Microbiological media and antibiotic disks were bought from HiMedia, Mumbai. Antibiotic powders were purchased from SRL fine chemicals, India. Chemical reagents for compound synthesis were purchased from Sigma–Aldrich, United States. LIVE/DEAD BacLight Bacterial Viability Kit (L-7012) was purchased from Thermo Fisher Scientific, United States. Qiagen RNeasy Mini kit was purchased from Qiagen, Germany and iScript cDNA synthesis kit was purchased from Bio-Rad, Hercules, CA, United States. SYBR Green PCR Master Mix was purchased from Thermo Fisher Scientific, United States. Primers used in this study were from Integrated DNA Technologies, United States.

The scheme of chemical synthesis is provided in Figure 1. Briefly, Salicylaldehyde (5 mmol) and methylamine (7 mmol) were stirred in methanol (5 ml) at room temperature overnight. The intermediate imine formed was reduced to amine by the addition of sodium borohydride (5 mmol) at ice-cold condition. The reaction mixture was stirred continuously at room temperature for an overnight period. Further, the reaction mixture was diluted with water and extracted thrice with dichloromethane. The organic phases were pooled together, dried over anhydrous sodium sulfate and evaporated to dryness under reduced pressure in a rotary evaporator. The crude product was purified on a silica column with a methanol: DCM system. The purity and identity of the recovered product were supported by ¹H-NMR, ¹³C-NMR and FTIR spectroscopy.

Two clinical isolates of S. aureus (P1966 and AB459) were received from a collaborator at JSS Medical College, Mysore, India. Received strains were cultured in mannitol salt agar to ascertain the viability and identity of S. aureus. Pure colonies obtained were maintained as glycerol stocks in -80°C. SarA deletion mutant, i.e., S. aureus ALC637 strain (ΔsarA::Tn917LTV1) was a kind gift from Professor Christiane Wolz. Strains were grown in tryptic soy broth (TSB) supplemented with 0.5% glucose and incubated at 37°C under the stationary condition for subsequent assays.

Staphylococcus aureus isolates were cultured to early exponential phase (1.4 × 10⁷ CFU/ml). Antibiotic susceptibility assay was performed to determine the drug resistance of S. aureus to various antibiotics that are commonly administered for S. aureus infections (Supplementary Table S1). A bacterial suspension of each strain was swabbed onto Mueller-Hinton agar plates and allowed to dry for 10 min. Antibiotic disks were placed on the plates and incubated for 24 h at 37°C. After the incubation period, the zone of inhibition diameter was measured and interpreted using the standard chart.

The overnight bacterial culture was inoculated in 10 ml of sterile TSB media and incubated at 37°C for 6 h to reach log phase culture. Ten microlitre of the diluted inoculum (1:200 dilutions, approximately containing 2.1 × 10⁶ CFU/ml) was inoculated into 96 well polystyrene microtitre plates containing different concentrations of the compound (0.075–1500 μM in twofold dilutions) in TSB supplemented with 0.5% (w/v) glucose. The plates were incubated at 37°C for 24 h and growth was measured at OD₅₉₅. The lowest concentration of the compound that prevents a visible growth was considered as minimum inhibitory concentration (MIC).

Similarly, biofilm inhibition was determined with another set of 96 well microtitre plate treated as above. Briefly, the planktonic cells were removed after the incubation period, and the wells were washed with 200 μl PBS twice to remove unbound or loosely adhered cells. The cells were then stained with 100 μl of 0.2% crystal violet for 20 min. After removing the excess stain by washing, the plates were air dried. The bound crystal violet was eluted with 33% acetic acid and the optical reading was taken at OD₅₉₅ in ELISA plate reader (BioRad i-Mark, Japan). Minimum biofilm-inhibiting concentration (MBIC) is the lowest concentration of the compound in which the formation of biofilm is limited to a level ≥90% (MBIC₉₀) or ≥50% (MBIC₅₀) in comparison with the untreated control cultures. All assays were carried out in triplicates.

