Rhodamine 6G Efflux Research Articles

Geraniol Potentiates the Effect of Fluconazole against Planktonic and Sessile Cells of Azole-Resistant Candida tropicalis: In Vitro and In Vivo Analyses.

Candida tropicalis is regarded as an opportunistic pathogen, causing diseases ranging from superficial infections to life-threatening disseminated infections. The ability of this yeast to form biofilms and develop resistance to antifungals represents a significant therapeutic challenge. Herein, the effect of geraniol (GER), alone and combined with fluconazole (FLZ), was evaluated in the planktonic and sessile cells of azole-resistant C. tropicalis. GER showed a time-dependent fungicidal effect on the planktonic cells, impairing the cell membrane integrity. Additionally, GER inhibited the rhodamine 6G efflux, and the molecular docking analyzes supported the binding affinity of GER to the C. tropicalis Cdr1 protein. GER exhibited a synergism with FLZ against the planktonic and sessile cells, inhibiting the adhesion of the yeast cells and the viability of the 48-h biofilms formed on abiotic surfaces. C. tropicalis biofilms treated with GER, alone or combined with FLZ, displayed morphological and ultrastructural alterations, including a decrease in the stacking layers and the presence of wilted cells. Moreover, neither GER alone nor combined with FLZ caused toxicity, and both treatments prolonged the survival of the Galleria mellonella larvae infected with azole-resistant C. tropicalis. These findings indicate that the combination of GER and FLZ may be a promising strategy to control azole-resistant C. tropicalis infections.

The anti-staphylococcal fusidic acid as an efflux pump inhibitor combined with fluconazole against vaginal candidiasis in mouse model

BackgroundCandida albicans is the most common fungus that causes vaginal candidiasis in immunocompetent women and catastrophic infections in immunocompromised patients. The treatment of such infections is hindered due to the increasing emergence of resistance to azoles in C. albicans. New treatment approaches are needed to combat candidiasis especially in the dwindled supply of new effective and safe antifungals. The resistance to azoles is mainly attributed to export of azoles outside the cells by means of the efflux pump that confers cross resistance to all azoles including fluconazole (FLC).ObjectivesThis study aimed to investigate the possible efflux pump inhibiting activity of fusidic acid (FA) in C. albicans resistant isolates and the potential use of Fusidic acid in combination with fluconazole to potentiate the antifungal activity of fluconazole to restore its activity in the resistant C. albicans isolates.MethodsThe resistance of C. albicans isolates was assessed by determination of minimum inhibitory concentration. The effect of Fusidic acid at sub-inhibitory concentration on efflux activity was assayed by rhodamine 6G efflux assay and intracellular accumulation. Mice model studies were conducted to evaluate the anti-efflux activity of Fusidic acid and its synergistic effects in combination with fluconazole. Impact of Fusidic acid on ergosterol biosynthesis was quantified. The synergy of fluconazole when combined with Fusidic acid was investigated by determination of minimum inhibitory concentration. The cytotoxicity of Fusidic acid was tested against erythrocytes. The effect of Fusidic acid on efflux pumps was tested at the molecular level by real-time PCR and in silico study. In vivo vulvovaginitis mice model was used to confirm the activity of the combination in treating vulvovaginal candidiasis.ResultsFusidic acid showed efflux inhibiting activity as it increased the accumulation of rhodamine 6G, a substrate for ABC-efflux transporter, and decreased its efflux in C. albicans cells. The antifungal activity of fluconazole was synergized when combined with Fusidic acid. Fusidic acid exerted only minimal cytotoxicity on human erythrocytes indicating its safety. The FA efflux inhibitory activity could be owed to its ability to interfere with efflux protein transporters as revealed by docking studies and downregulation of the efflux-encoding genes of both ABC transporters and MFS superfamily. Moreover, in vivo mice model showed that using fluconazole-fusidic acid combination by vaginal route enhanced fluconazole antifungal activity as shown by lowered fungal burden and a negligible histopathological change in vaginal tissue.ConclusionThe current findings highlight FA’s potential as a potential adjuvant to FLC in the treatment of vulvovaginal candidiasis.

Tyrosol induces multiple drug resistance in yeast Saccharomyces cerevisiae.

