35°C with 75% humidity. After gentle mixing, the broths were incubated for 5 days initially with the lids loose to allow the soil, compost, or manure to equally disperse in the broth. A negative control (10 ml of phosphate-buffered saline [PBS], pH 7.4, added to 90 ml BHIB-S) and a positive control (R20291 [RT027]) were included and processed in the same way as the test samples. After 5 days, a 5-ml aliquot of broth was subjected to alcohol shock by adding an equal amount of absolute ethanol and leaving at room temperature for ≥1 h. The mixture was centrifuged at 3,000 × g for 10 min, the pellet suspended in 100 μl PBS, and 10 μl of the suspension plated onto ChromID C. difficile agar (bioMérieux, Marcy l'Etoile, France), which was incubated anaerobically for 48 h. Presumptive identification of C. difficile was based on characteristic black, gray, or colorless umbonate colonies, with irregular edges (51). These were subcultured onto prereduced 10% horse blood agar (BA) plates. Further identification was based on colony morphology on BA, chartreuse fluorescence under UV light (∼360 nm), and the characteristic horse manure odor (52). If necessary, identification was confirmed by the presence of l-proline aminopeptidase activity (Rosco Diagnostica, Tasstrup, Denmark).

Quantitation of C. difficile.

Viable counts of C. difficile from soil, compost, and manure were made on a subset of samples. Approximately 1 g of soil/compost/manure was mixed with 1 ml of sterile 0.85% saline and then 2 ml ethanol added for spore selection. After 1 h, 100 μl of the mixture was inoculated onto ChromID C. difficile agar plates, which were incubated anaerobically for 48 h. Sterile 0.85% saline was used as a negative control. Colonies were counted at 48 h and converted to CFU per gram.

Processing of shoe sole sponges.

Each shoe sponge was processed for C. difficile using methods previously described (11) with slight modifications. The sponge was aseptically transferred into a Stomacher bag (Colworth, London, UK) containing 25 ml of PBS and homogenized for 60 s in Stomacher 400 (Colworth, London, UK). A volume of 100 μl of suspension was inoculated onto ChromID C. difficile agar, spread with the sterile hockey stick (InterPath Services Pty Ltd, West Victoria, Australia), and incubated anaerobically at 35°C for 48 h. The remaining 24.9 ml of suspension was concentrated by centrifugation at 3,000 × g for 10 min and the pellet resuspended in 200 μl of PBS. A 100-μl volume was placed onto ChromID agar plates, spread with a sterile hockey stick, and incubated anaerobically at 35°C for 48 h. The remaining fluid was inoculated into BHIB-S and incubated anaerobically at 35°C for 5 days, initially with the lids loose.

Molecular characterization.

All isolates were screened by PCR for detection of toxin A and B genes (tcdA and tcdB) and binary toxin genes (cdtA and cdtB) and by amplification of the 16S-23S rRNA intergenic spacer regions (PCR ribotyping) as described elsewhere (11). A comparator strain, R20291 (RT027) and ultrapure water were used as positive and negative controls, respectively. To assign RTs and assess diversity, ribotyping banding patterns were matched against our reference library comprising of reference strains from the European Centre for Disease Prevention and Control (ECDC) Brazier collection and a range of isolates from humans, animals, and the environment. Isolates that could not be determined from the reference library were designated using local nomenclature with the prefix QX.

Statistical analysis.

The diversity indices Simpson index (1–D) and Shannon index were measured (as described in the PAST, PAleontological STatistics reference manual v4.03 by Hammer, http://folk.uio.no/ohammer/past/) to analyze the diversity of RTs in garden samples. The chi-square test was used to analyze proportions. P values of ≤0.05 were considered to be statistically significant.