Synthesis and Investigation of Physicochemical and Biological Properties of Films Containing Encapsulated Propolis in Hyaluronic Matrix.

The dynamic development of nanotechnology has enabled the development of innovative and novel techniques for the production and use of nanomaterials. One of them is the use of nanocapsules based on biodegradable biopolymer composites. Closing compounds with antimicrobial activity inside the nanocapsule cause the gradual release of biologically active substances into the environment, and the effect on pathogens is regular, prolonged and targeted. Known and used in medicine for years, propolis, thanks to the synergistic effect of active ingredients, has antimicrobial, anti-inflammatory and antiseptic properties. Biodegradable and flexible biofilms were obtained, the morphology of the composite was determined using scanning electron microscopy (SEM) and particle size was measured by the dynamic light scattering (DLS) method. Antimicrobial properties of biofoils were examined on commensal skin bacteria and pathogenic Candida isolates based on the growth inhibition zones. The research confirmed the presence of spherical nanocapsules with sizes in the nano/micrometric scale. The properties of the composites were characterized by infrared (IR) and ultraviolet (UV) spectroscopy. It has been proven that hyaluronic acid is a suitable matrix for the preparation of nanocapsules, as no significant interactions between hyaluronan and the tested compounds have been demonstrated. Color analysis and thermal properties, as well as the thickness and mechanical properties of the obtained films, were determined. Antimicrobial properties of the obtained nanocomposites were strong in relation to all analyzed bacterial and yeast strains isolated from various regions of the human body. These results suggest high potential applicability of the tested biofilms as effective materials for dressings to be applied on infected wounds.

Preparation of Nano/Microcapsules of Ozonated Olive Oil in Hyaluronan Matrix and Analysis of Physicochemical and Microbiological (Biological) Properties of the Obtained Biocomposite.

Hydrogels, based on natural polymers, such as hyaluronic acid, are gaining an increasing popularity because of their biological activity. The antibacterial effect of ozone is widely known and used, but the instability the gas causes, severely limits its application. Ozone entrapment in olive oil by its reaction with an unsaturated bond, allows for the formation of stable, therapeutically active ozone derivatives. In this study, we obtained an innovative hydrogel, based on hyaluronic acid containing micro/nanocapsules of ozonated olive oil. By combination of the biocompatible polymer with a high regenerative capacity and biologically active ingredients, we obtained a hydrogel with regenerative properties and a very weak inhibitory effect against both bacterial commensal skin microbiota and pathogenic Candida-like yeasts. We assessed the stability and rheological properties of the gel, determined the morphology of the composite, using scanning electron microscopy (SEM) and particle size by the dynamic light scattering (DLS) method. We also performed Attenuated total reflectance Fourier transform infrared (FTIR-ATR) spectroscopy. The functional properties, including the antimicrobial potential were assessed by the microbiological analysis and in vitro testing on the HaCat human keratinocyte cell line. The studies proved that the obtained emulsions were rheologically stable, exhibited an antimicrobial effect and did not show cytotoxicity in the HaCat keratinocyte model.

Retrospective Analysis of the Ureaplasma spp. Prevalence with Reference to Other Genital Tract Infections in Women of Reproductive Age.

Ureaplasma spp. are frequently isolated from the genital tract of women of reproductive age. To date, it remains unclear whether they are commensal or pathogenic. In our study, we assessed the prevalence of Ureaplasma spp. in a group of 1,155 women of childbearing age. In addition, we assessed how often women with positive Ureaplasma spp. develop genital tract co-infections and how the vaginal pH changes. This study showed a relationship between colonization by Ureaplasma spp. and presenting symptoms. In fact, we showed that colonization of the genital tract by Ureaplasma spp. can affect the occurrence of co-infections such as Gardnerella vaginalis. We also observed a relationship between increased pH values and the presence of Ureaplasma spp.

Fungal contamination of ward furnishings and medical equipment used in the treatment and nursing of newborns.

