PHENOTYPICAL CHARACTERISTICS OF THE BIOLOGICAL PROPERTIES OF STAPHYLOCOCCI WITHDRAWN FROM PATIENTS WITH ALLERGIC DERMATITIS

Atopic dermatitis, eczema, allergic dermatitis occupy the main place among dermatoses, where the allergic component is leading in the onset and development of the disease. The most common complication of allergic dermatitis is the attachment of a secondary pyococcus infection, which is associated with a decrease in the antimicrobial resistance of the skin surface. Therapy of infectious lesions is complicated by the increasing resistance of the main pathogens of pyoderma – Staphylococcus aureus and Staphylococcus epidermidis – to widely used antibiotics. The aim of the research: to determine the phenotypic features of staphylococci extracted from patients with allergic dermatitis to assess their pathogenic potential. Materials and methods. The object of the study was 369 staphylococcus isolates removed from affected and intact skin sections of patients with allergic dermatitis, as well as from representative skin sections of healthy individuals undergoing inpatient treatment at the Department of Dermatology of “Institute of Dermatology and Venereology of NAMS of Ukraine”. Biochemical identification and biological properties of staphylococci were determined using methods of classical bacteriology. Results. As a result of the conducted researches, it is established that the complex of phenotypic traits of the removed staphylococcus cultures indicates the presence in the pathogen of factors related to the resistance of the host protection mechanisms and determines the intensity of the alterative action of the infectant in relation to the host organism, the phenotypic manifestation of the studied factors was higher in the staphylococcus isolates removed from the affected skin areas of patients with allergic dermatitis. Conclusions. The level and frequency of phenotypic expression of pathogenicity factors are more pronounced in microorganisms obtained from patients from affected and intact areas compared to controls, which confirms their pathogenetic role in the burden of the disease, which in turn can be used as an auxiliary differential diagnosis criterion.


Introduction
Atopic dermatitis, eczema, allergic dermatitis occupy the main place among dermatoses, where the allergic component is leading in the onset and development of the disease. Attention to these diseases is due to the high incidence; constantly recurrent course; increasing the number of trigger factors; insufficient effectiveness of traditional therapy [1].
According to a number of authors, about a tenth of the world's population is ill with eczema. In industrialized countries, this figure reaches 32-45 %, accounting for 51 % of all allergic dermatitis in some regions. According to various authors, the incidence of eczema in Ukraine is from 8 to 30 % of all skin diseases and is the most common pathology in the practice of a dermato-Original Research Article: full paper (2020), «EUREKA: Health Sciences» Number 1 venereologist [2]. According to the World Health Organization and national literature, the number of eczema patients has increased significantly in the last decade [1]. The eczema is characterized by the presence of acute inflammatory symptoms: intense itching of the skin, recurrent course, and increased sensitivity of patients to various exogenous and endogenous stimuli, tendency to spread and deepen the process. The most common complication of the eczematous process is the attachment of secondary urinary and fungal infections, which is associated with a decrease in antimicrobial resistance of the skin surface [3]. Treatment of infectious lesions is complicated by the increasing resistance of the main pathogens of pyoderma -Staphylococcus aureus and Staphylococcus epidermidis. Uncontrolled use of external antimicrobials, the sensitivity of which is lost, delays the process of rehabilitation of the infection and promotes the subsequent selection of resistant flora [4].
In atopic dermatitis (AD) also the course of the disease is often complicated by the adherence to a secondary infection. This feature reflects the anti-infective protection disorders inherent in patients with hypertension. In the case of S. aureus infection, the adverse effect of the microorganism is provided by T-lymphocyte-activating superantigens, stimulating the secretion of pro-inflammatory cytokines, and activating IgE antibody formation, which leads to the degranulation of caudal cells and the release of biologically active substances. In general, in addition to the classical scheme of appearance of IgE in the case of IgE-mediated AD, there is another mechanism of stimulation of its formation. This mechanism is associated with the action of superantigens, high molecular weight proteins, which include some antigens of bacterial or viral origin [5,6]. Some studies have shown that in patients with AD whose skin was colonized with S. aureus containing the superantigen genes, the mean score on the AD severity scale -SCORAD -was significantly higher than in patients whose skin was colonized with S. aureus isolates without superantigen genes [7].
Taking into account the above data on the negative effect of S. aureus on the course of blood pressure, it should be noted that although it is not a commensal skin, but it is known that the place of its vegetation may be nasal passages, from which шт 20 % of the population the microorganism is extracted, and its translocation to the skin may occur [8,9].
In addition, in recent years, a great deal of attention has been paid to the study of microbiocenosis and penetration of microorganisms into the skin in patients with chronic allergic skin diseases. The skin microbiota is considered as part of a meta-organism that includes a macro-organism and a set of all microorganisms -symbionts. This implies the existence of complex relationships between the microbiota and the human body, and, first, with its immune system, which not only regulates the interaction of the organism with the microbiota, but also itself formed under its influence [10,11]. At the same time, when investigating the microbiome of the skin of patients with allergic dermatitis, saprophytic species are often removed: S. epidermidis, S. haemolyticus, S. saprophyticus, S. capitis, S. warneri, S. hominis, S. simulans and S. cohnii harmful effects on the human body, but these properties are inherent in these microorganisms provided vegetation on healthy skin. However, it is often not possible to draw a clear line between saprophytes and pathogens that inhabit healthy human skin. Normal microflora play an important role in protecting the body against pathogenic microorganisms, although it may itself be a contributor to infectious disease [12,13]. However, it is known that pathogens of coagulase-negative staphylococci such as proteases, hemolysins, lipases, etc. provide storage of these microorganisms on the damaged skin [7,13]. That is why the study of the biological properties of coagulase-negative staphylococci with a definition of their pathogenic potential is a necessary diagnostic measure for the rational treatment of infectious complications of allergic dermatitis.
The aim of the research: to determine the phenotypic features of staphylococci extracted from patients with allergic dermatitis to assess their pathogenic potential.

