BIOFILM FORMATION AND ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS ISOLATED FROM DIFFERENT OBJECTS

Staphylococci have a wide range of pathogenic properties, among which stands increased resistance to antibiotics and the ability to form a biofilm. Being divided into coagulase-positive and coagulase-negative, they have different and, at the same time, common biological properties and may be a source of genetic material for each other. The aim of the study was to examine the properties of staphylococci, isolated from various objects (milk, pigs, companion animals, humans), their resistance to antibiotics, the ability to form a biofilm and the presence of genes, responsible for resistance to methicillin and biofilm formation. In this study we used 89 Staphylococci strains. 18 (20.2 %) strains were coagulase-positive, where 2 (2.2 %) strains, isolated from different animals and humans, had a complete set of phenotypic (plasma coagulation, biofilm formation, resistance to oxacillin and benzylpenicillin) and genotypic (mec A, fem B, ica A, ica D) signs of pathogenicity. Coagulase-negative staphylococci, isolated from various animals and humans, had resistance to oxacillin, benzylpenicillin and the ability to form biofilms, and also had the corresponding genes in their structure (mec A, ica AB, ica D) in 3.3 % of cases. Staphylococci, isolated from different animals and humans, were able to form a biofilm and had the appropriate set of genes (ica D, ica AB) in 5.6 % of cases.

Staphylococcal culture targets, used in this study

Type Milk Pigs Companion animals People
Coagulase-positive (CoPS)  2  3  2  11 Coagulase-negative (CoNS) 33 7 23 8 Antibiotic resistance of cultures was determined by the disc diffusion method (Kirby-Bauer) using antibiotic discs and a Müller-Hinton agar medium (HiMedia, India). The resistance of the obtained strains to Benzylpenicillin and Oxacillin was investigated. The results were evaluated in accordance with the recommendations of the European Committee for Antimicrobial Sensitivity EUCAST (version 10.0) [24] and the national criteria for the assessment of antibiotic resistance -the methodological guidance [25].
The ability to form biofilms in the derived isolates was determined and the results obtained were interpreted [26]. This study was performed using sterile polystyrene Petri dishes (Greiner Bio-One GmbH, Germany) of d=100 mm, which were added to 10 mL of Tryptone-soy broth (HiMedia, India) and added 1 ml of inoculum with a cell content of 0.5 to MacFarland daily culture of studied Staphylococci. The plates were cultured in a thermostat at a temperature of 37 °C for 24 hours, the residues of the nutrient medium were carefully removed, the planktonic forms were washed three times with a sterile phosphate buffer solution (KH 2 PO 4 ·Na 2 PO 4 ·H 2 O), pH 7.2-7.4. The Petri dishes were air-dried and 10 mL of Ethanol 96 % was added to fix the formed biofilms. The fixation exposure was 10 minutes. Then the fixing liquid was drained and after it, the Petri dishes were stained with 0.1 % alcohol solution of crystal violet for 10 min. Then washed the plates three times with a sterile phosphate buffer solution (pH 7.2) and dried. Contributed to 10 mL of Ethanol 96 % and placed on a shaker for shaking for 30 min. The contents of the Petri dishes were then pipetted and the amount of absorption of the biofilm of the dye was measured on an Evolution 300 spectrophotometer (Thermo Fisher Scientific, USA) at a wavelength of 570 nm. The density of the formed biofilm was determined by measuring the adsorption level of the dye with ethanol measured in units of optical density (OD) using a spectrophotometer.
When the value of an optical density is less than 0.1, it was considered that the strains do not form a biofilm, from 0.1 to 0.49 -the ability to form a film was considered as a low. When the value of optical density is from 0.5 to 1.0 -the medium density of the biofilm and the ability to form it. At values above 1.0 -the high ability to form a biofilm and its high density [26].
Polymerase Chain Reaction research. Studies of the obtained isolates for the presence of genes mecA, fem B, ica A, ica D, and ica AB were performed by PCR with detection of results by separation in agarose gel. As a negative control, a mixture for PCR without DNA was used.
Genomic DNA from Staphylococcus spp culture was isolated by the express method. To do this, the lyophilized mass of the culture of Staphylococcus spp was dissolved in 1 cm 3 of sterile buffered peptone water (HiMedia, India) and centrifuged at 13.5 thousand rpm for 2 min, and the supernatant was removed. The bacterial pellet was resuspended in 200 mm 3 of TE buffer and incubated in a thermostat at 95 °C for 5 minutes. Cell debris was precipitated by centrifugation at 5.0 thousand rpm. for 2 min and took 180 mm 3 of supernatant, which was used in PCR. The DNA concentration was measured on a Biofotomer spectrophotometer (Eppendorf, Germany) at a wavelength of 260 nm. The amplification reaction was performed in a reaction mixture with a volume of 25 μl, with the following composition: 1x PCR buffer, 2.5 mm MgCl 2 , 2.0 mm each of deoxynucleotide triphosphates, 10 pM each of the primers for detection and 1 Unit. DNA polymerase. DNA was added in an amount of 5.0 mm 3 (100-150 ng). The studies were performed on a thermal cycler 2720 (Applied Biosystems, USA) with the temperature profile, given in the relevant literature source. The nucleotide sequences and other characteristics of the primers that were used in the study are given in ( Table 2). The amplification products were separated in 1.5 % agarose gel.  Table 2 Characteristics of primers that were used in the studies

