Liquid chromatography–mass spectrometry (LC–MS)-grade methanol and acetonitrile were purchased from Scharlab Italia srl (Milan, Italy); distilled water was obtained via a Milli-Q system (Millipore, Bedford, MA, USA). MS-grade ammonium acetate, acetic acid, and formic acid from Fisher Chemical (Thermo Fisher Scientific Inc., San Jose, CA, USA) were also used. Ursolic and asiatic acid <98% (HPLC) were purchased from Sigma–Aldrich (Schnelldorf, Germany).

Leaves of Eucalyptus globulus (Labill., 1800) (EGL-L) were collected from eight different plants grown in the botanical garden of the INES Ruhengeri Institute of Applied Sciences, Musanze, Rwanda. Leaves were washed with tap water and air dried for 3 weeks. After drying, the leaves were ground using a grinder (particle size <800 μm).

Three aliquots of Eucalyptus leaves (20 g each) were shredded, placed into separate hermetic flasks and subjected to hydroalcoholic maceration with 70% ethanol. A 1:10 matrix-to-solvent ratio was used. The extraction process was carried out for 72 h at room temperature (RT) with constant stirring at 100 rpm. After extraction, the extracts were filtered, and the solvent was removed via a rotary evaporator (Buchi). The resulting extracts were frozen with liquid nitrogen and then freeze-dried. The lyophilization process was conducted under vacuum conditions for 36 h at −56°C and 1 mbar (1-DL alpha Plus freeze-drier). The dried extracts were divided into different aliquots and reconstituted in EtOH:water (70,30) prior to LC–MS analysis.

The quantification of UA and AA in EGL-L was conducted via HPLC–MS in SIM mode. The analyses were performed in triplicate to ensure accuracy and reproducibility. Calibration curves were constructed for both compounds, which were subsequently used to determine their concentrations in the extract samples. HPLC–MS analysis was performed using a 2,695 Alliance separation system (Waters Go, Milford, MA, USA) equipped with a QuattroTM API triple quadrupole mass spectrometer with an electrospray source (Micromass, Waters, Manchester, UK). Chromatographic conditions were the following: Column XSelect®HSST3 (250 mm × 2.1 mm, 5 μm), flow rate 0.2 mL/min, column temperature 30°C, injection volume 5 μL. A gradient profile was applied using water (eluent A) and acetonitrile (eluent B) as mobile phases both acidified with 0.1% formic acid. The initial conditions were set at 100% A, after 5 min of the isocratic step, a linear change to 100% B at 8 min, and holding for 7 min before returning to initial conditions. Columns recondition was achieved over 6 min, providing a total run time of 21 min. The column was maintained at 30°C and a flow rate of 0.20 mL/min was used. MSD parameter: ESI negative, capillary voltage 2.5 kV, cone voltage 25 V, extractor voltage 2, source block temperature 120°C, desolvation temperature 350°C. Cone-gas-flow nitrogen and argon were used as collision gas. Selective ion monitoring (SIM) in negative ion mode was used. Ursolic acid was recorded as m/z 455.4 [M-H]⁻ while asiatic acid as 487.7 [M-H]⁻. UA and AA could not be collided into fragments when collision energy was 40 eV, or no dominant product ions were detected if collision energy was higher than 50 eV, which indicated that MRM experiment was not suitable for UA and AA quantification. Analytes concentrations in the sample were calculated from the relation within slope line obtained by linear regression analysis of this calibration curve and multiplied for their dilution factor.

The following reference strains were tested: Staphylococcus aureus ATCC 25923, methicillin-resistant Staphylococcus aureus ATCC 43300 (MRSA), Streptococcus agalactiae ATCC 27956, Streptococcus uberis ATCC 19496 and Enterococcus faecium ATCC 19434.

Clinical isolates of Staphylococcus aureus (SA; n = 15), Streptococcus agalactiae (SAG; n = 17), Streptococcus uberis (SU; n = 18), and Enterococcus spp. (EN; n = 13), obtained from cows with subclinical or clinical mastitis, were tested.

Clinical strains were provided by the biobanks of the Animal Infectious Disease Laboratory of the University of Parma and by the IZSLER (Istituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia-Romagna) – Laboratory of Parma.

