Occurrence and Antimicrobial Resistance of Staphylococcus aureus Isolated from Healthy Pet Rabbits

Abstract

Background:

Staphylococcus aureus is a ubiquitous microorganism and an opportunistic pathogen responsible for numerous diseases in humans and animals, characterized by different clinical pictures with acute or subacute course. S. aureus, due to its great adaptability and versatility in terms of infections and hosts, can be considered a relevant pathogen because of the harmful effects on animal health and its potential for transmission from animals to humans and vice versa. In recent years, a marked increase in multidrug-resistant S. aureus has been reported, posing a serious threat for disease management, food safety, and animal and human health as they limit available therapeutic options. In light of a growing interest of the scientific community for this micro- organism and considering the limited data availability on the prevalence of this pathogen in pet rabbits, the purpose of this research was to evaluate the presence of S. aureus in pet rabbits.

Materials and Methods:

From November 2021 to December 2022, nasal swabs were collected from 50 pet rabbits from private households in the Campania Region, southern Italy, and underwent analysis for S. aureus detection. Samples were enriched in broth, then inoculated onto nutrient and selective media, including Blood agar base supplemented with 7% sheep blood and Baird-Parker Agar Base, following standard laboratory protocols. Incubations in aerobic conditions at 37°C were performed for 24/48h for colony identification. Antimicrobial susceptibility testing for all S. aureus isolates was conducted using the disc diffusion method.

Results:

Our results reported the presence of S. aureus in 16/50 (32%) rabbits examined, showing high levels of phenotypic resistance to different antibiotics, in particular penicillin 10U (81.2%) and erythromycin 15 μg (62.5%).

Conclusion:

The study demonstrated that pet rabbits represent a significant reservoir of S. aureus and contributes to the knowledge on the phenotypic antimicrobial resistance of these bacteria in rabbits raised in a domestic environment.

Introduction

The population of companion animals globally has grown substantially over the years: in fact, approximately half of household environments contain pets (Pomba et al., 2017; Westgarth et al., 2010). In Italy, a recent Assalco-Zoomark Report estimates the presence of 64.8 million pets in 2021, including nearly 30 million of fish, 13 million of birds, 10 million of cats, 8.7 million of dogs, and just over 3 million of small mammals (including rabbits) and reptiles (ASSALCO, 2023). In particular, rabbits are docile and attractive animals that require limited space and could easily be litter trained; especially those belonging to dwarf breeds are also suitable for children, because they can be house-trained, require minimal maintenance, and are considered safe and manageable (Chen and Quesenberry, 2006; Molnár et al., 2019).

In addition to this growing trend, it is important to note that there has also been a change in the social role of companion animals over time (Pomba et al., 2017), since they are more and more considered true family members and a close proximity and contact are very common. Studies on human–animal interaction have proposed that there are health benefits and risks associated with pet ownership (Overgaauw et al., 2020; Westgarth et al., 2010). There is evidence that companion animals can act as reservoirs of multitude of pathogenic microorganisms, including multidrug-resistant (MDR) bacteria (Kaspar et al., 2018). Several studies carried out on various pet species have detected the presence of zoonotic agents and opportunistic pathogens. These include Salmonella spp., thermotolerant Campylobacter, Pseudomonas aeruginosa (Dipineto et al., 2010; Santaniello et al., 2021; Teng et al., 2022), as well as antimicrobial resistance (AMR) bacteria such as Enterobacteriaceae producing extended-spectrum beta-lactamase, and Methicillin-resistant Staphylococcus aureus (MRSA) (Kaspar et al., 2018; Ruzauskas et al., 2015; Van Immerseel et al., 2004; Varriale et al., 2020).

Consequently, humans can acquire antimicrobial-resistant bacteria or their resistance genes not only from food-producing animals but also through contact with their pet or synanthropic animals (Pomba et al., 2017; Russo et al., 2021). Moreover, the increase of pets in urban areas has increased the use of antimicrobials for these animals, which has consequences for the carriage of AMR and environmental ecotoxicity (Domingo-Echaburu et al., 2021).

