Isolation and Identification of Methicilin Resistant Staphylcoccus aureus (MRSA) from Bovine Mastitic Milk in and around Wolaita Sodo, Southern Ethiopia

Tessema3 and Ashenafi Kiros Wubshet1

Ethiopia

Email: Binimanu10@gmail.com

Staphylococcus aureus, Significant; Therapeutic

Open Access Journal of Veterinary Science & Research

Abbrevations: AGR: Accessory gene regulator; CLSI: Clinical Laboratory Standard Institute; CMT California Mastitis Test; CNS: Coagulase Negative Staphylococci; DNA: Deoxyribo Nucleic Acid; EFSA European Food Safety Authority; MAR: Multiple Antibiotic Resistant; MIC: Minimum Inhibitory Concentration; MRSA: Methicilin Resistant Staphylococcus aureus; MSCRAMMs: Microbial Surface Components Recognizing Adhesive Matrix Molecules; NCCLS: National Committee for Clinical Laboratory Standards; NMC: National Mastitis Committee; OR: Odds Ratio; PBP: Penicillin Binding Protein: PCR: Polymerase Chain Reaction; SCC: Staphylococcus Chromosome Cassette; SE Staphylococcus Enterotoxin; SXT: Sulphamethoxazole-Trimethoprim

Introduction

Staphylococci are Gram-positive bacteria, with diameters of 0.5 – 1.5 μm and characterized by individual cocci, which divide in more than one plane to form grape- like clusters. The staphylococci are non-motile, non-spore forming facultative anaerobes that grow by aerobic respiration or by fermentation. Staphylococci are tolerant to high concentrations of salt and show resistance to heat. Pathogenic staphylococci are commonly identified by their ability to produce coagulase, and thus clot blood .This distinguishes the coagulase positive strains, S. aureus, S_. intermedius_ and S. hyicus from the other staphylococcal species such as S. epidermidis, that are coagulase-negative [1]. S. aureus is both commensal and pathogen. It is found as a commensal associated with skin, skin glands and mucous membranes. S. aureus affects skin, soft tissues, bloodstream and lower respiratory tract. It also causes severe deep-seated infections like endocarditis and osteolmyelitis [2]. S. aureus plays its most significant animal pathogenic role as cause of intra-mammary infections in cattle and small ruminants leading to considerable economic losses in cattle farming. The pathogen is frequent causative agent of clinical or subclinical mastitis in cattle [3]. Presence of S. aureus on the skin and mucosae of food producing animals, such as ruminants, and the frequent association of the pathogen with mastitis, often leads to contamination of milk [4]. Contamination of milk can also occur from environmental sources during handling and processing [5]. Milk is a good substrate for S. aureus growth and dairy products are common sources of staphylococcal food-poisoning [6]. S. aureus also causes severe animal diseases, such as suppurative disease, arthritis and urinary tract infections [7]. In recent years, there has been increased concern about antibiotic resistant strains of S. aureus. Development of resistance has been attributed to the extensive therapeutic use of antimicrobials or to their administration as growth promoters in food animal production [8]. Isolates of S. aureus are frequently resistant to methicillin and essentially all other β-lactam antibiotics. The resistance to methicillin in staphylococci is mediated by the mecA gene that encodes a modified penicillin-binding protein (PBP), the PBP2a or 2’, which shows reduced affinity to penicillins, such as methicillin and oxacillin and for all other beta-lactam antibiotics. An organism with this type of resistance is referred to as methicillin-resistant S. aureus (MRSA). The mecA gene resides on a staphylococcal chromosomal cassette (SCCmec) [9]. MRSA was initially reported as a nosocomial pathogen in human hospitals (hospital-associated MRSA) and was isolated from patients with compromised immune systems undergoing medical procedures. MRSA accounts for 30 to 40% of all hospital-acquired infections and for 40% to 70% of S. aureus infections in intensive care units [10]. In the 1990s, a major change in the epidemiology of MRSA has been observed, with the appearance of cases affecting people with no epidemiological connection to hospitals; strains that cause such infections are referred to as community-acquired or community associated MRSA [11]. Until recently, such strains were susceptible to many antibiotics other than β-lactams; however, resistance seems to be increasing, and multiple antibiotic resistant strains have started to emerge [12]. There is now increasing concern about the public health impact of MRSA associated with food producing animals, because MRSA and, consequently, their resistance genes can spread from animals to humans by direct contact or through the food chain [13]. MRSA strains have been isolated in many countries from cows’ or small ruminants’ milk and various dairy products [14]. There are also some studies on MRSA in some part of Ethiopia such as in Hawasa by Daka D, et al. [15], in Adama by Abera M, et al. [16], in and around Addis Ababa [17]. Concerning the study area (Wolaita Sodo), MRSA is poorly studied. Knowledge of MRSA is necessary to make decisions regarding antibiotic treatment and prerequisite for establishing control strategies. Without such local knowledge however, treatment measures fail and designing control programmes may be at best only guess- work and identification of MRSA data is needed to get some insight on their overall negative economic implications. Therefore, the present study was contemplated with the following. Thus, the objectives of this study were

