Molecular epidemiology of Clostridioides difficile obtained from fecal samples of wild animals in Brazil

Epidemiologia molecular do Clostridioides difficile obtido de amostras de fezes de animais selvagens no Brasil

ABSTRACT:

Clostridioides difficile is a strictly anaerobic, spore-forming Gram-positive bacterium associated with diarrhea, known as C. difficile infection (CDI). In domestic animals, C. difficile is considered an important pathogen mostly in pigs and horses, but there are also reports in other domestic species. In wild animals, the epidemiology of C. difficile is largely unknown, and the role of the bacterium as a cause of diarrhea is unclear. The aim of this study was to determine the prevalence of C. difficile in the feces of wild animals referred to the Center of Medicine and Research in Wild Animals (CEMPAS). Fecal samples obtained from 100 animals of 34 different species were subjected to qPCR for the detection of the C. difficile 16S rRNA gene and two major toxin genes (tcdA and tcdB) and to anaerobic bacterial isolation. A total of 63 animals (63%) were positive for C. difficile by qPCR, and 16 isolates were recovered. The opossum (Didelphis spp.) had the highest number of positive animals in both tests (from 21 samples, 19 were qPCR positive, and four isolates were recovered). Three toxigenic strains (RT 002, 004, and 014), all previously described as infecting humans and animals, were isolated in the following species: bearded dragon (Pogona vitticeps), pampas fox (Lycalopex vetulus), and marmoset (Callithrix sp.). The presence of C. difficile in the feces of wild animals highlights the importance of wildlife as potential carriers of infection for production animals or humans.

INDEX TERMS:
Didelphis spp.; qPCR; tcdB; tcdA; wild animals; Clostridioides difficile

RESUMO:

O Clostridioides difficile é uma bactéria Gram-positiva estritamente anaeróbica e formadora de esporos, associada à diarreia e conhecida como infecção por C. difficile (CID). Em animais domésticos, o C. difficile é considerado um patógeno importante principalmente em porcos e cavalos, mas também há relatos em outras espécies domésticas. Em animais selvagens, a epidemiologia do C. difficile é amplamente desconhecida, e o papel da bactéria como causa de diarreia não está claro. O objetivo deste estudo foi determinar a prevalência do C. difficile nas fezes de animais selvagens encaminhados ao Centro de Medicina e Pesquisa em Animais Selvagens (CEMPAS). Amostras de fezes obtidas de 100 animais de 34 espécies diferentes foram submetidas à qPCR para a detecção do gene 16S rRNA do C. difficile e dois principais genes de toxina (tcdA e tcdB), além de isolamento bacteriano anaeróbico. Um total de 63 animais (63%) foram positivos para C. difficile por qPCR, e 16 isolados foram recuperados. O gambá (Didelphis spp.) apresentou o maior número de animais positivos em ambos os testes (de 21 amostras, 19 foram positivas na qPCR, e quatro isolados foram recuperados). Três cepas toxigênicas (RT 002, 004 e 014), todas previamente descritas como infectando humanos e animais, foram isoladas nas seguintes espécies: dragão barbado (Pogona vitticeps), raposa-pampas (Lycalopex vetulus) e sagui (Callithrix sp.). A presença de C. difficile nas fezes de animais selvagens destaca a importância da vida selvagem como potencial portadora de infecção para animais de produção ou seres humanos.

TERMOS DE INDEXAÇÃO:
Didelphis spp.; qPCR; tcdB; tcdA; animais selvagens; Clostridioides difficile

Introduction

Clostridioides difficile is a major cause of diarrhea in human patients undergoing antibiotic therapy. C. difficile infection (CDI) rates have been increasing in patients even without a previous history of hospitalization (Anjewierden et al. 2020, Maslennikov et al. 2022). In domestic animals, it is currently considered an etiologic agent of neonatal diarrhea in piglets and enteritis in foals and adult horses (Gohari et al. 2014). In cats (Weese et al. 2001, Schneeberg et al. 2012) and dogs (Weese et al. 2001, Busch et al. 2015), its importance as a cause of diarrhea is still questionable.

