Occurrence and Antimicrobial Resistance among Staphylococci Isolated from the Skin Microbiota of Healthy Goats and Sheep

Staphylococci colonize the skin and mucous membranes of different animals. The purpose of this study was to determine the staphylococcal composition of the skin microbiota of healthy, non-vet visiting, and antimicrobially non-treated sheep and goats. In total, 83 strains (44 from goats and 39 from sheep) were isolated and identified using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). The diversity of the isolated Staphylococcus species was relatively high, and only coagulase-negative staphylococci (CoNS) were isolated. In sheep, S. vitulinus (9/39, 23.1%) was the most common species, followed by S. equorum (8/39, 20.5%), S. lentus (7/39, 17.9%), S. sciuri (6/39, 15.4%), S. xylosus (6/39, 15.4%), S. warneri (1/39, 2.6%), S. simulans (1/39, 2.6%), and S. nepalensis (1/39, 2.6%). In the goats, the most common species was S. sciuri, which was detected in 13 (29.5%) animals. The goat skin was also inhabited by S. equorum (7/44, 15.9%), S. vitulinus (6/44, 13.6%), S. cohnii (5/44, 11.4%), S. lentus (4/44, 9.1%), S. suscinus (3/44, 6.8%), S. caprae, (2/44, 4.5%), S. auricularis (2/44, 4.5%), S. warneri (1/44, 2.3%), and S. xylosus (1/44, 2.3%). Only one S. xylosus strain of goat origin carried the enterotoxin gene (sea). Antimicrobial resistance was not common among the isolated staphylococci. Only 31 (37.3%) strains were resistant to at least one antimicrobial agent, with the highest frequency of resistance to penicillin (16.8%), followed by clindamycin (9.6%), erythromycin (8.4%), moxifloxacin (8.4%), and tetracycline (7.2%). All isolates were susceptible to eight antibiotics (amikacin, gentamycin, ciprofloxacin, levofloxacin, rifampicin, chloramphenicol, trimethoprim-sulfamethoxazole, and tigecycline), representing six different classes. Three isolates displayed a multi-resistance phenotype (MDR): the goat isolates S. cohnii and S. sciuri, as well as the ewe isolate S. xylosus. The MDR S. cohnii isolate was found to be methicillin-resistant and carried the mecA gene. Moreover, the staphylococci isolated from the healthy animals carried genes conferring resistance to β-lactams (mecA, blaZ), tetracyclines (tetL, tetK), macrolides (ermB, ermC), lincosamides (lnu), and fluoroquinolones (grlA). However, the prevalence of these genes was low.


