Isolation, identification and antimicrobial profile of methicillin‐resistant Staphylococcus aureus from bovine mastitis in and around Adama, Central Ethiopia

Abstract Background Among bacterial pathogens, Staphylococcus aureus which lives in the mammary gland is the leading cause of bovine mastitis worldwide, which causes enormous economic losses to the dairy industry. Objectives and methods The study was carried out cross‐sectionally to determine the occurrence of methicillin‐resistant S. aureus (MRSA) and the risk factors for mastitis infection in dairy cows in and around Adama from October 2014 to June 2015. This particular study included 384 animals. Milk samples were collected and screened with California mastitis test. Then bacteria were cultured and identified using biochemical tests and disc diffusion test was used to determine the antimicrobial sensitivity for MRSA. Results The prevalence of mastitis was 121 (31.5%). Among them, 37 cases (30.6%) were clinical mastitis and 84 cases (69.4%) were subclinical mastitis. Among these positive cases, 37 cases (30.6%) of S. aureus were isolated. The prevalence of mastitis was significantly related to the breed, age, floor type and sanitation status of the milking (p <0.05). The Kirby–Bauer disc diffusion method was performed on Mueller Hinton agar medium according to NCCLS guidelines to test antibiotic sensitivity. The 32.4% of S. aureus isolates were resistant to oxacillin. Isolates of MRSA are more resistant to amoxicillin (75%), oxytetracycline (66.7%) and sulfa (50%). However, they were sensitive to kanamycin (75%), streptomycin (58.3%) and nalidixic acid (50%). Insufficient dosage, short treatment time and chronic infection in dairy cows in the herd are the main reasons for the large number of resistant strains. Conclusion and recommendation Generally, mastitis was prevalent in the area, and it was resistant to commonly used antibiotics. Therefore, hygienic, prevention and alternative treatment methods should be implemented.


INTRODUCTION
Milk produced by dairy cows is the food source for most rural and urban populations. The development of the Ethiopian dairy industry has helped to alleviate poverty and food insecurity in the country (Mohamed et al., 2004). However, this is limited by different reasons.
Regardless of the animal species, mastitis can lead to reduced milk production and remains one of the most economically important diseases in the global dairy industry (Bradley, 2002). Mastitis can cause a decrease in the quality and quantity of milk (Quinn et al., 1999). Mastitis is distributed in dairy cows globally and has been described as of extreme economic and zoonotic importance (Al-Majali et al., 2008). It is defined as clinical or subclinical and is a complex multifactorial disease. Its occurrence depends on different variables related to the host, the environment and the agent (Radostits et al., 2007).
Bacteria are the most common causes of mastitis which are found in the dairy cow and in the environment and are therefore a common threat to the mammary gland (Bradley, 2002). Among bacterial pathogens, Staphylococcus aureus is the leading cause of contagious bovine mastitis worldwide and it causes enormous economic losses to the dairy industry. Many species of staphylococci can produce a layer of extracellular polysaccharide and mucus, which are related to the virulence of the pathogen towards the host's defence mechanism.
Other Non-aures staphylococci are often found colonizing the teats and mammary glands. They rarely cause clinical mastitis and are called minor pathogens (Piepers et al., 2007;Radostits et al., 2007).
In addition to treatment, antibiotics are used in veterinary medicine for preventive purposes, to improve feed efficiency and promote growth particularly in developing countries. The large use of antimicrobials causes the emergence of antimicrobial-resistant pathogens.
Due to the emergence and spread of multidrug-resistant zoonotic pathogens, the public and scientific communities are increasingly concerned about the widespread use of antimicrobial agents. Antibioticresistant bacteria do not respond to conventional antibiotic treatment and prolongs the course of the disease. The resistance of S. aureus to antimicrobial agents can complicate the treatment of infections (Normanno et al., 2007). Shortly after the introduction of penicillin, MRSA is usually resistant to multiple drugs (Lee, 2003;Robinson & Enright, 2003).
MRSA has been increasingly reported as a new problem in veterinary medicine. The appearance of MRSA causes serious zoonotic diseases (Vanderhaeghen et al., 2010). It was first described as a hospital-based cause of infection, but it has received attention as a community pathogen (Said-Salim et al., 2003). It has been isolated from cattle, dogs, cats, pigs, horses and poultry all over the world (Leonard & Markey, 2008).
Therefore, the objectives of this study are to determine the occurrence of MRSA and determine the drug sensitivity pattern of MRSA.

