The Effects of Tormentic Acid and Extracts from Callistemon citrinus on Candida albicans and Candida tropicalis Growth and Inhibition of Ergosterol Biosynthesis in Candida albicans.

Candida albicans and Candida tropicalis are the leading causes of human fungal infections worldwide. There is an increase in resistance of Candida pathogens to existing antifungal drugs leading to a need to find new sources of antifungal agents. Tormentic acid has been isolated from different plants including Callistemon citrinus and has been found to possess antimicrobial properties, including antifungal activity. The study aimed to determine the effects of tormentic and extracts from C. citrinus on C. albicans and C. tropicalis and a possible mode of action. The extracts and tormentic acid were screened for antifungal activity using the broth microdilution method. The growth of both species was inhibited by the extracts, and C. albicans was more susceptible to the extract compared to C. tropicalis. The growth of C. albicans was inhibited by 80% at 100 µg/ml of both the DCM: methanol extract and the ethanol: water extract. Tormentic acid reduced the growth of C. albicans by 72% at 100 µg/ml. The effects of the extracts and tormentic acid on ergosterol content in C. albicans were determined using a UV/Vis scanning spectrophotometer. At concentrations of tormentic acid of 25 µg/ml, 50 µg/ml, 100 µg/ml, and 200 µg/ml, the content of ergosterol was decreased by 22%, 36%, 48%, and 78%, respectively. Similarly, the DCM: methanol extract at 100 µg/ml and 200 µg/ml decreased the content by 78% and 88%, respectively. A dose-dependent decrease in ergosterol content was observed in cells exposed to miconazole with a 25 µg/ml concentration causing a 100% decrease in ergosterol content. Therefore, tormentic acid inhibits the synthesis of ergosterol in C. albicans. Modifications of the structure of tormentic acid to increase its antifungal potency may be explored in further studies.

Antibiofilm Activity of Extract and a Compound Isolated from Triumfetta welwitschii against Pseudomonas aeruginosa.

Triumfetta welwitschii has been used as a traditional medicine in Africa. It is documented as a rich source of phytochemicals with antibacterial activities. To further explore the antibacterial potential of these phytochemical components, the phytochemical profile of the dichloromethane: methanol leaf extract from T. welwitschii was investigated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Compounds were isolated from the extract using column chromatography and thin-layer chromatography. Compound B1 was isolated from the fraction eluted by 90 hexane:10 ethyl acetate using column chromatography. The antibacterial activity of B1 against Pseudomonas aeruginosa was evaluated in vitro using the broth microdilution method and the iodonitrotetrazolium (INT) colorimetric assay. The antibiofilm activities of the extract and B1 against P. aeruginosa were determined by quantifying the biofilms using crystal violet. The effect of the extract and B1 on capsular polysaccharide and extracellular DNA content of biofilm formed by P. aeruginosa was determined using phenol-sulphuric acid and propidium iodide, respectively. A total of 28 peaks were detected and identified using UPLC-MS/MS. The three most abundant phytochemicals identified were catechin, umbelliferone, and a luteolin derivative. B1 showed antibacterial activity against P. aeruginosa with a minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) value of 25 µg/ml. Only 38% and 6% of the biofilms were formed in the presence of the extract and B1, respectively. The extract and B1 reduced the capsular polysaccharide content in biofilms formed in P. aeruginosa by 40% and 65%, respectively. The extract and B1 significantly reduced the extracellular DNA content of biofilms by 29% and 72%, respectively. The results of this study provide evidence of the antibacterial and antibiofilm activities of B1 and leaf extracts from T. welwitschii. Future work should identify the chemical structure of B1 using nuclear magnetic resonance and mass spectrometry.

Repurposing of Drugs for Antibacterial Activities on Selected ESKAPE Bacteria Staphylococcus aureus and Pseudomonas aeruginosa.

