Еlectrospun сellulose acetate membranes decorated with curcumin-PVP particles: preparation, antibacterial and antitumor activities.

Curcumin (Curc) exhibits anti-inflammatory, antibacterial and antitumor activity. However, its clinical application is limited by its poor bioavailability related to its extremely low water solubility. Novel materials allowing enhanced release of Curc in aqueous medium were obtained. The new materials consisted of electrospun fibers from cellulose acetate (CA) (mean fiber diameter ca. 780 nm ± 110 nm) with electrosprayed Curc/polyvinylpyrrolidone (Curc/PVP) particles. Scanning electron microscopy (SEM) showed that separated and evenly distributed particles of Curc/PVP were deposited on the surface of the mats and on the inner layers of the mat. X-ray diffraction studies showed that Curc was in amorphous state. In vitro studies demonstrated that Curc release was facilitated from Curc/PVP-on-CA mats (ca. 78% for 24 h) compared with the materials in which Curc was incorporated in CA fibers (17% for 24 h). Moreover, the curcumin-containing materials exhibited antibacterial activity against Gram-positive bacteria Staphylococcus aureus (S. aureus) and Gram-negative bacteria Escherichia coli (E. coli). Curc/PVP-on-CA fibrous mats exhibited high in vitro cytotoxicity towards HeLa tumor cells. Therefore, the obtained materials are promising for antibacterial wound dressing applications as well as for application in local treatment of cervical tumors.

Chitosan/Hyaluronate Complex-Coated Electrospun Poly(3-hydroxybutyrate) Materials Containing Extracts from Melissa officinalis and/or Hypericum perforatum with Various Biological Activities: Antioxidant, Antibacterial and In Vitro Anticancer Effects.

The present study aimed to fabricate innovative fibrous materials with various biological activities from poly(3-hydroxybutyrate), sodium hyaluronate (HA), chitosan (Ch), Melissa officinalis (MO), Hypericum perforatum (HP) extract, or a combination of both extracts. Electrospinning or electrospinning followed by dip coating and the subsequent formation of a polyelectrolyte complex were the methods used to prepare these materials. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), thermogravimetric analysis (TGA), and attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) were applied for investigating the morphology of materials, their thermal characteristics, and their surface chemical composition. The composition and design of the mats had an influence on the in vitro release behavior of the main bioactive compounds present in the MO and HP extracts incorporated in the materials. It was found that as-created materials comprising a combination of both extracts and a Ch/HA complex exerted higher antioxidant activity than that of (non-)coated MO-containing mats and Ch/HA-coated mats containing HP. The novel materials manifested antibacterial efficacy towards the pathogenic bacteria S. aureus and E. coli, as evidenced by the performed microbiological screening. Furthermore, the mats possessed a great growth inhibitory effect on HeLa cancer cells but had a less pronounced effect on the growth of normal mouse BALB/3T3 fibroblasts. The loading of both extracts in the mats and the formation of coating led to the enhancement of the in vitro anticancer and antibacterial activities of the materials. Thus, the novel materials have potential for use in local cancer therapy as well as for use as wound dressings.

Stress-induced L-forms of Mycobacterium bovis: a challenge to survivability.

This study addressed the ability of Mycobacterium bovis to produce unusual extreme morphologic forms (cell wall-deficient or L-forms) under stress conditions. Models using nutrient starvation and cryogenic stress treatments of Mycobacterium bovis, as well as the filtration technique followed by cultivation in semisolid medium, were used for isolation of L-form variants. Morphological transformations and developmental stages, typical for the bacterial L-cycle were observed by electron microscopy. Of special interest was the formation of giant filaments and common extremely thick membranous structures enveloping the entire L-form population. Following collapse of giant filamentous structures small viable cell elements, mainly granules and coccobacilli, were released and proved able to grow into large bodies or multiply by fission or budding. Derivation of viable filterable forms from L-form cultures and parental strain and their identification as Mycobacterium bovis based on specific IS6110 PCR was noteworthy. We suggest that formation of giant filaments and thick common membranous envelopes, observed under stress conditions, may serve a twofold purpose - protection against an unfavourable environment, and a role in reproduction of Mycobacterium bovis L-forms. The observed L-form conversion phenomenon in Mycobacterium bovis seems to be associated with an adaptive strategy of this pathogen for survival and reproduction in an unfavorable environment.

