Pharmacological activity of South African medicinal plants and mechanism of action against Staphylococcus aureus isolated from clinical bovine mastitis

Abstract

Bovine mastitis is an inflammation of the mammary parenchyma which is usually caused by an infection that could stem from microorganisms in an already infected udder of another cow, or from the environment. Bovine mastitis infection is mostly caused by Staphylococcus and Streptococcus species and has gained global importance due to the increase of multi-drug resistant bacteria, and resistance to common antibiotic therapy. Most staphylococci associated with bovine mastitis can express biofilm which can prevent and reduce the effects of antibacterial agents and the efficacy of the leucocytes. Antimicrobial resistance is a naturally occurring process that can be accelerated with the overuse or incorrect use of antibiotics. The World Health Organization has declared antimicrobial resistance as one of the top 10 most important public health threats. Antimicrobial resistance reduces the efficacy of treatment which may lead to an increase of chronic udder parenchyma infections/diseases with no cure, causing in some cases organ failure and death. Some mastitis pathogens developed resistance to antimicrobials due to their ability to form biofilm and spread this characteristic through the processes of transformation, conjugation and transduction. Gene replication during biofilm formation can lead to mutation (remodeling of the gene), and bacteria during this process protect themselves by producing extracellular polymeric substances leading to antibiotic resistance. Following a comprehensive literature survey, twelve medicinal plants growing in South Africa (Combretum molle, C. erythrophyllum, C. caffrum, C. elaeagnoides, C. padoides, Tithonia rotundifolia, Leonotis leonurus, L. nepetifolia, Rosmarinus officinalis, Bauhinia tomentosa, Maytenus peduncularis and Faurea saligna) used traditionally for the treatment of inflammation, fever, breast swelling, pain and wounds were selected in this study. The dried plant leaves were separately extracted with six different solvents, namely methanol, ethanol, acetone, dichloromethane: methanol (ratio 1:1), and cold and hot water following standard procedures. Antibacterial assays were conducted, including determination of the minimum inhibitory concentration (MIC), antibiofilm, and anti-quorum sensing activity. The antibacterial activity was investigated against six clinical isolates of Staphylococcus aureus from bovine mastitis cases (Ethics permission number V121_ 16, University of Pretoria) and two Staphylococcus ATCC strains (S. aureus ATCC 29213, S. epidermidis ATCC 35984) which were collected from the Milk Laboratory, Department of Production Animal Studies, Faculty of Veterinary Sciences, University of Pretoria, and Chromobacterium violaceum ATCC 12472 (available in the phytomedicine program bio-bank for the anti-quorum sensing activity. The MIC assay was carried out using the serial microdilution method. Antibiofilm activity of the extracts was determined at three different times, time zero (T0) which is at the planktonic stage of the bacterial cells for antiadhesion activity, time 24 (T24), which was done after 24 h of incubation to allow cell attachment and biofilm formation and at time 48 (T48) when the biofilm was at its full-grown matured stage using two different assays. The crystal violet assay was used to determine the anti-adhesion and reduction/inhibition of biofilm biomass production while the metabolic p-iodonitrotetrazolium violet (INT) assay was used to determine the bactericidal effect of the extracts on biofilm metabolic/respiratory activity. The antioxidant activity and anti-inflammatory effects of the extracts were determined using two antioxidant and inflammatory assays. For antioxidant activity the in vitro DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS (2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) electron reduction assays were used, and the 15-lipoxygenase enzyme (15-LOX) and nitric oxide (NO) inhibitory assays were employed to determine the anti-inflammatory activity. The cytotoxicity of the extracts was determined against bovine dermis and Vero cells using the 3-(4, 5-dimethylthiazolyl-2)-2.5-diphenyltetrazolium bromide (MTT) colorimetric assay. Finally, Synergistic screening of the extracts with the best antibacterial activity against the pathogens were tested in combination with different antibiotics using the checkboard method. From this study, of the six extractant, the acetone extract had good antibacterial activity against five of the pathogens compared with other extractants. The extracts had outstanding to weak MIC values ranging between 0.02 and 2.5 mg/mL. The extracts showed commendable inhibition against cell attachment and biofilm formation in the test organisms and inhibited the metabolic activity of the pathogens (bactericidal effect). The extracts at different concentrations exhibited good quorum quenching potential and had the ability to inhibit the production of nitric oxide and inhibited the 15-lipoxygenase enzyme. Most of the extracts were safe to both the Vero and bovine dermis cells. The Combretum padoides extracts (except the water extracts against some of the pathogens) had outstanding broad-spectrum antibacterial activity against all test organisms with MIC values of 0.02 – 0.16 mg/mL. Rosmarinus officinalis extracts also had good MIC values against the test organisms (MIC = 0.02 - 1.67 mg/ml). All extracts had varying antiadhesion and biofilm inhibition activity against the test organisms at different test times and showed bactericidal effects to the bacteria. The extracts of Leonotis leonorus had the best broad-spectrum antibiofilm activity (anti-adhesion and inhibition of the metabolic activity) at the three different test times with inhibition >50%. Combretum padoides inhibited the production of biofilm biomass in at least five of the test organisms and inhibited metabolic activity by more than 50% in six of the tested strains. Faurea saligna also inhibited the metabolic activity in at least seven (7) of the tested bacterial strains at the different test times. All extracts had violacein inhibitory activity, but the Combretum species as well as R. officinalis and F. saligna extracts had the best violacein inhibition while the extracts of C. padoides had outstanding anti-quorum sensing activity with violacein inhibition at all concentrations (0.08 to 2.5 mg/mL). The extracts of C. molle, C. padoides, F. saligna, R. officinalis, M. peduncularis, C. caffrum, and the methanol extract and fractions of C. elaeagnoides had good bactericidal effects against C. violaceum with the minimum bactericidal concentration (MBC) at 0.31 to 1.25 mg/mL respectively. This result suggests the anti-quorum sensing ability of the extracts, which have potential for further study in the management of disease virulence factors and may prevent the multiplication (biofilm formation) in organisms causing microbial infections. All extracts showed excellent to weak antioxidant potential using the DPPH and ABTS methods. Six of the extracts had the best DPPH scavenging activity (EC50 0.67 - 1.90 μg/mL respectively) when compared to the ascorbic acid and Trolox (EC50 7.11 and 6.20 μg/mL).