Introduction
Staphylococci are commonly found on the skin of humans, other mammals, and birds.There are differences in the composition of staphylococci in the skin microbiota between animals and humans.The coagulase-negative staphylococci (CoNS) Staphylococcus epidermidis is the most abundant species on human skin.The coagulase-positive staphylococci (CoPS) S. intermedius and S. pseudintermedius are the most prevalent bacteria in the skin microbiota of domestic animals (dogs and cats), whereas S. lentus is the most common species in healthy pigeons [1][2][3].Little is known about staphylococci isolated from the skin microbiota of healthy ewes and goats.Previous studies have focused on Staphylococcus strains associated with skin infections [4].Folliculitis, furunculosis, and impetigo are common staphylococcal infections in goats and sheep.Of note, folliculitis and furunculosis infections may be manifested by skin lesions in any location on the body [5].Staphylococcal infections usually occur upon bacterial entrance through the skin and breakdown of mucous membranes, e.g., during injuries or surgical incisions.The invading bacteria proliferate locally and produce enzymes and toxins that cause tissue damage [6].In sheep and goats, the most common cause of staphylococcal skin disease is S. aureus, although other bacteria, e.g., S. hyicus, S. haemolyticus, S. warneri, S. epidermidis, S. chromogenes, S. caprae, S. simulans, and S. xylosus, have also been isolated from some cases of disease [7].Furthermore, S. aureus and other coagulase-positive staphylococcal species: S. hyicus, S. intermedius, and S. schleiferi are associated with clinical mastitis in ewes [8].Also, coagulase-negative staphylococci, S. epidermidis, S. saprophyticus, S. simulans, S. xylosus, S. warneri, and S. chromogenes, have been isolated from cases of clinical mastitis [6] (Supplementary Materials Table S1).These bacteria can contaminate raw food and produce staphylococcal enterotoxins (SEs) that can cause food poisoning.Most staphylococcal food-borne diseases are caused by the production of classical enterotoxins (SEA, SEB, SEC, SED, and SEE).Generally, staphylococcal food poisoning (SFP) is a self-limited illness resolving within 24-48 h after the onset.However, the SFP symptoms in some groups may require hospitalization, especially in the case of infants, the elderly, or immunocompromised individuals [9].
Recent studies have reported increased resistance to β-lactams, tetracyclines, clindamycin, and less frequently, fluoroquinolones.The major threat to human and animal health associated with staphylococci is their methicillin resistance.Methicillin resistance associated with carriage of the mecA or mecC genes confers resistance to β-lactam antimicrobials.These genes are located on a mobile genetic element, i.e., the staphylococcal cassette chromosome mec (SCCmec), enabling horizontal transmission between isolates.Notably, methicillin-resistant staphylococci (MRS) are often multi-drug-resistant (MDR; resistant to three or more classes of non-beta lactam antibiotics), which extremely limits therapeutic options [10,11].Recent studies have shown cross-transmission of methicillin-resistant staphylococci between goats and humans [12].This aspect cannot be ignored, as foodproducing animals play an important role in the transmission of MDR bacteria through the food chain.Moreover, microbiome transfer may occur through direct contact between animals and humans.Therefore, livestock animals are a crucial part of One Health [13].
This study was focused on commensal staphylococci in the skin microbiota of healthy sheep and goats.Moreover, another aim was to determine whether the animal skin was colonized by methicillin-resistant Staphylococcus sp.(MRS).
Overall, 37.3% of the isolated strains were resistant to at least one antibiotic.The highest resistance rate was detected for penicillin (16.8%), clindamycin (9.6%), erythromycin (8.4%), moxifloxacin (8.4%), and tetracycline (7.2%) (Figure S1).The detailed results of antimicrobial resistance are presented in Tables 1 and 2. Of note, 100% of the strains exhibited susceptibility to gentamycin, amikacin, rifampicin, ciprofloxacin, levofloxacin, chloramphenicol, trimethoprim/sulfamethoxazole, and tigecycline.All of the penicillinresistant strains in the disk diffusion method were positive for the presence of the blaZ gene.Only one strain (S. cohnii isolated from the goats) presented a methicillin resistance phenotype.The strain was resistant to cefoxitin in the disk diffusion method and the harbored mecA gene.Three strains were multidrug resistant (MDR): S. cohnii was resistant to cefoxitin, penicillin, tetracycline, and tobramycin; S. sciuri was resistant to moxifloxacin, erythromycin, and clindamycin; and S. xylosus was resistant to penicillin, erythromycin, and clindamycin.The profiles of antimicrobial resistance and resistance genes detected in each strain are presented in Tables A1 and A2.Interestingly, resistance-coding genes were also detected in strains isolated from the goats presenting intermediate susceptibility to erythromycin (1 S. equorum strain with the ermB gene detected) and clindamycin (2 S. sciuri strains with the lnu gene detected).All the Staphylococcus strains tested negative for enterotoxin-coding genes except for one S. xylosus strain isolated from sheep where the sea gene was detected.

