Staphylococcus aureus (S. aureus) is a widely recognized bacterium that can spread to humans and animals resulting in life-threatening illnesses. It is also a major cause of bovine mastitis in cattle, buffalo, sheep and goats (Kløve et al., 2022). In dairy farms, S. aureus mastitis and its produced toxins cause major economic losses including decreased milk production, excessive drug residues contamination and chronic illness leading to deaths (Algammal et al., 2020). The antimicrobial resistance rates among mastitis S. aureus in Egypt are increased during the last few decades (El-Jakee et al., 2011; Ameen et al., 2019). Methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA), commonly called superbugs, are among the most significant pathogens that pose major threats to both human and animal health (Shrestha et al., 2021). Unfortunately, most β -lactam antibiotics are ineffective against S. aureus isolates, which harbors mecA/mecC genes (Hiramatsu et al., 2014; Saber et al., 2022). The fact that MRSA may produce biofilms on biotic and abiotic surfaces (Ascioferro et al., 2021) renders the issue even more challenging to be eradicated. It has been known for a considerable time that staphylococcal isolates are the most common causes of infections that are connected with biofilms (Lebeaux et al., 2013). The extracellular matrix, changing metabolic states and growth rate render biofilms more resistant to antibiotics than planktonic organisms (Silva et al., 2021). Notably, MRSA strains biofilm development and multidrug resistant (MDR) profile increase the possibility of chemotherapeutic failure (Abd-El-Hamid et al., 2020).

Moreover, virulence arrays are essential for overcoming the host defense power and increasing bacterial pathogenicity. Most S. aureus mastitis strains are multivirulent and are associated with biofilm production. The biofilm associated protein (BAP) is correlated with the presence of bap gene and ica operon, which control polysaccharide intercellular adhesin synthesis producing an extremely structured multicellular biofilm (Zhang et al., 2018). Moreover, S. aureus enterotoxins (SEs) and hemolysins (Hlα and Hlβ) promote pathogenicity by enhancing the pathogen’s adhesion, colonization and tissue invasion (Puah et al., 2016). Expression of S. aureus virulence genes is controlled via accessory gene regulatory (agr) system, which can be divided into four groups according to agrC and agrD gene sequences; agr I, II, III and IV. Although agr types vary in properties and prevalence according to geography, identifying the dominant type in each region may be beneficial (Javdan et al., 2019).

Intramammary infections caused by biofilm-producing S. aureus are common among cows with chronic mastitis. The incidence of these infections is increasing with poor management practices during milking. Milk is an excellent medium for the growth of bacterial species gaining access to the upper part of the gland with the production of virulence proteins and toxins leading to impairment of the host defense power and inflammation of the mammary gland (Foster and Höök, 1998). Of note, most available antibiotics are inefficient in eradicating these infections. To avoid treatment failure in S. aureus mastitis, new and alternative therapies in combination with available antimicrobial drugs must be formulated. In recent years, there has been growing interest in the utilization of naturally occurring substances such as essential oils (EOs) manufactured from different plant components owing to their biological effects including antioxidant, anti-inflammatory and anticancer (Krifa et al., 2015). Additionally, EOs have been widely reported in the scientific literature as potential antibacterial agents as they are efficient against a wide variety of pathogenic bacteria and yeast (Swamy et al., 2016; Puškárová et al., 2017). The leaves and inflorescence of Origanum vulgare are the main sources for carvacrol essential oil (EO). This plant was used in the ancient alternative medicine as antimicrobial, antidiabetic, anticancer and anti-inflammatory agent (Leyva-Lopez et al., 2017). The monoterpenic phenol such as carvacrol [2-methyl-5-(1-methylethyl) phenol] has significant effects on microbial cell membrane, respiratory metabolism and DNA (Cui et al., 2019). Additionally, it has antivirulence effects on foodborne pathogens, especially S. aureus (Cui et al., 2019). Interestingly, linalool, which has antimicrobial and antifungal properties is the main constituent of Lavandula officinalis and Citrus sinensis EOs (Kasper et al., 2010; Wang et al., 2023). In the same context, eugenol has potential activities against resistant pathogens including bacteria, fungi as well as viral infections. It is found in abundant amounts in clove buds (Syzygium aromaticum), cinnamon bark and leaves (Cinnamomum verum) (Taleuzzaman et al., 2021).

Relating to the abovementioned issues, the purpose of the current study was to investigate the antimicrobial resistance and virulence profiles of MRSA strains causing mastitis and to evaluate the antimicrobial and antibiofilm activities of different natural compounds including carvacrol, linalool, and eugenol against MRSA strains.

Bovine mastitis as one of the most important dairy cattle diseases affecting mammary tissue may be chronic, clinical or subclinical leading to great economic losses. Of note, serious zoonotic diseases were always associated with different form of mastitis (Hoe and Ruegg, 2005). Moreover, mastitis shares in the wide spread of antimicrobial resistance globally (Beyene et al., 2017). The resistant biofilm producing S. aureus is the most common cause of bovine mastitis; therefore, new therapeutic options including complementary and alternative therapies are urgently required (Ghaly et al., 2021; Mosallam et al., 2021; Elfaky et al., 2022; Ghaly et al., 2023). So, we aimed to find successful antimicrobial protocols to prevent the wide spreading of mastitis that is particularly associated with biofilm producing S. aureus in the endemic area.