In Brazil, the agent has already been described in dogs (Rainha et al. 2019), calves (Silva et al. 2015), horses (Silva et al. 2012), wildlife (Silva et al. 2014a, 2014b), and humans (Ferreira et al. 2017, Rizek et al. 2022, Carvalho et al. 2023). In Latin America, there are several reports of CDI in humans, mainly describing the occurrence of ribotypes (RT) 001, 014, 015, 017, 027, 106, 133, and 135 (Acuña-Amador et al. 2022).

In Brazil, cases of CDI in humans have been steadily increasing, especially due to the epidemic spread of RT BI/NAP1/027 (Trindade et al. 2019). Whole-genome analysis of strains isolated from hospitalized patients revealed the presence of a high number of virulence genes (Rizek et al. 2022) and vancomycin resistance genes (Saldanha et al. 2020).

The observation of the same strains of C. difficile in animals and rural workers effectively demonstrates the importance of this agent in the concept of One Health (O’Shaughnessey et al. 2019, Redding et al. 2021). The presence of C. difficile in animals (Weese et al. 2020), water treatment plants (Chisholm et al. 2022), food (Borji et al. 2023), soil (Marcos et al. 2023), and humans (Monaghan et al. 2022) demonstrates its ability to colonize various species and, consequently, facilitates its dissemination and the exchange of virulence genes (Mitchell et al. 2022).

Evidence of the presence of strains from the ST11 lineage (Ribotype 078) with clonal groups in various animal species and humans reinforces the concept of mutual exchange, both zoonotic and anthropozoonotic, of C. difficile (Knight et al. 2019). The identification of identical strains, using next-generation genetic sequencing techniques, in pigs and farm workers highlights the possibility of mutual and common environmental dissemination between humans and animals (Knetsch et al. 2014).

There is limited information on the infection and/or colonization by C. difficile in wild animals (Silva et al. 2014a, Bandelj et al. 2018, Weese et al. 2020). Some authors suggest that wild animals may act as a reservoir for C. difficile strains relevant to domestic animals and humans (Himsworth et al. 2014, Williams et al. 2018, Krijger et al. 2019, Weese et al. 2019). Thus, the aim of this study was to determine the presence of C. difficile in the feces of wild animals using qPCR and classical isolation techniques.

Materials and Methods

Animal Ethics. All procedures were approved by the Ethics Committee for the Use of Animals (CEUA) of the FMVZ-Unesp, Botucatu/SP (Protocol CEUA 0088/2022).

Study local and contextualization. Fecal samples were collected (directly from the rectal ampulla or immediately from the environment) during routine daily activities at “Centro de Medicina e Pesquisa em Animais Selvagens” (Center of Medicine and Research in Wild Animals - CEMPAS) in Botucatu, São Paulo (Southeast region), Brazil (22^o53’25’’ South, 48^o27”19”” West), and frozen at -80ºC. The samples were harvested between October and December 2020 from live animals located in Cempas, originating from regions within a 100km radius.

DNA extraction and qPCR. DNA extraction was performed from 200mg of feces using the E.Z.N. A^TM Stool DNA Kit (PROMEGA^® Madison/WI, USA) and homogenized continuously (Precellys^®, Bertin Technologies, Montigny-le-Bretonneux, França). qPCRs (GoTaq^® Probe Master Mix - PROMEGA^® Madison/WI, USA) were used to detect the 16S rRNA gene, and in the positive samples, the tcdA and tcdB genes were analyzed as previously described (Mutters et al. 2009, Kilic et al. 2015).

Classical isolation techniques. Samples positive for Clostridioides difficile by qPCR were submitted for bacterial culture as described previously (Silva et al. 2013a). Briefly, approximately 1g of each fecal sample was inoculated in brain heart infusion broth (BHI) (San Luis/MO, USA). After incubation under anaerobic conditions at 37°C for five days, the culture was subjected to alcohol shock and plated on selective agar (Silva et al. 2013b). Suspected C. difficile colonies based on characteristic colony appearance and smell were harvested and subjected to DNA extraction and multiplex PCR for detection of the constitutive gene (tpi) and tcdA, tcdB, and cdtB genes (Silva et al. 2011). Toxigenic strains (isolates positive for tcdA and/or tcdB) were also subjected to PCR ribotyping (Bidet et al. 1999). The C. difficile library from the “Escola de Veterinária” of the “Universidade Federal de Minas Gerais” (UFMG) was used and additionally, the band patterns were compared using the WEBRIBO⁴.

