India ranks first in milk production in the world, but mastitis has been considered the major constraint to milk production globally. Mastitis is an inflammation of the mammary gland, typically caused by dysbiosis- outgrowth of opportunistic pathogenic bacteria and simultaneous reduction of healthy commensals (Maity and Ambatipudi, 2021). Common antimicrobials used to treat mastitis in cattle include beta-lactams, tetracyclines, aminoglycosides and fluoroquinolones. However, the injudicious and sub-therapeutic usage of antibiotics poses significant risks due to the enrichment of resistant microorganisms, which compromise the effectiveness of antimicrobial therapy leading to economic losses in the dairy industry. This mastitis is caused by a cascade of pathogens viz. Staphylococcus aureus, Escherichia coli, Streptococcus dysgalactiae, Klebsiella pneumoniae, Enterobacter cloacae, Bacillus pumilus and Streptococcus agalactiae (Zhang et al., 2022; Panchal et al., 2024). There is growing evidence of antimicrobial resistance (AMR) among the mastitis-borne pathogens, posing a serious public health concern (Tripathi et al., 2022; Abdi et al., 2021). These pathogens produce β-lactamase and extended-spectrum β-lactamases (ESBL), which hydrolyse the β-lactam ring, rendering β-lactam antibiotics ineffective. High levels of β lactamase production have been reported among the multidrug-resistant (MDR) strains of Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae- all of which are currently listed as critical priority pathogens by the World Health Organization (Emon et al., 2024). 
       
Antibiotic resistance in these pathogens is often driven by the accumulation of antibiotic-resistant genes (ARGs), which may arise through spontaneous mutations or horizontal gene transfer, encoded by mobile genetic elements such as plasmids, staphylococcal cassette chromosomes (SCC) and jumping genes (Haq et al., 2024). Extended-spectrum β-lactamase (ESBL) genes (bla_TEM, bla_Z, bla_NDM, bla_SHV, bla_CTX-M) have been identified as the commonly occurring ARGs among the mastitis-borne pathogens from cattle (Hassani et al., 2022). Moreover, the highest resistance to beta-lactams is mediated by either β-lactamases encoded by blaZ gene that can hydrolyse β-lactam ring or by the mecA gene encoding the penicillin binding protein, PBP2a, which exhibits a low degree of affinity to the β-lactam antibiotics (Naranjo-Lucena and Slowey, 2023). 
       
Although several studies have investigated the etiology and AMR profiles of pathogens causing bovine mastitis, this study is the first report focusing on the AMR profiling of mastitis-associated pathogens in Chhattisgarh, a central bovine populated state of India. Notably, the highest prevalence of bovine mastitis was acknowledged in Chhattisgarh (Sub clinical mastitis- 63% and clinical mastitis-38%) in India (Krishnamoorthy et al., 2021). Furthermore, the prevalence and distribution of mastitis-borne pathogens exhibit spatio-temporal variations, underscoring the need for a region-specific approach for robust surveillance, prevention and control strategies against mastitis. There is a dearth in the literature on the critical antibiotic resistance genes (ARGs) among the MDR pathogens isolated from mastitis infected milk samples in this region. Therefore, the objectives of this study were (i) to isolate and characterise the pathogens causing mastitis in cattle through 16S rRNA gene sequencing, (ii) to perform antimicrobial resistance profiling of identified pathogens and (iii) to determine ARGs among the prevalent multidrug-resistant pathogens. This targeted approach not only enhances treatment efficiency but also serves as a critical intervention to mitigate the proliferation and transmission of resistant bacterial strains within the dairy production ecosystem.

Collection of milk samples and prevalence study of mastitis among cattle in Chhattisgarh
 
A total of 84 milk samples were collected from six organised dairy herds located at three districts in Chhattisgarh viz. Raipur (n=30), Durg (n= 24) and Bilaspur (n=30). Prior to sample collection, the teats of each cow were individually cleansed using cotton wool soaked with 70% ethanol. Approximately 15 ml of milk was aseptically collected from each cow, irrespective of the presence or absence of clinical signs of mastitis. Milk samples were kept in sterile airtight collection tubes and immediately transported to the laboratory under a cold chain to maintain sample integrity. All the samples were screened for mastitis using the californian mastitis test (CMT). For the test, 2 ml of milk was placed in each of the four cups of the CMT paddle to which an equal amount of CMT reagent was added and mixed well by a gentle circular motion for 10-15 s. The CMT result was interpreted as negative (0), trace (T), weakly positive (+), distinctly positive (++) and strongly positive (+++). Milk samples exhibiting a CMT score of +, ++ or +++ were classified as mastitis-positive milk samples. These positive samples were further processed for isolation and culture of pathogens. 
 
