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Virulence genes and antibiotic resistance profiling of staphylococcus species isolated from mastitic dairy cows in and around Bahir dar, Ethiopia

BMC Microbiology volume 25, Article number: 210 (2025) Cite this article

Abstract

Background

Mastitis is one of the primary causes of economic and production losses in the dairy cattle industry. Bacterial infections are the most significant contributors to bovine mastitis, with Staphylococcus species among the most prevalent and challenging pathogens. This issue is especially severe in low- and middle-income countries, including Ethiopia, where a comprehensive understanding of Staphylococcus species in clinical and subclinical mastitis remains poorly understood. This is particularly true in the regions surrounding Bahir Dar, where comprehensive data on the genetic determinants of virulence and resistance in Staphylococcus species causing bovine mastitis are notably lacking. The lack of such molecular insights hampers the development of targeted therapeutic and preventive strategies for managing mastitis in the region. Therefore, the present study aimed to investigate the virulence gene profiles and antimicrobial resistance (AMR) patterns of Staphylococcus species isolated from mastitic dairy cows in and around Bahir Dar, Ethiopia.

Methodology

A cross-sectional study was conducted from March 2023 to December 2023 to investigate the molecular characteristics of Staphylococcus species and their antimicrobial resistance profiles in dairy cows with mastitis. A total of 150 lactating cows from 21 farms were included in the study, with 600 milk samples collected from the four-quarters of each cow. The samples were screened via the California mastitis test and physical examination. Staphylococcus species isolates were identified and single-plex PCR was used to detect virulence genes. The antimicrobial resistance profile of the isolates was determined via the Kary–Bauer disk diffusion method.

Results

The overall quarter-level mastitis incidence was 19.83% (119/600). Among 119 mastitis-positive samples, 80 samples were bacteriologically confirmed to harbor Staphylococcus species with eight different Staphylococcus species, of which Staphylococcus chromogenes was the most prevalent isolate (19%), followed by S. aureus, S. hyicus and S. epidermidis (15%), S. hemolyticus (11%), S. simulans and S. xylosus (10%), and S. intermedius (5%). Seven distinct virulence genes were identified with varying frequencies: Coa (35%), seb (33.33%), mecA (31.67%), icaD (31.67%), Hla (20%), Hlb (10%), and sea (8.3%). The icaD and seb genes were observed in all 8 species with respective percentages (S. hemolyticus (62.5, 37.5), S. aureus (44.44, 55.55), S. hyicus (44.44, 44.44), S. epidermidis 2 (22.22, 44.44), chromogenes (9.1, 9.1), S. intermidius (33.33, 33.33), S. simulance (16.67, 16.67) and S. xylosus (16.67, 16.67). Both the Hla and Hlb genes were detected in the same three distinct species, with percentages of S. aureus (44.44; 22.22%), S. hemolyticus (42.5; 25%) and S. hyicus (55.55; 22.22). S. aureus exhibited the highest proportion of mecA-positive isolates, with 6 out of 9 isolates (66.67%) carrying the gene. All the isolated Staphylococcus species were 100% resistant to penicillin, and except for S. chromogenes and S. xylosus, the remaining 6 species of Staphylococcus also exhibited 100% resistance to tetracycline. Among all MDR isolates, 6/9 (66.7%) S. aureus, (5/8; 62.5%) S. hemolyticus, and (6/9; 66.7%) S. hyicus were resistant to up to seven classes of antibiotics. A lower frequency of MDR isolates was detected among S. simulans and S. xylosus (both at 2/6; 33.33%), resistant to up to five antibiotics.

Conclusions

Among the identified Staphylococcus species, S. chromogenes emerged as the dominant isolate. All eight isolated species harbored two or more virulence genes, with nearly one-third of the isolates carrying the mecA gene, underscoring their pathogenic potential in causing bovine mastitis. Furthermore, all the Staphylococcus isolates in this study were resistant to penicillin and were multidrug resistant.

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Introduction

Mastitis is one of the major causes of economic losses in dairy cattle industries. Bacteria are the most common cause of bovine mastitis; Staphylococcus species play a particularly significant role. The genus Staphylococcus is divided into 2 subgroups on the basis of its ability to coagulate rabbit plasma: coagulase-positive staphylococci and coagulase-negative staphylococci. The coagulase positive species tha couse bovine mastitis was listied by Kerro Dego O and Vidlund J [1] are: S. aureus [2], S. intermedius [3] and S. hyicus [4]. Among CPSs, S. aureus is the most frequent causative agent of clinical or subclinical mastitis in the dairy industry [5]. It is also a foodborne pathogen and the leading cause of foodborne food intoxication worldwide [6]. The CNS is difficult to identify at the species level in routine diagnostics and has often been of minor importance in the dairy industry. However, recent studies on mastitis incidence have revealed that the CNS is a major cause of mastitis in some countries [7]. Staphylococcus chromogens, S. epidermidis, S. hemolyticus, S. simulans, and S. xylosus are commonly isolated CNS species that are likely to cause mastitis [8].

The multidimensional problems of staphylococcus species are associated with various virulence factors used for colonization that affect the host [9, 10]. Among these genes, the toxic shock syndrome toxin-1 gene (TSST-1), enterotoxin gene [11], methicillin resistance gene (mecA), clumping factor A gene (clfa), intracellular adhesion or biofilm-forming gene (icaD), hemolysin toxin (Hlb), coagulation factor gene and panton-valentine leukocidin (PVL, encoded by the lukF/lukS-PV genes) [11, 12] are frequently reported in many studies because they are the most typical virulence factors responsible for the widespread pathogenesis of the udder and the teat canal [13].

The effective treatment of bovine mastitis depends on the antimicrobial resistance pattern of the pathogen, the type of mastitis, the cattle breed, and the treatment regimen [14]. The antimicrobial resistance of staphylococci is attributed to various resistance determinants, such as the genes blaZ and mecA for β-lactam resistance, the tetracycline resistance gene for tetracycline resistance, and erythromycin ribosome methylase for macrolide resistance. Therefore, antimicrobial resistance surveillance is important to ensure optimal results of antimicrobial use and minimize the risk of developing and spreading antimicrobial resistance [15]. Over the years, several researchers have studied the occurrence of mastitis in dairy herds in Ethiopia. According to the most recent published studies, the incidence of cow-level mastitis is estimated to be within the range of 23.2–81.1% for the country [16, 17]. Consequently, the disease has been documented as one of the major constraints of the dairy sector that needs attention. Most of the previous studies in Ethiopia focused on investigating the prevalence and few risk factors for mastitis at the cow level, and the molecular characteristics of virulence genes of staphylococcus species from dairy cows in and around Bahir Dar have not yet been thoroughly studied and documented. To provide evidence for the development of appropriate treatments and control measures for bovine mastitis, understanding the molecular characterization of the virulence genes of Staphylococcus species and their antibiotic resistance patterns is essential. Therefore, the present study was designed with the objectives of molecular detection of virulence genes and antimicrobial resistance profiles of Staphylococcus species isolated from bovine mastitis.

Materials and methods

Description of the study area

The study was conducted in and around Bahir Dar town. It is the capital city of the Amhara region and is located approximately 565 km northwest of Addis Ababa between 11°27´1.82 to 11°39´ 7.56 N and 37°16´ 36.75 E to 37°31´48.06 E. In the study area, there was a total of 84 dairy farms. According to the classification system proposed by Aweke and Mekibeb [18], these farms were categorized into three groups on the basis of herd size: 24 were classified as large-scale farms, each with 21 or more lactating cows; 38 as medium-scale farms, with herd sizes ranging from 11 to 20 lactating cows; and 22 as small-scale farms, each with 10 or fewer lactating cows.In a study conducted by Yayeh et al. [19], the average daily milk yield (DMY) was recorded as 7.8 L in the study area." According to Yeserah et al. [20], , there is a notable lack of standardized hygienic practices followed by producers, transporters, and processors throughout the milk production chain in the study area. The dairy value chain encompasses a broad array of interconnected stages, including the supply of inputs and services, milk production, aggregation, processing, marketing, and final consumption. It involves multiple stakeholders, such as input suppliers, veterinarians, extension agents, marketers, and financial institutions, each playing a critical role in ensuring the smooth functioning of the system.

Study animals

Dairy cows (n = 150) managed under intensive or semi-intensive systems in the study areas were selected for sample collection. Milk samples were collected from all lactating dairy cows from 21 dairy farms.

