Evaluation of the Effect of Plectranthus amboinicus L. Leaf Extracts on the Bacterial Antioxidant System and Cell Membrane Integrity of Pseudomonas aeruginosa PA01 and Staphylococcus aureus NCTC8325.

Plectranthus amboinicus (Indian borage) has been extensively studied for its medicinal properties, which can be exploited to develop new antimicrobial therapeutics. The current study investigated the effect of Plectranthus amboinicus leaf extracts on the catalase activity, reactive oxygen species, lipid peroxidation, cytoplasmic membrane permeability, and efflux pump activity in S. aureus NCTC8325 and P. aeruginosa PA01. As the enzyme catalase protects bacteria against oxidative stress, disruption of its activity creates an imbalance in reactive oxygen species (ROS) levels, which subsequently oxidizes lipid chains, leading to lipid peroxidation. In addition, bacterial cell membranes are a potential target for new antibacterial agents, as efflux pump systems play a crucial role in antimicrobial resistance. Upon exposure of the microorganisms to Indian borage leaf extracts, the observed catalase activity decreased by 60% and 20% in P. aeruginosa and S. aureus, respectively. The generation of ROS can cause oxidation reactions to occur within the polyunsaturated fatty acids of the lipid membranes and induce lipid peroxidation. To investigate these phenomena, the increase in ROS activity in P. aeruginosa and S. aureus was studied using H2DCFDA, which is oxidized to 2',7'-dichlorofluorescein (DCF) by ROS. Furthermore, the concentration of lipid peroxidation product (malondialdehyde) was assessed using the Thiobarbituric acid assay and was shown to increase by 42.4% and 42.5% in P. aeruginosa and S. aureus, respectively. The effect of the extracts on the cell membrane permeability was monitored using diSC3-5 dye and it was observed that the cell membrane permeability of P. aeruginosa increased by 58% and of S. aureus by 83%. The effect on efflux pump activity was investigated using Rhodamine-6-uptake assay, which displayed a decrease in efflux activity of 25.5% in P. aeruginosa and 24.2% in S. aureus after treatment with the extracts. This combination of different methods to study various bacterial virulence factors provides a more robust, mechanistic understanding of the effect of P. amboinicus extracts on P. aeruginosa and S. aureus. This study thus represents the first report of the assessment of the effect of Indian borage leaf extracts on bacterial antioxidant systems and bacterial cell membranes, and can facilitate the future development of bacterial resistance modifying agents derived from P. amboinicus.

Evaluation of the Effect of Leaf Development in Plectranthus amboinicus L. on Antimicrobial Activity and Virulence Factors of Pseudomonas aeruginosa PAO1 and Staphylococcus aureus NCTC8325.

Plectranthus amboinicus is widely recognized as a potential source of antimicrobial compounds due to the presence of bioactive components (essential oils) secreted by the glandular trichomes borne on the leaves. As such, an understanding of the effect of leaf development on the production of these essential oils (EOs) is of crucial importance to its medicinal applications. The current study represents the first comparative investigation of the effect of different stages of leaf development (lag, log, and stationary phase) upon the yield and bioactivity of phytochemicals produced. The effects of leaf extracts on the antimicrobial activity, cell surface hydrophobicity, biofilm formation, and motility of P. aeruginosa and Staphylococcus aureus were evaluated. Cryo-scanning electron microscopy was used to record the abundance and distribution of both glandular and non-glandular trichomes during leaf development. Gas chromatography-mass spectrometry analysis revealed that the potent phytochemical thymol is present primarily in log (30.28%) and stationary phase (20.89%) extracts. Log phase extracts showed the lowest minimum inhibitory concentration (25 mg/ml) when compared to other phases of development. Stationary phase extracts were shown to exhibit the highest biofilm dispersal activity against P. aeruginosa (80%), and log phase extracts against biofilms of S. aureus (59%). Log phase extracts showed the highest biofilm inhibitory activity against P. aeruginosa (66%) and S. aureus (63%). In conclusion, log phase leaf extracts of P. amboinicus exhibited a multimodal mechanism of action by displaying antimicrobial, antibiofilm activities and reducing the motility and hydrophobicity, which are important virulence factors in P. aeruginosa and S. aureus pathogenesis.

Staphylococcus aureus Biofilm: Morphology, Genetics, Pathogenesis and Treatment Strategies.

Staphylococcus aureus is a nosocomial bacterium causing different infectious diseases, ranging from skin and soft tissue infections to more serious and life-threatening infections such as septicaemia. S. aureus forms a complex structure of extracellular polymeric biofilm that provides a fully secured and functional environment for the formation of microcolonies, their sustenance and recolonization of sessile cells after its dispersal. Staphylococcus aureus biofilm protects the cells against hostile conditions, i.e., changes in temperature, limitations or deprivation of nutrients and dehydration, and, more importantly, protects the cells against antibacterial drugs. Drugs are increasingly becoming partially or fully inactive against S. aureus as they are either less penetrable or totally impenetrable due to the presence of biofilms surrounding the bacterial cells. Other factors, such as evasion of innate host immune system, genome plasticity and adaptability through gene evolution and exchange of genetic material, also contribute to the ineffectiveness of antibacterial drugs. This increasing tolerance to antibiotics has contributed to the emergence and rise of antimicrobial resistance (AMR), a serious problem that has resulted in increased morbidity and mortality of human and animal populations globally, in addition to causing huge financial losses to the global economy. The purpose of this review is to highlight different aspects of S. aureus biofilm formation and its overall architecture, individual biofilm constituents, clinical implications and role in pathogenesis and drug resistance. The review also discusses different techniques used in the qualitative and quantitative investigation of S. aureus biofilm and various strategies that can be employed to inhibit and eradicate S. aureus biofilm.