The dairy sector is important for nutritional wellbeing, as a support for livelihoods of many small holder dairy farmers and it makes a vital contribution to the economy of the country. Milk is considered as the most complete food in nature due to its rich nutrient composition that could be difficult to obtain from any other non-dairy food sources. In India most of the people consider milk and value-added dairy products as a source of protein in their daily diet Malar et al., (2025). As per the 20^th livestock census by government of India in 2019, total livestock population is 535.78 million. Among these, 35.94% is cattle and 20.45% is buffaloes which play a massive contribution to the total milk production and make India the highest milk producer in the world. A healthy and productive livestock herd is the pride of the small holder dairy farmer for which he has to overcome many hurdles.
       
Mastitis is the most common infectious disease in dairy cattle farming. It is responsible for the quantitative and qualitative milk deterioration (Kaouche-Adjlane and Mansour, 2020). The characteristic attributes of the disease include the reduction in the production, discarded milk, early culling, labour costs and veterinary services (Khan, 2019). A total economic loss of Rs. 7,824/- per month per cow has been reported, out of which a significant sum is of the treatment (Das et al., 2018). Mastitis is extremely contagious and there is high risk of spread among the whole herd through unhygienic practices.
       
Despite taking meticulous efforts in treatment of mastitis, cure rates are quite low due to various factors which include development of acquired as well as intrinsic antimicrobial resistance (Jadhav et al., 2010). Hence, Khasa et al., (2020) recommend that mastitis control should be the part of herd health programme and dairy management, as after development of infection, treatment can take a long course or even result in loss of milk in affected quarter permanently in some cases.
       
Although antibiotic treatment of mastitis is failing to give encouraging results, still today it is most important weapon in armamentarium of veterinarian when used judiciously with assistance of antimicrobial sensitivity testing (Jadhav et al., 2010). AMR in bacteria is a public health hazard and extensive use of antimicrobials is considered a potentially important driver of AMR (Chandrase- karan  et al., 2014).
       
Small holder dairy farmers often dont routinely test animals for mastitis which overlooks animals in subclinical stage. Alternatively they may follow empirical treatment by themselves or with help of veterinary clinician in subacute stage. This stage may respond to primary treatment but condition may recur later. Abdi et al., (2021) have observed that such non-prudent use of antimicrobials in dairy farms increased the development of antimicrobial resistant bacteria. Ultimately, if untreated or unresponsive to treatment, the udder goes towards chronic stage of mastitis where it becomes fibrinous and milk can no longer be produced. Such cows put economical strain on owner without contributing to income.
       
Antimicrobial sensitivity test helps to understand the resistance and susceptibility of bacteria towards a particular drug and thus helping in the choice of drug to be used for treatment (Jadhav et al., 2010, Singh et al., 2018 and Khan, 2019). Additionally, treatment of mastitis with antimicrobials reduces somatic cell count (SCC) and improves milk quality (Abdi et al., 2021). Most often a basic and inexpensive antibiotic is effective in early stage of mastitis and ABST can thus help to avoid expenses on the delayed infection when expensive and higher range antibiotics are required.
       
Regardless of substantial progress in study of pathogenesis, treatment and prevention of mastitis over several decades, both clinical mastitis (CM) and sub-clinical mastitis (SCM) remain a main challenge to dairy industry till today. Jadhav et al., (2010) mentioned that, low recovery rates of antibiotic therapy in subclinical mastitis cases especially has turned growth of livestock management towards preventive measures in area of infectious diseases. One major aspect of prevention is routine disease screening of animals. Thus, testing of milk samples for mastitis and further processing of positive samples for antimicrobial resistance testing is essential to implement proper mitigation measures (Abdi et al., 2021). Hence this study was conducted to determine efficacy as well as resistance to commonly used antibiotics in mastitis cases, among the samples presented at the laboratory.

