DEVELOPMENT OF RESISTANCE TO ANTIBIOTICS IN BACTERIA STAPHYLOCOCCUS SPP . ISOLATED FROM MILK SAMPLES IN THE SHEEP BREEDINGS ON EAST OF SLOVAKIA

During the last three years (2015 to 2017), the frequency of occurrence of bacteria Staphylococcus spp. were examined in total 3466 individual and 12 pool milk samples. Experiment was carried out in two herds of breed of sheep Improved Valaska in region of East Slovakia. Were isolated and taxonomically identified 15 species of the genus Staphylococcus spp. (n = 444). From the coagulase positive staphylococci (CPS), S. aureus was isolated during the reporting period, however, most often in the first year (45). The incidence of S. intermedius and S. hyicus has been irregular. From the coagulase negative staphylococci (CNS) (n = 288), were isolated S. epidermidis present in 37.5% (108), S. scheiferi 25.69% (74) and S. chromogenes 23.61% (68), while other species occurred only rarely. The bacteria S. aureus (n = 117) showed the highest resistance to novobiocine 14.5%, to erythromycin 12.8%, lincomycin 7.69% and also 7.69% to penicillin. In the framework of the CNS sensitivity we tested 108 strains of S. epidermidis, from which it was 11.1% resistant to novobiocine and 8.3% to erythromycin. Statistical comparison of the incidence of resistance to penicillin and novobiocine in S. aureus and S. epidermidis was performed using Chi square test. There was statistically significant dependence of tracked characters was confirmed on significance level α = 0.05. Also, the incidence of intermediate sensitivity of bacteria in the Staphylococcus spp., indicates the unfavourable development of resistance to the most commonly used antibiotics to treat the inflammation of the udder in sheep. It is therefore recommended to regularly check the resistance to antibiotics and often isolated bacteria CNS.


INTRODUCTION
The Staphylococcus spp.forms a group of microorganisms, which globally represents a significant proportion in the aetiology of the sheep mastitis (Contreras et al. 2007).
In particular, S. aureus as a coagulase positive species, were intensively studied for its pathogenicity in both, human and veterinary medicine.In recent decades however, highlighted the importance of coagulase negative staphylococci and (CNS), which were initially considered as a comensals and had a minority as the mammary gland pathogens.Their significance as a pathogen of ruminants, especially in subclinical forms of mastitis, were recorded by experts in many countries (Pyörälä and Taponen, 2009).Pitkälä et al. (2004) reported up to 50% market share of CNS from total isolation of bacterial species in Finland, while the main indicator of the subclinical mastitis is increasing of somatic cell count in milk of ruminants.
Prevention and control mastitis caused by CNS (controling CNS mastitis) are complex, since epidemiology is often unclear even for the fact that the group consists of more than 40 species, the characteristics of the CNS are diverse, they can be more or less virulent (Kiossis et al., 2007;Pyörälä and Taponen, 2009).Also, with reference to the specific conditions of ruminants, many authors in their works referred to as the most frequently isolated: S. epidermidis, S. chromogenes, S. simulans, S. xylosus, S. haemolyticus, S. warneri, and S. sciuri (Fthenakis 1994;Ergün et al., 2009).
Although CNS do not have a comparable range of the virulence factors, such as the S. aureus, one of the important factors of virulence is the ability to create resistance to the antibiotic, while some were described as multiresistant (Moniri et al., 2007).
Hidden intramammary infection half of the udder are referred to as sources of resistant staphylococci in sheep holdings, which in practice can be confirmed only by a bacteriological examination (Ergün et al., 2009;Kiossis et al., 2007).

Zigo et al. (2014)
in their work report that the coagulasenegative staphylococci were identified in 102 (65.4%) from all 156 positive isolates.The CNS and S. aureus caused subacute (5.1%), subclinical (3.9%) and acute (2.4%) forms of mastitis.The most frequently isolated were S. epidermidis, followed by S. chromogenes and S. No. 1/2018 xylosus from ewes with subacute and subclinical mastitis.From acute and chronical forms of mastitis were predominantly isolated S. aureus and S. epidermidis.
The aim of the work was to determine the occurrence and most common types of Staphylococcus spp. in the investigated individual pools and sheep's milk samples and comparison of the incidence of antibiotico-resistance of the most numerous tested species S. aureus and S. epidermidis.

Characteristics of experimental breeds of sheep
It was one of the breed of sheep with 330 Improved Valaska sheep, and another farm with 250 sheep with a program of gradual crossing with the "Lacaune" breed.Tracking the aetiology in mastitis in the findings of the pool samples was carried out during the three seasons of the machine milking, in the holdings with technological standards in Gelnica district.Have been performed a total of 12 comprehensive examination repeatedly from April to September.A significant measure in the course of the experiment was to treat all cases of clinical mastitis solely on the basis of proven susceptibility to a range of selected antibiotics.

Testing and sampling herds sheep's milk
At the beginning, and at the end of each season were carried out a clinical examination of the udder is supplemented by a Californian Mastitis Test individual sheep's milk, and bacteriological examination of samples according to the principles as stated by the authors Fthenakis (1994); Vasil (2004); Mørk et al. (2004).
Emphasis has been placed on aseptic sampling and transport of mixed pools samples and individual sheep's milk samples intended for bacteriological examination.

