Investigation of Antimicrobial Resistance and Virulence Genes among Methicillin-Resistant Staphylococcus aureus from Subclinical Mastitic Cows

Bovine mastitis continues to be the most frequent and costly disease affecting dairy cattle due to its detrimental impact on health, welfare, and productivity (Naranjo-Lucena and Slowey, 2023).

Two forms of mastitis, including subclinical and clinical, are frequently reported; however, overt subclinical mastitis (SCM) signs are rarely seen in the milk or udder. Mastitis led to decreased milk output and an increase in somatic cells counts Williamson et al. (2022). On the other hand, clinical mastitis (CM) is characterized by quick onset, changed milk content, reduced milk production, and the emergence of basic, immediately identifiable signs of inflammation in the infected udder (Bude and Mengesha, 2021). One of the primary bacteria that causes mastitis in dairy cattle is S. aureus (Ali et al., 2021). S. aureus is a facultative anaerobic, gram-positive bacteria that is coagulase-positive, catalase-positive, and oxidase-negative. It is a spherical, non-spore-forming, non-motile bacteia with a diameter of 0.5 to 1.5 µm. S. aureus is a significant infectious agent affecting the population and the health field (Bitrus et al., 2018). Methicillin-resistant Staphylococcus aureus (MRSA), also referred to as resistant staph or superbug, is regarded as one of the primary bacteria that infect patients in hospitals and the general public.

The World Health Organization ( WHO ) has designated MRSA as a high-priority bacterium for additional study and management (World Health Organization, 2017).

Methicillin-resistant Staphylococcus aureus (MRSA) appeared, probably due to acquiring the mecA gene (Sangappa and Thiagarajan, 2012). However, S. aureus pathogenicity involves the production of virulence factors that help the bacterium evade host defenses, allowing the microorganism to colonize the mammary glands of ruminants (Turner et al., 2019). The prevalence of (MRSA) is the most significant issue, making S. aureus infections especially problematic (Abril et al., 2020). Cell wall-associated adhesins and toxins are just two of many virulence factors that Staphylococci have that help the bacteria evade the immune system and make infections more severe. However, most of these factors were originally identified in S. aureus (González-Martín, et al., 2020). Hemolysins are among the several exoproteins produced by S. aureus, facilitating the organism to proliferate and cause disease in its mammalian hosts (Dinges et al., 2000). While most strains identified from intramammary infection in bovine express β-hemolysin, sphingomyelinase, and α-hemolysin is cytotoxic, both toxins are known to promote S. aureus adherence to mammary gland epithelial cells (Zhang et al., 2018; Algammal et al., 2020).

Two exfoliative toxins (ETs) with different immunological profiles were found to create some strains of S. aureus, namely eta and etb. Additionally, they have been linked to a group of impetiginous staphylococcal infections known as staphylococcal scalded skin syndrome. Many molecular epidemiological studies have already been conducted on enterotoxigenic S. aureus isolated from sheep milk and food (Scherrer et al., 2004).

The class of serine proteases that includes exfoliative toxins exhibits remarkable substrate selectivity. This hydrolysis causes keratinocytes to dissociate in human and animal skin (Nishifuji et al., 2008). This study aims to identify the nature and genetics of S. aureus in mastitis cases and to underpin the genes driving the antibiotic resistance. The finding would be valuable information for both animal handlers and milk consumers.

California Mastitis Test (CMT) was used to screen all 260 raw milk cows in Basrah province for subclinical mastitis from 21 November 2023 to 28 February. The milk was evaluated for Staphylococcus aureus using conventional and molecular methods.

All samples were primarily submitted to the (CMT ). The test relies on the reagent’s interaction with the DNA of somatic cells found in milk (Philpot and Nickerson, 1991). Briefly, a tiny sample of milk was extracted from each udder quadrant using the white plastic paddle and gently mixed with 2 mL of the scam reagent. Depending on the degree of gel, within 20 seconds, based on how much gel formation has occurred, the test is concluded positive. The positive CMT samples were transported immediately to the Central Research Unit utilizing an icebox.

Upon laboratory arrival, the milk samples were inoculated in brain heart infusion broth and incubated overnight at 37°C.