Bacterial strains were grown in Columbia blood agar after incubation of 24 h at 37°C. On individual colonies, Gram staining and catalase test were performed, followed by execution of API Staph® and API 20 Strep® System test (bioMérieux), as well as indicated by manufacturer (27).

AST was performed via the Kirby–Bauer disk diffusion method. Owing to the intrinsic resistance of Enterococcus spp., testing for this genus was limited to the following antibiotics: amoxicillin/clavulanic acid (20/10 μg), ampicillin (10 μg), enrofloxacin (5 μg), erythromycin (15 μg), florfenicol (30 μg), imipenem (10 μg), marbofloxacin (5 μg), oxytetracycline (30 μg), penicillin G (10 U), rifaximin (40 μg), and vancomycin (30 μg). For the strains belonging to the Streptococcus genus, in addition to the abovementioned antibiotics, the following antibiotics were also tested: cefazolin (30 μg), cefquinome (30 μg), ceftiofur (30 μg), cefuroxime (30 μg), and trimethoprim/sulphamethoxazole (1.25/23.75 μg). Staphylococcus aureus isolates were tested with all the antibiotics listed above, except vancomycin. Additionally, they were tested with fusidic acid (10 μg), gentamicin (10 μg), kanamycin (30 μg) and lincomycin (15 μg). The interpretation of the results followed the guidelines of the Clinical and Laboratory Standards Institute (CLSI) veterinary breakpoints. When veterinary breakpoints were not available, human breakpoints were adopted (28). To assess whether the microorganism was MDR or non-MDR, the categorization proposed in the literature was adopted (29).

Minimal inhibitory concentration (MIC) assays were performed on both field and reference bacterial strains. The bacterial inoculum was prepared according to the CLSI standard method (30). All microbiological assays were performed within 30 min after inoculum standardization. Briefly, for each strain, five bacterial colonies from fresh solid cultures were inoculated in sterile tubes with Müeller Hinton broth (MHB) and incubated at 37°C under aerobic conditions for 24 h for staphylococci, while streptococci and enterococci were incubated under microaerophilic conditions. After incubation, the bacterial suspension was centrifuged at 2000 rpm for 20 min at 4°C to separate the pellet containing bacteria from the supernatant. The pellet was subsequently resuspended in 10 mM phosphate buffer (PB), pH 7, to obtain an optical density (OD) of 0.08–0.13 at 600 nm in a 1 cm light path cuvette, corresponding to approximately 0.5 McFarland suspension (10⁸ CFU/mL). This suspension was further diluted 1:100 in sterile MHB. Then, 50 μL of the bacterial suspension, containing 10⁶ CFU/mL, was inoculated into each well to obtain a final concentration of 5×10⁵ CFU/mL. The bacterial suspensions were assessed via a Biophotometer plus (Eppendorf, Hamburg, Germany) spectrophotometer at 600 nm.

The MIC assay was performed following the methods outlined in the CLSI standard methods, with minor modifications. Briefly, EGL-L, AA and UA were prepared as stock solutions in DMSO at concentrations of 200 mg/mL for EGL-L and 25.6 mg/mL for UA and AA. Serial twofold dilutions of each compound in DMSO were performed in a 96-well microtiter plate (Greiner, Milan, Italy). Then, one microliter of each diluted compound was added to the wells of the plates, followed by the addition of 50 μL of bacterial suspension containing 10⁶ CFU/mL, resulting in a final bacterial concentration of 5×10⁵ CFU/ml. The final dilution range for EGL-L was 2000 to 3.9 μg/mL, whereas for AA and UA, it ranged from 256 to 0.5 μg/mL. Growth and sterility controls were included for each bacterial strain and compound tested. The plates were incubated at 37°C for 24 h as described above for each bacterial strain. For each assay, three experiments were performed, with three replicates each. After incubation, the MIC was determined by visual inspection. The minimal inhibitory concentration was considered the lowest concentration able to completely inhibit bacterial growth. Furthermore, the MIC₅₀ and MIC₉₀ were calculated: given a graded series of MICs starting with the lowest value, MIC₅₀ is the MIC value at which 50% of the isolates in a test population are inhibited (equivalent to the median MIC value), and the MIC₉₀ is calculated as n × 0.9, where n represents the test strains and represents the 90th percentile, as specified in the literature (31).

