*3.2. Antibacterial Spectrum of ADR1 Metabolites Against Significant Gram-Positive Pathogens*

The spectrum of activity of the ADR1 metabolites against different Gram-positive pathogens was determined as described under Section 2. The results presented in Table 2 showed that the metabolite extract possessed broad spectrum activity against Gram-positive pathogens as it effectively inhibited growth of all the reference strains. It may be noted that the activities of metabolite extract against MRSA strains were similar to those against *S. aureus* strains.

However, the above data provided more of a qualitative rather than quantitative estimate of the antibacterial activity. An accurate view of the potency of the metabolites was achieved by assessment of MIC90 values against the pathogenic strains used in this study. As presented in Figure 3a, the MIC90 values of the ADR1 metabolites against different strains of *S. aureus* were between 0.44 ± 0.07 − 0.84 ± 0.03 μg/mL, while against *S. epidermis* ATCC1222 it was even lower (0.23 ± 0.01 μg/mL). It may be noted that the potency against MRSA strains (Figure 3b) was as good as it was against *S. aureus* strains. These results can be considered as significant since the MIC90 values of the ADR1 metabolites stood better than the standard drug vancomycin (0.5 to 2 μg/mL) for MRSA strains [49]. Other than *S. aureus* strains, the MIC90 values of 1.92 ± 0.03 and 3.35 ± 0.18 μg/mL were observed against *E. faecium* strains AIIMS and ATCC 49214, respectively, which demonstrated good sensitivity of these strains to ADR1 metabolites. *E. faecalis* ATCC 29212 was the least sensitive to ADR1 metabolites among the reference pathogens since its MIC90 value (5.68 ± 0.20 μg/mL) stood at the maximum, but it was still better than some of the previously reported activities [50,51]. The MIC90 values of the metabolites from some of the recently reported *Streptomyces* spp. against the strains of *S. aureus* and MRSA were in the ranges of 2–125 μg/mL [52–54], which indicated wide variation in the anti-bacterial potency of the metabolites. The results of the present study clearly demonstrated that the anti-bacterial potency of ADR1 metabolites appeared at the bottom of the range or even lower, validating its promising potential for discovery of drugs against priority pathogens.

**Figure 3.** Potency of ADR1 metabolites for Gram-positive pathogens. Minimum inhibitory concentration (MIC90) values of the metabolites against the target pathogens were corelated with its antibacterial potency. Percent inhibition in growth at different concentration of the metabolites was measured against the reference strains as shown in (**a**) and in (**b**). The data represented mean ± SEM values of experiments done in triplicate (*p* value < 0.0001).


