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The occurrence and recurrence of mucosal biofilm-related Candida infections, such as oral and vulvovaginal candidiasis, are serious clinical issues. Vaginal infections caused by Candida spp., for example, affect 70 to 75% of women at least once during their lives. Miconazole (MCZ) is the preferred topical treatment against these fungal infections, yet it has only moderate antibiofilm activity. Through screening of a drug-repurposing library, we identified the quaternary ammonium compound domiphen bromide (DB) as an MCZ potentiator against Candida biofilms. DB displayed synergistic anti-Candida albicans biofilm activity with MCZ, reducing the number of viable biofilm cells 1,000-fold. In addition, the MCZ-DB combination also resulted in significant killing of biofilm cells of azole-resistant C. albicans, C. glabrata, and C. auris isolates. In vivo, the MCZ-DB combination had significantly improved activity in a vulvovaginal candidiasis rat model compared to that of single-compound treatments. Data from an artificial evolution experiment indicated that the development of resistance against the combination did not occur, highlighting the potential of MCZ-DB combination therapy to treat Candida biofilm-related infections.

The plant defensin HsAFP1 is characterized by broad-spectrum antifungal activity and induces apoptosis in Candida albicans. In this study, we performed a transcriptome analysis on C. albicans cultures treated with HsAFP1 to gain further insight in the antifungal mode of action of HsAFP1. Various genes coding for cell surface proteins, like glycosylphosphatidylinositol (GPI)-anchored proteins, and proteins involved in cation homeostasis, autophagy and in cell cycle were differentially expressed upon HsAFP1 treatment. The biological validation of these findings was performed in the model yeast Saccharomyces cerevisiae. To discriminate between events linked to HsAFP1's antifungal activity and those that are not, we additionally used an inactive HsAFP1 mutant. We demonstrated that (i) HsAFP1-resistent S. cerevisiae mutants that are characterized by a defect in processing GPI-anchors are unable to internalize HsAFP1, and (ii) moderate doses (FC50, fungicidal concentration resulting in 50% killing) of HsAFP1 induce autophagy in S. cerevisiae, while high HsAFP1 doses result in vacuolar dysfunction. Vacuolar function is an important determinant of replicative lifespan (RLS) under dietary restriction (DR). In line, HsAFP1 specifically reduces RLS under DR. Lastly, (iii) HsAFP1 affects S. cerevisiae cell cycle in the G2/M phase. However, the latter HsAFP1-induced event is not linked to its antifungal activity, as the inactive HsAFP1 mutant also impairs the G2/M phase. In conclusion, we demonstrated that GPI-anchored proteins are involved in HsAFP1's internalization, and that HsAFP1 induces autophagy, vacuolar dysfunction and impairment of the cell cycle. Collectively, all these data provide novel insights in the mode of action of HsAFP1 as well as in S. cerevisiae tolerance mechanisms against this peptide. ispartof: BBA - Biomembranes vol:1862 issue:8 ispartof: location:Netherlands status: Published online

The human fungal opportunistic pathogen Candida albicans resides in the human gut, genitourinary tract and on the skin. The majority of infections caused by C. albicans are biofilm-related. In the first part of this review, we discuss new insights into C. albicans biofilm characteristics, concentrating on the extracellular matrix, phenotypic switching, efflux pumps and persister cells. It is widely accepted that this multicellular lifestyle is more resistant to traditional antifungal treatment compared to free-living cells. Therefore, much effort is put in the search for combinations of drugs leading to synergistic interactions against microbial biofilms to achieve lower effective doses of the drugs. In the second part of this manuscript, we review all recently identified compounds that act synergistically with azoles, echinocandins and/or polyenes against C. albicans biofilms.

