CHANGING EPIDEMIOLOGY OF PRESUMPTIVE COMMUNITY-
ASSOCIATED-METHICILLIN-RESISTANT Staphylococcus aureus 
IN SLOVENIA IN 2014-2015 COMPARED TO 2010
Urška DERMOTA1*, Irena GRMEK KOŠNIK1,2, Sandra JANEŽIČ1,3, Maja RUPNIK1,3
1National Laboratory for Health, Environment and Food, Centre of Medical Microbiology,  
Prvomajska ulica 1, 2000 Maribor, Slovenia
2National Institute of Public Health, Gosposvetska ulica 12, 4000 Kranj, Slovenia
3Faculty of Medicine, University of Maribor, Taborska 8, 2000 Maribor
*Corresponding author: Tel. + 386 4 20 17 171; E-mail: urska.dermota@nlzoh.si
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
236
SPREMEMBA EPIDEMIOLOGIJE PRI PROTI METICILINU ODPORNEM 
Staphylococcus aureus DOMAČEGA OKOLJA V SLOVENIJI 
V LETIH 2014–2015 V PRIMERJAVI Z LETOM 2010
Dermota U, Grmek Košnik I, Janežič S, Rupnik M. Changing epidemiology of presumptive community-associated-methicillin-resistant Staphylococcus 
aureus in Slovenia in 2014-2015 compared to 2010. Zdr Varst. 2020;59(4):236-244. doi: 10.2478/sjph-2020-0030.
Received: Jan 28, 2020
Accepted: Sep 16, 2020 
Original scientific article
ABSTRACT
Keywords: 
Slovenia, CA-MRSA, 
LA-MRSA, mecC, 
spa types, clones
IZVLEČEK
Ključne besede: 
Slovenija, CA-MRSA, 
LA-MRSA, mecC, 
tipi spa, kloni
Introduction: Although the distinction between the Community-Associated-Methicillin-Resistant Staphylococcus 
aureus (CA-MRSA) and Hospital-Associated-Methicillin-Resistant S. aureus (HA-MRSA) has blurred in recent years, 
the CA-MRSA is an important group because of its potential to cause fulminant and severe infections. Its importance 
has further increased with the emergence of Livestock-Associated-Methicillin-Resistant S. aureus (LA-MRSA). 
Methods: In the present study we analysed clonal distributions and virulence factors in presumptive CA-MRSA 
isolated from January 2014 to December 2015 and compared the results with our previous study from 2010. 
Phenotypic definition for presumptive CA-MRSA was based on resistance to cefoxitin and oxacillin and susceptibility 
to at least two of the following four antibiotics: ciprofloxacin, erythromycin, clindamycin and gentamicin. 
Results: In 2014 and 2015 altogether 304 MRSA isolates fulfilled our screening phenotypic definition, 45 isolates 
were cultivated from clinical specimens and 259 from screening specimens. Sequence types ST398, LA-MRSA and 
mecC MRSA increased significantly in 2015 compared to 2010 (p-value <0.05) and were spread over Slovenia. 
Conclusion: The clonal distribution of presumptive CA-MRSA has changed within the study period in Slovenia. In 
2015 the most frequent clone among clinical and screening specimens was a pig-associated clone, ST398, but the 
number of confirmed ST398 infections remains low. While previously ST398 and mecC positive MRSA strains were 
geographically limited, they have spread throughout the country since 2010.
Uvod: Čeprav je ločevanje med proti meticilinu odporno Staphylococcus aureus domačega okolja (CA-MRSA) in 
proti meticilinu odporno Staphylococcus aureus bolnišničnega okolja (HA-MRSA) oteženo, je v zadnjih letih CA-
MRSA pomembna skupina, ki povzroča resne in ogrožajoče okužbe. Njen pomen se je povečal še s pojavom proti 
meticilinu odpornim Staphylococcus aureus rejnih živali (LA-MRSA).
Metode: V tej raziskavi smo analizirali zastopanost klonov in prisotnost virulenčnih dejavnikov pri sevih, sumljivih 
za CA-MRSA, ki so bili osamljeni od januarja 2014 do decembra 2015, in rezultate primerjali s študijo iz leta 
2010.  Fenotipska definicija za sumljive CA-MRSA je temeljila na odpornosti proti cefoksitinu in oksacilinu ter 
