Introduction
Bovine mastitis, an inflammation of the mammary gland, poses a significant concern for dairy herds worldwide due to its economic consequences, such as decreased milk production and increased culling rates [1]. In Ethiopia, which has the largest cattle population in Africa, bovine mastitis severely impacts dairy production, particularly in urban and peri-urban commercial farms. A systematic review and meta-analysis of studies on bovine mastitis in Ethiopia found that the prevalence of the disease varies between 3.9% and 73.7% across different regions, with an average prevalence of 43.6% [2].
Mastitis is caused by various bacterial species, with Staphylococcus aureus being the primary pathogen responsible for up to 76% of cases [3–5]. This pathogen reduces milk production and poses serious health risks to cattle [6]. Farmers often use antibiotics to treat mastitis [7], but overusing them can lead to antimicrobial-resistant (AMR) strains, financial losses, and diminished mastitis management benefits [8]. Studies have shown that S. aureus isolated from bovine mastitis often exhibits resistance to multiple antimicrobial agents, including penicillin, amoxicillin, tetracycline, amikacin, gentamicin, and erythromycin [1, 9–11]. The resistance of S. aureus to antimicrobials is a significant concern due to its implication for both animal and public health [10,12].
The ability of S. aureus to develop antibiotic resistance complicates treatment and increases culling rates in affected herds. This scenario not only heightens the economic burden on dairy farmers through rising veterinary costs and reduced milk production but also raises consumer concerns about dairy product safety. AMR in S. aureus related to bovine mastitis is a complex issue involving the production of β-lactamase enzymes [7], multiple resistance genes [13], virulence factors [7], biofilm [14], horizontal gene transfer [7] and the misuse and overuse of antibiotics in dairy farming [11].
Staphylococcus aureus is the primary cause of bovine mastitis in Ethiopia, responsible for up to 48.20% of cases [2]. However, research on the AMR profile of this bacterium in bovine mastitis is scarce. Recent studies revealed that S. aureus isolated from raw cow milk demonstrated resistance to oxacillin, amoxicillin, oxytetracycline, tetracycline, and sulfa [9,15,16]. There have been no recent investigations into the AMR profile of S. aureus in dairy farms around Hawassa, a key dairy production area in Ethiopia. Regular AMR testing is vital for identifying resistant strains, guiding effective treatments, minimizing treatment failures, and preventing the spread of resistant bacteria within herds and humans. Such testing enhances herd health management and promotes food safety and public health through responsible antibiotic use. An antibiotic sensitivity test is crucial for developing a careful and rational approach to the antimicrobial treatment of mastitis in animals [7]. Thus, this study aims to isolate S. aureus from bovine mastitis cases and assess the antimicrobial susceptibility of the isolates.
Materials and Methods
Milk sample collection
A total of 172 milk samples were collected from 95 dairy farms in Hawassa City and its suburbs in southern Ethiopia, all from cows diagnosed with mastitis. Of these, 18 samples were from cows with clinical mastitis, while 154 were from cows with subclinical mastitis. Aseptic procedures for sample collection, as outlined by the National Mastitis Council [17], were strictly followed. Samples were collected before milking; udders and teats were cleaned and dried, and each teat end was scrubbed with a cotton pledge soaked in 70% ethyl alcohol. To prevent recontamination, the teats on the far side of the udder were scrubbed first, using a separate pledge for each teat. The first few streams of milk were discarded, and approximately 10 ml of milk was collected into a sterile universal sample bottle held horizontally. After labeling, each sample was placed in an icebox and transported to the Microbiology Laboratory at the Faculty of Veterinary Medicine, Hawassa University, where they were either cultured immediately or stored at 4°C for up to 24 hours before culturing on standard bacteriological media.