**Table 2.** Spectrum of antibacterial activity of the metabolites produced by *S. californicus* ADR1.

#### *3.3. Anti-Biofilm Activity of ADR1 Metabolites Against S. aureus and MRSA*

Biofilm-associated infections posed a greater challenge in treatment of infectious diseases as it is one of the major contributing factors in enhancing antibiotic-resistance among *S. aureus* and its methicillin-resistant strains [3]. Therefore, the discovery of drugs with potent anti-biofilm activity is needed more than ever before to combat the ever-growing global challenge of antibiotic resistance against the notorious pathogens like *S. aureus* and MRSA. In view of this, the biofilm inhibitory potential of the ADR1 metabolites was investigated. The results (Figure 4a) suggested that the ADR1 metabolites were able to effectively inhibit formation of biofilm by the *S. aureus* and the MRSA strains. Up to 90% reduction in the formation of biofilm could be achieved at significantly lower concentration of the metabolites; the BIC90 values were noted to be in the range of 0.74 ± 0.08 to 4.59 ± 0.71 μg/mL. The effectiveness of the ADR1 metabolites in the inhibition of the biofilm was found to be better than the previously reported activity of biofilm inhibition by actinobacterial metabolites where maximum inhibition of 83% was recorded at 265 μg/mL [35]. However, when the activity against pre-formed biofilms (24 h) was examined, the BIC90 values for *S. aureus* strains increased by many folds, for example, up to 45.69 ± 3.32 and 89.54 ± 0.40 μg/mL for *S. aureus* ATCC 25923 and *S. aureus* ATCC 29213, respectively. The biofilms produced by MRSA proved even more resistant (Figure 4b). It was reported that some of the well-known antibiotics like pyrrolomycin and related compounds could inhibit the biofilm only up to 67%–87% [55]. Similarly, in a study on the effect of antibiotics like rifampicin, polymyxin B, kanamycin and doxycyclin on reduction of *S. aureus* biofilm formation, only rifampicin was found to inhibit the biofilm by about 50% [56]. The effect of ADR1 metabolites on inhibition of biofilm formation as well as on the preformed biofilms was better than some previously reported metabolite extracts [57–59]. Inhibition of biofilm formation strongly suggested that the metabolites prevented adherence of *S. aureus* and MRSA cell to the polystyrene surface. Further, their ability to disrupt pre-formed biofilms might limit the biofilm-associated drug resistance among the pathogens.

#### *3.4. Antioxidant Activity of the ADR1 Metabolites*

Antioxidants assume significance for therapeutic applications in view of their role in neutralizing reactive oxygen species in patients fighting diseases involving infectious agents or metabolic disorders [60–62]. Hence, it was prudent to examine if the ADR1 metabolites possessed any such activity. The standard assay, which measured the reduction of DPPH free radical, revealed the antioxidant properties of ADR1 metabolites (Figure 5). The free radical scavenging activity of the metabolite extract and of ascorbic acid (a well-known antioxidant agent), followed a different pattern, where a sharp increase in DPPH scavenging activity (from 40% to 80%) was observed when concentration of the metabolite was increased from 62.5 to 125 μg/mL. However, in the case of ascorbic acid, there

was no significant change in the activity over the above concentration range. The IC90 value for DPPH scavenging by ADR1 metabolite was achieved at the concentration of 217.24 ± 6.77 μg/mL, while that for the ascorbic acid it was 904.32 ± 12.93 μg/mL (Figure 5), which was approximately 4-fold higher compared to ADR1 metabolites. However, interestingly IC50 of the ascorbic acid was 4.617 ± 0.89 μg/mL while that of ADR1 extract was 77.41 ± 1.02 μg/mL (not plotted). It was recently reported that the endophytic actinobacterial strains BPSAC77, 101, 121 and 147 showed DPPH scavenging, with an IC50 value of 43.2 μg/mL, but the IC90 value was not reported in this study [63]. In another study, *Streptomyces* sp. had been found to scavenge DPPH free radicals at a much higher concentration, with an IC50 value at 435.31 ± 1.79 μg/mL [64]. In one of the recent studies, it was reported that IC50 values for DPPH scavenging activities of several isolates of actinobacteria varied from 12 ± 1.8 to 65 ± 3.2 μg/mL [65]. Such variations were also noted by others [54]. These reports indicated that there was a wide variation in the antioxidant activities of the metabolites produced by different actinobacterial strains. An in-depth characterization of relevant compounds in pure form may offer further insight into such differences in the activities.

**Figure 4.** Anti-biofilm activity of ADR1 metabolites. (**a**). Inhibition of biofilm formation and (**b**) inhibition of pre-formed biofilm of *S. aureus* ATCC 29213, 25923, MRSA 562 and ATCC 43300 are shown at various concentration of the ADR1 metabolites. The data represented mean ± SEM values of experiments done in triplicate (*p* value < 0.0001).

**Figure 5.** DPPH (1, 1-diphenyl-2-picrylhydrazyl) radical scavenging by ADR1 metabolites. Oxidation of DPPH free radicals were measured at the different concentrations of the metabolite. The data represented mean ± SEM values of the experiments done in triplicate (*p* value < 0.0001).