občutljivosti za vsaj dva od naslednjih štirih antibiotikov: ciprofloksacin, eritromicin, klindamicin in gentamicin.
Rezultati: V letih 2014 in 2015 je skupno 304 izolatov MRSA ustrezalo naši fenotipski definiciji. 45 izolatov je bilo 
osamljenih iz kliničnih kužnin, 259 iz nadzornih kužnin. Sekvenčni tip ST398, LA-MRSA in mecC MRSA, sta se v letu 
2015 v primerjavi z letom 2010 signifikantno povečala (p-vrednost <0,05) in se razširila po Sloveniji. 
Zaključki: Zastopanost klonov pri sevih, sumljivih za CA-MRSA, se je v obdobju raziskave v Sloveniji spremenila. V 
letu 2015 je bil najpogostejši klon med kliničnimi in nadzornimi kužninami klon ST398, MRSA rejnih živali, vendar 
je število potrjenih okužb s ST398 še vedno nizko. V letu 2010 so bili ST398 in mecC pozitivni sevi MRSA geografsko 
omejeni, v kasnejših letih pa so se razširili po vsej državi.
© Nacionalni inštitut za javno zdravje, Slovenija. 
1 INTRODUCTION
Methicillin-resistant Staphylococcus aureus (MRSA) is 
one of the major pathogens responsible for hospital-
associated (HA-MRSA) and community-associated (CA-
MRSA) infections. The epidemiological distinction between 
HA-MRSA and CA-MRSA is blurred, because CA-MRSA is 
increasingly invading into the health care setting and 
consequently the epidemiology of MRSA has changed (1-
3). MRSA infections in the community can also be caused 
by livestock-associated MRSA (LA-MRSA) (1, 4-7). In recent 
years, LA-MRSA, specifically sequence type (ST) ST398 has 
emerged in farm animals, and human infections caused by 
MRSA ST398 have been increasingly documented in Europe 
(1, 4-9). In 2011 a novel divergent mecA gene homologue 
(mecALGA251), designated mecC, was discovered. mecC MRSA 
was reported in humans, in animals (farm, companion and 
wildlife animals), and also from non-animal sources (e.g. 
water and urban wastewater), indicating the existence of 
mecC MRSA in several different reservoirs (1, 10, 11). 
No systematic surveillance of MRSA isolates and 
epidemiology of MRSA clone distribution in humans has 
been performed in Slovenia, and only the surveillance 
of presumptive CA-MRSA has been established. 
Antibiotic susceptibility patterns with partially available 
epidemiological data (colonization or infection with MRSA 
and hospitalization history in the previous year) was used 
as a screening tool to select the presumptive CA-MRSA 
in an earlier study (12), and isolates were analysed on a 
national level for the first time in 2010 (13). To be consistent 
with inclusion criteria and for comparative purposes we 
have retained the previous antibiotic susceptibility based 
criteria for strain selection and are referring to the strains 
as presumptive CA-MRSA.
Among 92 presumptive CA-MRSA in 2010 the most 
prevalent sequence types (STs) were ST45 (n=35, 38%), 
ST398 (n=14, 15.2%), ST5 (n=9, 9.8%) and ST22 (n=8, 8.7%) 
(13). mecC MRSA was confirmed previously in Slovenia and 
seven of 359 isolates (1.7%) were positive for mecC in a 
retrospective study covering the years 2006 to 2013 (14). 
The aim of the present study was to determine possible 
changes in the clonal distribution of presumptive CA-
MRSA in Slovenia. We performed a prospective study and 
compared the phenotypic and genotypic characteristics 
of presumptive CA-MRSA isolates collected from seven 
laboratories throughout Slovenia collected in 2014 and 
2015 with isolates obtained from the same laboratories in 
2010.
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
237
2 MATERIALS AND METHODS
2.1 Study Setting
