Age changes of foot microbiome estimated by microscopy method.

Despite of great number of investigations in the area of tinea pedis, question is opened: to what extent dermatophyte fungi are spread among modern population and does their occurrence interrelated with host age? Investigated group included 99 volunteers from 14 to 73 years old. Skin scales were collected from heel area of foot, and signs of heel skin trouble were expressed in points. In contrast to usual laboratory microscope magnification x900 we worked at x1750, what allowed to estimate not only fungal, but bacterial forms too. Average abundances of microbial morphotypes were expressed in points. Heel skin trouble increased in the process of aging (Pirsons` coefficient r=0.954). Bacilli occurred in all persons independently from age, but their abundance increased with aging (0.821). On the contrary cocci were more common and abundant in young person`s feet (-0.620). Occurrence of dermatophytes increased with age (0.891), at that relatively high values took place in young persons (10.5% with mycelium and 73.7% with spores) and in group without any heel skin trouble symptoms (7.7% and 76.9%), what allow to refer these fungi to normal habitats of foot skin.

Method of salivary histatins determination.

Histatins are the most significant antimicrobial peptides (AMP) of saliva and there are 12 types of such AMP. Histatin molecules contain relatively high percent of histidine and tyrosine residues. This property allows to use well known from organic chemistry Pauly reaction for detection of protein bounded histidine and tyrosine residues (BHT), which are in fact characterize the summary content of all histatins in saliva. Aim of the present study was comparison of BHT with antimicrobial activity of salivary AMP fraction in patients with inflammatory diseases of upper airways (IDUA). Group of examined persons include 28 patients with different diagnoses: chronic pharyngitis (n=11), chronic tonsillitis (n=7), nasopharyngitis (n=5), pollinosis (n=5). Degree of intensity of inflammatory symptoms was estimated in balls. The algorithm of BHT analysis include following steps: freezing - thawing of saliva; removal of microparticles by centrifugation; separation of fraction lower than100 kDa; dialysis for free amino acids removal; Pauly reaction carrying out. Antimicrobial activities of saliva and its low molecular fractions were estimated towards Candida albicans cells by the spectrophotometric method with bromocresol purpur. Analysis of saliva sediments for coccoid microbiota was carried out by PCR method. Pauly reaction for histatins estimation in saliva of IDUA patients use here for the first time. The histatins levels (BHT) were significantly correlated with the intensity of inflammatory symptoms (r=0,975) and activity of low molecular salivary fraction (AMP activity) (r=0,824). The AMP activity/ BHT ratio, i.e. antimicrobial activity of histatin unit, decreased together with growth of inflammatory symptoms intensity (r=-0,944). Any considerable differences in coccoid microbiota frequency of finding at different diagnoses were not detected. The S. aureus frequency of occurrence was connected neither with inflammatory symptoms intensity (r=0,118), nor with BHT concentration (r=0,318). However S. pyogenes and S. pneumoniae frequencies of occurrence demonstrated the invert correlation towards these indexes: (r=-0,627/-0,614) and (r=-0,827/-0,864). Probably at the exacerbation forms of IDUA the S. pyogenes and S. pneumoniae growth controlled by high levels of histatins.

Unfavorable Humoral Factors for Spermatozoon Survival in Vaginal Medium.

The effects of vaginal secretion, its antibacterial peptide fraction, albumin, and bacterial metabolites on the spermatozoon membranes were studied. Vaginal secretion was collected from healthy women; the fraction of antibacterial peptides was isolated with the use of membrane filter; electrophoretically pure BSA served as albumin; bacterial metabolites were isolated from supernatants of cultured Candida albicans yeast and E. coli and Staphylococcus aureus bacteria. Antibacterial activities of the preparations were evaluated by spectrophotometry and microscopy. Albumin in a concentration close to its serum concentration (50 mg/ml) destroyed spermatozoon membranes by 83.8±2.7% with the formation of vesicular debris. The native vaginal secretion and its antibacterial peptide fraction also destroyed spermatozoa by 50.8±4.4 and 18.0±9.7%, respectively. Activities of bacterial metabolites in concentrations close to the natural did not surpass 3%.

[New in clinical laboratory diagnostics dermatophyte fungi in children with atopic dermatitis.]

