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.

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.

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.

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.

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.

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.

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.

First report on echinocandin resistant Polish Candida isolates.

PURPOSE: Candida spp. are ranked as one of the four major causative agents of fungal infections. The number of infections caused by Candida species resistant to fluconazole, which is applied as the first line drug in candidiasis treatment, increases every year. In such cases the application of echinocandin is necessary. Echinocandin susceptibility testing has become a routine laboratory practice in many countries due to the increasing frequency of clinical failures during treatment with these drugs. METHODS: We performed anidulafungin, micafungin and caspofungin susceptibility testing according to the microdilution broth method on 240 Candida isolates collected in Polish hospitals. RESULTS: We identified 12 isolates resistant to all echinocandins within 240 examined isolates. Moreover, 6 of the examined samples were identified as rare Candida species and among them we observed very high echinocandin MIC values. CONCLUSION: Our research proves that in Poland there is a problem of echinocandin resistance. Moreover, we identified two species of Candida which are rare causative agents of human infections, and there was no reported incidence of such infections in Poland until now.

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.

Relation of the polymorphism of cyp51A sequence and the susceptibility of Aspergillus fumigatus isolates to triazoles determined by commercial gradient test (Etest) and by reference methods.

The aim of this study was to evaluate the accuracy of commercial gradient test (Etest) in the detection of triazole resistant Aspergillus fumigatus isolates using reference microdilution methods and the analysis of sequences of the cyp 51A gene. The study was performed on twenty clinical isolates which were identified as Aspergillus fumigatus based on the DNA sequences of the ITS1-2 fragment of ribosomal DNA and the ß-tubulin gene, out of them seventeen isolates showed wild-type cyp51A sequence and three were positive for the mutation TR34/L98H. All isolates were tested for the susceptibility to itraconazole (ITZ), voriconazole (VOR) and posaconasole (POS) using microdilution methods, according to EUCAST and CLSI protocols, as well as using Etest. The results of microdilution and Etests were analysed separately according to clinical breakpoints (CBP) defined by EUCAST version 7.0 and epidemiological cut off values (ECV). Etest as well as reference methods excellently recognised the WT isolates, which were susceptible to all tested triazoles, regardless of the method and CBP or ECV criteria used. The Etest recognized three non-WT isolates as resistant or intermediately sensitive to ITZ and POS and one as resistant to VOR. The categorical concordance between Etests and EUCAST and Etests and the CLSI method ranged from 90 to 100%. The interpretation of the results obtained from routine A. fumigatus Etests requires great caution. The use of the confirmative examinations with reference AST methods as well as with molecular tests is recommended.

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.

Examination of cyp51A and cyp51B expression level of the first Polish azole resistant clinical Aspergillus fumigatus isolate.

Aspergillus fumigatus is one of the most prevalent airborne fungal pathogens causing infections worldwide. Most A. fumigatus strains are susceptible to azoles, which are administered as the first line therapeutics. However, during last decade the acquired resistance to triazoles by these species has been described. There is a number of publications concerning the examination of clinical A. fumigatus strains from different countries, however there has been no report from Poland. Here, we describe for the first time, an examination of cyp51A and cyp51B expression level of 11 clinical A. fumigatus strains isolated during 2007-2014 period from the collection of Medical University in Wroclaw. Their susceptibility to itraconazole, voriconazole and posaconazole has been examined. The MIC values of triazoles for one of the examined isolates were respectively: > 8 mg/L for itraconazole, 2 mg/L for voriconazole and 0.5 mg/L for posaconazole. The cyp51A gene with its promoter region of all isolates was sequenced. It was found that the resistant isolate harbors the TR34/L98H mutation in the cyp51A gene and when cultured on media supplemented with voriconazole exhibits overexpression of both, cyp51A and cyp51B genes. The level of cyp51A gene expression was about 50 times higher than cyp51B.

PCR and real-time PCR assays to detect fungi of Alternaria alternata species.

