Isolation, Identification, and Antimicrobial Susceptibility Profiles of Staphylococcus aureus from Clinical Mastitis in Sebeta Town Dairy Farms

A cross-sectional study was carried out in and around Sebeta town dairy farms, Finfinne special zone, Ethiopia, from December 2019 to May 2020 to isolate, identify, and test antimicrobial susceptibility profile of Staphylococcus aureus from clinical mastitis. A total of 116 milk samples were purposively collected from 57 lactating cows with clinical mastitis. Isolation and identification of Staphylococcus aureus were carried out by using primary and secondary biochemical tests. Besides, Biolog was used for microbial identification systems. To know if the isolates develop resistance to antibiotics, the antimicrobial susceptibility test (ATS) was performed on Mueller-Hinton agar by the disk diffusion method. From a total of 57 lactating cows and 116 teat quarters examined, 21.05% (12/57) and 15.52% (18/116) were positive for S. aureus from clinical mastitis, respectively. From a total of 116 milk samples collected, 15.52% (18/116) Staphylococcus aureus were isolated, and from 11 farms surveyed, about 72.72% (8/11) overall farm prevalence of clinical mastitis due to S. aureus was recorded. All the 18 Staphylococcus aureus isolates were found susceptible to sulphamethoxazole/trimethoprim, erythromycin, gentamicin, ciprofloxacin, and chloramphenicol. However, high level of resistance was observed to common drugs such as penicillin (88.89%, 16/18) and tetracycline (61.11%, 11/18). The observed high level of resistance to penicillin and tetracycline also indicates the need to visit our treatment guidelines for mastitis caused by Staphylococcus aureus. Therefore, improved management and early treatment of the cases with drug of choice after the antimicrobial susceptibility test for each specific case can reduce chance of further development of resistance and are imperative to tackle clinical mastitis occurring at Sebeta and other similar farms in Ethiopia.


Introduction
In Ethiopia, the number of intensive and semi-intensive dairy farms has been increasing from time to time due to urbanization, increased human population, and income growth. However, the management practices of these dairy farms remained traditional. In such dairy farms, mastitis is the predominant disease. Mastitis is the inflammation of the mammary gland mainly due to a bacterial infection, and it is characterized by a variety of local and systemic symptoms. Mastitis could be prevented by implementing proper animal health management systems. However, most of the emerging dairy farms in Ethiopia lack optimum management practices and are predisposed to mastitis [1].
Milk is a major component in the human diet all over the world, but it also serves as a good medium for the growth of many microorganisms, especially pathogenic bacteria. However, health risk to consumers can be associated with milk, due to the presence of zoonotic pathogens and antimicrobial drug residues [2]. e quality of milk may be lowered by a number of factors such as adulteration, contamination during and after milking, and the presence of udder infections [3]. Pathogenic organisms in milk can be derived from the cow itself, the human hand, or the environment [2]. Staphylococcus aureus is a versatile pathogen of humans and animals that causes a wide variety of diseases [4]. e bacterium is a colonizer of the skin and mucosae from which it can invade multiple organs. In livestock, S. aureus is an important cause of mastitis, skin and soft tissue infections (SSTI), and to a lesser extent infection of the locomotory system. Surgical site infections (SSI) in which S. aureus is isolated have been increasingly reported in small companion animals and horses [5]. S. aureus is the most prevalent and economically significant pathogen causing inflammatory infections in dairy ruminants [6]. Approximately 30%-40% of all mastitis cases are associated with the bacterium [7].
Staphylococcus aureus can get access to milk either by direct excretion from udders with clinical or subclinical staphylococcal mastitis or by contamination from the environment during handling of raw milk [8,9]. When the udder is infected, S. aureus may be excreted through milk in variable amounts up to 108 CFU/mL [7]. e emergence of antibiotic-resistant bacteria has become a global public health concern affecting human and veterinary medicine [10]. e administration of antibiotics for the feeding of animals for curative purposes or as growth promoters may be a major factor in the selection of antimicrobial-resistant bacteria [11]. e indiscriminate use of antibiotics for the treatment of animal and human diseases as well as preservatives for milk has led to the development of multiple antibiotic resistances, thereby rendering the antibiotic treatment ineffective. S. aureus has been reported to frequently show multiple antimicrobial resistance patterns [12]. ere is no recent study which shows the current prevalence and antimicrobial resistance of S. aureus originated from cows' milk in Sebeta town, central area of Ethiopia. In addition, previous studies conducted in the area were also limited in number. us, this study was aimed to isolate, identify, and determine the antimicrobial susceptibility profiles of Staphylococcus aureus from clinical mastitis in the study area and finally recommend farm owners to choose genuine antibiotics which can cure S. aureus caused clinical mastitis.