INTRODUCTION AND OBJECTIVE: Newborn babies staying on hospital wards are likely to be colonized by microorganisms, including potentially pathogenic fungi. The aim of the study was to assess the mycological purity of hospital wards and medical equipment utilized in the treatment and nursing of newborns. MATERIAL AND METHODS: The study was conducted in Neonatal High Dependency Units (NHDU) and Neonatal Intensive Care Units (NICU). 539 samples were collected from 24 different sources, 130 from ward furnishings and 289 from medical equipment. The study was carried out following the microbiology research methods for sample collection. Subsequently, the samples (swabs, water from incubators, washings from respirator tubes and nasal cannulas (nCPAP)) were cultivated on Sabouraud agar plates. The stamps were collected with the application of Count-Tact method. The samples were incubated at the temperature of 25+/-2 o C and the number of fungi assessed (cfu/cm -2 of the surface area). The species were identified based on their morphological and biochemical features. RESULTS: Fungal growth was observed on 60% of samples collected from ward furnishings and 7% of samples collected from medical equipment. The average number of cfu/cm -2 ranged between 0-8.84 in the case of ward furnishings and between 0-1.22 cfu/cm -2 in the case of medical equipment. In 180 samples collected from the material which had direct contact with newborns no fungal growth was observed. CONCLUSIONS: The furnishings of the wards on which newborns were treated and nursed were contaminated with fungi to an extent which did not pose a threat to the life and health of the newborns. Medical equipment (respirators, incubators, nCPAP cannulas and masks) which came into direct contact with newborns was free from fungi.

Combined Biological Effects of N-Bromotaurine Analogs and Ibuprofen. Part II: Influence on a Local Defense System.

The stable N-bromotaurine analogs (N-dibromo-dimethyl taurine, N-monobromo-dimethyl taurine), and bromamine T (BAT) show anti-inflammatory and microbicidal properties. These bromamines are good candidates for a treatment of skin infectious/inflammatory diseases as local antiseptics. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), is commonly used in various infectious/inflammatory diseases due to its analgesic and antipyretic therapeutic effects. However, systemic administration of ibuprofen may also result in adverse side effects. It has been reported that ibuprofen enhances serum levels of TNF-α and worsens secondary skin infections caused by invasive streptococci (S. pyogenes). Recently we have demonstrated that bromamines inhibit the stimulatory effect of ibuprofen on the production of inflammatory cytokines (TNF-α, IL-6). The aim of this study was to examine the combined antibacterial actions of ibuprofen and bromamines against S. pyogenes and their joint effect on the generation of reactive oxygen species (ROS) by activated neutrophils and macrophages. We have shown that the microbicidal activity of bromamines against S. pyogenes was not altered by ibuprofen. On the other hand, co-administration of ibuprofen and bromamines markedly decreased the generation of ROS by activated neutrophils and macrophages. Finally, we discuss how the antioxidant combined effect of bromamines and ibuprofen may affect a local defense system.

Phagocytosis of live versus killed or fluorescently labeled bacteria by macrophages differ in both magnitude and receptor specificity.

Scavenger receptor (SR)-mediated opsonin-independent phagocytosis of bacteria by macrophages has been suggested to represent an important, early mechanism of anti-bacterial host defense. However, although the ability to bind bacteria has been demonstrated to be a shared feature of all types of SRs, in many cases the evidence is limited to the demonstration of increased binding of killed, fluorescently labeled bacteria to non-phagocytic cells transfected with these receptors. We sought to verify the ability of SRs to mediate non-opsonic phagocytosis of live Escherichia coli (Ec) and Staphylococcus aureus (Sa), model species of Gram-negative and -positive bacteria, respectively, and to assess the relative contributions of different SRs expressed on murine macrophages in this process. We found that the class A SR SR-A/CD204 was the major receptor mediating phagocytosis of fluorescently labeled Sa, whereas different SRs had highly redundant roles in the phagocytosis of live Sa. Conversely, different SRs contributed to the phagocytosis of fluorescently labeled Ec. In comparison, phagocytosis of live Ec was of much lower magnitude and was selectively mediated by SR-A. These results question the use of fluorescently labeled bacteria as valid replacements for live bacteria. The low magnitude of opsonin-independent phagocytosis of Ec and unimpaired phagocytosis of Sa in SR-A- or CD36-deficient macrophages indicate that the defect in this process might not be responsible for the reported impaired bacteria clearance in mice deficient in these receptors. We postulate that this impairment might result to a larger extent from inhibition of intracellular bacteria killing caused by pro-inflammatory cytokines, produced in excessive amounts by SR-deficient cells in response to bacterial products.