A total of 78 samples were analyzed during the period of April ’22 to March ’24 by Kirby Bauer disc diffusion method. All the mastitis milk samples were brought to the Animal Disease Diagnostic Laboratory (ADDL) by concerned small holder dairy farmers and veterinary clinicians. Firstly, the milk samples were incubated in nutrient broth at 37^oC for 24 hours. Later this culture was spread over Mueller Hinton Agar (MHA) plates uniformly. Two plates were used per sample for this purpose and then 12 antibiotic discs were placed at equidistant positions on the plates with 6 on each plate. These were incubated at 37^oC for 24 hours. Finally, the zone of sensitivity was measured for each antibiotic disc, observations were noted and interpreted as either susceptible or resistant to the exposed agent as suggested previously (Chandrasekaran et al., 2014). The antibiotics not confirming to both these categories were considered as having intermediate sensitivity. The antibiotics used for this purpose were Penicillin - G (10 units), Vancomycin (30 mcg), Ampicillin sulbactam (20 mcg), Cefazolin (30 mcg), Gentamicin (10 mcg), Amoxycillinclavulanate (30 mcg), Ceftriaxone (30 mcg), Erythromycin (15 mcg), Azithromycin (15 mcg), Tetracyline (30 mcg), Enrofloxacin (5 mcg) and Streptomycin (10 mcg) ( HiMedia®). These antibiotics were chosen based on their availability in local veterinary medical stores and common usage in the surrounding areas for treatment of mastitis.

Out of the 12 antibiotics against which the 78 samples were tested, the most effective antibiotic was Amoxycillin clavulanate (35.9 %) and least effective was Penicillin - G (2.6 %). Other effective antibiotics were ceftriaxone (26.9%) and tetracycline (23.1%). Maximum resistance was found in penicillin - G (78.1%) followed by cefazolin (67.9%) and azithromycin (62.8%). The results of the study are presented in Table 1 and also Fig 1, with respect to all antibiotics used and their responses. The zone of inhibition and sensitivity is shown in the Fig 2-6.














       
Similar to our study there is an earlier report of greatest resistance towards penicillin-G (100%) (Dharakwal et al., 2024). However, sensitivity to antibiotics used to treat mastitis, reported earlier is varied across many researchers. Singh et al., (2018) observed azithromycin and the third generation fluoroquinolones (ciprofloxacin, sparfloxacin and ofloxacin) to be most effective and amoxicillin, erythromycin, tetracycline and vancomycin as the most resistant drugs. In another report of mastitis by Staphylococcus sp. isolates, by Markos et al., (2023) 95.2% susceptibility was seen towards Chloramphenicol, similarly susceptibility to Gentamicin (91.9%), Cephalothin (90.3%), Kanamycin (88.7%) and Streptomycin (80.6%) were also recorded. However, 100% of the isolates were resistant to Penicillin-G, Polymyxin, Amoxicillin and Ampicillin while resistance to Tetracycline was 80.6%. This disparity could be based on pattern of antibiotics used in that region, lactation status of animal, environmental factors and breed factors among others.
       
The high resistance of pencillin-G could be attributed to the indiscriminate use of this drug in injectable and intramammary preparations used by the small holder dairy farmers without the prescription of the veterinary clinician as observed previously (Chandrasekaran et al., 2014). The use of penicillin and beta-lactams in general for mastitis prevention and treatment is widespread, explaining the resistance of S. aureus isolates against this antibiotic and its increase mainly in the last ten years (Molineri et al., 2021). Penicillin - G is a component of many commercial preparations available OTC.
       
The intramammary tubes which are used, commonly contain: cefoperazone, ceftiofur and cefquinome. Currently the cephalosporin group is widely administered through multiple routes since it is giving good results as is also seen in our study. However, unfortunately we can also observe a growing resistance to the same group after penicillin - G. More specific details of antimicrobial use patterns (such as dosage, type of antibiotic, time of administration etc.) of farms included in this study are not available to substantiate that farmers imprudently use antimicrobials. Hence more extensive data collection is required in future to arrive at a precise conclusion. Also, in future a larger field study is suggested for better planning of disease prevention and control in the area.
       