Study Area.
e study was conducted in and around Sebeta town, Oromia special zone around Finfinne, Ethiopia. Sebeta is located about 25 km south of Addis Ababa at 8°55′N and 38°37′E and an altitude of 2,356 meters above sea level ( Figure 1). e climate is good with an average annual temperature of 17.4°C and average annual rainfall of 1073 mm.

Study Animals.
is study was conducted in bovine. Lactating cows of both breeds, crossbreed (Holstein-Friesian-Zebu crosses) and local Zebu breeds, were included during the study period. e study population comprises lactating dairy cows that are managed under semi-intensive and intensive farming systems. A total of 57 lactating dairy cows in and around Sebeta town were sampled to isolate and identify S. aureus causing clinical mastitis in the area.

Study Design and Sampling.
A cross-sectional study was conducted from December 2019 to May 2020 to isolate, identify, and determine antimicrobial susceptibility profiles of Staphylococcus aureus in the study area. Eleven (11) dairy farms, 1 big, 4 medium, and 6 small size farms, were randomly selected from Sebeta town. A farm is considered small if the number of animals is <11, medium (11)(12)(13)(14)(15)(16)(17)(18)(19)(20), and large (>21). After proper clinical examination of the udder of the cows on the selected farms, cows showing only clinical signs of mastitis were purposively sampled as the aim were to isolate and identify S. aureus and to know its status on the occurrence of clinical mastitis. As a result, 57 cows were sampled and 116 milk samples from 116 quarters (teats) were collected. However, the remaining 112 quarters (228-116 � 112) were blind and some of them had puss and bloody milk during the sampling time.

Clinical Inspection of the Udder.
To identify a mastitis animal, the udders of the cows were examined by visual inspection and palpation for the presence of any lesion, pain, heat, and swelling. In addition, milk from each quarter was withdrawn and checked for any change in colour and consistency [13]. ese clinical mastitis cases were diagnosed on the basis of manifestation of visible signs such as inflammation of the udder characterized by heat and swelling with pain upon palpation and/or gross changes in milk, whereas clinical mastitis was diagnosed when misshaped, atrophied, hard, and fibrotic quarters were examined [14]. Accordingly, only cow and udder quarters with clinical cases were selected and from which milk was collected for laboratory testing.

Sample Collection and
Handling. Samples were collected aseptically as described in [15]. ey were collected before milking. Milk collection process was performed after cleaning the teats, initial stream of milk discarded, and teat tips scrubbed with cotton balls moistened with 75% alcohol. Samples were taken in sterile glass vials and closed with screw caps. e vials were marked with a permanent marker, so that the marking was easy to read when the vials were placed in racks. 10 ml of milk was collected into a horizontally held vial after the first streams of milk were discarded. After collection, the sample was placed in an icebox with +4°C and transported to the National Animal Health Diagnostic and Investigation Center (NAHDIC) for processing and stored at 4°C until inoculation for S. aureus isolation and identification was started.
2.6. Bacterial Identification. 1 ml of collected milk samples was pre-enriched in 9 ml of brain heart infusion broth incubated at 37°C for 24 hr. A loopful of the incubated culture was streaked onto mannitol salt agar (OXOID) selective and differential media and then incubated aerobically at 37°C for 24 hrs. S. aureus was identified according to their Gram reaction, morphology, hemolysis, and catalase test. Mannitol fermenting colonies were plated on 1% maltose purple agar base and tested for coagulase (4 h), pigment production (golden yellow) (B in Figure 2), and maltose fermentation (D in Figure 2). en, OmniLog/Biolog (fully automated coated microplate based bacterial identification system) using GEN III microplate with protocol A method was used to further confirm the species of suspected colonies.

Antimicrobial Susceptibility Test.
e antimicrobial susceptibility test (AST) was performed on Mueller-Hinton agar by the disk diffusion method (Figure 3(b)) [16]. ree to five isolated colonies of isolated S. aureus were transferred to 5 ml of 0.85% saline water. e turbidity was measured using densitometry and adjusted to 0.5 McFarland (Figure 3(a)). After measuring the turbidity, a sterile cotton swab was dipped into the suspension and then Mueller-Hinton agar plate was inoculated by rotating 60°. Antimicrobial discs were applied to the media using a disc dispenser and then incubated for 16-18 hrs.

Data Management and Analysis.
Microsoft Excel was used for data management and computation of descriptive statistics. Data were coded and entered on MS Excel spreadsheet. Percentage was calculated by dividing the total number of positive isolates per total number of cows and quarters examined.