Study design and sampling methods

A cross-sectional study design was employed from March 2023 to December 2023 to investigate virulence genes and antimicrobial resistance profiles of Staphylococcus species from mastitic dairy cows. In the study area, 84 dairy farms were managed in closed houses with different floor types, such as mud, wood, and stone; with different wall types, such as earthen, wood, cement, and stone; and with different roof types, such as iron sheets and plastic sheets. Dairy cows were also managed under zero grazing. Most dairy farms clean twice a day and have ventilation. The main feeds used by dairy farms are crop residue, hay, and concentrated feed. The cows were hand milked twice per day. The breeding method used in dairy farms is artificial insemination. From 84 dairy farms, 21 farms (25%) were selected by using a simple random technique for sample collection. From the selected farms, all lactating cows (n = 150) were included for sampling. This included both clinically symptomatic and asymptomatic (subclinical) cows that were assessed for mastitis. A total of 600 teat quarters were examined through clinical assessment and subclinical examination. Samples for clinical mastitis were collected on the basis of the presence of observable clinical signs of mastitis, including swelling, redness, heat, and abnormal milk characteristics such as clots, discoloration, or reduced yield, whereas for subclinical mastitis, the California mastitis test (CMT) was used as a screening tool.

Sample collection and transportation

Milk samples from all quarters except blind teats were collected from 150 dairy cows. Before collection, the quarters were soaked with 70.0% ethanol and dried via a towel. Initially, two drops of milk were discarded, and then 15 ml of milk from each of the four quarters was collected aseptically in sterile plastic tubes. Immediately after collection, milk samples were subjected to CMT to diagnose subclinical mastitis. The California mastitis test was conducted to identify the presence of subclinical mastitis according to the standard procedure described by Hoque et al. [21]. All positive samples were collected aseptically into sterile plastic tubes (15 mL/sample) following the procedures of the National Mastitis Council [22] and transported to Bahir Dar Animal Disease Surveillance, Investigation, and Diagnostic Laboratory Department of Microbiology via an ice box. A total of 119 bovine milk samples were collected and subjected to microbiological analysis to determine the presence of Staphylococcus species. Primary screening revealed that 80 of these samples tested positive for Staphylococcus species, 58 had subclinical mastitis, and 22 had clinical mastitis. After a two-month preservation period, the previously preserved samples (80 positive for Staphylococcus) were recultured for molecular techniques to detect virulence genes. However, during this reculturing process, 20 samples failed to demonstrate bacterial growth (6 samples from the clinical mastitis group and 14 samples from the subclinical mastitis group). Therefore, a total of 60 samples (80 initial positive samples minus the 20 that did not grow) were subsequently included for further molecular analysis.

Isolation and identification of Staphylococcus spp.

Mastitis-positive samples were further analyzed bacteriologically through culturing, Gram staining, and biochemical identification (e.g., catalase, coagulase, and Voges–Proskauer (VP) tests) to confirm the species-level identification of Staphylococcus. The bacteria were identified on the basis of morphology, hemolytic patterns, growth on mannitol salt agar, and other biochemical tests, as described by Thorberg and Branstrom [23] and Gebremedhin [24]. All presumptive isolates were stored at − 20 °C for further molecular identification. The isolates were subsequently transported to the Addis Ababa University Institute of Biotechnology for molecular detection via virulence gene profiling and antibiotic resistance testing.

Bacterial genomic DNA extraction

Genomic DNA was extracted from colonies of overnight cultures of Staphylococci on tryptone soy broth (TSB) via the boiling method [21]. In brief, a pure bacterial culture from mannitol salt agar was subcultured in tryptone soy broth (TSB). Each milliliter of broth culture was placed in a separate Eppendorf tube and centrifuged at 8,000 rpm for 5 min. The supernatant was discarded, and any closing liquid was eliminated by soaking with wipes. The pellet was collected and replenished with 200 µl of cold TE, followed by shaking with fingers to dissolve the pellet. Then, 400 µl of digestion solution was added to the sample containing 200 µl of TE sample from the previous step. After thorough mixing, 3 µl of proteinase K solution was added, and the mixture was incubated at 55 °C for 10 min. To obtain RNA-free genomic DNA, 5 µl of RNase was added. Then, 260 µl of 100% ethanol was added, and the mixture was transferred onto an EZ-10 spin column and spun at 10,000 rpm for 2 min. After discarding the supernatant, 500 µl of washing solution was added, and the mixture was spun at 10,000 rpm for 2 min twice. The spin column was placed into Eppendorf tubes, and 50 µl of elution buffer was added and incubated at 50 °C for 2 min. After incubation, the Eppendorf tube was spun at 10,000 rpm for 2 min. Finally, approximately 50 µl of bacterial genomic DNA was collected. DNA quantity and purity were determined with a Nanodrop 2000 (Thermo Fisher, USA) by measuring the 260/280 absorbance ratios, and the samples were stored at − 20 °C.

Molecular detection of virulence genes

The virulence genes of Staphylococcus species isolated from mastitic cows were detected via conventional single-plex PCR for the presence of virulence genes on the basis of methods described by Momtaz et al. [25], Wang et al. [26], and Kot et al. [27]. The target genes, primer sequences, and target fragments of the PCR products are indicated in Table 1. The PCRs were performed in a final volume of 20 μL of reaction mixture consisting of 3 μL [4] of genomic DNA, 0.5 μL of each primer, 11.6 μL of nuclease-free water, and 0.2 μL of qPCR (Tiangen Biotech, China: 0.1 U of Taq polymerase/μL), 0.2 μL of dNTPs, 2 μL of buffer, and 2 μL of MgCl2. A Prima 96 thermocycler (HiMedia, Laboratories Pvt. Ltd., India) was used for the amplification of virulence genes, and the cycling conditions were as follows: initial denaturation at 95 °C for 5 min; 35 cycles of denaturation at 95 °C for 30 s, annealing at a specific temperature for 45 s, and primer extension at 72 °C for 1 min; and a final extension at 72 °C for 8 min (Table 2).

Table 1 The sequences of primers used for molecular identification
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Table 2 Amount of PCR Component in µl and annealing temperature in ℃
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Electrophoresis and gel documentation

Agarose gel (2% w/v) (HiMedia Laboratories Pvt. Ltd., India) was made by heating the appropriate amount of agarose with 30 ml of 1X Tris–acetate EDTA (TAE) buffer in a 500 ml Erlenmeyer flask. The warm agarose mixture was poured into a plastic holder with a suitable comb and allowed to stand at room temperature for 30 min. A mixture of PCR product (5 ml) and 6X gel loading dye (2 ml) was loaded in separate wells on the submerged gel. The standard molecular weight marker 100 bp DNA Ladder was also loaded in one well. A voltage of 120 V was applied across the gel until the tracking dye migrated to the appropriate distance. The gel was removed, and the DNA bands were visualized under ultraviolet illumination and photographed with a gel documentation system (UVITEC, Cambridge, UK) under ultraviolet (UV) illumination. The molecular sizes of the DNA bands were analyzed according to the molecular weight of the DNA ladder [36].

Antimicrobial resistance profile of diverse Staphylococcus spp.

Antimicrobial resistance testing of staphylococcal isolates was performed via the disc diffusion method on Mueller‒Hinton agar (HiMedia, Mumbai) according to the guidelines of the Clinical Laboratory Standards Institute [37] against the 10 antibiotics. Antimicrobial discs (all from Oxoid, UK) with the following disc concentrations were used for antimicrobial susceptibility tests: gentamicin (GEN, 10 µg), ampicillin (AMP, 10 µg), erythromycin (E, 10 µg), trimethoprim (W, 5 µg), ciprofloxacin (CIP, 5 µg), norfloxacin (NOR, 10 µg), penicillin G (P10, 1U), cefoxitin (FOX, 30 µg), clindamycin (CD, 2 µg), and tetracycline (TE, 30 µg). The selection of antibiotics for testing was based on the guidelines of the Clinical and Laboratory Standards Institute (CLSI), which provides standard recommendations for antimicrobial susceptibility testing for staphylococcus species. The antibiotics chosen were also those commonly available on the local market and frequently used to treat bovine mastitis in Ethiopia. This approach ensures that the study findings are relevant to the actual practices and challenges faced by dairy farmers and veterinarians in the region. The standardized bacterial inoculum (approximately 1.5*108 cfu/mL) was prepared by adding the bacterial colony to 0.85% normal saline until the suspension was equivalent to the 0.5 McFarland standard. A sterile cotton swab was used to streak the standardized bacterial suspension onto Mueller–Hinton agar (MHA) plates.

