Iron-Limited Biofilms of Candida albicans and Their Susceptibility to Amphotericin B

Biofilms of Candida albicans were grown in vitro under iron limitation and at a low growth rate to simulate conditions for implant-associated biofilms in vivo. Their properties were compared with those of glucose-limited biofilms grown under analogous conditions. At steady state, the adherent cell populations of iron-limited biofilms were double those of glucose-limited biofilms, although the growth rates were similar (0.038 to 0.043 h⁻¹). Both biofilm types were resistant to amphotericin B, but daughter cells from iron-limited biofilms were significantly more susceptible to the drug than those from glucose-limited biofilms.     

Growth-rate-independent killing by ciprofloxacin of biofilm-derived Staphylococcus epidermidis; evidence for cell-cycle dependency.

Cell culture methods that allow culture of Staphylococcus epidermidis biofilms at controlled growth rates were used to examine susceptibility to ciprofloxacin. Changes in biofilm susceptibility, dependent upon growth rate, were compared with those for suspended populations grown in chemostat, and also for newly-formed daughter cells shed from the biofilm during its growth and development. Susceptibility increased for intact and resuspended biofilms, and also for planktonic cultures, with increases in growth rate. The dependence of susceptibility upon growth rate was greatest for slow growing cells (mu, 0.01-0.15/h). At any particular growth rate, biofilms appeared more susceptible than their planktonic counterparts. Newly-formed daughter cells were relatively tolerant to ciprofloxacin at all rates of growth. Lack of growth rate dependency for the newly-formed cells suggested a role for the cell-division cycle in determining resistance. This was confirmed by examining the susceptibility of S. epidermidis throughout batch cultures with cell division synchronized. Perfusion of various steady-state biofilms with ciprofloxacin demonstrated killing of the adherent population even at much reduced rates of growth.     

Susceptibility of Pseudomonas aeruginosa and Escherichia coli biofilms towards ciprofloxacin: effect of specific growth rate

Methods of cell culture which enable the control of specific growth rate and expression of iron-regulated membrane proteins within Gram-negative biofilms were employed for various clinical isolates of Pseudomonas aeruginosa taken from the sputum of cystic fibrosis patients and of a laboratory strain of Escherichia coli. Susceptibility towards ciprofloxacin was assessed as a function of growth-rate for intact biofilms, cells resuspended from the biofilms and also for newly formed daughter cells shed from the biofilm during its growth and development. Patterns of susceptibility with growth rate were compared to those of suspended cultures grown in a chemostat. In all instances the susceptibility of chemostat cultures was directly related to growth rate. Whilst little difference was observed in the susceptibility pattern for P. aeruginosa strains with different observed levels of mucoidness, such populations were generally more susceptible towards ciprofloxacin than those of E. coli. At fast rates of growth P. aeruginosa cells resuspended from biofilms were significantly more resistant than chemostat grown cells. Intact P. aeruginosa biofilms were significantly more resistant than cells resuspended from them. This is in contrast to E. coli, where cells resuspended from biofilm and intact biofilms were, at the slower rates of growth, equivalent and significantly more susceptible than chemostat-grown cells. At high growth rates all methods of E. coli culture produced cells of equivalent susceptibility. For all strains, daughter cells dislodged from the biofilms demonstrated a high level of susceptibility towards ciprofloxacin which was unaffected by growth rate. This sensitivity corresponded to that of the fastest grown cells in the chemostat.     

Inhibition of Nucleic Acid Biosynthesis Makes Little Difference to Formation of Amphotericin B-Tolerant Persisters in Candida albicans Biofilm

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 μg ml⁻¹; biofilm cells, 1 μg ml⁻¹) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 μg ml⁻¹ for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.     

Susceptibility of bacterial biofilms to tobramycin: role of specific growth rate and phase in the division cycle

