Antimicrobial Activity of Milk Whey in Different Mammals.

Antimicrobial activity of milk whey in different mammals against Candida albicans yeast cells was studied by a spectrophotometric method. The activity increased in the order goat→horse→camel→cow→human→mouse. The level of whey activity in mice was higher by 3 and 10 times than in humans and goats, respectively. Similar changes were noted for activity of the whey fraction <100 kDa containing a complex of antimicrobial polypeptides, and there was a direct correlation between these two parameters (r=0.881; p<0.05). The total activity of whey had a high degree of correlation with the content of serum albumin (r=0.992); in mice, the level of serum albumin in the milk whey was close to that in blood serum. Interspecific differences between the activity of whey in mammals may be associated with qualitative and quantitative variability of the antimicrobial polypeptide composition, as well as their synergistic or antagonistic interaction with each other.

Detection of Naganishia albida Yeasts in Dermatological Patients.

The yeasts Cryptococcus albidus (Naganishia albida) usually occur on natural substrates and rarely are the etiological factor of different mycoses. More than a half of mycosis cases described in the literature were reported during the period from 2004 to 2021. In this regard, evaluation of yeast sensitivity to antimycotic drugs is as important as their identification. In the present study, two yeast isolates from the skin of female patients (age 7 and 74 years) with infective dermatitis (ICD-10-CM Code L30.3) were studied. Common identification of the isolates, MALDI-TOF mass spectrometry, and analysis of the nucleotide sequences of the ITS1-5.8S-ITS2 rDNA region showed that they belong to the species N. albida. The sensitivity of the obtained strains to antimycotics of three different chemical groups, namely itraconazole, naftifine, and amphotericin B, determined by microdilution method in a synthetic medium showed the following minimum inhibitory concentrations: 64-128, 16, and 0.125-4 µg/ml, respectively. It was found that the sensitivity of this yeast to pooled human serum was 30-47%, i.e. lower by 1.9-2.9 times than the sensitivity of the collection strains of C. albicans and C. neoformans. This result could be explained by lower prevalence of N. albida in the human population in comparison with these species. However, the sensitivity of N. albida strains to the low-molecular-weight fraction of serum was approximately the same as in C. albicans and C. neoformans, which indicates their high sensitivity to antimicrobial peptides.

Antimicrobial Activity of the Serum before and after Vaccination with EpiVacCorona.

We evaluated the effect of vaccination with anti-COVID-19 vaccine EpiVacCorona on serum antimicrobial activity, formation of specific IgG antibodies, and expression of some antimicrobial peptides. Antimicrobial activity of the serum from 55 volunteers towards S. aureus cells was measured spectrophotometrically; IgG-antibodies against SARS-CoV-2 antigen were assayed by ELISA; expression of genes encoding antimicrobial peptides LL37, HBD1, and HBD2 was evaluated by PCR with reverse transcription. Total antimicrobial serum activity and activity of its low-molecular-weight fraction containing antimicrobial peptides demonstrated an inverse correlation. Both activities after vaccination increased in case of low initial values, but decreased in case of high initial values. The vector of change of specific IgG antibodies to coronavirus inversely correlated with the vector of change of activity of antimicrobial peptide fraction. The expression of genes of antimicrobial peptides LL37, HBD1, and HBD2 looked like normal distribution depending on activities of the antimicrobial peptides in the corresponding sera.

Alternative Methods of Evaluation of Antimicrobial Activity of Breast Milk Serum.

Antimicrobial properties are traditionally evaluated by the seeding technique, which is laborious, time-consuming, and rather imprecise. We studied the possibility of using microscopy and spectrophotometry methods for evaluation of cytotoxic activity of breast milk serum against opportunistic microbes. Activity of 50 breast milk samples obtained from healthy women at different lactation stages was tested against opportunistic yeast C. albicans. Microscopy showed that incubation of cell suspension with lactoserum led to destruction of cell walls and cytoplasmic membranes with the formation of vesicular debris that absorbed the dye from the medium. Spectrophotometric measurement of the dye remained in the medium revealed a dose-depended effect of the lactoserum on C. albicans cells and strong inverse correlation between the lactation period and cytotoxic activity of the lactoserum (r=-0.948). These methods can be used in veterinary and food processing for estimation of biological activity of milk.

