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

Livestock 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 and it largely contributes to the mainstream dairy milk production (Silanikove et al., 2010). Milk of goats has certain properties like better digestibility, alkalinity, buffer capacity and medicinal importance which make goat milk better than human and cow milk (Park, 2001). Mastitis can be illustrated as a result of pathological alterations in mammary glands resulting in elevation in somatic cell count of milk (Contreras et al., 2003). Mastitis occurs as clinical and/or subclinical form (Aqib et al., 2018). In clinical mastitis, signs of inflammation, redness, heat and pain are present, whereas there are no obvious indications of swelling in subclinical mastitis except decrease in milk production and increased somatic cell count (Sarker and Samad, 2011). 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 (over-prescription, 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.

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) was applied to collect milk samples (n= 200) from dairy farms located in district Faisalabad (Samundri, n= 62; Rasoolpur, n= 60; Livestock Farm of University of Agriculture Faisalabad, n= 36; Jhapal, n= 42) depending upon the willingness of the farmers to participate in the study and accessibility to Mastitis Research Laboratory, University of Agriculture Faisalabad, Pakistan. Milk samples were collected by strictly following the guidelines of National Mastitis Council of the USA (Reyher and Dohoo, 2011). Samples were screened by using Surf Field Mastitis test (SFMT) proposed by Muhammad et al. (2010). The SFMT positive milk samples, maintained in cold chain (4°C), were transferred to Mastitis Research Laboratory, University of Agriculture Faisalabad, Pakistan and preserved at -20°C till further process (Cengiz et al., 2015).

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).

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 colonies - no biofilm production; slight blackish -weak production; black sheeting - moderate production; and jet black dry sheeting - strong biofilm production (França et al., 2012; Mathur et al., 2006).

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), amoxicillin-clavulanic acid (20µg), amoxicillin (10µg), and oxacillin (1µg). Fresh cultures adjusted at 1.5×108 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.

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).

The present study found overall 42.0% (84/200) prevalence of subclinical mastitis from goats based on Surf Field Mastitis Test (SFMT). The prevalence of subclinical mastitis was found higher in Jhapal (59.5%) followed by Samundri (38.7%), Rasoolpur (38.3%) and UAF Livestock Farm (33.3%) while there was non-significant association (p> 0.05) among different areas. The overall prevalence of S. aureus was found 38.1% while among S. aureus there was 15.6% MRSA, 46.9% haemolytic S. aureus, 34.4% biofilm producing S. aureus during current study. The higher prevalence of S. aureus and MRSA was noted from livestock farm (50.0% and 33.3%) followed by Samundri (33.3% and 12.5%), Rasoolpur (39.1% and 11.1%) and Jhapal (36.0% and 11.1%). The percentage of haemolytic S. aureus and biofilm producing S. aureus was 83.3% and 83.3% from UAF livestock farm, 44.4% and 33.3% from Rasoolpur, 37.5% and 12.5% from Samundri, and 33.3% and 22.2% from Jhapal, respectively. The study found non-significant difference (p>0.05) for S. aureus, MRSA and haemolytic S. aureus while significant difference was noted for biofilm producing S. aureus among different areas of study (Table I; Fig. 1).

Table I.- Prevalence of subclinical mastitis, Staphylococcus aureus, methicillin resistant S. aureus, hemolytic and biofilm producing Staphylococci from different areas of Faisalabad.

p<0.05 indicate significant difference. Among different areas subclinical mastitis, *, p=0.072; #, p=0.799 and ‡, p=0.623; @, p=0.244; †, p=0.034.

C.I, confidence interval set at 95%; *p< 0.05 indicate significant difference.

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).

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).

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

Table III.- Comparative response of antibiogram of biofilm producing and non-biofilm producing S. aureus isolated from mastitic dairy goats.

NBPSA, non-biofilm producing S. aureus; BPSA, biofilm producing S. aureus; NA, not applicable.

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).

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).

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).

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.

The authors have declared no conflict of interest.