Biofilm inhibition was visually confirmed by confocal microscopy imaging. Clean sterile glass slides were placed in 15 ml of TSB medium in the absence (positive control) and the presence of the test compound (0.6, 1.25, and 2.5 μM) in a 50 ml centrifuge tube. One percent (100 μl) of the diluted bacterial culture (2.1 × 10⁶ CFU/ml) was added and incubated for 6 h at 37°C. After 6 h, the slides were rinsed with sterile PBS to remove non-adherent planktonic cells and stained with BacLight Bacterial Viability Kit (L7012) as per the kit protocol. Two and three-dimensional images were captured using a confocal laser scanning microscope with a 40× objective lens to show biofilm distribution (Olympus FLUOVIEW, FV1000).

Staphylococcus aureus isolates (P1966 and AB459) were grown in TSB in the presence (1.25 μM) and absence of test compound for 6 and 24 h at 37°C. After the incubation period, the walls of the test tubes were scrapped with a sterile cell scraper to bring the adhered cells into suspension. The total cell population (planktonic and biofilm) were harvested by centrifugation from grown cultures (0.5 ml) at log phase (6 h) and stationary phase (24 h) and immediately stored at -80°C. RNA was isolated using a Qiagen RNeasy Mini kit in accordance with the manufacturer’s instructions. RNA concentrations were determined by optical density measurements in a spectrophotometer at OD₂₆₀ (Thermo Scientific NanoDrop, United States). cDNA synthesis was carried out using the iScript cDNA synthesis kit according to the manufacturer’s instructions. Briefly, the reaction mixture was incubated for annealing at 25°C for 5 min, extension at 42°C for 30 min and inactivation of samples at 85°C for 5 min.

qRT-PCR was used to assess the transcriptional expression levels of SarA in the bacterial strains taken and down-regulation of representative virulence genes (RNAIII, fnbA, hla and hld) when treated with the compound. Sequences of primers used in this study are furnished in Table 1. The reaction mixture in a total volume of 20 μl, consisted 10 μl 2× SYBR Green PCR Master Mix, forward and reverse primers (1 μl each), 4 μl of nuclease free water and 4 μl of 20× diluted cDNA. PCR conditions included an initial denaturation at 95°C for 2 min, followed by 40 cycles of denaturation (95°C for 15 s), annealing (55.8°C for 15 s), and extension (72°C for 20 s). To ensure the samples were free from contamination, negative controls containing nuclease-free water instead of cDNA were run in parallel. The relative gene expression was analyzed using the 2^-ΔΔCT method with 16S rRNA as a reference gene.

The MTT assay was carried out to determine any cytotoxicity effect of 2-[(Methylamino)methyl]phenol. Eagle’s Minimum Essential Medium supplemented with non-essential amino acids, 10% fetal bovine serum and penicillin-streptomycin (100 U/ml–100 μg/ml)) was used as culture medium. In brief, 200 μl of viable Human epithelial type 2 (HEp-2) cells in a concentration of 1.5 × 10⁴ cells/ml were seeded in 96 well tissue culture plates. Varying concentrations (1.25, 12.5, and 125 μM) of the synthesized compound were added to each well when cells reached 80% confluence. The plate was incubated at 37°C for 24 h with 5% CO_2. Further 20 μl of MTT solution (5 mg/ml) were added to each well and incubated for 3 h. Following the incubation period, 200 μl of DMSO was added to the wells and OD was taken at OD₅₇₀. The percentage cell viability was calculated with reference to the untreated control cells using the below formula.

Percentage cell viability

Combinatorial studies were carried out between selected antibiotics that are commonly used for Staphylococcal infections and the test compound 2-[(Methylamino)methyl]phenol at the MBIC₅₀ concentration. The S. aureus strains (P1966 and AB459) were cultured in TSB along with test compound at the fixed MBIC concentration, 1.25 μM and varying concentration of antibiotics in twofold dilutions at 37°C for 24 h at the stationary condition. At the end of incubation, absorbance was measured at OD₅₉₅ and MIC was noted.