Placentа of BCG-Vaccinated Women in early Pregnancy is Colonized with Non-Immunogenic Mycobacterial L-forms.

PROBLEM: Long-lived mycobacterial L-forms (mL-forms) could be detected in the blood of BCG-vaccinated people. We have previously found mL-forms in term placentas and blood of neonates, delivered by healthy BCG-vaccinated mothers as first formal demonstration that BCG vaccination in the childhood of the woman could affect her placentobiome during pregnancy. Of note, the isolated mL-forms reverted to the cell-walled state of the parental BCG bacilli in vitro. METHOD OF STUDY: Here, we analyzed triple samples of blood, decidua and chorion taken from BCG-vaccinated pregnant women, directed to elective abortions (6-12 gestation weeks). The colonization of the primary samples with mycobacterial L-forms (mL-forms) was evaluated using microbiological isolation and subsequent identification by real time PCR and morphological characterization by light microscopy and SEM. The potential of early placenta-derived mL-forms to expand mycobacteria-reactive γδ T cells in vitro was assessed using FACS, whereas their immunogenicity in vivo was followed up after i.p. inoculation in rats. RESULTS: Our results showed two important findings: 1) viable filterable mL-forms varying in size, shape and proliferation modes are capable of colonizing the gestational tissues of BCG-vaccinated women early in pregnancy and 2) early placenta-derived mL-forms are not as immunogenic as walled M. bovis BCG bacilli, shown by lack of stimulation of mycobacteria-reactive γδ T cells co-cultured with early placenta-derived mL-forms and inefficient internalization of mL-forms by rat's peritoneal phagocytes in vivo. CONCLUSION: Although generally thought to be reduced in virulence, mL-forms could provide a reservoir, hidden from the immune system especially in an immune privileged niche like placenta.

5-Amino-8-hydroxyquinoline-containing Electrospun Materials Based on Poly(vinyl alcohol) and Carboxymethyl Cellulose and Their Cu2+ and Fe3+ Complexes with Diverse Biological Properties: Antibacterial, Antifungal and Anticancer.

Novel fibrous materials with diverse biological properties containing a model drug of the 8-hydroxyquinoline group-5-amino-8-hydroxyquinoline (5A8Q)-were fabricated using a one-pot method by electrospinning poly(vinyl alcohol) (PVA)/carboxymethyl cellulose (CMC)/5A8Q solutions. Experiments were performed to prepare Cu2+ (Fe3+) complexes of the crosslinked PVA/CMC/5A8Q materials. The formation of complexes was proven by using scanning electron microscopy (SEM), attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). The release of 5A8Q and 5A8Q.Cu2+ (Fe3+) was studied and their in vitro release profiles were mostly impacted by the hydrophilic/hydrophobic properties of the materials. The performed microbiological assays revealed that fibrous materials containing 5A8Q and their complexes exhibited good antibacterial and antifungal efficacy. Their activity was stronger against bacteria S. aureus than against bacteria E. coli and fungi C. albicans. Cell viability tests using MTT showed that the presence of 5A8Q and its complexes in the fibrous materials resulted in a significant decrease in the HeLa and MCF-7 cancer cell viability for the various times of cell incubation. Moreover, the observed cytotoxicity of the mats against cancer cells was greater than that against non-cancer HaCaT keratinocytes. All these properties make the novel materials potential candidates for the design of wound healing materials and as drug delivery systems for local therapy of cervical and breast cancer.

Electrospun Fibers of Biocompatible and Biodegradable Polyesters, Poly(Ethylene Oxide) and Beeswax with Anti-Bacterial and Anti-Fungal Activities.