• Isolation and identification of Staphylococcus aureus from mastitic cow’s milk.
• To estimate the occurrence of MRSA strains using antibiotic sensitivity test.

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• To asses associated risk factors leading to MRSA

mastitis infection.

Discussion

The study was conducted on bovine mastitis in Wolaita Sodo and its surroundings to determine the occurrence and major risk factors associated with MRSA in mastitis infection. The result revealed an overall occurrence of mastitis 40.9% and 21% was quarter level in the study area. This result agrees with the previous report by Kerro D [26] who recorded a finding of 40% in cows and 19 % in quarters in Southern Ethiopia. This report was also in agreement with the assertion by Radostits M, et al. [27] that, in most countries and irrespective of the cause, the prevalence of mastitis is about 50% in cows and 25% in quarters. The infection rate in cows was similar to the findings of [28], who found a prevalence of 45.8% in Sudan. The current finding of the study is higher than that of [29] and [30], who reported an overall prevalence of 28.8% at Bahir Dar and 29.5% around Wolaita Sodo, respectively. On the other hand, it is comparably low when compared with the work of [31] and [32] who reported a prevalence of 52.78% in and around Sebeta and 64.4% in Asella, respectively. Since mastitis is a complex disease and the difference in results could be due to difference in management system of the farm, the breeds of cattle considered and the geographical locations of the studies.

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The occurrence of clinical mastitis recorded in the present study is 4.66 % and that of subclinical mastitis 36.18%. The result is in agreement with 36.7% sub clinical mastitis prevalence reported by [16]. In the current study the rate of sub-clinical mastitis (36.18%) was higher than that of the clinical mastitis (4.66 %) as (36.7 versus 10 %) was reported by [16] in Adama town. In most reports including the present study, clinical mastitis is far lower than subclinical mastitis. This could be attributed to little attention given to subclinical mastitis, as the infected animal shows no obvious symptoms and secrets apparently normal milk and farmers, especially small holders, are not well informed about invisible loss from sub clinical mastitis. In Ethiopia, the subclinical forms of mastitis received little attention and efforts have been concentrated on the treatment of clinical cases [33]. The increasing occurrence of mastitis with increasing age was in agreement with the findings by Kerro D [26] who found that, the risk of clinical and subclinical mastitis increase significantly with the advancing age of the cow. This might be due to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without mastitis control program [34]. The study showed that breed was not significantly influenced the occurrence of mastitis [31]. In and around Sebeta showed that breed significantly influenced the occurrence of mastitis. Mastitis occurrence among breeds might reflect the differences in management rather than a true genetic difference [34]. In this study mastitis occurrence was associated with parity of the animal evaluated and found statistically significant. The increased occurrence of mastitis with parity in the current study is comparable with the previous reports of Mulugeta Y [30] in Wolaita Sodo town, Mekibib B, et al. [35] in Holota town and Haftu R, et al. [36] in northern Ethiopia. The association might be due to the increased opportunity of infection with time and the prolonged duration of infection, especially in a herd without mastitis control program [34]. The observed higher occurrence of mastitis during early lactation as compared to mid and late lactation stages was significant. The finding of higher infection in cows in early lactation stage followed by late and medium lactation stages in the study concurs with previous reports of Mulugeta Y [30], Biffa D, et al. [37] and Tamirat T [38] In cows most new infections occur during the early part of the dry period and in the first two months of lactation [34]. This may be due to an absence of dry period therapy and birth related influences. Radostits M, et al. [27] suggested that, the mammary gland is more Biniam T, et al. Isolation and Identification of Methicilin Resistant Staphylcoccus Aureus from Bovine Mastitic Milk in and around Wolaita Sodo, Southern Ethiopia. J Vet Sci Res 2017, 2(3): 000136.