Study area
Adama (Nazret) is one of the largest towns in Oromia region of Ethiopia. It is about 100 km away from Addis Ababa in the southeast direction at an altitude of 1650 m above sea level. Its annual temperature ranges from 13.9-29 • C. It is located at 8

Study population
The target dairy population is 50% local* Holstein cows with different age, lactation stage, feeding conditions and hygiene milking status.
Cows were selected randomly from the selected 15 large and 10 small dairy farms in and around Adama town. The age of the individual animals was determined based on their dentition. Breeds of animals were distinguished by their uniquely observable characteristics and body condition score was made and recorded as poor, medium and good according to Nicolson and Butterworth (1986

Sample size determination
The sample size required was determined, according to Thrusfield (2007), with defined precision of 5% and level of confidence of 95%.
where N is the required sample size, d is the desired absolute precision, Pexp is the expected prevalence, the previous prevalence in the area.
According to the formula, when there is no prior study in the area, it should be conducted that taking Pexp 0.5 for larger sample size, the calculated sample size was 384 lactating cows from the conveniently selected dairy farms in the study areas.

Study design
A cross-sectional study was conducted starting from October 2015 to June 2016.

Milk sample collection
Twenty-five dairy farms were selected randomly using list of farms in and around Adama as a sampling frame and lactating cows were selected randomly from the lists obtained from each selected farm.
Clinical mastitis was recognized by visual inspection and palpation of the udder for injuries, pain, heat and swelling. In addition, the milk of each quarter was withdrawn, confirmed the change of colour and consistency. A California mastitis test was performed. Mild circular movement was applied to the paddle. The positive samples indicate the gel formation in a few seconds. The results were qualified according to the gel formation and were classified as negative if there was no gel formation. The cows were considered positive if at least a quarter is positive per california mastitis test (CMT) (Radostits et al., 1994).
The milk samples were collected before milking, the teats were washed with tap water, dried and the teats were immersed in a cotton bar with 70% ethyl alcohol. After discarding the first three milking flows, the milk was recovered from the cows' aseptically (NMC, 1990

Isolation and identification of S. aureus
Final identification of staphylococci organisms and species assignment were done based on Gram staining, catalase test, sugar fermentation (Appendix B) and coagulase test (Mekonen, 2009).

Gram's staining
All cultures of suspected Staphylococcus were Gram stained and observed under an optical microscope to determine the Gram response, cell size, shape and arrangement. Gram-stained smears showing typical colonies of Gram positive cocci in irregular grape-like clusters are regarded as putative staphylococcal species (Quinn et al., 2002).

Catalase test
The pure culture was collected using a sterile loop and mixed with a drop of 3% H 2 0 2 on a clean glass slide. If the organism is positive, oxygen bubbles are released within a few seconds, while catalase negative isolates do not produce bubbles. Catalase positive cocci were considered as Staphylococcus (Quinn et al., 2002).

Oxidation and fermentation test
The oxidation and fermentation (OF) medium containing glucose is green. If the bacteria produce acid, the medium will turn yellow fermenting the glucose. Bacteria that can metabolize glucose under aerobic or anaerobic conditions are called facultative anaerobes, which are considered as Staphylococcus (Quinn et al., 1999).

Coagulase test
A test tube coagulase test was performed in a sterile test tube by adding 0.5 ml of selected S. aures isolates, grown in Tryptone Soy Broth (TSB) for 24 h at 37 • C, to 0.5 ml of citrated rabbit plasma, then gently swirling to mix and incubate the tube with a negative control tube containing a mixture of 0.5 ml sterile TSB and 0.5 ml rabbit plasma at 37 • C. Coagulation was assessed after the first 4 h of the test and the following 24 h of incubation. If any degree of solidification from loose clots to immovable solid clots was seen in the test tube, when the tube was inverted (tilted), the reaction was considered positive, and no degree of solidification were considered negative (Appendix C) (Quinn et al.,1999) Purple agar base