In yeast, multiple (pleiotropic) drug resistance (MDR) transporters efflux xenobiotics from the cytoplasm to the environment. Additionally, upon the accumulation of xenobiotics in the cells, MDR genes are induced. At the same time, fungal cells can produce secondary metabolites with physico-chemical properties similar to MDR transporter substrates. Nitrogen limitation in yeast Saccharomyces cerevisiae leads to the accumulation of phenylethanol, tryptophol, and tyrosol, which are products of aromatic amino acid catabolism. In this study, we investigated whether these compounds could induce or inhibit MDR in yeast. Double deletion of PDR1 and PDR3 genes, which are transcription factors that upregulate the expression of PDR genes, reduced yeast resistance to high concentrations of tyrosol (4-6 g/L) but not to the other two tested aromatic alcohols. PDR5 gene, but not other tested MDR transporter genes (SNQ2, YOR1, PDR10, PDR15) contributed to yeast resistance to tyrosol. Tyrosol inhibited the efflux of rhodamine 6G (R6G), a substrate for MDR transporters. However, preincubating yeast cells with tyrosol induced MDR, as evidenced by increased Pdr5-GFP levels and reduced yeast ability to accumulate Nile red, another fluorescent MDR-transporter substrate. Moreover, tyrosol inhibited the cytostatic effect of clotrimazole, the azole antifungal. Our results demonstrate that a natural secondary metabolite can modulate yeast MDR. We speculate that intermediates of aromatic amino acid metabolites coordinate cell metabolism and defense mechanisms against xenobiotics.

Abrogation of efflux pump activity, biofilm formation, and immune escape by candidacidal geraniol in emerging superbug, Candida auris.

During the last decade, Candida auris emerged as a threatening human fungal pathogen that notably caused outbreaks around the globe with high mortality. Considering C. auris species as newly discovered fungi, the evolutionary features remain elusive. The antifungal resistance which is a norm in C. auris underlines the need for innovative therapeutic options. ATP Binding Cassette (ABC) superfamily efflux pumps overexpression and biofilms are known to be major contributors to multidrug resistance (MDR) in C. auris. Therefore, herein, we investigated the antifungal potential of geraniol (Ger) as a promising naturalcompound in the fight against MDR C. auris. Our experiments proved that Ger was fungicidal in nature and impaired rhodamine 6G (R6G) efflux, confirming the specific effect on ABC transporters. Kinetic studies unravelled the competitive mode of inhibition by Ger for R6G efflux since the apparent Km increased with no change in Vmax value. Mechanistic insights also revealed that Ger depleted ergosterol content in C. auris. Furthermore, Ger led to inhibition in biofilm formation as evident from crystal violet staining, biofilm metabolic and biomass measurements. Additionally, enhanced survival of Caenorhabditis elegans model after C. auris infection demonstrated the in vivo efficacy of Ger. Lastly, the in vivo efficacy was confirmed from a THP-1 cell line model which depicted enhanced macrophage-mediated killing in the presence of Ger. Modulation of C. auris efflux pump activity and biofilm formation by Ger represents a promising approach to combatMDR. Together, this study demonstrated the potential therapeutic insights of Ger as a promising addition to the antifungal armamentarium required to treat emerging and resistant C. auris.

Digoxin Derivatives Sensitize a Saccharomyces cerevisiae Mutant Strain to Fluconazole by Inhibiting Pdr5p.

The poor outcome of treatments for fungal infections is a consequence of the increasing incidence of resistance to antifungal agents, mainly due to the overexpression of efflux pumps. To surpass this mechanism of resistance, a substance able to inhibit these pumps could be administered in association with antifungals. Saccharomyces cerevisiae possesses an efflux pump (Pdr5p) homologue to those found in pathogenic yeast. Digoxin is a natural product that inhibits Na+, K+-ATPase. The aim of this study was to evaluate whether digoxin and its derivatives (i.e., DGB, digoxin benzylidene) can inhibit Pdr5p, reversing the resistance to fluconazole in yeasts. An S. cerevisiae mutant strain that overexpresses Pdr5p was used in the assays. The effects of the compounds on yeast growth, efflux activity, and Pdr5p ATPase activity were measured. All derivatives enhanced the antifungal activity of fluconazole against S. cerevisiae, in comparison to fluconazole alone, with FICI values ranging from 0.031 to 0.500. DGB 1 and DGB 3 presented combined effects with fluconazole against a Candida albicans strain, with fractional inhibitory concentration index (FICI) values of 0.625 and 0.281, respectively The compounds also inhibited the efflux of rhodamine 6G and Pdr5p ATPase activity, with IC50 values ranging from 0.41 μM to 3.72 μM. The results suggest that digoxin derivatives impair Pdr5p activity. Considering the homology between Pdr5p and efflux pumps from pathogenic fungi, these compounds are potential candidates to be used in association with fluconazole to treat resistant fungal infections.