Following preincubation, the samples were subcultured on mannitol salt agar (Himedia, India) and Chromogenic media (Pioneer/France). The plates were incubated aerobically for 24 hours at 37°C. Gram’s staining was used to identify the suspected colonies on chromogenic agar.

The suspected isolates were confirmed by amplifying the nuc gene using PCR. By the instructions provided by the bacterial extraction kit manufacturer (Genaid, Korea), bacterial DNA was extracted. The primers were procured from Bioneer, Korea, and were specific to the nuc gene, which encodes a particular thermostable nuclease, Table 1.

Table 1: Sequence of primer for nuc gene with their manufacture.

Table 2: Sequence of primer for mecA gene with their manufacturer.

Table 3: Sequence of primers for hla,hlb, eta genes and their expected products.

For the nuc gene, a total of 20μl of the PCR reaction mixture was prepared. Ten microliters of Green Master Mix, one microliter of each oligonucleotide primer, one microliter of DNA template, and seven microliters of nuclease-free water was used .PCR program was applied with 35 cycles of PCR conducted at 94°C for 7 minutes and 58°C for 30 seconds ,72°C for 45 seconds and 72°C for 7 minutes).

To identify the MRSA phenotype, all identified S. aureus isolates were evaluated for antimicrobial sensitivity to cefoxitin (10μg) (NCCLS, 2023). The mecA gene was then found to molecularly validate MRSA isolates, Table 2.

Molecular detection of Methicillin-resistant staphylococcus aureus (MRSA) by (mecA) gene

By identifying the mecA gene using primer sequences shown in Table 2, MRSA isolates were molecularly confirmed.

The PCR reaction mixture for the mecA gene was prepared in a total amount of 20μl. One microliter of each oligonucleotide primer, one μl of DNA template, ten microliters of Green master mix, and seven microliters of nuclease-free water were combined. The thermocycling includes: 94°C for 5 minutes and 30 cycles of (94°C for 1 minute, 59°C for 1 minute, 72 °C for 1 minute, and final 72 °C for 7 minutes).

Eight antibiotics were chosen for the antibiotic sensitivity assays based on their common use in the field. This test was conducted according to the method described earlier.(Bauer et al., 1966). The antibiotic discs were provided by (Bioanalyse/ Turkey), including Erythromycin (30 μg), Gentamycin (30 μg), Ciprofloxacin (5 μg), Penicillin (10 μg), Tetracycline (10 μg), Vancomycin (10 μg), Sulfamethoxazole (10 μg), and Chloramphenicol(10 μg).

Primers were employed to identify the virulence genes (including hla, hlb, and eta) in MRSA-positive isolates (Table 3).

A total of 20μl of the PCR reaction mixture for the hla and hlb genes was produced. With each of the oligonucleotide primers (0.5 μl), DNA template (2 μl), Green master mix (10 μl), and nuclease-free water (6 μl).

For the hla, hlb gene, a total of 20μl of the PCR reaction mixture was prepared, carrying 2 μl of DNA template, 0.5 μl of each oligonucleotide primer, 10 μl of Green Master Mix, and 6 μl of nuclease-free water.

The reaction mixture of (eta) consisted of 1 microliter of DNA template, 0.5 microliter of each oligonucleotide primer, 10 microliters of Green master mix, and 8 microliters of nuclease-free water.

The hla and hlb genes were amplified under the following conditions: 94°C for 5 minutes, 35 cycles (94°C for 30 seconds, 56°C for 30 seconds, 72°C for 30 seconds, and 72°C for 5 minutes). Whereas, the (eta) gene was subjected to 30 cycles of PCR conditions, which included 95 °C for 5 minutes, 54 °C for 30 seconds, 72 °C for 30 seconds, and 72 °C for 5 minutes.

Based on CMT, a total of 120 (46.15%) samples showed positive results (Table 4; Figure 1).

Table 4: Percentage of subclinical mastitis according to CMT.

Table 5: Illustrate the number of S. aureus isolates detected by molecular detection, traditional microbiological methods, and cultural methods.

The percentage of suspected S. aureus isolates was confirmed using mannitol salts agar and CHROMagar™ Staph aureus was positive in 140 (53%) and 80 (31%), respectively.

Table 3 all suspected isolates were subjected to PCR targeting the nuc gene (Table 5, Figures 2 and 3).The molecular technique-based assay characterized S. aureus in 41 (16%) of samples.

