Abstract
BACKGROUND: Microbial carotenoids have gained industry interest due to their safety and diverse biological activities; however, the low yield of carotenoids hinders their applications. Hence, this study focused on optimizing carotenoid pigment production from Micrococcus luteus strains by studying 54 physical and chemical independent conditions. The chronic infections by Enterococcus faecalis are related to its ability to form biofilms on the surface of several implanted medical devices, such as urinary catheters. Therefore, the potential antibacterial and antibiofilm activities of the purified pigment against E. faecalis were investigated in our study. RESULTS: Using one-factor-at-a-time experiments, the top-examined parameters were tryptic soya broth (TSB), agitation, temperature, pH, incubation time, inoculum size (IS), sodium chloride, tryptophan, glycerol, tryptone, glutaric acid, toluene, ferric sulphate, and disodium hydrogen phosphate. The data from the Plackett-Burman design showed that temperature, sodium chloride, tryptophan, and toluene were fundamental factors controlling carotenoid production. The conditions for the upstream process were determined via response surface methodology design, which included TSB medium, agitation speed of 120 rpm, temperature of 32.5 °C, pH = 7, incubation time of 96 h, 2% IS, sodium chloride (12.5 g/l), tryptophan (12.5 g/l), glutaric acid (5 g/l), toluene (12.5%), and disodium hydrogen phosphate (5 g/l). Submerged fermentation model validation using the M6 isolate (accession number of PP197163) revealed an increase in carotenoid production up to 6-fold (1.2 g/l). The produced pigment was purified and characterized as β-carotene, and the stability study showed that the extracted β-carotene was stable for a year in dimethyl sulfoxide at 4 °C. The MTT test data proved that the pigment was safe on human dermal fibroblasts with an IC(50) equal to 542.7 µg/ml. For the first time, it was reported that the stable purified β-carotene exhibited powerful antibacterial activity against multidrug-resistant (MDR) E. faecalis, with inhibition zones ranging from 13 to 32 mm and minimum inhibitory concentrations (MICs) ranging from 3.75 to 30 µg/ml at safe concentrations. In addition, it was found that our stable purified β-carotene showed up to 94% inhibition in biofilm formation by strong biofilm-forming E. faecalis. In addition, the β-carotene-coated catheter manifested a lower biofilm formation by E. faecalis by up to 75.3%. Moreover, crystal violet staining, dual staining, and fluorescence staining techniques displayed immature biofilms of E. faecalis when treated with 0.25 and 0.5 MICs of β-carotene. The mechanistic pathway for the purified β-carotene's antibiofilm activity was strongly linked to the inhibition of gelatinase enzyme production (up to 100% inhibition) as manifested phenotypically, genotypically, and by molecular docking. CONCLUSION: This work provided a deeper insight into optimizing carotenoid production from M. luteus by investigating the influence of 54 diverse conditions. Also, this is the first time to report the antibacterial and antibiofilm actions of the stable purified microbial β-carotene against strong biofilm-forming MDR E. faecalis colonizing urinary catheters.