Antimicrobial resistance profiles on Staphylococcus aureus and Escherichia coli in mastitis affected lactating cattle

Introduction

Mastitis in cattle is still a very complicated condition that affects productivity. It presents a significant economic challenge to the global dairy industry. Numerous pathogen groups, including fungi, bacteria, and mycoplasma, have been identified as its causative agents (Ashraf and Imran, 2020) [3] . Contamination source of these organism includes hands of milking people, contaminated towels, water, dust, air, skin of animals, glandular tissues etc. Therefore, consumption of raw or under-pasteurized milk and their products from dairy cows constitutes potential microbiological hazards and affects the human health. The main strategy to treat mastitis is through the use of antibiotics (penicillin, ampicillin, tetracycline, gentamicin, etc.) (Ruegg, 2018) [12] . Even though bacteria quickly develop resistance to βlactam antibiotics, such as penicillin and cephalosporins, they are still the most often used antibiotics (Royster and Wagner, 2015) [11] . Staphylococcus aureus and E. coli are the most common causes of contagious and environmental clinical mastitis, respectively. Antimicrobial treatment is frequently used to prevent and control mastitis. Unfortunately, bacteriological cure failures occur frequently even with the best antimicrobial therapies available; this is especially prevalent for S. aureus mastitis and antimicrobial resistance (AMR), which is considered to be one of the main causes of low recovery rates (Barkema et al., 2006) [4] .

Materials and Method 2.1 Study population

The current investigation was carried out for a period of one year from November 2022 to December 2023, in the Department of Veterinary Medicine, College of Veterinary and Animal Science, Navania, Udaipur (RAJUVAS, Bikaner). Cattle of different age and breed were examined.

Milk sampling

After the confirmation of both clinical and subclinical mastitis, 70% ethyl alcohol was used to disinfect the cattle's teats. The first milk squirt was discarded before sampling, and around 5 mL of milk was aseptically collected into a sterile test tube for microbiological examination. Finally, all of the samples were moved to the lab by using an icebox to keep an appropriate cooling chain going. Before being streaked onto the agar plate, all milk samples were stored at room temperature.

Diagnosis of clinical and sub-clinical mastitis

Clinical mastitis was diagnosed based on observable symptoms such as a firm and inflamed udder, sensitivity to touch, abnormal size and texture of the mammary gland, as well as the production of abnormal milk, including flakes, clots, watery consistency, pus, and the presence of bloodstained milk. Additionally taken into consideration are related general clinical symptoms such fevers, depression and lethargy, appetite loss, and abruptly decreased milk production. The California Mastitis Test (CMT) was conducted to screen for sub-clinical mastitis in each cattle. Following the procedure outlined by Rana et al. (2020) [10] , 2 mL of milk was collected from each quarter of the cow's udder and placed into individual wells on CMT paddles. Equal amounts of CMT reagent were added, and the mixture was gently swirled for 30 seconds. Sub-clinical mastitis was diagnosed based on the CMT results, specifically the coagulation and viscosity of the mixture. The test outcomes were interpreted as negative (0 or trace), weakly positive (+), clearly positive (++), or strongly positive (+++) following the guidelines provided by Abebe et al. (2016) [2] .

Isolation and identification of S. aureus and E. coli from mastitic milk

In general, 20 µL of milk was streaked from the appropriate samples onto a 5% bovine blood agar (Hi Media) plate. The plate was then incubated at 37 °C for up to 48 hours, and the plate was checked every 24 hours to ensure that the desired bacteria were growing as effectively as possible. Staphylococcus colonies have a distinct look that includes elevated, smooth, yellow, pigmented, and either full or partial hemolysis. Suspect colonies were further subcultured on MSA (Hi Media) or Mannitol Salt Agar (Hi Media) and incubated for 48 hours at 37 °C. Finally, bacterial colonies that fermented MSA were subjected to the tube coagulase, catalase, and Gram's staining tests. Likewise, Eosin methylene blue (EMB) agar and MacConkey agar (Hi Media) were used to further subculture and incubate suspected E. coli colonies, which were spherical, thick, moist, smooth, and greyish white. Gram's staining, along with the Triple Sugar Iron (TSI) test, Indole, Methyl Red, Voges-Proskauer, and Citrate utilization (IMViC) tests, were conducted to biochemically confirm the presence of E. coli.

Antibiotic susceptibility pattern of S. aureus and E. coli

The Kirby-Bauer disk diffusion method was utilized to assess the antibiotic sensitivity of all S. aureus and E. coli isolates against a total of twelve antibiotics. Ampicillin, Ceftriaxone, Ciprofloxacin, Erythromycin, Gentamicin, Oxacillin, Penicillin-G, Streptomycin, Sulfamethoxazole-Trimethoprim, and Tetracycline were among these antibiotics. A pure culture of every identified strain of S. aureus and E. coli was eliminated from nutrient broth for the susceptibility test. The bacterium was then streaked along the surface of Muller-Hinton agar using a cotton swab, and the solution was allowed to dry for three to five minutes. Using clean, sterile forceps, antibiotic disks were then placed on the agar surface and gently squeezed to ensure attachment. The plates were then incubated aerobically for 24 hours at 37 °C. Furthermore, using the meter scale to measure the diameters of the zone of inhibition surrounding the discs to the nearest millimeter, the isolates were categorized as sensitive and resistant to the medications tested based on the manufacturer's chart.

