Occurrence of Methicillin Resistant Staphylococcus aureus Carriage in Different Animal Species in Eastern Algeria

Livestock is an important agricultural activity in the world that plays a fundamental role in the economic, ecological, cultural and environmental level. As a result, the animal health is an important factor. However, bacterial pathogen is consistently striking this sector (Srour, 2006).
       
Staphylococcus aureus is a bacterium of significant importance because of its ability to cause a wide range of diseases and capacity to adapt to diverse environmental forms (Grema et al., 2015). It is an important opportunistic pathogen that frequently colonizes the skin, the nose and mucous membranes of healthy humans and animals and can cause multiple infectious diseases of diverse severity ranging from minor skin infections, such as furunculosis to severe and highly debilitating conditions such as pneumonia and endocarditis (Jensen and Lyon, 2009; Lozano et al., 2016). In addition, S. aureus causes inflammation of the mammary gland in bovine and the lower part of the foot in poultry (Quinn et al., 2000).
       
Antibiotics are commonly used in farms for the treatment and prevention of various diseases, to improve food and to increase milk production (Sharma et al., 2011). Nevertheless, zoonotic bacteria like S. aureus develop resistance to these antimicrobial agents. The emergence of this bacterial resistance in animals and their products has highlighted the role that plays the resistance transfer to the human population via the food chain and to the environment by a direct contact with animals (Mesfin, 2015).
       
Among Staphylococcus aureus isolates, meticillin-resistant S. aureus (MRSA) represents the major cause of antibiotic-resistance For many years, MRSA was considered as a specific pathogen of human (Baptiste et al., 2005; Leonard et al., 2008). In recent years, a clone of MRSA has appeared in farm animals (MRSA-L) and isolated in veterinarians, farmers and in foods of animal origin (Chairat et al., 2015). Nowadays, it has been detected in other pets (dogs, horses, cats, pet birds, cattle and pigs) associated with serious infections and become public health concern since it could not be treated easily with many antibiotics (Habibullah et al., 2017).
       
In Algeria, the problem of antimicrobial resistance in veterinary medicine is poorly documented. This study highlights the antimicrobial resistance status resulting from S. aureus and meticillin-resistant S. aureus (MRSA) carriage in animals.

Samples collection
 
A total of 754 nasal and rectal swab samples were collected from randomly selected farms including cattle, sheep, goats, rabbits, cats, poultry and dogs from Batna governorate while horses were sampled from Setif governorate, eastern Algeria. The study included 377 animals from both genders. This investigation was performed between January 2018 to April 2019 in Microbiology laboratory, Batna 1 University, Algeria. All samples were aseptically collected and examined for detection of S. aureus (El Seedy et al., 2017).
 
Isolation and identification of S. aureus
 
First, samples were incubated in a nutrient broth for enrichment (Schmidt et al., 2014). After overnight incubation at 37°C, 1μl of this broth was streaked into mannitol salt agar (MSA) which is a selective medium for S. aureus. The plates were then incubated for 24 hours. The incubation was extended to 48 hours if the result of the culture was negative or uncertain. Morphologically distinguishable staphylococcal colonies were cultured again on MSA plate to obtain pure cultures. The appearance of yellow­ish colonies on MSA was presumed to be S. aureus, subcultured and identified by conventional methods (gram’s staining, catalase test, free coagulase production and biochemical tests (API Staph galery) according to Giacitini et al., (2017).
 
Antimicrobial sensitivity testing
 
Disc diffusion testing was performed on all staphylococcal isolates in accordance with CLSI (2011) against the following panel of 10 antimicrobial agents; Pénicillin (10 µg), Oxacillin (5 µg), Cefoxitin (30 µg), Erythromycin (15 µg), Gentamicin (10 µg), Ciprofloxacin (5 µg), Rifampicin (5 µg), Vancomycin (30 µg), Tetracyclin (30 µg), Amoxicillin and Clavulanic acid (20 µg /10 µg).
       