S. aureus is an ubiquitous microorganism and an opportunistic pathogen responsible in humans and animals for numerous diseases, characterized by different clinical pictures with acute or subacute course (Peton and Le Loir, 2014). The presence of S. aureus is not always a direct cause of disease: in fact, in humans S. aureus is present on the skin and mucous membranes in 20–30% of healthy subjects and is commonly found in numerous animal species both farmed and companion, even in the absence of clinical manifestations (Argudín et al., 2017; Flenghi et al., 2023; Peton and Le Loir, 2014). S. aureus, due to its great adaptability versatility in terms of infections and hosts, can be considered a relevant harmful effect on animal health and its potential for transmission from animals to humans and vice versa. S. aureus can trigger a wide range of diseases that vary in severity, from superficial skin infections to severely life-threatening conditions like pneumonia, endocarditis, sepsis, and toxic shock syndrome. S. aureus also exhibits high virulence, attributed to an array of factors it generates to intensify the severity of diseases. This spectrum includes toxins like Panton-Valentine leukocidin and alpha-toxin which damage host tissues and successfully evade immune responses.

In addition, recent years have seen a marked increase in MRSA strains. Such types pose a serious threat as they limit available therapeutic options for disease management, directly impacting animal morbidity and mortality (Costa et al., 2022), food safety, and human and public health. AMR poses a major threat to public health around the world and it is estimated that it could cause the death of 10 million people per year by 2050 (Murray et al., 2022). S. aureus was associated with more than 1 million deaths in 2019 and was the leading bacterial cause of death in 135 countries (Ikuta et al., 2022).

Although the prevalence of S. aureus is reported in companion animals in the literature, the majority of data concern dogs and cats (Costa et al., 2022; Drougka et al., 2016), while surveys of this pathogen in the rabbit species are scarce (Jangsangthong et al., 2022; Loncaric and Künzel, 2013; Rougier et al., 2006). Therefore, the aim of this study was to determine the prevalence of S. aureus healthy pet rabbits in Campania region (Italy) and to characterize the isolates according to their phenotypic AMR.

Materials and Methods

Sampling

Between November 2021 and December 2022, nasal swabs were collected from 50 pet rabbits and analyzed for detection of S. aureus. All the animals examined were selected from private household located in the Campania Region (southern Italy). The samples were then collected by sterile cotton-tipped swabs (both sides using one swab). Each swab sample was inoculated in tubes containing Buffered Peptone Water (BPW) (Oxoid, ThermoFisher, Italy), stored, and transported under refrigeration condition to the microbiological laboratory for the analysis, within 2 h of collection. Specific rabbit-handling procedures were performed by specialist veterinary and with informed consent of the owners. To reduce possible variability in nasal microbial communities, only clinically healthy and individually housed rabbits were used for the purpose of trying to emphasize the asymptomatic reservoir role of these animals and to assess the presence of S. aureus in the absence of clinical signs of disease.

The age of animals sampled for the study ranged from 8 months to 7 years, and all rabbits had not received antibiotic treatment in the 6 months before sampling.

Bacterial isolation and identification

All nasal swabs were analyzed for detection of S. aureus following the laboratory protocols according to Attili et al. (2020), including the use of control organisms for quality check. Each sample was inoculated in sterile tubes containing 10 mL of BPW, vortexed, and incubated at 37°C for 24 h. In brief, after an enrichment in broth, samples were streaked onto nutrient and selective media as Blood agar base supplemented with 7% sheep blood (Liofilchem s.r.l., Italy) and the Baird-Parker Agar Base (Oxoid, ThermoFisher, Italy); plates were incubated under aerobic conditions at 37°C for 24/48 h and inspected for colonies identification after incubation.

