Role of Fungi in Biodegradation of Imidazolium Ionic Liquids by Activated Sewage Sludge.

Ionic liquids (ILs), due to their specific properties, can play the role of persistent water contaminants. Fungi manifest the ability to decompose hardy degradable compounds, showing potential in the biodegradation of ILs, which has been studied extensively on sewage sludge; however, attention was drawn mainly to bacterial and not fungal species. The aim of the research was to determine the significance of fungi in ILs' biodegradation to extend the knowledge and possibly point out ways of increasing their role in this process. The research included: the isolation and genetic identification of fungal strains potentially capable of [OMIM][Cl], [BMIM][Cl], [OMIM][Tf2N], and [BMIM][Tf2N] degradation, adjustment of the ILs concentration for biodegradability test by MICs determination and choosing strains with the highest biological robustness; inoculum adaptation tests, and finally primary biodegradation by OECD 301F test. The study, conducted for 2 mM [OMIM][Cl] as a tested substance and consortium of microorganisms as inoculum, resulted in an average 64.93% biodegradation rate within a 28-day testing period. For the individual fungal strain (Candida tropicalis), the maximum of only 4.89% biodegradation rate was reached in 10 days, then inhibited. Insight into the role of fungi in the biodegradation of ILs was obtained, enabling the creation of a complex overview of ILs toxicity and the possibilities of its biological use. However, only an inoculum consisting of a consortium of microorganisms enriched with a selected strain of fungi was able to decompose the IL, in contrast to that consisting only of an individual fungal strain.

PCR-RFLP assays for species-specific identification of fungi belonging to Scopulariopsis and related genera.

Fungi of the Scopulariopsis genus, commonly found in the environment, are opportunistic pathogens that can cause various types of human infections. So far, no efficient molecular method has been developed for species differentiation among Scopulariopsis and related genera. In order to advance this field, we have evaluated performance of polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assays, based on cytochrome c oxidase subunit 1 and ß-tubulin genes. The assays resulted in 2-10 restriction patterns, depending on the gene amplified and restriction enzyme applied. Pooled analysis of the patterns allowed to propose an algorithm, that can be successfully used for an accurate species-specific identification of 21 species of the Scopulariopsis-like fungi.

Detection of Polish clinical Aspergillus fumigatus isolates resistant to triazoles.

We studied the presence of triazole resistance of 121 Aspergillus fumigatus clinical isolates collected in two Polish cities, Warsaw and Wroclaw, to determine if resistance is emerging in our country. We identified five itraconazole resistant isolates (4.13%) carrying the TR34/L98H alteration in Cyp51A gene, four of which were cross-resistant to posaconazole and one to voriconazole. One isolate was intermediate susceptible to itraconazole and harbored no Cyp51A alterations. The study confirms the presence of azole resistant A. fumigatus strains in Poland at a level that is comparative to other European countries.

Rapid Assays for Specific Detection of Fungi of Scopulariopsis and Microascus Genera and Scopulariopsis brevicaulis Species.

PURPOSE: Fungi of Scopulariopsis and Microascus genera cause a wide range of infections, with S. brevicaulis being the most prevalent aetiological agent of mould onychomycosis. Proper identification of these pathogens requires sporulating culture, which considerably delays the diagnosis. So far, sequencing of rDNA regions of clinical isolates has produced ambiguous results due to the lack of reference sequences in publicly available databases. Thus, there is a clear need for the development of new molecular methods that would provide simple, rapid and highly specific identification of Scopulariopsis and Microascus species. The objective of this study was to develop simple and fast assays based on PCR and real-time PCR for specific detection of fungi from Scopulariopsis and Microascus genera, and separately, S. brevicaulis species. METHODS: On the basis of alignment of ß-tubulin gene sequences, Microascus/Scopulariopsis-specific primers were designed and S. brevicaulis-specific primers were reevaluated. DNA from cultured fungal isolates, extracted in a two-step procedure, was used in Microascus/Scopulariopsis-specific and S. brevicaulis-specific PCR and real-time PCR followed by electrophoresis or melting temperature analysis, respectively. RESULTS: The specificity of the assays was confirmed, as positive results were obtained only for Scopulariopsis spp. and Microascus spp. isolates tested in Microascus/Scopulariopsis-specific assay, and only for S. brevicaulis and S. koningii (syn. S. brevicaulis) isolates in a S. brevicaulis-specific assay, respectively, and no positive results were obtained neither for other moulds, dermatophytes, yeast-like fungi, nor for human DNA. CONCLUSIONS: The developed assays enable fast and unambiguous identification of Microascus spp. and Scopulariopsis spp. pathogens.

