Isolation, characterization and antimicrobial resistance profiling of mastitogenic Escherichia coli from dairy cows

Introduction

Bovine mastitis, an inflammatory condition of mammary tissue is one of the important infectious diseases, significantly hampers the sustainability and productivity of dairy sector (Fesseha et al., 2021; Kour et al., 2023) [10,17] . It incurred substantial economic losses reflected as decreased quantity and quality of milk, increased culling rate and veterinary services (Sharun et al., 2021; Morales-Ubaldo et al., 2023) [27,23] . Being one of the costliest diseases of dairy sector, mastitis accounts for 20-30 billion USD annual economic loss (direct/indirect losses) worldwide (Gayathri et al., 2024; Bose and Siva Kumar, 2025) [12,4] . As per a study, the economic loss/cow due to gram negative pathogen mastitis (211.03 USD) was significantly higher than gram positive mastitis (133.73 USD) (Fu et al., 2022) [11] . As per Bansal and Gupta (2009) [2] , the financial loss incurred due to clinical and subclinical mastitis in India is approximately INR 7165.51 crores. Mastitis in dairy animals caused by wide variety of pathogens including contagious (Staphylococcus aureus, Streptococcus agalactiae, Corynebacterium pyogens and Mycoplasma spp.), environmental (E. coli, K. pneumoniae, S. dysagalactiae and S. uberis) and opportunistic (Coagulase negative Staphylococci) agents (Ndahetuye et al., 2019) [25] . Environmental mastitis pathogens (mainly E. coli and K. pneumoniae) predominantly present in animal surroundings and gained access through teat canal during or one to two hours of milking (Hogan and Smith, 2012; Cobirka et al., 2020) [14,7] . E. coli predominantly associated with acute mastitis, characterized by abnormal changes in milk and udder accompanied by systemic signs (fever, anorexia and depression) (Bobbo et al., 2017) [3] . Based on the presence or absence of apparent clinical signs, bovine mastitis is classified into two types: clinical mastitis (CM) and sub-clinical mastitis (SCM) (Abebe et al., 2016) [1] .

CM characterized by physical and biochemical changes in milk, pathological changes in mammary tissue and systemic signs. In contrast, no evident clinical signs were observed in cases of subclinical mastitis (Jesse et al., 2023). As per Kour and Co-worker (2023), 70-80% share of production losses are attributed to subclinical mastitis. The high incidence of mastitis is closely related to high lactation yield of animal with approximately 15-20% cow population affected annually (Haider et al., 2023) [13] . In a meta-analysis study of bovine mastitis, the pooled prevalence of CM and SCM is 15% and 42% in world and respectively and 18% and 45% in India respectively (Krishnamoorthy et al., 2021) [18] . Despite E. coli being a major cause of clinical bovine mastitis, information on the current antimicrobial susceptibility patterns of mastitis-associated isolates remains limited. The emergence of antimicrobial-resistant E. coli strains has further complicated treatment outcomes, highlighting the need for continuous surveillance. Therefore, isolation and characterization of E. coli from clinical mastitis cases, along with antimicrobial susceptibility testing, are essential to identify effective therapeutic agents, monitor resistance trends, and support evidence-based mastitis control and antimicrobial stewardship programs. The aim of the study was to investigate the bacteriological characteristics and antibiotic resistance patterns of E. coli associated with clinical bovine mastitis for supporting effective mastitis management and antimicrobial stewardship.

Material and methods Sample Collection and Isolation of E. coli

Milk samples were aseptically collected from bovine quarters clinically affected with mastitis presented at the Referral Veterinary Polyclinic and Teaching Veterinary Clinical Complex (RVP-TVCC), ICAR-Indian Veterinary Research Institute (ICAR-IVRI), Izatnagar, Bareilly, Uttar Pradesh, India. Samples were transported to the laboratory under refrigerated conditions for bacteriological examination. For isolation of E. coli, milk samples were centrifuged at 2,670 × g for 10 min and the sediment was inoculated into Brain Heart Infusion (BHI) broth followed by incubation at 37 °C for 24-48 h. Enriched cultures were streaked onto MacConkey Lactose Agar (MLA) and incubated at 37 °C for 24-48 h. Presumptive lactosefermenting colonies were further sub-cultured onto Eosin Methylene Blue (EMB) agar for selective isolation of E. coli. The isolates were subsequently subjected to biochemical.