The bactericidal kinetic curves (BKC) of EGL-L, AA and UA were determined following the methods reported in literature (32). For each compound, three concentrations were selected based on their MIC values against the reference bacteria: 1 × MIC, 2 × MIC, and 4 × MIC. A growth control was also included. Each compound was subjected to serial twofold dilutions in DMSO in a 96-well microtiter plate (Greiner, Milan, Italy). Subsequently, 1 μL of each diluted solution was transferred to the respective wells, followed by the addition of 50 μL of bacterial suspension at a concentration of 10⁶ CFU/mL, resulting in a final bacterial concentration of 5 × 10⁵ CFU/mL. BKC were monitored by measuring the optical density at 620 nm using a spectrophotometer reading plates (Biophotometer Plus, Eppendorf, Hamburg, Germany). Measurements were taken at the following time points (T): 0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 24, and 48 h.

The biofilm-forming ability of the reference and field strains was evaluated via methods described in the literature (33), with slight modifications. The bacterial strains were cultivated in MHB at 37°C for 24 h. After incubation, the bacterial suspension was centrifuged at 2000 rpm for 20 min at 4°C, and the supernatant was removed. The pellet was resuspended in 10 mM phosphate buffer (PB), pH 7, and adjusted spectrophotometrically to obtain a final concentration of 10⁷ CFU/mL. Each well of a 96-well flat-bottomed microtiter plate was filled with 180 μL of tryptic soy broth (TSB) supplemented with 1% glucose. Thereafter, 20 μL of the previously prepared bacterial suspension was added to each well, and the plates were incubated at 37°C for 24 h. The sterility control consisted of 180 μL of TSB supplemented with 1% glucose and 20 μL of PB. Three replicates were carried out for each bacterial strain. The tests were repeated three times for each bacterial strain. After incubation, the wells were emptied and washed three times with sterile phosphate-buffered saline (PBS; pH 7.2). The residual liquid was removed by gentle flicking, and the remaining biofilm was fixed with 150 μL of methanol for 15 min. After the methanol was removed, the plates were air-dried for 2 h at room temperature. The biofilms were stained with 0.1% Hucker crystal violet (150 μL for each well) for 30 min. After staining, the crystal violet solution was removed by sequential washing with sterile deionized water. The wells were then air-dried, and 150 μL of 95% ethanol was added to each well for 30 min to solubilize the stain.

The solubilized stain was transferred to a new flat-bottomed microtiter plate, and the OD was measured at 620 nm. The biofilm-forming ability was classified on the basis of the criteria described in the literature (33, 34). Briefly, the cutoff OD (ODc) was defined as three times the standard deviation above the mean OD of the negative control, and the strains were classified as follows:

OD ≤ OD c nonadherent ( NA )

OD c < OD ≤ 2 X OD c weakly adherent ( WA )

2 X OD c < OD ≤ 4 X OD c moderately adherent ( MA )

4 x OD c ≤ OD strongly adherent ( StA ) .

The minimal biofilm inhibitory concentrations (MBICs) of all the tested compounds were determined for reference and field strains classified as MA or StA. Each well of a 96-well flat-bottomed microtiter plate was filled with 178 μL of TSB supplemented with 1% glucose. Twofold dilutions of each stock solution of EGL-L, AA and UA in DMSO were prepared in a separate microtiter plate, and 2 μL of each dilution was then added to the respective wells (Greiner, Milan, Italy). The final dilution range for EGL-L was 2000 to 3.9 μg/mL, whereas for AA and UA, it ranged from 256 to 0.5 μg/mL.

The bacterial pellet was prepared as described above, resuspended in PB and adjusted spectrophotometrically to obtain a final concentration of 10⁷ CFU/mL. Thereafter, 20 μL of the bacterial suspension was added to each well. The growth control consisted of 180 μL of TSB supplemented with 1% glucose and 20 μL of bacterial suspension (10⁷ CFU/mL). The sterility control was prepared with 200 μL of TSB with 1% glucose. The plates were incubated at 37°C for 24 h. Three replicates were carried out for each bacterial strain. After incubation, biofilm staining was performed as described above.

For each tested compound and concentration, the percent inhibition was calculated via the following formula: OD inhibition = ( 1 − OD GC / OD x ) %

where:

OD GC = Mean OD of the growth control .

OD x = Mean OD of the tested concentration .

The minimal biofilm inhibitory concentration 50 (MBIC₅₀) and minimal biofilm inhibitory concentration 80 (MBIC₈₀) values were calculated for each bacterial strain.