Discussion
This is the first study incorporating MALDI-TOF-MS to successfully characterize commensal staphylococcal populations in healthy sheep and goats.It should be emphasized that these were no-vet visiting and antimicrobially non-treated animals.We isolated 83 staphylococci and were able to assign all the isolates to 18 different species.The diversity of the Staphylococcus species isolated from the skin microbiota of these animals was relatively high.Ten different staphylococcal species (i.e., S. sciuri, S. equorum, S. vitulinus, S. cohnii, S. lentus, S. caprae, S. suscinus, S. auricularis, S. warneri, and S. xylosus) were detected in the goats, while eight species (i.e., S. vitulinus, S. equorum, S. lentus, S sciuri, S. xylosus, S. warneri, S. simulans, and S. nepalensis) were identified in the sheep.A previous study reported only two species, i.e., S. aureus and S. epidermidis, isolated from the body sides of healthy sheep and goats [14].In another study, S. lentus, S. aureus, S. epidermidis, S. xylosus, and S. caprae were isolated from the nasal cavity, vagina, udder, and anus of dairy goats.However, these animals suffered from mastitis.The most prevalent species was S. lentus [15].In this study, S. sciuri was the dominant bacterial skin colonizer of goats, whereas S. vitulinus was the most frequently isolated from the ewe skin microbiota.Of note, the phylogenomic analyses of the Staphylococcaceae family suggest the taxonomic reassignment of five Staphylococcus species, i.e., S. sciuri, S. fleurettii, S. lentus, S. stepanovicii, and S. vitulinus, to the novel genus Mammaliicoccus, with Mammaliicoccus sciuri as the type species [16].As mentioned above, S. sciuri, S. vitulinus, and S. lentus (proposed to be reclassified as Mammaliicoccus) were common species of the animal skin microbiota.Additionally, S. equorum were isolated frequently from the ewe and goat skin.We observed differences in the staphylococcal composition between sheep and goat.S. cohnii, S. suscinus, S. caprae and S. auricularis were present only on the goat skin.In contrast, S. simulans and S. nepalensis were found only in the ewe's skin.Noteworthy, the healthy skin was not colonized by CPS.It is well known that S. aureus and other CPS, particularly S. schleiferi and S. pseudintermedius, as well as coagulase-variable S. hyicus, are important veterinary pathogens responsible for infections in a number of different animal species [7].Nasal carriage of S. aureus was documented among sheep and goats treated at the University of Veterinary Medicine in Austria [17].Also, a study from France showed that 29% of ewes carried S. aureus in their nares [18].
Another focus of this study was the identification of enterotoxin genes (sea) in staphylococci colonizing non-hospitalized animals.We found the enterotoxin gene only in one S xylosus strain, isolated from the goats.In a previous study, no enterotoxin genes were detected in any CNS strains isolated from goats with mastitis; however, sec and see were identified in S. aureus [15].In another study, the see gene was found in a single S. lentus strain isolated from goats' milk [19].More recently, Kotzamanidis et al. [20] reported the presence of enterotoxin genes in S. aureus strains recovered from clinical mastitis and subclinical mastitis cases in goats, sheep, and bovines.
In this study, the majority of commensal CoNS isolates from the skin microbiota of healthy animals were susceptible to a broad range of antimicrobials.All the isolates were susceptible to eight antibiotics (amikacin, gentamycin, ciprofloxacin, levofloxacin, rifampicin, chloramphenicol, trimethoprim-sulfamethoxazole, and tigecycline), representing six different classes.Overall, we found that 41% of the isolates were susceptible to all the tested antimicrobials, and only 3.6% of the isolates exhibited multi-resistance.Among all the isolates, 26 (37.34%) were found to be resistant to at least one antimicrobial agent, with the highest frequency of resistance to penicillin, followed by clindamycin, erythromycin, moxifloxacin, and tetracycline.A single S. cohnii isolate was methicillin-resistant.Of note, the ewe isolates exhibited the highest frequency of resistance to penicillin and tetracycline compared with the goat isolates.A higher frequency of resistance to penicillin and tetracycline was observed in a previous study in Greece [20], but these investigations focused on S. aureus strains isolated from mastitis.A study from Taiwan also reported a higher rate of resistance to penicillin among staphylococci strains isolated from cases of mastitis in dairy goats [15].These strains showed the high frequency of resistance to tetracycline, neomycin, and gentamycin.A study from Tunisia indicated that the majority of S. aureus strains isolated from healthy sheep were susceptible to tested antimicrobial agents, with the following exceptions: penicillin, tetracycline, and fusidic acid [21].
Screening for genes encoding resistance to many classes of antimicrobial agents, such as penicillin, cephalosporins, tetracyclines, macrolides, lincosamides, and aminoglycosides, indicated their presence in only 11 staphylococcal strains.Among sheep isolates, tek(K) gene encoding membrane-associated efflux proteins conferring resistance to tetracycline was detected in two S. lentus strains.The tetL gene was present in a single S. cohnii isolate from the goats.Moreover, the erm (B) and erm (C) genes, which code for methylases that modify the target site in 23S rRNA and inhibit the binding of macrolides, lincosamides, and streptogramin B MLS B to the bacterial ribosome, were present in the goat S. equorum and S. sciuri isolates.The presence of the ermC gene in a S. sciuri isolate from sheep has previously been reported [22].In another study, the resistance gene mphC-coding macrolide phosphotransferase was found in a S. lentus isolate of ewers origin [23].In this study, the gene lnu(A) encoding a lincosamide nucleotidyltransferase was detected in two S. lentus isolates from goats.Only one S. vitulinus isolate carried grlA, which codes for resistance to phenicols.The blaZ gene encoded β-lactamases were identified in three strains of goat's origin, i.e., S. cohnii, S. sciuri, and S. warneri and in ten strains of ewe's origin, i.e., S. equorum, S. lentus, S. sciuri, S. vitulinus, and S. xylosus.Gharsa et al. [21] reported the presence of blaZ as well as tet(K) genes in Staphylococcus aureus isolated from nasal swabs of healthy sheep.Importantly, methicillin resistance conferred by the gene mec A was found in one S. cohnii isolate colonizing the goats.A study focused on S. aureus reported the presence of the mecA gene in five strains isolated from healthy sheep [23].Another study from Austria reported the presence of the mecA gene in 12 strains isolated from hospitalized goats [17].More recently, El-Deep et al. [12] identified seven methicillin-resistant S. aureus and two methicillin-resistant S. epidermidis strains.
To summarize, resistance to tetracyclines and penicillin was found among the staphylococci isolated from healthy sheep and goats.It is important because these antibiotics are used as first-line treatments in veterinary medicine.However, no resistance to trimethoprim-sulfamethoxazole, which is commonly used in veterinary practice, was detected.Also, all staphylococci strains were susceptible to gentamycin, amikacin, and ciprofloxacin, i.e., antibiotics that are clinically effective in veterinary medicine.Moreover, the majority of stains were susceptible to erythromycin, which is used to treat staphylococcal infections in animals.
In the One Health concept, the health of humans is closely connected to the health of animals.In addition to direct contact between animals and humans, antimicrobial-resistant bacteria and resistance genes may be transmitted via the food chain.The probability of transferring staphylococci to milk is high.Moreover, farm animals can be a source of microorganisms moving into their environment, which may lead to significant changes in the farm dust microbiome.The present study indicated that non-vet visiting goats and sheep from small farms are colonized by antibiotic-resistant staphylococci, including multidrug-resistant strains.However, the prevalence of antibiotic resistance genes among these strains was low.This study showed that healthy sheep and goats are colonized by a variety of CoNS staphylococci.

