Epidemiological investigation and typing of Candida glabrata clinical isolates by FTIR spectroscopy

Candida glabrata has emerged as one of the leading agents of fungal infections and strain typing is essential for epidemiological investigation that is generally achieved by molecular techniques. In this work, we studied twenty-nine C. glabrata strains isolated from different patients, using a phenotypic approach based on Fourier Transform Infrared (FTIR) spectroscopy, which has been in a previous study successfully applied as a rapid typing method for Candida albicans. A two-step procedure was used for the analysis. The first step included sixteen strains for the internal validation phase, which aimed at finding the spectral windows that would provide the best differentiation between strains. In this phase, hierarchical cluster analysis (HCA) carried out using three spectral windows (900-1200, 1540-1800, 2800-3000 cm(-1)) allowed to obtain the best classification, where each patient strains could be clustered together. A genotypic technique based on randomly amplified polymorphic DNA-analysis (RAPD) confirmed these results. In a second step, the external validation phase, thirteen other clinical strains of C. glabrata isolated from multiple sites in four ICU patients, were tested by FTIR spectroscopy. The analysis was based on the spectral regions previously found in the first step. HCA classification of the strains gave four groups, one group per patient. These results suggest that no inter-human transmission took place. This study shows the potential of FTIR approach for typing of C. glabrata with several advantages compared to other techniques. FTIR typing is fast, effective, and reagent free. Moreover, it is applicable to all micro-organisms and requires a small quantity of biomass.     

Eficacia terapéutica del voriconazol en candidiasis vulvovaginal crónica por Candida glabrata

BackgroundVulvovaginal candidosis is a common infection in young women, and it is associated with high morbidity and high health costs.AimsVulvovaginal candidosis caused by Candida glabrata is a therapeutic challenge due to the acquired resistance of many strains of this species to azole antifungals.MethodsWe present two cases of vaginal candidosis complicated by fluconazole-resistant Candida glabrata, and treated with voriconazole.ResultsBoth patients improved after administration of voriconazole, 400 mg/12 h the first day and then 200 mg every 12 h for 14 days. Their symptoms disappeared and cultures became negative.ConclusionsThese results suggest voriconazole can be used as a therapeutic alternative for this type of candidosis which, although not life threatening, is associated with a high morbidity.     

Fungicidal activity of the human peptide hepcidin 20 alone or in combination with other antifungals against Candida glabrata isolates

Candida glabrata infections are often difficult to eradicate due to the intrinsically low susceptibility to azoles of this species. In addition, C. glabrata has also been shown to be insensitive to several cationic peptides, which have been shown to be promising novel therapeutic candidates for the treatment of fungal infection. In this study, the in vitro fungicidal activity of the human cationic peptide hepcidin 20 (Hep-20) was evaluated against clinical isolates of C. glabrata with different levels of fluconazole susceptibility. Interestingly, all isolates were susceptible to Hep-20 (100–200 μg/ml) at pH 7.4, whereas the fungicidal effect of the peptide was higher (50 μg/ml) at acidic pH values. In addition, an increased antifungal activity was observed for Hep-20 with amphotericin B and a synergistic effect was demonstrated for the Hep-20/fluconazole and Hep-20/caspofungin combinations.     

Synthesis and antimicrobial activity of novel amphiphilic aromatic amino alcohols

We report in this work the preparation and in vitro antimicrobial evaluation of novel amphiphilic aromatic amino alcohols synthesized by reductive amination of 4-alkyloxybenzaldehyde with 2-amino-2-hydroxymethyl-propane-1,3-diol. The antibacterial activity was determined against four standard strains (Staphylococcus aureus, Staphylococcus epidermidis, Escherichia coli, Pseudomonas aeruginosa) and 21 clinical isolates of methicillin-resistant Staphylococcus aureus. The antifungal activity was evaluated against four yeast (Candida albicans, Candida tropicalis, Candida glabrata and Candida parapsilosis). The results obtained showed a strong positive correlation between the lipophilicity and the antibiotic activity of the tested compounds. The best activities were obtained against the Gram-positive bacteria (MIC = 2–16 μg ml^?1) for the five compounds bearing longer alkyl chains (4c–g; 8–14 carbons), which were also the most active against Candida (MIC = 2–64 μg ml^?1). Compound 4e exhibited the highest levels of inhibitory activity (MIC = 2–16 μg ml^?1) against clinical isolates of MRSA. A concentration of twice the MIC resulted in bactericidal activity of 4d against 19 of the 21 clinical isolates.     