Increasing cases of multidrug-resistant pathogens have evolved into a global health crisis. ESKAPE group of bacteria are associated with antibiotic resistance, and infections caused by these pathogens result in high mortality and morbidity. However, de novo synthesis of antibiotics is expensive and time-consuming since the development of a new drug has to go through several clinical trials. Repurposing of old drugs for the treatment of antimicrobial resistant pathogens has been explored as an alternative strategy in the field of antimicrobial drug discovery. Ten non-antimicrobial compounds were screened for antibacterial activity on two ESKAPE organisms, Staphylococcus aureus and Pseudomonas aeruginosa. The drugs used in this study were amodiaquine an antimalarial drug, probenecid used to prevent gout, ibuprofen a painkiller, 2-amino-5-chlorobenzaxazole used as a tool for assessing hepatic cytochrome P450 activity in rodents, ellargic acid an antioxidant, quercetin an antioxidant and anti-inflammatory drug, N-N diacryloylpiperazine used to crosslink polyacrylamide gel in 2D-protein electrophoresis, epicatechin an antioxidant and antiviral drug, curcumin an anticancer drug, and quinine an antimalarial drug. Antibacterial susceptibility tests were carried out for the 10 compounds. Curcumin exhibited the most potent antimicrobial activity against both bacteria, with MICs of 50 µg/ml and 100 µg/ml for P. aeruginosa and S. aureus, respectively. Ellargic acid was found to have an MIC of 100 µg/ml against S. aureus. Curcumin caused protein and nucleic acid leakage from the bacterial cell membrane in both bacterial species. When curcumin was combined with ciprofloxacin, it was found to enhance the antibacterial effects of ciprofloxacin. The combination with ciprofloxacin reduced the MIC for ciprofloxacin from 0.5 µg/ml to 0.0625 µg/ml on P. aeruginosa and 0.25 µg/ml to 0.0625 µg/ml on S. aureus. The results obtained show that curcumin has antibacterial activity against S. aureus and P. aeruginosa and may enhance the antibacterial activity of ciprofloxacin.

Effects of Tormentic Acid and the Extracts from Callistemon citrinus on the Production of Extracellular Proteases by Staphylococcus aureus.

Staphylococcus aureus is among the common nosocomial pathogens. Antibiotics have been used to treat S. aureus infections. However, there has been increased mortality associated with drug-resistant strains of S. aureus. Extracellular proteases have been implicated to be responsible for the transition of S. aureus from an adhesive pathogen to an invasive pathogen. The development of resistant strains has necessitated the search for new sources of drugs. Plants have been traditionally used as sources of therapeutic molecules. The objective of this study was to determine the effect of tormentic acid and the extracts from Callistemon citrinus on the production of extracellular proteases by S. aureus. The broth microdilution antibacterial susceptibility assay was used to determine the antibacterial effects of tormentic acid and the extracts on S. aureus. Both extracts showed a minimum inhibitory concentration (MIC) value of 50 µg/ml. The water : ethanol (50 : 50) and the dichloromethane : methanol (50 : 50) extracts were found to be bactericidal against S. aureus at a concentration of 100 µg/ml and 50 µg/ml, respectively. The effect of tormentic acid and extracts on extracellular protease production was investigated using the protease assay. A zone of proteolytic activity (Pr) was measured as the ratio of the diameter of the colony to the total diameter of colony plus zone of hydrolysis. The extracts reduced the production of extracellular proteases, while tormentic acid completely inhibited the production of extracellular proteases by S. aureus. The Pr value for tormentic acid was found to be 1. The Pr values of the dichloromethane : methanol extract and the water : ethanol extract were 0.92 and 0.84, respectively. In conclusion, tormentic acid was shown to inhibit extracellular protease production; therefore, there is need to explore its use in antivirulence therapy to combat S. aureus infections.

