Emergence of Pathogenic Strains of Staphylococcus aureus in Goat Milk and Their Comparative Response to Antibiotics

Iqra Muzammil1, Muhammad Ijaz Saleem1, Amjad Islam Aqib2,*, Ambreen Ashar3, Syed Ashar Mahfooz1, Sajjad ur Rahman4, Muhammad Shoaib4, Muhammad Aamir Naseer1, Imran Khan Sohrani1, Javeed Ahmad1, Razaullah Saqi1, Fizzah Laeeq Lodhi1 and Qaisar Tanveer5 1Department of Clinical Medicine and Surgery, Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000 2Department of Medicine, Faculty of Veterinary Science, Cholistan University of Veterinary and Animal Sciences, Bahawalpur-63100 3Government College for Women University, Faisalabad-38000 4Institute of Microbiology, Faculty of Veterinary Science, University of Agriculture, Faisalabad-38000 5Institute of Pharmacy, Physiology and Pharmacology, University of Agriculture, Faisalabad-38000 Article Information Received 24 October 2019 Revised 22 May 2020 Accepted 20 August 2020 Available online 02 October 2020


INTRODUCTION
L ivestock plays an important role in the agriculture sector of Pakistan. The total goat population of Pakistan is up to 74.1 million to produce 0.915 million tons of milk and 0.717 million tons of mutton annually (Anonymous, 2018). Milk production in goats is an active and emergent business in harsh climate areas where large ruminants cannot be reared or are difficult to rear O n l i n e F i r s t A r t i c l e . Sub-clinical mastitis has occurrence of 45% in goats in Punjab whereas 53.3% in Khyber-Pakhtunkhwa (KPK), Pakistan (Najeeb et al., 2013;Ali et al., 2010). Subclinical mastitis in goats is predominantly caused by transmissible bacteria e.g. Staphylococcus spp., Streptococcus spp., Pasteurella spp. and E. coli (Persson and Olofsson, 2011;Contreras et al., 2007). Staphylococcus aureus is the major causative agent where its frequency of isolation and identification vary from 4-40% of the entire isolated pathogens (Leitner et al., 2007). Antimicrobial resistance is reported in S. aureus probably due to excessive administration of antimicrobials (overprescription, suboptimal termination of treatment regimen and/or insufficient dose administration of antimicrobials) resulting in lateral gene transfer (transformation, transduction and conjugation) of DNA from resistant strain for survivability (Castro-Sánchez et al., 2016). S. aureus can produce biofilm which act as a protective layer for the pathogen and provides continuous persistence via development of resistant genes e.g. mecA, vanA, icaA, icaB etc. (Jyothi et al., 2018). Production ability of coagulase enzyme is considered a significant phenotypic determinant in S. aureus linked with pathogenicity (Moreillon et al., 1995). Multiple drug resistance (MDR) is one of additional challenges in bacterial mastitis (Hameed et al., 2007). In 1972, first MRSA was isolated from dairy mastitic milk (Devriese et al., 1972). MRSA is now becoming major bacterial etiology of mastitis in addition to its isolation from vaginal and nasal swabs of animals (Cortimiglia et al., 2015).
Treatment with broad spectrum antibiotics along with anti-inflammatory drugs is used to treat mastitis in goats. Mechanism of development of drug resistance in bacteria associated with goat mastitis is very important to understand transmission frequency, better management strategies and developing valuable remedial interference (Aqib et al., 2018a;Merz et al., 2016). Therefore, epidemiological studies of pathogenic strains of S. aureus along with their response to antibiotics are necessary for prevention and treatment protocols. Subclinical mastitis in goats remained as neglected issue despite of its increasing prevalence and antimicrobial resistance of bacterial etiologies. Current study was thus designed to investigate the occurrence of different pathogenic strains of S. aureus, associated risk factors, and in-vitro antibiogram of biofilm positive and biofilm negative S. aureus in mastitic goats from Faisalabad, Pakistan.

Sampling plan and screening for subclinical mastitis
Faisalabad is the second biggest city of province Punjab and the third most populated city of Pakistan having a total area of 5,856km². Purposive sampling technique (Thrusfield, 2007) (Cengiz et al., 2015).

Risk factor analysis
A questionnaire comprising information like age of animal, type of housing, type of drainage system, type of floor, condition of floor, farm hygiene, source of drinking water, feeding system, vaccination against diseases, deworming, mastitis control program, parity number, stage of lactation, body condition score, milk consistency, milk yield, and teat injury was filled at the time of sampling to assess risk factors associated with mastitis. The risk factors were assumed based on the previous studies conducted by Amin et al. (2011) and Megersa et al. (2010).