Multiple-locus variable-number tandem repeat fingerprinting as a method for rapid and cost-effective typing of animal-associated Staphylococcus aureus strains from lineages other than sequence type 398.

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

[Identification of yeasts isolated from the genital tract of women by mass spectrometry MALDI-TOF].

INTRODUCTION: A fungal infection of vagina is one of the most common gynecological problems of women of childbearing age. There has been a gradual increase in yeast- like fungi which are problematic to identify. There is thus a need for searching new identification methods, which would allow for fast recognition and selection of the proper course of treatment. The aim of study was to evaluate the usefulness of MALDI-TOF mass spectrometry technique in the diagnosis of pathogenic yeast-like fungi isolated from the genital tract of women. METHODS: There were identified 484 yeast-like fungi strains isolated from the genital tract of patients treated at Centrum Badah Mikrobiologicznych i Autoszczepionek im. Jana Bobra in Cracow (CBMiA) in the age group 20 - 50. The material was analyzed using streak plating on the culture medium: Sabouraud agar (CBMiA) and chromogenic media chromID Candida (bioMdrieux). The strains were identified using MALDI-TOF mass spectrometer (MALDI Biotyper MicroflexTM Series, Bruker Daltonics, Germany) operating as IVD (In Vitro Diagnostic). The procedure of proteins extraction with the use of ethanol and formic acid was used. RESULTS: All 484 yeast-like fungi strains isolated from the genital tract of women were correctly identified to the species level. CONCLUSIONS: MALDI-TOF mass spectrometry method may be successfully used for routine identification of yeast-like fungi and in particular the strains of non-albicans Candida, which have a lower susceptibility to antifungal agents.

Molecular methods of diagnosing allergic sensitization in patients with atopic dermatitis and infected by with Staphylococcus aureus.

The severity of allergic symptoms in patients with atopic dermatitis (AD) intensifies when the number of colonies patient's of Staphylococcus aureus on patents' skin increases. The basic feature determining the quality of any diagnostic test for S. aureus is its credibility. Performing a test always carries the risk of obtaining false positive and/or false negative results. Furthermore, producing material for microbiological analysis of internal body cavities is sometimes difficult. Therefore, in our study, we compared the results of three tests to determine if their results were mutually compatible and if they confirmed whether S. aureus was present in patients with AD and what was its role in the development of the disease in those patients. Infection with S. aureus was tested in patients with AD and healthy volunteers using the API Staph system. The specific IgE antibodies for staphylococcal enterotoxin A (SEA) and B (SEB) were measured using the UniCAP system. The secretion of IFN-γ, IL-2, IL-13 by peripheral blood mononuclear cells (PBMCs) after stimulation with SEA and SEB were studied with Elispot assay. We found that only certain patients with AD and S. aureus produced antibodies against SEA and SEB in the acute phase of AD. The secretion of IFN-γ was low in patients with exacerbated AD and S. aureus. Testing for the presence of S. aureus in the mucous membrane of the nasal vestibule and skin lesions is not sufficient for complex diagnosis of the role of S. aureus in the pathomechanism of AD. Measuring the presence of antibodies against bacterial components in patients' serum and the reactivity of patients' immune cells against these bacterial components is required in order to accurately diagnose this role of S. aureus in a patient.

Effectiveness of Polyvalent Bacterial Lysate and Autovaccines Against Upper Respiratory Tract Bacterial Colonization by Potential Pathogens: A Randomized Study.