PCR Detection of Scopulariopsis brevicaulis.

Scopulariopsis brevicaulis is known as the most common etiological factor of the mould toenail infections. There are also reports indicating that S. brevicaulis could cause organ and disseminated infections. Nowadays microscopic observations from the direct sample and culture are crucial for the appropriate recognition of the infection. In this paper a PCR-based method for S. brevicaulis detection is presented. The specificity of the reaction was confirmed, as positive results were obtained only for tested S. brevicaulis isolates and no positive results were obtained for other moulds, dermatophytes, yeast-like fungi, and human DNA.

Gold(III) complexes with chloride and cyanopyridines: Facilitated hydrolysis of nitrile ligand to amide and antibacterial activity.

A range of novel simple gold(III) compounds has been synthesized in their monocrystalline form, including two previously unknown chloro-complexes of Au3+ with 2-cyanopyridine or 3-cyanopyridine, respectively. Our investigations have revealed the intricate nature of the reaction between 2-cyanopyridine and tetrachloroauric acid, yielding at least three distinct products. The main product, obtained in high yield, is a salt featuring a tetrachloroauric anion and a pyridinium cation stabilized by a hydrogen bond to a further 2-cyanopyridine molecule. Moreover, we observed the in-situ formation of a 2-cyanopyridine-AuCl3 complex, which undergoes hydrolysis of the nitrile bond to yield a picolinamide-Au(III) complex. The complexes were characterized by IR and Raman spectroscopies, NMR spectroscopy, and single-crystal XRD studies. Additional computational studies were conducted to explain unusual spectral features, the observed disparities in the complexation reactions of the three isomeric cyanopyridine ligands and the distinct reactivity of the complex with 2-cyanopyridine. Based on these studies, we propose a mechanism for the catalyzed hydrolysis of the nitrile bond within the Au(III) complex. Finally, we assessed the antimicrobial efficacy of the synthesized gold(III) complexes against a spectrum of bacteria and fungi.

PCR test for Microsporum canis identification.

Microsporum canis, for which the natural hosts are cats and dogs, is the most prevalent zoophilic agent causing tinea capitis and tinea corporis in humans. We present here a diagnostic PCR test for M. canis, since its detection and species identification is relevant to the choice of treatment and to the understanding of a probable source of infection. An M. canis-specific PCR was evaluated using 130 clinical isolates of dermatophytes (including M. canis [n = 15] and 13 other species), 10 yeast or mold isolates, 12 hair and skin samples from animals with or without experimental M. canis infection, and 35 patient specimens, including seven specimens positive for M. canis and 15 dermatophyte negative samples. All pure cultures, animal specimens and clinical samples with M. canis were detected by the PCR test, whereas none of the other fungal isolates or samples without M. canis was negative. This study indicates that the PCR test for M. canis identification applied directly to patient specimens or animal hair, as well as to clinical isolates had 100% specificity and sensitivity.

New PCR test for detection of Candida glabrata based on the molecular target chosen by the RAPD technique.

Rapid, reliable diagnosis is a necessary condition for the successful treatment of infections. Such diagnostic assays are continually being developed. The paper presents a method for selecting the molecular target for PCR-based diagnostics based on the comparison of RAPD patterns. A sequence encoding Candida glabrata CBS138 hypothetical protein was selected. The limit of detection for PCR and real-time PCR reactions with DNA extracted from blood samples spiked with Candida glabrata was estimated at 1 CFU/ml. The application of the assays developed in this study would thus seem to be promising as a complementary method in the diagnostics of C. glabrata infections.