Efecto inhibitorio in vitro de ajoeno sobre aislamientos de Candida recuperados de secreciones vaginales

AimsThe main purpose of this work was to evaluate the in vitro activity of ajoene of the Candida, obtained from vaginal discharges.MethodsFor this, 136 samples were analyzed. The yeasts were recovered and identified by conventional mycological methods. The susceptibility to ajoene (at 20, 15, 12.5, 10, 6.25 and 3.125 μg/ml) was performed according to the CLSI M27-A2 document with the EUCAST modifications. The ATCC reference strains 90028 (Candida albicans), 22019 (Candida parapsilosis), and 6258 (Candida krusei) were included in this study. The minimal inhibitory concentration (MIC) was considered as the minimal concentration of ajoena able to inhibit 80% of the fungal growth.ResultsFifty five yeasts were recovered, 36 (65.4%) of them were causing candidosis and 19 (34.5%) were colonizing. C. albicans was the most frequent (81.8%) of the six isolated species, prevailing on the patients with candidosis (54.5%). The non-albicans species were less frequently isolated (18.2%), and Candida glabrata was the prevailing agent (7.3%) followed by Candida tropicalis (3.6%), C. krusei, C. parapsilosis, Candida guilliermondii and Candida sp. (1.8% each of them). The susceptibility tests to ajoeno showed inhibition of fungal growth in 98.2% of the isolates, showing MIC values ?15 μg/ml, and in (one isolate of C. glabrata) (1.8%) this value was >20 μg/ml. The reference strains showed MIC values of 3.125 and 10 μg/ml.ConclusionsThe results here presented, obtained from a significant number of isolates, mainly C. albicans, demonstrate, once more, the potential of ajoeno as an antifungal agent.     

Antifungal activity of amniotic fluid against different Candida species

BackgroundAlthough Candida is a commensal of the urogenital tract, intrauterine fungal infections are extremely uncommon in clinical practice.AimsIn the present work we evaluated whether amniotic fluid (AF) possesses direct antifungal activity against clinical isolates of Candida albicans and other Candida species.MethodsA total of 23 AF samples from pregnant women with gestational age of 38–41 weeks were obtained under aseptic conditions by the aspiration of the amniotic sac during cesarean section. Different Candida species were inoculated in amniotic fluid and Sabouraud broth, used as control, and were incubated at 37 °C for 48 h. Quantitative cultures of test samples and controls were performed at 0, 4, 8, 12, 24, and 48 h.ResultsAF collected from 23 pregnant women had consistent and significant inhibitory activity against all Candida isolates tested. Nonetheless, a complete inhibition of growth by all 23 AF samples tested was observed only against Candida glabrata.ConclusionsIt is likely that the antifungal activity of the AF against C. albicans, C. glabrata and Candida parapsilosis observed in vitro also exists in vivo, contributing to protect against intrauterine fungal infections.     

Metabolic engineering of Torulopsis glabrata for improved pyruvate production

During pyruvate production, ethanol is produced as a by-product, which both decreases the amount of pyruvate and makes the recovery of pyruvate more difficult. Pyruvate decarboxylase (PDC, EC 4.1.1.1), which degrades pyruvate to acetaldehyde and ultimately to ethanol, is a key enzyme in the pyruvate metabolism of yeast. Therefore, to order to increase the yield of pyruvate in Torulopsis glabrata, targeted PDC-disrupted strains were metabolically engineered. First, T. glabrata ura3 strains that were suitable for genetic transformation were isolated and identified through ethyl methansulfonate mutagenesis, 5-fluoroortic acid media selection, and Sacchramyces cerevisiae URA3 complement. Next, the PDC gene in T. glabrata was specifically disrupted through homologous recombinant with the S. cerevisiae URA3 gene as the selective marker. The PDC activity of the disruptants was about 33% that of the parent strain. Targeted PDC gene disruption in T. glabrata was also confirmed by PCR amplification and sequencing of the PDC gene and its mutants, PDC activity staining, and PDC Western blot. The disruptants displayed higher pyruvate accumulation and less ethanol production. Under basal fermentation conditions (see Section 2), the disruptants accumulated about 20 g/L of pyruvate with 4.6 g/L of ethanol, whereas the parental strain (T. glabrata IFO005) only accumulated 7–8 g/L of pyruvate with 7.4 g/L of ethanol. Under favorable conditions in jar fermentation, the disruptants accumulated 82.2 g/L of pyruvate in 52 h.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Synthesis and carbonic anhydrase inhibitory properties of sulfamides structurally related to dopamine