Results

qPCR and classical isolation techniques

A total of 100 fecal samples from 34 different species were analyzed from June to November 2020. qPCR detected the presence of Clostridioides difficile DNA in 63 (63%) fecal samples, of which 19 were positive for toxins A and B (tcdA/tcdB) (Table 1). C. difficile was isolated from 16 samples, of which three isolates (Bearded dragon - P. vitticeps, Pampas fox - Lycalopex vetulus, and Marmoset - Callithrix sp.) were toxigenic (all positive for the tcdA and tcdB genes) and classified as ribotypes 004, 014 and 002, respectively.

Table 1.
Molecular detection of Clostridioides difficile, toxin genes tcdA and tcdB, and selective bacterial isolation in fecal samples of wild animals

	Species	Animals	qPCR		Culture
		N	16	tcdA + tcdB +	Isolation	tcdA + tcdB +
Synanthropic mammals	Opossum (Didelphis spp.)	21	19	8	4	0
	Rabbit (Oryctolagus cuniculus)	3	0	0	0	0
	Rat mouse (Mus musculus)	1	1	1	0	0
	Hare (Lepus europaeus)	1	0	0	0	0
	TOTAL	26	20	9	4	0
Carnivorous mammals	Crab-eating fox (Cerdocyon thous)	9	7	1*	1	0
	Pampas fox (Lycalopex vetulus)	3	3	1	2	1
	Maned wolf (Chrysocyon brachyurus)	3	3	0	0	0
	Puma (Puma concolor)	3	3	1	1	0
	Wild cat (Leopardus tigrinus)	2	2	0	0	0
	Jaguarundi (Puma yagouaroundi)	1	1	0	1	0
	Ocelot (Leopardus pardalis)	1	1	1**	0	0
	TOTAL	22	20	4	5	1
Other mammals	Howler monkey (Alouatta guariba)	2	0	0	0	0
	Capybara (Hydrochoerus hydrochaeris)	1	0	0	0	0
	Water opossum (Chironectes minimus)	1	1	1	1	0
	Capuchin monkey (Sapajus apella)	6	4	0	1	0
	Crab-eating raccoon (Procyon cancrivorus)	1	1	1	0	0
	Hairy dwarf porcupine (Erinaceus roumanicus)	3	2	0	0	0
	Hairy dwarf porcupine (Coendou prehensilis)	2	0	0	0	0
	Marmoset (Callithrix sp.)	7	1	1	1	1
	Giant anteater (Myrmecophaga tridactyla)	7	5	1**	2	0
	Nine-banded armadillo (Dasypus novemcinctus)	1	1	0	0	0
	Armadillo (Euphractus sexcinctus)	1	0	0	0	0
	Brown deer (Mazama gouazoubira)	2	1	0	0	0
	TOTAL	34	16	4	5	1
Reptiles	Leopard gecko (Eublepharis macularius)	1	0	0	0	0
	Red-fronted tortoise (Chelonoidis carbonaria)	7	4	0	1	0
	Boa constrictor (Boa constrictor constrictor)	7	2	2	0	0
	Bearded dragon (Pogona vitticeps)	1	1	1**	1	1
	Python (Python molurus)	1	0	0	0	0
	Teiu (Tupinambis merianae)	1	0	0	0	0
	TOTAL	18	7	3	2	1
TOTAL %		100	63	19	16	3

• * Only tcdA positive, ** only tcdB positive.

Discussion

The frequency of Clostridioides difficile in opossum (Didelphis spp.) deserves to be highlighted: 19 out of the 21 sampled opossum (90.48%) were positive for C. difficile by qPCR, nine of which were classified as toxigenic by qPCR. Additionally, four strains (21.05%) were isolated. One hypothesis for this high frequency is the cohabitation of these animals in areas with domestic animals and humans, including in urban centers (Silva et al. 2014a, Zlender et al. 2022).