Recombinant thermostable AP exonuclease from Thermoanaerobacter tengcongensis: cloning, expression, purification, properties and PCR application.

Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity, especially at high temperatures. The gene encoding a homologue of AP exonuclease was cloned from the thermophilic anaerobic bacterium Thermoanaerobacter tengcongensis and transformed into Escherichia coli. The protein product showed high identity (80%) to human Ape1 nuclease, whereas to E. coli exonuclease III - 78%. This is the first prokaryotic AP nuclease that exhibits such high identity to human Ape1 nuclease. The very high expression level (57% of total soluble proteins) of fully active and soluble His6-tagged Tte AP enzyme with His6-tag on C-terminal end was obtained in Escherichia coli Rosetta (DE3) pLysS. The active enzyme was purified up to 98% homogeneity in one chromatographic step using metal-affinity chromatography on Ni(2+)-IDA-Sepharose resin. The yield was 90 mg (14000 kU) of pure His6-tagged Tte AP (153 kU/mg) from 1 liter of culture. The optimal conditions of Tte AP endo-, exonuclease and 3'-nuclease activity were investigated using fluorescein labeled dsDNA with inserted AP sites and ssDNA. Optimal Tte AP endonuclease activity was observed at 70-75 degrees C, pH 8.0 and at low Mg2+ concentration (0.5 mM). Higher Mg2+ concentration (> 1 mM) enhanced 3'-5' exonuclease activity and at Mg2+ concentration > 2.0 mM 3' nuclease activity was observed. Because of the endonuclease activity of Tte AP exonuclease, the enzyme was applied in PCR amplification of long DNA templates. Tte AP exonuclease eliminated AP-sites in DNA template and improved the efficiency of DNA amplification.

Fungal co-culture improves the biodegradation of hydrophobic VOCs gas mixtures in conventional biofilters and biotrickling filters.

The present study systematically evaluated the potential of Candida subhashii, Fusarium solani and their consortium for the abatement of n-hexane, trichloroethylene (TCE), toluene and α-pinene in biofilters (BFs) and biotrickling filters (BTFs). Three 3.2 L BFs packed with polyurethane foam and operated at a gas residence time of 77 s with an air mixture of hydrophobic volatile organic compounds (VOCs) were inoculated with C. subhashii, F. solani and a combination of thereof. The systems were also operated under a BTF configuration with a liquid recirculating rate of 2.5 L h-1. Steady state elimination capacities (ECs) of total VOCs of 17.4 ± 0.7 g m-3 h-1 for C. subhashii, 21.2 ± 0.8 g m-3 h-1 for F. solani and 24.4 ± 1.4 g m-3 h-1 for their consortium were recorded in BFs, which increased up to 27.2 ± 1.6 g m-3 h-1, 29.2 ± 1.9 g m-3 h-1, 37.7 ± 3.3 g m-3 h-1 in BTFs. BTFs supported a superior biodegradation performance compared to BF, regardless of the VOCs. Moreover, a more effective VOC biodegradation was observed when C. subhashii and F. solani were grown as a consortium. The microbial analysis conducted revealed that the fungi initially introduced in each BF represented the dominant species by the end of the experiment, with C. subhashii gradually overcoming F. solani in the system inoculated with the fungal consortium.

Solvent influence on the crystal structures of new cadmium tri-tert-butoxysilanethiolate complexes with 1,4-bis(3-aminopropyl)piperazine: luminescence and antifungal activity.