Bacterial Isolation and Identification.
From a total of 57 lactating cows and 116 teat quarters examined, 21.05% (12/ 57) and 15.52% (18/116) were positive for S. aureus caused clinical mastitis, respectively (Table 1). From a total of 116 milk samples collected, 15.52% (18/116) Staphylococcus aureus were isolated from lactating cows having a clinical form of mastitis revealing active cases of mastitis with visible signs of inflammation on the udder and changes in milk quality.
During this study, a total of 11 farms were surveyed and about 8 farms were found positive with S. aureus (Table 2). Hence, the overall herd prevalence of clinical mastitis due to S. aureus was recorded to be 72.72%.
Out of the resistant S. aureus isolates, 2 (11.11%) were found to be multidrug resistant against 3 antibiotic discs primarily to penicillin G, tetracycline, cefoxitin, and amoxicillin + clavulanic acid and 8 (44.44%) isolates were resistant to penicillin G and tetracycline.  In the current study, it was noticed that 15.52% of S. aureus were isolated and identified from quarters with clinical mastitis. Similar results were reported by Esron et al. [4], who reported 15.5% S. aureus prevalence of clinical mastitis and 13.3% S. aureus quarter isolates in Addis Ababa dairy farms. e finding of this study was higher than that of previous reports. is higher prevalence might be due to the poor management practiced in the farms. During sample collection time, we observed that they do not follow strict farm biosecurity, proper cleaning of the floor, and washing of animal bodies. Moreover, they do not also exercise the proper milking order which is milking healthy cows (cows with no mastitis), then followed by milking of healthy quarters, and then at last, milking quarters and cows having clinical mastitis.

Discussion
However, a higher detection rate was reported by other authors [19,20] who isolated S. aureus with an incidence of 73.3% and 40.5% at quarter and animal level, respectively, from Addis Ababa dairy farms. High prevalence of S. aureus is due to its contagious nature and has been adapted to survive in the udder and establish chronic and subclinical infections. From the udder, there is shed of the milk, which serves as a source of infection for healthy cows during the milking process [21].
It is difficult to eliminate the bacteria from the mammary gland due to the very low rate of self-cure and a number of factors affect the rate of cure after treatment, which is in general low [22]. e herd/farm prevalence was also recorded to be high (72.72%) that means from the eleven (11) farms sampled, eight (8) of them were found positive for S. aureus. is is also due to the poor management practiced in the farms and due to the treatment of clinical mastitis with antibiotics (penicillin G and tetracycline) which have developed resistance to S. aureus.

Veterinary Medicine International
Similarly, the current study showed a very high level of resistance at 88.89% and 61.11% for Staphylococcus aureus isolates to penicillin G and tetracycline, respectively (Table 3). is high S. aureus resistance to penicillin G and tetracycline antibiotics might be due the fact that animal health experts in the farm/area do not have other drugs of choice for the treatment of mastitis, instead, they repeatedly use penicillin G and tetracycline during treatment. e finding of this study is similar to [23], who reported 96.7% resistance, and [24], who reported 87.2% resistance. is high level of resistance was due to the isolates producing a penicillinase enzyme (a type of β-lactamase) that hydrolyses the beta-lactam ring of penicillin [25].

Conclusion and Recommendations
e present study showed the occurrence of higher S. aureus caused clinical mastitis at animal level and very high prevalence at farm/herd levels considering mastitis can be also occurred in subclinical form in farms. Isolates of S. aureus from clinical mastitis demonstrated the existence of alarming levels of resistance to commonly used antimicrobial agents such as penicillin G and tetracycline suggesting a possible development of resistance to prolonged and indiscriminate usage of those antibiotics. On the other hand, S. aureus was also found susceptible to antibiotics such as trimethoprim, erythromycin, gentamicin, ciprofloxacin, and chloramphenicol, suggesting that these antibiotics have not been commonly used as the treatment of choice for mastitis in farms.
On the basis of the above findings, the following recommendations are forwarded: (i) Farm management such as farm biosecurity, good farm hygiene, and milking order should be practiced and improved to prevent and control S. aureus mastitis and to maximize the milk production of the farm (ii) As a short-term solution, cows detected positive for S. aureus with clinical mastitis should be treated with erythromycin or gentamicin or ciprofloxacin antibiotics to cure from mastitis and minimize the risk of transmission to other healthy cows (iii) Implement a systemic application of an in vitro antibiotic susceptibility test prior to the use of antibiotics in both treatment and prevention of intramammary infections, which can be considered as a long-term solution to control mastitis (iv) Regular screening and strip cup examination of the cows and cultural and other bacteriological examination of infected quarters should be conducted so that proper therapy is administered.
Data Availability e data sets used and analyzed during the current study are available from the corresponding author upon reasonable request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.