Role of bacteria Staphylococcus spp., yeasts of Candida spp., Malassezia spp. genera in pathogenesis of atopic dermatitis (AD) in infant patients is well known. However, no data concerning the incidence of dermatophytes in such disease entity were obtained. Aim of the study was estimation of dermatophytes carriage in children with AD. Group of patients involved 44 persons 1 to 18 years old with clinically diagnosed acute AD. Especially for the study a method of skin scales collection was created: painless for patients and safe for medical personnel. The method is based on following stages - rubbing of lichenificated skin zones with sterile swab which was preliminary saturated with phosphate/Tween 80 buffer pH 7,9 and centrifuging of the suspension for sedimentation of skin scales. Microscopic examination of hydrolyzed scales was carried out at maximal magnification x1750, at that different dermatophyte and yeast fungal forms were registered. Spores of dermatophyte fungi were detected with 67,0% frequency, whereas dermatophyte mycelium - with 18,3% frequency. No correlation between dermatophyte spores and mycelium was found out (Pirson`s coefficient r=0,236), however no mycelium without spores was detected, but in 73,3% of cases spores occurred without mycelium. Yeast spores were detected in 45,2% of patients - this fact is agreed with data obtained earlier by the seeding method, but yeast mycelium was found out in 3,5% of patients.Therefore frequent occurrence of dermatophyte fungi on the skin of AD infants is revealed for the first time. At that dermatophytes were found out to exist more often in the form of spores. These results are important for choice of the further therapy.

[The use of real time PCR for quantitative determination of some propionic bacteria residing on human skin].

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Propionibacterium species: P. acnes, P. granulosum and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen.

[The use of real time PCR for quantitative determination of some propionic bacteria residing on human skin].

A test system has been developed for determination of propionic bacterial species residing on human skin. This system developed in the real time PCR format is applicable for quantitative determination and also detection of genomes of the following Propionibacterium species: P. acnes, P. granulosum and P. avidum. This system was used for analysis of wash samples from the skin of 17 pentathlon sportsmen and 16 students. All three species of propionic bacteria were found in all skin wash samples. However, contamination with P. acnes was two times higher in control group than in the group of pentathlon sportsmen.

[Isolation and study of perspective probiotic strain of spore-forming bacteria from Bacillus genus].

AIM: To study physiologic, biochemical as well as antagonistic characteristics of isolated strain of Bacillus pumilus in comparison with known spore-forming bacteria from B. subtilis species. MATERIALS AND METHODS: Four strains of Bacillus spp. were used in the study: B. subtilis 3H, B. subtilis 534, B. subtilis 1719, B. pumilus isolated from the environment. The following strains from the collection of Tarasevich State Institute of Standardization and Control for Immunobiologicals were used for determining of antagonistic activity: Staphylococcus xylosus 25, Proteus mirabilis 24a, S. aureus "Nikiforov", S. aureus "Filippov", P. vulgaris 177, Shigella flexneri 337, Escherichia coil O111:H55, S. sonnei 170, Pseudomonas aeruginosa 9022, Candida albicans 690; clinical test-strains of yeast fungi: C. albicans (3 strains), C. haemuloni, C. tropicalis, Rhodotorula spp., Debaryomyces hansenii. RESULTS: Identification of isolated strain on combination of morphologic, tinctorial, cultural and biochemical characteristics showed that studied strain belonged to B. pumilus species. It did not have hemolytic and lecitinase activities, was resistant to several groups of antibiotics and had index of adhesion 1.76 +/- 2.5. In experiments in vivo the strain was non-toxic, non-toxigenic and avirulent. In preparations of isolated DNA, plasmids were not found. B. pumilus had high antagonistic effect against opportunistic and pathogenic bacteria as well as yeast fungi. Comparative assessment of tested strains from Bacillus genus showed that the isolated strain was not inferior than other representatives of spore-forming bacteria on spectrum and intensity of aforementioned characteristics. CONCLUSION: Low-adhesive, safe, plasmid-less strain of B. pumilus, which have intensive antagonistic properties, could be considered as a candidate for the development of new probiotic drugs for medical or veterinary use.

Antagonistic activity of Malassezia Spp. towards other clinically significant yeast genera.