A series of novel sulfamides incorporating the dopamine scaffold were synthesized. Reaction of amines and tert-butyl-alcohol/benzyl alcohol in the presence of chlorosulfonyl isocyanate (CSI) afforded sulfamoyl carbamates, which were converted to the title compounds by treatment with trifluoroacetic acid or by palladium-catalyzed hydrogenolysis. Inhibition of six α-carbonic anhydrases (CAs, EC 4.2.1.1), that is, CA I, CA II, CA VA, CA IX, CA XII and CA XIV, and two β-CAs from Candida glabrata (CgCA) and Mycobacterium tuberculosis (Rv3588) with these sulfamides was investigated. All CA isozymes were inhibited in the low micromolar to nanomolar range by the dopamine sulfamide analogues. K_is were in the range of 0.061–1.822 μM for CA I, 1.47–2.94 nM for CA II, 2.25–3.34 μM for CA VA, 0.041–0.37 μM for CA IX, 0.021–1.52 μM for CA XII, 0.007–0.219 μM for CA XIV, 0.35–5.31 μM for CgCA and 0.465–4.29 μM for Rv3588. The synthesized sulfamides may lead to inhibitors targeting medicinally relevant CA isoforms with potential applications as antiepileptic, antiobesity antitumor agents or anti-infective.     

Carbonic anhydrase inhibitors: Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with sulfonamides,sulfamates and sulfamides

The fungal pathogen Candida glabrata encodes for a β-carbonic anhydrase (CA, EC 4.2.1.1), CgNce103, recently discovered. Only anions have been investigated as CgNce103 inhibitors up until now. Here we report the first sulfonamides inhibition study of this enzyme. Simple sulfonamides showed weak or moderate CgNce103 inhibitory properties, whereas acetazolamide, and a series of 4-substituted ureido-benzene-sulfonamides, sulfamates and sulfamides showed effective CgNce103 inhibitory properties, with K_Is in the range of 4.1–115 nM, being also ineffective as human CA II inhibitors. As there is significant resistance of C. glabrata clinical isolates to many classical antifungal agents, inhibition of the β-CA from this organism may allow an interesting means of controlling the pathogen growth, eventually leading to antifungals with a novel mechanism of action.     

Primer caso de fungemia asociada a catéter por Candida nivariensis en la Península Ibérica

BackgroundIn recent years the incidence of candidemia caused by non-albicans Candida species has been increasing. Two cryptic species have been described within the Candida glabrata complex, Candida nivariensis and Candida bracarensis, which may be troublesome in laboratory identification and have lower susceptibility to fluconazole.AimsTo describe the first isolation of C. nivariensis in the Iberian Peninsula from a patient suffering from a catheter-related fungemia.Case reportAn 81-year-old man was hospitalized for surgical treatment of an intestinal fistula that was associated to a severe malnutrition. Cultures of the patient's central venous catheter tip and blood yielded white colonies in BD CHROMagar Candida^® medium, which could not be identified by conventional microbiological methods. Although intravenous fluconazole was administered, blood cultures continued being positive 5 days later. The MIC values of the isolate were as follows: 1 μg/ml for amphotericin B, 0.015 μg/ml for anidulafungin, 0.125 μg/ml for caspofungin, 0.015 μg/ml for micafungin, 4 μg/ml for fluconazole, 0.25 μg/ml for itraconazole, 0.25 μg/ml for posaconazole, and 0.03 μg/ml for voriconazole. Antifungal treatment was changed to intravenous caspofungin for 2 weeks. The intestinal fistula was surgically treated. There was no evidence of relapse during the following month, and the patient was discharged. The isolate was identified as C. nivariensis based on DNA sequencing of the ITS regions of rRNA.ConclusionsC. nivariensis should be regarded as an emerging pathogen which requires molecular methods for a definitive identification. Our patient was successfully treated with caspofungin.     

Isolation of Candida auris in large hospitals in the Autonomous Community of Valencia; population-based study (2013–2017)