Isolation and identification of pathogens through 16S rRNA sequencing
 
A total of 45 mastitis-positive milk samples were subjected to pathogen isolation. A loopful of each milk sample was spread onto three different culture media: Brain heart infusion (BHI) agar, MacConkey agar (MLA) and mannitol salt agar (MSA) (HiMedia Laboratories, India). The inoculated plates were incubated at 37°C for 18-24 h in a BOD incubator to facilitate growth of bacteria. Following incubation, colonies were examined for morphological characteristics including size, shape, edge, elevation, tex­ture and pigmentation. Based on these morphological traits, distinct colonies were selected and sub cultured onto BHI agar to get the pure isolates. Gram’s staining was performed to determine the Gram reaction and the morphological characteristics of the isolates.
       
Further, the pure bacterial cultures were grown in BHI broth at 37°C for 24 hours. Approximately 200 µl of the broth culture was transferred into sterile microcentrifuge tubes and centrifuged at 14,000 × g for 4 minutes to pellet the bacterial cells. Genomic DNA was extracted from the pellet using the DNeasy® Blood and Tissue kit (Qiagen, Germany) following the manufacturer’s instructions. The concentration of the extracted DNA was measured using a Qubit 4.0 fluorometer (Thermo fisher scientific, USA) and the integrity of DNA was evaluated on a 1% agarose gel. Full-length 16S rRNA gene, encompassing the V1-V9 hypervariable region (~1.6 kb) was amplified via PCR. The amplified full-length 16S PCR products were subsequently sequenced using the PacBio sequel II platform (Nucleome Informatics, India).
 
Antimicrobial susceptibility testing of bovine mastitis pathogens
 
Antimicrobial susceptibility testing of the isolated bacterial strains was performed using the Kirby-Bauer disc diffusion technique and the results were interpreted according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2020). A uniform suspension of each bacterial isolate was prepared in Brain Heart Infusion (BHI) broth and the inoculum was evenly spread onto muller-hinton agar medium with sterile cotton swabs. The antibiotic discs were placed on it followed by overnight incubation at 37°C. In total, thirteen commercially available antibiotic discs across six major antibiotic classes such as amoxyclav (30 mcg/disc), ampicillin (10 mcg/disc), ceftriaxone (30 mcg/disc), cefotaxime (30 mcg/disc), cefixime (5 mcg/disc), streptomycin (10 mcg/disc), gentamicin (10 mcg/disc), amikacin (30 mcg/disc), tetracycline (30 mcg/disc), doxycycline (10 mcg/disc), co-trimoxazole (25 mcg/disc), ciprofloxacin (5 mcg/disc) and ofloxacin (5 mcg/disc) were used in the present study (HiMedia Laboratories, India). Zones of inhibition were measured in millimetres and interpreted as sensitive (S), intermediately resistant (I), or resistant (R) categories based on CLSI breakpoints. Isolates displaying resistance to at least three different antibiotic classes were classified as multidrug-resistant (MDR). 
 
Identification of antibiotic-resistant genes (ARGs) using PCR
 
The top 32 multidrug-resistant (MDR) bacterial isolates were screened for the presence of the β-lactam-resistant genes (bla_TEM, bla_Z, bla_NDM1, bla_NDM2) as these isolates had average antibiotic resistance rate above 85%. Four sets of primers, the expected amplicon size and the references are provided in Table 1. PCR amplification was carried out in a 25 µl reaction volume comprising the following components: 12.5 µl of PCR master mix, 2 µl of forward primer, 2 µl of reverse primer, 4 µl of genomic DNA template and 4.5 µl of nuclease-free water. The PCR was performed under the following thermal cycling conditions: Initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation at 94°C for 1 min, primer annealing at 58°C (52°C-59°C, determined by specific primers) for 30 s, extension at 72°C for 1min and a final extension at 72°C for 10 min. Further, amplified PCR products were visualised on a 1.8% agarose gel using a gel documentation system under UV illumination.