Biochemical Characterization of E. coli

Presumptive E. coli isolates were biochemically characterized using the KB010 HiE. coli™ Identification Kit (HiMedia Laboratories, India). The kit comprised four carbohydrate utilization tests (glucose, sorbitol, sucrose, and lactose) and eight conventional biochemical tests including methyl red, Voges-Proskauer, citrate utilization, indole production, β-glucuronidase activity, nitrate reduction, ONPG, and lysine utilization. Pure bacterial cultures were inoculated into Brain Heart Infusion broth and incubated until turbidity matched the 0.5 McFarland standard. Subsequently, 50 µL of inoculum was dispensed into each well of the kit and incubated at 35±2 °C for 18-24 h. Test results were interpreted according to the manufacturer's instructions.

Antimicrobial Susceptibility Testing

Antimicrobial susceptibility testing of confirmed E. coli isolates was performed by the Kirby-Bauer disk diffusion method on Mueller-Hinton agar (MHA) following standard guidelines. Bacterial suspensions equivalent to a 0.5 McFarland standard were prepared and uniformly spread on MHA plates. Antibiotic discs were placed aseptically on the inoculated agar surface, and plates were incubated at 35±2 °C for 16-18 h. The diameter of inhibition zones was measured and interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines. The antibiotic exhibiting the highest susceptibility against the isolates was selected for subsequent in vivo studies.

Results

Isolation and Biochemical characterization of Escherichia coli

A total of 54 milk samples were collected from clinical cases of bovine mastitis. Following enrichment and selective culturing, 25 (46.3%) samples produced characteristic pink lactose-fermenting colonies on MacConkey Lactose Agar (MLA). These isolates were subsequently streaked onto Eosin Methylene Blue (EMB) agar, where 14 isolates exhibited the characteristic green metallic sheen suggestive of E. coli. Biochemical characterization using the KB010 HiE. coli Identification Kit revealed that 12 isolates showed positive reactions in 9 out of 12 biochemical tests, whereas the remaining two isolates exhibited positive reactions in 6 out of 12 tests.

Antimicrobial Susceptibility Profile of E. coli Isolates

The antimicrobial susceptibility pattern of the 12 confirmed E. coli isolates is presented in Table X. The isolates exhibited a high level of resistance to sulphonamides (93.75%), followed by amoxicillin (87.50%), erythromycin (81.25%), and tetracycline (75.00%). Intermediate susceptibility was observed for co-trimoxazole (31.25%), amoxicillin-clavulanate (31.25%), and ampicillin (25.00%). Among the tested antimicrobials, the isolates showed the highest susceptibility to ceftiofur (87.50%), followed by ciprofloxacin (81.25%), ceftriaxone (81.25%), gentamicin (75.00%), and enrofloxacin (68.75%). Based on the antimicrobial susceptibility profile and its widespread therapeutic use against bovine coliform mastitis, ceftriaxone was selected as the reference antibiotic for subsequent evaluation in the E. coli-induced murine mastitis model.