The bacterial isolates were classified as susceptible, intermediate, or resistant according to the manufacturer’s instructions. Resistant Staphylococcus species are defined as those resistant to at least one antimicrobial drug, whereas those resistant to three or more antimicrobial categories are defined as multidrug-resistant Staphylococcus species [38]. The selected antibiotic discs were placed 24 mm apart on MHA plates and incubated at 37 °C for 24 h. The zones of inhibition produced by the antimicrobial discs were measured, and the zones of inhibition measured were compared with the standards established at the Clinical Laboratory Standards Institute [37].

Data management and analysis

After coding, the data were entered into an MS Excel spreadsheet and checked for accuracy. The data were recorded in a Microsoft Excel spreadsheet at each step of the laboratory work, from the isolation and identification process to the molecular and antibiotic resistance tests. STATA version 16 software was used to analyze the results of the study. Descriptive statistics were used to analyze the isolation rates, proportions of antimicrobial resistance, and virulence gene profiles of the Staphylococcus species isolates.

Results

Prevalence of Staphylococcus species

A total of 600 teat quarters of cows were examined via clinical examination and CMT methods. In this study, a total of 119 (19.83%) quarter-level mastitis-positive samples were analyzed, comprising 33 clinically positive samples (5.5% of the total) and 86 subclinically positive samples (14.33% of the total). Among the 33 clinically positive samples, 22 samples (3.67%) were identified as being caused by Staphylococcus species. Similarly, of the 86 subclinical positive samples, 58 samples (9.67%) were also found to be associated with Staphylococcus species. Consequently, the incidence of cow-level clinical mastitis was 16%, and that of subclinical mastitis was 32.7%. The aggregate cow-level mastitis prevalence was thus 48.7%, indicating that nearly half of the cows in the herd were affected by some form of mastitis.

Among the 119 mastitis-positive samples, 80 samples were bacteriologically confirmed to harbor Staphylococcus species. Among the isolated Staphylococcus species, S. chromogenes was the most prevalent species (19%), whereas S. intermedius was the least prevalent isolate (5%) (Fig. 1).

Fig. 1
figure 1

The frequency of Staphylococcus spp. in milk samples

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Detection of virulence genes

The detection of virulence genes was conducted by using gene-specific primers (Table 1) in 60 Staphylococcus isolates. Among the tested isolates, seven toxin genes were detected: Coa, icaD, Hlb, Hla, sea, seb, and mecA. From eight isolated species of Staphylococcus, S. hyicus contained all seven toxin genes, and from S. hemolyticus and S. aureus, six toxin genes were extracted. On the other hand, S. simulans and S. chromogens contained only 2 toxin genes (Table 4). Thirty-six (60%) of the isolates were positive for one or more virulence genes, and 24 (40%) of the isolates carried two or more virulence genes. The Coa gene was the most predominant toxin gene (35%), followed by seb (33.33%). The prevalence of the methicillin resistance genes (mecA) and icaD was 31.67%. Other virulence genes detected included Hla, present in 12 isolates (20%), and Hlb, found in 6 isolates (10%). The sea gene was the least frequently observed gene and was detected in only 5 isolates (8.33%).

In terms of the coagulation status of the tested isolates, coagulase-positive Staphylococcus species presented higher percentages of mecA (18.33%) and Hla (11.67%) than coagulase-negative species did (13.33% and 8.33%, respectively). Coagulase-negative Staphylococcus species, however, presented a greater presence of the icaD gene (18.33%) than did coagulase-positive species (13.33%). The icaD gene was most prevalent in subclinical isolates, with 14 isolates (23.33%) testing positive. The sea was also more predominant in subclinical isolates. However, the seb and mecA genes were more commonly detected in subclinical isolates and coagulase-positive Staphylococcus than in clinical isolates and coagulase-negative Staphylococcus.

On the other hand, the study revealed the absence of the following virulence genes across all 60 isolates: Panton-Valentine Leukocidin (PVL), Toxic Shock Syndrome Toxin 1 (TSST-1), mecC (a variant of methicillin resistance), and Enterotoxin C (seC). In general, as shown below (Tables 3 and 4), icaD and sea bacteria were more predominant in subclinical isolates and coagulase-negative isolates than in clinical isolates and coagulase-positive isolates, whereas seb and mecA genes were more common in subclinical isolates and coagulase-positive isolates than in clinical isolates and coagulase-negative isolates.

Table 3 Toxin gene profiles of clinical and subclinical milk samples (N = 60)
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Table 4 Distribution of virulence genes among different Staphylococcus spp. (N = 60)
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The Coa gene, which encodes for coagulase production, was examined in all 60 Staphylococcus isolates, and the results revealed that 21 isolates (35%) were positive for the gene. The Coa gene was amplified from three distinct Staphylococcus species: S. aureus 9 (100%), S. intermedius 3 (100%), and S. hyicus 9 (100%).

The amplification of the icaD gene was observed in all 8 different species, with an overall value of 31.67%; S. hemolyticus 5 (62.5%) S. aureus and S. hyicus 4 (both at 44.44), S. epidermidis 2 (22.22%), followed by one isolate belonging to each of the four species named S. chromogenes (9.1%), S. intermidius (33.335), S. simulance (16.67%) and S. xylosus (16.67%).

The investigation into antibiotic resistance genes in the present study focused on the detection of mecA and mecC, both of which are crucial markers for methicillin resistance in Staphylococcus species. The mecC gene, which confers resistance through an alternative penicillin-binding protein (PBP2c), was absent in all the isolates, indicating that this less common variant of methicillin resistance was not present in the bacterial strains examined. Conversely, the mecA gene, which encodes the altered penicillin-binding protein PBP2a, which is responsible for methicillin resistance, was detected in 19 isolates (31.67%). These isolates were distributed across five distinct Staphylococcus species, with varying frequencies of mecA prevalence: S. aureus exhibited the highest proportion of mecA-positive isolates, with 6 out of 9 isolates (66.67%) carrying the gene. S. hemolyticus also demonstrated a significant prevalence of mecA-positive isolates, with 4 out of 8 isolates (50%) showing resistance. S. epidermidis, another CNS species, presented 4 out of 9 isolates (44.4%) with the mecA gene. S. hyicus, which is typically regarded as less clinically significant in terms of antibiotic resistance, presented 4 out of 9 isolates (44.4%) with mecA. S. chromogenes, a species frequently implicated in bovine mastitis, presented the lowest prevalence of mecA positivity, with only 1 out of 11 isolates (9%) exhibiting the gene.

Among the three enterotoxin genes, the seb and sea genes were detected at percentages of 33.33% and 8.33%, respectively, whereas the sec gene was not detected. The amplification of the seb gene was observed in 8 different species: S. aureus (n = 5 (55.55%)), S. hyicus (n = 4 (44.44%)), S. epidermidis (n = 4 (44.44%)), and S. hemolyticus (n = 3 (37.5%)), followed by one isolate belonging to each of the following four species: S. chromogenes (9.1%), S. intermidius (33.335), S. simulance (16.67%) and S. xylosus (16.67%). The amplification of the sea gene was observed in four distinct species: S. epidermidis (n = 2 (22.22%)), followed by one isolate belonging to each of the following three species: S. hemolyticus (n = 1 (12.5%)), S. hyicus (n = 1 (11.11%)), and S. xylosus (16.67%).

Among the total 80 presumptive isolated Staphylococcus species, 36 (45%) presented hemolytic characteristics on blood agar. Among these compounds, 27 (33.755%) were alpha-hemolytic, whereas 9 (11.75%) were beta-hemolytic. . Among the 12 (20%) Hla virulence genes, 8 were from alpha agar, and 4 were from beta-hemolytic reactions on blood agar. A total of 6 (10%) Hlb virulence genes, 4 from alpha agar, and 2 from beta-hemolytic production on blood agar, but none of the hemolytic virulence genes were detected via the gamma hemolytic reaction. Both the Hla and Hlb genes were detected in the same three distinct species, with percentages of S. aureus (44.44%; 22.22%), S. hemolyticus (42.5%; 25%) and S. hyicus (55.55%; 22.22%).

Antimicrobial resistance profiles of Staphylococcus species

The antimicrobial resistance profiles of various Staphylococcus species are presented in Table 5. The antimicrobial resistance of the staphylococcal isolates to 10 antimicrobial agents was evaluated. In this study, all the isolated Staphylococcus species were found to be resistant to penicillin (100%). All the S. aureus, S. intermedius, S. epidermidis, S. hyicus, S. simulans, and S. hemolyticus isolates exhibited 100% resistance to tetracycline. Trimethoprim resistance occurred in 100% of the S. simulans, S. hemolyticus, and S. xylosus isolates.