Identification of pathogenic strains of S. aureus
SFMT positive samples were cultured on blood agar at 37°C for 24 h. Characteristic pinpoint colonies were further cultured on Mannitol Salt Agar, selective and differential medium for S. aureus, following the same incubation conditions. Series of biochemical tests were performed following guidelines of Bergey's Manual of Determinative Bacteriology (Bergey and Holt, 1994).
Isolates were identified for their expression as α, β and γ haemolysis on blood agar by inoculating S. aureus on blood agar at 37°C for 24 h. Methicillin resistant S. aureus were identified by their resistance against oxacillin disc following standard protocol described in clinical and laboratory standard institute. Biofilm was identified by Congo Red Agar (CRA) method, a previously established method (Freeman et al., 1989). For biofilm identification, fresh culture of S. aureus was grown on CRA and incubated for 24 h at 37°C. After incubation, colour of colonies indicated strength of biofilm i.e. pinkish red coloniesno biofilm production; slight blackish -weak production; black sheeting -moderate production; and jet black dry O n l i n e

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sheeting -strong biofilm production (França et al., 2012;Mathur et al., 2006).
In-vitro drug response against bpSA and nbpSA In-vitro drug response was evaluated using the Kirby Bauer disc diffusion test. The positive isolates were subjected to testing against various antibiotics such as vancomycin (30µg), chloramphenicol (10µg), oxytetracycline (30µg), trimethoprim+sulphamethoxazole (25µg), gentamicin (10µg), linezolid (30µg), amoxicillinclavulanic acid (20µg), amoxicillin (10µg), and oxacillin (1µg). Fresh cultures adjusted at 1.5×10 8 CFU were swabbed on Muller Hinton Agar whereas antibiotic discs were aseptically placed at equal distances from each other following the guidelines of CLSI (2015). Incubation was given at 37°C for 18-20 h and zone of inhibitions were measured by Vernier Callipers in millimetres and compared with provided standards.

Statistical analysis
The obtained data were analysed by descriptive statistics for occurrence of S. aureus and antibacterial activity of antibiotics whereas risk factor analysis was assessed by odd's ratio at 5% probability using IBM SPSS (version 20).

Risk factor analysis
The findings of the current study presented type of drainage system, type of feeding, vaccination against diseases, and teats injury as potential risk factors (p< 0.05) of subclinical mastitis. Poor type of drainage system (p= 0.0002) and stall feeding (p< 0.0001) showed higher odds of getting mastitis compared to partially controlled drainage system (p= 0.0018) and mixed feeding (p= 0.0572), respectively. The risk factor analysis revealed backyard type of housing showing higher odds of getting mastitis (p= 0.0843) as compared to open type of housing (p= 0.8289). Similar findings were found in case of earthen type of floor (p= 0.0996) as compared to brick floor (p= 0.9393). Mid stage of lactation (p= 0.8414), 1-2 kidding (p= 0.7900), normal body condition scoring (p= 0.8096) were not proved to be potential risk factors of subclinical mastitis (Table II).

Antibiotic Susceptibility testing against S. aureus and biofilm producing S. aureus
The in-vitro findings of current study revealed 100% sensitivity of S. aureus against gentamicin, oxytetracycline, amoxicillin, and linezolid while 80% of biofilm negative S. aureus (nbpSA) showed sensitivity against amoxicillin + clavulanic acid (Table III; Fig. 1). Higher percentages of goat milk based resistant isolates were noted from bpSA and nbpSA against vancomycin, chloramphenicol, oxacillin, amoxicillin+clavulanic acid and amoxicillin. None of the isolate from bpSA and nbpSA was resistant against linezolid, gentamicin, and oxytetracycline in this study. In case of oxacillin, amoxicillin clavulanate and trimethoprim+sulphamethoxazole there was significant (p<0.05) difference at intermediate cadre, and same was observed at sensitive cadre of isolates. The antibiotics did not differ significantly in efficacies between sensitive bpSA strains and sensitive nbpSA strains (Table III). Fig. 1. Biofilm producing strains, response of S. aureus against antibiotics and haemolytic strains. A, arrows point out different kinds of biofilm: yellow arrow, no biofilm; blue arrow, weak biofilm; green arrows, moderate biofilm; black arrow, strong biofilm. B, zone of inhibitions produced by antibiotics against S. aureus (yellow arrows, zones of inhibition). C, haemolysis on blood agar produced by S. aureus (yellow arrow, partial haemolysis).

