Sessile Legionella pneumophila is able to grow on surfaces and generate structured monospecies biofilms

Currently, models for studying Legionella pneumophila biofilm formation rely on multi-species biofilms with low reproducibility or on growth in rich medium, where planktonic growth is unavoidable. The present study describes a new medium adapted to the growth of L. pneumophila monospecies biofilms in vitro. A microplate model was used to test several media. After incubation for 6 days in a specific biofilm broth not supporting planktonic growth, biofilms consisted of 5.36 ± 0.40 log (cfu cm^?2) or 5.34 ± 0.33 log (gu cm^?2). The adhered population remained stable for up to 3 weeks after initial inoculation. In situ confocal microscope observations revealed a typical biofilm structure, comprising cell clusters ranging up to ～300 μm in height. This model is adapted to growing monospecies L. pneumophila biofilms that are structurally different from biofilms formed in a rich medium. High reproducibility and the absence of other microbial species make this model useful for studying genes involved in biofilm formation.     

Continuous power generation and microbial community structure of the anode biofilms in a three-stage microbial fuel cell system

A mediator-less three-stage two-chamber microbial fuel cell (MFC) system was developed and operated continuously for more than 1.5 years to evaluate continuous power generation while treating artificial wastewater containing glucose (10 mM) concurrently. A stable power density of 28 W/m³ was attained with an anode hydraulic retention time of 4.5 h and phosphate buffer as the cathode electrolyte. An overall dissolved organic carbon removal ratio was about 85%, and coulombic efficiency was about 46% in this MFC system. We also analyzed the microbial community structure of anode biofilms in each MFC. Since the environment in each MFC was different due to passing on the products to the next MFC in series, the microbial community structure was different accordingly. The anode biofilm in the first MFC consisted mainly of bacteria belonging to the Gammaproteobacteria, identified as Aeromonas sp., while the Firmicutes dominated the anode biofilms in the second and third MFCs that were mainly fed with acetate. Cyclic voltammetric results supported the presence of a redox compound(s) associated with the anode biofilm matrix, rather than mobile (dissolved) forms, which could be responsible for the electron transfer to the anode. Scanning electron microscopy revealed that the anode biofilms were comprised of morphologically different cells that were firmly attached on the anode surface and interconnected each other with anchor-like filamentous appendages, which might support the results of cyclic voltammetry. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Distribution Coefficients of Dietary Sugars in Artificial Candida Biofilms

Candida species are the most important fungal pathogens in humans and cause a variety of superficial and systemic diseases. Biofilm formation is a major virulence attribute contributing to Candida pathogenicity. Although the concentration and distribution of nutrients as well as antifungals across the biofilm thickness play a pivotal role in the development and persistence of Candida biofilms, only limited information is available on the latter aspects of Candida biofilms. Therefore, we attempted to characterize the diffusion coefficient (De) of common dietary sugars such as glucose, galactose, and sucrose in Candida albicans biofilms using horizontal attenuated total reflection-Fourier transform infrared spectroscopy (HATR-FTIR). Artificial Candida biofilms were formed using agarose polymers. De of three sugars tested, glucose, galactose, and sucrose in this artificial Candida biofilm model was found to be 4.08E-06 ± 3.63E-08, 4.08E-06 ± 3.70E-08, and 5.38E-06 ± 4.52E-08 cm² s⁻¹, respectively. We demonstrate here the utility of HATR-FTIR for the determination of diffusion of solutes such as dietary sugars across Candida biofilms.     

Enhanced electrode-reducing rate during the enrichment process in an air-cathode microbial fuel cell

The improvement in electricity generation during the enrichment process of a microbial consortium was analyzed using an air-cathode microbial fuel cell (MFC) repeatedly fed with acetate that was originally inoculated with sludge from an anaerobic digester. The anodic maximum current density produced by the anode biofilm increased from 0.12 mA/cm² at day 28 to 1.12 mA/cm² at day 105. However, the microbial cell density on the carbon cloth anode increased only three times throughout this same time period from 0.21 to 0.69 mg protein/cm², indicating that the biocatalytic activity of the consortium was also enhanced. The microbial activity was calculated to have a per biomass anode-reducing rate of 374 μmol electron g protein⁻¹ min⁻¹ at day 28 and 1,002 μmol electron g protein⁻¹ min⁻¹ at day 105. A bacterial community analysis of the anode biofilm revealed that the dominant phylotype, which was closely related to the known exoelectrogenic bacterium, Geobacter sulfurreducens, showed an increase in abundance from 32% to 70% of the total microbial cells. Fluorescent in situ hybridization observation also showed the increase of Geobacter-like phylotypes from 53% to 72%. These results suggest that the improvement of microbial current generation in microbial fuel cells is a function of both microbial cell growth on the electrode and changes in the bacterial community highly dominated by a known exoelectrogenic bacterium during the enrichment process.     