Table 5 Antimicrobial resistance profiles of different Staphylococcus spp (N = 60)
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Despite their high resistance to penicillin and tetracycline, norfloxacin, gentamicin, ciprofloxacin, and clindamycin remain effective for certain species, although with some variability: Staphylococcus intermedius isolates are 100% susceptible to cefoxitin, norfloxacin, gentamicin, ciprofloxacin, and clindamycin, indicating that these antibiotics remain highly effective against this species. Furthermore, the S. intermedius, S. epidermidis, S. hyicus, S. simulans, S. hemolyticus, and S. xylosus isolates were 100% susceptible to gentamicin. Notably, norfloxacin was 100% effective against S. intermedius, S. chromogenes, S. epidermidis, and S. xylosus. Clindamycin demonstrated 100% effectiveness against S. hemolyticus, S. intermedius, and S. chromogenes (Table 5).

Detection rate of MDR in Staphylococcus species

The present study identified multidrug-resistant isolates among all eight different Staphylococcus species. Among all MDR isolates, 6/9 (66.7%) S. aureus, (5/8; 62.5%) S. hemolyticus and (6/9; 66.7%) S. hyicus strains were resistant to up to seven classes of antimicrobials. A lower frequency of MDR isolates was detected among S. simulans and S. xylosus (2/6; 33.33%), which were resistant to up to five classes of antimicrobials (Table 5).

Detection of inducible clindamycin resistance in Staphylococcus species

In a study of 60 Staphylococcus isolates, a significant prevalence of erythromycin resistance was observed, with 48 isolates (80%) demonstrating resistance to this macrolide antibiotic. Further characterization of these erythromycin-resistant isolates via the "D" test revealed varying phenotypic expressions of macrolide–lincosamide–streptogramin B (MLSB) resistance. A total of 33 isolates (68.75%) presented the MS phenotype. Seven isolates (14.58%) presented an inducible MLSB phenotype. These isolates exhibit resistance to both erythromycin and clindamycin when induced but remain susceptible to clindamycin in the absence of induction. Eight isolates (16.67%) displayed the constitutive MLSB phenotype, where resistance to both erythromycin and clindamycin was present regardless of induction. This phenotype is often associated with a chromosomal mutation leading to the continuous expression of the erm gene.

Discussion

This study, which was conducted on dairy farms in and around Bahir Dar town, aimed to explore the molecular detection of virulence genes and antimicrobial resistance profiles of Staphylococcus species isolated from bovine mastitis cases. Bovine mastitis is a significant and prevalent disease in dairy cattle, with profound economic repercussions for the dairy industry worldwide [25, 39], including reduced milk yield, altered milk quality, and increased veterinary and management costs. Therefore, the identification of mastitis-associated pathogens and their virulence determinants via advanced molecular techniques is vital for effective control and prevention strategies [40].

In the present study, the most frequently isolated coagulase-negative staphylococcal species included S. chromogenes, S. epidermidis, S. hemolyticus, S. simulans, and S. xylosus. These findings are consistent with those of a Danish study that investigated mastitis in early lactation heifers across multiple farms, where similar CNS species were also implicated [41]. However, in contrast, a Canadian study of herds with a high prevalence of CNS infection revealed a slightly distinct species distribution, with S. hominis, S. sciuri, and S. xylosus emerging as the most frequently isolated CNS species, followed by S. epidermidis and S. warneri [12]. The observed differences in the dominant CNS species isolated in this study compared with those reported in other regions, such as Canada, may be attributed to several factors, including regional variations in microbial reservoirs, farm management practices, and the use of antimicrobial agents. For example, S. hominis and S. sciuri were more prevalent in the Canadian study, which might reflect differences in livestock handling, antibiotic usage patterns, or even genetic factors that influence the colonization and virulence potential of specific CNS species in different geographic locations.

On the other hand, the predominant findings of S. chromogenes, S. epidermidis, and S. hemolyticus agreed with those of previous studies in the USA, Canada, and Argentina reported by Hasan et al., [42]; Jenkins et al., [43]; De Visscher et al., [44]; Raspanti et al., [45], regardless of their percentage. These coagulase-negative staphylococci (CNS) are opportunistic pathogens commonly found as part of the normal skin microbiota and mucosal flora of cattle. While generally considered less virulent than coagulase-positive staphylococci, CNS species can still cause significant intramammary infections, particularly when host immune defenses are compromised or when environmental conditions favor bacterial colonization.

The present study revealed that the isolation rates of the CPS and CNS species were 35% and 65%, respectively. These results were greater than those reported by Tarekgne et al. [46] in the Tigray region and by Zeryehun and Abera [47] in selected districts of Eastern Harrarghe, who reported CNS prevalence rates of 51.6% and 34.2%, respectively. The higher isolation rate of CNS species from milk might be associated with the CNS being a prominent biofilm producer [48, 49], which enhances bacterial resistance to antibiotics and evades the immune system. In addition, the CNS is isolated mainly from subclinical mastitis; this helps the pathogen remain undetected and not be treated with antimicrobials.

Several virulence factors are produced by Staphylococcus species, including the coagulase protein, which is encoded by the Coa gene, which is important for pathogenicity [50]. Fibrinogen is converted to fibrin, which leads to abscessation and persistence of microorganisms in host tissue. Furthermore, Coagulase is a virulence factor in intermarry infection. Therefore, we examined the coa genes of the isolates via PCR. In our study, 21/60 (35%) Coa were observed. Staphylococcus enterotoxin B is one of the most potent bacterial superantigens and contributes to the fatal exacerbation of methicillin-resistant S. aureus infection [51]. In this study, 20 (33.3%) seb were detected among all the Staphylococcus species. The occurrence of seb (33.33%) was greater in the present study than in the findings of Kumar et al. [52], Rodrigues et al. [53], and Grispoldi et al. [54], who detected this gene in 0.90%, 4.10% and 5.88% of S. aureus isolates, respectively.

With respect to the hemolysin genes, a differential distribution between subclinical and clinical isolates was observed; Hla was found to be more predominant in subclinical isolates and coagulase-positive staphylococci, regardless of their percentage; similarly, the highest prevalence in subclinical and coagulase-positive staphylococci was reported (82%) in Pakistan [20]. In contrast, Hlb was more commonly detected in clinical isolates than in subclinical isolates, but this finding contradicts the findings of Shahzad et al. [20]. Interestingly, Hlb was detected at comparable frequencies in both coagulase-negative and coagulase-positive isolates, suggesting that Hlb may be an important virulence factor in both groups during the clinical phase of mastitis. The Hla and Hlb genes were detected in 20% and 10% of the Staphylococcus isolates, respectively. These findings are lower than those reported in similar studies conducted in other geographic regions. Specifically, studies from Brazil [55] and India [56] reported much higher prevalence rates for these hemolysin genes, with Hla detected in 91.7% of isolates in Brazil and 47.3% in India. Similarly, Hlb was found in 94.6% of the Brazilian isolates and 92.9% of the Indian isolates, suggesting a considerable discrepancy between our results and those reported in these other studies. Several factors, such as geographic and environmental variability, sample source and size, laboratory methods, and bacterial strain variability, may explain the significant differences observed in the prevalence of the Hla and Hlb genes between our study and those conducted in Brazil and India.

The mecA gene was detected in 19 out of 60 isolates (31.67%) of Staphylococcus species in this study, making it the third most frequently detected virulence factor. This significant prevalence of the mecA gene highlights the growing concern regarding methicillin-resistant staphylococci (MRSA) in both veterinary and human medicine. These findings are broadly consistent with reports from other regions, underscoring the global relevance of methicillin resistance in staphylococcal species. For example, a study conducted in the UK [57] and another in Nigeria [58] reported 29.5% and 30.5% carriage of the mecA gene, respectively, in Staphylococcus species isolated from bovine milk, which aligns closely with the results of the present study. In contrast, a higher prevalence of the mecA gene was reported in Nepal, where a study indicated that 70.7% of Staphylococcus species isolates carried the gene [59]. This substantial difference may be attributed to varying environmental factors, management practices, or selective pressures on antimicrobial use in the regions studied. These disparities in resistance patterns underscore the need for region-specific surveillance and antimicrobial stewardship programs.