Effect of Albumin on Tumor Cells In Vitro.

The study examined the effect of BSA on cultured cells of breast cancer, erythroleukemia, and ovarian carcinoma. BSA cytotoxic activity was examined with light microscopy and spectrophotometry in the concentration range of 6.25 to 50 mg/ml. The high concentrations of BSA disrupted the tumor cells in parallel with formation of vesicular debris. The destruction parameters were similar in diverse types of cells: BSA (50 mg/ml) disrupted 75.5±0.7% breast cancer cells, 75.8±0.2% erythroleukemia cells, and 78.8±0.1% cells of ovarian carcinoma (p≥0.05). The effect of BSA was dose-dependent in each type of cells. The data attest that BSA can disrupt various tumor cells in vitro.

Antimicrobial Effect of Albumin on Bacteria and Yeast Cells.

We studied the effect of albumin (human serum, bovine serum, and ovalbumin) on Candida albicans, Cryptococcus neoformans, E. coli, and Staphylococcus aureus cells. Antimicrobial activity was evaluated by microscopy, inoculation, and spectrophotometry. All three albumins showed antimicrobial activity against all studied cultures and their effect was dose-dependent. At concentrations of serum albumins close to physiological (50 mg/ml), the cells of microorganisms were destroyed with the formation of debris vesicles, while at lower concentrations (10 mg/ml), only cell membrane integrity was impaired. According to spectrophotometry, activity of the human serum albumin in a physiological concentration against the studied microorganisms was close to the activity of native human serum.

Congenital immune defense of the hair.

Studies of two recent decades provided ample data on antibacterial peptides protecting animal and human epithelium of different types; however, no reports about the presence of these compounds in hair keratinocytes appeared up to the present time. Peptides were extracted from specimens of normal intact hair with citric acid solution in 50% ethanol. Antibacterial activity of the resultant extracts was evaluated on Candida albicans test culture by staining and microscopy, by evaluation of growth inhibition zones, and by inoculations. Direct contact with the extract destroyed the greater part of Candida albicans cells up to complete destruction of membranes. Application of the extract to dishes with agar with Candida albicans led in the formation of apparent zones of growth inhibition. The percentage of killed cells increased with prolongation of incubation with the extract. Electrophoresis of hair extract showed bands characteristic of antibacterial peptides RNase, psoriasin, and ß-defensins. Removal of the peptides from the extract by filtration through membrane filter led to loss of its activity. The results indicate that human hair keratinocytes possess congenital antibacterial immunity.

Creation of a new synthetic medium for culturing Helicobacter pylori.

We developed a scheme of consecutive replacement of complex components of a known Brucella medium containing peptones and blood with simple analogs and created a synthetic medium for Helicobacter pylori culturing. H. pylori cells require hemic iron for their growth; an appreciable increment in biomass was ensured by hemoglobin, but not simpler hemocontaining compounds (hemin and cytochrome C). Glutamine (20 g/liter) was used as the main nitrogen-containing component, and other amino acids were added in trace amounts. Adhesion was provided by adding agarose gel (0.1%) also promoting the increase in biomass. The proposed medium of a certain chemical composition differs from the known foreign analogs by the presence of hemocontaining component (hemoglobin), short period of exponential growth, and appreciable accumulation of cell protein.

IgE and IgG antibodies to Malassezia spp. yeast extract in patients with atopic dermatitis.