Archaea-based microbial fuel cell operating at high ionic strength conditions

In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC’s performance. Without any added redox mediator, maximum power (P _max) and current at P _max were 11.87/4.57/0.12 μW cm⁻² and 49.67/22.03/0.59 μA cm⁻² for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P _max was 50.98 and 5.39 μW cm⁻² for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.     

A Biobattery Capsule for Ingestible Electronics in the Small Intestine: Biopower Production from Intestinal Fluids Activated Germination of Exoelectrogenic Bacterial Endospores

Functioning ingestible capsules offer tremendous promise for a plethora of diagnostic and therapeutic applications. However, the absence of realistic and practical power solutions has greatly hindered the development of ingestible electronics. Microbial fuel cells (MFCs) hold great potential as power sources for such devices as the small intestinal environment maintains a steady internal temperature and a neutral pH. Those conditions and the constant supply of nutrient-rich organics are a perfect environment to generate long-lasting power. Although previous small-scale MFCs have demonstrated many promising applications, little is known about the potential for generating power in the human gut environment. Here, this work reports the design and operation of a microbial biobattery capsule for ingestible applications. Dormant Bacillus subtilis endospores are a storable anodic biocatalyst that will provide on-demand power when revived by nutrient-rich intestinal fluids. A conductive, porous, poly(3,4-ethylenedioxythiophene) polystyrene sulfonate hydrogel anode enables superior electrical performance in what is the world's smallest MFC. Moreover, an oxygen-rich cathode maintains its effective cathodic capability even in the oxygen-deficit intestinal environment. As a proof-of-concept demonstration in stimulated intestinal fluid, the biobattery capsule produces a current density of 470 µA cm⁻² and a power density of 98 µW cm⁻², ensuring its practical efficacy as a novel and sole power source for ingestible applications in the small intestine.     

Biofilm growth of individual and dual strains of <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> from the dairy industry on ultrafiltration membranes

Formation of biofilms in dairy membrane plants causes membrane pore blocking, product contamination and subsequent economic loss. To investigate the biofilm growth, two Klebsiella oxytoca strains, K. B006 and TR002, previously isolated from New Zealand dairy membrane plants, were grown both individually and combined on three types of ultrafiltration (UF) membranes in different concentrations of whey medium in biofilm reactors (CBR 90, BioSurface Technologies, Bozeman, USA). Biofilms of both the individual and combined strains grew on the membrane surfaces to levels of 4.9–7.99 log colony-forming units (CFU) cm⁻² measured by standard plate counting after removing the cells by sonication. More biofilm grew on used polyethersulfone (PES) membranes than on new PES and polyvinylidene fluoride (PVDF) membranes. Both strains formed good biofilms, although K. B006 formed a denser biofilm than TR002. This corresponded to our previous study on the attachment of these organisms, where K. B006 attached in greater numbers than K. TR002. The dual strains produced a higher biofilm density than single strains on the new membranes. Biofilm density tended to increase with increased whey concentration. The saturated biofilm was approximately 10⁸ CFU cm⁻². PES membranes appeared to support biofilm growth less readily than did PVDF membranes and therefore may be the preferred material for UF membranes to reduce problems with microbial colonisation. Used membranes were more readily colonised with biofilm than were new membranes. Therefore, selecting a membrane type and monitoring membrane age will help manage biofilm development during UF.     

Synthesis of benzoylthiourea derivatives and analysis of their antibacterial performance against planktonic Staphylococcus aureus and its biofilms

Following the appearance of several antimicrobial agents to control the spread of infections, two major challenges have emerged: (i) the occurrence and blowout of multiresistant bacteria and the increase of chronic diseases and (ii) difficult-to-eradicate infections. In this study, we tested five benzoylthiourea derivatives for their ability to inhibit and stop bacterial growth and evaluated the possible influence of 1,2,4-triazolyl-benzoylthiourea derivative 4 on the formation and eradication of Staphylococcus aureus biofilms. Benzoylthiourea derivatives 4 , 6 , 10 , 11 and 13 were obtained in one or two steps with low cost and subjected to tests to identify their minimum inhibitory concentration (MIC) and minimum bactericidal concentration. In vitro tests were also performed to assess their effects on biofilm formation and in preformed biofilms and scanning electron microscopy was used to visualize the effects on biofilm formation. The 1,2,4-triazolyl-benzoylthiourea derivative 4 showed bacteriostatic activity against the S. aureus HU25 clinical strain with an MIC of 16 µg ml⁻¹, which is below the toxic concentration (at 2500 µg ml⁻¹, 62·25% of the cells remained viable). Compound 4 also effectively prevented biofilm formation at the three subinhibitory concentrations tested (1/2 MIC, 1/4 MIC and 1/8 MIC) as confirmed by scanning electron microscopy. For breakdown of formed biofilms, the main influence was at a subinhibitory concentration (1/2 MIC). These findings make compound 4 a strong candidate for studies on the development of new antimicrobial and antibiofilm agents.     