Monocrystals of dinuclear µ-1,4-bis(3-aminopropyl)piperazine-κ4N1,N1':N4,N4'-bis[bis(tri-tert-butoxysilanethiolato-κS)cadmium(II)], [Cd2(C12H27O3SSi)4(C10H24N4)] or [Cd2{SSi(OtBu)3}4(µ-BAPP)], 1, and polynuclear catena-poly[[bis(tri-tert-butoxysilanethiolato-κS)cadmium(II)]-µ-1,4-bis(3-aminopropyl)piperazine-κ2N1':N4'], [Cd(C12H27O3SSi)2(C10H24N4)]n or [Cd{SSi(OtBu)3}2(µ-BAPP)]n, 2, with 1,4-bis(3-aminopropyl)piperazine (BAPP) and tri-tert-butoxysilanethiolate ligands, were obtained from the same ratio of reactants, but with different solvents used for the crystallization processes. The structures and properties of both complexes were characterized using elemental analysis, X-ray diffraction and FT-IR, 1H NMR and luminescence spectroscopy. Applied density functional theory (DFT) computational methods and noncovalent interaction (NCI) analysis were used for geometry optimization and visualization of the interactions between the metallic centres and their surroundings. The X-ray analysis revealed four-coordinate CdII centres bound to two S atoms of the silanethiolate groups and two N atoms of the BAPP ligand; however, it chelates to tertiary and primary N atoms in 1, whilst in 2 it does not chelate and bonds only to RNH2. The photoluminescence properties of complexes 1 and 2 result from free-ligand emission and differ significantly from each other with respect to emission intensity. Additionally, antifungal activity was investigated against 18 isolates of fungi. Compound 1 strongly inhibited the growth of three dermatophytes: Epidermophyton floccosum, Microsporum canis and Trichophyton rubrum.

Virulence of Clinical Candida Isolates.

The factors enabling Candida spp. infections are secretion of hydrolytic enzymes, adherence to surfaces, biofilm formation or morphological transition, and fitness attributes. The aim of this study was to investigate the correlation between known extracellular virulence factors and survival of Galleria mellonella larvae infected with clinical Candida. The 25 isolates were tested and the activity of proteinases among 24/24, phospholipases among 7/22, esterases among 14/23, hemolysins among 18/24, and biofilm formation ability among 18/25 isolates was confirmed. Pathogenicity investigation using G. mellonella larvae as host model demonstrated that C. albicans isolates and C. glabrata isolate were the most virulent and C. krusei isolates were avirulent. C. parapsilosis virulence was identified as varied, C. inconspicua were moderately virulent, and one C. palmioleophila isolate was of low virulence and the remaining isolates of this species were moderately virulent. According to our study, virulence of Candida isolates is related to the expression of proteases, hemolysins, and esterases.

Composite Polyurethane-Polylactide (PUR/PLA) Flexible Filaments for 3D Fused Filament Fabrication (FFF) of Antibacterial Wound Dressings for Skin Regeneration.

This paper addresses the potential application of flexible thermoplastic polyurethane (TPU) and poly(lactic acid) (PLA) compositions as a material for the production of antibacterial wound dressings using the Fused Filament Fabrication (FFF) 3D printing method. On the market, there are medical-grade polyurethane filaments available, but few of them have properties required for the fabrication of wound dressings, such as flexibility and antibacterial effects. Thus, research aimed at the production, characterization and modification of filaments based on different TPU/PLA compositions was conducted. The combination of mechanical (tensile, hardness), structural (FTIR), microscopic (optical and SEM), degradation (2 M HCl, 5 M NaOH, and 0.1 M CoCl2 in 20% H2O2) and printability analysis allowed us to select the most promising composition for further antibacterial modification (COMP-7,5PLA). The thermal stability of the chosen antibiotic-amikacin-was tested using processing temperature and HPLC. Two routes were used for the antibacterial modification of the selected filament-post-processing modification (AMI-1) and modification during processing (AMI-2). The antibacterial activity and amikacin release profiles were studied. The postprocessing modification method turned out to be superior and suitable for wound dressing fabrication due to its proven antimicrobial activity against E. coli, P. fluorescens, S. aureus and S. epidermidis bacteria.

Multi-Technique Investigation of Grave Robes from 17th and 18th Century Crypts Using Combined Spectroscopic, Spectrometric Techniques, and New-Generation Sequencing.