On the other hand, a lower prevalence of the mecA gene was reported in Finland, with only 5% of bovine mastitis isolates carrying the gene [60]. This lower rate could reflect differences in livestock management practices, stricter regulations on antibiotic use, or a lower prevalence of MRSA in the Finnish agricultural system, where stringent measures for controlling antimicrobial resistance are often implemented. Our study revealed mecA in 66.67% of S. aureus isolates associated with cattle mastitis. Xu et al. [61] reported mecA in 35.70% and 73.08% of S. aureus isolates respectively. A lesser percentage of mecA was reported by Dan et al. [62] as compared to the findings of the present study.

In this study, 19 (31.67%) isolates were detected resistant to the cefoxitin disks. All isolates detected to be resistant to cefoxitin were tested genotypically using PCR to detect the presence of the gene encoding mecA and the result was indicates all phynotypically cefoxitin resistance (31.67%) Staphylococcus species isolates were shown resistance as its genotypic methicillin resistace.; These results are similar to those from research conducted by Ramandinianto et al. [63]. The antibiotic cefoxitin is a good inducer for the expression of the mecA gene because it can increase the expression of PBP2a, which is encoded by the mecA gene [64]. This also agrees with Reichmann and Pinho [65] and Anand et a [66].

Biofilm formation encoded by an intracellular adhesion gene is the major cause of drug resistance and long-term intramammary infections in bovines [67, 68]. Among the 60 Staphylococcus species isolates tested, 31.67% (19/60) were found to harbor the icaD gene. This result was greater than that reported by Gajewska [69] in central Poland, who reported carriage of 21.4% of the icaD genes. However, this finding was lower than that of Felipe et al. [70], who reported that 73.2% of icaD genes were present in Staphylococcus species isolates from Argentina. The difference in virulence gene detection in staphylococcus species between the present study and other reports may be due to differences in the epidemiology of the study sites. Variations in the virulence gene acquisition of staphylococcus species via horizontal gene transfer might also be another reason for the difference in the number of virulence genes detected from staphylococcus species isolates.

The present study revealed 100% resistance to penicillin in all the tested isolates, followed by trimethoprim, tetracycline, and erythromycin. Moreover, low resistance rates to gentamicin, norfloxacin, ciprofloxacin, and clindamycin were also detected in the staphylococcal isolates tested in this study. The pattern of resistance to ampicillin and penicillin-G was in line with that reported by [71, 72], who reported that staphylococcus species isolated from mastitic milk in Turkey were 100% resistant to penicillin-G and ampicillin.

The greater degree of resistance to the beta-lactam class of antimicrobials might be related to their prolonged usage in veterinary medicine. This result is also similar to previous findings reported in Brazil by [73, 74], who reported that CNS isolates from mastitic milk were resistant to penicillin and ampicillin and that oxacillin-resistant Staphylococcus was resistant to all beta-lactam antibiotics, which carry an altered penicillin-binding protein, which is encoded by the mecA gene [74]. Resistance to these antimicrobials has also been frequently reported by other authors [28, 29]. In agreement with other recent studies [60, 75], resistance to the tested antimicrobials was greater in the CNS than in S. aureus in the present study. Our results were similar to previously reported findings of low-level resistance to gentamicin and ciprofloxacin in both CPS and CNS isolates from bovine mastitis patients [76, 77], which might be due to the low frequency of the use of these antimicrobials on dairy farms compared with the use of penicillin, erythromycin, and tetracycline.

In our study, 19 out of 60 isolates (31.67%) were positive for the mecA gene, and these isolates exhibited resistance to several antibiotics, including ampicillin, penicillin, tetracycline, and trimethoprim. The detection of methicillin-resistant Staphylococcus isolates carrying the mecA gene that also display resistance to multiple other antibiotics is a concerning finding. This multidrug resistance pattern underscores the growing challenge posed by methicillin-resistant staphylococci (MRSA) in both veterinary and human healthcare. The multidrug resistance observed in these mecA-positive isolates is consistent with findings from other studies reporting similar resistance profiles in farm animals [78, 79].

In the present study, four staphylococcal species exhibited multidrug resistance (MDR), defined as resistance to ≥ 3 antimicrobial classes. This finding aligns with previous studies conducted on dairy farms, particularly those focused on mastitic cows, which have similarly documented the emergence of MDR in Staphylococcus species [80, 81]. Excessive exposure to and repetitive use of similar groups of antimicrobials and antimicrobials with similar modes of action may induce the development of MDR [80, 82]. On the other hand, intrinsic bacterial factors, biofilm formation, and the encoding of multiple resistance genes have also been reported to affect MDR expression [83].

This study revealed that the frequency of inducible clindamycin resistance (the iMLSB phenotype, which results in a positive D-zone test) among erythromycin-challenged isolates was 14.58% (7/48). The present findings are lower than those of studies from Chandigarh and Bangalore, which reported inducible resistance rates of 26.1% and 22.2%, respectively, among erythromycin-resistant isolates [84, 85]. In the present study, 68.75% of the erythromycin-resistant staphylococcal isolates exhibited true clindamycin resistance. From the current study, we can conclude that a lower frequency of inducible clindamycin resistance was recorded among the staphylococcal isolates.

Conclusions

Mastitis, especially subclinical mastitis, is a major problem for dairy cows in the study area, with about half of the cows affected. Among the identified Staphylococcus species, S. chromogenes emerged as the dominant isolate. All the staphylococcal species isolated in the study carried two or more genes linked to their ability to cause disease. A key concern is that almost one-third of the isolates carried the mecA gene, which makes the bacteria resistant to methicillin. This raises alarms about the growing problem of methicillin-resistant staphylococci (MRSA), which is not only a threat to animal health but also to human health. The bacteria were also found to be resistant to penicillin and were multidrug-resistant, meaning they don't respond to many commonly used antibiotics. This limits treatment options and makes controlling the disease more difficult. As a result, it is crucial to monitor these resistant bacteria, especially in cases of subclinical mastitis, which can go unnoticed but still affect milk production and cow health. The study recommends using stronger antibiotics, such as norfloxacin and gentamicin, to treat mastitis, while avoiding antibiotics like penicillin and tetracycline, which the bacteria are resistant to.

Limmtitation of the study

In this study, the identification of Staphylococcus species was carried out using conventional methods due to the unavailability of species-specific primers. Additionally, antimicrobial resistance patterns were verified phenotypically, as molecular detection of resistance genes was not conducted, which presents another limitation of this study.

Data availability

The raw data collected during this study has been included as a supporting file alongside this manuscript. Additional information regarding the data can be obtained by contacting the corresponding author."

Abbreviations

blaZ:

B-lactamase resistance encoding gene

CLSI:

Clinical Laboratory Standard Institute

CMT:

California Mastitis Test

CNS:

Coagulase-Negative Staphylococcus

CPS:

Coagulase-Positive Staphylococcus

DNA:

Deoxy Ribonucleic Acid

Hla:

Alpha Hemolysin Toxin

Hlb:

Beta Hemolysin Toxin

icaD:

Intracellular Adhesion

MSA:

Mannitol Salt Agar

MecA:

Methicillin Resistance Gene

MDRS:

Multidrug resistance Staphylococcus

MHA:

Mueller–Hinton Agar

PCR:

Polymerase Chain Reaction

STATA:

Statistics And Data

TSST-1:

Toxic Shock Syndrome Toxin-1 Gene

References

  1. Kerro Dego O, Vidlund J. Staphylococcal mastitis in dairy cows. Front Vet Sci. 2024;11: 1356259. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fvets.2024.1356259.

    Article  PubMed  PubMed Central  Google Scholar 

  2. Markey Bk. Clinical veterinary microbiology. 2nd ed. Edinburgh: Mosby Elsevier; 2013.

    Google Scholar 

  3. Sasaki T, Kikuchi K, Tanaka Y, Takahashi N, Kamata S, Hiramatsus K. Reclassification of phynotypically identified staphylococcus intermius strain. J Clin Microbiol. 2007;45:2770–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/JCM.00360-07.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  4. De Buck J, Ha V, Naushad S, Nobrega DB, Luby C, Middleton JR, et al. Non-aureus staphylococci and bovine udder health: current understanding and knowledge gaps. Front Vet Sci. 2021;8:360. https://doiorg.publicaciones.saludcastillayleon.es/10.3389/fvets.2021.658031.

    Article  Google Scholar 

  5. Simojoki H, Salomäki T, Taponen S, Iivanainen A, Pyörälä S. Innate immune response in experimentally induced bovine intramammary infection with Staphylococcus simulans and S. epidermidis. Veterinary Research. 2011;42(1):1–10.