The MBIC₅₀ and MBIC₈₀ were defined as the minimal concentrations of compounds able to inhibit 50 and 80% of biofilm formation, respectively, compared with the negative control (31).

The data were analyzed via SPSS v29.1 software (IBM SPSS Statistics for Windows, Version 29.1; IBM Corp., Armonk, NY, USA). The variables (MICs) were checked for a normal distribution via the Kolmogorov–Smirnov normality test. Differences in MICs were subsequently assessed via the Kruskal–Wallis test. Post hoc analysis was performed to compare bacterial groups via pairwise comparisons of mean ranks, with Bonferroni correction.

The quantification of UA and AA in EGL-L was conducted using HPLC-MS in SIM mode. The analyses were performed in triplicate to ensure accuracy and reproducibility. Calibration curves were constructed for both compounds (calibration range: UA from 3 to 15 μg/g; AA from 5 to 20 μg/g) and subsequently used to determine their concentrations in the extract samples (calibration equation: UA y = 175.55x + 3002.1, R² = 0.97; AA y = 1157.5x + 35,920, R² = 0.98). Results revealed that in 1 g of EGL-L, the concentration of UA was 16.12 ± 0.32 μg/g, while AA was present at 47.29 ± 0.13 μg/g. Chromatograms of EGL-L and the two compounds under study are shown in Figure 1. The chemical composition of Eucalyptus globulus Labill., particularly its ethanolic extract and volatile fraction, is well-documented in the literature. Previous studies have reported a high yield of triterpenes in ethanolic and methanolic extracts, especially oleanolic acid, betulin, and betulonic acid, along with their acetylated derivatives (35). Additionally, LC-MS analysis of ethanol-extracted E. globulus leaves has identified antioxidant and bioactive phytochemicals, including salicylic acid β-D-glucuronide, chlorogenic acid, epicatechin, 2″-O-galloylhyperin, isoquercitrin, isorhapontin, quercitrin, and quercetin-3-O-glucuronide (36). Given the extensive literature on E. globulus composition, we opted for a targeted approach, focusing solely on the identification and quantification of ursolic acid and asiatic acid. These two compounds were selected due to their well-documented biological activity and were also used as pure standards in subsequent experimental studies (22).

The AST results are reported in Supplementary Tables S1–S3. The highest prevalence of MDR strains was observed in the SU group, with 14 out of 18 strains. Within this group, imipenem was the only antibiotic active against all the strains, whereas trimethoprim/sulphamethoxazole was ineffective in all the strains. In the SAG group, none of the isolates were classified as MDR. All the SAG isolates were susceptible to cefquinome, cefuroxime, imipenem, and penicillin G but resistant to trimethoprim/sulphamethoxazole. In the EN group, only one strain was identified as MDR. This strain was susceptible to amoxicillin/clavulanic acid, florfenicol, imipenem, penicillin G and vancomycin. Notably, many EN-resistant strains resistant to ampicillin (8 out of 13) were detected. The SA group presented a greater number of MDR strains than non-MDR (10 vs. 6) strains. In particular, 3 out of 15 strains were resistant to cefoxitin, a phenotypic marker for MRSA. On the other hand, cefquinome, fusidic acid, florfenicol, imipenem and rifaximin were the most active antibiotics in this group of bacteria, with 14 out of 15 strains being susceptible to these antibiotics.

As reported in Table 1, all the compounds involved in this study showed antimicrobial activity against the reference bacterial strains. EGL-L showed the highest antimicrobial activity against SA ATCC 25923 and SU ATCC 19496, with an MIC of 250 ± 0 μg/mL, while its lowest activity was observed against SAG ATCC 27956, with an MIC of 500 ± 0 μg/mL. Ursolic acid had the lowest MIC value against SU ATCC 19496 (4 ± 0 μg/mL) and the highest MIC values against SAG ATCC 27956 and MRSA ATCC 43300, both at 24 ± 0 μg/mL. Finally, AA had the highest activity against SU ATCC 19496, with an MIC of 4 ± 0 μg/mL, and the lowest activity against MRSA ATCC 43300 and SA ATCC 25923, with an MIC of 32 ± 0 μg/mL.