BACKGROUND: Polyvalent bacterial lysate (PBL) is an oral immunostimulating vaccine consisting of bacterial standardized lysates obtained by lysis of different strains of bacteria. Autovaccines are individually prepared based on the results of smears obtained from the patient. Both types of vaccine can be used to treat an ongoing chronic infection. This study sought to determine which method is more effective against nasal colonization by potential respiratory tract pathogens. MATERIAL AND METHODS: We enrolled 150 patients with aerobic Gram stain culture and count results indicating bacterial colonization of the nose and/or throat by potential pathogens. The participants were randomly assigned to each of the following groups: 1. administration of PBL, 2. administration of autovaccine, and 3. no intervention (controls). RESULTS: Reduction of the bacterial count in Streptococcus pneumoniae-colonized participants was significant after the autovaccine (p<0.001) and PBL (p<0.01). Reduction of the bacterial count of other ß-hemolytic streptococcal strains after treatment with the autovaccine was significant (p<0.01) and was non-significant after PBL. In Haemophilus influenzae colonization, significant reduction in the bacterial count was noted in the PBL group (p<0.01). Methicillin-resistant Staphylococcus aureus colonization did not respond to either treatment. CONCLUSIONS: The autovaccine is more effective than PBL for reducing bacterial count of Streptococcus pneumoniae and ß-hemolytic streptococci, while PBL was more effective against Haemophilus influenzae colonization.

Methicillin-resistant Staphylococcus pseudintermedius infection in a bone marrow transplant recipient.

Staphylococcus pseudintermedius is a veterinary pathogen that has seldom been described as an agent of human disease. Features of this probably underreported coagulase-positive Staphylococcus species are depicted here through the description of a graft-versus-host disease-related wound infection caused by a multidrug-resistant strain.

[Efficacy of taurine haloamines and chlorhexidine against selected oral microbiome species]. / Porównanie mikrobójczych wlasciwosci haloamin tauryny i chlorheksydyny wobec wybranych drobnoustrojów mikrobiomu jamy ustnej.

INTRODUCTION: Uncontrolled bacteria of dental plaque generate formation of oral biofilm located on teeth and subgingival surfaces. It may induce local inflammation (gingivitis) with further development of periodontal diseases. A variety of oral bacteria such as Streptococcus mutans and Porhyromonas gingivalis are involved in pathogenesis of dental carries and periodontitis. Very often bacterial infections are associated with candidiasis (Candida albicans). Chlorhexidine (CHX) is the most commonly used antiseptic in dentistry due to its strong antibacterial activity and capacity to reduce the accumulation of oral biofilms. However, other antiseptics, especially endodontic irrigants, are still tested to improve their preventive and therapeutic effects in oral cavity infections. In this in vitro study we have compared antimicrobial activity of CHX with that of taurine chloramine (TauC1) and taurine bromamine (TauBr), natural taurine derivatives with known antibacterial and anti-inflammatory properties. METHODS: Antimicrobial activity of CHX, TauC1 and TauBr was tested by incubation of the compounds with S. mutans, P gingivalis and C. albicans. The agents were incubated in low (105/ml) and high (108/ml) density microbe suspensions, related to early and late biofilm infections, respectively. In some experiments bacteria were incubated with a combination of CHX + NaOCl and CHX + TauBr. MIC was determined by the pour-plate method. RESULTS: CHX showed the strongest antimicrobial activity against all tested pathogens. On the contrary, TauC1 was the weakest antiseptics used without effect on the growth of C. albicans. TauBr at non-cytotoxic concentrations inhibited the growth of S. mutans and P gingivalis with slight effect on the low density C. albicans. All tested agents showed weaker antiseptic properties in the presence of serum. Moreover, we have shown that interactions between CHX and sodium hypochlorite (NaOC1), the main endodontic irrigant, but not between CHX and TauBr,resulted in precipitation. Therefore, it may restrict their simultaneous application in root canal treatment. However, in spite of this unwanted reaction, the mixture of CHX with NaOCl kills pathogens more effectively then CHX alone. CONCLUSIONS: The results confirmed CHX exceptional potential as primary antiseptic in dentistry, especially in prevention and treatment of dental carries, periodontal diseases and mouth candidiasis. Moreover, our study shows that TauBr may be used alternatively or in combination with CHX in killing of oral pathogens, due to its strong antibacterial and anti-inflammatory properties.