Statistical analysis was carried out using GraphPad Prism software version 6.05 (GraphPad Software Inc., San Diego, CA, United States). One-way ANOVA and multiple comparisons were carried out to test the significance. The minimum level of significance was set at p ≤ 0.05. All the assays were carried out in triplicates and the results were expressed as mean ± SD.

2-[(Methylamino)methyl]phenol was synthesized as shown in the reaction scheme (Figure 1). The obtained compound had a yield of 82%, golden yellow in appearance. ¹H-NMR (300 MHz, CDCl₃) δ2.48 (s, 3H), 3.97(s, 2H), 6.75–6.80 (m, 1H), 6.83 (d, J = 8.1 Hz, 1H), 6.99 (d, J = 7.2 Hz, 1H), 7.14–7.29 (m, 1H). ¹³C-NMR (75 MHz, CDCl₃) 35.1, 54.5, 122.2, 128.5, 116.3, 118.9, 128.8, 158.3. IR (Neat) 3430.7, 3001.7, 1646.0, 1434.8, 1411.6, 1261.2, and 1023.1 cm^-1. Spectra of ¹H-NMR, ¹³C-NMR and FTIR are provided in Supplementary Figures S1–S3, respectively.

The resistance pattern to various conventionally used antibiotics against S. aureus infections was shown in Supplementary Table S1. The clinical isolates exhibited multidrug resistance to most of the tested antibiotics that include gentamicin, azithromycin, erythromycin, co-trimoxazole, ciprofloxacin, doxycycline, methicillin, oxacillin, cloxacillin, cefuroxime, cephalexin, vancomycin and trimethoprim.

The expression level of SarA in the clinical isolates taken in this study was evaluated by qRT-PCR which confirms that the strains express SarA in the log and late phases of growth and is valid for further gene expression studies with the drug at corresponding phases (Figure 2A). The SarA deletion mutant ALC637 did not show any significant difference in biofilm when treated with different concentrations of the SarA targeted compound, 2-[(Methylamino)methyl]phenol (Figure 2B). Antibiofilm activity was evaluated for the SarA target-specific compound 2-[(Methylamino)methyl]phenol against the SarA positive clinical S. aureus strains and the results were presented in Figures 2C,D. Significant biofilm inhibition (>70%) was observed at a concentration of 1.25 μM. Moreover, 2-[(Methylamino)methyl]phenol did not show any antibacterial activity in the entire test range of concentrations up to 1500 μM. The compound did not exhibit disruption of preformed biofilm of both the strains (data not shown).

Visual confirmation of the biofilm inhibition by the compound was checked by Live/Dead staining in a confocal laser scanning microscope (Figure 3). The maximum biofilm inhibited concentration of the compound in microtitre plate assays (1.25 μM), also showed more inhibition in the captured images compared to that of the lower (0.6 μM) and higher (2.5 μM) concentrations. The results were in concordance with our crystal violet biofilm inhibition studies. The absence of dead cells (presented by red fluorescence) in Live/Dead stained images indicate the exclusion of any antibacterial activity possessed by the compound.

Representative genes took in the present study (RNAIII, fnbA, hla and hld) under the control of quorum regulator SarA were down-regulated significantly except hld at both the log and late phases of growth in the case of S. aureus P1966. Whereas, in the case of S. aureus AB459, downregulation was more in the log growth phase compared to late growth phase (Figure 4).

MTT assay data showed that the drug neither inhibits the proliferation of cells nor gives toxic effect to the HEp-2 cells even after incubating the cells with the compound at its MBIC concentration. Percentage of cell viability was equal to that of control (untreated cells) even after cells were exposed to 10× and 100× MBIC concentrations of the compound (Figure 5).

Effect of the test compound in reducing the MIC values of conventionally used antibiotics against S. aureus was tested by combinatorial usage of both. In the case of S. aureus P1966, except for Azithromycin and Cephalexin, all other antibiotics taken showed a marked reduction in MIC values when used in combination with 1.25 μM of the test compound. Whereas in the case of S. aureus AB459 only a few antibiotics (Cloxacillin, Cephalexin and Vancomycin) showed marked MIC reduction and other antibiotics MICs were as same as that of when used alone (Table 2).