In this study, we recorded relative higher prevalence of bovine mastitis with S. aureus infections (20.6%). Other studies announced lower prevalence rates of S. aureus (16.1, 10 and 3%) among mastitic cows (Schukken et al., 2009; Tenhagen et al., 2009; Beyene et al., 2017). The variations in the prevalence rates of S. aureus among mastitic cows in this study and other previous studies may be attributed to differences in the standard hygienic practices applied in different countries (Getaneh and Gebremedhin, 2017). Therefore, proper control practices should be directed to prevent the wide spreading of bovine mastitis through segregations or selective culling of infected animals in some cases, which did not respond to any type of treatment protocols side by side with proper milking procedures (Ruegg, 2017). Interestingly, poor prognosis is always associated with the bacterial pathogens with multivirulence arrays (Ammar et al., 2020). Biofilm production is one of the main causes of antimicrobial resistance and it is a leading trait for increasing the sharpness and frequency of bovine mastitis treatment failure. Biofilm producing S. aureus is the causative agent of severe mastitis cases that respond very slowly to treatment (Szweda et al., 2012; Abd-El-Hamid et al., 2020). In this study, all our S. aureus isolates were phenotypically and genotypically identified as biofilm producers. The phenotypic identification of biofilm producers matched with the genetic detection of icaA gene among all S. aureus isolates. Previously, almost all S. aureus isolates causing bovine mastitis were able to produce biofilms (de Castro Melo et al., 2013; Bendary et al., 2016; Notcovich et al., 2018).

Of note, all our isolates were identified as MRSA, which showed complete resistance to cefoxitin and harbored mecA gene. Unfortunately, VRSA were detected among our isolates with a relative higher prevalence rate (40.5%). Convergently, previous reports announced that MRSA were the most prevalent recovered strains among bovine mastitis (Holmes and Zadoks, 2011; Krukowski et al., 2020). Moreover, 73% (27/37) of the tested MRSA isolates and all VRSA ones were MDR. Of note, the antimicrobial resistance and the wide spreading of MDR isolates were increasingly noticeable among bovine mastitis cases worldwide (Hoque et al., 2018; Salauddin et al., 2020). The antimicrobial resistance is a global multifaceted phenomenon and the increasing rates of this problem may be attributed to several factors including the inappropriate use of antibiotics, especially in veterinary fields as growth enhancers, self-medication and the poor application of antimicrobial stewardship programs (Prestinaci et al., 2015).

Surprisingly, most of our isolates were multivirulent harboring three or more virulence genes, which may compound the severity of the diseases. Therefore, bovine mastitis associated with biofilm producing MRSA showing MDR patterns is a common crisis globally. Therefore, there is an urgent need for additional efforts and researches to address this issue. Poor prognosis is always associated with biofilm producing MRSA strains owing to the extreme resistance of microbial cells in biofilms. For that, finding new alternatives are important research approaches, which have attracted the interest of many researchers. One of innovative approaches to treat S. aureus biofilm-related infections was evaluated in previous studies using non-antibiotics drugs (Kiedrowski and Horswill, 2011; Richter et al., 2017). Some detergent such as cis-2-decanoic acid could disperse staphylococcal biofilms (Davies and Marques, 2009). Moreover, EOs such as carvacrol, linalool and eugenol have been used in food industry owing to their preservative potency against foodborne pathogens. In this report, we evaluated the antimicrobial and antibiofilm activities of these EOs against S. aureus, the predominant contagious pathogens causing bovine mastitis and we found great anti-MRSA activities of the investigated EOs, especially linalool. This finding goes parallel with the published results for linalool antimicrobial and antibiofilm activities (Bagamboula et al., 2004; Aelenei et al., 2019). The cell membrane, especially mesosomes and cell wall integrity are the targeting sites of linalool (Gao et al., 2019). Interestingly, the synergistic interactions between the investigated EOs and other antimicrobial drugs were announced in this study. Several authors stated that treatment failure owing to antimicrobial resistance could be solved via using combinations of the available antibiotics and other natural compound such as EOs (Lahmar et al., 2017; Aelenei et al., 2019; Özel et al., 2022). These EOs could increase the rates of antimicrobial susceptibility and reversal of antimicrobial resistance. The antibiotic actions could be rescued when used in combination with EOs. In accordance with our results, the resistances of MRSA to β-lactam antibiotics were highly reduced in the presence of EOs (Lahmar et al., 2017). Although the exact synergistic interactions between antibiotics and EOs have still not been exactly clarified, several authors used time-kill curve analysis to confirm the efficacy of these combinations (Aleksic et al., 2014; Abd El-Hamid et al., 2022).

Multidrug resistance and multivirulence were the common phenotypes among MRSA strains incriminated in bovine mastitis. Despite the great difficulties to control and eradicate these phenotypes with common used antimicrobial drugs, EOs, especially linalool as proven in this study give us the bright hope to increase the therapeutic options and the possibility of treatment success. Therefore, we recommended using combination therapies between the available antibiotics and the natural compounds such as EOs.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

Collection of milk samples in this study was approved by the Animal Ethics Review Committee of Suez Canal University (AERCSCU2023029), Egypt.

MA: Methodology, Software, Writing – review & editing. RE: Investigation, Formal Analysis, Writing – review & editing. MBa: Data curation, Project administration, Writing – review & editing. MA: Software, Visualization, Writing – review & editing. AS: Data curation, Formal Analysis, Writing – review & editing. KA: Funding acquisition, Data curation, Writing – review & editing. FS: Supervision, Writing – review & editing. AM: Supervision, Formal Analysis, Writing – review & editing. NA: Conceptualization, Validation, Writing – review & editing. MG: Resources, Supervision, Writing – review & editing. AE: Methodology, Software, Writing – review & editing. HA: Formal Analysis, Writing – review & editing. MBe: Methodology, Writing – original draft, Writing – review & editing.