[A comparison of biochemical and genetic classification of propionibacterium acnes strains isolated from skin lesions of patients with acne]. / Porównanie biochemicznej i genetycznej klasyfikacji szczepów Propionibacterium acnes izolowanych ze zmian skórnych od osób z tradzikiem mlodzienczym.

INTRODUCTION: Propionibacterium acnes is dominating group of resident bacteria in skin biocenosis. These bacteria participate in autosterilisation of skin with the process of decomposition of triglycerides into free fatty acids and by keeping the pH of skin on the level 5.5. When the process goes out of control the excess of fatty acids in sebaceous glands leads to necrosis and inflammation. Apart of the presence on the skin P acnes also are present in mucous membranes of intestinal tract, eyes, internal ears channels, and in upper respiratory tracts. Traditionally they are regarded as anaerobes, but they tolerate oxygen atmosphere and are resistant to phagocytosis, surviving even in macrophages. These bacteria produce a number of enzymes and proinflammatory factors activating monocytes, stimulating mitogenic activity of lymphocytes T. According to common opinion they are responsible for disease acne vulgaris, but there are also researchers claiming their low pathogenicity. The list of the P acnes related diseases is not short, some of these diseases may be facilitated by predisposing factors as surgery interventions, diagnostic, or cosmetic procedures. The aim of the study was to compare standard biochemical analysis of P acnes strains to genotypic typing basing on the results from MP-PCR analyses. Relations of hemolysis activity to biochemical types or genetic types were also analysed. METHODS: The analysis of 66 P acnes strains isolated from skin lesions of patients with acne vulgaris was performed. A collection of the strains was analysed biochemically according to Pulverer, Sourek and Hoffler method modified by Kasprowicz, and typed genetically by MP-PCR method. Relations of biochemical and genetic types to beta-hemolysis of strains were studied. RESULTS: Dominating biochemical type was type number I grouping 79% of all isolates, and dominant genotype was A which was detected in 75% of all collected strains. Beta-hemolysis was a feature present in 34% of strains, more frequently in type I (40%) than in type II (12%). beta-hemolysis was present only in A-genotype strains, but A-genotype by itself does not determine that reaction: 53% of A-genotype strains does not exhibit beta-hemolysis. All type I strains represent A-genotype. Type II was genotypicaly differentiated: all genotypes A, A', B, and C were present. CONCLUSIONS: The results obtained show genotypic heterogenity of P acnes strains and relations to biochemical types. Hemolysis was detected independently to biotype or genotype representation. The results confirm biochemical and genetic heterogenities of P acnes, but the observations also indicate necessity of further microbiological-molecular investigation of that bacteria group using other molecular techniques to the study.

The occurrence and comparative phenotypic characteristics of Staphylococcus spp. from healthy and diseased, household and shelter dogs, based on routine biochemical diagnostic methods.

To determine the staphylococcal colonization pattern in healthy and diseased dogs, living in two particular environments, a number of microbiological samples were taken. Overall, twenty dogs, either healthy or with infected skin lesions, were examined. In each case bacterial swabs were collected from the nasal mucosa, ear, perineum, lumbo-sacralis triangle, and from the infection sites if such were present. A total number of 104 isolates representing different staphylococcal species were isolated and identified using routine biochemical methods applied in diagnostic laboratories. Among 17 isolated staphylococcal species, Staphylococcus intermedius was the most common species isolated from both healthy or diseased dogs living either in animal shelter or household environments. The pattern of Staphylococcus sp. colonization differs considerably for animals living in the two tested habitats. In particular, S. aureus MRSA and MSSA isolates were detected only in infected skin lesion samples from animals that dwelled in the animal shelter. As could be expected, S. intermedius was found to be a predominant causative agent in canine skin infections. In our study, we demonstrated that S. intermedius in its carrier-state, inhabits mainly the mucosal membrane of the nasal vestibule. It was also found in the samples taken from the skin, the lumbo-sacralis triangle and perineum, but was rarely isolated from the ears.