A novel method of cell culture, enabling growth rate control of sessile Gram-negative populations, has been employed to assess the sensitivity of Escherichia coli towards the aminoglycoside antibiotic, tobramycin. Changes in sensitivity, dependent on the growth rate, were compared with those for suspended populations grown in a chemostat and also those for newly-formed daughter cells shed from the biofilm during its growth and development. At specific growth rates up to 0.3 h-1 the susceptibility both of the resuspended biofilm cells and of their planktonic, chemostat grown controls increased in proportion to the growth rate. As the growth rate was increased further (up to 0.7h-1), the susceptibility of the resuspended biofilm cells remained high, whilst that of the planktonic controls decreased. Newly-formed daughter cells, dislodged from the biofilm, demonstrated a uniformly high sensitivity to the antibiotic at all growth rates. This sensitivity corresponded to that of the fastest-growing cells resuspended from biofilms. Lack of growth rate dependency of killing for the newly-formed daughter cells and their high sensitivity to tobramycin suggested that tobramycin activity might vary during the cellular division cycle. Indeed, when synchronous populations were exposed to tobramycin at various times during their division cycle, sensitivity decreased markedly 20 min before the onset of septation, and increased as septation began. Regulation of the cellular division cycle might therefore account, at least partly, for the observed effects of growth rate on susceptibility.     

Activities of Fluconazole,Caspofungin, Anidulafungin,and Amphotericin B on Planktonic and Biofilm Candida Species Determined by Microcalorimetry

We investigated the activities of fluconazole, caspofungin, anidulafungin, and amphotericin B against Candida species in planktonic form and biofilms using a highly sensitive assay measuring growth-related heat production (microcalorimetry). C. albicans, C. glabrata, C. krusei, and C. parapsilosis were tested, and MICs were determined by the broth microdilution method. The antifungal activities were determined by isothermal microcalorimetry at 37°C in RPMI 1640. For planktonic Candida, heat flow was measured in the presence of antifungal dilutions for 24 h. Candida biofilm was formed on porous glass beads for 24 h and exposed to serial dilutions of antifungals for 24 h, and heat flow was measured for 48 h. The minimum heat inhibitory concentration (MHIC) was defined as the lowest antifungal concentration reducing the heat flow peak by ≥50% (≥90% for amphotericin B) at 24 h for planktonic Candida and at 48 h for Candida biofilms (measured also at 24 h). Fluconazole (planktonic MHICs, 0.25 to >512 μg/ml) and amphotericin B (planktonic MHICs, 0.25 to 1 μg/ml) showed higher MHICs than anidulafungin (planktonic MHICs, 0.015 to 0.5 μg/ml) and caspofungin (planktonic MHICs, 0.125 to 0.5 μg/ml). Against Candida species in biofilms, fluconazole''s activity was reduced by >1,000-fold compared to its activity against the planktonic counterparts, whereas echinocandins and amphotericin B mainly preserved their activities. Fluconazole induced growth of planktonic C. krusei at sub-MICs. At high concentrations of caspofungin (>4 μg/ml), paradoxical growth of planktonic C. albicans and C. glabrata was observed. Microcalorimetry enabled real-time evaluation of antifungal activities against planktonic and biofilm Candida organisms. It can be used in the future to evaluate new antifungals and antifungal combinations and to study resistant strains.     

Effect of growth-rate on resistance of gram-negative biofilms to cetrimide

A method of cell culture, which allows control of growth rate for sessile Gram-negative populations, has been employed to assess the sensitivity of Escherichia coli biofilms to the antiseptic compound, cetrimide. Growth-rate-dependent changes in sensitivity were compared for chemostat-grown, planktonic cells, for cells resuspended from the biofilm and also for newly formed daughter cells shed from the biofilm during its growth and development. Susceptibility to cetrimide decreased in all instances with increases in growth-rate up to mu = 0.15 h-1. As growth rate was increased beyond this value then sensitivity increased in proportion to the rate of division. At rates of growth less than mu = 0.15 h-1 the susceptibility of the biofilm-derived cells and their offspring was significantly less than that of cells of planktonic origin.     

Effects of lactoferricin B against keratitis-associated fungal biofilms

Biofilms are considered as the most important developmental characteristics in ocular infections. Biofilm eradication is a major challenge today to overcome the incidence of drug resistance. This report demonstrates the in vitro ability of biofilm formation on contact lens by three common keratitis-associated fungal pathogens, namely, Aspergillus fumigatus, Fusarium solani, and Candida albicans. Antifungal sensitivity testing performed for both planktonic cells and biofilm revealed the sessile phenotype to be resistant at MIC levels for the planktonic cells and also at higher concentrations. A prototype lens care solution was also found to be partially effective in eradication of the mature biofilm from contact lenses. Lactoferricin B (Lacf, 64 μg/ml), an antimicrobial peptide, exhibited almost no effect on the sessile phenotype. However, the combinatory effect of Lacf with antifungals against planktonic cells and biofilms of three fungal strains that were isolated from keratitis patients exhibited a reduction of antifungal dose more than eightfold. Furthermore, the effect of Lacf in lens care solution against biofilms in which those strains formed was eradicated successfully. These results suggest that lactoferricin B could be a promising candidate for clinical use in improving biofilm susceptibility to antifungals and also as an antibiofilm-antifungal additive in lens care solution.     

