INVESTIGATION OF PRESENCE OF METHICILLIN RESISTANT STAPHYLOCOCCI IN STUDENTS OF THE FACULTY OF VETERINARY MEDICINE AT THE UNIVERSITY OF BELGRADE

Resistance to methicillin in staphylococci is considered to be one of the most dangerous forms of bacterial resistances to antibiotics. Methicillinresistant staphylococci (MRS) are zoonotic agents which cause local and systemic infections in humans and animals, oft en with a fatal outcome due to the absence of adequate antibiotic therapy. People colonized with strains of MRS are asymptomatic carriers and reservoirs of these strains in human populations. Th e aim of this research was to determine the prevalence of strains of MRS among clinically healthy students of the Faculty of Veterinary Medicine in Belgrade. Th e study was conducted on 100 volunteers: 62 males and 38 females. Given that staphylococci are expected to be found in the highest percentage in the nose and on the armpit skin, the swabs were taken from these regions of each person. Blood agar was innoculated immediately on taking the swabs Aft er the incubation and isolation, the staphylococci were identifi ed to species level. Th eir susceptibility to methicillin was tested in a disk-diff usion test with cefoxitin. All strains which were found to be resistant to cefoxitin were investigated for the presence of mecA gene with PCR. Staphylococci were isolated in 146 out of the 200 swabs taken: there were 79 nose swabs and 67 axillar swabs positive for these bacteria. Seventeen isolates were resistant to cefoxitin and the presence of the mecA gene was confi rmed in seven, four of which were taken from the nose and three from the axillary region. Th e results of this research show that, being 6%, the prevalence of mecA-positive staphylococci in the 1* E-mail: dusan@vet.bg.ac.rs Arhiv veterinarske medicine, Vol. 9, No. 2, 17 28, 2016 Rađenović M. ... et al.: Investigation of presence...

population of clinically healthy students of veterinary medicine is significant. Th e percentage of methicillin-resistant staphylococci was higher in nose than in the axillar region of the students.

INTRODUCTION
Th e resistance to methicillin was fi rst detected in Staphylococcus aureus in Great Britain in 1961 (Ašanin et al., 2012;Ćirković et al., 2015;Kluytmans et al., 1997). Th is type of resistance emerged only two years aft er methicillin, a semisynthetic penicillin preparation resistant to an inducible enzyme -penicillinase, was introduced into clinical practice. Th e same year the term MRSA (methicillin-resistant Staphylococcus aureus) was coined, which is still in use, although methicillin is not used for the detection of this type of resistance any more. Th e most frequent mechanism of resistance to methicillin is the acquisition of mecA gene (2.1 kb), which is part of the mobile genetic element of the staphylococcal cassette chromosome mec (SCCmec) (Oliveira et al., 2002;Schito, 2006, De Brito et al., 2015. MecA gene codes for the synthesis of the new penicillin-binding protein, PBP 2a (78 kD), which has low affi nity towards methicillin (Oliveira et al., 2002). Th e production of PBP 2a results in the resistance of staphylococci towards all beta-lactam antibiotics (penicillins, betalactams with beta-lactamase inhibitors, cephems excluding cephalosporins which are active against MRSA strains -ceft aroline and ceft obiprole -and carbapenems) (CLSI, 2016; EUCAST, 2016). Besides mecA genes, staphylococcal cassette chromosome carry many resistance genes to other antibiotics, which is why methicillin-resistant staphylococci (MRS) are considered multidrugresistant pathogens that cause local and systemic infections in humans and animals with frequent fatal outcome due to the absence of effi cacious antibiotics. In 2011 in MRSA strains isolated from cattle and humans another form of mecA gene, mecA LGA251 homologue (García-Álvarez et al., 2011), named mecC gene (Ito et al., 2012) was detected, which was impossible to be done earlier with the protocols for the detection of mecA genes. Th is discovery has raised a question of relevancy of data about the prevalence of MRS on the earth, since it has been clear that there must be more than it was thought.
Although the application of strict control measures in certain countries of the EU, such as Denmark and Sweden, has led to the decrease in the incidence of infections caused by MRSA in humans to less than 1%, it is estimated that the global epidemiological situation concerning the emergence and spread of MRSA strains in the 20th century has dramatically worsened. Th us the resistance of staphylococci to methicillin is considered to be one of the most dangerous and most widespread resistances to antibiotics of all (David et al., 2010). Besides well-known categories, HA-MRSA (healthcare-associated MRSA) and CA-MRSA (community-associated MRSA), there is a third one -LA-MRSA (livestock-associated MRSA). Th e existence of asymptomatic carriers is of utmost importance in the epidemiology of MRS. Th ese are people and animal which are not diseased, but are colonised by MRS strains, which means that these bacteria are part of the microbiota of the skin and mucous membranes. Asymptomatic MRS carriers are sources of infection for people who are most at risk of developing disease. Given that these asymptomatic carriers are clinically healthy people, it is diffi cult to identify them and impossible to treat by any means. Asymptomatic carriers may be colonised by any of the listed MRSAs (HA-MRSA, CA-MRSA and LA-MRSA). Due to the increasing number of reports concerning the transmission from animals to people, MRSA strains have been considered zoonotic infective agents and are in many countries governed by legislation, which precisely defi ne procedures of their detection, prevention of emergence and spread, and how to combat infections. Special attention was paid to the MRSA strain which belongs to the clonal complex 398 (CC398), known by its jargon name -'the pig clone' . Clone MRSA CC398 colonises pigs, which almost never become ill and as asymptomatic carriers are source of human infection (Lewis et al., 2008). It is thought that CC 398 strain has spread through farm workers and their family members to people in cities and can now be found in hospitals in the EU, in intensive-care units, where it causes 15% of the MRSA infections.

