Dairy mastitis is currently the most important challenge facing the dairy industry in various countries throughout the world. This reduces milk production and thereby adversely affects animal well-being while also exacting an economic toll on the dairy industry (Khan et al., 2019). Subclinical mastitis is most prevalent in North America and Uganda, while clinical mastitis is most common in the United Kingdom (Krishnamoorthy et al., 2021). Dairy mastitis is a form of intramammary inflammation that is most often caused by certain pathogenic bacterial species including Escherichia coli, Streptococcus uberis, Staphylococcus aureus, and Klebsiella pneumonia (Bar et al., 2008; Klaas and Zadoks, 2018; Machado and Bicalho, 2018). While antibiotics are generally regarded as the first choice approach to treating this devastating disease, the excessive use of antibiotics can result in the presence of residual antibiotics in foods from these animals, thus contributing to the emergence of antibiotic-resistant bacteria (ARBs). These ARBs can move from the food chain into humans and the environment, with multidrug-resistant bacteria causing an estimated 700,000 deaths per year worldwide (Krömker and Leimbach, 2017). Without appropriate measures to curb antibiotic use, it is estimated that global antimicrobial consumption in the context of the production of food animals will rise from 63,151 tons in 2010 to 105,596 tons as of 2030 (Van Boeckel et al., 2015).

These factors underscore the urgent need to explore alternatives to traditional antibiotics for use in the post-antibiotic era. Plant-derived compounds such as polyphenols (Manso et al., 2021), flavonoids (Biharee et al., 2020), terpenoids (Sycz et al., 2022), plant essential oils (Lopes et al., 2020), and alkaloids (Othman et al., 2019) have all been reported to exhibit antimicrobial activity. The natural plant derivative ursolic acid (UA, C₃₀H₄₈O₃) is present in the stem bark, leaves, and peels of many Chinese herbs and fruits. UA exhibits diverse antioxidant, anti-inflammatory, antimicrobial, antitumor, hepatoprotective, and other activities (Hussain et al., 2017; Liu et al., 2023). UA or derivatives prepared therefrom exhibit robust antibacterial efficacy against Gram-positive microbes including Streptococcus mutans (Park et al., 2015), S. aureus (ATCC 6538), and methicillin-resistant S. aureus (MRSA) (Kefi et al., 2023; Du et al., 2024). Gram-negative bacteria are surrounded by a phospholipid-and LPS-rich outer membrane that restricts UA entry into these cells such that minimum inhibitory concentration (MIC) values for UA when used to treat Gram-negative bacteria tend to be higher than those for Gram-positive bacteria (Sycz et al., 2022).

The UA has also been reported to exhibit synergistic efficacy when applied in combination with β-lactam antibiotics such as ampicillin and benzacillin, enhancing the antibiotic sensitivity of S. aureus and Staphylococcus epidermidis (Kurek et al., 2012). UA (32 μg/mL) also reportedly synergizes with colistin when used to treat clinical Klebsiella pneumoniae BC936 and E. coli U3790 isolates (Sundaramoorthy et al., 2019). Despite the promising in vitro bacteriostatic activity of UA, it is not commonly used in the context of poultry or livestock production. The utilization of UA as a feed additive for animal husbandry warrants consideration as an alternative to antibiotic administration.

The antibacterial actions of UA are reported to be associated with morphological changes in the bacterial cells, inhibition of biofilm formation, and impairment of the bacterial cell membrane (Sycz et al., 2022). UA reduces biofilm synthesis by S. mutans by competitive inhibition of glucosyltransferases and reducing the expression of the gft gene that inhibits the binding of extracellular polymeric substance (EPS) in the biofilm (Liu et al., 2021; Lyu et al., 2021). According to Ren et al. (2005), UA isolated from Diospyros dendo leaves reduces biofilm formation by E. coli (including ATCC 25404), which contributes to the overexpression of motAB genes. UA damages the membrane integrity of MRSA and increases the concentration of Alkyl hydroperoxide reductase subunit C (AhpC) that related to the oxidative response (Wang et al., 2016).

The present study details the antibacterial activity of UA preparations of different purity levels (98, 50, 30, and 10% pure) and the synergistic effects of combining UA and antibiotics when treating S. aureus strains. Scanning and transmission electron microscopy (SEM and TEM) analyses were also performed, as were flow cytometry and S. aureus biofilm formation assays, thereby enabling the detailed examination of the mechanistic basis for the antimicrobial effects of UA. Together, these results will provide a valuable foundation for efforts to apply UA as an alternative to antibiotics when seeking to treat dairy mastitis.