Susceptibility of Cryptococcus neoformans biofilms to antifungal agents in vitro

Microbial biofilms contribute to virulence and resistance to antibiotics by shielding microbial cells from host defenses and antimicrobial drugs, respectively. Cryptococcus neoformans was demonstrated to form biofilms in polystyrene microtiter plates. The numbers of CFU of disaggregated biofilms, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide reduction, and light and confocal microscopy were used to measure the fungal mass, the metabolic activity, and the appearance of C. neoformans biofilms, respectively. Biofilm development by C. neoformans followed a standard sequence of events: fungal surface attachment, microcolony formation, and matrix production. The susceptibilities of C. neoformans cells of the biofilm and planktonic phenotypes to four antifungal agents were examined. The exposure of C. neoformans cells or preformed cryptococcal biofilms to fluconazole or voriconazole did not result in yeast growth inhibition and did not affect the metabolic activities of the biofilms, respectively. In contrast, both C. neoformans cells and preformed biofilms were susceptible to amphotericin B and caspofungin. However, C. neoformans biofilms were significantly more resistant to amphotericin B and caspofungin than planktonic cells, and their susceptibilities to these drugs were further reduced if cryptococcal cells contained melanin. A spot enzyme-linked immunosorbent assay and light and confocal microscopy were used to investigate how antifungal drugs affected C. neoformans biofilm formation. The mechanism by which amphotericin B and caspofungin interfered with C. neoformans biofilm formation involved capsular polysaccharide release and adherence. Our results suggest that biofilm formation may diminish the efficacies of some antifungal drugs during cryptococcal infection.     

Role for cell density in antifungal drug resistance in Candida albicans biofilms

Biofilms of Candida albicans are less susceptible to many antifungal drugs than are planktonic yeast cells. We investigated the contribution of cell density to biofilm phenotypic resistance. Planktonic yeast cells in RPMI 1640 were susceptible to azole-class drugs, amphotericin B, and caspofungin at 1 x 10(3) cells/ml (standard conditions) using the XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide sodium salt] assay. As reported by others, as the cell concentration increased to 1 x 10(8) cells/ml, resistance was observed with 10- to 20-fold-greater MICs. Biofilms that formed in microtiter plate wells, like high-density planktonic organisms, were resistant to drugs. When biofilms were resuspended before testing, phenotypic resistance remained, but organisms, when diluted to 1 x 10(3) cells/ml, were susceptible. Drug-containing medium recovered from high-cell-density tests inhibited low-cell-density organisms. A fluconazole-resistant strain showed greater resistance at high planktonic cell density, in biofilm, and in resuspended biofilm than did low-density planktonic or biofilm organisms. A strain lacking drug efflux pumps CDR1, CDR2, and MDR1, while susceptible at a low azole concentration, was resistant at high cell density and in biofilm. A strain lacking CHK1 that fails to respond to the quorum-sensing molecule farnesol had the same response as did the wild type. FK506, reported to abrogate tolerance to azole drugs at low cell density, had no effect on tolerance at high cell density and in biofilm. These observations suggested that cell density has a role in the phenotypic resistance of biofilm, that neither the drug efflux pumps tested nor quorum sensing through Chk1p contributes to resistance, and that azole drug tolerance at high cell density differs mechanistically from tolerance at low cell density.     

Cell density and cell aging as factors modulating antifungal resistance of Candida albicans biofilms

Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 10⁸ cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.     

Quinacrine Inhibits Candida albicans Growth and Filamentation at Neutral pH

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 μg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.     