Methicillin-resistant Staphylococcus aureus can be identified phenotypically utilizing cefoxitin antimicrobial disk. All verified S. aureus isolates were resistant to a cefoxitin disk 41/41 (100 %) (Figure 4).

All S. aureus isolates were submitted to molecular detection for the (mecA) gene using gene specific primers of 41 S.aureus isolates, only 30 isolates were mecA positive. The results are shown in Table 6 and Figure 5.

Table 6: Percentage of (mec A) gene in S. aureus isolates among subclinical mastitis.

Eight antimicrobials were used to assess antibiotic sensitivity of S. aureus isolates, as shown in Table 7 and Figure 6. The isolates exhibited resistant to Vancomycin, Gentamycin, Penicillin, Erythromycin, and Tetracycline with percentages of ( 80%, 63.3%, 37%, 20 %, and 17%), respectively. In contrast, the isolates were vulnerable to Sulfamethoxazole, Chloramphenicol, Tetracycline, Penicillin, Ciprofloxacin, and Gentamycin, with a ratio of 83%, 83%, 67%, 60%, 47%, and 27%, respectively.

The majority of prevalent virulence gene found in this investigation was hlb 70% (21/30), which was followed by hla 63.3% (19/30), as shown in Figures 7 and 8 the eta gene percentage in this investigation was (30/30) 100%.

Table 7: Antibiogram of S. aureus isolates (n=30) against (8 antimicrobial agents).

The infection of the mammary gland, known as bovine mastitis, is one of the most serious diseases affecting dairy herds globally because of its financial implications. It causes considerable losses in decreased production and culling rates (Azooz et al., 2020; Sharun et al., 2021). Based on clinical features, clinical and subclinical mastitis can be differentiated. The former is characterized by watery discharges, clots, or flakes in milk, and the affected areas are usually swollen, hot, and unpleasant.

It is more difficult to diagnose subclinical mastitis because there are no outward signs in animals or milk. Its primary signs are decreased milk supply and an increased somatic cell count. Subclinical duration lasts longer than clinical, and infection allows the spread of pathogens within the herd (Cobirka et al., 2020). In this study, the California mastitis test results revealed that 140 (41.3%) samples tested positive for CMT.

In Iraq, studies including (Al-Iedani and Ghazi, 2016) recorded that the incidence of subclinical mastitis among cattle was 38.89% in Al-Sulaimaniyah Province. Compared with other studies conducted in Basrah, the detection rate of SCM using CMT ranged from 38% to 56.6% (Al-Iedani, 2016; Boss et al., 2016). The traditional microbiological methods used for S. aureus detection rely on enriching the milk sample in Brain heart infusion broth (BHI), differentiating it on Mannitol salts agar, and subculturing on Chromogenic agar. According to several studies, the pathogens most frequently present in mastitis cases are Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Escherichia coli, and Klebsiella pneumonia (Morales-Ubaldo et al., 2023). The S. aureus isolation rate in this study from SCM was 16%. This rate was in line with a previous study in Nepal, which reported that 15.2% of CMT-positive milk was S. aureus (Király et al., 2024). S. aureus in our study is higher than in a study conducted by (Dive et al., 2013), 6(2.8%) cows with SCM were positive for S. aureus. Variations in the sample’s attributes (size, season, type), isolation technique, and geographic locations may account for the discrepancy in S. aureus prevalence between this study and earlier research.

MRSA strains have emerged as formidable nosocomial pathogens and have disseminated globally due to their ability to develop antimicrobial chemotherapy resistance (Shittu et al., 2011). Although using antibiotics in modern medicine to treat infections caused by bacteria has changed the field, the indiscriminate, improper, and frequently abusive use of antibiotics has led bacteria to develop drug resistance (Chakraborty et al., 2022). Most of the world is plagued with bacteria and superbugs resistant to many drugs. Pathogens with antibiotic resistance dramatically increase the rates of morbidity and death.

Antimicrobial resistance is growing quickly and threatens to outpace the introduction of new antimicrobials (Christaki et al., 2020). Cefoxitin is phenotypically an alternative marker and potentially more sensitive for detecting methicillin resistance, as recorded in (Mohammed and Al-Iedani, 2020).