The presumptive S. aureus colonies were identified by standard microbiological methods such as morphology, pigmentation, Gram's staining technique, and catalase production (Loncaric and Künzel, 2013; UK SMI ID 7, 2020). In addition, all isolated colonies were subcultured in Nutrient Agar (NA) (Oxoid, ThermoFisher, Italy) for 24 h at 37°C and subjected to latex agglutination test and coagulase test according to the manufacturer's criteria (Liofilchem s.r.l, Italy) (Coagulase Test Protocol, 2010). S. aureus standard reference strain (ATCC 25923) was used to verify the condition of incubation and the performance of the culture media.

Collected isolates grown on NA were finally identified by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight. One colony (two spots per colony) of fresh and pure culture on each NA plate was applied in a 96-well steel target plate (Bruker, Bremen, Germany) using a toothpick overlaid with 1 μL of α-cyano-4-hydroxycinnamic acid–matrix solution (Bruker Daltonik GmbH, Bremen, Germany) and air-dried at room temperature (18–27°C). For each isolate, two replicates were performed. Mass spectra obtained from each isolate were processed with MALDI Biotyper 4.1.100 software (Bruker, Germany) according to the manufacturer's criteria. Identifications were obtained by comparing the mass spectra to the Bruker MSP database (version DB5989) using the Bruker Compass software (Bruker Daltonics) at default settings. The score threshold for explicit determination on species level was set to 2.0.

Determination of AMR

All isolates of S. aureus were tested for antimicrobial susceptibility using the disc diffusion method according to the recommendations of Clinical Laboratory Standards Institute (CLSI) M100 30th edition (CLSI, 2020). The antibiotics selected (CLSI, 2020; Loncaric and Künzel, 2013; Ramana et al., 2009) and tested were: penicillin (P, 10 U), gentamicin (CN, 10 μg), erythromycin (E, 15 μg), tetracycline (TE, 30 μg), ciprofloxacin (CIP, 5 μg), clindamycin (DA, 2 μg), trimethoprim–sulfamethoxazole (SXT, 25 μg), chloramphenicol (C, 30 μg), nitrofurantoin (F, 300 μg), and rifampicin (RIF, 5 μg). Inducible resistance to methicillin was detected by cefoxitin disc (FOX, 30 μg). The inhibition zones were measured and scored as sensitive, intermediate susceptibility and resistant according to CLSI (CLSI, 2020). Isolates categorized as intermediate were considered resistant. The reference strain S. aureus ATCC 25923 was used as a quality control. Isolates resistant to at least three classes of antibiotics were considered MDR (Sweeney et al., 2018; Zulkeflle et al., 2016).

Data analysis

The data collected during sampling and the results obtained from laboratory analysis were cataloged using Microsoft Excel spreadsheet software; the results obtained, once checked, were analyzed using VassarStats free statistical software.

Results

The results of the present investigation show the presence of S. aureus in 16 out of 50 rabbits examined (32%; CI 95% 19.9–46.8). All strains isolated were resistant to at least one of the antibiotics tested. Respectively, 81.2% (n = 13) and 62.5% (n = 10) of the S. aureus isolates were resistant to penicillin and erythromycin. Nine isolates (56.2%) were resistant to clindamycin, and 37.5% (n = 6) showed resistance to chloramphenicol and rifampicin. Resistance for sulfamethoxazole–trimethoprim (n = 4; 25%), gentamicin, tetracycline, and ciprofloxacin (n = 3; 18.7%) was less frequent. Two of the isolates (12.5%) showed resistance to nitrofurantoin. Ten out of 16 strains (62.5%) were considered MDR (Sweeney et al., 2018; Zulkeflle et al., 2016). In addition, five isolates (31.2%) showed resistance to cefoxitin (screening test for methicillin resistance). All AMR results are reported in Table 1.

Table 1.