Investigation of multidrug efflux pumps in relation to fluconazole resistance in Candida albicans biofilms

A main characteristic associated with microbial biofilms is their increased resistance to antimicrobial chemotherapies. However, at present very little is known about the phenotypic changes that occur during the transition from the planktonic to the biofilm mode of growth. Candida albicans biofilms displayed an organized three-dimensional structure, and consisted of a dense network of yeasts and filamentous cells deeply embedded in exopolymeric matrix. These biofilms were intrinsically resistant to fluconazole. Moreover, the resistance phenotype was maintained by sessile cells when resuspended as free-floating cells, thus demonstrating that biofilm integrity and the presence of exopolymeric material are not the sole determinants of biofilm resistance. Under planktonic conditions, one of the main mechanisms of azole resistance in C. albicans is through active efflux of these drugs mediated by ATP-binding cassette (ABC) transporters and major facilitators. In this study we used northern hybridization to monitor expression of genes belonging to two different types of efflux pump, the ABC transporters and major facilitators (encoded by CDR and MDR genes, respectively), in C. albicans populations under both planktonic and biofilm growth. It was demonstrated that expression of genes encoding both types of efflux pump were up-regulated during the course of biofilm formation and development. Moreover, antifungal susceptibilities of biofilms formed by a set of C. albicans mutant strains deficient in efflux pumps were investigated to determine their contribution to biofilm resistance. Remarkably, mutants carrying single and double deletion mutations in Delta(cdr)1, Delta(cdr)2, Delta(mdr)1, Delta(cdr)1/Delta(cdr)2 and Delta(mdr)1/Delta(cdr)1 were hypersusceptible to fluconazole when planktonic, but still retained the resistant phenotype during biofilm growth. These analyses demonstrate that C. albicans biofilm resistance is a complex phenomenon that cannot be explained by one mechanism alone, instead it is multifactorial and may involve different molecular mechanisms of resistance compared with those displayed by planktonic cells.     

Candida albicans biofilms produce antifungal-tolerant persister cells

Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.     

Interaction of biofilm bacteria with antibiotics in a novel in vitro chemostat system.

Pseudomonas aeruginosa was cultivated at low growth rates under iron-limiting conditions on acrylic tiles. Biofilm cells exhibited increased tobramycin resistance compared with that of planktonic cells, and in old biofilms were more resistant than were cells in young biofilms. However, on suspension of the biofilm bacteria, glycocalyx-mediated resistance was lost.     

Derivatives of the Mouse Cathelicidin-Related Antimicrobial Peptide (CRAMP) Inhibit Fungal and Bacterial Biofilm Formation

We identified a 26-amino-acid truncated form of the 34-amino-acid cathelicidin-related antimicrobial peptide (CRAMP) in the islets of Langerhans of the murine pancreas. This peptide, P318, shares 67% identity with the LL-37 human antimicrobial peptide. As LL-37 displays antimicrobial and antibiofilm activity, we tested antifungal and antibiofilm activity of P318 against the fungal pathogen Candida albicans. P318 shows biofilm-specific activity as it inhibits C. albicans biofilm formation at 0.15 μM without affecting planktonic survival at that concentration. Next, we tested the C. albicans biofilm-inhibitory activity of a series of truncated and alanine-substituted derivatives of P318. Based on the biofilm-inhibitory activity of these derivatives and the length of the peptides, we decided to synthesize the shortened alanine-substituted peptide at position 10 (AS10; KLKKIAQKIKNFFQKLVP). AS10 inhibited C. albicans biofilm formation at 0.22 μM and acted synergistically with amphotericin B and caspofungin against mature biofilms. AS10 also inhibited biofilm formation of different bacteria as well as of fungi and bacteria in a mixed biofilm. In addition, AS10 does not affect the viability or functionality of different cell types involved in osseointegration of an implant, pointing to the potential of AS10 for further development as a lead peptide to coat implants.     

Fungal β-1,3-Glucan Increases Ofloxacin Tolerance of Escherichia coli in a Polymicrobial E. coli/Candida albicans Biofilm

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.     

Pseudomonas aeruginosa PAO1 Preferentially Grows as Aggregates in Liquid Batch Cultures and Disperses upon Starvation