The presence of immunoglobulins to Malassezia spp. surface proteins in the sera from patients with atopic dermatitis and healthy subjects was studied. It was found that 28% of 25 examined patients with atopic dermatitis had IgE antibodies to Malassezia spp. surface protein preparation. All patients and 5 healthy subjects had IgG antibodies to this preparation. The presence and concentration of specific IgE antibodies in patients with atopic dermatitis correlated with reverse titers of IgG antibodies to this preparation (r=0.782). The medians of values reciprocal to IgG antibody titers in patients with atopic dermatitis with and without specific IgE antibodies to the preparation and in healthy subjects were 64, 1024, and 16, respectively. The preparation derived from Candida albicans (candidine) and previously derived preparation from Malassezia did not cross-react. According to immunoblotting data, the preparation contains allergens presented by proteins with molecular weights 15, 36, 52-56, and 78.4 kDa.

Modified method for evaluation of plasma membrane integrity in eukaryotic cell.

We propose a method for evaluation of the number of viable cells by the content of bromcresol purple dye absorbed by dead cells from the incubation medium. Myramistin was used as a pore-forming agent. The number of live cells in yeast suspension inversely correlated with the percentage of dye absorbed by cells. The method is simple and requires no special equipment. The effect of myramistin on Candida albicans and Malassezia sympodialis cells and on epitheliocytes was evaluated. Two-hour treatment with myramistin in a concentration commonly used in clinical practice (100 mug/ml) decreased the number of viable cells by 2 and 1 order of magnitude, respectively. Epitheliocytes under the same conditions absorbed approximately the same mount of the dye as Candida cells.

A method for estimation of urease activity in gastric mucosa biopsy specimens and Helicobacter pylori cell suspensions.

A method for measuring urease activity in biopsy specimens and Helicobacter pylori cultures from these specimens is proposed. The method is based on measurement (with a portable pH-meter) of the rate of pH changes in a reaction mixture consisting of buffer, substrate (urea), and biopsy specimen or bacterial cells. This method revealed that urease activity of biopsy specimens correlated with that of H. pylori suspension in the same experiment. High urease activity was found in biopsy specimens containing the greatest number of Helicobacter cells; only one of 14 specimens free of H. pylori cells showed no urease activity. Introduction of this method into clinical practice will help to evaluate the contribution of H. pylori to the pathological process.

Drug sensitivity of Candida yeast isolated from patients with allergic diseases.

Viability of 40 Candida spp. cultures was studied after long-term exposure to antifungal drugs in minimum inhibitory concentrations. The fungicidal effect decreased in the series: pimafucin-nitrofungin-diflucan-orungal-levorine-clotrimazole-exoderil. Nizoral in a concentration of 4 microg/ml was ineffective; in the rest cultures the effect was either fungistatic (of different degree) or null. Pimafucin, diflucan, nitrofungin, orungal, levorine, and exoderil possessed individual fungicidal effects.

Yeast fungi in patients with allergic diseases: species variety and sensitivity to antifungal drugs.

Yeast microflora was studied in the skin of 91 patients with atopic dermatitis, in bronchial secretion of 13 patients with bronchial asthma and 8 patients with allergic bronchopulmonary mycosis. Forty-eight isolates were obtained. Alypophilic yeast fungi isolated from the skin were presented mainly by genera Candida spp. (48%) and Rhodotorula spp. (29%), while the cultures isolated from bronchial secretion mainly by Candida albicans. The sensitivity of yeast cultures to the antifungal drugs diflucan, clotrimazole, nizoral, orungal, exoderil, levorin, pymafuzin, and nitrofungin was determined. The most efficient drugs were diflucan, clotrimazole, nizoral, and orungal. More than half isolates were sensitive only to the high concentrations of levorin (48%), pymafuzin (75%), and nitrofungin (82%); 64% isolates were insensitive even to high concentrations of exoderil. Preliminary in vitro selection of the antifungal drugs is required for efficient elimination of the yeasts.

Allergen-containing drug from Malassezia spp. yeast.

Malassezia spp. yeast habituating the skin of healthy humans can be a source of allergens for patients with atopic dermatitis. We proposed a method for obtaining allergen-containing preparation by trimming outer cell wall proteins with 1% sodium dodecyl sulfate. The resultant preparation contained 36 and 67 kD proteins known as Malassezia allergens. IgE antibodies to these proteins were detected in the sera of young people with atopic dermatitis.