The UA has been reported to exhibit a wide range of promising uses as an antibacterial agent and antioxidant in settings including the cosmetic industry (López-Hortas et al., 2018). Sekandi et al. (2023) demonstrated that UA from Spermacoce princeae (K. Schum) showed effective bactericidal and antifungal activity against C. albicans, S. aureus, and P. aeruginosa strains (zone of inhibition diameter, 12–20 mm), while exhibiting weaker bacteriostasis against E. coli and K. pneumoniae (zone of inhibition diameter, <10 mm). Here, UA extracted from Rosmarinus officinalis L. (98% purity) was found to effectively inhibit the growth of S. aureus ATCC25923, S. dysgalactiae ATCC27957, E. faecalis ATCC29212, and S. mutans ATCC25175 with MIC values ranging from 9.75–39 μg/mL, while also inhibiting S. agalactiae ATCC13813 at a MIC of 156 μg/mL. UA was unable to inhibit the growth of E. coli ATCC 25922 or clinical Gram-negative isolates (Klebsiella pneumoniae, Fecal coliform, and Salmonella), which exhibited MIC values greater than 5,000 μg/mL. The bacteriostatic activity of UA was positively correlated with its purity in this study, and the MBC values for 98% UA ranged from 78–156 μg/mL when used to treat clinical S. aureus isolates. These results confirmed that UA was better able to suppress the growth of Gram-positive bacteria relative to Gram-negative bacteria, in line with prior evidence (Horiuchi et al., 2007; Wrońska et al., 2022). This is likely attributable to the fact that Gram-negative bacteria exhibit an outer membrane and express efflux pumps that prevent the intracellular entry and accumulation of UA and related compounds (Briers and Lavigne, 2015; Opperman and Nguyen, 2015). UA was able to inhibit the growth of Gram-positive S. aureus and S. epidermidis by 80% at a 20 μg/mL dose in a prior study, whereas higher concentrations (50 μg/mL) only suppressed the growth of E. coli, P. hauseri, and C. jejuni by 20–30% (Wrońska et al., 2022). The MIC for UA derived from the aerial portions of Sambucus australis against S. aureus (ATCC 6538) was previously measured at 32 μg/mL, whereas it exhibited moderate levels of activity against E. coli (ATCC 25922), K. pneumoniae (ATCC 10031), and Shigella flexneri (ATCC 12022) with a MIC of 64 μg/mL (do Nascimento et al., 2014). There is also prior evidence supporting the activity of UA extracted from Alstonia scholaris leaves against E. faecalis, L. monocytogenes, and Bacillus cereus (MIC: 1–8 μg/mL), whereas it failed to adversely affect E. coli, S. enterica, and P. aeruginosa (MIC >128 μg/mL) (Wang et al., 2016). UA derived from D. melanoxylon and S. australis, when used to treat a range of Gram-negative bacteria (E. coli, K. pneumoniae, P. syringae, S. typhi, and S. flexneri) with good antibacterial efficacy (MIC: 25–64 μg/mL) (Mallavadhani et al., 2004; do Nascimento et al., 2014).

In an effort to enhance the sensitivity of Gram-negative bacteria to UA, derivatives thereof with altered structural properties can be used, or UA can be applied in a synergistic manner with antibiotics (Cunha et al., 2010; Mlala et al., 2019; Wu et al., 2021; Pereira et al., 2022). For example, the MIC of a UA derivative in which the C-3 OH group had been modified to produce 3β-acetoxy-urs-12-en-28-oic acid was lower than that of unmodified UA when used to treat S. flexneri (ATCC 12022), Vibrio cholerae (ATCC 15748), Listeria monocytogenes (ATCC 19117), and E. coli (ATCC 25922). Moreover, the combination of UA and kanamycin increased E. coli (ATCC 25922) susceptibility, reducing MIC values from 128 to 16 μg/mL (do Nascimento et al., 2014). Here, the administration of 1/4x MIC UA plus 1/2x MIC ceftiofur hydrochloride or 1/8x MIC ampicillin was sufficient to suppress the growth of clinical S. aureus isolates more readily than either UA or antibiotics alone. The 98% pure UA extracted from Rosmarinus officinalis L. and these tested antibiotics thus exhibited good synergistic activity against dairy cow-derived S. aureus isolates.

Staphylococcus aureus demonstrated a high degree of susceptibility to UA treatment, with MIC values at the lowest tested concentrations of UA (9.75–78 μg/mL) and MBC values ranging from 9.75–156 μg/mL. The bacterial growth of three S. aureus strains was fully inhibited by treatment with 1x and 2x MIC doses of 98% UA, and even those bacteria treated with a 1/2x MIC dose of UA exhibited an extended lag phase followed by a less pronounced exponential growth phase from 14 to 24 h, with this prolongation of the lag phase being attributable to the inhibition of DNA replication (Rolfe et al., 2012), consistent with the ability of UA to delay bacterial growth. Biofilm formation by bacteria growth on abiotic and biotic sources is a complex process that entails adhesion, the production of extracellular polymeric substances, the formation of matrix and microcolonies, and dispersal (Rabin et al., 2015). The ability of bacteria to establish biofilms is closely related to the resistance of these microbes. Here, UA concentrations above the MIC were associated with reduced biofilm formation, with no differences in biofilm levels between the control group and S. aureus S5 and S6 treated with a 1/2x, 1/4x MIC dose of UA. Zhou et al. (2013) previously showed that UA from Sigma-Aldrich was able to inhibit S. mutans and S. gordonii biofilm formation at a sub-inhibitory 1/4x MIC concentration (64 μg/mL), while UA extracted from Arctium lappa leaves can reportedly significantly suppress P. aeruginosa biofilm formation at a 500 μg/mL dose (Lou et al., 2015).