Discussion

In the current study, pathogenic E. coli was isolated from the clinical cases of mastitis presented to RVP-TVCC and cattle and buffalo farm, IVRI. On screening 54 mastitis milk samples, 12 (22.2%) samples were found positive for E. coli by culture and biochemical test. In a study conducted in Maya city, Ethiopia, the prevalence rate of E. coli mastitis was 22.6% which is in agreement to our study finding (Dahesa et al., 2025) [8] . Researcher reported similar finding of E. coli prevalence (22.45%) in an investigation done at Romania dairy farm from 2018-2022 (Drugea et al., 2025) [9] . A nine years (2016-2024) study in Eastern Iran also documented the comparable prevalence (19.92%) of E. coli mastitis cases, closely aligning with our findings (Moradi et al., 2025) [22] . The positivity of E. coli in current study was consistent with Xu and co-workers (2022) [29] , who reported that the prevalence of E. coli mastitis was 26.99% in certain parts of Jiangsu province, China. In Contrary to our finding, Rana and co-worker (2022) [26] reported E. coli prevalence (30.43%) which is higher than our study prevalence. In a meta-analysis study from 1995-2019, the pooled prevalence of S. aureus and E. coli was 41% and 15% respectively in India (Krishnamoorthy et al., 2021) [18] . Manasa and coworkers (2019) [21] reported that 13 (16.6%) out of 78 samples were found positive for E. coli in clinical cases of bovine mastitis. In another study conducted between 2005-2016, the prevalence of mastitis due to E. coli was 14% (Krishnamoorthy et al., 2017) [18] . A study undertaken in Free State Province of South Africa reported that, the prevalence of SCM was 39.1% with majority of cases (88.23%) attributed to E. coli infection (Khasapane et al., 2025) [16] . The variation in prevalence of E. coli mastitis could be attributed to unhygienic practices in the farm as well as suboptimal managemental conditions. Twelve confirmed isolates of E. coli from clinical cases of bovine mastitis were used for ABST. Findings suggested high degree of resistance to sulphonamides (93.75%), amoxicillin (87.5%), erythromycin (81.25%) and ampicillin (75.00%) followed by co-trimoxazole (37.50%) and ampicillin (31.25%). The E. coli strains were quiet susceptible to ceftiofur (87.50%), cefriaxone (81.25%), ciprofloxacin (81.25%) followed by gentamicin (75.00%).

In a study carried out in North America between 2011-22 reported that E. coli exhibited low level of resistance to ceftiofur (2.8%) and cefoperazone (3.4%) with intermediate level for ampicillin (9.2%) (Sweeney et al., 2024) [28] . The 6.25% resistant level by E. coli strains for ceftiofur was slightly higher than reported (2.8%) by Sweeney and coworkers (2024) [28] . Contrary to our findings, Khasapane and co-worker (2025) [16] reported alarming level of resistant in E. coli with degree of resistance 71.6%, 70% and 30% for penicillins, ciprofloxacin and gentamicin respectively. In contrast to our findings, antimicrobials like streptomycin (17.7%), tetracycline (15.93%) and ampicillin (11.5%) showed lower degree of resistant against E. coli, whereas none E. coli isolate exhibited resistant to gentamicin and ciprofloxacin (Majumder et al., 2021) [20] . In a study in Brazil, the resistant to ceftriaxone was slightly lower (7.3%) than our study (Campos et al., 2022) [5] . In a study conducted in Bangladesh, the susceptibility rate (>70%) of cefriaxone and ciprofloxacin was in agreement with our finding (Rana et al., 2022) [26] . In contrast to our finding, My et al. (2023) [24] reported low level of resistance (32%) in mastitogenic E. coli isolates to tetracycline. The resistant to ceftiofur (4%) was slightly lower than our findings, which might be due to small sample size of our study (My et al., 2023) [24] . As per a study, the sensitivity rate (89.5%) of ceftiofur against E. coli strains is in alignment with our findings (Zhao et al., 2024) [29] . The findings of Li and coworker (2022) were also in agreement with current study showing almost similar level of resistant (52.5%) to ampicillin. Rana et al. (2022) [26] documented somewhat higher level of resistance in mastitogenic E. coli to ampicillin (58.82%) and ceftriaxone (26.47%). The findings of our study revealed alarming data of antimicrobial resistance among mastitogenic E. coli isolates, with only 6/14 antibiotics exhibited >75% sensitivity rate. Majumder and co-worker (2021) [20] documented that, out of 32 isolates of mastitogenic E. coli, 19 isolates showed multi drug resistance, while 13 isolates exhibited resistant to single antibiotic. Chowdhury and co-worker (2025) [6] also highlighted the severity of resistance, reported 31.9% multi drug resistance and 8.9% extensively drug resistance E. coli strains.

Conclusion

The present study confirmed the occurrence of Escherichia coli in clinical bovine mastitis through cultural and biochemical characterization. The isolates exhibited high resistance to commonly used antibiotics, particularly sulphonamides, amoxicillin, erythromycin, and tetracycline. In contrast, ceftiofur, ceftriaxone, ciprofloxacin, and gentamicin demonstrated high antimicrobial efficacy against the isolates. These findings emphasize the importance of routine antimicrobial susceptibility testing for effective therapeutic decision-making and prudent antimicrobial use in bovine mastitis management.

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