BackgroundCandida auris is an emerging multidrug-resistant and highly virulent yeast that spreads easily among patients.AimsTo describe the characteristics of candidemia caused by C. auris in the southeast of Spain (Autonomous Community of Valencia – ACV) through a 5-year population-based study.MethodsAn analysis of all the episodes of candidemia diagnosed in the ACV, with approximately 4,500,000 inhabitants, during 2013–2017, was done. Data were obtained from the Epidemiological Surveillance Valencian Network, a network that collects all the microbiological data from the hospitals in the study region.ResultsBased on the records, 1.9% of the isolates recovered from the positive blood cultures (corresponding to 1789 patients) were yeasts. This implies an annual rate of 7.09 cases/100,000 inhabitants. Of the 23 yeast species isolated, Candida albicans was the most frequent (37.3%), showing a higher frequency than Candida parapsilosis (28.4%) and Candida glabrata (15.6%) (p < 0.0001). It is remarkable the emergence of C. auris during 2016 and 2017, as this species became the fourth more prevalent in 2016 (9.2%), and the third in 2017 (15.7%). Fungemia was more common in hospitals with >500 beds (63.3% versus 36.7% in small hospitals) (p < 0.0001), and C. auris was mostly isolated in large hospitals (8.5% versus 0.3%); its incidence was higher in autumn and among the age group of 65–84 years.ConclusionsThe information about the local epidemiology of candidemia is essential in order to decide the best empirical treatment approach. This study reports the novel presence of C. auris in large hospitals. This pathogen has usually resistance to several antifungals and causes severe fungemia, so the results of this work reveal the need to monitor the presence of this species systematically.     

Carbonic anhydrase inhibitors. Inhibition of the β-class enzyme from the pathogenic yeast Candida glabrata with anions

A β-carbonic anhydrase (CA, EC 4.2.1.1), the protein encoded by the NCE103 gene of Candida glabrata which also present in Candida albicans and Saccharomyces cerevisiae, was cloned, purified, characterized kinetically and investigated for its inhibition by a series simple, inorganic anions such as halogenides, pseudohalogenides, bicarbonate, carbonate, nitrate, nitrite, hydrogen sulfide, bisulfite, perchlorate, sulfate and some isosteric species. The enzyme showed significant CO₂ hydrase activity, with a k_cat of 3.8 × 10⁵ s^?1 and k_cat/K_M of 4.8 × 10⁷ M^?1 s^?1. The Cà glabrata CA (CgCA) was moderately inhibited by metal poisons (cyanide, azide, cyanate, thiocyanate, K_Is of 0.60–1.12 mM) but strongly inhibited by bicarbonate, nitrate, nitrite and phenylarsonic acid (K_Is of 86–98 μM). The other anions investigated showed inhibition constants in the low millimolar range, with the exception of bromide and iodide (K_Is of 27–42 mM).     

Etiología de la candidiasis en pediatría: análisis comparativo con el paciente adulto

BackgroundCandida spp. represents a group of commensal yeasts that can act as pathogens and cause candidiasis in different anatomical locations.AimsThe aim of this study was to perform an epidemiological and comparative analysis between the isolates of Candida spp. in clinical specimens during a three year-period (2010-2012) from children (0-14 years) and adults (15-99 years) in the Valencian Community (RedMIVA).MethodsThe microbiological surveillance network of Valencian Community was used as the information source.Results and conclusionsCandida was isolated in 52,436 patients (1,604 [3.1%] children and 50,832 [96.9%] adults). Candida albicans was significantly (p < 0.05) the predominant species in both age groups, and in almost every type of clinical specimen. The distribution of other species varied depending on the sample type and age group. In blood specimens, Candida parapsilosis followed by C. albicans, Candida famata and Candida lusitaniae were the main species found in children, whereas C. albicans followed by C. parapsilosis, Candida glabrata and Candida tropicalis were the predominant species in adults. In sterile fluids, urine and lower respiratory tract samples, C. parapsilosis was the second most prevalent species in the children group, while C. glabrata and C. tropicalis were the main second species in adults.     

Probing the gel to liquid-crystalline phase transition and relevant conformation changes in liposomes by 13C magic-angle spinning NMR spectroscopy

A straightforward way to visualize gel to liquid-crystalline phase transition in phospholipid membranes is presented by using ¹³C magic-angle spinning NMR. The changes in the ¹³C isotropic chemical shifts with increasing temperature are shown to be a sensitive probe of the main thermotropic phase transition related to lipid hydrocarbon chain dynamics and relevant conformational changes. The average value of the energy difference between trans and gauche states in the central C4–11 fragment of the DMPC acyl chain was estimated to be 4.02 ± 0.2 kJ mol^− 1 in the liquid crystalline phase. The reported spectral features will be useful in ¹³C solid state NMR studies for direct monitoring of the effective lipid chain melting allowing rapid uniaxial rotation of membrane proteins in the biologically relevant liquid-crystalline phase.     