Materials and methods
The object of the study were 369 isolates of staphylococci, removed from affected and intact areas of patients' skin for allergic dermatitis, as well as from representative areas of skin of healthy individuals undergoing inpatient treatment at the Department of Dermatology of "Institute of Dermatology and Venereology of NAMS of Ukraine" in 2017-2019 years. Biochemical identification and biological properties of staphylococci were determined using bacterioscopic and bacteriological methods of investigation [14][15][16].
Statistical processing of the results was performed using the software package for Microsoft Excel 2003. The arithmetic mean values for a series of data (M) and the error of averages (m) were calculated. The reliability of the data obtained was evaluated by pairwise comparison and determination of the confidence interval based on the Student's t-factor calculation (t). The differences were considered statistically significant at p<0.05. The degree of correlation was estimated using Pearson correlation analysis (R) to determine statistical significance (p). If the value of the correlation coefficient R per module is closer to 1, then it means that there is a strong connection, and if closer to 0 -the connection is weak or missing.

Results of the research
As a result of the analysis of bacteriological studies, comparative data were obtained regarding the staphylococcal component of the skin microbiocenosis of 114 patients with allergic dermatitis and 20 practically healthy persons. From these groups, 386 strains of microorganisms (346 strains from patients and 40 strains from practically healthy persons) were isolated, which were assigned to 4 genera: Staphylococcus, Streptococcus, Micrococcus and Corynebacterium spp., with predominance of varieties of staphylococci -369 strains (95.6 %), which were removed both individually and in interspecies and intraspecific associations (predominantly).
In the beginning, the composition of the skin microbiota of patients with atopic dermatitis, eczema, and a group of practically healthy individuals was analyzed. In Fig. 1, 2 presents the ratio of the most common types of staphylococci in the skin biotopes of patients with allergic dermatitis.  In the next phase of the study, the obtained data from pathogenic staphylococcal cultures were analyzed to determine the enzymatic activity of staphylococcus cultures ( Table 1). The next stage of the study was to investigate the antibiotic resistance of the extracted laboratory strains of S. aureus to antibacterial preparations of different chemical groups, as the most pathogenic representative of the genus, to identify such antibacterial agents that led to the most complete elimination of the pathogen. The obtained data are shown in Fig. 3.   Fig. 3. Resistance rates of S. aureus strains isolated from patients with allergic dermatitis

