Screening of Milk Borne Staphylococcus aureus for Resistance against Beta Lactam Antibiotics

Clinical and sub clinical mastitis are significantly reported diseases in dairy cows causing severe economic losses to dairy farmers by way of reduced milk yield and quality (Kumar et al., 2017; Zeryehun et al., 2017). In dairy cows, mastitis caused by Staphylococcus aureus is commonly subclinical, manifested by elevated concentrations of leucocytes (primarily neutrophils) in milk (elevated somatic cell counts, SCC) (Rainard et al., 2017). Failure of treatment in mastitis is due to indiscriminate use of antibiotics without testing in vitro sensitivity (Awandkar et al., 2013). There is a greater need to emphasize the importance of judicious usage of antibiotics to reduce the development of resistance posing massive challenge in the treatment of bacterial diseases. In view of the growing incidence of anti-microbial resistance in large animals, the present study has been undertaken to screen sensitivity pattern of commonly used antibiotics in mastitis treatment, followed by isolation, microbiological and molecular characterization of Staphylococcus organisms.

Study area
 
The study was carried out in the Department of Veterinary Pharmacology and Toxicology and Department of Veterinary Public Health, Madras Veterinary College, Tamil Nadu University of Veterinary and Animal Sciences, Chennei- 7, India.
 
Antibiotic sensitivity testing
 
A total of 45 milk samples were collected from mastitis affected cows and screened for the presence of Staphylococcus organisms. Antibiotic sensitivity pattern was carried out by disc diffusion method on Mueller-Hinton agar plates (Balouiri et al., 2016).
 
Isolation of Staphylococcus organisms
 
The organisms in the milk sample were subjected to growth on Mannitol Salt Agar. Each isolate was cultured on Mannitol Salt Agar and incubated at 37°C for 24 hours. The procedure used in the preparation of Mannitol Salt Agar is standardized (Shields and Yang, 2006).
 
Minimum inhibitory concentration
 
MICs of Ceftriaxone and Cloxacillin against Staphylococcus aureus were determined using broth dilution method. The method described by Andrews, (2001) was followed by Wiegand et al., (2008).
 
Polymerase chain reaction
 
PCR amplification of nuc gene was carried out for molecular characterization of Staphylococcus aureus. Detection of mec A in the presumptive isolates was done using PCR (Brakstad et al., 1992; Pournajaf et al., 2014).The primer sequences for mecA gene and nuc gene and their cyclic temperatures for amplification are given in Table 1 and Table 2 respectively.
 

 

Antibiotic sensitivity testing
 
Results indicated a sensitivity pattern of antibiotics in the following order: Levofloxacin (87.5%), Ciprofloxacin (82.5%), Co-trimoxazole (77.5%), Amoxyclav (72.5%), Gentamicin (72.5%), Amikacin (67.5%), Tetracycline (42.5%), Cephalothin (42.5 %), Cefuroxime (37.5%), Azithromicin (25%) Ceftriaxone (72.5%), Cloxacillin (67.5%). Verma et al., (2018) conducted a study to see the antibiotic sensitivity pattern in mastitis affected cows and results indicate 65.96% and 63.83% sensitivity for Ceftriaxone and Amoxicillin, respectively. The results of Antibiotic Sensitivity testing are depicted in Table 3 and Fig 1.
 

 

 
Isolation of Staphylococcus aureus
 
Among the total number of milk samples collected, 88.88% of the samples were confirmed to be Staphylococcus organisms by virtue of growth of yellow coloured colonies on MSA plates. The colonies were aseptically picked and transferred to 50% glycerol stock solution for the purpose of storage. Mannitol Salt Agar (MSA) has been used since 1945 as a selective medium for the isolation of pathogenic Staphylococci (Blair et al., 1967; Chapman, 1945). So, it possibly indicates that some of the samples that were found positive for Staphylococcus organism belonged to genus other than Staphylococcus aureus (Sheilds and Tsang, 2006). The growth of yellow coloured colonies is depicted in Fig 2.
 

 
Polymerase chain reaction
 
Among the positive isolates of Staphylococcus organisms that were subjected to nuc gene amplification, 25% of the isolates were confirmed to be positive for Staphylococcus aureus as indicated by PCR results. The positive Staphylococcus organisms were also subjected to amplification of mecA gene using PCR and results revealed the presence of mec A gene in 12.5% of Staphylococcus isolates that were subjected to amplification of the concerned gene. Amplification of mecA gene was done with the help of PCR using mec A primer. The agarose gel electrophoresis of mec A and nuc gene PCR products are depicted in Fig 3.
 

 
Minimum inhibitory concentration
 
MIC₅₀ and MIC₉₀ for ceftriaxone against Staphylococcus aureus were recorded to be 10.95 µg/ml and 87.5 µg/ml, respectively. Similarly, MIC₅₀ and MIC₉₀ of Cloxacillin were found to be 43.75 µg/mland 87.5 µg/ml, respectively against Staphylococcus aureus. Current FDA Ceftriaxone breakpoints are <4 µg/ml (susceptible), 8 µg/ml (intermediate) and >16 µg/ml (resistant). The FDA Cloxacillin breakpoints are < 2µg/ml (Susceptible) and > 4µg/ml (Resistant). The MIC study for Cloxacillin was found to be 43.75 µg/ml - 87.5 µg/ml against Staphylococcus aureus. The results of the present study suggest that the isolates of S. aureus were resistant to Ceftriaxone which would involve a mechanism of PBP mutation. MIC values indicate resistance of Staphylococcus aureus to both ceftriaxone and cloxacillin. However, study on a greater number of isolates is required to characterize the extent and type of resistance.

The MIC values clearly indicate that Staphylococcus aureus is resistant to both Ceftriaxone and Cloxacillin. There is a heightened need to be judicious in our choice of antibiotics and also work out the right combination of antibiotics to stem the development of resistance. Also, further studies are required involving larger geographical area to arrive at a better conclusion.

Authors are thankful to the Professor and Head, Department of Clinics, Madras Veterinary College Teaching Hospital, Tamil Nadu Veterinary and Animal Sciences University, Chennai, Tami Nadu, India for providing the facilities required to conduct this study.