Isolation and identification of S. aureus
Staphylococcus aureus was isolated and identified following the National Mastitis Council guidelines [17]. In refrigerated milk samples, bacteria may concentrate in the cream layer and clump with fat globules. To disperse these, samples were warmed to 25°C for 15 minutes and shaken before plating on standard bacteriological media. A 0.01-ml loop of each milk sample was streaked onto 7% sheep blood agar (Oxoid, Hampshire, England) and Mannitol Salt Agar (MSA) using the quadrant streaking method, and the plates were incubated aerobically at 37°C for 24 to 48 hours. After incubation, the plates were examined for morphological traits, including colony size, shape, color, and hemolytic properties. Staphylococcus aureus was identified by golden-yellow, round, smooth, and shiny colonies exhibiting beta-hemolysis on blood agar while yellow colonies on MSA due to mannitol fermentation. Presumptive colonies from MSA and blood agar were selected, sub-cultured on Tryptic Soya Agar (Oxoid, Hampshire, England), and incubated at 37°C for 24–48 hours to obtain pure cultures. Further confirmation was done based on biochemical assays, including gram staining (+ coccus), catalase test (+), and coagulase test (+). Gram-positive cocci that were catalase and coagulase-positive were confirmed as S. aureus. A sample was deemed positive for S. aureus if at least one colony was identified as such.
Antimicrobial susceptibility test
An antimicrobial susceptibility test was performed on 44 randomly selected S. aureus isolates from a total of 88. The isolates were assessed against 11 antimicrobials commonly used to treat bovine mastitis in Ethiopia, employing the Kirby–Bauer disk diffusion method following the Clinical and Laboratory Standards Institute guidelines [18]. Briefly, bacteria previously identified were inoculated onto blood agar (Oxoid, Hampshire, England) and incubated at 37°C for 24 hours. The isolated colonies were then transferred to 4–5 ml of tryptone soya broth (Oxoid, Hampshire, England) and incubated at 37°C until slight turbidity was observed, typically within 2–8 hours. The bacterial suspension’s turbidity was adjusted to a McFarland standard of 0.5. A 100-μl suspension was spread on Mueller Hinton agar (HIMEDIA, India) using a swab, after which antimicrobial disks were placed aseptically on the agar. Incubation continued at 35°C for 24 hours, with S. aureus ATCC 25923 serving as a control. The following antimicrobial disks (HIMEDIA, India) and concentrations were tested: amoxicillin–clavulanic acid (30 μg), ampicillin (10 μg), cefotaxime (30 μg), ceftriaxone (30 μg), erythromycin (15 μg), gentamicin (10 μg), kanamycin (5 μg), nitrofurantoin (100 μg), penicillin (10 μg), streptomycin (10 μg), and tetracycline (10 μg). The sizes of the zones of inhibition were interpreted as R (resistant), I (intermediate), and S (susceptible), taking into account the breakpoints reported by “Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals” [18].
Results
Bacterial isolation
Of 172 milk samples cultured, S. aureus was confirmed to grow in 51.2% of the samples, amounting to 88 cases. This bacterium was detected in 22.2% of clinical mastitis samples (4 of 18) and 54.5% of subclinical samples (84 of 154). Staphylococcus aureus was isolated from 50 of the 95 herds tested, representing 52.6% of the herds (Table 1).
Antimicrobial susceptibility test
Of the 88 S. aureus isolates from clinical and subclinical mastitis, antimicrobial susceptibility tests were conducted on 44 (50%) isolates against 11 antimicrobials. The results indicated that all the isolates exhibited resistance to one or more of the antimicrobials tested. Staphylococcus aureus was completely susceptible to ceftriaxone and gentamicin (100% each) and highly susceptible to erythromycin (88.6%), streptomycin (88.6%), cefotaxime (72.7%), and nitrofurantoin (72.7%). Conversely, the isolates showed high resistance to ampicillin (84.1%) and penicillin (81.8%), along with tetracycline (36.4%) and amoxicillin–clavulanic acid (34.1%) (Table 2).
A total of 18 distinct patterns of AMR were identified. Of the 44 S. aureus isolates, 19 (43.2%) showed multi-drug resistance (MDR), which involved three or more different classes of antimicrobials. The MDR patterns observed were penicillin-streptomycin-tetracycline, ampicillin-penicillin-streptomycin-tetracycline, ampicillin-kanamycin-penicillin-streptomycin, ampicillin-amoxicillin-streptomycin-tetracycline, ampicillin-amoxicillin-streptomycin-kanamycin-penicillin, ampicillin-erythromycin-penicillin-streptomycin-tetracycline, and ampicillin-amoxicillin-penicillin-streptomycin-tetracycline (Table 3).