    Article  Google Scholar 

  6. Grace, D. and Fetsch, A. Staphylococcus aureus—A foodborne pathogen: Epidemiology, detection, characterization, prevention, and control: An overview. Staphylococcus aureus. 2018:3–10. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/B978-0-12-809671-0.00001-2.

  7. Pyörälä S, Taponen S. Coagulase-negative staphylococci—emerging mastitis pathogens. Vet Microbiol. 2009;134(1–2):3–8.

    Article  PubMed  Google Scholar 

  8. Leroy F, Van Coillie E, Braem G, Piessens V, Verbist B, De Vuyst L, et al. Subtyping of Staphylococcus hemolyticus isolates from milk and corresponding teat apices to verify the potential teat-skin origin of intramammary infections in dairy cows. J Dairy Sci. 2015;98(11):7893–8.

    Article  PubMed  CAS  Google Scholar 

  9. Argemi X, Hansmann Y, Prola K, Prévost G. Coagulase-negative staphylococci pathogenomics. J Int J Mol Sci. 2019;20(5):1215.

    Article  PubMed  CAS  Google Scholar 

  10. Andrade NC, Laranjo M, Costa MM, Queiroga MCJA. Virulence Factors in Staphylococcus Associated with Small Ruminant Mastitis: Biofilm Production and Antimicrobial Resistance Genes. 2021;10(6):633.

    CAS  Google Scholar 

  11. Holmes A, Ganner M, McGuane S, Pitt T, Cookson B, Kearns A. Staphylococcus aureus isolates carrying Panton-Valentine leucocidin genes in England and Wales: frequency, characterization, and association with clinical disease. J Clin Microbiol. 2005;43(5):2384–90.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Davidson TJ, Dohoo IR, Donald A, Hariharan H, Collins K. A cohort study of coagulase-negative staphylococcal mastitis in selected dairy herds in Prince Edward Island. Can J Vet Res. 1992;56(4):275.

    PubMed  PubMed Central  CAS  Google Scholar 

  13. Costa FN, Belo NO, Costa EA, Andrade GI, Pereira LS, Carvalho IA, et al. Frequency of enterotoxins, toxic shock syndrome toxin-1, and biofilm formation genes in Staphylococcus aureus isolates from cows with mastitis in the Northeast of Brazil. Trop Anim Health Prod. 2018;50(5):1089–97.

    Article  PubMed  CAS  Google Scholar 

  14. Bhosale RR, Osmani RA, Ghodake PP, Shaikh SM, Chavan SR. Mastitis: an intensive crisis in Veterinary Science. Int J Pharma Res Health Sci. 2014;2(2):96–103.

    Google Scholar 

  15. Piessens V, De Vliegher S, Verbist B, Braem G, Van Nuffel A, De Vuyst L, et al. Characterization of coagulase-negative staphylococcus species from cows‘ milk and environment based on bap, icaA, and mecA genes and phenotypic resistance to antimicrobials and teat dips. J Dairy Sci. 2012;95(12):7027–38.

    Article  PubMed  CAS  Google Scholar 

  16. Abera M, Demie B, Aragaw K, Regassa F, Regassa A. Isolation and identification of Staphylococcus aureus from bovine mastitic milk and their drug resistance patterns in Adama town, Ethiopia. Afr J Dairy Farm Milk Prod. 2013;1:19–23.

    Google Scholar 

  17. Zeryehun T, Aya T, Bayecha R. Study on prevalence, bacterial pathogens and associated risk factors for bovine mastitis in small-holder dairy farms in and around Addis Ababa. Ethiopia J Anim Plant Sci. 2013;23:50–5.

    CAS  Google Scholar 

  18. Aweke S, Mekibib B. Major Production Problems of Dairy Cows of Different Farm Scales Located in the Capital City Addis Ababa, Ethiopia. Journal of Veterinary Science & Technology. 2017;8:6–11.

    Google Scholar 

  19. Yayeh Z, Kassa A, Dagnew E. Milk Production, Marketing and Processing Practices of Dairy Cattle in Debremarkos Woreda of East Gojjam Zone, Amhara Regional State. J Nutr Food Sci. 2017;7: 607. https://doiorg.publicaciones.saludcastillayleon.es/10.4172/2155-9600.1000607.

    Article  Google Scholar 

  20. Yeserah B, Tassew T, Mazengia H. Handling Practices of Raw Cow’s Milk and Major Constraints of Clean Milk Production in and around Bahir Dar City. Ethiopia J Adv Dairy Res. 2020;8: 234. https://doiorg.publicaciones.saludcastillayleon.es/10.35248/2329-888X.19.8.2.234.

    Article  Google Scholar 

  21. Hoque M, Das ZC, Talukder AK, Alam MS, Rahman ANM. Different screening tests and milk somatic cell count for the prevalence of subclinical bovine mastitis in Bangladesh. Trop Anim Health Prod. 2015;47(1):79–86.

    Article  PubMed  Google Scholar 

  22. NMC. Microbiological procedures for the diagnosis of bovine udder infection and determination of milk quality. 4th ed. USA: National Mastitis Council; 2004.

  23. Thorberg BM, Brändström B. Evaluation of Two Commercial Systems and a New Identification Scheme Based on Solid Substrates for Identifying Coagulase-Negative Staphylococci from Bovine Mastitis. J Vet Med Ser B. 2000;47(9):683–91.

    Article  CAS  Google Scholar 

  24. Gebremedhin EZ, Ararso AB, Borana BM, Kelbesa KA, Tadese ND, Marami LM, Sarba EJ. Isolation and identification of Staphylococcus aureus from milk and milk products, associated factors for contamination, and their antibiogram in Holeta, Central Ethiopia. Vet Med Int. 2022;2022:6544705.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Momtaz H, Rahimi E, Tajbakhsh E. Detection of some virulence factors in Staphylococcus aureus isolated from clinical and subclinical bovine mastitis in Iran. Afr J Biotech. 2010;9(25):3753–8.

    CAS  Google Scholar 

  26. Wang D, Wang Z, Yan Z, Wu J, Ali T, Li J, et al. Bovine mastitis Staphylococcus aureus: antibiotic resistance profile, resistance genes and molecular typing of methicillin-resistant and methicillin-sensitive strains in China. Infect Genet Evol. 2015;31:9–16.

    Article  PubMed  Google Scholar 

  27. Kot B, Szweda P, Frankowska-Maciejewska A, Piechota M, Wolska K. Virulence gene profiles in Staphylococcus aureus isolated from cows with subclinical mastitis in eastern Poland. J Dairy Res. 2016;83(2):228–35.

    Article  PubMed  CAS  Google Scholar 

  28. Vasudevan P, Nair MKM, Annamalai T, Venkitanarayanan KS. Phenotypic and genotypic characterization of bovine mastitis isolates of Staphylococcus aureus for biofilm formation. Vet Microbiol. 2003;92(1–2):179–85.

    Article  PubMed  CAS  Google Scholar 

  29. Goh S-H, Byrne S, Zhang J, Chow A. Molecular typing of Staphylococcus aureus based on coagulase gene polymorphisms. J Clin Microbiol. 1992;30(7):1642–5.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. Xie Y, He Y, Gehring A, Hu Y, Li Q, Tu S-I, et al. Genotypes and toxin gene profiles of Staphylococcus aureus clinical isolates from China. PLoS ONE. 2011;6(12): e28276.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Løvseth A, Loncarevic S, Berdal KG. Modified multiplex PCR method for detection of pyrogenic exotoxin genes in staphylococcal isolates. J Clin Microbiol. 2004;42(8):3869–72.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Sabat A, Krzyszton-Russjan J, Strzalka W, Filipek R, Kosowska K, Hryniewicz W, et al. A new method for typing Staphylococcus aureus strains: multiple-locus variable-number tandem repeat analysis of polymorphism and genetic relationships of clinical isolates. J Clin Microbiol. 2003;41(4):1801–4.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  33. Booth MC, Pence LM, Mahasreshti P, Callaghan MC, Gilmore MS. Clonal associations among Staphylococcus aureus isolates from various sites of infection. Infect Immun. 2001;69(1):345–52.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  34. Pitcher D, Saunders N, Owen R. Rapid extraction of bacterial genomic DNA with guanidium thiocyanate. Lett Appl Microbiol. 1989;8(4):151–6.

    Article  CAS  Google Scholar 

  35. Okolie CE, Wooldridge KG, Turner DP, Cockayne A, James R. Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes. BMC Microbiol. 2015;15:1–7.