Prevalence of mastitis in dairy cows in Chhattisgarh
 
Prevalence of mastitis was investigated across six organized dairy farms located at Raipur, Durg and Bilaspur districts in Chhattisgarh. Out of 84 milk samples collected, 39 samples were found negative for mastitis while 45 samples were tested positive for mastitis through the California mastitis test (CMT). Overall prevalence of mastitis in Chhattisgarh was 53.6%, while district-wise prevalence rates were estimated as 70%, 41.7% and 46.7% for Raipur, Durg and Bilaspur districts, respectively (Table 2). Meta-analysis on the incidence of mastitis in India revealed comparable results, however a higher prevalence of mastitis (clinical-38% and subclinical-63%) had been observed at Chhattisgarh and Madhya Pradesh states in the central zone of India (Krishnamoorthy et al., 2021). 


       
Dairy farming serves as a primary source of livelihood and income generation in Chhattisgarh, second only to agriculture. Mastitis is a common and globally recognised health problem in dairy animals, contributing significantly to the economic losses due to reduced milk yield, emergence of antimicrobial resistance and reduced animal welfare (Mutua et al., 2020). Therefore, it strongly advocates surveillance of bovine mastitis pathogens, which constitute the major driving force for spreading antimicrobial resistance, to formulate future-ready strategic interventions for effective disease management and antibiotic stewardship in veterinary practice. Implementation of antimicrobial stewardship ensures reduced antibiotics use, correct drug dosage and duration, promotes targeted therapy and preserves the effectiveness of existing antibiotics and limit the propagation of AMR through milk.
 
Molecular identification of mastitis-borne pathogens
 
The 45 mastitis-positive milk samples were processed to get 125 isolates, distributed across Raipur (n=72), Durg (n=19) and Bilaspur (n=34) districts, respectively. Full-length 16S rRNA gene sequencing (V1-V9 region) using the PacBio sequel II sequencing platform identified 37 distinct bacterial species, comprising 12 Gram-negative and 25 Gram-positive pathogens. The predominant bacterial species were identified as Escherichia coli (19.2%), Enterobacter cloacae (8.8%), Klebsiella pneumoniae (7.2%), Staphylococcus chromogenes (6.4%) and Staphylococcus aureus (4.8%) of the total isolates. The abundance of 37 different species of pathogens associated with bovine mastitis is represented in Table 3. Recent advances in high-throughput sequencing technologies have revealed a broader spectrum of bacterial diversity with targeted amplicon (16S) sequencing (Gao et al., 2017). To the best of our knowledge, this is the first study to use PacBio SMART technology for full-length 16S rRNA sequencing to identify bacterial pathogens from mastitis-positive milk samples from cattle. Gram-negative Enterobacteriaceae constitute a major group of pathogens, representing approximately 30-40% of those associated with mastitis in cattle (Abdel-Hameed et al., 2025). These diverse groups of microbes colonise at the mammary gland to produce pathogenic invasion and mammary dysbiosis (Maity and Ambatipudi, 2021).


 
Antibiotic resistance pattern of bacterial isolates from bovine mastitis
 
Antimicrobial resistance profile of the 125 bacterial isolates against 13 antibiotics, determined through the disc diffusion method, is illustrated in Fig 1. The isolates showed highest resistance against cefixime (87.2%, n=109/125) followed by cefotaxime (82.4%, n=103/125), ciprofloxacin (68.8%, n=86/125), ceftriaxone (65.6%, n=82/125), amikacin (52.8%, n=66/125), ampicillin (52%, n=65/125), streptomycin (44%, n=55/125), tetracycline (37.6%, n=47/125), co-trimoxazole (36%, n=45/125), doxycycline (34.4%, n=43/125), amoxyclav (33.6%, n=42/125), ofloxacin (27.2%, n=34/125) and gentamicin (24%, n=30/125), respectively. Antibiotic resistance pattern of bacterial isolates as sensitive (S), intermediately resistant (I), or resistant (R) categories are presented in supplementary file. These results indicate that the mastitis-associated bacterial isolates exhibited the highest resistance to β-lactams (especially cephalosporins and penicillins) as well as to the quinolone group (notably ciprofloxacin). In contrast, the isolates were relatively more susceptible to aminoglycosides, particularly gentamicin. Furthermore, 75.2% of isolates (94/125) were identified as multidrug-resistant (MDR). A similar trend was reported by Hole et al., (2026), where Staphylococcus aureus isolates from clinical bovine mastitis exhibited marked resistance to ampicillin and methicillin antibiotics and moderate resistance to tetracycline, gentamicin and enrofloxacin antibiotics, indicating the existence of resistance strains in the dairy milk.   