Discussion
Global studies consistently identify S. aureus as a common cause of bovine mastitis [1,3–5]. In this study, S. aureus was isolated from over half (51.2%) of the cultured milk samples, including clinical and subclinical cases. This rate exceeds those in previous studies: 15.52% [19], 30.6% [15], 46.5% [20], 5.5% [21], 33.05% [9], and 28.1% [22]. It also surpasses findings from other countries, such as 28% in India [23], 30.32% in Pakistan [11], 11.3% in China [24], and 50% in Italy [14]. However, a higher isolation rate of 60% and 76% was reported in Mexico [25] and South Africa [4], respectively. The variation in S. aureus isolation rates between the present study and previous ones can be attributed to various factors, including differences in cow breed and parity, lactation stage, udder and teat hygiene, herd size, milking practices, and contamination of bedding materials used [7,13,26,27]. In addition, S. aureus is known for its robustness in different environmental conditions, enabling it to survive extreme temperatures and moisture levels, which greatly contributes to its persistence in dairy farm environments [13].
Table 1.
Isolation rate of S. aureus from clinical and subclinical mastitis.
| Form of mastitis | Number of milk samples cultured | Number of positive samples | Isolation rate (%) |
|---|---|---|---|
| Clinical | 18 | 4 | 22.2 |
| Subclinical | 154 | 84 | 54.5 |
| Total | 172 | 88 | 51.2 |
Table 2.
Antimicrobial susceptibility of S. aureus isolated from bovine mastitis (n=44).
| Antimicrobial | Disc content | Disk diffusion inhibition zone diameters (mm) | ||
|---|---|---|---|---|
| Resistant (%) |
Intermediate (%) |
Susceptible (%) |
||
| Ampicillin | (10 μg) | 84.1 | _ | 15.9 |
| Amoxicillin–clavulanic acid | (30 μg) | 34.1 | _ | 65.9 |
| Cefotaxime | (30 μg) | 0 | 27.3 | 72.7 |
| Ceftriaxone | (30 μg) | 0 | 0 | 100 |
| Erythromycin | (15 μg) | 2.3 | 9.1 | 88.6 |
| Gentamicin | (10 μg) | 0 | 0 | 100 |
| Kanamycin | (5 μg) | 9.1 | 36.4 | 54.5 |
| Nitrofurantoin | (100 μg) | 0 | 27.3 | 72.7 |
| Penicillin | (10 μg) | 81.8 | _ | 18.2 |
| Streptomycin | (10 μg) | 0 | 11.4 | 88.6 |
| Tetracycline | (10 μg) | 36.4 | 13.6 | 50 |
| Mean | 28.2 | 11.2 | 60.6 | |
Table 3.
Percentage and frequency of AMR pattern of S. aureus (n=44) for selected antimicrobials agents.
| No. of antimicrobials | Name of antimicrobials | Frequency | Percentage |
|---|---|---|---|
| 1 | Strep | 2 | 4.55 |
| 2 | Amp, Strep Amp, Amox Amp, Strep Amp, Pen Pen, Strep |
2 1 1 1 1 |
4.55 2.27 2.27 2.27 2.27 |
| 3 | Amp, Pen, Strep Pen, Strep, Tetra Amp, Amox, Pen Amp, Pen, Tetra Amp, Amox, Strep |
6 4 2 1 1 |
13.64 9.09 4.55 2.27 2.27 |
| 4 | Amp, Amox, Pen, Strep Amp, Pen, Strep, Tetra Amp, Kan, Pen, Strep Amp, Amox, Strep, Tetra |
7 8 2 1 |
15.91 18.18 4.55 2.27 |
| 5 | Amp, Amox, Kan, Pen, Strep Amp, Ery, Pen, Strep, Tetra Amp, Amox, pen, Strep, Tetra |
2 1 1 |
4.55 2.27 2.27 |
The study revealed significant variability in the antimicrobial susceptibility of S. aureus isolates from bovine mastitis. The isolates showed varying resistance to nine of the 11 antimicrobials tested. However, all of the S. aureus isolates were found to be 100% susceptible to gentamicin and ceftriaxone. The high rate of susceptibility to gentamicin is in quite agreement with other studies that reported 100% susceptibility of S. aureus to gentamicin [9,21]. However, other studies have reported varying level of resistance to this drug from 36% to as high as 85.8% [15,28].