Result and Discussion

Overall samples

In total, 250 cattle were screened for clinical and subclinical mastitis. Of them, 89 (35.60%) (89/250) cattle were found positive for the CMT test and identified as having sub-clinical mastitis. In addition, 12 out of 250 cattle (4.80%) (12/250) were found affected with clinical mastitis showing clinical signs and symptoms (Table 1).

Grading of bovine clinical and sub-clinical mastitis based of CMT

Among 250 lactating cows, 149 animals were found CMT negative, 22 were identified as weakly positive (+) and 35 were distinctly positive (++) and 32 were diagnosed as strongly positive (+++) for sub-clinical mastitis. However, all of the 12 clinical mastitis cases were found to be strongly positive (+++) for the CMT.

Prevalence of S. aureus and E. coli in clinical and sub-clinical mastitis affected cattle

In total, 101 clinical and sub-clinical mastitic samples were cultured with S. aureus being the most prevalent pathogen in both cases, which were 5/12 (41.66%) and 43/89 (48.31%), respectively (Table 1). The prevalence of E. coli was found to be 4/23 (33.33%) and 14/89 (15.73) in clinical and subclinical mastitis affected cows, respectively.

Antimicrobial susceptibility profiles

All the S. aureus and E. coli isolates were found to be multidrug resistant (MDR). Among the S. aureus isolates, the highest resistance was observed for tetracycline (77.08%), oxacillin and ampicillin (70.83%) and gentamicin (64.58%) (Table 2). More than 58% of the isolates exhibited resistance against amoxicillin-clavulanic acid, erythromycin and streptomycin. In the case of E. coli, this exhibits the highest resistance against ampicillin and sulfamethoxazole-Trimethoprim (61.11%) antibiotics. More than 70% of E. coli isolates were sensitive to ceftriaxone, ciprofloxacin and erythromycin (Table 3).

Discussion

In the present study, the overall prevalence of subclinical mastitis at cattle level was 35.60% and the smallest proportion (4.80%) was suffered from clinical mastitis. Finding of present investigation is in agreement with that of Rana et al. (2022) [9] . Zeryehun and Abera (2017) [14] showed comparatively higher prevalence of sub-clinical and clinical mastitis in Eastern Ethiopia, which was 51.8% and 12.5%, respectively. The occurrence of mastitis in lactating cattle varies due to the geographical location, breed, age, stages of lactation, status of the udder, number of parity, immunity, management, hygiene and milking practices in dairy farms (Ashraf et al., 2018 and Rana et al., 2022) [1,9] . However, the predominance of sub-clinical mastitis in the current study area is a concerning issue that negatively impacts the human food chain in addition to causing economic losses due to decreased milk production. Worldwide, S. aureus and E. coli are the most common causative agents of clinical and subclinical mastitis in cattle (Haubert et al., 2017) [6] . However, in both clinical and sub-clinical cases, the most frequently isolated organism was S. aureus, which represented 41.66% and 48.31%, respectively. Finding of present investigation is in agreement with that of Traversa et al. (2015) [13] in Italy and Rana et al. (2022) [9] in Bangladesh and slightly higher in Brazil (56%) (Gundogan et al., 2014) [5] . Moreover, the incidence of environmental fatal mastitis caused by Escherichia coli is dependent on a number of parameters, including farm management, hygienic techniques, and the presence of virulence factors. Surprisingly, any type of mastitis in the mammary gland always first attempts to be treated with antimicrobials to reduce the fatal consequence, especially to control the contagious infections (Rana et al., 2022) [9] . Since the screening of antimicrobial resistance test, both S. aureus and E. coli isolates harboured multiple types of resistance to bacteria. According to different mastitis research, it is focused that β-lactams antimicrobials are most frequently used in both parental and intramammary routes in lactating cows for treatment of mastitis infection (Priyantha et al., 2021) [8] . In the present study, high antimicrobial resistance frequency to the β-lactams penicillin (52.08%), amoxycillinclavulanic acid (58.33%), ampicillin and oxacillin (70.83%) was observed, which is closely related by previous findings of Priyantha et al. (2021) [8] , Haubert et al. (2017) [6] and Rana et al. (2022). According to previous findings, S. aureus and E. coli resistance to penicillin, ampicillin, amoxycillin, and oxacillin is the most common type of antimicrobial resistance among mastitis-causing bacteria (Rana et al., 2020) [10] . Many earlier investigations have revealed the high resistance frequency of E. coli to sulfamethoxazole-trimethoprim (58.33%) (Priyantha et al., 2021) [8] . It was observed that a significant percentage of S. aureus and E. coli strains were susceptible to ceftriaxone (70.83% and 72.22%) and ciprofloxacin (64.58% and 72.22%). These results compatible with those of earlier research studies (Rana et al., 2022; Rana et al., 2020) [9,10] . The broad-spectrum nature of these antimicrobials combined with reduced exposure in dairy farm conditions may account for the high rate of sensitivity to ceftriaxone and ciprofloxacin.

Conclusion

It was concluded that, there was considerable, variations in the prevalence of clinical and sub clinical type mastitis in cattle. AMR prevalence was more common in bovine mastitis pathogens namely S. aureus and E.coli. Overall, antimicrobial resistance to amphicillin, oxacillin and tetracycline was found in mastitis cases. The determination of AMR will facilitate the selection of an appropriate therapeutic approach.

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