Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species

Gbelská, Yvetta; Hervay, Nora Tóth; Dzugasova, Vladimira; Konecna, Alexandra

doi:10.21769/bioprotoc.2428

Cited by 11 publications

(8 citation statements)

References 6 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Cells are then washed to remove R6G on the outer surface of cells. Subsequently, glucose is added to the culture as a source of energy, and the efflux of R6G is measured by fluorimetry [35]. A known EPI (e.g reserpine) is included as a positive control for inhibition of the efflux of R6G.…”

Section: Discussionmentioning

confidence: 99%

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

Mombeshora

Mukanganyama

2019

BMC Complement Altern Med

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Mombeshora

Mukanganyama

2019

BMC Complement Altern Med

View full text Add to dashboard Cite

show abstract

“…Procedure was based on the protocol in Gbelska’s article [ 53 , 54 ]. C. albicans SC5314 cells were added to YEPD medium and incubated at 28 °C for 20 h with shaking at 150 rpm.…”

Section: Methodsmentioning

confidence: 99%

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives

et al. 2021

View full text Add to dashboard Cite

A newly synthetized series of N-phenacyl derivatives of 2-mercaptobenzoxazole, including analogues of 5-bromo- and 5,7-dibromobenzoxazole, were screened against Candida strains and the action mechanism was evaluated. 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanone (5d), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichloro-phenyl)ethanone (5i), 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,4,6-trichlorophenyl)ethanone (5k) and 2-[(5-bromo-1,3-benzoxazol-2-yl)sulfanyl]-1-phenylethanone (6a) showed anti-C. albicans SC5314 activity, where 5d displayed MICT = 16 µg/mL (%R = 100) and a weak anti-proliferative activity against the clinical strains: C. albicans resistant to azoles (Itr and Flu) and C. glabrata. Derivatives 5k and 6a displayed MICP = 16 µg/mL and %R = 64.2 ± 10.6, %R = 88.0 ± 9.7, respectively, against the C. albicans isolate. Derivative 5i was the most active against C. glabrata (%R = 53.0 ± 3.5 at 16 µg/mL). Benzoxazoles displayed no MIC against C. glabrata. Benzoxazoles showed a pleiotropic action mode: (1) the total sterols content was perturbed; (2) 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(3,4-dichlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(2,3,4-trichlorophenyl)ethanol (8h–i) at the lowest fungistatic conc. inhibited the efflux of the Rho123 tracker during the membrane transport process; (3) mitochondrial respiration was affected/inhibited by the benzoxazoles: 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-chlorophenyl)ethanol and 2-(1,3-benzoxazol-2-ylsulfanyl)-1-(4-bromophenyl)ethanol 8c–d and 8i. Benzoxazoles showed comparable activity to commercially available azoles due to (1) the interaction with exogenous ergosterol, (2) endogenous ergosterol synthesis blocking as well as (3) membrane permeabilizing properties typical of AmB. Benzoxazoles display a broad spectrum of anti-Candida activity and action mode towards the membrane without cross-resistance with AmB; furthermore, they are safe to mammals.

show abstract

“…ABC transporter-mediated efflux was determined using rhodamine 6G (Sigma-Aldrich, USA) as previously described [33]. The fluorescence of the released R6G was measured at 530 nm, with an emission at 560 nm in an automated plate reader (Model 550 Microplate Reader Bio-Rad, Milan, Italy).…”

Section: Analysis Of Rhodamine 6g Effluxmentioning

confidence: 99%

Circulation of Fluconazole-ResistantC. albicans, C. aurisandC. parapsilosisBloodstream Isolates Carrying Y132F, K143R or T220L Erg11p Substitutions in Colombia

Ceballos-Garzón

Penuela

Beltran

et al. 2022

Preprint

View full text Add to dashboard Cite

Although Candida spp., is a common cause of bloodstream infections and is often associated with high mortality rates, its resistance to antifungal drugs, and the molecular mechanisms involved have been poorly studied in Colombia. Here, 123 bloodstream isolates of Candida spp. were collected. MALDI-TOF MS identification and fluconazole (FLC) susceptibility patterns were assessed on all isolates. Subsequently, sequencing of ERG11, TAC1 or MRR1, and efflux pumps were performed for resistant isolates. Out of 123 clinical strains, C. albicans accounted for 37.4%, followed by C. tropicalis 26.8%, C. parapsilosis 19.5%, C. auris 8.1%, C. glabrata 4.1%, C. krusei 2.4% and C. lusitaniae 1.6%. Resistance to FLC reached 18%. Erg11 amino acid substitutions associated with FLC-resistance (Y132F, K143R or T220L) were found in 58% of 19 FLC-resistant isolates. Furthermore, novel mutations were found in all genes studied. Regarding efflux pumps, 42% of 19 FLC-resistant Candida spp strains showed significant efflux activity. Finally, six of the 19 FLC-resistant isolates neither harbored resistance-associated mutations nor showed efflux pump activity. Although C. albicans remain the most predominant species, non-C. albicans species comprise a high proportion (62.6%). Among FLC-resistant species, C. auris (70%) and C. parapsilosis (25%) displayed the highest percentages of resistance. In 68% of FLC-resistant isolates, a mechanism that could explain their phenotype was found (e.g. mutations, flux pump activity or both). We provide evidence that endemic isolates harbor amino acid substitutions related with resistance to one of the most used molecules in the hospital setting, with Y132F being the most frequently detected one.

show abstract

Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species

Cited by 11 publications

References 6 publications

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

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

In Vitro Anti-Candida Activity and Action Mode of Benzoxazole Derivatives

Circulation of Fluconazole-ResistantC. albicans, C. aurisandC. parapsilosisBloodstream Isolates Carrying Y132F, K143R or T220L Erg11p Substitutions in Colombia

Contact Info

Product

Resources

About