Antibacterial activities, proposed mode of action and cytotoxicity of leaf extracts from Triumfetta welwitschii against Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa has become a main cause of Gram-negative infection, particularly in patients with compromised immunity. High rates of resistance to antibiotics are associated with nosocomial infections caused by P. aeruginosa strains. The search for novel antimicrobials has been necessitated by the emergence of antimicrobial resistance in some bacteria Plant-based antimicrobials has great potential to combat microbial infections using a variety of mechanisms. Triumfetta welwitschii plant roots are traditionally used to treat symptoms of diarrhoea and fever, suggesting that it possess antimicrobial and immunomodulatory effects. Since research investigating antimicrobial properties of the roots of Triumfetta welwitschii has been explored, there is need to investigate the antimicrobial activity of its leaf extracts in order to probe their prospective use as new antimicrobial agents that can be used to combat nosocomial infections. The objective of this study was to evaluate the antibacterial activities, the mode of action and cytotoxicity of T. welwitschii leaf extracts. METHOD: Extracts of T. welwitschii leaves were obtained using eight different solvents, the serial exhaustive extraction method and the cold maceration technique. In vitro antibacterial activity evaluation of the extracts was done on eight bacterial isolates using the broth microdilution method. The mode of action for the most potent extracts was investigated using the rhodamine 6G efflux assay and the propidium iodide-based membrane damage assay. Toxicity of the extracts was evaluated using the haemolytic and MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assays. RESULTS: The results showed that acetone, ethanol and dichlorometane: methanol extracts had the most potent antibacterial activities against Pseudomonas aeruginosa (ATCC 27853). All three extracts caused membrane disruption of P. aeruginosa as shown by nucleic acid leakage. All three extracts were unable to inhibit efflux pumps. CONCLUSION: The presence of antibacterial activities and low toxicity shown by the extracts indicates that the plant may be a source of effective antibacterial against some bacterial infections caused by P. aeruginosa. The disruption of membrane integrity is one possible mode of action of antibacterial activity of the potent extracts.

Development of an accumulation assay and evaluation of the effects of efflux pump inhibitors on the retention of chlorhexidine digluconate in Pseudomonas aeruginosa and Staphylococcus aureus.

BACKGROUND: Chlorhexidine digluconate (CHG) is used as a disinfectant. The emergence of pathogens resistant to the biocide raises health concern. Information on specific efflux mechanisms utilised by bacteria to confer reduced susceptibility to the biocide, may be used to develop ways of preventing the efflux of the biocide from nosocomial pathogens resulting in higher disinfection activity. The aim of the study was to evaluate the role of ATP-binding cassette transporters on the transport of CHG in bacteria. METHODS: Clinical strains of Pseudomonas aeruginosa, Staphylococcus aureus and their respective laboratory strains ATCC 27853 and ATCC 9144 were used for susceptibility tests. The minimum inhibitory concentration (MIC) of CHG with or without an efflux pump inhibitor [reserpine or carbonyl cyanide m-chlorophenylhydrazone (CCCP)] was determined using the broth microdilution method. A spectrophotometric method to quantify the amount of chlorhexidine in a sample was developed, validated and used to quantify CHG within P. aeruginosa and S. aureus cells. RESULTS: In the presence of reserpine, the MIC of CHG against the clinical strains of P. aeruginosa and S. aureus decreased from 6.3 to 3.2 µg/ml but showed no change against both ATCC isolates. The MIC of CHG in the presence of CCCP for both strains of P. aeruginosa remained unchanged but showed a reduction for both isolates of S. aureus. The suitability of the spectrophotometric method developed for quantifying the amount of CHG accumulated in microbial cells was validated and used successfully to quantify CHG accumulated within bacterial cells. CONCLUSION: The spectrophotometric determination of CHG within microbial cells may be used to quantify CHG in microbial cells. Only the clinical strain of P. aeruginosa showed significant efflux of CHG suggesting the participation of efflux transporters in the pumping out of CHG from this isolate. The use of efflux pump inhibitors together with the biocide may be explored to preventing the efflux of the biocide from P. aeruginosa resulting in order to increase disinfection activity.