Ceftriaxone is a broad-spectrum third-generation cephalosporin antibiotic that effectively targets various bacterial infections in humans, including Gram-positive and Gram-negative bacteria [29]. Although it is seldom used in veterinary medicine for bovine mastitis, this study found all S. aureus isolates to be susceptible, indicating that ceftriaxone could serve as a potential treatment. It has demonstrated efficacy in treating staphylococcal mastitis in crossbred cows with a single intravenous dose of 20 mg/kg body weight [30].
Erythromycin and streptomycin were the second most effective drugs in this study, clearing 88.6% of the tested S. aureus isolates. This aligns with Dabele et al. [21] who reported 100% susceptibility to erythromycin. However, it contrasts with an Italian study [14] that found a high resistance rate of 98.7% for erythromycin. Staphylococcus aureus isolated from bovine mastitis shows varying levels of susceptibility to streptomycin. The high susceptibility rate to streptomycin found in this study is in line with the study conducted by Ahmed et al [16] who reported that 71.8% of the isolates were found to be susceptible to streptomycin.
While some studies report that S. aureus showed resistance to cefotaxime ranging from 58.8% to 100% [16,25], we found it to be the third most effective antimicrobial, with 72.7% of isolates susceptible. Cefotaxime, such as ceftriaxone, is a third-generation broad-spectrum cephalosporin commonly used in veterinary medicine for treating bacterial infections in animals, particularly dogs and cats. The high rate of intermediate to full susceptibility indicates its potential as an antibiotic for treating bovine mastitis, especially given the prevalence of MDR bacteria, even though it is not routinely used for such infections in large animals [31].
Nitrofurantoin, which ranked as the third most effective antimicrobial, demonstrated the same susceptibility to S. aureus as cefotaxime. This synthetic chemotherapeutic agent is commonly used in veterinary medicine to treat urinary tract infections caused by bacteria such as S. aureus in small animals such as dogs and cats [32]. However, its effectiveness in treating bovine mastitis has not yet been established. Nevertheless, the rarity of clinical resistance to nitrofurantoin [32], coupled with the observation that S. aureus isolated from mastitic milk samples in this study exhibited a high rate of sensitivity, suggests that it may hold promise as an antimicrobial for bovine mastitis treatment.
Our investigation revealed a significant resistance of S. aureus to key beta-lactam antibiotics, with 84.1% of isolates resistant to ampicillin and 81.8% to penicillin. These findings align with prior studies in Ethiopia, which reported 97.4% to 100% resistance to penicillin [9,16], and in Mexico showing 100% resistance to both penicillin and ampicillin [26]. Our results are consistent with the global systematic reviews of AMR in S. aureus from bovine mastitis, highlighting widespread resistance to penicillin [33]. Factors contributing to this high resistance include the production of β-lactamase (BlaZ) enzymes that inactivate these antibiotics, genetic mutations in the bacterial strains that affect antibiotic susceptibility [34], and the selective pressure from the extensive use of these antibiotics in veterinary practices. In addition, the biofilm formation by S. aureus contributes to its persistence and resistance in the mammary gland tissue [35].
The study found that 34.1% of S. aureus isolates were resistant to amoxicillin–clavulanic acid, which is lower than the resistance rates reported in other studies on S. aureus in milk samples from bovine mastitis: 75% by Yimana and Tesfaye [15], 42.5% by Haq et al. [11], and 64% by Shahzad et al. [28] for amoxicillin alone. Staphylococcus aureus from bovine mastitis often shows significant amoxicillin resistance, mainly due to β-lactamase production, an enzyme that degrades β-lactam antibiotics. This enzyme is usually encoded by transferable plasmids among bacteria, promoting the spread of resistance [36]. The lower resistance in this study is likely due to combining amoxicillin with clavulanic acid, a β-lactamase inhibitor that enhances the efficacy of the drug [34]. Supporting this, another Ethiopian study reported 100% susceptibility of S. aureus isolates to amoxicillin combined with clavulanic acid [21].
