All analytical grade chemicals were procured from the Sigma Aldrich Co.(St Louis, MO, USA). Starch casein agar was purchased from Hi-media Lab Pvt. Ltd, Mumbai, India. Pseudomonas aeruginosa and Staphyloccoucs aureus (Clinical isolates and Standard cultures procured from ATCC and MTCC (Chandigrah).

2.2 Screening of antibacterial activity of naturally occurring plant and microbial compounds for efflux pump inhibition in MDR strains:

To check the mechanism of antibiotic resistance and inhibition of efflux pump in MDR Pseudomonas aeruginosa and Staphylococcus aureus was analysed by agar based Ethidium Bromide cartwheel assay [16]. For this purpose, a nutrient agar plates containing the EtBr (700 µg/L) were prepared. The 24 h old culture of P. aeruginosa clinical isolate M and MTCC 27583 and S. aureus ATCC 6538 and clinical isolates was diluted up to turbidity standard McFarland 0.5 in 0.9% saline and then inoculated in cartwheel pattern on agar plates. After 24 h of incubation the plates were observed under UV trans-illuminator for the presence of fluorescence in each organism. Similarly, to evaluate the inhibition of efflux pump activity, freshly growing cultures of Pseudomonas aeruginosa MTCC 27583 and Staphylococcus aureus (Clinical isolate denoted as M) and S. auerus ATCC 6538 treated with the piperine (positive control), curcumin, prodgiosin, narigenin and blue pigment isolated from Streptomyces sp. at 100 µg/ml concentration for 2 h at room temperature under mild shaking. Treated cultures were swabbed on EtBr containing agar plates and observed under UV trans-illuminator for the presence of fluorescence after 24 h of incubation. The intensity of fluorescence emission was measured by Image J software.

The procedure was carried out for all the test organisms (Staphyloccocus aurues and Pseudomonas aeruginosa) and each test was carried out in triplicate by micro dilution method [17]. Stock solution of the test agent curcumin (dissolved in DMSO (dimethylsulfoxide; Merck, Germany), blue pigment (BP) and piperine (Pip) according to [17]. 200 µL of the nutrient broth was added in each of fourteen MIC tubes per bacterial strain. In the first MIC tube containing 200 µL broth, 200 µL of stock was added. After mixing well, 200 µL was transferred to the second MIC tube. This was continued till the last (14th ) tube. From the last tube 200 µL of the final solution was discarded. By following this serial dilution, the concentrations of the aqueous extract achieved was the following – 512, 256,128, 64, 32, 16, 8, 4, 2,, 0.5, 0.25, 0.12, 0.06 µg /ml. respectively. To each of the fourteen such prepared MIC tubes with varying concentrations, 200 µL (5 X 10⁵organisms/ml) of the earlier prepared strain of organism was added such that the final volume per tube was 400 µL. The tubes were then incubated for 24 hours at 37^oC. Similarly, the MIC for standard antibiotics ciprofloxacin and penicillin against S. auerus ATCC 6538 were determined. After the incubation, the MIC values were determined by visual inspection of the tubes. In each series of tubes, the last tube with clear supernatant was considered to be without any growth and taken as MIC value. Turbidity in the MIC tube indicated growth of the bacteria implying that the bacteria are resistant to test compound and existing antibiotics

From the above results, the synergistic effect of blue pigment and curcumin, with ciprofloxacin and penicillin against S. auerus ATCC 6538 respectively, were determined by well diffusion assay. For this purpose the ciprofloxacin and penicillin at its 2/3 MIC concentration and blue pigment and curcumin at its 1/2 MIC concentration were used. The synthesized conjugates were written as Blue pigment-Ciprofloxacin (BP-Cipr), Curcumin-Ciprofloxacin (Cur-Cipr). Similarly the conjugates with penicillin were written as Blue pigment-penicillin (BP-Peni) and Curcumin-penicillin (Cur-Peni).Fold increase in antibacterial activity was calculated as per the protocol described by Birla et al. [18].

The effect of blue pigment and its antibiotic conjugates on uptake and release of EtBr studied by fluorescence emission assay and modified spectrophotometric real time efflux assay as reported earlier respectively [19, 20]. Overnight grown culture of Staphylococcus aureus (Clinical isolate) and S. auerus ATCC 6538 was used to prepare bacterial suspension (turbidity adjusted according to standard McFarland) in uptake buffer (110 mM NaCl, 7 mM KCl, 50 mM NH4Cl, 0.4 mM Na₂HPO₄, 52 mM Tris base and 0.2% glucose, adjusted to pH 7.5 with HCl). The cells were pelleted out by centrifugation at 5000 G for 5 min and suspended in uptake buffer containing blue pigment, conjugated Penicillin-blue pigment and Ciprofloxacin-blue pigment (at its MIC concentration), Piperine (at its MIC concentration) and EtBr at 10 µg/mL concentration. After 1 h of incubation at 37°C (for EtBr uptake) the cells with treatment and without treatment (control) were again separated, washed two times with uptake buffer and suspended in same buffer. Immediately, the suspension was assayed for the release of EtBr using spectrophotometer (Shimadzu, UV 1800) and accumulation of EtBr by Spectrophotoflurometer (Shimadzu, RF-5301) and TLC method.