    Article  CAS  Google Scholar 

  36. Younis G, Sadat A, Maghawry M. Characterization of coa gene and antimicrobial profiles of staphylococcus aureus isolated from bovine clinical and subclinical mastitis. Adv Anim Vet Sci. 2018;6(4):161–8.

    Article  Google Scholar 

  37. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 30th ed. Supplement M100. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2021.

  38. Phophi L, Petzer IM, Qekwana DN. Antimicrobial resistance patterns and biofilm formation of coagulase- negative Staphylococcus species isolated from subclinical mastitis cow milk samples submitted to the Onderstepoort Milk Laboratory. BMC Vet Res. 2019;15(1):420.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  39. Atasever, S. Estimation of Correlation Between Somatic Cell Count and Coagulation Score of Bovine Milk. Int J Agric Biol. 2012;14(2):315–7.

  40. El-Behiry A, Elsayed M, Marzouk E, Bathich Y, Alves D, Mario N. Detection of virulence genes in Staphylococcus aureus and Streptococcus agalactiae isolated from mastitis in the Middle East. Br Microbiol ResJ. 2015;10(3):1–9.

    Article  CAS  Google Scholar 

  41. Aarestrup FM, Jensen N. Prevalence and duration of intramammary infection in Danish heifers during the peripartum period. J Dairy Sci. 1997;80(2):307–12.

    Article  PubMed  CAS  Google Scholar 

  42. Hasan M, Kober A, Rana E, Bari M. Association of udder lesions with subclinical mastitis in dairy cows of Chattogram. Bangladesh Adv Anim Vet Sci. 2022;10(2):226–35.

    Google Scholar 

  43. Fazal MA, Rana EA, Akter S, Alim MA, Barua H, Ahad A. Molecular identification, antimicrobial resistance and virulence gene profiling of Staphylococcus spp. associated with bovine subclinical mastitis in Bangladesh. Vet Anim Sci. 2023;21:100297.

    Article  PubMed  PubMed Central  Google Scholar 

  44. De Visscher A, Piepers S, Haesebrouck F, Supré K, De Vliegher S. Coagulase-negative Staphylococcus species in bulk milk: Prevalence, distribution, and associated subgroup-and species-specific risk factors. J Dairy Sci. 2017;100(1):629–42.

    Article  PubMed  Google Scholar 

  45. Raspanti CG, Bonetto CC, Vissio C, Pellegrino MS, Reinoso EB, Dieser SA, et al. Prevalencia y sensibilidad an antibióticos de especies de estafilococos coagulase-negatives provenientes de mastitis subclinical bovines de tambos de la región central de Argentina. Rev Argent Microbiol. 2016;48(1):50–6.

    PubMed  Google Scholar 

  46. Tarekgne E, Skeie S, Rudi K, Skjerdal T, Narvhus JA. Staphylococcus aureus and other Staphylococcus species, in milk and milk products from Tigray region, Northern Ethiopia. Afr J Food Sci. 2015;9(12):567–76.

    Article  Google Scholar 

  47. Zerihun T, Abera G. Prevalence and bacterial isolates of mastitis in dairy farms in selected districts of Eastern Harrarghe zone, Eastern Ethiopia. J Vet Med. 2017;2017:6498618.

    Google Scholar 

  48. Goetz C, Tremblay YD, Lamarche D, Blondeau A, Gaudreau AM, Labrie J, et al. Coagulase-negative staphylococci species affect the biofilm formation of other coagulase-negative and coagulase-positive staphylococci. J Dairy Sci. 2017;100(8):6454–64.

    Article  PubMed  CAS  Google Scholar 

  49. Lee YJ, Lee YJ. Characterization of biofilm-producing coagulase-negative staphylococci isolated from bulk tank milk. Veterinary Sciences. 2022;9(8):430.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Barkema H, Schukken Y, Zadoks R. Invited review: the role of cow, pathogen, and treatment regimen in the therapeutic success of bovine Staphylococcus aureus mastitis. J Dairy Sci. 2006;89(6):1877–95.

    Article  PubMed  CAS  Google Scholar 

  51. Bae JS, Da F, Liu R, He L, Lv H, Fisher EL, et al. Contribution of staphylococcal enterotoxin B to Staphylococcus aureus systemic infection. J Infect Dis. 2021;223(10):1766–75. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/infdis/jiaa584. PMID: 32937658.

    Article  PubMed  CAS  Google Scholar 

  52. Kumar R, Yadav BR, Anand SK, Singh RS. Prevalence of adhesin and toxin genes among isolates of Staphylococcus aureus obtained from mastitic cattle. World J Microbiol Biotechnol. 2011;27(3):513–21.

    Article  CAS  Google Scholar 

  53. Rodrigues MX, Silva NC, Trevilin JH, Cruzado MM, Mui TS, Duarte FR, et al. Antibiotic resistance and molecular characterization of Staphylococcus species from mastitic milk. Afr J Microbiol Res. 2017;11(3):84–91.

    Article  CAS  Google Scholar 

  54. Grispoldi L, Massetti L, Sechi P, Iulietto MF, Ceccarelli M, Karama M, et al. Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows. J Dairy Sci. 2019;102(2):1059–65. https://doiorg.publicaciones.saludcastillayleon.es/10.3168/jds.2018-15373. PMID: 30591337.

    Article  PubMed  CAS  Google Scholar 

  55. Pinheiro L, Ivo Brito C, De Oliveira A, Yoshida Faccioli Martins P, Cataneli Pereira V, de Souza Ribeiro, da Cunha MdL. Staphylococcus epidermidis and Staphylococcus hemolyticus: molecular detection of cytotoxin and enterotoxin genes. Toxins. 2015;7(9):3688–99.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  56. Nasaj M, Saeidi Z, Asghari B, Roshanaei G, Arabestani MR. Identification of hemolysin encoding genes and their association with antimicrobial resistance pattern among clinical isolates of coagulase-negative Staphylococci. BMC Res Notes. 2020;13:1–6.

    Article  Google Scholar 

  57. Xu Z, Mkrtchyan HV, Cutler RR. Antibiotic resistance and mecA characterization of coagulase-negative staphylococci isolated from three hotels in London, UK. Front microbiol. 2015;6:947.

    Article  PubMed  PubMed Central  Google Scholar 

  58. Ibadin EE, Enabulele IO, Muinah F. Prevalence of mecA gene among staphylococci from clinical samples of a tertiary hospital in Benin City, Nigeria. Afr Health Sci. 2017;17(4):1000–10.

    Article  PubMed  PubMed Central  Google Scholar 

  59. Shrestha LB, Bhattarai NR, Rai K, Khanal B. Antibiotic resistance and mecA gene characterization of coagulase-negative staphylococci isolated from clinical samples in Nepal. Infection and Drug Resistance. 2020;Volume 13:3163–9.

    Article  Google Scholar 

  60. Taponen S, Nykäsenoja S, Pohjanvirta T, Pitkälä A, Pyörälä S. Species distribution and in vitro antimicrobial resistance of coagulase-negative staphylococci isolated from bovine mastitic milk. Acta Vet Scand. 2015;58:1–13.

    Article  Google Scholar 

  61. Xu J, Tan X, Zhang X, Xia X, Sun H. The diversities of staphylococcal species, virulence and antibiotic resistance genes in the subclinical mastitis milk from a single Chinese cow herd. MicrobPathog. 2015;88:29–38. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.micpath.2015.08.004. PMID: 26276706.

    Article  CAS  Google Scholar 

  62. Dan M, Yehui W, Qingling M, Jun Q, Xingxing Z, Shuai M, et al. Antimicrobial resistance, virulence gene profile and molecular typing of Staphylococcus aureus isolates from dairy cows in Xinjiang Province, northwest China. J Glob Antimicrob Resist. 2019;16:98–104. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jgar.2018.08.024. PMID: 30213718.

    Article  PubMed  Google Scholar 

  63. Ramandinianto SC, Khairullah AR, Effendi MH. MecA gene and methicillin-resistant Staphylococcus aureus (MRSA) isolated from dairy farms in East Java. Indonesia Biodiversitas. 2020;21(8):3562–8.

    Google Scholar 

  64. Koupahi H, Jahromy SH, Rahbar M. Evaluation of different phenotypic and genotypic methods for detection of Methicillin Resistant Staphylococcus aureus (MRSA). Iran J Pathol. 2016;11(4):370–6.

    PubMed  PubMed Central  Google Scholar 

  65. Reichmann NT, Pinho MG. Role of SCCmec type in resistance to the synergistic activity of oxacillin and cefoxitin in MRSA. Sci Rep. 2017;7:6154.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Anand KB, Agrawal P, Kumar S, et al. Comparison of cefoxitin disk diffusion test, oxacillin screen agar, and PCR for mecA gene for detection of MRSA. Indian J Med Microbiol. 2009;27(1):27–9.