Electron microscopy examinations demonstrated that UA extracted from Rosmarinus officinalis L. significantly altered the shapes of treated S. aureus cells such that they became irregularly shaped with a rough surface, in contrast to the smooth, rounded appearance of control cells. Cell wall and membrane damage were evident in treated cells, with cytoplasmic irregularities. This is similar to what has been reported previously with respect to the morphological changes evident in S. aureus cells treated with vine tea extract and its active ingredient, 2R, 3R-Dihydromyricetin (Liang et al., 2020). These compounds were shown to be capable of disrupting membrane permeability via increasing extracellular β-galactosidase content and decreasing total protein levels by 15.5 and 9.9%, respectively (Liang et al., 2020). UA treatment has also been demonstrated to suppress the viability of carbapenem-resistant Klebsiella pneumoniae (CRKP), inhibiting biofilm formation and inducing the downregulation of genes associated with biofilm production (pgaA, luxS, wbbM, and wzm) while also disrupting the membranes of these cells such that cytoplasmic contents were able to leak out and the overall size of the cytosol was reduced (Qian et al., 2020). These results align well with the observed electron microscopy findings for UA-treated bacteria cells, which exhibited clear evidence of cell wall damage and the leakage of intracellular contents. Therefore, the numbers of S. aureus proteins were decreased by UA treatment in this study detected by SDS-PAGE. Using two-dimensional (2D) proteomic analysis, it demonstrated that UA damaged the membrane integrity of MRSA, and induced the proteins involved in the bacterial phosphoenolpyruvate sugar phosphotransferase system and the oxidative response (Wang et al., 2016).

The present data support the ability of UA to suppress biofilm-related activity in S. aureus as previously reported by Jyothi et al. (2018). The triterpenoid madecassic acid has been shown to disrupt cell wall and cytoplasmic membrane integrity to trigger the release of contents from the cytoplasm, while also inhibiting TCA cycle activity, decreasing the activity of malate dehydrogenase and succinate dehydrogenase, and separating DNA base pairs (Wei et al., 2023). These compounds may also promote hydrophobic phenolic group accumulation within the lipid bilayer, thereby mediating lipid-protein interactions that increase the overall permeability of the membrane and destroy its integrity (Zore et al., 2011; Wu et al., 2016; Park et al., 2018). UA can impact many different genes involved in key metabolic processes in S. mutans, inhibiting glycolysis, fatty acid synthesis, amino acid synthesis, and peptidoglycan synthesis, thus mediating antimicrobial effects (Park et al., 2018).

It has been reported that the cell membrane integrity is crucial to prevent the excessive accumulation and production of reactive oxygen species (Wang et al., 2016; Yang et al., 2023). Prior studies have highlighted a correlation between increased ROS biogenesis and bacterial apoptosis (Li et al., 2021). Isobavachalcone flavonoids, when used to treat S. aureus at a 2x MIC dose, can reduce the ΔpH aspect of the proton motive force, interfering with bacterial membrane homeostasis such that ROS accumulation occurs (Song et al., 2021). To further clarify the mechanisms whereby UA kills bacterial, intracellular ROS content was assessed with DCFH-DA, revealing that 98% UA treatment at doses of 78–625 μg/mL was sufficient to significantly increase ROS levels within S. aureus cells by 291.4, 149.8, 114.9, and 43.8%, with a negative correlation between 98% UA concentrations and ROS levels.

In summary, these findings highlight that 98% UA from Rosmarinus officinalis L. exhibits robust antibacterial effects against Gram-positive bacteria without any corresponding impact on Gram-negative bacteria. Clinical S. aureus isolates associated with bovine mastitis, when treated with 98% UA at 78 μg/mL, exhibited irregularly shaped cells and the dissolution of cell wall and membrane layers, with the pronounced inhibition of intracellular protein synthesis and high levels of ROS production (Figure 5). Based on these data, UA represents a promising alternative to traditional antibiotics that can be used alone or in combination with extant antibiotic drugs in an effort to treat S. aureus infections.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: the NGS sequences for the S. aureus S4, S. aureus S5, and S. aureus S6 isolates were submitted to the NCBI database (BioSample accession number SAMN38649273, SAMN38673743 and SAMN38673744).

GL: Writing – original draft, Writing – review & editing. PQ: Data curation, Project administration, Writing – original draft. XC: Investigation, Supervision, Writing – review & editing. LW: Investigation, Resources, Writing – review & editing. WZ: Software, Writing – review & editing. WG: Funding acquisition, Supervision, Writing – review & editing.