On the basis of above MIC results, the minimum inhibitory concentrations of test compound and antibiotics were used for synthesis of conjugates. Simple method in which the test compound and antibiotics were mixed in 1:1 ratio in phosphate buffer (pH 7). Briefly, 2 ml of), blue pigment (10 µg/ml) and curcumin ( 200 µg/ml) mixed with 2 ml of Ciprofloxacin (0.06 µg/ml)/Penicillin (2 µg/ml) and incubate for 1h at 37°C with continuous shaking. The synthesis of conjugated antibiotic was confirmed by UV visible spectrophotometer (UV 1800, Shimadzu). Again antimicrobial activity of synthesized conjugated were checked by disc diffusion assay.

Statistical analyses were performed using Graph Pad Prism (Version 2.1, USA). All experiments were performed in triplicates and data represents as a mean ± standard deviation. The differences among means were determined using One-Way ANOVA and multiple comparisons at significance and confidence levels of 0.05 (95% confidence interval) was determined. The p value of < 0.05 was considered significant.

The all-natural products prodigiosin, naringenin, curcumin, orange and blue pigment of Streptomyces were tested for its efflux pump inhibition activity. For this purpose first we confirmed the presence of active efflux pumps in selected strains of S. aureus by simple, instrument free agar based EtBr cartwheel [16]. EtBr act as a substrate for many efflux pumps and it expel out from the bacterial cells [16]. Among the tested bacterial strains of Staphylococcus, S. aureus ATCC 6538 showed highest efflux pump activity i.e. no fluorescence was observed even at high concentration like 700 µg/ml EtBr. Meanwhile, S. aureus M (a clinical isolate) showed slight efflux pump activity, indicated by reduced fluorescence. Therefore, S. aureus ATCC 6538 was selected for the further efflux pump inhibition study (Fig. 1).

For the confirmation of efflux pump inhibitory potential of the compounds, effluxing activity of test organism were evaluated by exposing a test organism S. aureus ATCC 6538 to natural products. The test organism S. aureus ATCC 6538 was treated with prodigiosin, naringenin, blue, orange pigment, curcumin, EtBr (negative control) and piperine (positive control) and then swabbed on EtBr agar plate. After 24 h incubation the plates were observes for the fluorescence emission (Fig. 2) under UV transilluminator (Camag, Swiss). The bacterial suspension without treatment was used as a growth control, which showed no fluorescence (Fig. 2a). Amongst the tested natural products it was observed that, only blue pigment and curcumin showed an efflux pump inhibition potential in S. aureus ATCC 6538 while, in light of EtBr (negative control) and piperine (positive control) none of the other tested natural products showed efflux pump inhibition (Fig. 2b, c, d and e). The retention of EtBr was confirmed by fluorescence emission, piperine, blue pigment and curcumin showed a higher peak (red colour intensity) as compare to growth control and EtBr (Fig. 2f, g, h, i and j). The treatment of blue pigment leads to accumulate higher EtBr, which resulted in maximum fluorescence as compare to curcumin treatment (Fig. 2i). This indicates the higher efflux pump inhibition potential of blue pigment against S. aureus ATCC 6538. Thus, both the products blue pigment and curcumin were shortlisted for the further studies.

Multidrug resistance phenotype of bacteria is due to the active efflux activity, hence inhibition of efflux pump activity proven to be an excellent way to tackle resistance [20]. In this investigation it was proved that the blue pigment act as an efflux pump inhibitor in S. aureus ATCC 6538. Similarly, Kalia et al. [20] reported capsaicin as a Nor A efflux pump inhibitor in the Staphylococcus aureus. The copper nanoparticles also reported as potent inhibitor of efflux pumps in Staphylococcus aureus and P. aeruginosa [21]. Similarly, Martin et al. [16] reported the EtBr cartwheel assay was a very good tool for identification of MDR phenotype in clinical strains and also, for determination of MIC of antibiotics, which was a substrate for efflux pump thereby, confirmed the activity of overexpressed efflux system. Also, coumarin from Mesua ferrea showed the inhibition of EtBr efflux by arresting overexpressed Nor A efflux pump in Staphylococcus aureus [11]

On the basis of screening results of efflux pump inhibition, the minimum inhibitory concentration (MIC) of blue pigment, curcumin and standard antibiotics i.e. ciprofloxacin and penicillin was determined against S. aureus ATCC 6538 by microdilution method. MICs for all screened compound and standard antibiotics are summarized in Table S1. From the Table 1, it was observed that blue pigment and curcumin showed high MIC values against S. aureus as compare to standard antibiotics penicillin, ciprofloxacin (Table 1).