Antagonistic activity of Malassezia yeast towards clinically significant yeast species was studied. Ten Malassezia strains exhibited this activity. M. furfur strain exhibited maximum activity and the least sensitivity to "foreign" metabolites. M. globosa proved to be the most sensitive and the least active. M. furfur metabolites exhibited pronounced activity towards 6 Basidiomycetes strains. This effect was significantly higher in comparison with antagonistic activity towards 13 Ascomycetes species. Studies of a complex of M. furfur antagonistic metabolites showed that it has at least two components: thermolabile proteins with molecular weights of 33 and 35 kDa and a thermostable one, proteinase-resistant. In contrast to metabolites of many other yeast species, this substance is more effective against related Basidiomycetes microorganisms (Cryptococcus albicans), while antagonistic proteins are active mainly towards Ascomycetes, such as Candida albicans. It was found that mycocin-like activity of Malassezia is encoded by chromosomes, but not plasmids.

[Assessment of rate of hydroxyl anions production by Helicobacter pylori in stomach].

Production of hydroxyl anions by tissue samples of pylorus mucous membrane obtained from 45 patients with gastric or duodenal ulcers was investigated. The production was estimated using the recently developed method based on measurement of rate of pH change in urea-containing reaction mixture. The rate of [OH-] generation as a result of H. pylori metabolism accounted on pylorus square varied from 0.4 to 1318.9 mcmol [OH-]/min, and in 90.2% of cases it did not exceed 128.1 mcmol [OH-]/min. This rate is comparable to mean rate of [H+] generation in stomach of healthy man--114.2-238.4 mcmol [H+]/min. Obtained results allow to conclude that this bacterium may participate in regulation of stomach acid-base balance.

[Influence of the degree of aeration on halotolerance of yeasts of the genera Candida, Rhodotorula, and Malassezia]. / Vliianie stepeni aératsii sredy na galotolerantnost' drozhzhei rodov Candida, Rhodotorula i Malassezia.

The biochemical mechanisms were studied that determine different reactions of yeasts of different genera to two simultaneously imposed stressors, hypoxia and osmotic shock. For Candida lipolytica, these two stressors were antagonistic, which resulted in stimulation of yeast growth by NaCl (in a wide range of concentrations) under microaerobic conditions. The reaction of Malassezia sp. was different: the degree of halotolerance of this microorganism was lower under microaerobic conditions. An intervening reaction pattern was characteristic of Rhodotorula aurantiaca. These differences were found to be determined, above all, by the induction of a salt-resistant respiratory system (oxidase) in Candida lipolytica, which could not be induced in Malassezia sp. In addition, the synthesis of catalase was enhanced in Candida lipolytica, which provided for neutralization of the active forms of oxygen accumulating as a result of inhibition of other protective enzymes by salt.

[Killer toxins of clinically important yeasts]. / Killernye toksiny klinicheski znachimykh drozhzhei.

The review deals with some theoretical and applied aspects of the capacity of yeasts for synthesizing toxins. Similarly to antibiotic formation in micellar fungi and actinomycetes and the synthesis of bactericins in prokaryotes, yeast cells also have their mechanism of protection from other microorganisms. The substances, essentially of the same nature, synthesized by yeast are known for more than 30 years as mycocins or killer toxins. They are proteins or glycoproteins, active mainly against yeast microorganisms. Mycocins are not active against bacteria and protozoa exhibiting only fungicidal or fungistatic action. The formation of mycocins may be determined by nucleus or plasmid DNA. In this review information on killer toxins produced by clinically important yeasts of the genera Candida, Cryptococcus and Rhodotorula is systematized.

[Minimum inhibitory concentrations of various antifungal agents against Basidiomycetes clinical isolates]. / Minimal'nye podavliaiushchie kontsentratsii nekotorykh protivogribkovykh preparatov dlia klinicheski znachimykh bazidiomitsetnykh drozhzhei.

The basidiomycete yeasts are often isolated from clinical samples. A minimal inhibiting concentrations (MIC) of ten antifungals of different groups--azols, allilamines, polyens etc.--against isolates of Rhodotorula, Cryptococcus [symbol: see text] Trichosporon yeasts genera were estimated. No one of these cultures was sensitive to azoles at concentrations 0-256 mcg/ml. A vitality of cultures after incubation during 10 days with antifungals was investigated. Miramistin was the most potent fungicidal agent against all cultures.

[Antagonistic interactions between stress factors during the growth of microorganisms under conditions simulating the parameters of their natural ecotopes]. / Antagonistitseskoe vzaimodeistvie stressornykh faktorov pri roste mikroorganizmov v usloviiakh, imitiruiushchikh parametry ikh prirodnykh ékotopov.