The Centre of Medical Microbiology at the National 
Laboratory for Health, Environment and Food (NLZOH) 
covers seven laboratories throughout Slovenia. The same 
NLZOH laboratories that participated in the study in 2010 
were invited to collect prospective MRSA isolates from 
routine diagnostics that fulfilled the screening phenotypic 
pattern for presumptive CA-MRSA during a two-year 
sampling period (1 January 2014 to 31 December 2014 
and 1 January 2015 to 31 December 2015). Isolates were 
classified as presumptive CA-MRSA if they were resistant 
to oxacillin and cefoxitin and susceptible to at least two of 
the following four antibiotics: ciprofloxacin, erythromycin, 
clindamycin and gentamicin. Each MRSA isolate, with 
additional information (gender, sample material and date 
of sampling), was sent to the NLZOH Kranj for molecular 
analyses. Information was extracted from the laboratory 
information system (MBL, Src Infonet, Kranj). All isolates 
were identified by MALDI-TOF mass spectrometry (Bruker 
Daltonic GmBH, Bremen, Germany). 
2.2 Antimicrobial Susceptibility Testing
The susceptibility patterns of the MRSA isolates were 
performed by the agar disk diffusion method according 
to the guidelines of the European Committee on 
Antimicrobial Susceptibility Testing (EUCAST) (15, 
16). The antibiotics tested were penicillin, cefoxitin, 
gentamicin, tobramycin, kanamycin, erythromycin, 
clindamycin, tetracycline, ciprofloxacin, trimethoprim-
sulfamethoxazole, chloramphenicol, rifampin, linezolid, 
mupirocin and fusidic acid (BD, Sparks, USA). Minimal 
inhibitory concentration (MIC) determination of oxacillin, 
cefoxitin and vancomycin was performed using the E-test 
(bioMerieux, Marcy-l’Etoile, France). 
2.3 Molecular Characterisation
The presence of mecA or mecC and Panton-Valentine 
leukocidin (PVL) genes was tested by PCR using Genotype 
MRSA (Hain Lifescience GmbH, Nehren, Germany). 
Genes encoding for staphylococcal enterotoxins (sea, 
seb, sec, sed and see), toxic shock syndrome toxin (tst), 
locus enterotoxin gene cluster (egc) and staphylococcal 
exfoliative toxins (eta, etb, etd) were detected by 
multiplex PCR (17-19). SCCmec typing was performed by a 
multiplex PCR strategy previously described by Chen et al. 
and Petersdorf et al. (20, 21). 
The DNA sequence-based methods, spa typing, is based 
on the number of tandem repeats and sequence variation 
in region X of the S. aureus-specific staphylococcal 
protein A gene. Amplification, sequencing and analysis 
of spa typing were performed according to a method 
described previously and analysed with Ridom SpaServer 
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
238
(22). Spa clustering was performed using the spa-plugin in 
BioNumerics, and the results displayed with a minimum 
spanning tree. Multilocus sequence typing (MLST) is a 
highly discriminatory method of DNA sequence-based 
typing that relies on analysis of relatively conserved 
genes that encode essential proteins (seven housekeeping 
genes). MLST was performed for 19 MRSA isolates 
according to a method described by Enright et al. (23) and 
sequence types (ST) were determined with BioNumerics 
MLST-plugin (version 7.6; Applied Maths). All other STs 
were assigned based on spa types as published on http://
spaserver.ridom.de and Kinross et al. (6).
2.4 Statistical Analyses
Data were analysed with the free online software MedCal’c 
(www.medcalc.org). Categorical variables were compared 
using the Chi-square test. A p-value ≤0.05 was considered 
as statistically significant.
3 RESULTS 