Results of Antimicrobial Susceptibility Test of 16 Staphylococcus aureus Strains Isolated from Rabbit Nasal Swabs

ID	P	FOX	CN	E	TE	CIP	F	DA	SXT	C	RD
16	R	S	S	R	S	S	S	R	S	R	S	^a
23	R	R	S	R	S	S	S	R	S	S	S	^a
4	R	R	S	R	R	S	R	R	R	R	R	^a
7	R	S	S	R	S	S	S	R	R	R	R	^a
3	R	R	R	R	S	R	S	R	R	S	R	^a
5	R	S	S	R	S	S	S	R	S	R	S	^a
8	R	S	S	S	S	S	S	S	S	S	S
2	R	S	R	S	S	S	S	S	S	S	S
5S	R	S	S	S	S	S	S	S	S	S	S
11	S	S	S	R	S	S	S	S	S	S	S
9	R	S	S	R	S	S	S	S	S	S	S
6	S	S	R	R	R	S	S	R	S	S	S	^a
12	R	S	S	S	S	R	S	R	S	S	R	^a
31	S	S	S	S	R	S	S	S	R	S	S
16S	R	R	S	R	S	S	R	R	S	R	R	^a
29	R	R	S	S	S	R	S	S	S	R	R	^a

MDR strains.

C, chloramphenicol, 30 μg; CIP, ciprofloxacin, 5 μg; CN, gentamicin, 10 μg; DA, clindamycin, 2 μg; E, erythromycin 15 μg; F, nitrofurantoin, 300 μg; FOX, cefoxitin, 30 μg; MDR, multidrug-resistant; P, penicillin, 10 U; R, resistant; RD, rifampin, 5 μg; S, susceptible; SXT, sulfamethoxazole–trimethoprim, 1.25 + 23.75 μg; TE, tetracycline, 30 μg.

Discussion

The results obtained in the present survey suggest that clinically healthy pet rabbits can be considered important reservoirs of MDR Staphylococcus aureus.

As in other studies conducted mainly on intensively reared rabbits, our research showed that clinically healthy rabbits had high colonization in the nose, suggesting that it is a stable colonization in equilibrium between bacterium and host (Attili et al., 2020). Rabbits from an ethological point of view spend a lot of time cleaning and feeding, as well as if cooped up in groups enact affiliation behaviors, including resting in physical contact and allogrooming (DiVincenti and Rehrig, 2016). In this way they can carry the microorganism from the nose to other body sites. In fact, several studies report that S. aureus can be isolated from many parts of rabbits' bodies, including the ear, the skin of the interdigital spaces, from the forelimbs, axillary and inguinal regions, nipples, perineum, vagina, and foreskin (Attili et al., 2020). Therefore, the finding that is reported in this study could increase knowledge about the epidemiology of S. aureus to consider the risk and role of these increasingly popular pets. The identified strains displayed resistance to several antibiotics, including critically important antibiotics for humans (Scott et al., 2019). The presence of S. aureus is not always a direct cause of disease, in fact this microorganism is commonly found in numerous animal species, even asymptomatic (Peton and Le Loir, 2014).

In dogs and cats, S. aureus can act as an opportunistic pathogen causing pyoderma, abscesses, otitis externa, upper respiratory tract infections, and endocarditis. In rabbits, it causes skin infections and mastitis as reported by Costa et al. (2022), who isolated S. aureus in 7.8% of rabbits with skin infections. Other studies reported a 5.3% prevalence of methicillin-resistant Staphylococcus infection in pets, rabbit included. Clinical infections caused by MRSA in lagomorphs have been documented in the United Kingdom, Ireland, Germany, and Austria (Loncaric and Künzel, 2013). Among different species of companion animals, rabbits are poorly studied and limited data are available in the existing literature (Loncaric and Künzel, 2013; Rougier et al., 2006). Several studies have reported the presence of MDR S. aureus and MRSA in healthy pets (Drougka et al., 2016; Jangsangthong et al., 2022).

Recent years have seen a significant increase in the number of S. aureus strains resistant to beta-lactam antibiotics, such as penicillin, commonly used to treat infections. Such strains pose a serious threat as they limit available therapeutic options for disease management, directly impacting animal morbidity and mortality (Costa et al., 2022), food safety, and human and public health.