Fibrous materials composed of core-sheath fibers from poly(ethylene oxide) (PEO), beeswax (BW) and 5-nitro-8-hydroxyquinoline (NQ) were prepared via the self-organization of PEO and BW during the single-spinneret electrospinning of a homogeneous blend solution of the partners. Additionally, the application of the same approach enabled the preparation of fibrous materials composed of core-double sheath fibers from PEO, poly(L-lactide) (PLA) and NQ or 5-chloro-7-iodo-8-hydroxyquinoline (CQ), as well as from PEO, poly(ε-caprolactone) (PCL) and NQ. The consecutive selective extraction of BW and of the polyester with hexane and tetrahydrofuran, respectively, evidenced that core-double sheath fibers from PEO/polyester/BW/drug consisted of a PEO core, a polyester inner sheath and a BW outer sheath. In order to evaluate the possibility of the application of fibrous materials from PEO/BW/NQ, PEO/PLA/BW/NQ, PEO/PCL/BW/NQ and PEO/PLA/BW/CQ for plant protection, microbiological studies were performed using both phytopathogenic microorganisms (Pseudomonas corrugata, Fusarium graminearum and Fusarium avenaceum) and beneficial microorganisms (Pseudomonas chlororaphis, Bacillus amyloliquefaciens and Trichoderma asperellum). It was found that the fibrous materials had anti-bacterial and anti-fungal activity against both phytopathogenic and beneficial microorganisms. This is the first report on the activity of fibrous materials loaded with 8-hydroxyquinoline derivatives not only against phytopathogenic but also against beneficial microorganisms that are of importance in agriculture.

Innovative Fibrous Materials Loaded with 5-Nitro-8-hydroxyquinoline via Electrospinning/Electrospraying Demonstrate Antioxidant, Antimicrobial and Anticancer Activities.

A new type of fibrous mat based on a cellulose derivative-cellulose acetate (CA) or CA and water-soluble polymers (polyvinylpyrrolidone, PVP or poly(vinyl alcohol), PVA)-loaded with the model drug 5-nitro-8-hydroxyquinoline (5N) was fabricated via electrospinning or electrospinning in conjunction with electrospraying. Scanning electron microscopy (SEM), X-ray diffraction analysis (XRD), Fourier-transform infrared spectroscopy (FTIR), water contact angle measurements and ultraviolet-visible spectroscopy (UV-Vis) were used for the complex characterization of the obtained novel material. The decoration of CA fibers with a water-soluble polymer containing the drug resulted in the facilitation of wetting and fast drug release. The 5N-containing fibrous material showed antioxidant activity. Moreover, the proposed materials' antibacterial and antifungal properties were tested against S. aureus, E. coli, P. aeruginosa and C. albicans. Well-distinguished, sterile zones with diameters above 3.5 cm were observed around all 5N-containing mats. The mats' cytotoxicity toward HeLa carcinoma cells and normal mouse BALB/c 3T3 fibroblasts was assessed. The 5N-in-CA, PVP,5N-on-(5N-in-CA) and PVA,5N-on-(5N-in-CA) fibrous mats possessed anticancer efficacies and much lower levels of toxicity against normal cells. Therefore, the as-created novel electrospun materials, which are based on polymers loaded with the drug 5N via electrospinning/electrospraying, can potentially be applied for topical wound healing and for local cancer therapy.

Unique biological properties of Mycobacterium tuberculosis L-form variants: impact for survival under stress.

Bacteria can, under certain conditions, enter into a cell-less state known as L-form conversion. This phenomenon is universal, but also recognized with difficultly by microbiologists. The current study addresses several aspects concerning the ability of tubercle bacilli to use L-form conversion as a unique adaptive strategy to survive and reproduce under unfavorable conditions. Nutrient starvation of M. tuberculosis in vitro followed by passages in Middlebrook 7H9 semisolid medium was used for stress induction and the selective isolation of mycobacterial L-form variants. Light and electron microscopy images evidence the peculiar characteristics of mycobacterial L-forms. For example, mycobacterial L-forms were observed to lose their acid-fastness and change their morphology. In addition, wide morphological variability, the presence of large and elementary bodies, coccoids and small granular forms, as well as the appearance of unusual modes of irregular cell division were observed. Unlike classical tubercle bacilli, L-form variants grew and developed typical "fried-egg" colonies faster. L-forms were verified as M. tuberculosis by spoligotyping. The results provide insights into the nature of L-form phenomena in M. tuberculosis and link them to the mechanisms allowing mycobacterial survival under stress.