Bacteriological examination
The inoculum of each sample of milk was inoculated on the plates with 5% blood agar, incubated at 37 °C, and after 24 hours of reading from.When the growth were more than 7 colonies from one type of colony were inoculated and cultured on selective nutrient soils.Identification of Staphylococcus spp.bacterial cultures was carried out according to the assessment growth of suspected bacteria on nutrient agars (5% of blood Agar, N° 110, Baird-Parker agar, Brilliance ž UTI Clarity Agar (OXOID Ltd., Basingstoke, Hants, UK).The pigment formation, haemolysis, catalase positivity, Gram positivity, creation of free or coupled coagulase, and other characters, were determined.The identification of each species was made by STAPHYtest 24 and evaluated by TNW ProAuto 7.0 (Erba-Lachema, Brno, CZECH REPUBLIC) with a probability of correct designations of the kind above 90%.The functionality of the set was controlled using a strain of Staphylococcus aureus CCM 7113 (CCM, Masaryk University, Brno, CZECH REPUBLIC).

Testing of the sensitivity on antibiotics of the most numerous species of Staphylococcus
Bacteria isolated from various forms of mastitis (n = 432) and pools milk samples (n = 12), were tested in vitro by a disc method (EUCAST, 2014) by evaluation of the zones of inhibition to grow on Mueller-Hinton agar after 24 hours incubation at 37 °C.
To the test of sensitivity of staphylococci to fourteen antibiotics (ampicilin, amoxicillin, cefoperazone, cefoxitin, cloxacilin, erythromycin, lincomycin, neomycin, penicillin, novobiocine, oxacilin, methicilin, streptomycin and tetracycline) have been use test discs (OXOID Ltd., Basingstoke, Hants, UK) as shown in table 1.The choice of antibiotics reflects the range of which is contained in a number of intramammary products to treat mastitis, which are available in Slovakia.Sensitivity or resistance of the bacteria tested were interpreted according to the reference zones in accordance with the instructions of the EUCAST (2014).
In the tests were used as control the tribes S. aureus CCM 5973 and S. epidermidis 4418.In view of the abundance of the species of the CPS, or CNS was only possible for species S. aureus and S. epidermidis in practical terms, to evaluate resistance as a percentage: a negligible (< 0.1%), very low (0.1 -1%), low (1 -10%), moderate (10 -20%), high (20 -50%) or very high (50 -70%).

Statistical analyses
Statistical analysis we performed using software Microsoft Excel 2007.Chi square test ( test) we used to compare the individual proportions (Kabrt 2013).The dependence of the individual signs was tested at a significance level α = 0.05, with critical value = 5.991.

RESULTS AND DISCUSSION
Table 2 gives an overview of the types of the bacteria Staphylococcus spp., which we have been isolated from sheep's milk, during the three years on holdings in Eastern Slovakia.In the reporting period, a total of 156 coagulase positive staphylococci were isolated of which 75% (117) was S. aureus, in 14% ( 22) has been isolated S. intermedius, and 10.9% (17) S. hyicus.
S. aureus was isolated during the reporting period, but most frequently at the beginning of the reference period (45).S. intermedius was isolated in the first two years of tracking, however, most in the second year (19).S. hyicus has been isolated only in the first year of follow-up (17).During the period considered from 288 coagulase negative staphylococci S. epidermidis was isolated in 37.5% (108), S. schleiferi 25.69% (74), S. chromogenes 23.61% (68), S. cohnii, ssp.urealyticum 3,82%, S. xylosus 2.43%, and other species occurred only rarely (Table 2).Table 3 provides an overview of the incidence of resistance to 14 tested antibiotics in the four species of staphylococci (n = 367), which were the most frequently isolated from sheep's milk, during the three years of follow-up.The table 4 is showed the occurrence of resistance to 14 antibiotics in S. aureus (from CPS group), and S. epidermidis (most commonly occurring from 12 kinds of CNS), which have been isolated from sheep's milk in the course of three years.In the evaluation of the tests of sensitivity of the two most numerous species were numerically expressed as numbers of (S) -sensitive, (IM)intermediate, (R)resistant as well as the values of the resistance in percentage (%).

Bogdanovičová et al. (2014)
reported that the antimicrobial resistance profile of the tested S. aureus strains to different antibacterial agents revealed that 17.8% (n = 11) of the strains were resistant to at least one antibiotic.
In the framework of the coagulase negative staphylococci (tables 3 and 4) was on the sensitivity tested 108 strains from these S. epidermidis in 11.1% was resistant to novobiocine, and 8.3% to erythromycin, moreover, can be identified with intermediate sensitivity to the adverse ampicillin (13), lincomycin (11), erythromycin (10), amoxicilin, novobiocine and penicillin (9 tribes).
At work we are comparing the incidence of following characters (S, IS, R) in two groups, the most numerous of staphylococci S. aureus and S. epidermidis using statistical method Chi-squared test.On the significance level α = 0.05 (5%), was recorded in twelve antibiotic substances test value (G < χ2), the statistically independence of tracked characters was confirmed.The antibiotic substance penicillin and novobiocine when applied, G > χ 2 , in the test groups of S. aureus and S. epidermidis statistically dependence of the observed characters was confirmed, which means that the occurrence of the characters was not random.
Table 2 The types of the bacteria Staphylococcus spp., which have been isolated from sheep's milk, during the three years on holdings in eastern Slovakia.
Note: Ddose of antibiotics in µg, content of one disc; Z -reference zones in mm.

Table 3
Total overview of the incidence of resistance to 14 tested antibiotics in the four species of staphylococci (n = 367), which were the most frequently isolated from sheep's milk, during the three years of follow-up.

Table 4
An overview of the sensitivity and the occurrence of resistance to 14 tested antibiotics in two types: S.aureus as representative of the CPS, and S.epidermidis as most frequently isolated from 12 species of CNS, isolated from sheep's milk in the years 2015 to 2017.