Two type strains of S. aureus (ATCC 35556: methicillin-sensitive; ATCC 700699: methicillin-resistant) were purchased from American Type Culture Collection (Manassas, VA). Fourteen clinical isolates of S. aureus were obtained from Pathology Department of Jacobi Medical Center (Bronx, NY). All S. aureus strains were routinely cultured on Luria-Bertani (LB) agar or LB broth medium. All chemicals and antibiotics were purchased from Sigma Aldrich (St. Louis, MO).

Antibacterial activity and synergism of some bioactive compounds such as GSH, polyamines, and epigallocatechin-3-gallate (green tea extract) have been previously studied by several investigators as described [23] [24] [25] [29] [30]. To understand antibacterial activity and synergism of GSH minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and bacterial growth rate were performed. Fresh overnight cultures of S. aureus were diluted in saline to an optical density at 600 nm of 0.1 to 0.15 (approximately 1 × 10⁹ viable cells per mL). A portion of the diluted cell suspension (~10⁶ cells) was inoculated into 1 mL of divalent cation-adjusted Mueller-Hinton (MH) (Oxoid, Ogdensburg, New York) broth (pH 7.0) supplemented with GSH at 0, 5, 10, 15, 20, 25, 30, 35, 40, 45 mM. The inoculum was then incubated overnight (16 to 18 hrs) at 37˚C without shaking. MIC levels were defined as the lowest concentration of GSH that completely inhibited cellular growth. MBC levels were determined by plating the cellular growth of the inoculums used for the MIC measurement on plain LB agar plates with incubation at 37˚C for 24 hours. MBC levels were defined as the lowest concentration of GSH that killed 100% of the inoculated cells. MIC and MBC levels were confirmed by three independent experiments. Bacterial growth rate with or without GSH was determined by conventional growth curves at an optical density of 600 nm. Briefly, overnight cultured cells were diluted 100-fold into MH broth (30 mL) supplemented with GSH at the subinhibitory concentration or without any additional compound. Then, the cells were cultured at 37˚C with shaking (300 rpm) and the cellular growth was measured at time intervals.

Antibiotic susceptibility with or without GSH was determined by MIC measurement as described [29]. Briefly, each antibiotic was added to MH broth (pH 7.0) to achieve serial two-fold dilutions between 0.25 and 256 µg/mL with subinhibitory concentrations of GHS or without GSH using sterile 17 × 100 mm snapped-cap Falcon culture tubes (1 mL/tube; Fisher Scientific). Fresh overnight culture of the diluted cells (~10⁶ cells) was inoculated to each MH broth supplemented with antibiotics and GSH. The inoculum was then incubated overnight at 37˚C without shaking. MIC levels were defined as the lowest concentration that completely inhibited the growth of the inoculum. MIC levels were confirmed by three independent experiments. Antibiotic resistant breakpoints were followed by Clinical Laboratory Standards Institute (CLSI; M100, 32^nd edition, 2022; https://clsi.org/standards/products/free-resources/access-our-free-resources/).

A checkerboard assay was performed to determine the interaction between GSH and antibiotics as described by White et al. [31]. Briefly, an array of combinations was made between 0 to 20 mM of GSH and 0 to 1024 μg/mL of antibiotics (chloramphenicol, methicillin, and tetracycline). Fresh overnight culture of the diluted cells (~10⁶ cells) as described above was inoculated to each GSH/antibiotic combination in the array. The inoculum was incubated at 37˚C for 18 hours. Fractional inhibitory concentration (FIC) was calculated as follows: FIC of GSH = lowest MIC of GSH in combination with antibiotic/MIC of GSH alone, FIC of each antibiotic = lowest MIC of each antibiotic in combination with GSH/MIC of each antibiotic alone. FIC index = FIC of GSH + FIC of each antibiotic. Synergism was defined as a FIC index of ≤0.5; indifference defined as a FIC index of ≥0.5 by ≤4; antagonism defined as a FIC index of ≥4.