Core/Double-Sheath Composite Fibers from Poly(ethylene oxide), Poly(L-lactide) and Beeswax by Single-Spinneret Electrospinning.

The conventional approach for preparation of core-sheath fibers is coaxial electrospinning. Single-spinneret electrospinning of emulsions is a much less common method to obtain core-sheath fibers. Core-sheath structure may be generated by electrospinning of homogeneous blend solutions; however, reports on such cases are still scarce. Herein, the preparation of nanofibrous composites from poly(ethylene oxide) (PEO), poly(L-lactide) (PLA) and beeswax (BW) by single-spinneret electrospinning of their homogeneous blend solutions in chloroform is reported. The produced fibers had core/double-sheath structure with a PEO core, PLA inner sheath and BW outer sheath. This original fiber structure was evidenced by transmission electron microscopy, selective extraction of BW or PEO, and X-ray photoelectron spectroscopy. The PLA/BW double sheath led to hydrophobicity of the PEO/PLA/BW mats. The tensile tests revealed that PEO/PLA/BW mats had substantially improved mechanical behavior as compared to PEO, PLA and PEO/BW mats. PEO/PLA/BW mats can be used as drug carriers as evidenced by the one-pot incorporation of the model drug 5-nitro-8-hydroxyquinoline (NQ) into the fibrous materials. Microbiological tests showed that PEO/PLA/BW/NQ had antimicrobial activity. Therefore, the new materials are promising for wound healing applications.

Core-Sheath-Like Poly(Ethylene Oxide)/Beeswax Composite Fibers Prepared by Single-Spinneret Electrospinning. Antibacterial, Antifungal, and Antitumor Activities.

Composite fibrous materials are prepared from poly(ethylene oxide) (PEO) and beeswax (BW) by single-spinneret electrospinning using chloroform as a common solvent. The obtained fibers have core-sheath-like structure, as evidenced by the water contact angle values and corroborated by the results on the elemental composition of the fiber's surface determined by X-ray photoelectron spectroscopy (XPS) and by analyses with scanning electron microscopy of fibers before and after selective extraction of PEO or BW. Furthermore, the core-sheath-like structure is proven by transmission electron microscopy. This is attributed to self-assembly of BW molecules on the surface of the formed fibers driven by the incompatibility between PEO and BW. 5-Nitro-8-hydroxyquinoline (NQ) is embedded as a model drug with antibacterial, antifungal, and anticancer properties in the PEO/BW fibrous materials. XPS analyses reveal that NQ is present on the surface of the PEO/BW/NQ materials. Using a purposely designed cell for fixation of the fibrous materials the NQ release in phosphate buffer solution with рН 7.4 is followed. The new PEO/BW/NQ fibrous materials exhibit antibacterial activity against S. aureus and E. coli, antifungal effect against C. albicans, and selective anticancer activity against HeLa (human cervical adenocarcinoma cells) and SH-4 (human melanoma cells) cell lines.

Bio-Based Electrospun Fibers from Chitosan Schiff Base and Polylactide and Their Cu2+ and Fe3+ Complexes: Preparation and Antibacterial and Anticancer Activities.

The Schiff base derivative (Ch-8Q) of chitosan (Ch) and 8-hydroxyquinoline-2-carboxaldehyde (8QCHO) was prepared and fibrous mats were obtained by the electrospinning of Ch-8Q/polylactide (PLA) blend solutions in trifluoroacetic acid (TFA). Complexes of the mats were prepared by immersing them in a solution of CuCl2 or FeCl3. Electron paramagnetic resonance (EPR) analysis was performed to examine the complexation of Cu2+(Fe3+) in the Ch-8Q/PLA mats complexes. The morphology of the novel materials and their surface chemical composition were studied by scanning electron microscopy (SEM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS). The performed microbiological screening demonstrated that in contrast to the neat PLA mats, the Ch-8Q-containing mats and their complexes were able to kill all S. aureus bacteria within 3 h of contact. These fibrous materials had efficiency in suppressing the adhesion of pathogenic bacteria S. aureus. In addition, Ch-8Q/PLA mats and their complexes exerted good anticancer efficacy in vitro against human cervical HeLa cells and human breast MCF-7 cells. The Ch-8Q-containing fibrous materials had no cytotoxicity against non-cancer BALB/c 3T3 mouse fibroblast cells. These properties render the prepared materials promising as wound dressings as well as for application in local cancer treatment.