Unique roles of aminophospholipid translocase Drs2p in governing efflux pump activity, ergosterol level, virulence traits, and host-pathogen interaction in Candida albicans.

Infections caused by Candida albicans are rising due to increment in drug resistance and a limited arsenal of conventional antifungal drugs. Thus, elucidating the novel antifungal targets still represent an alternative that could overcome the problem of multidrug resistance (MDR). In this study, we have uncovered the distinctive effect of aminophospholipid translocase (Drs2p) deletion on major MDR mechanisms of C. albicans. We determined that efflux activity was diminished in Δdrs2 mutant as revealed by extracellular rhodamine 6G (R6G) efflux and flow cytometry. Moreover, we further unveiled that Δdrs2 mutant displayed decreased ergosterol content and increased membrane fluidity. Furthermore, Drs2p deletion affects the virulence attributes and led to inhibited hyphal growth and reduced biofilm formation. Additionally, THP-1 cell lines' mediated host-pathogen interaction studies revealed that Δdrs2 mutant displayed enhanced phagocytosis and altered cytokine production leading to increased IL-6 and decreased IL-10 production. Taken together, the present study demonstrates the relevance of Drs2p in C. albicans and consequently disrupting pathways known for mediating drug resistance and immune recognition. Comprehensive studies are further required to authenticate Drs2p as a novel antifungal drug target.

Novel ABC Transporter Associated with Fluconazole Resistance in Aging of Cryptococcus neoformans.

Cryptococcus neoformans causes meningoencephalitis in immunocompromised individuals, which is treated with fluconazole (FLC) monotherapy when resources are limited. This can lead to azole resistance, which can be mediated by overexpression of ABC transporters, a class of efflux pumps. ABC pump-mediated efflux of FLC is also augmented in 10-generation old C. neoformans cells. Here, we describe a new ABC transporter Afr3 (CNAG_06909), which is overexpressed in C. neoformans cells of advanced generational age that accumulate during chronic infection. The Δafr3 mutant strain showed higher FLC susceptibility by FLC E-Test strip testing and also by a killing test that measured survival after 3 h FLC exposure. Furthermore, Δafr3 cells exhibited lower Rhodamine 6G efflux compared to the H99 wild-type cells. Afr3 was expressed in the Saccharomyces cerevisiae ADΔ strain, which lacks several drug transporters, thus reducing background transport. The ADΔ + Afr3 strain demonstrated a higher efflux with both Rhodamine 6G and Nile red, and a higher FLC resistance. Afr3-GFP localized in the plasma membrane of the ADΔ + Afr3 strain, further highlighting its importance as an efflux pump. Characterization of the Δafr3 mutant revealed unattenuated growth but a prolongation (29%) of the replicative life span. In addition, Δafr3 exhibited decreased resistance to macrophage killing and attenuated virulence in the Galleria mellonella infection model. In summary, our data indicate that a novel ABC pump Afr3, which is upregulated in C. neoformans cells of advanced age, may contribute to their enhanced FLC tolerance, by promoting drug efflux. Lastly, its role in macrophage resistance may also contribute to the selection of older C. neoformans cells during chronic infection.

In vitro and in vivo analysis of monotherapy and dual therapy with ethyl caffeate and fluconazole on virulence factors of Candida albicans and systemic candidiasis