2.6
Data analysis prevalence. The reported prevalence was higher than the report of 36.67% by Hundera et al. (2005), 38.2% by Workneh et al. (2002) and 22.3% by Getahun et al. (2008). In this study, the general prevalence of clinical mastitis was less than that of subclinical mastitis. Similarly, Bekele et al. (2012) and Workneh et al. (2002) indicated the higher prevalence of subclinical mastitis than clinical mastitis. The variation of morbidity between the sub-clinical and clinical mastitis may be due to the fact that the cow's defence mechanism reduces the severity of the disease. In, Ethiopia, nonclinical mastitis has been ignored and strives are made to treat clinical cases (Kerro & Tareke, 2003). However, it was higher than the reports made by Hussein (1999) (10.6%). Relative prevalence of S. aureus might be related to ineffective udder and hand washing, the lack of hand washing, the use of dirty clothes for teat disinfection and milking materials disinfection. The high incidence rate of S. aureus could also be due to the wide spread of organisms in the mammary gland, allowing it to survive in the udder and establish chronic and low-level clinical infections (Radostits et al., 1994).
MRSA is recognized as one of the main causes of hospital infection and it is also responsible for a wide range of infections (Gould et al., 2012;Wang et al., 2012). The results of this study showed that 32.4% of the isolates were MRSA. This result is lower than those  (Mollering, 2012). As a result, penicillin and other beta-lactam antibiotics cannot be used to treat MRSA infection. The most important resistant mechanism for β-lactamase antibiotic is the production of beta-lactamase that inactivates β-lactams (e.g., amoxicillin) by hydrolysing the beta-lactam ring. In the present study, amoxicillin disc diffusion zone of the edge test was adopted to detect β-lactamase production, and most MRSA isolates were positive for β-lactamase production.
This is due to the indiscriminate use of antibiotics/antimicrobial agents for prevention and treatment purposes. In our study, most MRSA strains are multidrug resistant (Figure 1). These results are consistent with the overall findings of Kesah et al., 2003, that show MRSA strains are multidrug resistant (Peng et al., 2010;).

CONCLUSION AND RECOMMENDATIONS
Increasingly, MRSA has been isolated from bovine mastitis. Therefore, Therefore, the emergence of MRSA poses a serious threat to livestock and public health.

S. aureus
Based on the above concluding remarks, the following recommendations are proposed: • Antimicrobial susceptibility test should be carried out at a regular interval with a view to selecting appropriate therapy is needed. • Proper hygienic and improved managemental practices should be introduced at farm level.
• Creating public awareness about transmission, prevention and control of MRSA is paramount important.
• Future studies should focus on determining the antimicrobial resistance mechanisms MRSA isolates to fix the problem.

CONFLICT OF INTEREST
The authors declare no conflict of interest.

ETHIC STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to and the appropriate ethical review committee approval has been received.

DATA AVAILABILITY STATEMENT
The data supporting the present result will be available up on request from the first author.

PEER REVIEW
The peer review history for this article is available at https://publons.
Direction: Suspend 31.02 g in 1000 ml distilled water. Add 5-10 g of the carbohydrate to be tested. Boil to dissolve the medium completely.
Dispense in tubes as desired and sterilize by autoclaving at 15 lbs pressure (121 • C) for 15 min. Alternatively sterilize the basal medium prepared using 900 ml distilled water, and add 100 ml separately sterilized 5-10% solution of the desired carbohydrate to it.
Direction: Suspend 38 g of the powder in 1 L of distilled water.
Mix throughly, heat with frequent agitation and boil for 1 min to completely dissolve the powder; autoclave at 121 • C for 15 min.

VIII Tryptone soya broth (Oxoid, England)
4. Examine periodically for coagulation by gently tipping the tube after the first hour and once every hour thereafter until 4 h have elapsed. If no clot is observed at the end of this period, examine at 24 h. Avoid shaking or agitating the tube during reading. Doubtful or false-negative results may occur due to breakdown of the clot.
5. Record results: Positive for any degree of clotting-from a loose clot suspended in plasma to a solid clot that is immovable when the tube is inverted, and negative for no degree of clotting.