3.1 Bacterial Isolates – Distribution Between Clinical 
and Screening Specimens
In 2010, 2014 and 2015, 92, 122 and 182 MRSA isolates had 
a positive susceptibility pattern of presumptive CA-MRSA, 
respectively. Of those, 335 MRSA isolates were cultivated 
from screening specimens and 61 from clinical specimens 
(year 2010, n=16, 17.4%; year 2014, n=20, 16.4%; year 2015, 
n=25, 13.7%) (Table 1). 
Table 1. Distribution of presumptive CA-MRSA isolates among clinical specimens.
a value is number (%) 
Skin and soft tissue
Aspirate tracheae
Knee joint puncture
Urine 
Nose swab
Blood culture
Bone 
Faeces 
Nasopharynx 
Ear swab
12 (75)
1 (6.3)
-
1 (6.3)
-
-
-
-
-
2 (12.4)
13 (52)
4 (16)
1 (4)
2 (8)
1 (4)
1 (4)
1 (4)
1 (4)
1 (4)
-
13 (65)
4 (20)
1 (5)
1 (5)
1 (5)
-
-
-
-
-
Specimen
Resultsa for MRSA patients from
2010
(n=16)
2015
(n=25)
2014
(n=20)
3.2 Distribution of Spa Types among Presumptive CA-
MRSA
Presumptive CA-MRSA strains isolated during 2014 
were associated with 46 different spa types and two 
(1.6%) strains were non-typable. Strains from 2015 were 
associated with 71 different spa types and one (0.5%) 
strain was non-typable. Based on spa types were strains 
in 2014 and 2015 were assigned to 18 MLST STs (Table 2, 
Figure 1). 
Figure 1. Distribution of CA-MRSA spa types circulating 
in Slovenia in 2014 and 2015.
Cluster analysis was performed using the spa typing plug-in tool 
of the BioNumerics program. Nodes indicate spa types and the 
number of sections within the node indicate number of strains for 
each spa type. Tree is color-coded according the year of isolation.  
239
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
Table 2. Most frequent spa types and sequence types of presumptive CA-MRSA isolates from year 2010, 2014 and 2015. 
Legend: *Previously published (9); **New spa type; aAssociated multilocus sequence types published on http://spaserver.ridom.de; 
bPreviously published (15). Underlined spa type multilocus sequence types are those determined in this study.
1
2
3
4
5
6
7
8
9
10
>10
t011
t034
t127
t728
t002
t015
t359
t1048
t091
t223
t003, t005,  
t008, t010,  
t021, t022, t044, 
t050, t051, t067, 
t122, t131, t177, 
t216, t304, t316, 
t331, t334, t447, 
t448, t583, t589, 
t595, t685, t786,  
t843, t976, t991, 
t1344, t1451, t1510, 
t1523, t1689,  
t1943, t2032, t2135, 
t2289, t2576, t2970, 
t3002, t3213, t3256, 
t3380, t3673, t3992, 
t4272, t4335, t4960, 
t5168, t5510, t9397, 
t10765, t12135, 
t15607**, t15608, 
t15609**, t15610**, 
t15628**, t15629**, 
t15773**, t15823** 
t011
t359
t002
t127
t034
t003
t015
t050
t008
t2164
t026, t032, 
t091, t095, 
t116, t189, 
t216, t223, 
t230, t267, 
t331, t355, 
t448, t622, 
t693, t728, 
t808, t843,  
t1081, t1094, 
t1192, t1340, 
t1451, t1689, 
t1842, t1943, 
t4335, t5032, 
t5500, t7268, 
t14168, 
t14540, 
t14541, 
t14542, 
t14546**
t015
t011
t728
t091
t008
t002
t005
t026
t127
t020
t003, t006, 
t034, t041, 
t105, t108, 
t116, t174, 
t355, t595, 
t737, t791, 
t843, t846, 
t1079, t1094, 
t1111, t1179, 
t1218, t1231, 
t1509, t1911, 
t2032, t3824, 
t4365,  
t13070 
ST398b
ST398b
ST1
ST45
ST5
ST45
ST97
ST130
ST7
ST22
ST1, ST5, 
ST8, ST22, 
ST30, ST45, 
ST59, ST80, 
ST88, ST97, 
ST152/377, 
ST130, 
ST188, 
ST247, 
ST398, 
ST913, 
ST3138
ST398b
ST97
ST5
ST1
ST398b
ST5
ST45
ST45
ST8
ST5
ST5, ST7, 
ST8, ST15, 
ST22, ST30, 
ST45, ST59, 
ST88, ST97, 
ST152/377, 
ST130, ST188, 
ST398, ST627, 
ST1774, 
ST3138
ST45
ST398b
ST45
ST7
ST8 
ST5
ST22
ST45
ST1
ST22
ST1, ST5, 
ST7, ST8, 
ST22, ST30, 
ST45, ST72, 
ST88, ST130, 
ST152/377, 
ST225, 
ST228, ST398
17.0 (31)
6.6 (12)
5.5 (10)
5.5 (10)
4.9 (9)
