ANTIMICROBIAL RESISTANCE AND BIOLOGICAL PROPERTIES OF STAPHYLOCOCCUS SPP. ISOLATED FROM PIGS

of Interpretation of Results. 77 strains of Staphylococcus spp. Collected from sows at the farm No. 2, were isolated: hemolytic properties were detected in 90.6% (39 coagulases positive and 19 coagulase negative); 22 (56.4%) strains of coagulase-negative Staphylococcus spp.; 13.6% of isolates had hemolytic properties. The results of antibiograms of crops from the farm No.1: 51.8% of strains were susceptibile to penicillin, 47.6% – resistant; 13.62 – susceptibile to fluoroquinolones, 80.9% – resistant; 96.7% – susceptibile to chloramphenicol, no resistant strains were detected. Conclusions. Acquired resistance of Staphylococcus spp. to certain groups of antibiotics


INTRODUCTION
Staphylococcus carriers were an important source of contamination for food, raw materials, birds, pigs and pork products (1,2). It has been repeatedly proven that pigs were sources of staphylococci, in particular methicillin-resistant S. aureus (MRSA) (3). Such cases were especially common in Denmark (4), Canada (4), Germany (6) and Switzerland (7). In young pigs, the disease manifests itself in the form of exudative epidermitis (EE), caused by strains of Staphylococcus hyicus, Staphylococcus aureus and Staphylococcus chromogenes, which produce exfoliative toxins. However, pigs in most cases were hidden carriers (7). Experimental transfer of methicillin-resistant S. aureus to minks during feeding of pork waste contaminated with MRSA is also known (9). In 2017, 80 methicillin-resistant staphylococcus strains were isolated in Ukraine, including 77.5% from domestic animals, 11.3% from poultry, 6.3% from cattle and 5% from pigs (10).
The purpose of the study was to investigate the biological properties and antibiotic resistance of Staphylococcus spp. isolated from pigs of two industrial pig farms located in Kyiv (No.1) and Vinnytsa regions (No.2).

MATERIAL AND METHODS
Isolation and identification of Staphylococcus spp. was conducted in accordance with: DSTU EN 6888:2003 "Microbiology of food and animal feed" standard was cultivated to the Baird Parker agar (Merck) medium and were incubated at 37°C for 24-48 hours. Isolates produced on Baird Parker agar at the end of this period were defined by morphology colony, Gram stain, catalase test, and coagulation test. The hemolytic properties of staphylococci were studied on Columbia blood agar (BioMerieux). Selected colonies from Columbia blood agar introduced into Tryptone-soy broth and cultured at t 35°C for 2-4 hours. The optical density was determined using a densitometer DEN-1(Biosan) and McFarland standard (HiMedia). The susceptibility of strains to antibiotics was determined by disc-diffusion method using Müller-Hinton agar, inoculum: 0.5 according to the McFarland turbidity standard and were incubated at 35°C, for 18±2 hours. The results were assessed in accordance with the re-commendations of version 8 of the European Committee on Antimicrobial Susceptibility Testing EUCAST (11) and Guidelines "Determination of susceptibility of microorganisms to antibacterial drugs" (12).