Time-killing assay was performed to determine synergism between GSH and antibiotics as described by White et al. [31]. Briefly, three conditions of MH broth (1 mL for each) supplemented with a subinhibitory concentration of GSH alone, the subinhibitory concentration of antibiotics alone, and both GSH and the antibiotics at the same subinhibitory concentrations were prepared in sterile 17 × 100 mm snapped-cap Falcon culture tubes. Fresh overnight cultures of each isolate (~10⁶ cells) were inoculated in each condition of the three prepared MH broth. The inoculated cells were incubated at 37˚C without shaking, and aliquots (100 μL) were withdrawn at specific time intervals (0, 4, 8, and 12 hours) and spread the cells on plain LB agar plates with an appropriate dilution in case. Colony forming units (CFUs) on the LB agar plates were counted after 24 hours’ incubation at 37˚C. Synergism was defined as a ≥2log10 decrease in CFUs per milliliter during the time-period (12 hours).

The bacterial killing pattern was determined by CFUs per milliliter at different concentrations of GSH and at different pH levels. Briefly, fresh overnight culture of cells was diluted to about ~10⁸ in 1 mL of MH broth containing GSH (0, 5, 10, 15, 20, and 25 mM) and HCl to be pH levels of 7.1, 5.9, 5.2, 4.4, 4.1, and 4.0 which are equivalent pH levels of the concentrations of GSH, respectively. The cells with GSH or HCl were incubated without shaking for 18 hours at 37˚C and spread on plain LB agar plates with an appropriate dilution in case. CFUs on the LB agar plates were counted after 24 hours’ incubation at 37˚C.

Antibacterial activity of GSH was evaluated by determining MIC and MBC levels for fourteen clinical isolates of S. aureus including 10 MRSA and two type strains of ATCC35556 (methicillin-sensitive) and ATCC700699 (methicillin-resistant). Results revealed that MICs and MBCs were 15 to 20 mM and 25 to 40 mM of GSH, respectively (Table 1). To clarify the results, bacterial growth rate with or without GSH was determined for the type strains. The growth rate of the type strains significantly decreased with subinhibitory (10 mM) and inhibitory (20 mM) concentrations of GSH when compared to that without GSH (data not shown here). These results suggest that GSH exhibits antibacterial activity on both methicillin sensitive and resistant S. aureus.

^aMethicillin susceptibility was based on CLSI breakpoints (M100, 32nd edition, 2022; https://clsi.org/standards/products/free-resources/access-our-free-resources/). R: methicillin-resistance (MIC ≥ 4 μg/mL); S: methicillin-sensitive (MIC ≤ 2 μg/mL). ^bBacterial growth was measured as MIC and MBC as described in Materials and Methods and repeated three times with identical results. MIC values denote no growth at the indicated or higher concentrations in MH broth; MBC values denote no visual colony at the indicated or higher concentrations in MH agar plate.

To test the effect of GSH on antibiotic susceptibility subinhibitory concentrations of GSH were combined with several antibiotics for MIC measurement of the type strain S. aureus ATCC 700699. Results showed that, without GSH, the type strain exhibited high levels MICs (≥32 μg/mL) for all tested β-lactam antibiotics and MICs of 8 μg/mL (chloramphenicol), 32 μg/mL (ciprofloxacin), 256 μg/mL (gentamicin), 32 μg/mL (tetracycline), and 4 μg/mL (vancomycin). In contrast, MIC levels of all tested-antibiotics in combination with subinhibitory concentrations of GSH (1, 6, and 12 mM) were decreased in a dosage dependent manner up to 1000-fold (MIC falls from ≥256 to 0.25 μg/mL methicillin at 12 mM GSH). Cellular growth with GSH alone (12 mM) was slightly lower than that without GSH; MIC levels to methicillin, oxacillin, chloramphenicol, ciprofloxacin, gentamicin, tetracycline, and vancomycin were all less than 1.0 μg/mL at 12 mM GSH (Table 2). To corroborate the effect of GSH fourteen clinical isolates including 10 MRSA (MIC ≥4 μg/mL) were used to determine methicillin susceptibility with or without GSH. As shown in Table 3, methicillin resistant isolates were also resistant to other class(s) of antibiotic(s). MIC levels of clinical isolates both methicillin-sensitive and -resistant were all decreased to ≤1 μg/mL of methicillin in combination with subinhibitory concentrations of GSH (7.5 to 10 mM). These results suggest that the subinhibitory concentration of GSH enhances methicillin susceptibility regardless of antibiotic susceptibility in S. aureus.