susceptible to new infection during the early and late dry period, which may be due to the absence of udder washing and teat dipping, which in turn may have increased the presence of potential pathogens on the skin of the teat. Moreover, during a dry period due to the low bactericidal and bacteriostatic qualities of milk, the pathogens can easily penetrate into the teat canal and multiply; this can be carried over into the post parturient period and ultimately develop into mastitis. In agreement with Abera M, et al. [16] in Adama town and Fekadu B, et al. [39] in southern Ethiopia, the finding of a high prevalence of mastitis in farms with muddy floors when compared with concrete floor types shows the occurrence of mastitis is significantly associated with the housing (bedding) type or condition of the farm. This is due to association with poor sanitation and cows which were maintained in dirty and muddy common barns with bedding materials that favor the proliferation and transmission of mastitis pathogens. Occurrence of mastitis was significantly associated with milking hygienic practice. Cows at farms with poor milking hygiene standard are severely affected than those with good milking hygiene practices Mulugeta Y, et al. [30], Sori H, et al. [31] and Lakew M [32]; This might be due to absence of udder washing, milking of cows with common milkers’ and using of common udder cloths, which could be vectors of spread especially for contagious mastitis [40]. The quarter level mastitis occurrence recorded in the current study is comparable with the finding of 19% in quarters by Kerro D, et al. [26]. The hind quarters were affected higher than the front quarters. This is due to the fact that the hind quarters are highly predisposed for contamination with dirt. In addition to this, large amount of milk is produced from hind quarters and as a result the pressure on the teat canal forces the canals to be opened widely which allows entrance of microbes. The observation of blind quarters in this study might be an indication of a serious mastitis problem on the farms and of the absence of culling that should have served to remove a source of mammary pathogens for the cows. Microbiological examination of milk from lactating dairy cows shows the presence for S. aureus in the present study. The present finding is in line with the findings of Abebe M, et al. [17] who reported 15.5% at Addis Ababa. It also shows similarity with the findings observed in Egypt (17.2%) [41]. This is a low result when it is compared with that of Bedad A, et al. [42], Workeneh S, et al. [43] and Kerro D, et al. [26] who reported 39.1%, 39.2% and 40.3% S. aureus isolates at Assela, Addis Ababa and Southern Ethiopia, respectively. The possible Copyright© Biniam T, et al.

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explanation for the variation might be that S. aureus is a contagious pathogen transmitted from one cow to another or individual by contact with animals during unhygienic milking procedures [44]. The presence of isolates S. aureu in subclinical mastitis was significantly higher than that of clinical mastitis and this is due to S. aureus has adapted to survive in the udder and establish chronic and subclinical infections. From there it shed into the milk, which serves as a source of infection for healthy cows during the milking process [40]. The present study showed the resistance of S. aureus to penicillin G, cefoxitin, amoxicillin-clavulinic acid, tetracycline, streptomycin, chloramphenicol, sulphamethoxazole-trimethoprim and vancomycin. This is in accordance with the findings of Abebe M, et al. [17] who reported resistance of S. aureus to penicillin (94%), tetracyclin (73.8%) around Addis Ababa. In line with the current finding, 94.4% and 58.3% resistance were also recorded around Adama [16] to penicillin and sulphamethoxazole trimethoprim respectively. Moreover, the present study has demonstrated the existence of alarming level of resistance of S. aureus to commonly used antimicrobials (pencillin G and tetracycline) in the study farms for dairy cows. The results were in accordance with reports from earlier studies in other countries Jakee J, et al. [45], Edward M, et al. [46] and Gentilini E, et al. [47] suggesting a possible development of resistance from prolonged and indiscriminate usage of some antimicrobials. Absolute resistance to Penicillin G must be of a great concern; since this antibiotic represents the main antibiotic group recommended for Staphylococcal mastitis treatment and regular use of antibiotics for the treatment of cows may result in the spread of resistant strains. Antibiotic resistance is carried on plasmids and transposons which can pass from one Staphylococcal species to another [48]. This is not surprising because penicillin G and tetracycline are the most commonly used antimicrobials for the treatment of infection or mastitis in veterinary practice in Ethiopia. The resistance of S. aureus to penicillin and cefoxitin may be attributed to the production of beta lactamase, an enzyme that inactivates penicillin and closely related antibiotics. It is believed that around 50% of mastitis causing S. aureus strains produces beta-lactamase [49]. Similar with the finding of Abebe M, et al. [17] in Adama town, lower resistance was recorded for chloramphenicol and sulphamethoxazole trimethoprim. The reason why chloramphenicol and sulphamethoxazole-trimethoprim were less resistant might be that they are not frequently used in the study area in veterinary services, and perhaps Biniam T, et al. Isolation and Identification of Methicilin Resistant Staphylcoccus Aureus from Bovine Mastitic Milk in and around Wolaita Sodo, Southern Ethiopia. J Vet Sci Res 2017, 2(3): 000136.