The MIC₅₀ and MIC₉₀ results for EGL-L, UA and AA are reported in Table 2. The Eucalyptus globulus leaf extract had the highest MIC₅₀ and MIC₉₀ values against SA strains (500 μg/mL and 1062.50 μg/mL, respectively). An MIC₅₀ value of 250 μg/mL was obtained for EGL-L against all the other bacterial groups, while the lowest MIC₉₀ (500 μg/mL) was observed against the EN group. The lowest MIC₅₀ and MIC₉₀ values for UA were observed in the SU group (2.0 μg/mL) and SAG group (8 μg/mL), respectively. UA had the highest MIC₅₀ and MIC₉₀ values against the SA group (12 and 64 μg/mL, respectively). The lowest MIC₅₀ and MIC₉₀ values for AA were detected in the SU and SAG groups (6 and 16 μg/mL, respectively), whereas the highest MIC₅₀ and MIC₉₀ values were detected in the SA group (24 μg/mL and 187.50 μg/mL, respectively). Overall, the MIC₅₀ and MIC₉₀ values for EGL-L were significantly greater than those observed for UA and AA (p < 0.001). Moreover, the Kruskal–Wallis nonparametric test revealed significant differences in the median MIC values between the different bacterial groups for UA (<0.001) and AA (<0.001), whereas no significant difference was detected for EGL-L (p = 0.056) (Figures 2a–c). The pairwise comparison among each bacterial group revealed that for UA, the SA group was significantly different from the SU group (p < 0.05). For AA, the pairwise comparison revealed a significant difference between the SA group and the SU/SAG group (p < 0.05) (Figure 3). In Table 3 are showed the MIC average of EGL-L, UA and AA against MDR field strains of S. aureus and S. uberis.

Figure 4 presents the results of BKC of EGL-L against reference bacterial strains. For E. faecium and S. agalactiae, the tested concentrations were 2000, 1,000, and 500 μg/mL, while for MRSA, S. aureus, and S. uberis, the concentrations tested were 1,000, 500, and 250 μg/mL. For E. faecium, all tested concentrations showed a bacteriostatic effect; however, the 2000 μg/mL concentration demonstrated the highest antimicrobial activity at all experimental time points. This concentration exhibited similar results against S. agalactiae. In contrast, for MRSA and S. aureus, all tested concentrations showed similar OD values to the growth control up to 6 h. However, during the logarithmic growth phase, a strong antimicrobial activity was observed, inhibiting bacterial growth at the remaining time points. For S. uberis, the 500 and 250 μg/mL concentrations showed lower OD values than the growth control throughout the experiment, while the 1,000 μg/mL concentration only exhibited this effect at 24 and 48 h.

Figure 5 shows the results of the BKC of UA against reference bacterial strains. Specifically, concentrations of 64, 32, and 16 μg/mL were tested against S. agalactiae and MRSA, while 32, 16, and 8 μg/mL were tested against S. aureus. Finally, concentrations of 16, 8, and 4 μg/mL were tested against S. uberis and E. faecium. Regarding S. agalactiae, all tested concentrations demonstrated bactericidal activity. In contrast, against MRSA, bactericidal effects were observed only at 64 and 32 μg/mL, while 16 μg/mL exhibited bacteriostatic activity up to 8 h. After the logarithmic phase, at 24 h, the OD values of the 16 μg/mL concentration were similar to those of the growth control, indicating bacterial growth. A similar pattern was observed against S. aureus, with only 32 μg/mL exhibiting bactericidal activity. In contrast, concentrations of 16 and 8 μg/mL demonstrated bacteriostatic effects up to 8 h, prior to the logarithmic phase. For E. faecium, 8 and 4 μg/mL concentrations exhibited bacteriostatic effects up to 24 h, while the 16 μg/mL concentration showed a bactericidal effect. Finally, against S. uberis, the 16 and 8 μg/mL concentrations showed bactericidal effects.

Figure 6 displays the results of the BKC of AA against reference strains. Specifically, concentrations of 128, 64, and 32 μg/mL were tested against MRSA and S. aureus, while 32, 16, and 8 μg/mL were tested against E. faecium and S. agalactiae. Lastly, concentrations of 16, 8, and 4 μg/mL were tested against S. uberis. Asiatic acid exhibited strong antimicrobial activity with bactericidal effect against MRSA and S. aureus at all tested concentrations. In contrast, against E. faecium and S. agalactiae, bactericidal effect was observed only at 32 and 16 μg/mL, while 8 μg/mL showed a bacteriostatic effect up to 24 h. A similar pattern was observed against S. uberis, where 16 and 8 μg/mL showed bactericidal effects, while the 4 μg/mL concentration exhibited a bacteriostatic effect up to 24 h.