Candida albicans is the most clinically prevalent cause of systemic fungal infections in the immunocompromised population. The biofilm-forming ability of C. albicans confers resistance to conventional antifungal agents. The main aim of this study was to investigate the antifungal effects of ethyl caffeate (EC) alone and in combination with fluconazole (FLU) against C. albicans isolates. The single and combined antifungal activities of EC and FLU were evaluated against planktonic and biofilm cells of C. albicans by the checkerboard assay, time-kill test, crystal violet assay, live/dead staining, rhodamine 6G (R6G) efflux analysis and hydrolase activity. Monotherapy and dual therapy of EC and FLU against systemic candidiasis in a mouse model was also evaluated. The results showed that EC+FLU displayed synergism in 14/26 planktonic C. albicans isolates and 11/26 C. albicans biofilms with fractional inhibitory concentration index (FICI) values ranging between 0.06-0.49 and 0.02-0.38, respectively. Compared with monotherapy, the combination of EC+FLU can markedly inhibit adhesion, yeast-to-hyphae transition, premature and mature biofilm metabolism, hydrolase secretion and drug efflux function of C. albicans Z1407 and Z4935. Moreover, EC can potentiate the antifungal activity of FLU to improve mouse survival, reduce fungal burden and alleviate pathological damage in both C. albicans isolates compared with EC or FLU used alone. EC exhibits a moderate antifungal potential but can be a strong synergist with FLU against C. albicans, highlighting the potential of EC in clinical antifungal therapy as a sensitiser.

In vitro and in vivo Study of Antifungal Effect of Pyrvinium Pamoate Alone and in Combination With Azoles Against Exophiala dermatitidis.

Infections of Exophiala dermatitidis are often chronic and recalcitrant. Combination therapies with novel compounds and azoles could be an effective solution. Previously, we have demonstrated that pyrvinium pamoate exerted antifungal activity alone and favorable synergy with azoles against planktonic E. dermatitidis. Herein, the underlying antifungal mode of action were investigated. Pyrvinium alone showed sessile MIC50 (SMIC50) of 8->16 μg/ml against E. dermatitidis biofilms. However, synergism of PP with itraconazole, voriconazole, and posaconazole were observed against 16 (88.9%), 9 (50%), and 13 (72.2%) strains of E. dermatitidis biofilms. In accordance with in vitro susceptibilities, pyrvinium alone at concentration of 2 μg/ml resulted in significant growth restriction of planktonic E. dermatitidis. Pyrvinium alone resulted in reduction of biofilm formation. Higher concentration of pyrvinium was associate with more progressive reduction of biofilm mass. The in vivo activity of pyrvinium alone and combined with azoles was evaluated using Galleria mellonella model. Pyrvinium alone significantly improved the survival rate of larvae (P < 0.0001). The combination of pyrvinium and voriconazole or posaconazole acted synergistically in vivo (P < 0.05). Fungal burden determination revealed significant reduction of numbers of colony forming unit (CFU) in larvae treated with pyrvinium-itraconazole and pyrvinium-posaconazole compared to itraconazole or posaconazole alone group, respectively. The effect of pyrvinium on apoptosis, expression of TOR and HSP90, and drug efflux reversal were evaluated by PI/Annexin V staining, Real-Time Quantitative PCR and Rhodamine 6G assay, respectively. Pyrvinium alone or combined with azoles significantly (P < 0.05) increased late apoptosis or necrosis of E. dermatitidis cells. Pyrvinium combined with posaconazole significantly decreased the expression of TOR and Hsp90 compared to posaconazole alone group (P < 0.05). Pyrvinium resulted in significant (P < 0.05) decrease of the efflux of Rhodamine 6G. These findings suggested pyrvinium could be a promising synergist with azoles. The underlying mechanisms could be explained by inducing apoptosis/necrosis, inhibition of drug efflux pumps, and signaling pathways related with stress response and growth control.

The H741D mutation in Tac1p contributes to the upregulation of CDR1 and CDR2 expression in Candida albicans.

The wide use of antifungal agents has led to the development of resistance in the pathogenic yeast strain Candida albicans. Gain-of-function mutations in transcription factors such as Tac1p demonstrated their ability to control expression of the ABC transporter genes CDR1 and CDR2, and mediation of azole resistance. Previously, we obtained a series of azole-resistant isolates with high-level expression of CDR1 or/and CDR2, and identified the novel H741D mutation in Tac1p. In the present study, the TAC1 alleles from isolate C13 were introduced into tac1Δ/Δ mutant. The H741D change was seen in TAC1C13 in addition to several other amino acid differences. Hyperactive alleles TAC1C13 exhibited higher minimum inhibitory concentrations (MICs) of fluconazole and itraconazole than that observed in SN152 containing the wild-type TAC1 allele. And alleles TAC1C13 conferred constitutively high levels of Cdr1p and Cdr2p. Moreover, the importance of H741D in conferring hyperactivity to TAC1 was also confirmed by site-directed mutagenesis. Compared with SN152, the presence of H741D resulted in > 2-fold increase in CDR1 and CDR2 gene and protein expression, > 4-fold increase in fluconazole and itraconazole MICs and higher rates of Rhodamine 6G efflux by 43.24%.