Using the commercially prepared disc, freshly sub-cultured isolates were emulsified in 3-4 ml of sterile distilled water. The turbidity of the suspension was adjusted to the turbidity of standard equivalent to 0.5 McFarland (Mustapha et al., 2016). A sterile swab was dipped in the bacterial suspension and then smeared on Mueller-Hinton agar media surface. Five discs of antibiotics commercially available were dispensed into each inoculated plate and incubated at 37°C for 24 h. Strains were classified as resistant in accordance with EUCAST (2019) for all antibiotics tested.
 
Cefoxitin disk diffusion test
 
Resistance to methicillin was tested using a cefoxitin disc whose load is 30 µg and by an oxacillin disc of 5 µg load. The reading has been done in accordance with the recommendations of the antibiogram committee of CA-SFM (Comité de l’Antibiogramme de la Société Française de Microbiologie).

In veterinary medicine, the study of the carriage of S. aureus in animals remains limited to a few pets such as cats, horses and pigs (Labrecque, 2007). The interest about S. aureus and MRSA in livestock, domestic and wild animals has signiûcantly increased (Lozano et al., 2016); Methicillin resistant Staphylococcus aureus emerged 50 years ago, as a nosocomial pathogen but in the last decade it has become also a major cause of serious infections in both human and animals (Stefani et al., 2012).
       
Our study was focused on the prevalence of S. aureus and methicillin resistant strains in eight categories of farm animals (Horse, sheep, cattle, goats, rabbits, poultry, dogs and cats) (Table 1).
 

 
Occurrence of Staphylococcus aureus
 
The results of S. aureus and MRSA strains prevalence on the monitored farms are summarized in Table 2.
 

       
S. aureus was recovered from all examined animal species. Of the 754 samples, 43.61% samples were positive for S. aureus. Wang et al., (2019) reported a lower isolation rate with 40.66%. The highest recovery rate was noted in rabbits (92%). It was followed by goats (31.91%), bovine (25%), dogs (23.75%), cats (21.25%), poultry and horses (15% both) and lowest with sheep (10%). Radwan et al., (2015) reported the same result for low isolation rate in sheep while, Giacinti et al., (2017) reported a higher prevalence with 53.5% in dairy sheep farms. Recovery of S. aureus from nasal and rectal swabs in goats was lower than those found in the investigation of Daaloul-Jedidi et al., (2016) (86.6%). The isolation rates from bovine and poultry was lower than those of Akkou et al., (2016) in Algeria who reported 29.81% and 55%, respectively.
       
This investigation revealed that nasal swabs were found more infected by S. aureus than rectal swabs in goats, rabbits, cats, dogs and poultry except in sheep in which equal rate (50%) was observed in both nasal and rectal swabs. In bovine and horses, S. aureus isolates were more from rectal than nasal swabs. This result does not corroborate with those of Linhares et al., (2015) and Daaloul-Jedidi et al., (2016) who reported a predominance of this pathogens in nasal samples than in rectal ones in pigs and goats respectively. This portage can constitute a potential risk of transmission to people who are in contact with these animals (Carlet and Shlemmer, 2015). The frequency of S. aureus in nasal swabs was higher than that reported by Mai-siyama et al., (2014) as 10% for sheep, 20% for cattle and 08% for goats. For rectal samples, Al-Thani et al., (2012) reported a total absence of S. aureus in these samples, especially for sheep and goats. This can be explained by the difference in breeding conditions such as the presence of several animals on the same farm (birds, pets) which facilitates the transmission of this germ.
       
The present study showed the influence of age in the carriage of S. aureus. It was more isolated in animals aged more than one year old, which is explained by the skin modifications which are considered as the first barrier against pathogens with decrease in elasticity and thickness of subcutaneous cell tissue and dry skin (Branchet et al., 2012). In addition, some anomalies of the respiratory system, more often observed in elderly animals, will cause the aggression of the bronchial epithelium, expressed by a decrease in bronchial secretions which are the second barrier against the entry of many airborne infectious agents (Vareille et al., 2011). Our findings are in consistent with data of Branchet et al., (2012) who also described age as a factor associated with the carriage of S. aureus. The results reported that females are most infected by S. aureus in all animal species. Weese (2005) confirmed that sex is a risk factor, the female seems to be more susceptible to colonization by S. aureus than males. 
 