The first case of a Staphylococcus pseudintermedius infection after joint prosthesis implantation in a dog.

We have reported a bacterial infection in a dog with progressive dysplasia of the hips. Orthopedic surgery was performed. Seven weeks prior to the surgery, the patient was bitten by another dog. The postimplantation wound exuded for four days after the surgery. Microbiological analysis performed by standard identification techniques showed the presence of Staphylococcus intermedius, but an additional molecular analysis indicated S. pseudintermedius. This was followed by an evaluation of antibiotic susceptibility of the strain which showed cefoxitin, ciprofloxacin, clindamycin, trimethoprim/sulfamethoxazole, doksycycline, erythromycin, and gentamicin resistance. Minimal inhibitory concentration (MIC) values for selected antibiotics were reported. Resistance for cefoxitin indicates that methicillin resistant S. pseudintermedius (MRSP) strains were present in individual macroorganisms, but they can expand and persist the colonization of other hosts.

[Differentiation of Staphylococcus aureus isolates based on phenotypical characters]. / Róznicowanie izolatów Staphylococcus aureus w oparciu o cechy fenotypowe.

Typing of Staphylococcus aureus isolates is a necessary procedure for monitoring the transmission of S. aureus among carriers and in epidemiology. Evaluation of the range of relationship among isolates rely on epidemiological markers and is possible because of the clonal character of S. aureus species. Effective typing shows the scheme of transmission of infection in a selected area, enables identifying the reservoir of the microorganism, and may enhance effective eradication. A set of typing methods for use in analyses of epidemiological correlations and the identification of S. aureus isolates is presented. The following methods of typing are described: biotyping, serotyping, antibiogram, protein electrophoresis, cell protein profiles (proteom), immunoblotting, multilocus enzyme electrophoresis (MLEE), zymotyping, and standard species identification of S. aureus in the diagnostic laboratory. Phenotyping methods for S. aureus isolates used in the past and today in epidemiological investigations and in analyses of correlations among S. aureus isolates are presented in this review. The presented methods use morphological characteristics, physiological properties, and chemical structures of the bacteria as criteria for typing. The precision of these standard methods is not always satisfactory as S. aureus strains with atypical biochemical characters have evolved recently. Therefore it is essential to introduce additional typing procedures using molecular biology methods without neglecting phenotypic methods.

Topical taurine bromamine, a new candidate in the treatment of moderate inflammatory acne vulgaris: a pilot study.

Taurine bromamine (TauBr), the product of taurine and hypobromous acid (HOBr), exerts anti-inflammatory and antibacterial properties. Recently we have shown that Propionibacterium acnes, a potential pathogenic agent of acne, is extremely sensitive to TauBr. As topical antibiotics are associated with the emergence of resistant bacteria, TauBr seems to be a good candidate for topical therapy for acne vulgaris. In our double blind investigation, the efficacy and safety of 3.5 mM TauBr cream was evaluated. 1% Clindamycin gel (Clindacin T), one of the most common topical agents in the treatment of acne vulgaris, was used as a control. Forty patients with mild to moderate inflammatory facial acne vulgaris were randomly treated with either TauBr or clindamycin for 6 weeks, twice-a-day. More than 80% of the patients markedly improved with both treatments, without any adverse effects observed. Both TauBr and clindamycin produced a significant reduction in inflammatory skin lesion counts (papules/ pustules). After 6 weeks, comparable reductions of acne lesions, 65% and 68%, were observed in the TauBr and clindamycin groups, respectively. In conclusion, these data support our concept that TauBr can be used as a topical agent in the treatment of acne vulgaris, especially in patients who have already developed antibiotic resistance.