Hypervirulence and cross-resistance to a clinical antifungal are induced by an environmental fungicide in Cryptococcus gattii

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.

Octyl gallate reduces ABC multidrug transporter CaCdr1p expression and leads to its mislocalisation in azole-resistant clinical isolates of Candida albicans

ObjectivesFungal pathogens pose a serious threat to public health. Widespread and prolonged use of antifungal drugs has led to the development of multidrug resistance in the human fungal pathogen Candida albicans. Among several mechanisms leading to drug resistance in C. albicans, overexpression of drug efflux transporters remains by far the leading cause of multidrug resistance, facilitated by overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) transporters. Hence, targeting efflux pumps still represents a promising approach to combat multidrug resistance. In this study, the effect of octyl gallate (OG), a natural food additive, on drug efflux pump activity of C. albicans was analysed. MethodsDrug efflux pump activity was determined by rhodamine 6G (R6G) efflux and Nile red accumulation assay in a Candida drug resistance protein 1 (CaCdr1p)-overexpressing strain. Gene expression and protein expression and localisation were studied by RT-PCR, Western blot and confocal microscopy. Ergosterol content was measured by the alcoholic KOH method. ResultsOG specifically inhibits the activity of CaCdr1p, belonging to the ABC superfamily. The underlying mechanism was confirmed as competitive mode of inhibition by OG as revealed by Lineweaver–Burk plot. Furthermore, OG leads to reduced expression of CDR1 and CaCdr1p and mislocalisation of CaCdr1p. Additionally, OG sensitises azole-susceptible and -resistant clinical matched-pair isolates Gu4 & Gu5 and leads to impeded R6G efflux and depleted ergosterol content. ConclusionThe ability of OG as a potent inhibitor of CaCdr1p that chemosensitises drug-resistant C. albicans warrants further studies to be exploited as an effective antifungal agent.

Characterization of the Efflux Capability and Substrate Specificity of Aspergillus fumigatus PDR5-like ABC Transporters Expressed in Saccharomyces cerevisiae.

This research analyzed six Aspergillus fumigatus genes encoding putative efflux proteins for their roles as transporters. TheA. fumigatus genes abcA, abcC, abcF, abcG, abcH, and abcI were cloned into plasmids and overexpressed in a Saccharomyces cerevisiae strain in which the highly active endogenous ABC transporter gene PDR5 was deleted. The activity of each transporter was measured by efflux of rhodamine 6G and accumulation of alanine β-naphthylamide. The transporters AbcA, AbcC, and AbcF had the strongest efflux activities of these compounds. All of the strains with plasmid-expressed transporters had more efflux activity than did the PDR5-deleted background strain. We performed broth microdilution drug susceptibility testing and agar spot assays using an array of compounds and antifungal drugs to determine the transporter specificity and drug susceptibility of the strains. The transporters AbcC and AbcF showed the broadest range of substrate specificity, while AbcG and AbcH had the narrowest range of substrates. Strains expressing the AbcA, AbcC, AbcF, or AbcI transporter were more resistant to fluconazole than was the PDR5-deleted background strain. Strains expressing AbcC and AbcF were additionally more resistant to clotrimazole, itraconazole, ketoconazole, and posaconazole than was the background strain. Finally, we analyzed the expression levels of the genes by reverse transcription-quantitative PCR (RT-qPCR) in triazole-susceptible and -resistant A. fumigatus clinical isolates. All of these transporters are expressed at a measurable level, and transporter expression varied significantly between strains, demonstrating the high degree of phenotypic variation, plasticity, and divergence of which this species is capable.IMPORTANCE One mechanism behind drug resistance is altered export out of the cell. This work is a multifaceted analysis of membrane efflux transporters in the human fungal pathogen A. fumigatus Bioinformatics evidence infers that there is a relatively large number of genes in A. fumigatus that encode ABC efflux transporters. However, very few of these transporters have been directly characterized and analyzed for their potential role in drug resistance.Our objective was to determine if these undercharacterized proteins function as efflux transporters and then to better define whether their efflux substrates include antifungal drugs used to treat fungal infections. We chose six A. fumigatus potential plasma membrane ABC transporter genes for analysis and found that all six genes produced functional transporter proteins. We used two fungal systems to look for correlations between transporter function and drug resistance. These transporters have the potential to produce drug-resistant phenotypes in A. fumigatus Continued characterization of these and other transporters may assist in the development of efflux inhibitor drugs.