4.4 (8)
3.8 (7)
3.3 (6)
2.2 (4)
2.2 (4)
44.6 (81)
17.1 (21)
9.8 (12)
8.1 (10)
6.5 (8)
4.9 (6)
4.9 (6)
4.1 (5)
3.3 (4)
2.4 (3)
2.4 (3)
36.5 (45)
21.8 (20)
13.0 (12)
6.5 (6)
6.5 (6)
5.4 (5)
4.3 (4)
4.3 (4)
4.3 (4)
3.3 (3)
2.2 (2)
28.4 (26)
Rank
201720142010*
spa type spa type spa typeMLSTa MLSTa MLSTaFrequency % 
(number)
Frequency % 
(number)
Frequency % 
(number)
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
240
The distribution of the most prevalent STs varied in 
different geographic regions (Figure 2). In 2014 12 spa 
types were determined among clinical specimens (t002, 
t008, t011, t015, t034, t050, t091, t223, t267, t359, t1842, 
t2164) while in 2015 20 spa types were determined (t002, 
t005, t008, t010, t011, t015, t034, t051, t122, t127, t131, 
t359, t1048, t1344, t2576, t3213, t4335, t15608, t15610, 
t15773). 
The first major clone, ST45, detected in 2010 (n=35, 
38%) decreased significantly overall to 15.5% (n=19) 
in 2014 (p-value=0.0003) and to 15.9% (n=29) in 2015 
(p-value=0.0001). 
The second major clone confirmed in 2010 and associated 
with LA-MRSA, ST398 (spa types t011, t034, t1344, t1451, 
t2576, t2970), increased significantly (p-value <0.05) from 
15.2% (n=14) in 2010 to 27.5% (n=50) in 2015 (Figure 3). The 
association of these LA-MRSA with infections increased, 
as in 2010 it was reported for only one isolate from skin 
and soft tissue infection, while in 2015 LA-MRSA were 
associated with skin and soft tissue infection (n=3) and 
lower respiratory tract infection (n=1). 
mecC positive MRSA, ST130 and ST3138 increased 
significantly (p-value <0.05) from 1.1% (n=1) in 2010 to 8.2% 
(n=15) in 2015 (Figure 3). In 2010 mecC was isolated from 
skin/mucosal (screening) swabs for MRSA colonization, in 
2015 mecC MRSA were recovered from sputum (n=1) and 
blood culture (n=1), and all the remaining strains were 
recovered from skin/mucosal (screening) swabs for MRSA 
colonization. 
2010*
2010
2014
2014
A-NLZOH Laboratory Kranj                  E- NLZOH Laboratory Maribor
B-NLZOH Laboratory Nova Gorica        F- NLZOH Laboratory Celje
C-NLZOH Laboratory Koper                 G- NLZOH Laboratory Novo mesto
D-NLZOH Laboratory Murska Sobota
*Data already shown by Dermota et al. 2015
A-NLZOH Laboratory Kranj                    E- NLZOH Laboratory Maribor
C-NLZOH Laboratory Koper                   F- NLZOH Laboratory Celje
D-NLZOH Laboratory Murska Sobota      G- NLZOH Laboratory Novo mesto
ST45
Others
prevalence ST398 is ≤1%                                                
prevalence ST398 is between > 10 and ≤ 30%               
prevalence ST398 is between > 1 and ≤ 10%      
prevalence ST398 is ≥ 30%
mecC MRSA
ST398        
ST5
ST1
2015
2015
Figure 2.
Figure 3.
The distribution of presumptive CA-MRSA clones circulating in Slovenia for 2010, 2014 and 2015.        
The distribution and prevalence of ST398 clone and mecC MRSA in Slovenia for 2010, 2014 and 2015.
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
241
Table 3. Frequencies of virulence associated genes and spa types in presumptive CA-MRSA isolates from 2010, 2014 and 2015.
Legend: *Data already shown by Dermota et al. 2015; sea staphylococcal enterotoxin gene type A, seb staphylococcal enterotoxin gene 
type B, sec staphylococcal enterotoxin gene type C, sed staphylococcal enterotoxin gene type D, see staphylococcal enterotoxin gene 
type E, locus egc locus enterotoxin gene cluster, PVL Panton-Valentine leukocidin, tst toxic shock syndrome toxin gene, eta exfoliative 
toxin type A, etb exfoliative toxin type B, etd exfoliative toxin type D
sea 
 