    Article  PubMed  CAS  Google Scholar 

  67. Seng R, Kitti T, Thummeepak R, Kongthai P, Leungtongkam U, Wannalerdsakun S, et al. Biofilm formation of methicillin-resistant coagulase-negative staphylococci (MR-CoNS) isolated from community and hospital environments. PLoS ONE. 2017;12(8): e0184172.

    Article  PubMed  PubMed Central  Google Scholar 

  68. Shrestha LB, Bhattarai NR, Khanal B. Antibiotic resistance and biofilm formation among coagulase-negative staphylococci isolated from clinical samples at a tertiary care hospital of eastern Nepal. Antimicrob Resist Infect Control. 2017;6:1–7.

    Article  Google Scholar 

  69. Gajewska J, Chajęcka-Wierzchowska W. Biofilm formation ability and presence of adhesion genes among coagulase-negative and coagulase-positive staphylococci isolates from raw cow‘s milk. Pathogens. 2020;9(8):654.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  70. Felipe V, Morgante CA, Somale PS, Varroni F, Zingaretti ML, Bachetti RA, et al. Evaluation of the biofilm- forming ability and its associated genes in Staphylococcus species isolated from bovine mastitis in Argentinean dairy farms. Microb Pathog. 2017;104:278–86.

    Article  PubMed  CAS  Google Scholar 

  71. Kim S-J, Moon DC, Park S-C, Kang HY, Na SH, Lim S-K. Antimicrobial resistance and genetic characterization of coagulase-negative staphylococci from bovine mastitis milk samples in Korea. J Dairy Sci. 2019;102(12):11439–48.

    Article  PubMed  CAS  Google Scholar 

  72. Achek R, El-Adawy H, Hotzel H, Tomaso H, Ehricht R, Hamdi TM, et al. Diversity of staphylococci isolated from sheep mastitis in northern Algeria. J Dairy Sci. 2020;103(1):890–7.

    Article  PubMed  CAS  Google Scholar 

  73. Soares LC, Pereira IA, Pribul BR, Oliva MS, Coelho SM, Souza M. Antimicrobial resistance and detection of mecA and blaZ genes in coagulase-negative Staphylococcus isolated from bovine mastitis. Pesquisa Veterinária Brasileira. 2012;32:692–6.

    Article  Google Scholar 

  74. Miragaia M. Factors contributing to the evolution of mecA-mediated β-lactam resistance in staphylococci: update and new insights from whole-genome sequencing (WGS). Front Microbiol. 2018;9:2723.

    Article  PubMed  PubMed Central  Google Scholar 

  75. Dos Santos FF, Mendonça LC, de Lima Reis DR, de Sá GA, Lange CC, Ribeiro JB, et al. Presence of mecA-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil. J Dairy Sci. 2016;99(2):1374–82.

    Article  Google Scholar 

  76. Frey Y, Rodriguez JP, Thomann A, Schwendener S, Perreten V. Genetic characterization of antimicrobial resistance in coagulase-negative staphylococci from bovine mastitis milk. J Dairy Sci. 2013;96(4):2247–57.

    Article  PubMed  CAS  Google Scholar 

  77. Klibi A, Maaroufi A, Torres C, Jouini A. Detection and characterization of methicillin-resistant and susceptible coagulase-negative staphylococci in milk from cows with clinical mastitis in Tunisia. Int J Antimicrob Agents. 2018;52(6):930–5.

    Article  PubMed  CAS  Google Scholar 

  78. Monecke S, Gavier-Widen D, Mattsson R, Rangstrup-Christensen L, Lazaris A, Coleman DC, et al. Detection of mecC-positive Staphylococcus aureus (CC130-MRSA-XI) in diseased European hedgehogs (Erinaceus europaeus) in Sweden. PLoS ONE. 2013;8(6): e66166.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  79. Schabauer A, Pinior B, Gruber C-M, Firth CL, Käsbohrer A, Wagner M, et al. The relationship between clinical signs and microbiological species, spa type, and antimicrobial resistance in bovine mastitis cases in Austria. Vet Microbiol. 2018;227:52–60.

    Article  PubMed  Google Scholar 

  80. Rana EA, Islam MZ, Das T, Dutta A, Ahad A, Biswas PK, et al. Prevalence of coagulase-positive methicillin-resistant Staphylococcus aureus and Staphylococcus pseudintermedius in dogs in Bangladesh. Veterinary medicine and science. 2022;8(2):498–508.

    Article  PubMed  CAS  Google Scholar 

  81. Dabele DT, Borena BM, Admasu P, Gebremedhin EZ, Marami LM. Prevalence and Risk Factors of Mastitis and Isolation, Identification and Antibiogram of Staphylococcus Species from Mastitis Positive Zebu Cows in Toke Kutaye, Cheliya, and Dendi Districts, West Shewa Zone, Oromia, Ethiopia. Infect Drug Resist. 2021;14:987–98. https://doiorg.publicaciones.saludcastillayleon.es/10.2147/IDR.S295257.

  82. Das T, Rana EA, Dutta A, Bostami MB, Rahman M, Deb P, et al. Antimicrobial resistance profiling and burden of resistance genes in zoonotic Salmonella isolated from broiler chicken. Veterinary Medicine and Science. 2022;8(1):237–44.

    Article  PubMed  CAS  Google Scholar 

  83. Christaki E, Marcou M, Tofarides A. Antimicrobial resistance in bacteria: mechanisms, evolution, and persistence. J Mol Evol. 2020;88:26–40.

    Article  PubMed  CAS  Google Scholar 

  84. Gupta V, Datta P, Rani H, Chander J. Inducible clindamycin resistance in Staphylococcus aureus: A study from North India. J Postgrad Med. 2009;55(3):176.

    Article  PubMed  CAS  Google Scholar 

  85. Sasirekha B, Usha M, Amruta J, Ankit S, Brinda N, Divya R. Incidence of constitutive and inducible clindamycin resistance among hospital-associated Staphylococcus aureus. Biotech. 2014;4:85–9.

    CAS  Google Scholar 

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Acknowledgements

The authors of this manuscript are grateful to Bahir Dar University, Bahir Dar Animal Disease Surveillance, Investigation and Diagnostic Laboratory, and the Institute of Biotechnology, Addis Ababa University, for providing materials and technical support during the laboratory work. I also gratefully acknowledge the cooperation and support of the dairy holders during data collection and animal examination.

Funding

The authors did not receive any funds for this work.

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Authors and Affiliations

  1. School of Veterinary Medicine, Bahir Dar University, Bahir dar, Ethiopia

    Dessie Debeb Getahun, Habtamu Tassew Tarekegn & Bizuneh Tsehayneh Azene

  2. Bahir Dar Animal Disease Surveillance Investigations and Diagnostic Laboratory, Bahir dar, Ethiopia

    Laikemariam Teshome Abebe

  3. Department of Veterinary Laboratory Technology, College of Agriculture and Natural Resource, Debre Markos University, Debre Markos, Ethiopia

    Mequanint Addisu Belete

  4. Institute of Biotechnology, Addis Ababa University, Addis Ababa, Ethiopia

    Tesfaye Sisay Tessema

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  1. Dessie Debeb Getahun
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Contributions

DD- conceptualized the study, designed the methodology, conducted the tests, and wrote the original paper. LT and MA participated while doing my laboratory work. HT, BT & TS- contributed to manuscript editing and final preparation. All authors contributed to the article and approved the submitted version.

Corresponding author

Correspondence to Dessie Debeb Getahun.

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Ethics approval and consent to participate

Bahir Dar University's ethical review committee evaluated ethical issues related to research work and approved its ethical soundness and acceptability. Thus, Bahir Dar University sent a letter of cooperation to the study area. The purpose and procedures of the study were properly explained to the farm owner and study participants, and milk samples were collected from dairy farms whose owners were willing to participate. All methods were performed according’ to the relevant guidelines and regulations.

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Not applicable.

Competing interests

The authors declare no competing interests.

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Getahun, D.D., Tarekegn, H.T., Azene, B.T. et al. Virulence genes and antibiotic resistance profiling of staphylococcus species isolated from mastitic dairy cows in and around Bahir dar, Ethiopia. BMC Microbiol 25, 210 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12866-025-03886-9

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Keywords

BMC Microbiology

ISSN: 1471-2180

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