Structural changes that occur upon photolysis of the Fe(II)a3–CO complex in the cytochrome ba3-oxidase of Thermus thermophilus: A combined X-ray crystallographic and infrared spectral study demonstrates CO binding to CuB

The purpose of the work was to provide a crystallographic demonstration of the venerable idea that CO photolyzed from ferrous heme-a₃ moves to the nearby cuprous ion in the cytochrome c oxidases. Crystal structures of CO-bound cytochrome ba₃-oxidase from Thermus thermophilus, determined at ~ 2.8–3.2 Å resolution, reveal a Fe–C distance of ~ 2.0 Å, a Cu–O distance of 2.4 Å and a Fe–C–O angle of ~ 126°. Upon photodissociation at 100 K, X-ray structures indicate loss of Fe_a3–CO and appearance of Cu_B–CO having a Cu–C distance of ~ 1.9 Å and an O–Fe distance of ~ 2.3 Å. Absolute FTIR spectra recorded from single crystals of reduced ba₃–CO that had not been exposed to X-ray radiation, showed several peaks around 1975 cm^? 1; after photolysis at 100 K, the absolute FTIR spectra also showed a significant peak at 2050 cm^? 1. Analysis of the ‘light’ minus ‘dark’ difference spectra showed four very sharp CO stretching bands at 1970 cm^? 1, 1977 cm^? 1, 1981 cm^? 1, and 1985 cm^? 1, previously assigned to the Fe_a3–CO complex, and a significantly broader CO stretching band centered at ~ 2050 cm^? 1, previously assigned to the CO stretching frequency of Cu_B bound CO. As expected for light propagating along the tetragonal axis of the P4₃2₁2 space group, the single crystal spectra exhibit negligible dichroism. Absolute FTIR spectrometry of a CO-laden ba₃ crystal, exposed to an amount of X-ray radiation required to obtain structural data sets before FTIR characterization, showed a significant signal due to photogenerated CO₂ at 2337 cm^? 1 and one from traces of CO at 2133 cm^? 1; while bands associated with CO bound to either Fe_a3 or to Cu_B in “light” minus “dark” FTIR difference spectra shifted and broadened in response to X-ray exposure. In spite of considerable radiation damage to the crystals, both X-ray analysis at 2.8 and 3.2 Å and FTIR spectra support the long-held position that photolysis of Fe_a3–CO in cytochrome c oxidases leads to significant trapping of the CO on the Cu_B atom; Fe_a3 and Cu_B ligation, at the resolutions reported here, are otherwise unaltered. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Purification of a recombinant heavy chain fragment C vaccine candidate against botulinum serotype C neurotoxin [rBoNTC(H(c))] expressed in Pichia pastoris

A purification process for the manufacture of a recombinant C-terminus heavy chain fragment from botulinum neurotoxin serotype C [rBoNTC(H_c)], a potential vaccine candidate, has been defined and successfully scaled-up. The rBoNTC(H_c) was produced intracellularly in Pichia pastoris X-33 using a three step fermentation process, i.e., glycerol batch phase, a glycerol fed-batch phase to achieve high cell densities, followed by a methanol induction phase. The rBoNTC(H_c) was captured from the soluble protein fraction of cell lysate using hydrophobic charge induction chromatography (HCIC; MEP HyperCel?), and then further purified using a CM 650M ion exchange chromatography step followed by a polishing step using HCIC once again. Method development at the bench scale was achieved using 5–100 mL columns and the process was performed at the pilot scale using 0.6–1.6 L columns in preparation for technology transfer to cGMP manufacturing. The process yielded approximately 2.5 g of rBoNTC(H_c)/kg wet cell weight (WCW) at the bench scale and 1.6 g rBoNTC(H_c)/kg WCW at the pilot scale. The purified rBoNTC(H_c) was stable for at least 3 months at 5 and ?80 °C as determined by reverse phase-HPLC and SDS–PAGE and was stable for 24 months at ?80 °C based on mouse potency bioassay. N-Terminal amino acid sequencing confirmed that the N-terminus of the purified rBoNTC(H_c) was intact.     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

Benzophenone derivatives from the plant endophytic fungus,Pestalotiopsis sp.

Two new (1–2) and one known (3) benzophenone derivatives, along with five known ambuic acid analogues (4–8) were isolated from the solid cultures of a Pestalotiopsis sp. Compound 2 represented both enantiomeric and atropisomeric isomers, and the absolute configurations of enantiomers [(−)-2 and (+)-2] were determined by electronic circular dichroism (ECD) calculations. All the isolates were evaluated for their antimicrobial and cytotoxic activities. Chlorinated compounds 2 and 3 showed potent antimicrobial activities against four pathogenic bacteria, and compound 3 also displayed strong antifungal activity against Candida glabrata (ATCC 90030) with an MIC₅₀ value of 2.6 ± 0.1 μg/mL. Compound 1 exhibited moderate cytotoxicity against U2OS and MCF-7 with IC₅₀ values of 11.6 and 16.8 μM, respectively.