^aMIC measurement was repeated three times with identical results. ^b+++: growth levels as an optical density of ≥1.5 at OD₆₀₀; ++: growth levels as an optical density of ≥1.0 at OD₆₀₀.

^aMIC measurement was repeated three times with identical results. ^bSubinhibitory concentrations of GSH (7.5 to 10 mM). Note: Cip: ciprofloxacin; Ery: erythromycin; Meth: methicillin; Tet: tetracycline; Van: vancomycin.

The type strain, ATCC 700699, was used for interaction between GSH and antibiotics by determining FIC indices. As shown in Table 4, FIC indices for GSH in combination with chloramphenicol, methicillin, and tetracycline were 0.126, 0.066, and 0.258, respectively, which were all less than 0.5 ranged in synergism as described [31]. These results suggest that GSH synergistically enhances the susceptibility of antibiotics. The synergism of GSH on the antibiotics was confirmed by time-killing assays for the same type strain with the same antibiotics. Results showed that the type strain grew with the subinhibitory concentration of either GSH or antibiotics similar as the same strain without any additional compound within 12 hours. In contrast, the same subinhibitory concentrations of both GSH and each of the antibiotics killed ≥99% of initial inoculums within 12 hours (Figure 1). The killed-cell numbers were ≥2log10 within the time-period, which also ranged in synergism between GSH and the antibiotics as described [31].

GSH is composed of three amino acids with two free α-carboxyl groups from

^aFIC (fractional inhibitory concentration) was defined as the ratio of the MIC of a compound used in combination to the MIC of the compound tested alone. The values represent the majority of three independent tests. ^bFICI (FIC index) was calculated from summing the FIC of each compound as described in Materials and Methods.

glutamate and glycine [16]. The free α-carboxyl groups of exogenous GSH may decrease pH levels of the culture medium (MHB), which is possibly associated with the antibacterial activity of GSH. To test this possibility pH levels of MHB supplemented with GSH at different concentrations were measured. MHB containing GSH at concentrations of 0, 5, 10, 15, 20, 25, and 30 mM exhibited pH levels of 7.1, 5.9, 5.2, 4.4, 4.1, 4.0, and 4.0, respectively. These results indicate that exogenous GSH acidifies the culture medium in a dosage dependent manner. To understand the pH effect of GSH on antibacterial activity, bacterial killing assays of the type strain ATCC 700699 were performed by MHB supplemented with GSH at 0, 5, 10, 15, 20, and 25 mM; in parallel, bacterial killing assays of the same strain were performed by the same medium adjusted to the equivalent pH levels with HCl (pH; 7.1, 5.9, 5.2, 4.4, 4.1, and 4.0). As shown in Figure 2, the killing effect of GSH was significantly increased at ≥5 mM (equivalent to ≥pH 5.9) and killed ≥ 99% of all inoculum at 25 mM (equivalent to pH 4.0). The killing effect of pH (by HCl) was also increased at ≥ pH 5.2; however, a significant amount of cells (~10⁴ cells per mL) survived at pH 4.0 (equivalent to 25 mM GSH) (Figure 2). These results indicate that the killing effect of GSH is higher than that of HCl-mediated acidity.