Operational temperature regulates anodic biofilm growth and the development of electrogenic activity

The operational temperature of microbial fuel cell reactors influences biofilm development, and this has an impact on anodic biocatalytic activity. In this study, we compared three microbial fuel cell (MFC) reactors acclimated at 10°C, 20°C and 35°C to investigate the effect on biomass development, methanogenesis and electrogenic activity over time. The start-up time was inversely influenced by temperature, but the amount of biomass accumulation increased with increased temperatures, the 10°C, 20°C and 35°C acclimated biofilms resulted in 0.57, 0.82 and 5.43 g biomass (volatile suspended solids) per litre respectively at 56 weeks of operation. Biofilm build-up on the 35°C anode was further demonstrated by scanning electron microscopy, which showed large aggregations of biomass accumulating on the anode when compared to 10°C and 20°C biofilms. Biomass accumulation had a direct impact on biocatalytic performance, with the maximum power at 35°C after 60 weeks of operation being 2.14 W m⁻³ and power densities for the 10°C and 20°C reactors being and 4.29 W m⁻³. Methanogenic activity was also shown to be higher at 35°C, with a rate of 10.1 mmol CH₄ biofilm per gram of volatile suspended solid (VSS) per day, compared to 0.28 mmol CH₄ per gram of VSS per day produced at 20°C. These results demonstrate that higher MFC operating temperatures could be detrimental to the biocatalytic performance of electrochemically active bacteria in anodic biofilms due to biomass accumulation with enhanced development of non-electrogenic communities (e.g. methanogens and fermenters), meaning that, over time, psychro- or mesophilic operation can have beneficial effects for the development of electrogenically active populations in the reactor.     

Quantification of water and biomass in small colony variant PAO1 biofilms by confocal Raman microspectroscopy

The Raman spectra, water content, and biomass density of wild-type (WT) Pseudomonas aeruginosa PAO1, small colony variant (SCV) PAO1, and Pseudoalteromonas sp. NCIMB 2021 biofilms were compared in order to determine their variation with strain and species. Living, fully submerged biofilms were analyzed in situ by confocal Raman microspectroscopy for up to 2 weeks. Water to biomass ratios (W/BRs), which are the ratios of the O–H stretching vibration of water at 3,450 cm⁻¹ to the C–H stretching band characteristic of biomass at 2,950 cm⁻¹, were used to estimate the biomass density and cell density by comparison with W/BRs of protein solutions and bacterial suspensions, respectively, on calibration curves. The hydration within SCV biofilm colonies was extremely heterogeneous whereas W/BRs were generally constant in young WT biofilm colonies. The mean biomass in biofilm colonies of WT or colony cores of SCV was typically equivalent to 16% to 27% protein (w/v), but was 10% or less for NCIMB 2021. The corresponding cell densities were 7.5 to >10 × 10¹⁰ cfu mL⁻¹ for SCV, while the maximum cell density for NCIMB biofilms was 2.8 × 10¹⁰ cfu mL⁻¹.     

The impact of electron donors and anode potentials on the anode-respiring bacteria community

Both anode potentials and substrates can affect the process of biofilm formation in bioelectrochemical systems, but it is unclear who primarily determine the anode-respiring bacteria (ARB) community structure and composition. To address this issue, we divided microbial electrolysis cells (MECs) into groups, feeding them with different substrates and culturing them at various potentials. Non-turnover cyclic voltammetry indicated that the extracellular electron transfer components were uniform when feeding acetate, because the same oxidation peaks occurred at − 0.36 ± 0.01 and − 0.17 ± 0.01 V (vs. Ag/AgCl). Illumina MiSeq sequencing revealed that the dominating ARB was Geobacter, which did not change with different potentials. When the MECs were cultured with sucrose and mixed substrates, oxidation peak P3 (− 0.29 ± 0.015 V) occurred at potentials of − 0.29 and 0.01 V. This may be because of the appearance of Unclassified_AKYG597. In addition, oxidation peak P4 (− 0.99 ± 0.01 V) occurred at high and low potentials (0.61 and − 0.45 V, respectively), and the maximum current densities were far below those of the middle potentials. Illumina MiSeq sequencing showed that fermentation microorganisms (Lactococcus and Sphaerochaeta) dominated the biofilms. Consequently, substrate primarily determined the dominating ARB, and Geobacter invariably dominated the acetate-fed biofilms with potentials changed. Conversely, different potentials mainly affected fermentable substrate-fed biofilms, with dominating ARB turning into Unclassified_AKYG59.