As the blue pigment and curcumin showed comparatively less antimicrobial potential, their combinational effect was evaluated. During the combination study it was observed that both blue pigment and curcumin were significantly increased the antimicrobial potential of penicillin (Table 2). The conjugate of curcumin and blue pigment with penicillin showed 1.1 to 1.9 folds increase in antibacterial activity of penicillin (Cur-Peni, BP-Peni) respectively, against S. aureus ATCC 6538 (Table 2, Fig. 3a). Similarly, about 1.2 to 1.5 folds increase in antibacterial activity of ciprofloxacin when conjugated with curcumin and blue pigment (Cur-Cipro, BP-Cipro,) respectively (Table S2, Fig. 3b).

To confirm this combination effect and mode of actions, these all natural products were further screened for its efflux pump inhibition potential by fluorescence emission assay. The efflux pump inhibition was one of the major mechanism describe before for high antimicrobial potential in such combinations i.e. antibiotics and natural products [19, 20].

To confirm the inhibitory action of blue pigment antibiotic conjugate against efflux pump of S. aureus ATCC 6538 fluorescence emission of EtBr and real time efflux assay was carried out. The increase in fluorescence intensity is proportional to accumulation of EtBr [19]. The increase in accumulation of EtBr within the cell confirmed the inhibition of efflux pump. Staphylococcus aureus ATCC 6538 showed the high fluorescence intensity which directly related to accumulation of EtBr within the cell (Fig. 4a). The penicillin-Blue pigment (BP-Peni) conjugates showed maximum inhibition of efflux pump as compared to positive control piperine -EtBr a known efflux pump inhibitor (Fig. 4a).

Blue pigment showed significant inhibition of efflux pump in S. aureus ATCC 6538 confirmed by fluorescence emission assay, hence we checked the effect of blue pigment and its conjugate with penicillin on uptake and release of EtBr in S. aureus ATCC 6538 by real time efflux assay. The efflux of EtBr again confirmed by the spectrophotometric analysis. It was found that in control (without treatment) set the high amount of EtBr release by cells (high intensity of characteristic peak of EtBr, Fig. 4b, peak 2) as compared with standard peak of EtBr (Fig. 4b, peak 1). Concentration of EtBr released by penicillin treated cells was lower as peak intensity was decreased followed by blue pigment treated cells (Fig. 4b, peak 3 and 4, respectively) as compared to control. In blue pigment-penicillin (BP-Pen) conjugate treatment the characteristic peak of EtBr was not observed, which proved that the EtBr completely retain by cells (Fig. 4b, peak 5). Hence, it confirmed the complete inhibition of efflux pump activity due to our synthesized conjugate BP-Pen in S. aureus ATCC 6538. Similarly, the released EtBr confirmed by TLC method (inset of Fig. 4b). It was found that in S. aureus ATCC 6538 treated with the BP-Penicillin EtBr band in TLC was not observed (less intensity as compared to control) which again confirmed that the EtBr was retained by cells due to inhibition of efflux pump and released by cells (inset of Fig. 4b). Similarly, Christane et al. [21] quantified inhibition of efflux pump by Cu- nanoparticles in S. aureus and P. aeruginosa on the basis of residual fluorescence by real time efflux assay. The results are in accordance with the Christane et al. [21].

Synthesized natural product antibiotic conjugates studied by UV-visible spectrophotometer (Shimadzu UV -1800, Tokyo, Japan). The spectra of natural product antibiotic conjugates were represented in Fig. 5. It was observed that standard penicillin showed the maximum absorption at 264 nm. The UV visible spectra of curcumin-penicillin showed the characteristics peaks of both curcumin (at 405nm) and penicillin (at 310 and 264 nm), respectively (Fig. 5a). Also, UV- Visible spectra Blue pigment –penicillin conjugates (BP-Peni) showed all characteristic peak of pigment (at 600 nm) and penicillin which confirmed the synthesis of natural product antibiotic conjugates (Fig. 5b).

Similarly, Blue pigment (BP)-Cipro conjugates showed the strongest peak at 600 nm and 271 nm, respectively which is the characteristics peak of blue pigment and ciprofloxacin (Fig. 5c) which confirmed the successful synthesis of antibiotic conjugates. Also, in Curcumin-Cipro (Cur-Cipro) conjugate the characteristic peak of both curcumin (at 571 nm) and ciprofloxacin (at 271 nm) (Fig. 5d) was observed. Such results are reported in case of silver, gold, copper, selenium and chitosan nanoparticles conjugated with the different antibiotics which enhanced the activity of antibiotics [21, 22, 23, 24].