In both natural and artificial environments, bacteria predominantly grow in biofilms, and bacteria often disperse from biofilms as freely suspended single-cells. In the present study, the formation and dispersal of planktonic cellular aggregates, or ‘suspended biofilms’, by Pseudomonas aeruginosa in liquid batch cultures were closely examined, and compared to biofilm formation on a matrix of polyester (PE) fibers as solid surface in batch cultures. Plankton samples were analyzed by laser-diffraction particle-size scanning (LDA) and microscopy of aggregates. Interestingly, LDA indicated that up to 90% of the total planktonic biomass consisted of cellular aggregates in the size range of 10–400 µm in diameter during the growth phase, as opposed to individual cells. In cultures with PE surfaces, P. aeruginosa preferred to grow in biofilms, as opposed to planktonicly. However, upon carbon, nitrogen or oxygen limitation, the planktonic aggregates and PE-attached biofilms dispersed into single cells, resulting in an increase in optical density (OD) independent of cellular growth. During growth, planktonic aggregates and PE-attached biofilms contained densely packed viable cells and extracellular DNA (eDNA), and starvation resulted in a loss of viable cells, and an increase in dead cells and eDNA. Furthermore, a release of metabolites and infective bacteriophage into the culture supernatant, and a marked decrease in intracellular concentration of the second messenger cyclic di-GMP, was observed in dispersing cultures. Thus, what traditionally has been described as planktonic, individual cell cultures of P. aeruginosa, are in fact suspended biofilms, and such aggregates have behaviors and responses (e.g. dispersal) similar to surface associated biofilms. In addition, we suggest that this planktonic biofilm model system can provide the basis for a detailed analysis of the synchronized biofilm life cycle of P. aeruginosa.     

Delicate Metabolic Control and Coordinated Stress Response Critically Determine Antifungal Tolerance of Candida albicans Biofilm Persisters

Candida infection has emerged as a critical health care burden worldwide, owing to the formation of robust biofilms against common antifungals. Recent evidence shows that multidrug-tolerant persisters critically account for biofilm recalcitrance, but their underlying biological mechanisms are poorly understood. Here, we first investigated the phenotypic characteristics of Candida biofilm persisters under consecutive harsh treatments of amphotericin B. The prolonged treatments effectively killed the majority of the cells of biofilms derived from representative strains of Candida albicans, Candida glabrata, and Candida tropicalis but failed to eradicate a small fraction of persisters. Next, we explored the tolerance mechanisms of the persisters through an investigation of the proteomic profiles of C. albicans biofilm persister fractions by liquid chromatography-tandem mass spectrometry. The C. albicans biofilm persisters displayed a specific proteomic signature, with an array of 205 differentially expressed proteins. The crucial enzymes involved in glycolysis, the tricarboxylic acid cycle, and protein synthesis were markedly downregulated, indicating that major metabolic activities are subdued in the persisters. It is noteworthy that certain metabolic pathways, such as the glyoxylate cycle, were able to be activated with significantly increased levels of isocitrate lyase and malate synthase. Moreover, a number of important proteins responsible for Candida growth, virulence, and the stress response were greatly upregulated. Interestingly, the persisters were tolerant to oxidative stress, despite highly induced intracellular superoxide. The current findings suggest that delicate metabolic control and a coordinated stress response may play a crucial role in mediating the survival and antifungal tolerance of Candida biofilm persisters.     

In Vitro Analysis of Finasteride Activity against Candida albicans Urinary Biofilm Formation and Filamentation

Candida albicans is the 3rd most common cause of catheter-associated urinary tract infections, with a strong propensity to form drug-resistant catheter-related biofilms. Due to the limited efficacy of available antifungals against biofilms, drug repurposing has been investigated in order to identify novel agents with activities against fungal biofilms. Finasteride is a 5-α-reductase inhibitor commonly used for the treatment of benign prostatic hyperplasia, with activity against human type II and III isoenzymes. We analyzed the Candida Genome Database and identified a C. albicans homolog of type III 5-α-reductase, Dfg10p, which shares 27% sequence identity and 41% similarity to the human type III 5-α-reductase. Thus, we investigated finasteride for activity against C. albicans urinary biofilms, alone and in combination with amphotericin B or fluconazole. Finasteride alone was highly effective in the prevention of C. albicans biofilm formation at doses of ≥16 mg/liter and the treatment of preformed biofilms at doses of ≥128 mg/liter. In biofilm checkerboard analyses, finasteride exhibited synergistic activity in the prevention of biofilm formation in a combination of 4 mg/liter finasteride with 2 mg/liter fluconazole. Finasteride inhibited filamentation, thus suggesting a potential mechanism of action. These results indicate that finasteride alone is highly active in the prevention of C. albicans urinary biofilms in vitro and has synergistic activity in combination with fluconazole. Further investigation of the clinical utility of finasteride in the prevention of urinary candidiasis is warranted.