Disclosing azole resistance mechanisms in resistant Candida glabrata strains encoding wild-type or gain-of-function CgPDR1 alleles through comparative genomics and transcriptomics.

The pathogenic yeast Candida glabrata is intrinsically resilient to azoles and rapidly acquires resistance to these antifungals, in vitro and in vivo. In most cases azole-resistant C. glabrata clinical strains encode hyperactive CgPdr1 variants, however, resistant strains encoding wild-type CgPDR1 alleles have also been isolated, although remaining to be disclosed the underlying resistance mechanism. In this study, we scrutinized the mechanisms underlying resistance to azoles of 8 resistant clinical C. glabrata strains, identified along the course of epidemiological surveys undertaken in Portugal. Seven of the strains were found to encode CgPdr1 gain-of-function variants (I392M, E555K, G558C, and I803T) with the substitutions I392M and I803T being herein characterized as hyper-activating mutations for the first time. While cells expressing the wild-type CgPDR1 allele required the mediator subunit Gal11A to enhance tolerance to fluconazole, this was dispensable for cells expressing the I803T variant indicating that the CgPdr1 interactome is shaped by different gain-of-function substitutions. Genomic and transcriptomic profiling of the sole azole-resistant C. glabrata isolate encoding a wild-type CgPDR1 allele (ISTB218) revealed that under fluconazole stress this strain over-expresses various genes described to provide protection against this antifungal, while also showing reduced expression of genes described to increase sensitivity to these drugs. The overall role in driving the azole-resistance phenotype of the ISTB218 C. glabrata isolate played by these changes in the transcriptome and genome of the ISTB218 isolate are discussed shedding light into mechanisms of resistance that go beyond the CgPdr1-signalling pathway and that may alone, or in combination, pave the way for the acquisition of resistance to azoles in vivo.

Mechanistic Insights Underlying Tolerance to Acetic Acid Stress in Vaginal Candida glabrata Clinical Isolates.

During colonization of the vaginal tract Candida glabrata cells are challenged with the presence of acetic acid at a low pH, specially when dysbiosis occurs. To avoid exclusion from this niche C. glabrata cells are expected to evolve efficient adaptive responses to cope with this stress; however, these responses remain largely uncharacterized, especially in vaginal strains. In this work a cohort of 18 vaginal strains and 2 laboratory strains (CBS138 and KUE100) were phenotyped for their tolerance against inhibitory concentrations of acetic acid at pH 4. Despite some heterogeneity has been observed among the vaginal strains tested, in general these strains were considerably more tolerant to acetic acid than the laboratory strains. To tackle the mechanistic insights behind this differential level of tolerance observed, a set of vaginal strains differently tolerant to acetic acid (VG281∼VG49 < VG99 < VG216) and the highly susceptible laboratory strain KUE100 were selected for further studies. When suddenly challenged with acetic acid the more tolerant vaginal strains exhibited a higher activity of the plasma membrane proton pump CgPma1 and a reduced internal accumulation of the acid, these being two essential features to maximize tolerance. Based on the higher level of resistance exhibited by the vaginal strains against the action of a ß-1,3-glucanase, it is hypothesized that the reduced internal accumulation of acetic acid inside these strains may originate from them having a different cell wall structure resulting in a reduced porosity to undissociated acetic acid molecules. Both the vaginal and the two laboratory strains were found to consume acetic acid in the presence of glucose indicating that metabolization of the acid is used by C. glabrata species as a detoxification mechanism. The results gathered in this study advance the current knowledge on the mechanisms underlying the increased competitiveness of C. glabrata in the vaginal tract, a knowledge that can be used to guide more suitable strategies to treat infections caused by this pathogenic yeast.

Permeation of intrinsic water into ethanol- and water-saturated, monomer-infiltrated dentin bond interfaces.

OBJECTIVES: The aim of this study was to evaluate the formation of dentin bonding interfaces using the water-wet and the ethanol-wet techniques under simulated pulpal pressure, and to assess the effect of adhesive solvent and thermomechanical loading. METHODS: Flat dentin surfaces were restored under 20mm-simulated pulpal pressure following two bonding approaches (water-wet and ethanol-wet bonding) in combination with dental adhesives containing ethanol (Single Bond Plus and Scotchbond Multi-Purpose) or acetone (One-Step Plus and All-Bond 2) as solvent. Half of the restorations of each subgroup were subjected to thermocycling followed by cyclic loading (three teeth per group). Bond strength was measured using the microtensile bond strength test and fitted to a Weibull distribution (α=0.05). Ultrastructural analyses of the interface and leakage/nanoleakage evaluation were performed using confocal scanning microscopy (CLSM) and transmission electron microscopy (TEM). RESULTS: Water permeation through dentin tubules during adhesive application prevented adequate penetration of adhesive monomers into the demineralized collagen matrix in both bonding techniques, but more severely for water-wet bonding. Acetone-solvated adhesives showed worse bonding performance and hybridization than ethanol-based systems when applied in the ethanol-wet mode, both before and after thermomechanical challenge. SIGNIFICANCE: The ethanol-wet bonding technique helps to compensate for water permeation from dentin tubules during the bonding procedures to form more stable dentin bonds, especially when used in conjunction to ethanol-solvated systems.