Pegylated Curcumin Derivative: Water-Soluble Conjugates with Antitumor and Antibacterial Activity.

During the past years, the synthesis of polymer prodrug structures, based on natural phytochemical compounds with a great range of valuable biological properties, has become a promising solution in cancer prevention, imaging, and detection. Curcumin (Curc) remains one of the most studied natural products, due to the impressive palette of biological properties and the possibility to be easily loaded in various micro- and nanostructures and chemically modified. In this study, pegylated curcumin derivatives were prepared by a direct esterification reaction between poly(ethylene glycol)diacid (PEG of 600 g/mol molar mass, PEG600) and Curc in the presence of N,N'-dicyclohexylcarbodiimide (PEG600-Curc). The successful reaction resulted in a water-soluble stable product that was characterized by infrared spectroscopy (Fourier transform infrared (FT-IR)) and proton (1H) and carbon (13C) NMR. The effect of the pH values of buffer solutions on PEG600-Curc spectral properties (absorption and photoluminescence) was investigated by UV-vis and fluorescence spectrophotometry. Based on the biological tests, it was confirmed that PEG600-Curc exhibits cytotoxic activity against Graffi cell lines, as a function of the Curc concentration in the conjugate and the incubation time. PEG600-Curc antibacterial activity was validated in microbiological tests against pathogenic microorganisms such as Staphylococcus aureus. Most importantly, despite the covalent attachment of Curc to PEG and the slight reduction in the therapeutic index of the conjugate, both the anticancer and antimicrobial activities remain the highest reported, thus opening the gate for further, more clinically oriented studies.

Cellulose Acetate-Based Electrospun Materials with a Variety of Biological Potentials: Antibacterial, Antifungal and Anticancer.

Novel eco-friendly fibrous materials with complex activities from cellulose acetate and cellulose acetate/polyethylene glycol (CA,PEG) containing 5-chloro-8-hydroxyquinoline as a model drug were obtained by electrospinning. Several methods, including scanning electron microscopy, X-ray diffraction analysis, ultraviolet-visible spectroscopy, water contact angle measurements, and mechanical tests, were utilized to characterize the obtained materials. The incorporation of PEG into the fibers facilitated the drug release. The amounts of the released drug from CA/5-Cl8Q and CA,PEG/5-Cl8Q were 78 ± 3.38% and 86 ± 3.02%, respectively (for 175 min). The antibacterial and antifungal activities of the obtained materials were studied. The measured zones of inhibition of CA/5-Cl8Q and CA,PEG/5-Cl8Q mats were 4.0 ± 0.18 and 4.5 ± 0.2 cm against S. aureus and around 4.0 ± 0.15 and 4.1 ± 0.22 cm against E. coli, respectively. The complete inhibition of the C. albicans growth was detected. The cytotoxicity of the obtained mats was tested toward HeLa cancer cells, SH-4 melanoma skin cells, and mouse BALB/c 3T3 fibroblasts as well. The CA/5-Cl8Q and CA,PEG/5-Cl8Q materials exhibited anticancer activity and low normal cell toxicity. Thus, the obtained fibrous materials can be suitable candidates for wound dressing applications and for application in local cancer treatment.

8-Hydroxyquinoline-5-Sulfonic Acid-Containing Poly(Vinyl Alcohol)/Chitosan Electrospun Materials and Their Cu2+ and Fe3+ Complexes: Preparation, Antibacterial, Antifungal and Antitumor Activities.

Novel poly(vinyl alcohol) (PVA)/chitosan (Ch)-based fibrous materials containing an ionizable model drug, 8-hydroxyquinoline-5-sulfonic acid (SQ), were successfully fabricated by electrospinning. Complexes between the components of the crosslinked PVA/Ch/SQ mats and Cu2+ and Fe3+ ions were formed. The coordination of these ions in the mats was examined by electron paramagnetic resonance spectroscopy (EPR). The microbiological screening against S. aureus and C. albicans revealed that both the incorporation of SQ in the mats and the complexation with Cu2+ and Fe3+ imparted to these materials antibacterial and antifungal activities. Moreover, the SQ-containing mats and their complexes displayed good cytotoxicity against human cervical HeLa tumor cells. The most prominent was the cytotoxicity of the Cu2+ complex of the mats. The combined antibacterial, antifungal and in vitro antitumor activities render these novel materials promising candidates for wound dressing applications and for application in the local treatment of cervical tumors.