Two stress factors, hypoxia (microaerobic conditions) and a high salt concentration, if applied simultaneously to aerobic microorganisms, display an antagonistic mode of interaction. As a result, the NaCl level that is usually optimal for moderate halophiles (5-6%) becomes optimal for the growth of weak halophiles (Rhodococcus erythropolis and Shewanella sp. CN32); the halotolerant yeast Yarrowia lypolytica acquires halophilic properties (with a growth optimum at a NaCl concentration of 10%), and the growth rate of the extremely halophilic Halobacterium salinarum increases at supraoptimal salt concentrations (25-34%). This phenomenon is apparently due to multiple changes in metabolic reactions. In particular, high salt concentrations suppress respiration and the formation of enzymes (superoxide dismutase and catalase) that protect the cell from toxic oxygen species. Therefore, establishment of microaerobic conditions compensates for the loss of these protective mechanisms and enables cell growth at higher salt concentrations than under aerobic conditions. Of some importance can also be the increase in the intracellular concentrations of osmoprotectants caused by the suppression of their intracellular oxidation. The implications of this phenomenon for the ecophysiology of microorganisms (including oiloxidizing species) and for the classification of weak and moderate halophiles are discussed.

[The yeast malassezia on the skin of healthy individuals and patients with atopic dermatitis]. / Griby malassezia na kozhe zdorovykh liudei i bol'nykh atopicheskim dermatitom.

The lipophilic yeast Malassezia spp. from the skin of 32 healthy individuals and 21 patients with atopic dermatitis was isolated and identified. Malassezia sympodialis was shown to be most common in both groups, M. Globosa was less frequently encountered. Moreover, no Malassezia was found in 34 and 53% of cases, respectively. There was a relationship of the skin colonization of Malassezia to the patients' age. Malassezia spp. cultures whose appurtenance was impossible to identified by the Guillot-Gueho method were detected.

[Synthetic media for cultivation of lipophilic yeast malassezia spp]. / Sinteticheskie sredy dlia kul'tivirovaniia lipofil'nykh drozhzhei malassezia spp.

The capacity of the lipophilic yeast Malassezia spp. (Pityrosporum spp.) to grown in a synthetic nutritional media has been studied. The modified Dixon's medium was taken as a prototype for preparing the medium containing amino acid, Tween, and an emulsifier as carbon and energy sources. The isolates of M. sympodialis have demonstrated to relatively more rapidly grow in the medium than those of M. globosa.

[Clinico-microbiological assessment of therapeutic antifungal drugs therapeutic activity in patients with atopic dermatitis sensitized to yeast fungi]. / Kliniko-mikrobiologicheskaia otsenka terapevticheskoi éffektivnosti protivogribkovykh preparatov u bol'nykh atopicheskim dermatitom, sensibilizirovannykh k drozhzhepodobnym gribam.

AIM: To study of therapeutic activity of local (natamycine) and systemic (fluconasol) antifungal drugs in patients with atopic dermatitis (AD) with mycogenic sensitization and candida carriage. MATERIALS AND METHODS: 29 patients with severe AD and diagnosed contamination of the skin lesions with yeast fungi have received antifungal treatment with fungicidal drugs in low-dose long-term course. RESULTS: The drugs were effective in 83% of patients: poor effect was in 28%, moderate--in 21% and good--in 34% of cases. 5 (17%) patients did not respond. Mild, moderate and severe AD responded in 87, 36 and 60% of patients, respectively. The highest response was registered in patients without sensitization to yeast fungi (78%) or low sensitization (83%). In high sensitization the effect occurred in 57% of the cases. CONCLUSION: The authors have developed indications for treatment with antifungal drugs in AD patients with mycogenic sensitization.

[Yeast-like fungi on the skin of patients with atopic dermatitis]. / Drdozhzhepodobnye griby na kozhe bol'nykh atopicheskim dermatitom.

Colonization by yeast-like flora and its composition on the skin of patients with atopic dermatitis (AD) in comparison with those on the skin of patients with skin diseases of nonatopic etiology and healthy subjects were studied. Inoculations from affected and unaffected sites of the skin were made by the contact method with the use of bacteriological plates with antibiotic-containing solid agar medium. The study revealed that 39% of AD patients, 45% of patients with other skin diseases and 3% of healthy subjects had yeast-like fungi of their skin, Candida spp. (53%) and Rhodotorula spp. (21%) occurring most frequently. In patients with a severe course of AD the tendency towards the preferable colonization of affected sites of the skin was observed: in these cases yeasts occurred 2.5-5 times more frequently than in patients with the mild and moderate course of AD.