     

Enhancement of hexavalent chromium reduction and electricity production from a biocathode microbial fuel cell

Enhancement of Cr (VI) reduction rate and power production from biocathode microbial fuel cells (MFCs) was achieved using indigenous bacteria from Cr (VI)-contaminated site as inoculum and MFC architecture with a relatively large cathode-specific surface area of 340–900 m² m⁻³. A specific Cr (VI) reduction rate of 2.4 ± 0.2 mg g⁻¹VSS h⁻¹ and a power production of 2.4 ± 0.1 W m⁻³ at a current density of 6.9 A m⁻³ were simultaneously achieved at an initial Cr (VI) concentration of 39.2 mg L⁻¹. Initial Cr (VI) concentration and solution conductivity affected Cr (VI) reduction rate, power production and coulombic efficiency. These findings demonstrate the importance of inoculation and MFC architecture in the enhancement of Cr (VI) reduction rate and power production. This study is a beneficial attempt to improve the efficiency of biocathode MFCs and provide a good candidate of bioremediation process for Cr (VI)-contaminated sites.     

Characterization of biofilm formation on a humic material

Biofilms are major sites of carbon cycling in streams. Therefore, it is crucial to improve knowledge about biofilms’ structure and microbial composition to understand their contribution in the self-purification of surface water. The present work intends to study biofilm formation in the presence of humic substances (HSs) as a carbon source. Two biofilm flowcells were operated in parallel; one with synthetic stream water, displaying a background carbon concentration of 1.26 ± 0.84 mg L⁻¹, the other with added HSs and an overall carbon concentration of 9.68 ± 1.00 mg L⁻¹. From the biofilms’ results of culturable and total countable cells, it can be concluded that the presence of HSs did not significantly enhance the biofilm cell density. However, the biofilm formed in the presence of HSs presented slightly higher values of volatile suspended solids (VSS) and protein. One possible explanation for this result is that HSs adsorbed to the polymeric matrix of the biofilm and were included in the quantification of VSS and protein. The microbial composition of the biofilm with addition of HSs was characterized by the presence of bacteria belonging to beta-Proteobacteria, Cupriavidus metallidurans and several species of the genus Ralstonia were identified, and gamma-Proteobacteria, represented by Escherichia coli. In the biofilm formed without HSs addition beta-Proteobacteria, represented by the species Variovorax paradoxus, and bacteria belonging to the group Bacteroidetes were detected. In conclusion, the presence of HSs did not significantly enhance biofilm cell density but influenced the bacterial diversity in the biofilm.     

In vitro and ex vivo biofilms of dermatophytes: a new panorama for the study of antifungal drugs

Abstract

This study describes an ex vivo model that creates an environment for dermatophyte biofilm growth, with features that resemble those of in vivo conditions, designing a new panorama for the study of antifungal susceptibility. Regarding planktonic susceptibility, MIC ranges were 0.125-1?µg ml^?1 for griseofulvin and 0.000097-0.25?µg ml^?1 for itraconazole and terbinafine. sMIC50 ranges were 2->512?µg ml^?1 for griseofulvin and 0.25->64?µg ml^?1 for itraconazole and terbinafine. CLSM images demonstrated a reduction in the amount of cells within the biofilm, but hyphae and conidia were still observed and biofilm biomass was maintained. SEM analysis demonstrated a retraction in the biofilm matrix, but fungal structures and water channels were preserved. These results show that ex vivo biofilms are more tolerant to antifungal drugs than in vitro biofilms, suggesting that environmental and nutritional conditions created by this ex vivo model favor biofilm growth and robustness, and hence drug tolerance.     