DISCUSSION
Prevalence of subclinical mastitis, S. aureus, MRSA, hemolytic and biofilm producing S. aureus The prevalence of subclinical mastitis in current study was in line with findings of Najeeb et al. (2013) who reported 45% subclinical mastitis from goats. On the other hands, 37.5% and 53% subclinical mastitis in goats was also noted in previous studies by Abo-Shama (2014) and Ali et al. (2010). S. aureus has been formerly described  as one of the most significant causative agent in caprine mastitis (Ali et al., 2010;Najeeb et al., 2013). Higher biofilm positive S. aureus in current study was in contradiction with findings of França et al. (2012) who reported 7.6% bpSA based on CRA from caprine milk. Hemolysins are involved in various pathological processes. Kenny et al. (1992) reported that haemolytic toxins can develop clinical signs in mastitis cases, and Ebrahimi et al. (2007) reported that the udder of mastitic goats contain hemolytic Staphylococci. In the current study, 15.6% of S. aureus were found to be resistant to methicillin which was in line with the previous results of 9.2% as discussed by El-Deeb et al. (2018), 20% by Bochev and Russenova (2005), and 28.57% by Ebrahimi et al. (2007). The methicillin-resistant Staphylococci cannot be successfully treated with beta-lactam antibiotics as discussed by previous studies (Aqib et al., 2018b;Dar et al., 2006).

Risk factors
Potential risk factors of current study were in line with findings of previous studies conducted in Pakistan. Feeding system is significant factor for subclinical mastitis. Poor drainage system or farm hygiene can lead to occurrence of mastitis (Ali et al., 2010;Aqib et al., 2019;Najeeb et al., 2013). Teat injury is also strongly associated with mastitis (Ferdous et al., 2018). Wound on the teats and udder facilitates the entry of microbes into the glands, leading to mastitis (Gebrewahid et al., 2012). The findings of current study were in line with those of previous trials conducted on prevalence of subclinical mastitis in goats by Ali et al. (2010) and Najeeb et al. (2013).

Antibiogram
The results of current study were in line with those reported by Ali et al. (2010) and Saleem et al. (2018) who found 80-100% of S. aureus sensitive against these antibiotics. The decreased use of gentamicin in the late 1990's and obvious shift in strains of clinical isolates of S. aureus were major factors for increased gentamicin sensitivity (Klevens et al., 2006). Oxytetracycline is used as first line treatment by field workers. Oppliger et al. (2012) also suggested that S. aureus isolated from farm workers were 100% sensitive to oxytetracycline.
Vancomycin resistance is a rising problem in S. aureus isolates and their number is increasing day by day which may be due to the acquired resistance as occurred in case of methicillin (Marques et al., 2013). Glycopeptide antibiotics such as vancomycin are last choice for the severe clinical infections of MRSA throughout the world. But the continuous use of vancomycin for handling of MDR S. aureus infections has caused a decrease in vancomycin sensitivity in many countries (Hiramatsu et al., 1997;Rağbetli et al., 2016). Vancomycin resistance in S. aureus when studied at genomic level shows that the development of vanA gene is associated with this behaviour (Akpaka et al., 2017). Mastitis is well known for its deterioration and lack of response to treatment chiefly due to resistance by bacteria against antibiotics (Shamila-Syuhada et al., 2016).

CONCLUSION
The present study found overall higher prevalence of subclinical mastitis (42%) in goats with increased percentage of S. aureus (3.8.1%) and pathogenic strains of S. aureus (MRSA 15.6%, hemolytic S. aureus 46.9%, and biofilm producing S. aureus 34.4%). Risk factor analysis revealed type of drainage system, type of feeding, and teats injury as potential risk factors of mastitis. The in-vitro drug trial indicated higher sensitivity of S. aureus against oxytetracycline, trimethoprim + sulphamethoxazole, gentamicin and linezolid against S. aureus and biofilm producing S. aureus. Biofilm producing S. aureus were highly resistant against amoxicillin and vancomycin. Current study reports higher prevalence of pathogenic strains of S. aureus, larger number of potential risk factors, and diversified response of antibiotic susceptibilities which suggest extensive molecular studies and development of effective preventive measures.

Statement of conflict of interest
The authors have declared no conflict of interest.

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Emergence of Pathogenic Strains of S. aureus in Goat Milk