The textile fragments of the funeral clothes found in the 17th and 18th century crypts were subjected to spectroscopic, spectrometric, and microbial investigation. The next-generation sequencing enabled DNA identification of microorganisms at the genus and in five cases to the species level. The soft hydrofluoric acid extraction method was optimized to isolate different classes of dyes from samples that had direct contact with human remains. High-performance liquid chromatography coupled with diode matrix and tandem mass spectrometry detectors with electrospray ionization (HPLC-DAD-ESI-MS/MS) enabled the detection and identification of 34 colourants that are present in historical textiles. Some of them are thus far unknown and uncommon dyes. Indigo, madder, cochineal, turmeric, tannin-producing plant, and young fustic were identified as sources of dyes in textiles. Scanning electron microscopy with energy-dispersive X-ray detector (SEM-EDS) and Fourier transform infrared spectroscopy (FT-IR) were used to identify and characterize fibres and mordants in funeral gowns. Of the 23 textile samples tested, 19 were silk while the remaining four were recognized as wool. The presence of iron, aluminium, sodium, and calcium suggests that they were used as mordants. Traces of copper, silica, and magnesium might originate from the contaminants. The large amount of silver indicated the presence of metal wire in one of the dyed silk textiles. SEM images showed that textile fibres were highly degraded.

Diagnostic PCR tests for Microsporum audouinii, M. canis and Trichophyton infections.

Since traditional diagnosis of dermatophyte infections is slow, we present a rapid new PCR test for detection of Trichophyton spp., Microsporum canis and M. audouinii infections. The performance of the test was evaluated with: 58 dermatophyte isolates; 10 yeast, mould and human DNA control samples; 25 routine specimens from patients suspected of having dermatophytosis; 10 hair specimens from guinea pigs experimentally infected with M. canis; and two samples from un-infected control animals. DNA was prepared by a 10-min procedure from pure cultures as previously described. The 302 bp PCR product was obtained for 35/35 Trichophyton isolates (10 species included) and the 279 bp for 3/3 M. canis and 4/4 M. audouinii samples. None of the 2 E. floccosum, 11 M. gypseum, 3 M M. persicolor or 12 control samples (yeast, mould, human DNA) were positive with either of the two PCR tests. Among the patient specimens, seven were T. rubrum positive, two for T. mentagrophytes, one was positive for T. tonsurans and 15 were dermatophyte negative by routine investigation (culture and/or pan-dermatophyte + T. rubrum multiplex PCR). The PCR results with our procedures were in 100% agreement with these results. Finally, the Microsporum PCR was positive for 10/10 guinea pig specimens from infected animals but for 0/2 of the control animal samples. The evaluation of the two PCR tests indicated excellent sensitivity and specificity.

Optimized 5-hour multiplex PCR test for the detection of tinea unguium: performance in a routine PCR laboratory.

We recently reported the development of a 5-hour multiplex PCR test for the detection of tinea unguium and the optimization of this test by the inclusion of an inhibition control. Here we report the performance of this procedure as used in a routine clinical laboratory as compared to conventional microscopy and culture-based techniques performed in a mycology reference laboratory. We found in processing 109 samples that 22 (20.2%) yielded fungi in culture while the suspected etiologic agents were noted microscopically in 15 (13.8%) that were negative in culture. Fungi were detected by PCR in 37 (33.9%) samples, of which only three were positive in culture. Since the majority of PCR positive but culture negative samples were positive in microscopic examinations, the increased sensitivity was not due to contamination. PCR inhibitors were present in 5% of the samples, but this was overcome by re-running the samples with a 50% reduction of sample DNA. In conclusion, the PCR test performance in the routine setting was excellent and provided a markedly reduced time to diagnosis with a higher sensitivity.

Typing of Candida isolates from patients with invasive infection and concomitant colonization.

We investigated the relationship between colonizing and invasive isolates from patients with candidaemia. Molecular typing was performed using random amplification of polymorphic DNA (RAPD) and multilocus sequence typing (MLST). We found MLST to be sufficient for typing Candida isolates, and that surveillance cultures are helpful in predicting concomitant invasive isolates, but not necessarily the pathogen involved in subsequent episodes.