Results
Manifestation of phenotypic resistance to benzylpenicillin and oxacillin are shown in Table 4. Staphylococci, isolated from milk, were resistant to benzylpenicillin in 37.1 % and oxacillin in 25.7 % of cases. Among staphylococci, isolated from pigs, 3 coagulase-positive (30 %) and 1 coagulase-negative (10 %) strains were simultaneously resistant to benzylpenicillin and oxacillin. 10 coagulase-negative (40 %) and 1 coagulase-positive (4 %) staphylococci, isolated from companion animals, were resistant to benzylpenicillin and oxacillin. In the study of staphylococci, isolated from humans, it was found that 3 (15.7 %) coagulase-negative and 1 coagulase-positive (5.2 %) strains were simultaneously resistant to benzylpenicillin and oxacillin. The results of detection of pathogenicity genes in staphylococcal cultures by polymerase chain reaction are shown in Table 5.   In particular, in 1 strain of coagulase-positive staphylococcus, isolated from milk, simultaneous phenotypic resistance to oxacillin and benzylpenicillin was established and the presence of mecA and femB genes was detected, which was confirmed by polymerase chain reaction.
Staphylococci, isolated from milk, formed a biofilm of high (20.0 %), medium (34.3 %) and low (28.7 %) density. Genes, which determine the formation of the biofilm, were found in 7 studied strains. In particular, icaD and isaA were detected simultaneously in 2 coagulase-positive strains, and the icaD gene was also detected in 3 coagulase-negative strains, isolated from milk.
Among staphylococci, isolated from pigs, the presence of mecA and femB genes was detected in 1 (10 %) coagulase-positive staphylococcus. The mecA gene was also present in 1 coagulase-negative staphylococcus, isolated from pigs.
In 100 % of cases, staphylococci, isolated from pigs, showed the ability to biofilm formation. In particular, a high-density biofilm, formed by 10 % of staphylococci, 90 % formed a medium-density biofilm. At the same time, the presence of genes, responsible for the formation of the biofilm (icaD, isaA), were detected only in 1 (10 %) of coagulase-positive staphylococcus.
Staphylococci, isolated from companion animals, that showed phenotypic resistance to benzylpenicillin and oxacillin, did not contain the structural genes mecA and femB. 88 % of the studied strains showed the ability to form a high-density biofilm, the remaining 12 % formed medium-density biofilms. The icaD biofilm gene was found in 3 (12.0 %) coagulase-negative staphylococci, and the icaA biofilm gene was isolated in 1 (4 %) coagulase-positive staphylococcus.