In vitro bonding performance of self-etch adhesives: II--ultramorphological evaluation.

OBJECTIVE: To study the interfacial ultra-morphology formed by "all-in-one" self-etch adhesives. METHODS: Forty-nine extracted molars were assigned to one of five all-in-one adhesives: Adper Prompt L-Pop (AP, 3M ESPE); Clearfil S3 Bond (S3, Kuraray); G-Bond (GB, GC America); iBond (iB, Heraeus Kulzer) and Xeno IV (XE, Dentsply Caulk). Adper Single Bond Plus (SB, 3M ESPE), a two-step etch&rinse adhesive, and Clearfil SE Bond (SE, Kuraray), a two-step self-etch adhesive, were used as controls. Dentin, unground enamel and ground enamel were used as bonding substrates. Dentin specimens were processed for FESEM and TEM analyses. Enamel specimens were processed for FESEM. RESULTS: Dentin: GB, iB, S3, SE and XE resulted in a submicron-thick hybrid layer (0.2-0.7 microm), but only S3 and SE did not result in interfacial gaps. AP resulted in the thickest hybrid layer (1.7-2.9 microm) among the self-etch adhesives. SB resulted in a 3.4-5.2 pm thick hybrid layer. Unground enamel-GB, iB and SE resulted in a mostly featureless morphology resembling that of untreated enamel with areas in which the superficial enamel layer was removed without dissolving the subsurface enamel. XE resulted in areas of intra-prismatic etching and areas without any etching pattern. S3 resulted in frequent shallow intra-prismatic etching, while AP was able to unveil the enamel crystallites across the entire enamel surface. Phosphoric acid in SB resulted in the deepest intra- and inter-prismatic demineralization. Ground enamel--AP resulted in a well-defined inter-prismatic etching pattern. iB, GB, SE and S3 resulted in islands of superficially dissolved enamel within areas without evidence of enamel dissolution. XE resulted in etched enamel areas with mild intraprismatic exposure of crystallites. Phosphoric acid in SB resulted in deep enamel etching. CONCLUSIONS: Only AP, an aggressive self-etch adhesive, showed enamel morphological features that resemble those created by etch & rinse adhesives. S3 and SE were the only self-etch adhesives that did not result in dentin interfacial debonding.

Interfacial adaptation of adhesive materials to root canal dentin.

Extracted single-rooted maxillary teeth were endodontically treated and filled with gutta-percha/AH-26 (GP), Resilon points/RealSeal (RS), AdheSE DC/Multicore Flow (ADH, self-etch control), or Excite DSC/Multicore Flow (EXC, total-etch control). Specimens were analyzed with electron microscopy using three methods: (a) field emission scanning electron microscopy (FESEM) of the interface; (b) transmission electron microscopy (TEM) of the interface; and (c) FESEM of the material fitting surface. The three adhesive materials (RS, ADH, and EXC) formed a dentin hybrid layer, which nonetheless resulted in interfacial separation. Gaps were more frequent for GP, which did not hybridize dentin. The fitting surfaces exhibited resin tags at all levels for EXC. Tags were less frequent with ADH, especially in the apical third. For RS, resin tags were rare and virtually absent from the apical half, whereas GP did not form tags. Despite the hybridization, a tight seal of the root canal is difficult to achieve because of the complexity and the mechanical challenge of the substrate.

Advances in dentin adhesion.

While bonding to enamel has been a dependable technique, bonding to dentin is still an overwhelming task--dentin is a naturally soaked organic tissue. The enamel bonding agents of the 1960s and 1970s progressively evolved into complex multibottle or universal adhesives in the early 1990s. These multibottle systems were formulated to bond to enamel, dentin, composite, amalgam, porcelain, and nonprecious metal. More recently, simplified dentin adhesives were introduced following one of two adhesion strategies: total-etch adhesives that include an acid gel to remove the smear layer and dissolve hydroxyapatite (generally 30% to 40% phosphoric acid for 15 to 30 seconds); and self-etching primers that treat the dentin and enamel with a non-rinsing solution of acidic monomers in water without removing the smear layer. Recent attempts to simplify the bonding procedure have led to the introduction of adhesives that do not use a separate acid-etch step and, therefore, do not etch enamel to the same depth as phosphoric acid-based dental adhesives.