Eubiotic vs. dysbiotic human blood microbiota: the phenomenon of cell wall deficiency and disease-trigger potential of bacterial and fungal L-forms.

The current review provides data and focuses on blood as a niche for the presence of cell wall-deficient microbes (L-forms). The hypothesis for the existence of L-form microbiota in humans was tested by us using an innovative methodology for the isolation of L-form cultures from human blood. Criteria were conceived for the individual assessment of blood microbiota and recognition of two types of states -- "eubiotic" and "dysbiotic" blood microbiota. Cell wall-deficient microbes (CWD) that inhabit blood in healthy people are in natural balance with the host homeostasis, which corresponds to the "eubiotic" state. When interacting with a host, CWD bacteria or fungi employ a strategy distinctive for a latent lifestyle. In contrast to "eubiotic," "dysbiotic" blood microbiota manifests when the balance is disrupted and there is an excess of L-form variants of opportunistic microbes that invade from the external microbiota, i.e., from all body sites in contact with the external environment. Our case studies on people with multiple sclerosis (MS), Parkinson's disease, psoriasis, thyroid cancer, and diabetes revealed the appearance of "dysbiotic" blood microbiota that outlined the disease-trigger potential of opportunistic bacteria and fungi existing in blood as CWD variants. Blood microbiota assessment could be of diagnostic and prognostic importance for the pathological processes occurring within the body, as well as for understanding the microbial pathogenesis.

Molecular characterization of Mycobacterium tuberculosis isolates from different regions of Bulgaria.

Mycobacterium tuberculosis isolates from different regions of Bulgaria were studied by a variety of molecular typing tools. Based on spacer oligonucleotide typing (spoligotyping), the 113 strains were subdivided into 35 spoligotypes: 5 unique profiles and 15 profiles shared by two to 29 strains; the Hunter-Gaston diversity index (HGI) was 0.9. Comparison with the international database SITVIT2 at the Institut Pasteur de Guadeloupe showed the presence of two globally distributed shared types, ST53 (25.7%) and ST47 (6.2%). Nineteen (16.8%) and six (5.3%) strains belonged to the ST125 (LAM/S subfamily) and ST41 (LAM7_TUR subfamily) types described in SITVIT2 as ubiquitous/rare and ubiquitous/common types, respectively. Seven spoligoprofiles (12 strains) were not found in the database; two of them constituted new shared types. The Beijing genotype strains were not found in the studied collection in spite of close contacts with Russia in the recent and historical past. Additional subtyping by IS6110-restriction fragment length polymorphism (RFLP) and 12-locus mycobacterial interspersed repetitive unit (MIRU)-variable number of tandem repeat analyses were performed within selected spoligotypes. In particular, MIRU typing showed better discrimination within ST125 than IS6110-RFLP typing (HGI = 0.83 versus 0.39). A high gradient for ST125 in Bulgaria compared to its negligible presence in the global database and neighboring countries leads us to suggest a Bulgarian phylogeographic specificity of this spoligotype. To conclude, this first study of the Bulgarian M. tuberculosis population demonstrated its heterogeneity and predominance of several worldwide-distributed and Balkan-specific spoligotypes.

Utility of new 24-locus variable-number tandem-repeat typing for discriminating Mycobacterium tuberculosis clinical isolates collected in Bulgaria.