Samples were collected from seven carnivorous species (Cerdocyon thous, Leopardus tigrinus, Puma yagouaroundi, Leopardus pardalis, Chrysocyon brachyurus, Puma concolor, and Lycalopex vetulus) (n=22). C. difficile DNA was detected by qPCR in 20 (91%) of the samples, of which five were culture-positive. This frequency suggests that wild canids and felids can harbor and disseminate C. difficile strains, similar to those previously reported by Silva et al. (2014b). In addition to its possible role as an asymptomatic carrier, C. difficile can also cause disease in these animals, which was previously observed in Brazil, where this pathogen has already been confirmed to cause fatal diarrhea in an ocelot (L. pardalis) (Silva et al. 2014b).

C. difficile DNA was detected in primates (Sapajus apella and Callithrix sp.) and, for the first time, in giant anteaters (Myrmecophaga tridactyla). There are very few studies on the prevalence of C. difficile in primates, and a previous study in Brazil (Carvalho et al. 2022) failed to isolate C. difficile from 24 capuchin monkeys (Sapajus spp.). On the other hand, CDI has already been described in several primate species, including marmoset (Callithrix sp.) (Armstrong et al. 2019). Among reptiles, seven were positive by qPCR, and two isolates were recovered. C. difficile was previously reported to colonize apparently healthy reptiles (Andrés-Lasheras et al. 2018, Ramos et al. 2019). Interestingly, the red-faced tortoise (Chelonoidis carbonaria), which is commonly kept as a pet, stands out with four positive samples (4/7, 57%).

Synanthropic animals and reptiles had the highest number of positive fecal samples, and therefore, they may be a reservoir for C. difficile strains (Andrés-Lasheras et al. 2018, Williams et al. 2018, Ramos et al. 2019). In this context, the identification of 13 non-toxigenic strains (Didelphis spp., Cerdocyon thous, Lycalopex vetulus, Puma concolor, Puma yagouaroundi, Chironectes minimus, Sapajus apella, Myrmecophaga tridactyla, Chelonoidis carbonaria), even though they are not associated with the development of CDI cases, can hold epidemiological significance. This group of strains, despite lacking the pathogenicity locus (PaLoc), has the potential to disseminate resistance genes through mobile genetic elements such as plasmids and transposons, as documented by Kartalidis et al. (2021). Non-toxigenic strains can also be capable of encoding virulence factors and acquiring the PaLoc, becoming virulent (Chowdhury et al. 2016).

Interestingly, the detection of ribotypes 002, 006, and 014, which are commonly associated with CDI in humans (Dauby et al. 2017), suggests that these toxigenic strains could infect humans or vice versa (Himsworth et al. 2014, Krijger et al. 2019). Ribotypes 002 and 014 have already been described in captive and wild animals (Zlender et al. 2022). Ribotype 014 is the most frequently observed, being the primary driver of the global spread of this agent (Berger et al. 2020, Wen et al. 2022). This ribotype exhibits significant genetic diversity, facilitating the transmission of antimicrobial resistance genes, such as tetracycline, erythromycin, and aminoglycosides (Knight et al. 2017, Andino-Molina et al. 2019). Recently, the presence of RT014/20 strains in metronidazole-resistant dogs was demonstrated (Leite et al. 2023).

The difference between the qPCR results and isolation can be explained by the high sensitivity of the technique (Maestri et al. 2022). We believe that the use of BHI without a previous alcohol shock would have reduced the sensitivity of the isolation method. It´s also possible that qPCR was able to detect strains that were not viable or in enough quantity to be cultured (Jia et al. 2023). This technique is highly practical due to its rapid execution and greater feasibility under anaerobic conditions compared to isolation (Okanda et al. 2020). The use of primers for constituent genes (tpi) (Kilic et al. 2015) and for the gene encoding the 60kDa chaperonin protein (cpn60) (Kohler et al. 2022) can improve the specificity of the qPCR results.

The primary limitation of this study is the inability to gather data regarding the animals’ location, habitat, interactions with humans and other animal species, as well as their clinical history.

Conclusion

Considering the number of isolates recovered, the ribotypes found and the rate of qPCR positivity in the feces of the species included, it is important to consider wild animals as possible reservoirs or carriers of Clostridioides difficile and the possibility of transmission to humans and other domestic animals.

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• ⁴
  Available at <https://webribo.ages.at/> Accessed on May 11, 2022>

Publication Dates

• Publication in this collection
  26 Feb 2024
• Date of issue
  2024

History

• Received
  20 Sept 2023
• Accepted
  20 Oct 2023