In the present study, we demonstrated that the majority of S. aureus isolated were resistant to penicillin (81.2%). Out of the 16 isolates, 10 (62.5%) were resistant to erythromycin and 9 (56.2%) to clindamycin. Resistance to sulfamethoxazole–trimethoprim (25%), gentamicin, tetracycline, and ciprofloxacin (18.7%) was less frequently recorded. Two isolates (12.5%) showed resistance to nitrofurantoin, 10 strains presented a MDR phenotype, and 5 isolates (31.2%) showed resistance to cefoxitin (screening test for methicillin resistance).

Our findings are partially in line with the results of Costa et al. (2022), who reported that the prevalence of penicillin resistant S. aureus strains in companion animals in Portugal was 81.8%. In contrast, the frequency of resistance recorded for erythromycin, clindamycin, chloramphenicol, rifampicin, sulfamethoxazole–trimethoprim, gentamicin, and tetracycline in the same study was low, ranging from 0% to 14.5%. Similarly, the percentage of isolates with the MDR phenotype was much lower than that recorded by us, 14.5% versus 62.5%.

The frequency of resistance to ciprofloxacin and cefoxitin reported by these authors was high compared to results recorded in our study: 54.5% versus 18.7% and 56.4% versus 31.2%, respectively. However different results were obtained in Zambia (Youn et al., 2014), where 100% of strains isolated were sensible to cefoxitin, 63.6% to penicillin, 36.4% to sulfamethoxazole–trimethoprim, and 9.1% to tetracycline with a percentage of MDR strains of 10.4%. In contrast, in a cross-sectional study conducted in Greece (Drougka et al., 2016), the authors report that among the S. aureus strains isolated from dogs and cats, 35.8% were resistant to tetracycline, 26.4% to kanamycin, 24.5% to rifampin and erythromycin, 22.6% to clindamycin, gentamycin, and fusidic acid, and 13.2% to sulfamethoxazole–trimethoprim.

The emergence of AMR Staphylococcus spp. represents a global threat, because animal origin Staphylococci harbor a wide variety of resistance genes; at least 44 AMR genes in staphylococci have been detected in human and animal isolates (Argudín et al., 2017).

Although the transmission mechanism of community-associated S. aureus is still unclear, several epidemiological studies reported that MRSA may be also transmitted through pets contact. Given the prevalence of MDR S. aureus isolated from pet rabbits, and the possibility of its carriage not only to other sites in the body but also in the domestic environment, this study is a first step toward understanding pet rabbit-related risks that may increase S. aureus infections. Moreover, Zhu et al. (2021) revealed that household environment was an important key reservoir for MRSA transmission. A narrative review reports that, until the late 1990s, cases of MRSA in domestic animals were sporadic and usually due to infections.

The clonal types discovered among pets were connected to those infecting humans living in the same house location (Aires-de-Sousa, 2017). The close contacts between humans and their animals, such as sleeping and sharing beds or behavior like licking the face or wounds and accidental biting, can be some of the main risk factors for the exchange of microorganisms, including MDR pathogens (Aires-de-Sousa, 2017; Overgaauw et al., 2020). From Global Health holistic perspective, it is important to encourage further efforts by the scientific community to better understand the contribution of pets in the diffusion and transmission to humans and the environment of zoonotic agents and AMR microorganisms.

Limits

This study is just a first step toward understanding pet rabbit-related risks that may increase S. aureus infections. Molecular characterization of the isolated strains has not been performed. Other limitations of our present study were that we had a relatively small number of animals (50 rabbits).

Conclusions

Our results suggest that healthy pet rabbits are potential carriers of MDR S. aureus. We therefore emphasize the need for much larger epidemiological studies in companion animals to assess the role of these species in the spread of potentially pathogenic bacteria to humans and the dissemination of antibiotic resistance. The One Health approach would be desirable to achieve global health especially considering the intimate relationship of humans with animals and their environment and the broad spectrum of determinants that emerge from this relationship.