In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli / C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

Mucosal biofilm-related fungal infections are very common, and the incidence of recurrent oral and vulvovaginal candidiasis is significant. As resistance to azoles (the preferred treatment) is occurring, we aimed at identifying compounds that increase the activity of miconazole against Candida albicans biofilms. We screened 1,600 compounds of a drug-repositioning library in combination with a subinhibitory concentration of miconazole. Synergy between the best identified potentiators and miconazole was characterized by checkerboard analyses and fractional inhibitory concentration indices. Hexachlorophene, pyrvinium pamoate, and artesunate act synergistically with miconazole in affecting C. albicans biofilms. Synergy was most pronounced for artesunate and structural homologues thereof. No synergistic effect could be observed between artesunate and fluconazole, caspofungin, or amphotericin B. Our data reveal enhancement of the antibiofilm activity of miconazole by artesunate, pointing to potential combination therapy consisting of miconazole and artesunate to treat C. albicans biofilm-related infections.

The spread of antibiotic resistance and the challenges associated with antiseptics such as chlorhexidine have necessitated a search for new antibacterial agents against oral bacterial pathogens. As a result of failing traditional approaches, drug repurposing has emerged as a novel paradigm to find new antibacterial agents. In this study, we examined the effects of the FDA-approved anticancer agent toremifene against the oral bacteria Porphyromonas gingivalis and Streptococcus mutans . We found that the drug was able to inhibit the growth of both pathogens, as well as prevent biofilm formation, at concentrations ranging from 12.5 to 25 μM. Moreover, toremifene was shown to eradicate preformed biofilms at concentrations ranging from 25 to 50 μM. In addition, we found that toremifene prevents P. gingivalis and S. mutans biofilm formation on titanium surfaces. A time-kill study indicated that toremifene is bactericidal against S. mutans . Macromolecular synthesis assays revealed that treatment with toremifene does not cause preferential inhibition of DNA, RNA, or protein synthesis pathways, indicating membrane-damaging activity. Biophysical studies using fluorescent probes and fluorescence microscopy further confirmed the membrane-damaging mode of action. Taken together, our results suggest that the anticancer agent toremifene is a suitable candidate for further investigation for the development of new treatment strategies for oral bacterial infections.

Summary Botrytis cinerea is a necrotrophic fungal pathogen causing disease in many plant species, leading to economically important crop losses. So far, fungicides have been widely used to control this pathogen. However, in addition to their detrimental effects on the environment and potential risks for human health, increasing fungicide resistance has been observed in the B. cinerea population. Biological control, that is the application of microbial organisms to reduce disease, has gained importance as an alternative or complementary approach to fungicides. In this respect, the genus Trichoderma constitutes a promising pool of organisms with potential for B. cinerea control. In the first part of this article, we review the specific mechanisms involved in the direct interaction between the two fungi, including mycoparasitism, the production of antimicrobial compounds and enzymes (collectively called antagonism), and competition for nutrients and space. In addition, biocontrol has also been observed when Trichoderma is physically separated from the pathogen, thus implying an indirect systemic plant defence response. Therefore, in the second part, we describe the consecutive steps leading to induced systemic resistance (ISR), starting with the initial Trichoderma–plant interaction and followed by the activation of downstream signal transduction pathways and, ultimately, the defence response resulting in ISR (ISR-prime phase). Finally, we discuss the ISR-boost phase, representing the effect of ISR priming by Trichoderma spp. on plant responses after additional challenge with B. cinerea.

Oral infections are among the most common diseases worldwide. Many protocols for the prevention and treatment of oral infections have been described, yet no golden standard has been developed so far. The antiseptic chlorhexidine and antibiotics are often used in these treatment procedures. However, long-term use of chlorhexidine can lead to side effects and extensive use of antibiotics can promote the development of antibiotic-resistant bacteria, which in turn can compromise the effectiveness of the treatment. Consequently, it remains important to search for new antibacterial agents for the treatment of oral infections. In this study, we report on the antibacterial activity of the anti-asthma drug zafirlukast against oral pathogens Porphyromonas gingivalis and Streptococcus mutans. Furthermore, its activity against oral biofilms grown on titanium surfaces was confirmed. In addition, we demonstrated that zafirlukast displays no cytotoxicity against human osteoblasts. Combined, this study paves the way for further research to determine the potential of zafirlukast to be used as a new antibiotic against oral pathogens. ispartof: FEMS Microbiology Letters vol:364 issue:2 ispartof: location:England status: published