MATERIAL AND METHODS
Th e research was conducted on a hundred students of the Faculty of Veterinary Medicine in Belgrade. Th ere were 62 males and 38 females aged from 19 to 31 years. Swabs were taken from students of all years, from the 1st to the 6th, as well as from interns. From each student two swabs were taken: one from the nose and the second from the skin of the axillary region. In addition, each person was asked to fi ll in the questionnaire connected with the research. Th e questions were divided into fi ve groups. Th e fi rst group dealt with the student's pet (if they had any). Th e second group of questions included anamnestic data on the previous use of antibiotics, and the third one information about hospitalisation and visits to the hospital. Th e last two question groups concerned contact with farm animals (cattle and/or pigs) which are potential carriers of LA-MRSA.
Blood agar (Columbia agar with 5% sheep blood, bioMérieux, France) plates were inoculated for the isolation of staphylococci immediately aft er the swabs were taken and the petri dishes were incubated for 18-24 hours at 37°C. Commercial tests, ID32 STAPH (bioMérieux, France) and BBL Crystal Gram-Positive ID Kit (Becton Dickinson, USA) were used for the identifi cation of the isolates.
Phenotypic research on the resistance to methicillin was performed by disc diff usion method on Mueller Hinton agar (bioMérieux, France) using antimicrobial-susceptibility test discs of cefoxitin (30 μg) (Becton Dickinson, USA). Th e results were interpreted according to the guidelines proposed by EUCAST (EUCAST, 2016). Th e inoculum density of the strains tested was approximately 1-2x10 8 CFU/mL, which is equivalent to 0.5 McFarland standard.
All isolates resistant to cefoxitin were tested for the presence of mecA and mecC genes using the PCR technique. Staphylococcus spp. DNA extraction was performed following the protocol proposed by the European Union's reference laboratory for antimicrobial resistance -EU Reference Laboratory-Antimicrobial resistance, Faculty of Veterinary Medicine, Lisbon, Portugal.
According to the protocol for the detection of mecA gene (Isenberg, 2004), the length of PCR product was 533 bp. Th e sequence of the primers (Invitrogen, USA) for the amplifi cation of 533 bp region of mecA gene was the following: primer 1 (5'-AAA ATC GAT GGT AAA GGT TGG C-3') and primer 2 (5'-AGT TCT GCA GTA CCG GAT TTG C-3') each in fi nal concentration of 0.25 μΜ. All deoxyribonucleoside triphosphates (Th ermo Scientifi c) were used in concentration of 200 μM. In addition, the following reagents were used: PCR buff er 1x, 1.5 mM MgCl 2 and 5 U TaqDNA polymerase (Th ermo Scientifi c).
Th e procedure started by initial denaturation at 94°C for 5 minutes, followed by 40 cycles of denaturation at 94° C for 30 seconds, annealing at 55°C for 30 seconds and extension at 72°C for 1 minute, and, fi nal extension at 72°C for 5 minutes in the thermocycler (Eppendorf, Germany). Th e Aft er electrophoresis in 1.5% agarose gel with ethidium bromide in 1x TBE buff er at 100V, PCR products were visualised on the UV transilluminator (Vilber Lourmat, Germany). Two reference strains were used for quality control: Staphylococcus aureus ATCC 43300 as the positive control, and as the negative one Staphylococcus aureus ATCC 25923.
Isolates which were negative for the presence of mecA genes were assessed

RESULTS
Staphylococci were isolated from 146 out of 200 swabs (79 isolates from the nose and 67 from the axilla). Using the disc diff usion method, resistance to cefoxitin was detected in 17 isolates, i.e. in 12 nose and 5 axillary swabs. PCR technique detected mecA gene in 7 isolates, which were identifi ed as S. epidermidis (n=4), S. aureus (n=2) and S. haemolyticus (n=1). Four mecA-positive isolates were from the nose and three from the armpit. Six out of seven mecApositive isolates originated from males. Five MRS isolates was identifi ed in 3rd-year students and one in a 1st-year and a 4th-year student.
Th e diameters of the growth inhibition zone around the cefoxitin discs in the disc diff usion test, which were the criteria for the suspicion that the isolates tested were methicillin-resistant, are presented in Table 1. In addition, in Table  1 the results of the biochemical identifi cation of the isolates and the molecular (PCR) research. In one student, methicillin-resistant S. epidermidis was found both in the nose and in the axillary region. Th e prevalence of MRS isolates detected in the students of the Faculty of Veterinary Medicine was 6% (Figure 1). Concerning ten isolates, the diameter of growth inhibition zone around the cefoxitin disc was less than 25 mm for coagulase-negative staphylococci and less than 22 mm for S. aureus, which is why they were, in accordance with the recommendations of EUCAST, considered resistant to methicillin. However, with the PCR technique in those isolates the presence of mecA and mecC genes was not detected.