Evaluation of antibacterial activity
 
Results of antibiotic sensitivity test on S. aureus iso­lates recovered from nasal and rectal swabs from different animal species revealed that all the isolates were completely resistant (100%) to ciprofloxacin and gentamycin in sheep and horses and to penicillin in dogs (Table 3).
 

       
The overall results of resistance to penicillin, gentamicin, ciprofloxacin and erythromycin was highly prevalent with 64.81%, 50.53%, 48.16 and 47.79 respectively. Moreno-Grúaet_al(2018) gave the same resistance rate to penicillin.
       
Table 3 shows a high resistance to vancomycin. Effectively, this may be due to the fact that the clinical isolates of S. aureus with intermediate and complete resistance to vancomycin have emerged within the past two decades and have become a serious public health problem (Cong et al., 2020). Noble et al., (1992) demonstrated that there is Vancomycin resistance transfer, mediated by transposons mainly found on plasmids, which raised considerable worry about the risk of dissemination of vancomycin-resistant determinants to universally susceptible microorganisms of medical importance, especially S. aureus. This concern was subsequently confirmed by the successful transfer of the van element from Enterococcus faecalis to a MRSA strain in mix-infected mice (Cong et al., 2020). Haaber et al., (2015) discovered that susceptibility of S. aureus to vancomycin is reduced by concurrent exposure to colistin, a cationic peptide antimicrobial employed to treat infections by gram-negative pathogens.
       
Giacinti et al., (2017) revealed in their investigation that S. aureus isolates were phenotypically resistant to all the β-lactams tested and to erythromycin, streptomycin, kanamycin and tetracycline. Same results were revealed in our study for erythromycin (47.79%).
       
S. aureus strains (29.46%) showed a resistance to cefoxitin and were thus identified as methicillin-resistant S. aureus (MRSA) strains as described by Hachemi et al., (2019). Radwan et al., (2015) reported high rate of methicillin resistance with 58.3%.
       
According to Table 1, cats were more affected by SARM strains followed by horses and sheep (55.55%, 46.66% and 40% respectively). Faires et al., (2009) gave a little lower rate for cats (10%). For Sarhan and Mohammed (2019), MRSA isolates prevalence in sheep (92.30%) were significally higher than our results.
       
Weese (2005) reported same MRSA isolation rate in horses with 42.5% while most published reports indicated that MRSA colonization in horses ranged from 0 to 11% (Grema et al., 2015). The previously prevalence might under-represent true carriage as this study samples were only from the nasopharynx.
       
Bounar-Kechih et al., (2018) isolated also MRSA from poultry at 53.5% which was higher than our results with 26.66%.
       
Dogs and rabbits were the least affected by S. aureus carriage (12.5% and 7.41% respectively). Loeffler et al., (2005) and Moreno-Grúa et al., (2018) gave also a low rate with 9% for dogs and 12.5% for rabbits, respectively.
              
All the isolated MRSA strains showed cross-resistance to antibiotics used in the study. They were more resistant towards two or multiple antibiotic combinations; therefore, they were cross-resistant to many other antibiotic families other than β-lactam antibiotics. In Algeria, selling antibiotics is totally uncontrolled what means abusive and indiscriminate use in human but also in the animal.

The main finding of this study indicates clearly that S. aureus and MRSA were highly isolated from all animal species joining the results of other studies. They were also resistant to β-lactam antibiotics (Penicillin and Oxacillin). This result also suggests that it may be a potential risk for human health because of a possible transmission from animals. Because of the few studies done in this area in Algeria, it will be interesting to realize sampling from multiple anatomic sites when screening animals for MRSA and to undertake molecular studies are needed in order to understand the cross transmission from animal to human or from human to animals.