in human medicine. Similar suggestion was given by Jaims E [50] that the development of antimicrobial resistance is nearly always as a result of repeated therapeutic and/or indiscriminate use of them. The resistance of S. aureus isolates to beta-lactams such as penicillin, cefoxitin, ampicillin and tetracycline was evident. High percentage of S. aureus was resistant to penicillin, cefoxitin, ampicillin, tetracycline and streptomycine. In agreement with the finding of by Daka D, et al. [15], the study showed cefoxitin resistant isolates was obtained from the milk in this study area. All cefoxitin resistant S. aureus were also resistanct to penicillin G. Out of the 28 cefoxitin resistant S. aureus isolates, 85.71% of were also resistant to amoxicillin. This is an indicator of MRSA [15]. MRSA strains have developed multidrug resistance worldwide with broad diversity in prevalence rate in different regions [8]. In the present observation, S. aureus isolates showed multidrug resistance primarily to penicillin G, cefoxitin, tetracycline and streptomycin because of resistance to β-lactams and frequent usage of the drugs. This is comparable with findings of Sharma D, et al. [51] who reported a 70% prevalence of multidrug resistant S. aureus from raw milk of dairy cattle in India. The study shows most of the MRSA isolates were from previously treated animals. This showed a strong association between previous treatment and occurrence of MRSA. A causal relationship between the use of antimicrobial drugs and MRSA has been demonstrated in LA-MRSA and often co-resistant to several other antimicrobial agents [52]. The association between MRSA and age of cows was significant. All of the isolated MRSA were from adult and old age category. The possible explanation for this may be as the age of cow’s advances, the chance of having treatment with antibiotic increases. Though vancomycin is the drug of choice for the treatment of MRSA, vancomycin resistance recorded in the study is in agreement with the finding of 57.1% by Daka D, et al. [15] in Hawasa town. It has also been documented that MRSA isolates that are resistant to beta- lactam antibiotics may develop induced resistance to Vancomycin [53].

Conclusion and Recommendations

Different risk factors are associated with mastitis in the study area. Among these, miliking hygiene, floor type and age of the animal were critical. Mastitis caused by S. aureus is one of the major problems of dairy cows in milk Copyright© Biniam T, et al.

production. It was found that the majority of the tested isolates were resistant to the various antimicrobial agents especially penicillin G, cefoxitin, tetracycline, streptomycin and amoxicillin. It was also observed that large proportions of the isolates were susceptible to sulphamethoxazole-trimethoprim and chloramphenicol. In the present observation, S. aureus isolates showed multidrug resistance primarily to penicillin G, cefoxitin, tetracycline and streptomycin because of resistance to β-lactams and frequent use. The findings suggest that multi drug resistances S. aureus are present in higher concentrations in dairy farms of the study area which indicated that MRSA spread within the farm. The possible explanations for the high record of multi drug resistant S. aureus in dairy farms may be due to the unrestrictive and uncontrolled use of antibiotics in dairy farms.

Based on the above conclusion the following points are forwarded

• Staphylococcus aureus mastitis control strategy should be initiated and promoted in the study area;
• Awareness should be created among dairy farm owners and dairy workers on the effect of MRSA;
• Veterinarians should be aware of the concerns regarding MRSA and should educate the owners/handlers the risks;
• There should be regular antimicrobial sensitivity testing to select effective antibiotics and also help to reduce the problems of drug resistance development towards commonly used antibiotics;
• Risk factor controlling mechanism should be implemented
• Furthermore, impacts and dynamics of genetic antibiotic determinants should also be investigated using molecular methods.

Acknowledgement

I would like to express my deepest gratitude to my academic advisors Dr. Biruk Tesfaye (DVM, MSc, Assist. Prof.) and Dr. Tesfaye Sisay (DVM, MSc, PhD) for their overall guidance and advices, sharing his knowledge from the beginning to the end of my thesis paper.

My great thanks go to my friend Dr. Ashenafi Kiros, for his interesting supports and advices.

I am highly indebted to members of Sodo Regional Veterinary Laboratory, especially Dr. Ergate, Ato Endashaw and W/ro Muluemebete for their kind reception, preparing equipments and materials for the project work and good willing to use their laboratory with unfavorable condition.

Much of the acknowledgement goes to my staff members and veterinary medicine students of Wolaita Sodo University for their kind approach and cooperative where ever and whenever I was in need of their Support.