Fungi of the Alternaria genus are mostly associated with allergic diseases. However, with a growing number of immunocompromised patients, these fungi, with A. alternata being the most prevalent one, are increasingly recognized as etiological agents of infections (phaeohyphomycoses) in humans. Nowadays, identification of Alternaria spp. requires their pure culture and is solely based on morphological criteria. Clinically, Alternaria infections may be indistinguishable from other fungal diseases. Therefore, a diagnostic result is often delayed or even not achieved at all. In this paper we present easy to perform and interpret PCR and real-time PCR assays enabling detection of A. alternata species. On the basis of alignment of ß-tubulin gene sequences, A. alternata-specific primers were designed. DNA from fungal isolates, extracted in a two-step procedure, were used in PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The assays specificity was confirmed, since positive results were obtained for all A. alternata isolates, and no positive results were obtained neither for other molds, dermatophytes, yeast-like fungi, nor human DNA. The assays developed here enable fast and unambiguous identification of A. alternata 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.

Real-time PCR approach in dermatophyte detection and Trichophyton rubrum identification.

Dermatophytes are keratinophilic molds that infect human hair, nails and skin. Diagnosis of dermatophytosis is based on morphological, serological and biochemical features. However, identification is difficult and laborious due to similarities between microorganisms. Thus, there is considerable interest to develop mycological diagnostic procedures based on molecular biology methods. In this study, fast, two-step DNA extraction method and real-time PCR was used for detection of dermatophytes DNA using pan-dermatophyte primers and identification of Trichophyton rubrum from pure cultures. The applied method allowed correct detection of all dermatophytes and correct identification of Trichophyton rubrum in less than 2 hours.

The extended version of restriction analysis approach for the examination of the ability of low-molecular-weight compounds to modify DNA in a cell-free system.

One of the primary requirements in toxicology is the assessment of ability of chemicals to induce DNA covalent modification. There are several well-established methods used for this purpose such as (32)P-Postlabeling or HPLC-MS. However, all of these approaches have difficult to overcome limitations, which prevents their use in genotoxin screening. Here, we describe the simple protocol exploiting specificity of restriction enzymes for the detection of DNA modification. It uses a specifically designed DNA amplicon, which contains two restriction sites recognized by Tru1I or MspI/HapII endonucleases. Modification of a restriction site abolishes its recognition and thus cleavage by the corresponding enzyme. The inhibition of cleavage indicates the occurrence of DNA modification of the restriction site(s), simultaneously pointing at the kind of base pairs (AT or GC) involved in DNA adduct formation. Previously, the application of this method was demonstrated for two antitumor compounds. Current study shows the extended version, that includes different ways of activation of tested compounds. Moreover, we propose an array of applications being of interest in toxicological research such as monitoring the kinetics of DNA adduct formation, detection of oxidative DNA damage, as well as assessment of the ability of antioxidative phytochemicals to prevent the latter DNA lesions.

The use of a one-step PCR method for the identification of Microsporum canis and Trichophyton mentagrophytes infection of pets.

INTRODUCTION: Dermatophytes are a closely related group of keratinophilic fungi. They encompass important etiological agents of superficial fungal infections. These fungi are able to invade keratinized tissues of humans and animals, causing dermatophytosis (ringworm) of hair, nails or skin. THE AIM: Traditional diagnostics of ringworm is based on morphological identification of cultured fungi and is time-consuming. MATERIALS AND METHODS: In this study, we applied a method patented by Brillowska-Dabrowska and coworkers (Brillowska-Dabrowska A, Saunte DM, Arenderup MC, 2007, Five-hour diagnosis of dermatophyte nail infections with specific detection of Trichophyton rubrum. J Clin Microbiol 45: 1200-1204) which involves extraction of fungal DNA and PCR amplification with pan-dermatophyte primers to confirm the presence of dermatophytes. RESULTS: The method used here is able to confirm the presence of dermatophyte DNA in pure cultures in less than 5 hours.