Efficacy of selected South African plants against multidrugresistant staphylococci isolated from clinical cases of bovine mastitis

Abstract

Among cattle diseases, bovine mastitis remains a serious problem with financial implications to the farmer as it adversely affects milk production. Staphylococcus aureus and non-aureus staphylococcus (NAS) species are often isolated from milk samples in cases of bovine mastitis. Various antimicrobial agents have been used for treatment of mastitis pathogens in veterinary medicine with limited success because of increasing prevalence of resistance to commonly used antibiotics. Some bacteria causing bovine mastitis are known for their ability to form biofilms, which helps them resist antibiotic effects. The use of natural plant products is being explored as an alternative antimicrobial treatment for mastitis and a template for new mastitis drug development. The primary aim of this study was to investigate antibacterial and antibiofilm activities of selected South African plants against drug-resistant staphylococci organism isolated from clinical cases of bovine mastitis. The study also examined the cytotoxic, anti-inflammatory and antioxidant activities of some of the plants. Plants for this study were selected based on known antimicrobial activity, chemotaxonomic relationships to plant species with antibacterial activity, availability, and/or the existence of traditional uses against infectious diseases. The nine selected South African plants include Antidesma venosum, Elaeodendron croceum, Erythrina caffra, Indigofera frutescens, Pleurostylia capensis, Searsia lancea, Searsia leptodictya, Trichilia emetica and Ziziphus mucronata. Eight S. aureus isolates, three Staphylococcus chromogenes isolates, one Staphylococcus haemolyticus isolate and one ATCC strain of S. aureus were used to determine the antibacterial activity of extracts of the plants prepared with acetone and ethanol using standard methods, while one S. aureus isolate and one ATCC strain of Staphylococcus epidermidis were used in the antibiofilm assay. The leaf extracts of the plants with good antimicrobial activity were also assessed for their antibiofilm, antioxidant, anti-inflammatory and cytotoxic activities using standard methods. The range of antibacterial minimum inhibitory concentration (MIC) values obtained in this study was relatively low and promising. All the plant extracts had good to weak antibacterial activity against all the drug resistant isolates with MIC ranging between 0.01 – 1.41 mg/ml. The lowest MIC value of 0.01 mg/ml obtained in this study was shown by the acetone extract of S. lancea while the highest MIC value of 1.41 mg/ml was recorded with the acetone extract of T. emetica. Generally, the acetone extracts of all the plants showed better activity than their ethanol counterparts except for E. caffra. Ethanol is generally preferred as an extraction solvent in industrial applications, such as large-scale preparation of bioactive plant extracts, owing to its relative safety compared to acetone, which is more flammable. The S. aureus strains appeared to be more susceptible to the extracts than the NAS strains. An interesting finding was that the drug resistant isolates used in this study were generally more susceptible to the extracts than the ATCC strain which was in turn more susceptible to gentamicin, the positive control. All the plant extracts tested had LC50 values higher than the recommended cytotoxic cut-off concentration of 0.02 mg/ml. The ethanol extract of E. caffra had the best mean selectivity index (SI), calculated as LC50/MIC against all the pathogens, of 8.30. The antibiofilm investigation revealed that most of the plant extracts had very good inhibitory activity, inhibiting more than 50% of the test organism biofilm biomass, with E. caffra and A. venosum showing outstanding activities. The results also suggest that the S. epidermidis (NAS) ATCC strain was more susceptible to the antibiofilm activities of the plant extracts than the S. aureus isolate. The antioxidant activity investigation of the crude extracts of three of the plants showed that the ethanol extract of S. lancea had the best antioxidant activity against the 2, 2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radicals when compared to the other extracts. The ethanol extract of S. lancea had better anti-inflammatory activities than other extracts prepared using different solvents. The results reported in this dissertation demonstrate the potential of the selected South African plants to manage bovine mastitis as the plant extracts showed good antibacterial activities at relatively non-cytotoxic concentrations. The results further reveal the abilities of the plants to disrupt the biofilm-forming activities of drug-resistant Staphylococcus spp. implicated in bovine mastitis. The anti-inflammatory and antioxidant activities demonstrated by some of the plants also showed the potential of the plants to arrest oxidative stress and anti-inflammatory activities involved in the pathophysiology of the disease. S. lancea and E. caffra appeared to show great potential in the overall management of infectious bovine mastitis. Potential for synergistic antibacterial activity of the plant extracts needs to be examined for potential formulation of a polyherbal product. The active compounds need to be isolated and identified from the most active plant extracts to provide possible marker compounds for quality control if an active extract or fraction is to be developed for use against mastitis pathogens. The mechanism of action of the active plant preparations (extracts, fractions or isolated compounds) needs to be elucidated to identify how they can be most useful in preventing or treating diseases caused by mastitis pathogens for possible complementary applications in the management of bovine mastitis.