Vanillin confers antifungal drug synergism in Candida albicans by impeding CaCdr2p driven efflux

AimAmong the most common mechanisms of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, overexpression of drug efflux pumps remains the predominant mechanism. Hence to inhibit efflux pumps and chemosensitize C. albicans against traditional antifungal drugs still represents an attractive approach. The present study aimed to analyze the effect of Vanillin (Van), a natural food flavoring agent, on drug efflux pump activity of Candida albicans. Methods and resultsWe observed that Van specifically inhibits Candida drug resistance protein 2 (CaCdr2p) activity belonging to ATP Binding Cassette (ABC) superfamily as revealed by abrogated rhodamine 6G efflux and nile red accumulation assay with CaCdr2p over expressing strain. Insight studies into the mechanisms suggested that abrogated efflux by CaCdr2p is due to competitive mode of inhibition by Van as depicted by Lineweaver-Burk plot. RT-PCR, western blot and confocal microscopy further unraveled that Van leads to reduced expression of CDR2 and CaCdr2p mislocalization respectively. Furthermore, Van sensitizes the azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated rhodamine 6G efflux and depleted ergosterol. Furthermore, Van synergizes with membrane targeting drugs fluconazole and amphotericin B as their fractional inhibitory coefficient index was less than 0.5. ConclusionVan being a potent inhibitor of CaCdr2p and chemosensitizing of drug resistant C. albicans warrants further studies to be exploited as effective antifungal agent.

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

BackgroundPseudomonas 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.MethodExtracts 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.ResultsThe 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.ConclusionThe 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.

Voriconazole-Based Salts Are Active against Multidrug-Resistant Human Pathogenic Yeasts.

Voriconazole (VOR) hydrochloride is unequivocally converted into VOR lactates and valinates upon reaction with silver salts of organic acids. This study found that the anticandidal in vitro activity of these compounds was comparable or slightly better than that of VOR. The Candida albicans clinical isolate overexpressing CaCDR1/CaCDR2 genes, highly resistant to VOR, was apparently more susceptible to VOR salts. On the other hand, the susceptibility of another C. albicans clinical isolate (demonstrating multidrug resistance due to the overexpression of CaMDR1) to VOR salts was comparable to that to VOR. Comparative studies on the influence of VOR and its salts on Rhodamine 6G efflux from susceptible and multidrug-resistant C. albicans cells revealed that VOR salts are poorer substrates for the CaCdr1p drug efflux pump than VOR.

The efflux pump inhibitor tetrandrine exhibits synergism with fluconazole or voriconazole against Candida parapsilosis.

In the last two decades, with the wide use of azoles, antifungal resistance among Candida parapsilosis has considered a matter of concern worldwide. The aim of this study is to evaluate the antifungal potentials of tetrandrine (TET) alone and in combination with fluconazole (FLC)/voriconazole (VRC) against C. parapsilosis. Susceptibility tests were performed by microdilution method, checkerboard assay, time-kill test, spot assay. Subsequently, rhodamine 6G efflux test and the expressions of transporter related genes, namely CDR1 and MDR1 for C. parapsilosis were analyzed by qRT-PCR. The susceptibility test showed that TET presented strong synergism with FLC and VRC with fractional inhibitory concentration index (FICI) in a range of 0.094-0.562. The susceptibility results were also confirmed by spot assay and time-kill studies. With TET treatment, a vast quantity of rhodamine 6G could not be pumped out from the cells as considerably intracellular red fluorescence was accumulated. Meanwhile, the expressions of efflux-associated genes presented varying degrees of inhibition. These results indicated that TET was a decent antifungal synergist to promote the antifungal efficacy of FLC/VRC, and the underlying antifungal mechanism might be associated with the inhibition of efflux pump and the elevation of intracellular drug content.