Porphyromonas gingivalis is a major pathogen involved in oral diseases such as periodontitis and peri‐implantitis. Management of these diseases typically includes mechanical debridement of the colonized surfaces followed by application of antiseptics or antibiotics. Disadvantages associated with the use of antiseptics and the growing worldwide problem of antibiotic resistance have necessitated the search for alternative agents. In this study, the antibacterial and antibiofilm properties of AM404, an active metabolite of paracetamol, were tested against P. gingivalis and other bacterial pathogens. The activity of AM404 was tested against 10 bacteria, including both oral and nonoral human pathogens. The minimal inhibitory concentration (MIC) of AM404 was determined by measuring optical density (OD) values. The minimum biofilm inhibitory concentration (MBIC) was detected by crystal violet staining. The activity of structural analogs of AM404 was tested by MIC determinations. The effect of AM404 on P. gingivalis biofilms formed on titanium disks as a model for dental implants was evaluated by colony forming unit counting. Potential cytotoxicity of AM404 towards HEK‐293 (human embryonic kidney cells), HepG2 (human hepatoma cells), IEC‐6 (rat intestinal cells), and Panc‐1 cells (pancreatic cancer cells) was assessed by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide assays. To get more insight in the mode of action of AM404, we used the fluorescent dyes N‐phenyl‐1‐napthylamine and SYTOX green to investigate outer and inner membrane damage of P. gingivalis induced by AM404, respectively. Of all tested pathogens, AM404 only inhibited growth and biofilm formation of P. gingivalis. Moreover, it showed potent activity against P. gingivalis biofilms formed on titanium surfaces. A structure–activity analysis demonstrated that the unsaturated carbon chain is essential for its antibacterial activity. Importantly, AM404 was not toxic towards the tested mammalian cells up to concentrations approaching 4× the MIC. Membrane damage assays using fluorescent probes N‐phenyl‐1‐napthylamine and SYTOX green revealed that membrane permeabilization presumably is the primary antibacterial mode of action of AM404. Collectively, our results suggest that AM404 has the potential to be used for the development of new drugs specifically targeting P. gingivalis‐related infections.

The plant-derived decapeptide OSIP108 increases tolerance of yeast and human cells to apoptosis-inducing agents, such as copper and cisplatin. We performed a whole amino acid scan of OSIP108 and conducted structure-activity relationship studies on the induction of cisplatin tolerance (CT) in yeast. The use of cisplatin as apoptosis-inducing trigger in this study should be considered as a tool to better understand the survival-promoting nature of OSIP108 and not for purposes related to anti-cancer treatment. We found that charged residues (Arg, His, Lys, Glu or Asp) or a Pro on positions 4–7 improved OSIP108 activity by 10% or more. The variant OSIP108[G7P] induced the most pronounced tolerance to toxic concentrations of copper and cisplatin in yeast and/or HepG2 cells. Both OSIP108 and OSIP108[G7P] were shown to internalize equally into HeLa cells, but at a higher rate than the inactive OSIP108[E10A], suggesting that the peptides can internalize into cells and that OSIP108 activity is dependent on subsequent intracellular interactions. In conclusion, our studies demonstrated that tolerance/survival-promoting properties of OSIP108 can be significantly improved by single amino acid substitutions, and that these properties are dependent on (an) intracellular target(s), yet to be determined.

In contrast to planktonic cultures of the human fungal pathogen Candida albicans, C. albicans biofilms can contain a persister subpopulation that is tolerant to high concentrations of currently used antifungals. In this chapter, the method to determine the persister fraction in a C. albicans biofilm treated with an antifungal compound is described. To this end, a mature biofilm is developed and subsequently treated with a concentration series of the antifungal compound of interest. Upon incubation, the fraction of surviving biofilm cells is determined by plating and plotted versus the used concentrations of the antifungal compound. If a persister subpopulation in the biofilm is present, the dose-dependent killing of the biofilm cells results in a biphasic killing pattern.