Discussion
Staphylococci -representatives of normal microflora of the skin, mucous membranes of the respiratory and digestive tract, but they can cause severe infectious complications in patients with allergic dermatitis. The phenomenon of competitive relationships between S. aureus and S. epidermidis is also described in the literature. Substances that produce S. epidermidis (autoinducers) are known to block the formation of toxins in many strains of S. aureus, whereas enzymes synthesized by S. aureus do not inhibit the proliferation of S. epidermidis [13,17].
According to the research results, the bacterial flora in persons with allergic dermatitis was significantly different from the microflora of healthy people and had its own characteristics, due to the formation of new microbial associations, which changed the habitat and the relationship between the associates. The study of microbial constituents of biotopes showed the dominance of microorganisms of the genus Staphylococcus in the skin cells of both patients and healthy individ- uals. The difference was observed in the species composition of staphylococci and the degree of insemination of individual lesions. The appearance of non-resident staphylococcus species with higher pathogenic potential in affected and intact skin areas was a distinctive feature for most patients (Fig. 1, 2). As can be seen from Fig. 1, lesions of S. aureus strains (58.8 %) were observed in the lesions of the affected skin with a gradual decrease in the areas of intact skin and skin of healthy individuals (55.2 % and 7.5 %, respectively). The number of S. epidermidis strains found was inversely related to S. aureus strains (10.6 %, 13.1 %, and 77.5 %, respectively). No correlation relationships were found for S. haemolyticus strains depending on the extraction site.
As can be seen from Fig. 2, the prevalence of S. aureus strains (43.1 %) was observed in the lesions of the affected skin of eczema patients with a gradual decrease in the areas of intact and healthy skin (40.2 % and 7.5 %, respectively). The number of found S. epidermidis strains was inversely related to S. aureus strains (20.6 %, 19.3 %, and 77.5 %, respectively). The density of colonization in the centers of damaged skin of patients with allergic dermatitis averaged 10 7 -10 8 CFU/ml, in intact areas and skin of healthy individuals -10 3 -10 5 CFU/ml.
The study of the biological properties of staphylococci showed that all isolates had typical morphology (gram-positive cocci) and biochemistry, in the catalase test -catalase-positive, all the extracted S. aureus strains had coagulase activity.
Haemolytic activity (β-type) was possessed by 72.5±3.5 % of coagulase-negative staphylococci, of which 55.4±6.6 % of S. epidermidis cultures ( Table 1). Lipolytic activity was found in 71.9±3.5 % of coagulase-negative staphylococci, more frequently in S. haemolyticus (82.7±4.4 %) and S. epidermidis (76.8±5.6 %). When determining this trait in S. aureus cultures, it was found that 74.7±3.3 % of the strains were positive in this test. It is important to note that lipolytic activity was more commonly observed in strains of staphylococci extracted from affected lesions of patients with allergic dermatitis than from intact or control areas.
Staphylococci with proteolytic activity are more aggressive and, in the body, cause toxic tissue damage [11]. In the study of proteolytic activity, it was noted that about half of the coagulase-negative staphylococcus cultures fermented milk more often (50.9±3.9 %), with S. epidermidis strains making up 73.2±5.9 % and less frequently diluting gelatine (19.2±3.0 %), regardless of the locus of crop selection. A similar trend was observed among laboratory isolates of S. aureus: most cultures fermented milk (95.3±1.6 %) and infrequently diluted gelatine (12.3±2.5 %).
A direct statistically significant relationship was established in S. haemolyticus and S. aureus between haemolytic activity and lipolytic activity (R=0.99, p=0.0001 and R=0.9, p=0.0001, respectively), and between haemolytic activity and fermentation of milk of designated varieties of staphylococci -R=0.95, p=0 and R=0.99, p=0.0001, respectively. For S. epidermidis cultures, these values were slightly lower -between haemolytic activity and lipolytic activity (R=0.46, p=0.01) and between hemolytic and fermentation of milk -R=0.49, p=0.04. The obtained data indicate the importance of individual pathogens of these microorganisms both in the development and maintenance of the inflammatory process of infectious genesis, and of the combined influence of these factors at a certain stage of the infectious process.
In determining the resistance of S. aureus to antibacterial preparations of different chemical groups (Fig. 3), the detection of 73.3 % of penicillin-resistant strains draws attention, with 27.5 % of them being so-called MRSA strains, which makes it impossible prescribing to the patient any β-lactam antibiotics. Isolated strains showed moderate sensitivity to aminoglycosides, lincosamides and tetracyclines -ranging from 49 % to 52 %, and quite low to macrolides -up to 37.3 %. Also noteworthy is the appearance in the structure of antibiotic resistance of vancomycin-resistant strains -4.1 %, which indicates an increase in the aggressive potential of the pathogens obtained. The sensitivity of the extracted pathogens was highest for the preparations of fusidic acid, oxazolidinone and fluoroquinolones -92.8 %, 93.3 % and 67.5 % of the strains, respectively. It is known that the use of one class of antibiotics may increase the risk of developing resistance to another class, so at the next stage of the study, a comprehensive assessment of the resistance of the extracted strains of S. aureus was made, taking into account the prevalence of polyresistant strains. Based on the monitoring, 54 % of MDR strains and 4 % of XDR strains were detected. Significant is the complete absence of PDR strains, i.e. microorganisms resistant to all classes of known antibiotics.

Study limitations.
The study included adult patients with allergic dermatitis diagnosed with atopic dermatitis (L 20.0) and eczema (L 30.0 -other dermatitis) according to the International Classification of Diseases 10.
Prospects for further research. Further studies include the study of correlations between phenotypic and genotypic traits of extracted pathogen isolates in order to develop algorithms for predicting the severity of marked allergic dermatitis.

Conclusions
Thus, the complex of phenotypic features of the removed staphylococcus cultures indicates the presence in the pathogen of factors associated with the resistance of host protection mechanisms, on the one hand, and high pathogenic potential, on the other, which promotes active colonization of both affected and intact areas of the skin, which provides the conditions for long-term persistence and determines the intensity of the alterative action of the infectious agent against the host organism.
The level and frequency of phenotypic expression of pathogenicity factors are more pronounced in microorganisms obtained from patients from affected and intact areas compared to controls, which confirms their pathogenetic role in the burden of the disease, which in turn can be used as an auxiliary differential diagnosis criterion.