Biomarkers of Alzheimer′s Disease in the Cerebrospinal Fluid of Spanish Patients With Mild Cognitive Impairment

The study of biomarkers in the cerebrospinal fluid (CSF) of patients with mild cognitive impairment (MCI) is a technique used with increasing frequency in the early diagnosis of Alzheimers disease (AD). Our objectiv was to gain an own experience while evaluating the reliability, sensitivity, and reproducibility of this technique in Spanish patients. Thirty-seven patients with MCI and twenty-four control subjects were studied by means of AD biomarker analysis in CSF. xMAP Luminex and INNO-BIA Alzbio3 reagents of Innogenetics were used. The study variables assessed were levels of Aβ_1–42, T-tau and P-tau_181p proteins as well as the ratios of T-tau/Aβ_1–42 and P-tau_181p/Aβ_1–42. Samples from nineteen patients were examined twice. Intra-class correlation coefficients for the three biomarkers used showed values higher than 0.95. We observed significant differences between the control group and the MCI groups. In the 6 months following lumbar puncture (LP), eleven (29%) patients with MCI developed AD. These patients showed significant lower levels in Aβ_1–42 protein (276.35 ± 78 vs. 367.13 ± 123.49, P < 0.03) and higher ratios (T-tau/Aβ_1–42 [0.38 ± 0.2 vs. 0.22 ± 0.14, P < 0.01] and P-tau_181p/Aβ_1–42 [0.27 ± 0.13 vs. 0.16 ± 0.1, P < 0.008]) to those in the same group who remained stable. We obtained similar results to those in the most recent reliable literature with our ROC curves, especially with our P-tau_181p values and T-tau/Aβ_1–42 ratio in order to differentiate between control and AD groups. Our experience showed that the analysis of CSF-AD biomarkers in patients with MCI is reliable, sensitive and reproducible. In our knowledge, this is the first experience in Spanish patients.     

Fate of three bioluminescent pathogenic bacteria fed through a cascade of urine microbial fuel cells

Microbial fuel cell (MFC) technology is currently gaining recognition as one of the most promising bioenergy technologies of the future. One aspect of this technology that has received little attention is the disinfection of effluents and the fate of pathogenic organisms that find their way into the waste stream. In this study, three independent trials were carried out to evaluate the fate of three bioluminescent pathogenic bacteria (Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus) introduced into the anodic chamber of a urine-fed cascade of 9 MFCs with matured, electroactive biofilms. These are common examples of enteric human pathogens, which could contaminate urine or waste streams. The results showed that the average power generation in the closed circuit cascade reached 754 ± 16 µW, with an average pathogen log-fold reduction of 6.24 ± 0.63 compared to 2.01 ± 0.26 for the open circuit cascade for all three pathogens. The results suggest that the bio-electrochemical reactions associated with electricity generation were the primary driving force for the inactivation of the introduced pathogens. These findings show that pathogenic organisms introduced into waste streams could be inactivated by the power-generating process within the MFC cascade system, thereby preventing propagation and thus rendering the effluent safer for possible reuse.

     

Colonisation and succession of marine biofilm-dwelling ciliate assemblages on biocidal antifouling and fouling-release coatings in temperate Australia

Ciliate assemblages are often overlooked, but ubiquitous components of microbial biofilms which require a better understanding. Ciliate, diatom and bacterial colonisation were evaluated on two fouling-release (FR) coatings, viz. Intersleek 970 and Hempasil X3, and two biocidal antifouling (AF) coatings, viz. Intersmooth 360 and Interspeed 5640, in Port Phillip Bay, Australia. A total of 15 genera were identified during the 10 week deployment. Intersleek 970 displayed the most rapid fouling by ciliates, reaching 63.3(± 5.9) cells cm^?2. After 10 weeks, all four coatings were extensively fouled. However, the toxicity of the AF coatings still significantly inhibited microbial fouling compared to the FR coatings. On all treatments, colonies of sessile peritrichs dominated the ciliate assemblage in the early stage of succession, but as the biofilm matured, vagile ciliates exerted more influence on the assemblage structure. The AF coatings showed selective toxic effects, causing significant differences in the ciliate species assemblages among the treatments.     

Enhanced germicidal effects of pulsed UV‐LED irradiation on biofilms

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low‐frequency pulsed ultraviolet A (UVA)‐light‐emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA‐LED irradiation (365 nm, 0·28 mW cm^?2, in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony‐forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5‐min irradiation, over 90% of viable micro‐organisms in biofilms had been killed, and pulsed irradiation (1–1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro‐organisms in biofilm (>99·9%), and 20‐min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH^?), significantly protected viable micro‐organisms in biofilms from UVA‐LED irradiation. Conclusions: The study demonstrated that pulsed UVA‐LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5‐min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low‐frequency pulsed UVA‐LED system to be applied to pathogenic biofilms for disinfection.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.