       
Emergence and transmission of antimicrobial resistance (AMR) through bovine mastitis milk is a critical global health issue, which needs an urgent attention and immediate containment. Cephalosporins are widely used in veterinary medicine to treat bacterial infections; however, their extensive use has significantly increased the relative abundance of β-lactam resistance genes (bla_TEM), which further confer resistance to cephalosporins (Dong et al., 2022). Staphylococcus aureus isolates from bovine mastitis cases also showed resistance to cephalosporins including cefoxitin, cefotaxime, cefepime and ceftazidime, which is in consistent with the present results (Roshan et al., 2022). Similarly, a high level of resistance to β-lactam antibiotics such as penicillin, amoxicillin and ceftazidime up to 98.1% was observed in the isolates from Chinese dairy cows with clinical mastitis symptoms (Han et al., 2022).

Profiling of antibiotic resistance genes (bla_TEM, bla_Z, bla_NDM2 and bla_NDM1) among MDR pathogens
 
Prevalence of four antibiotic-resistant genes (ARGs) viz. bla_TEM, bla_Z, bla_NDM2 and bla_NDM1 was evaluated among the 32 multi-drug resistant (MDR) mastitis pathogens through PCR assay. bla_TEM gene was most frequently detected, among 81.3% (26/32) of the isolates, followed by bla_Z in 75% (24/32), bla_NDM2 in 50% (16/32) and bla_NDM1 among 21.9% (7/32) of the mastitis-borne pathogens of dairy cows (Table 4). Notably, three isolates, Enterobacter cloacae, Enterococcus hirae and Acinetobacter baumanii were found to carry all the four studied ARGs. Electrophoresis patterns of the PCR-amplified resistance genes are shown in Fig 2 and 3. Occurrence of multidrug resistance to β-lactams is attributed to the major gene families encoding Extended- spectrum β-lactamases (ESBLs) (bla_TEM, bla_SHV and bla_{CTX M}) and carbapenemases (bla_KPC, bla_NDM) among the mastitis pathogens (Bajaj et al., 2016).  These findings are in accordance to the previous studies by Hassani et al., (2022) who reported the existence of bla_TEM and bla_Z genes among 50% and 12% of foodborne bacteria isolated from bovine milk. Moreover, a high prevalence of the bla_Z gene among 55.31% of the bacterial isolates from raw milk of cows infected with subclinical mastitis was reported by Haq et al., (2024). On the contrary, a lower prevalence of the bla_TEM gene (38.9%) was reported by Bag et al., (2021) on the Escherichia coli isolates from bovine clinical mastitis in Bangladesh.In India, the blaZ gene coding for beta-lactam gene was targeted among Staphylococcus aureus from subclinical mastitis milk and the results revealed 81.4% of these isolates carried blaZ gene as comparable to our study (Solanki et al., 2023). 






       
A limitation of this study is that alternative ESBL-encoding genes as well as tetracycline-and fluoroquinolone- resistant genes need to be assessed to understand the MDR phenotype of the bacterial isolates from bovine mastitis cases. Furthermore, the risk factors affecting mastitis in cattle, including genetic (age, parity, milk yield, stage of lactation) and non-genetic factors (Season, hygiene and sanitation, herd strength and general management and care practices), were not considered which could strengthen this study and also highlight the need to improve management practices for better control of mastitis in dairy cattle.

The complete analysis of the abundance, diversity and dissemination of resistance genes in bacteria is extremely crucial for strategic management and effective control of mastitis, which ultimately helps to curb the emergence and spread of antibiotic-resistant pathogens within the dairy food chain. Enterobacter cloacae, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus and Staphylococcus chromogenes emerged as the predominant pathogens from bovine mastitis in Chhattisgarh. These pathogens exhibited widespread resistance to β-lactams and fluoroquinolones, highlighting the reduced efficacy of the common antibiotics. The phenotypic resistance profile is correlated with the existence of ESBL-producing four β-lactam antibiotic resistance genes (bla_TEM, bla_Z, bla_NDM2 and bla_NDM1) among the multidrug-resistant mastitis pathogens. These findings underscore the urgent need to characterise the genetic determinants of antimicrobial resistance, whether plasmid- or chromosome-mediated, along with the mechanisms contributing to virulence and disease progression.

The present study was supported by the Indian Council of Agricultural Research, Department of Agricultural Research and Education, Government of India. 

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.

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.