RESULTS
Farm No. 1 proved to be a satisfactory farm in terms of safety against contagious diseases; number of pigs was up to 2 500 heads. There were 210 pigs. Purulent diseases, abscesses and boils were not reported in piglets. Of the 77 sows sampled from the nose Staphylococcus spp. were isolated in 64 cases (83.1%). All strains grown on the Beard Parker agar had typical colonies of black and grey, shiny and convex with a diameter of 1 mm to 1.5 mm after incubation for 24 hours and a diameter of 1.5-2.5 mm after 48 hours of incubation. The 39 isolates (60.94%) were coa-gulated with rabbit plasma. The ability to hemo-lysis of sheep erythrocytes showed only 58 (90.6%) of strains. Of these, 39 coagulase-positive strains and 19 coagulase-negative strains did not show hemolytic properties of 6 coagulase-negative strains.
The farm No. 2 proved to be a satisfactory farm in terms of contagious diseases; number of pigs was up to 3 500 heads. Diseases caused by Sta-phylococcus spp. we're not registered. In 22 samples (56.4%), isolates of Staphylococcus spp. were isolated from 39 sows from farm No.2.
The isolates grew in the form of shiny black and grey colonies with a narrow white margin. The isolates did not coagulate plasma. The 3 strains (13.6%) from the isolated strains had hemolytic properties.
Staphylococcus had a natural susceptibility to penicillins, but subsequently acquired resistance to them (13). In the study, were used: Natural Penicillin, Benzylpenicillin; Semisynthetic Oxacillin, Gentamicin, Tobramycin from the aminoglycosides group, and Erythromycin from the macrolids group (13). The susceptibility to natural Tetracycline and Semisynthetic Doxycycline; Lincomycin and Clindamycin from the group of lincosamides was investigated. Susceptibility for norfloxacin was recommended for screening susceptibility for all fluoroquinolones (11). There were studied Norfloxacin, Ciprofloxacin, Ofloxacin, Pefloxacin, Lomefloxacin, Levofloxacin, Sparfloxacin and Gatifloxacin (12,13). Susceptibility to Chloramphenicol, and Rifampicin was investigated (11). 51.87% of the selected strains obtained at the farm No.1 were susceptible to Penicillin antibiotics, and 47.59% were resistant. 78.48% of bacterial strains showed susceptibility to aminoglycosides, and 21.52% of the strains were resistant. 87.1% of strains showed susceptibility to macrolides. 70.45% of the isolates were susceptible to tetracyclines. 70% of isolates showed susceptibility, and 30% were resistant to lincosamides. 13.62% were susceptible, and 80.93% -resistant to fluoroquinolones. 96.72% of revealed strains were susceptible to chloramphenicol. 73.53% of the studied isolates were susceptible to rifampicin.
10 strains from 22 pigs, on farm No. 2, were isolated and an antibiogram was determined (tab. 1). It was established that 10 isolated strains were resistant to oxacillin and clindamycin. 3 strains were susceptible, and 7 -resistant to benzylpenicillin and norfloxacin. 1 culture was susceptible, and 9 -resistant to tetracycline and erythromycin. 4 strains were susceptible, and 6 -resistant to chloramphenicol.

DISCUSSIONS
The obtained results indicate a significant colonization of coagulase-positive and coagulase-negative staphylococci in the nasal passages of sows. A significant percentage of isolated crops possesses "pathogenicity factors". Plasma coagulation and hemolysis were particularly important. The high circulation of staphylococci in the herd leads to an increase of antibiotic resistance. Natural penicillin is known to have little effect on staphylococci, while synthetic and semisynthetic penicillin antibiotics of other groups inhibit their growth.
The results showed that in the farm No. 1 polyresistant strain of coagulase-positive and coagulase-negative staphylococci was present. A major component of beta-lactam resistance is the socalled mecA gene, which encodes the formation of modified penicillin-binding protein and thus interferes with the incorporation of beta-lactam into the cell wall. When the cell is methicillin-resistant Staphylococcus aureus in contact with β-lactam antibiotics, the additional β-lactam-resistant penicillin binding protein (PBP2a) takes on the biosynthetic functions of normal PBPs.
Staphylococcus aureus resistance to methicillin (Oxacillin) may be due to the production of additional PBP-2a, which is encoded by the chromosomal mecA gene, inactivation through hyper production of β-lactamases and modification of normal PBPs. The presence two resistant strains of coagulase-positive staphylococci and one intermediate resistanse in pigs may indicate the possible presence of the mecA gene. Also, the resistance of the isolated strains to Ampicillin indicates the synthesis of staphylococcal penicillinase. The higher resistance to Tetracycline was shown by coagulase-positive staphylococci, which was confirmed by the resistance of these strains to Doxycycline. Also, indicative is the resistance of some coagulase-positive staphylococci to both Lycomycin and Clindamycin, which is not observed among coagulase-negative strains. Practically all staphylococci have been shown to be resistant to Fluoroquinolones of different generations, including Gatifloxacin. Two intermediate resistanse strains of coagulase-negative staphylococci were detected for Chloramphenicol while all coagulase-positive strains were susceptible.
As a result, the circulation of polyresistant coagulase-positive staphylococcus strains proved to be resistant to almost all antibiotic groups, especially to Fluoroquinolones -4.6% susceptible, 6.0%intermediate resistanse, and 89.4% -resistant. Among coagulase-negative staphylococci, 20% were susceptible, 5.0% were intermediate resistanse, and 75% were resistant to Fluoroquinolines. Coagulase-negative strains from farm No. 2 also had poly resistant properties in tested antibiotics of all groups.