 
seb 
sec 
 
 
 
 
 
 
 
sed 
 
 
 
 
see
Locus egc 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
PVL 
 
 
 
tst 
 
 
eta
etb
etd
6 
 
 
2 
33 
 
 
 
 
 
 
 
14 
 
 
 
 
none
50 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8 
 
 
 
8 
 
 
none
none
none
2.5 
 
 
1.6 
10.7 
 
 
 
 
 
 
 
14.7 
 
 
 
 
0
43.4 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10.7 
 
 
 
1.6 
 
 
0.8
-
-
t008 (4), t041 
(1), t2032 (1) 
 
t002 (1), t026 (1) 
t002 (1), t015 
(18), t026 (3), 
t116 (1), t728 
(5), t737 (1), 
t791 (1), t1079 
(1), t1231 (1), 
t13070 (1) 
 
t002 (2), t003 
(1), t008 (4), 
t015 (2), t020 
(1), t846 (1), 
t1094 (1), t1179 
(1), t2032 (1)
-
t002 (4), t003 
(1), t005 (3), 
t006 (1), t015 
(20), t020 (1), 
t026 (4), t041 
(1), t105 (1), 
t116 (1), t728 
(5), t737 (1), 
t791 (1), t1079 
(1), t1094 (1), 
t1111 (1), t1231 
(1), t3824 (1), 
t13070 (1) 
 
 
 
 
 
 
t002 (2), t005 
(1), t008 (1), 
t091 (1), t355 
(1), t791 (1), 
t4335 (1)
t026 (1), t105 
(1), t728 (5),  
t1111 (1) 
-
-
-
15 
 
 
6 
41 
 
 
 
 
 
 
 
17 
 
 
 
 
none
74 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10 
 
 
 
23 
 
 
1
none
3
6.5 
 
 
2.2 
35.8 
 
 
 
 
 
 
 
15.2 
 
 
 
 
0
54.3 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
8.7 
 
 
 