Retrograde signaling disruption influences ABC superfamily transporter, ergosterol and chitin levels along with biofilm formation in Candida albicans

ObjectiveThe rise in fungal infections is alarming due to emergence of multidrug drug resistance (MDR). Hence elucidating novel drug targets to circumvent the problem of MDR warrants immediate attention. This study analyzes the effect of retrograde (RTG) signaling disruption on major MDR mechanisms and virulence of the human pathogenic fungal species Candida albicans. Material and methodsDrug transporter activity was measured by rhodamine 6G (R6G) efflux. Membrane damage was studied by propidium iodide intake and ergosterol level determination. Cell wall effect was estimated by quantifying chitin levels and cell sedimentation rate. Biofilm formation was visualized by calcoflour white and crystal violet staining and measured by dry mass and MTT assay. Cell adherence to buccal epithelial cell was determined by trypan blue staining and MTT assay. Virulence was studied using nematode model Caenorhabditis elegans. ResultWe demonstrated that mutant of transcription factor CaRTG3 leads to impaired efflux activity of ATP Binding Cassette (ABC) superfamily multidrug transporters. We further uncover that rtg3 mutant exhibited a disrupted membrane, decreased ergosterol levels and increased chitin content. Furthermore, RTG signaling disruption leads to inhibited biofilm formation and cell adherence to buccal epithelial cells. Lastly, rtg3 mutant displayed a reduced infectivity in C. elegans illustrating its vulnerability as antifungal target. Interestingly, all the abrogated phenotypes could be rescued in the revertant strain of rtg3 mutant. ConclusionPresent study establishes a link between RTG signaling, drug efflux and biofilm formation and validates CaRTG3 as antifungal target. Intricate studies are needed to further understand and exploit this therapeutic opportunity.

Decreasing Cell Population of Individual Candida Species Does Not Impair the Virulence of Candida albicans and Candida glabrata Mixed Biofilms.

Candida albicans and Candida glabrata are two commonly seen opportunistic fungi in clinical settings and usually co-isolated from the population inflicted with denture stomatitis and oropharyngeal candidiasis. Although C. albicans and C. glabrata mixed biofilm is deemed to possess enhanced virulence compared with their individual counterparts (especially C. albicans single biofilm), the relevant descriptions and experimental evidence on the relationship of Candida virulence with their individual cell number in mixed biofilms are contradictory and insufficient. In this study, two standard C. glabrata isolate and eight C. albicans ones were used to test the cell quantities in their 24- and 48-h single and mixed biofilms. A series of virulence factors including antifungal resistance to caspofungin, secreted aspartic proteinase (SAP) and phospholipase (PL) levels, efflux pump function and β-glucan exposure were evaluated. Through this study, the declines of individual cell counting were observed in the 24- and 48-h Candida mixed biofilms compared with their single counterparts. However, the antifungal resistance to caspofungin, the SAP and phospholipase levels, the rhodamine 6G efflux and the efflux-related gene expressions were increased significantly or kept unchanged accompanying with reduced β-glucan exposure in the mixed biofilms by comparison with the single counterparts. These results reveal that there is a competitive interaction between C. albicans and C. glabrata strains in their co-culture without at the expense of the mixed biofilm virulence. This study presents a deep insight into the interaction between C. albicans and C. glabrata and provides new clues to combat against fungal infections caused by Candida mixed biofilms.

Eucalyptal D Enhances the Antifungal Effect of Fluconazole on Fluconazole-Resistant Candida albicans by Competitively Inhibiting Efflux Pump.

The frequent emergence of azole-resistant strains has increasingly led azoles to fail in treating candidiasis. Combination with other drugs is a good option to effectively reduce or retard its incidence of resistance. Natural products are a promising synergist source to assist azoles in treating resistant candidiasis. Eucalyptal D (ED), a formyl-phloroglucinol meroterpenoid, is one of the natural synergists, which could significantly enhance the anticandidal activity of fluconazole (FLC) in treating FLC resistant C. albicans. The checkerboard microdilution assay showed their synergistic effect. The agar disk diffusion test illustrated the key role of ED in synergy. The rhodamine 6G (R6G) efflux assay reflected ED could reduce drug efflux, but quantitative reverse transcription PCR analysis revealed the upregulation of CDR1 and CDR2 genes in ED treating group. Efflux pump-deficient strains were hyper-susceptible to ED, thus ED was speculated to be the substrate of efflux pump Cdr1p and Cdr2p to competitively inhibit the excretion of FLC or R6G, which mainly contributed to its synergistic effect.