We performed a whole-transcriptome analysis of miconazole-treated Candida albicans biofilms, using RNA-sequencing. Our aim was to identify molecular pathways employed by biofilm cells of this pathogen to resist action of the commonly used antifungal miconazole. As expected, genes involved in sterol biosynthesis and genes encoding drug efflux pumps were highly induced in biofilm cells upon miconazole treatment. Other processes were affected as well, including the electron transport chain (ETC), of which eight components were transcriptionally downregulated. Within a diverse set of 17 inhibitors/inducers of the transcriptionally affected pathways, the ETC inhibitors acted most synergistically with miconazole against C. albicans biofilm cells. Synergy was not observed for planktonically growing C. albicans cultures or when biofilms were treated in oxygen-deprived conditions, pointing to a biofilm-specific oxygen-dependent tolerance mechanism. In line, a correlation between miconazole’s fungicidal action against C. albicans biofilm cells and the levels of superoxide radicals was observed and confirmed both genetically and pharmacologically using a triple superoxide dismutase mutant and a superoxide dismutase inhibitor N-N′-diethyldithiocarbamate, respectively. Consequently, ETC inhibitors that result in mitochondrial dysfunction and affect production of reactive oxygen species can increase miconazole’s fungicidal activity against C. albicans biofilm cells.

Invasive fungal infections caused by opportunistic fungal pathogens are associated with high mortality rates, mainly due to the occurrence of genotypic and/or phenotypic resistance. One of the causes of phenotypic resistance is the preferred growth of various fungal pathogens as biofilms, which are tolerant or resistant to most classes of antifungal agents. Moreover, increasing evidence points to biofilm formation as a general prerequisite for the development of systemic infections. Therefore, new antibiofilm agents are urgently needed to reduce the incidence of biofilm-associated infections. Nowadays, antimicrobial peptides (AMPs) are considered as valuable alternatives for or complements to the classical antifungal agents to combat fungal infections. Many review reports describe activity of AMPs against free-living planktonic fungal pathogens. In contrast, this review summarizes the antibiofilm properties of natural or synthetic AMPs against fungal biofilms and their potential to enhance the antibiofilm activity of existing antifungal agents.

We here report on the in vitro activity of toremifene to inhibit biofilm formation of different fungal and bacterial pathogens, including Candida albicans , Candida glabrata , Candida dubliniensis , Candida krusei , Pseudomonas aeruginosa , Staphylococcus aureus , and Staphylococcus epidermidis . We validated the in vivo efficacy of orally administered toremifene against C. albicans and S. aureus biofilm formation in a rat subcutaneous catheter model. Combined, our results demonstrate the potential of toremifene as a broad-spectrum oral antibiofilm compound.

The fungal pathogen Botrytis cinerea establishes a necrotrophic interaction with its host plants, including lettuce (Lactuca sativa), causing it to wilt, collapse and eventually dry up and die, which results in serious economic losses. Global expression profiling using RNAseq and the newly sequenced lettuce genome identified a complex network of genes involved in the lettuce―B, cinerea interaction. The observed high number of differentially expressed genes allowed us to classify them according to the biological pathways in which they are implicated, generating a holistic picture. Most pronounced were the induction of the phenylpropanoid pathway and terpenoid biosynthesis, whereas photosynthesis was globally down-regulated at 48 h post-inoculation. Large-scale comparison with data available on the interaction of B. cinerea with the model plant Arabidopsis thaliana revealed both general and species-specific responses to infection with this pathogen. Surprisingly, expression analysis of selected genes could not detect significant systemic transcriptional alterations in lettuce leaves distant from the inoculation site. Additionally, we assessed the response of these lettuce genes to a biotrophic pathogen, Bremia lactucae, revealing that similar pathways are induced during compatible interactions of lettuce with necrotrophic and biotrophic pathogens.