Review on Current Status of Echinocandins Use.

Fungal infections are rising all over the world every year. There are only five medical compound classes for treatment: triazoles, echinocandins, polyenes, flucytosine and allylamine. Currently, echinocandins are the most important compounds, because of their wide activity spectrum and much lower sides effects that may occur during therapy with other drugs. Echinocandins are secondary metabolites of fungi, which can inhibit the biosynthesis of ß-(1,3)-D-glucan. These compounds have fungicidal and fungistatic activity depending on different genera of fungi, against which they are used. Echinocandin resistance is rare-the major cause of resistance is mutations in the gene encoding the ß-(1,3)-D-glucan synthase enzyme. In this review of the literature we have summarized the characteristics of echinocandins, the mechanism of their antifungal activity with pharmacokinetics and pharmacodynamics, and the resistance issue.

The Effect of Posaconazole, Itraconazole and Voriconazole in the Culture Medium on Aspergillus fumigatus Triazole Resistance.

Triazoles are the only compounds used as antibiotics in both medicine and agriculture. The presence of triazoles in the environment can contribute to the acquisition of azole resistance among isolates of Aspergillus fumigatus. The objective of this study was to investigate the effect of A. fumigatus exposure to triazoles on susceptibility to these compounds. Seventeen triazole-resistant and 21 triazole-sensitive A. fumigatus isolates were examined. The isolates were transferred 20 times on the Sabouraud medium supplemented with posaconazole, itraconazole or voriconazole, followed by five times on the medium not supplemented. The minimum inhibitory concentrations of antimycotics were examined according to the EUCAST broth microdilution method after the 20th transfer and also the 25th transfer. In addition, the expression levels of genes mdr1, mdr2, mdr3, atrF, cyp51A and cyp51B were determined. Cultivation of A. fumigatus on media supplemented with posaconazole, itraconazole and voriconazole resulted in the acquisition of resistance to the tested triazoles of all examined isolates. After recultivation on Sabouraud without azoles, most of the isolates lost their acquired resistance. The long-term use of triazole compounds in agriculture may result in the occurrence of triazole resistant A. fumigatus isolates in the environment, not only by induction or selection of mutations in the cyp51A gene, but also by contribution to changes in the gene expression.

Processing of Polyester-Urethane Filament and Characterization of FFF 3D Printed Elastic Porous Structures with Potential in Cancellous Bone Tissue Engineering.

This paper addresses the potential of self-made polyester-urethane filament as a candidate for Fused Filament Fabrication (FFF)-based 3D printing (3DP) in medical applications. Since the industry does not provide many ready-made solutions of medical-grade polyurethane filaments, we undertook research aimed at presenting the process of thermoplastic polyurethane (TPU) filament formation, detailed characteristics, and 3DP of specially designed elastic porous structures as candidates in cancellous tissue engineering. Additionally, we examined whether 3D printing affects the structure and thermal stability of the filament. According to the obtained results, the processing parameters leading to the formation of high-quality TPU filament (TPU_F) were captured. The results showed that TPU_F remains stable under the FFF 3DP conditions. The series of in vitro studies involving long- and short-term degradation (0.1 M phosphate-buffered saline (PBS); 5 M sodium hydroxide (NaOH)), cytotoxicity (ISO 10993:5) and bioactivity (simulated body fluid (SBF) incubation), showed that TPU printouts possessing degradability of long-term degradable tissue constructs, are biocompatible and susceptible to mineralization in terms of hydroxyapatite (HAp) formation during SBF exposure. The formation of HAp on the surface of the specially designed porous tissue structures (PTS) was confirmed by scanning electron microscope (SEM) and energy-dispersive X-ray spectroscopy (EDS) studies. The compression test of PTS showed that the samples were strengthened due to SBF exposure and deposited HAp on their surface. Moreover, the determined values of the tensile strength (~30 MPa), Young's modulus (~0.2 GPa), and compression strength (~1.1 MPa) allowed pre-consideration of TPU_F for FFF 3DP of cancellous bone tissue structures.