Bacterial Isolation and Identification
Samples were collected from animals bred on small farms by private owners.The animals were not exposed to antimicrobial treatment prior to this study.Swabs were taken from the skin in the perianal area using Amies transport medium swabs (Sarstedt, Hildesheim, Germany).One swab was collected from each animal.The samples were inoculated on Chapman Agar (BIomeriux, Marcy-l'Étoile, France).After 24 h of incubation at 37 • C, bacterial colonies with proper morphological features were isolated on Columbia Agar (BIomeriux, Marcy-l'Étoile, France ) and then cultivated according to standard microbiological procedures.Staphylococcal strains were identified to the species level using MALDI TOF-MS (Matrix-assisted laser desorption/ionization-time of flight mass spectrometry) following the manufacturer's procedure in the research mode (MALDI Biotyper ® , Bruker, Billerica, MA, USA).

Susceptibility Testing
Susceptibility to all antimicrobial agents was tested using the disk-diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, with the exception of amikacin, tobramycin, and tigecycline, which followed EUCAST guidelines.The following antimicrobial agents were tested: cefoxitin, ciprofloxacin, levofloxacin, moxifloxacin, clindamycin, chloramphenicol, erythromycin, gentamicin, penicillin, rifampicin, tetracycline, trimethoprim/sulfamethoxazole, amikacin, tobramycin, and tigecycline.
Appendix A Table A1.Antimicrobial resistance profiles and distribution of resistance genes in staphylococcal isolates of goat origin.Table A2.Antimicrobial resistance profiles and distribution of resistance genes in isolated staphylococcal isolates of ewe origin.

Table 1 .
Antimicrobial resistance of staphylococcal strains isolated from the skin of goats.

Table 2 .
Antimicrobial resistance of staphylococcal strains isolated from the skin of sheep.