Footnotes

Authors' Contributions

T.P.R.: conceptualization (lead), methodology (equal), investigation (lead), validation (equal), data curation (lead), writing—original draft preparation (lead), writing—review and editing (supporting). L.B.: conceptualization (lead), methodology (equal), investigation (supporting), validation (equal), data curation (lead), writing—original draft preparation (lead), writing—review and editing (lead). A.M.: methodology (equal), investigation (equal), validation (equal). A.F.: supervision (lead), writing—review and editing (supporting). L.D.: supervision (lead), writing—review and editing (lead). All authors have read and agreed to the published version of the article.

Ethical Approval and Informed Consent Statement

This study did not require approval by an ethics committee since all procedures involving animals were included in the standard clinical examination and veterinary diagnostic investigations.

Author Disclosure Statement

No conflicting financial interests exist.

Funding Information

No funding was received for this article.

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ID	P	FOX	CN	E	TE	CIP	F	DA	SXT	C	RD
16	R	S	S	R	S	S	S	R	S	R	S	^a
23	R	R	S	R	S	S	S	R	S	S	S	^a
4	R	R	S	R	R	S	R	R	R	R	R	^a
7	R	S	S	R	S	S	S	R	R	R	R	^a
3	R	R	R	R	S	R	S	R	R	S	R	^a
5	R	S	S	R	S	S	S	R	S	R	S	^a
8	R	S	S	S	S	S	S	S	S	S	S
2	R	S	R	S	S	S	S	S	S	S	S
5S	R	S	S	S	S	S	S	S	S	S	S
11	S	S	S	R	S	S	S	S	S	S	S
9	R	S	S	R	S	S	S	S	S	S	S
6	S	S	R	R	R	S	S	R	S	S	S	^a
12	R	S	S	S	S	R	S	R	S	S	R	^a
31	S	S	S	S	R	S	S	S	R	S	S
16S	R	R	S	R	S	S	R	R	S	R	R	^a
29	R	R	S	S	S	R	S	S	S	R	R	^a

ID	P	FOX	CN	E	TE	CIP	F	DA	SXT	C	RD
16	R	S	S	R	S	S	S	R	S	R	S	^a
23	R	R	S	R	S	S	S	R	S	S	S	^a
4	R	R	S	R	R	S	R	R	R	R	R	^a
7	R	S	S	R	S	S	S	R	R	R	R	^a
3	R	R	R	R	S	R	S	R	R	S	R	^a
5	R	S	S	R	S	S	S	R	S	R	S	^a
8	R	S	S	S	S	S	S	S	S	S	S
2	R	S	R	S	S	S	S	S	S	S	S
5S	R	S	S	S	S	S	S	S	S	S	S
11	S	S	S	R	S	S	S	S	S	S	S
9	R	S	S	R	S	S	S	S	S	S	S
6	S	S	R	R	R	S	S	R	S	S	S	^a
12	R	S	S	S	S	R	S	R	S	S	R	^a
31	S	S	S	S	R	S	S	S	R	S	S
16S	R	R	S	R	S	S	R	R	S	R	R	^a
29	R	R	S	S	S	R	S	S	S	R	R	^a

ID	P	FOX	CN	E	TE	CIP	F	DA	SXT	C	RD
16	R	S	S	R	S	S	S	R	S	R	S	^a
23	R	R	S	R	S	S	S	R	S	S	S	^a
4	R	R	S	R	R	S	R	R	R	R	R	^a
7	R	S	S	R	S	S	S	R	R	R	R	^a
3	R	R	R	R	S	R	S	R	R	S	R	^a
5	R	S	S	R	S	S	S	R	S	R	S	^a
8	R	S	S	S	S	S	S	S	S	S	S
2	R	S	R	S	S	S	S	S	S	S	S
5S	R	S	S	S	S	S	S	S	S	S	S
11	S	S	S	R	S	S	S	S	S	S	S
9	R	S	S	R	S	S	S	S	S	S	S
6	S	S	R	R	R	S	S	R	S	S	S	^a
12	R	S	S	S	S	R	S	R	S	S	R	^a
31	S	S	S	S	R	S	S	S	R	S	S
16S	R	R	S	R	S	S	R	R	S	R	R	^a
29	R	R	S	S	S	R	S	S	S	R	R	^a