Consequently, (100%) of S. aureus isolates in the recent study were resistant to the cefoxitin disk. This result is higher than reported previously. Mohammed and Al-Iedani (2019), who clarified that the detection rate of MRSA using the cefoxitin disc diffusion method was 66.66%.

When it comes to MRSA isolation, one of the best methods is the detection of mecA. The mecA encodes the lowest β-lactam potential alters protein (PBP2a) found on SCCmec-resistant genomes (Jani et al., 2017). In the current study, the MRSA ratio according to the mecA detected by PCR was 73%. This rate agrees with a previous study (Ibrahim et al., 2023), where it is reported that the rate of MRSA according to the mecA detected by PCR was 86.66%.

In addition, Hammadi and Yousif (2013) have reported that 88% of S. aureus cases were MRSA; in contrast, Al-Jebouri and Mdish (2013) found only 10% of S. aureus cases to be MRSA.

By applying the disc diffusion technique, 30 S. aureus isolates were submitted to antimicrobial susceptibility tests against eight antimicrobial agents. According to the findings of the current study. S. aureus isolates were resistant to Vancomycin (80%) and 83% susceptible to Sulfamethoxazole and Chloramphenicol. These results are higher than reported earlier (Tassew et al., 2017), where it was reported that S. aureus isolates were resistant to Vancomycin in percentage (56.8%). Our findings are in agreement with another study Hoque et al. (2023), where it is reported that S. aureus isolates were susceptible to sulfamethoxazole in a higher percentage (77%).

Furthermore, this study found resistance against Gentamycin, Penicillin, and Erythromycin with decreasing rates of (63.3%, 37%, and 20%), respectively. These results are lower than those reported before Tassew et al. (2017), who have found resistance to penicillin (95.6 %), and in line with gentamycin (59.4%), On the other hand, the isolates were susceptible to Sulfamethoxazole, Chloramphenicol, Tetracycline, Penicillin, Ciprofloxacin and Gentamycin, with a ratio of 83%, 83%, 67%, 60%, and 47% and 27%, respectively.

These results are higher than those reported earlier (Shrestha et al., 2021), who found that Tetracycline was sensitive ( 48.3%).

In contrast, it has been found that maximum susceptibility to Ciprofloxacin is higher than in this study (92% ) and similar to the percentage of Gentamycin in this study (69.23%) (Tassew et al., 2017). The source of isolates may cause minor variations in resistance, and usage of these antibiotics may be the cause of the ongoing rise in resistance.

Few studies investigated hemolysin genes in S. aureus strains isolated from mastitis milk of dairy species. (Aslantas et al., 2022). In the current study, hla (70.3%) and hlb (78%) genes in S. aureus isolates are lower than reported before (Acosta et al., 2018), with a higher percentage of hla (95.93% ) and hlb 93.50%. In contrast, another study. Moraveji et al. (2014) reported the presence of hlb only in one of 20 isolates of S. aureus from bovine mastitis milk. Moreover, the percentage of exfoliative toxin (eta) in this study was 100%. This result disagrees with a previous study Yang et al. (2020), where it is clarified that none of the MRSA isolates carried the eta gene.

Our findings emphasize the critical presence of S. aureus on a wider scale and propose the importance of monitoring antibiotic resistance profiles before the application of antibiotics in animals.

I would like to express my sincere gratitude to my supervisors for their invaluable guidance, support, and encouragement throughout this research. I also appreciate the support of my family and friends, for their patience and motivation.

The nature and genetics of S. aureus in mastitis cases, as well as the presence of virulence genes, underpin the driving of antibiotic resistance. The finding would be valuable information for both animal handlers and milk consumers.

NNJ, RAF: Supervision of writing review and editing.

ZAF: Data collection, formal analysis, writing original draft.

All authors contributed significantly to the research, discussed the results, and approved the final version of the manuscript.

The authors have carefully reviewed all AI-assisted outputs to ensure accuracy, integrity, and compliance with the ethical standards of research and publication. Responsibility for the content of this manuscript remains entirely with the authors.

The author declares that there are no conflicts of interest related to this research. The study was conducted independently, without any financial, commercial, or personal relationships that could be construed as potential sources of bias. All experimental design, data collection, analysis, and interpretation were carried out solely by the author and collaborators within the academic framework.