The present study evaluated new markers for molecular typing of Mycobacterium tuberculosis with a collection of strains circulating in Bulgaria. A study sample included 133 strains from epidemiologically unlinked patients from different regions of the country. Spoligotyping was used as a primary typing tool; it subdivided these strains into 37 types, including 15 clusters and 22 singletons. Traditional IS6110-restriction fragment length polymorphism (RFLP) typing and novel 24-locus variable number tandem-repeat (VNTR) typing methods were applied to the selection of 73 strains. Discriminatory power (Hunter-Gaston index [HGI]) of these methods was found to be 0.983 and 0.997, respectively. The 73 strains were subdivided into 66 types by a 24-locus mycobacterial interspersed repetitive unit (MIRU)-VNTR scheme, 62 types by a classical 12-locus MIRU-VNTR scheme, 51 types by IS6110-RFLP typing, and 31 types by spoligotyping. A combination of the five most polymorphic loci (MIRU40, Mtub04, Mtub21, QUB-11b, and QUB-26) was shown to achieve a high discrimination (HGI = 0.984). To conclude, a complete 24-locus scheme excellently differentiated strains in our study, whereas a reduced 5-locus set provided a sufficiently high differentiation and may be preliminarily suggested for the first-line typing of M. tuberculosis isolates in Bulgaria.

Molecular snapshot of drug-resistant and drug-susceptible Mycobacterium tuberculosis strains circulating in Bulgaria.

We report results of the first study on the molecular basis of drug resistance in Mycobacterium tuberculosis strains currently circulating in Bulgaria. The study panel consisted of 133 (including 37 drug-resistant) isolates recovered from newly diagnosed, adult pulmonary TB patients from different regions of Bulgaria in 2005--2006. Three types of the rpoB mutations were found in 20 of 27 RIF-resistant isolates; rpoB S531L was the most frequent. Eleven (48%) of 23 INH-resistant isolates had katG S315T mutation. inhA -15C > T mutation was detected in one INH-resistant isolate (that also had katG315 mutation) and three INH-susceptible isolates. A mutation in embB306 was found in 7 of 11 EMB-resistant isolates. Comparison with spoligotyping and 24-VNTR locus typing data suggested that emergence and spread of drug-resistant and MDR-TB in Bulgaria are not associated with any specific spoligotype or MIRU-VNTR genotype.

Cell wall deficiency and its effect on methicillin heteroresistance in Staphylococcus aureus.

Clinical strains of Staphylococcus aureus with different phenotypic methicillin susceptibility characteristics, bearing or lacking the mecA gene, were tested for their ability to transform into a cell wall-deficient state under special conditions of cultivation. Conversion to L-form growth with formation of typical L-form 'fried egg' colonies and expression of oxacillin resistance was observed in sensitive (mecA-negative) and heteroresistant (mecA-positive) strains. Transmission electron microscopy observation of these strains revealed pleomorphic populations of cell wall-deficient cells with ultrastructure morphology similar to that of a control stable L-form strain of S. aureus. The results demonstrate that expression of phenotypic methicillin resistance could be associated with cell wall deficiency in S. aureus strains and could underlie the phenomenon of heteroresistance.

Antibacterial and antioxidant electrospun materials from poly(3-hydroxybutyrate) and polyvinylpyrrolidone containing caffeic acid phenethyl ester - "in" and "on" strategies for enhanced solubility.

Caffeic acid phenethyl ester (CAPE) possesses a set of valuable biological properties: antioxidant, antibacterial, antitumor, anti-inflammatory, antiviral, etc. However, CAPE is poorly soluble in aqueous environment which is limiting its possible therapeutic applications. In the present study novel fibrous materials enhancing CAPE solubility and accelerating CAPE release were developed. The materials were prepared from poly(3-hydroxybutyrate) (PHB) by electrospinning and by electrospinning combined with dip-coating. The effects of the composition - without/with addition of polyvinylpyrrolidone (PVP) and of the design of fiber (CAPE in the bulk of the fiber or incorporated in the PVP coating) on some of the properties of these materials were studied. X-ray diffraction and differential scanning calorimetry analyses revealed that CAPE was in the amorphous state in CAPE-loaded fibers and in the PVP coating. The new CAPE-containing materials exhibited good antioxidant activity. The microbiological screening demonstrated that incorporation of CAPE in the fibers or in the coating induced complete killing of Gram-positive S. aureus and led to inhibition of the growth of Gram-negative E. coli by the fibrous materials. Moreover, pathogenic S. aureus did not adhere onto CAPE-containing fibrous mats. Therefore, the obtained materials are promising candidates for use as wound dressing materials.