In this study, 36.4% of S. aureus isolates showed resistance to tetracycline, lower than the 66.7% [15] and 69.2% [16] reported in Ethiopia. Global research has identified varying resistance levels in S. aureus from bovine mastitis, with some regions reporting rates as high as 40%–86.5% [14,25,33,37], all exceeding the current finding. The emergence of resistance genes and the misuse of antibiotics in veterinary medicine significantly contribute to this issue. Conversely, a very high susceptibility rate of 100% to tetracyclines has been noted in S. aureus from bovine mastitis [21]. This significant variation in tetracycline susceptibility rates across studies is likely attributed to the differing frequency of the drug’s use in treating bovine mastitis in various regions.
MDR is defined as an isolate that is not susceptible to at least one agent in at least three antimicrobial classes [38 ]. Based on this definition, MDR was observed in 43.2% S. aureus in the present study that is alarming. This finding is considerably higher from the 11.6% finding in China [24], although it is somewhat lower than the 50% [10] and 52% [28] findings reported from Pakistan. In contrast, Dabele et al [21] from Ethiopia reported that none of the S. aureus isolates tested were MDR in their study.
Limitations
MDR in S. aureus causing bovine mastitis poses a significant issue in veterinary medicine. Various factors contribute to this antibiotic resistance. Research shows that S. aureus isolates often harbor multiple resistance genes, including mecA, tetK, blaZ, and aacA-aphD, leading to significant AMR [7,9,11,13]. In addition, S. aureus produces several virulence factors, such as hemolysins, leukocidins, enterotoxins, and superantigens, enabling it to evade the host’s immune system and establish infections [7]. Of note, its strong biofilm formation further protects it from antibiotics and immune responses [14,23]. Furthermore, the misuse and overuse of antibiotics in dairy farming create selection pressure that fosters MDR strains [11]. Staphylococcus aureus can also acquire resistance genes from other bacteria via horizontal gene transfer methods such as conjugation, transformation, and transduction [7]. The present study found that a significant proportion of S. aureus isolates exhibited MDR. However, the use of culture methods and the absence of advanced techniques such as molecular assays, due to financial constraints, hindered the detection of resistance genes and biofilm-producing strains. Addressing these limitations is crucial for future research on S. aureus antimicrobial susceptibility.
Conclusion
This study highlights that S. aureus is a significant cause of mastitis in dairy farms, isolating it from over half of mastitis-positive milk samples analyzed. The high isolation rate raises public health concerns regarding the consumption of raw milk and its products. Among the isolates tested, 39.4% showed varying resistance to nine of eleven antimicrobials, with MDR observed in 43.2% of S. aureus isolates. The highest resistance was noted against ampicillin and penicillin, while S. aureus was susceptible to gentamicin and ceftriaxone, followed by erythromycin and streptomycin. The significant isolation rate of S. aureus, coupled with considerable MDR to commonly used antimicrobials, underscores critical implications for veterinary practices and public health. Thus, a comprehensive approach integrating improved management, ongoing education for veterinarians and dairy farmers, prudent antimicrobial usage, and regular resistance monitoring is essential to combat the growing threat of AMR posed by S. aureus and other bacteria in bovine mastitis.
Acknowledgments
The authors are grateful to the owners of all dairy farms who participated in the study for their willingness and cooperation during sample collection.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Funding
The authors didn’t receive funding for this research.
Author contributions
This work was conducted with the contribution of all authors. RA conceptualized and designed the study, supplied the antimicrobial drug discs, and wrote the manuscript. HH collected field data, performed S. aureus isolation and antimicrobial susceptibility tests, and conducted data analysis and interpretation. All authors have reviewed and approved the final manuscript for publication.
Data availability statement
The raw data supporting the conclusions of this article will be made available by the authors on request.