The evaluation of biofilm formation by reference strains revealed that only SA ATCC 25923 was moderately adherent, whereas MRSA ATCC 43300 was strongly adherent. All the other reference strains exhibited weak adherence. For the field strains, nonbiofilm producers (nonadherent) belonged to the EN (5 out of 13; 38.5%) and SU (8 out of 18; 44.4%) groups. Weak biofilm producers were most common in the SU group (8 out of 18; 44.4%) and EN group (7 out of 13; 53.8%) but least common in the SA group (1 out of 15; 6.7%). Conversely, the SA group presented the highest proportion of moderate biofilm-producing strains (6 out of 15; 40.0%), followed by the SAG group (5 out of 17; 29.5%), SU group (2 out of 18; 11.1%) and EN group (1 out of 13; 7.7%) (Table 4).

Finally, strong biofilm-producing bacteria were found only in the SA group (8 out of 15; 53.3%) and SAG group (8 out of 17; 47.0%).

The MBIC₅₀ and MBIC₈₀ results are reported in Table 4.

For Staphylococcus aureus ATCC 25923, EGL-L had an MBIC₅₀ and MBIC₈₀ of 3.96 μg/mL and 7.81 μg/mL, respectively. The UA MBIC₅₀ and MBIC₈₀ values were identical (32 μg/mL). The MBIC₅₀ and MBIC₈₀ for AA were both 16 μg/mL. With respect to EGL-L, an MBIC₅₀ was found for all the field MA strains, except for S. uberis BV2 (>2000 μg/mL). The lowest MBIC₅₀ value was observed for Enterococcus sp. BV21 (7.81 μg/mL), while the highest value was found for S. agalactiae IZS5 and IZS12 (2000 μg/mL). Most of the MA field strains reported an MBIC₈₀ > 2000 μg/mL for EGL-L (9 out of 14).

Ursolic acid had the lowest MBIC₅₀ for S. agalactiae IZS5 and IZS11 (0.50 μg/mL), while the highest value was observed for S. uberis BV2 (256 μg/mL). An MBIC ₈₀ of 8 μg/mL was obtained for UA on all SAG field strains identified as moderately adherent, except for S. agalactiae IZS4 (128 μg/mL), whereas an MBIC₈₀ > 256 μg/mL was reported for S. uberis BV2, BV16 and S. aureus IZS5.

Finally, AA had the lowest MBIC₅₀ for S. aureus BV16 (< 0.5 μg/mL) and the lowest MBIC ₈₀ for S. agalactiae IZS10 (4 μg/mL).

The results of the MBIC₅₀ and MBIC₈₀ values for the StA reference and field strains are reported in Table 5. For MRSA ATCC 43300, the MBIC₅₀ and MBIC₈₀ values of EGL-L were 125 μg/mL and 250 μg/mL, respectively. For UA, the MBIC₅₀ and MBIC₈₀ were 32 μg/mL and 64 μg/mL, respectively. For AA, both the MBIC₅₀ and the MBIC₈₀ were 16 μg/mL. For the field strains, the lowest MBIC₅₀ for EGL-L was observed for S. aureus BV44 (3.9 μg/mL), whereas the highest was observed for S. agalactiae IZS13 (500 μg/mL). Notably, 8 out of 16 StA strains presented an MBIC₈₀ > 2000 μg/mL. Ursolic acid had the lowest MBIC₅₀ (0.5 μg/mL) against S. aureus BV44, S. agalactiae IZS2 and IZS6, whereas S. aureus BV5 and S. agalactiae IZS7 presented MBIC₈₀ values > 256 μg/mL.

For AA, the lowest MBIC₅₀ value (2 μg/mL) was observed for S. aureus BV43 and BV44 and S. agalactiae IZS6 and IZS8; the latter also presented the lowest MBIC₈₀ at the same value. The highest MBIC₅₀ was observed for S. agalactiae IZS2 (128 μg/mL). Finally, the highest MBIC₈₀ for AA was observed for S. agalactiae IZS7 (>256 μg/mL).