Discussion
In this study we used 89 strains, of which 18 (20.2 %) were coagulase-positive. 2 strains of coagulase-positive staphylococci, isolated from different animals and humans, had a complete set of phenotypic (plasma coagulation, biofilm formation, resistance to oxacillin and benzylpenicillin) and genotypic (mecA, femB, icaA, icaD).
Coagulase-negative staphylococci, isolated from various animals and humans, were resistant to oxacillin, benzylpenicillin (according to EUCAST [24] and Guidelines for [25], and had the ability to form biofilms with mecA, icaAB, icaD in their structure in 3.3 % of cases. Staphylococci, isolated from different animals and humans, were able to form a biofilm and had the appropriate set of genes (icaD, icaAB) in 5.6 % of cases.
The data, obtained by us, are consistent with the publications [26,29,30], that S. aureus, isolated from various samples of raw materials and products of animal origin [29], milk and washes from milking equipment forms biofilms of high density [26] and has polyresistance to various antibiotics, and has the presence of mecA [30].

Immunology and microbiology
Thus, according to [31], the average level of pathogenic staphylococci, accompanied by their significant polyresistance in pigs in Ukraine for the period 2013-2018 was 1.3 %.
The obtained results indicate that the studied strains of staphylococci, regardless of the source of isolation, were phenotypically resistant to oxacillin in 31.4 % of cases and formed biofilms in 100 % of cases.
The analysis of the obtained data gives grounds to believe that staphylococci, regardless of the source of isolation, may be carriers of genetic determinants of pathogenicity, in particular genes that cause resistance to methicillin (mec) and biofilm formation (femB, icaA, icaD).
However, according to the obtained data, the strains, isolated from the hospital patients with nosocomial staphylococcus, had the highest pathogenic potential.
The studied strains of staphylococci have a rich variety of pathogenic properties and are represented by both coagulase-positive and coagulase-negative representatives. The vast majority of studied staphylococci have certain pathogenic symptoms depending on the object of excretion. Staphylococci, isolated from animals, were almost at the level with staphylococci, isolated from humans, by pathogenic properties and, in some aspects, even exceeded.
At the same time, it should be noted, that the phenotypic manifestation of resistance to oxacillin, as well as the ability to form biofilms, in our studies, did not coincide with the results of the detection of relevant genetic markers. Indirectly, this fact indicates the need for in-depth study of this phenomenon in order to identify previously unknown genetic loci that cause the phenotypic manifestation of resistance and the ability to form biofilms, which is important for understanding the pathogenesis of diseases, caused by staphylococci and their timely diagnosis.
These studies have some limitations. Namely, a small number of staphylococcal isolates studied. Also, financial constraints did not allow DNA sequencing of isolated staphylococci and comparative analysis of DNA nucleotide sequences with NCBI data and other databases of DNA nucleotide sequences.
The prospect of further research is to identify previously unknown genetic loci that cause the phenotypic manifestation of resistance and the ability to form biofilms, which is important for understanding the pathogenesis of diseases, caused by staphylococci and their timely diagnosis. To this end, it is advisable to create a collection of isolates of staphylococci, characterized by resistance to antibiotics and the formation of a biofilm, and to sequence their DNA.

Conclusion
The studied strains of staphylococci, regardless of the source of isolation, were phenotypically resistant to oxacillin in 31.4% of cases and formed biofilms in 100% of cases.
Coagulase-negative staphylococci, isolated from various animals and humans, were resistant to oxacillin, benzylpenicillin, had the ability to form biofilms and had in their structure the genes mecA, icaAB, icaD in 3.3% of cases.
Staphylococci, regardless of the source of isolation, both coagulase-positive and coagulase-negative, can be carriers of genetic determinants of pathogenicity, in particular genes that cause resistance to methicillin (mec) and biofilm formation (femB, icaA, icaD).