DISCUSSION
Th e prevalence of staphylococci resistant to methicillin can vary significantly among various studies and may result from genuine diff erences in the prevalence of MRSA in the populations tested (Ćirković et al., 2015). Th e complete control of the presence of certain clones of MRS in the people of the whole country is virtually impossible. However, monitoring the presence of MRS in certain populations may be the fi rst step in the battle against the high prevalence of methicillin-resistant staphylococci. Th is research was based on that principle, with the aim of detecting the number of asymptomatic carriers of MRS strains in a small population of the students of the Faculty of Veterinary Medicine in Belgrade. From the investigation conducted on 100 students, the data on the prevalence of staphylococci resistant to methicillin in our nearest surrounding can be obtained.
Th e nasal mucous membrane is considered the primary location colonised by MRS strains (Rohr et al., 2004), which has been confi rmed in our study. MRS may colonise other locations on the human body, despite not having been found on the nasal mucosa. In research performed in England, it was discovered that the colonisation of the pharynx with MRSs (72/635, or 11.3%) occurs more frequently than that of the nose (59/635, 9.3%) or other body parts (Bignardi et al., 2009). Some authors consider that there are three types of asymptomatic carriers of CA-MRSA in a population (Kluytmans et al., 1997). According to these, 20% of asymptomatic carriers are colonised incessantly, whilst in 60% the presence of MRSA happens periodically. Th e rest of the asymptomatic carriers, 20%, are rarely colonised with MRS. Th e research done by Lim et al. (2006) suggests that persistent asymptomatic MRS carriers who have these in the nose are protected from the colonisation with new S. aureus strains.
Statistically, MRSs were detected in 6% of the students of the Faculty of Veterinary Medicine in Belgrade. Th is is signifi cantly lower prevalence in comparison to the countries with high prevalence of MRS, such as in India, where it has been detected to be 41% (Joshi et al., 2013). Th e highest prevalence of MRS was detected in hospital environments, which was confi rmed by multiple studies. For example, in a hospital in Portugal 54 strains of S. aureus were isolated and in 14 (25.9%) the mecA gene was detected (Espadinha et al., 2013). Out of the 1,195 patients of the Saint Louis hospital in USA, 31.8% were colonised with MRS (Fritz et al., 2009).
Th e investigation completed by researchers in Serbia (Ćirković et al., 2013) revealed a low prevalence of MRSA strains: it was 0.37% in 533 second-, third-and fourth-year students of the School of Medicine in Belgrade. It is to be underlined that in this research coagulase-negative staphylococci (CoNS) were not taken into consideration. However, our investigation was performed on fewer people but the prevalence of MRSA was 2%. Due to direct contacts with hospital patients, higher-year students of the School of Medicine were at considerably higher risk of being colonised by MRSA strains. By contrast, according to the survey, students of the Faculty of Veterinary medicine had no risky contacts, thus it remains unclear what is the reason of higher prevalence of MRSA strains in this population compared to medical students.
Investigation into the presence of MRSA strains in the nose of healthy students of the Medical Faculty in Czech Republic detected as many as 32% 1styear and 30% 5th-year students asymptomatically colonised with MRSA (Holý et al., 2015).
Similar research to ours was undertaken in Mexico, when nasal and pharyngeal swabs were collected from 21-year old clinically healthy volunteers According to the survey, 5 out of the 6 examined people from whom MRS were isolated, had immediate contact with farm animals (cattle and swine) and domestic pets (dogs and cats). None of them came into either permanent or periodic contact with people who work at hospitals or some other healthcare facilities. One person had contact neither with pets nor with farm animals. As stated in the questionnaires, none of the persons with MRS had taken any antibiotics or had visited hospitals in the previous six months. Th ree persons harbouring MRS were in direct contact with family members or friends who had taken antibiotics in the recent past. Th us, it is confi rmed that all people are susceptible to MRS (Mišić, 2013).
More than 50% isolates which proved to be resistant to methicillin in the disc diff usion test were not mecA-positive, which points to the unreliability of the disc diff usion method in the investigation into the resistance of staphylococci towards methicillin.

CONCLUSIONS
Th e diameter of growth inhibition zone in the disc diff usion method may serve only as a tool for arising suspicion that there is resistance to methicillin in routine diagnostic. Th e prevalence of MRS in clinically healthy students of the Faculty of Veterinary Medicine, which was 6%, was considered to be signifi cant. Th e incidence of methicillin-resistant staphylococci was higher on the nasal mucous membrane than in the axillary region.