Often samples come to the laboratory after the animal has been treated previously for a prolonged term with multiple medications and these include chronic cases as well. So the ABST shows mixed cultures or multiple drug resistant cultures. Among the 78 samples 15 showed resistance to all drugs used. Also, 5 samples showed no growth on the MHA plates and these are not included in this data.  As reported earlier by Singh et al., (2018) this absence of growth could be because they were treated recently resulting in antibiotic residues in the milk or because causative organisms require specific media and highly enriched media for growth. Ghadge (2023) also states that this can complicate the interpretation of ABST results since the chemical residues may disturb the antibiotic sensitivity pattern of the bacteria. Additionally persistent low grade antibiotic treatment may lead to development of resistant strains which will not respond to any antibiotic in the test. Khalifa et al., (2024) mention that drug residues can be found in food, the environment, or animals, causing major health concerns to people, including antibiotic resistance development and can originate from variety of sources, including extra-label drug use and ignoring drug withdrawal periods. Busal et al., (2020) have reported that 6% (n=6) samples during their study, were positive for antibiotic residues in the fresh milk for gentamycin, streptomycin and sulfonamide groups Zubair Ahmad Akhoon et al. (2025) observe that election of treatment requires cost/benefit analysis especially in food animals. Its efficacy, safety with minimal harmful effects and minimal residues in food animals also requires due attention as irrational and indiscriminate use of drugs leads to the increased probability of drug residues in food animal products like milk, meat, eggs etc.
       
Jadhav et al., (2010) note that sub clinical mastitis (SCM) during lactation is not treated because of high treatment cost, economic losses due to milk discard during milk withholding period and very low cure rates. Treatment of SCM during lactation is undertaken only if the causative organisms are highly contagious with aim to curb the spread of infection to healthy cows of herd from cows with SCM. This could be the reason why small holder dairy farmer and veterinary clinicians continue with empirical treatment leading to antibiotic resistance in the microbes. Also most often samples are brought to the laboratory once the organism/s stop responding to the medication.
       
Largely, animal disease surveillance is less developed in India and infrastructure to support delivery of veterinary services is inadequate. Several groups are known to offer animal health services. The untrained “animal health workers” and para-veterinarians are more popular with farmers as they charge less for consultations (compared to veterinarians who are few and charge more). Over-the-counter access of antibiotics, without prescription and direct marketing of drugs to small holder dairy farmers are common. Because of this, small holder dairy farmers are able to treat their animals by themselves and only consult when cases become non- responsive to treatment. Drug withdrawal periods are rarely observed and occurrence of antibiotic- contaminated milk has been reported. Awareness on AMR is low and antimicrobial stewardship in livestock is yet to be developed (Mutua et al., 2020).
       
Small holder dairy farmers and veterinary clinicians also follow dry cow therapy and infuse antibiotics into the udder via teats at the time of drying - off. This is usually irrespective of the health status of i.e. infected or not infected and adds to increase resistance of organisms towards antibiotics.
       
Kumar et al., (2024) have observed few reasons which affect the choice of drug and its usage in cases of mastitis, these are as below:
•   There is varying level of pressure on the veterinary clinicians to comply with the implicit or explicit demand of farmers regarding animal treatment.
•   Withdrawal period of antibiotic is critical because the use of products with shorter withdrawal periods could reduce the losses of small holder dairy farmers due to discarded produce.
•   Most small holder dairy farmers (54.17%) do not discuss previous experience of disease treatment with the veterinary clinicians.
•   Ultimately, small holder dairy farmers are the final decision makers to decide whether diagnostic tests will be performed or not.
•   Small holder dairy farmers are also responsible for the administration of antimicrobials
•   The extent of antibiotic sensitivity testing is influenced by the attitudes of small holder dairy farmers up to the some extent.
•   Large holder dairy farmers request veterinary clinicians to prescribe prophylactic treatment for their livestock. They feel that if disease with high probability of occurrence was not prevented, it could cause high morbidity and mortality rates with accompanying financial losses and result in subsequent treatment with expensive antibiotics.

Early diagnosis of mastitis followed by ABST and judicious administration of antibiotics should be practiced to prevent such high rate of resistance in mastitis causing organisms. This along with good herd planning and hygienic practices can definitely help to reduce incidence, complexity and severity of mastitis in years to come.

The authors express their gratitude to the President, Senior Vice President and colleagues at Animal Disease Diagnosis Laboratory of BAIF Development Research Foundation for their unwavering support throughout the study.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
Written informed consent was obtained from all participants prior to inclusion in the study and confidentiality of personal data was maintained throughout.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.