8.7 
 
 
-
-
-
t008 (2), t003 (1) 
 
 
t216 (1), 
t1340 (1)
t015 (2), t026 
(1), t050 (1), 
t095 (1), t116 
(2), t230 (1), 
t331 (3), t622 
(1), t14540 (1) 
 
 
t002 (9), t003 
(3), t015 (1), 
t026 (1), t1192 
(1), t2164 (2), 
t14541 (1) 
-
t002 (10), t003 
(6), t015 (5), 
t026 (2), t032 
(1), t050 (4), 
t095 (1), t116 
(2), t223 (2), 
t230 (1), t331 
(3), t359 (2), 
t448 (1), t622 
(1), t728 (1), 
t808 (1), t1081 
(1), t1192 (2), 
t1340 (1), t1842 
(1), t2164 (2), 
t5032 (1), t14540 
(1), t14541 (1) 
 
 
 
t002 (9), t050 
(1), t127 (1), 
t2164 (1),  
t355 (1) 
t015 (1), t728 (1) 
 
 
t015 (1)
-
-
3 
 
 
2 
13 
 
 
 
 
 
 
 
18 
 
 
 
 
none
53 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
13 
 
 
 
2 
 
 
1
none
none
8.2 
 
 
3.3 
22.5 
 
 
 
 
 
 
 
9.3 
 
 
 
 
0
40.7 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
5.5 
 
 
 
12.6 
 
 
0.5
-
1.6
t002 (5), t021 (1), 
t122 (1), t127 (1), 
t304 (3), t976 (2), 
t4335 (1), t12135 (1)
t216 (3), t976 
(2), t2289 (1)
t002 (4), t011 (1), t015 
(6), t050 (1), t223 (2), 
t331 (2), t359 (1), t448 
(1), t583 (2), t589 (1), 
t728 (10), t786 (1), 
t1048 (2), t1510 (1), 
t1523 (1), t3213 (1), 
t4335 (1), t5168 (1),  
t15628 (1), t15773 (1) 
t002 (4), t003 (2), t008 
(2), t010 (2), t011 (1), 
t050 (1), t067 (1), t359 
(1), t447 (1), t2032 
(1), t15607 (1) 
-
t002 (9), t003 (2), t005 
(2), t010 (1), t011 (3), 
t015 (7), t021 (1), t022 
(1), t050 (1), t067 
(1), t091 (1), t122 (1), 
t127 (1), t216 (1), t223 
(4), t331 (2), t359 (1), 
t447 (1), t583 (2), 
t589 (1), t685 (1), 
t728 (10), t1048 (1), 
t1510 (1), t1523 (1), 
t2135 (1), t2289 (1), 
t3002 (2), t3213 (1), 
t3673 (1), t4335 (1), 
t5168 (1), t5510 (1), 
t15607 (1),  t15608 
(1),  t15609 (1),  
t15610 (1), t15628 (1), 
t15629 (1),  t15773 
(1),  t15823 (1)  
t002 (4), t010 (1), t044 
(1), t067 (1), t127 (1), 
t131 (1), t595 (1) 
 
t002 (4), t015 (1), t122 
(1), t223 (3), t359 (1), 
t685 (1), t728 (10), 
t4335 (1), t15610 (1)
t991 (1)
-
t044 (1), t131 
(1), t991 (1)
Rank
201720142010*
Number  
of isolates
Number  
of isolates
spa type 
(number)
spa type 
(number)
spa type 
(number)
Frequency 
%
Frequency 
%
Frequency 
%
Number 
of isolates
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
242
3.3 Virulence Factors Among All Presumptive CA-MRSA
Two out of 11 tested virulence genes were not detected 
in the studied strain collection (see, etb). Enterotoxin 
genes (sec, sed and egc) were commonly present, while 
exfoliative toxins were rarely detected (Table 3). PVL 
was more common in 2014 (n=13, 10.7%) than in 2015 
(n=10, 5.5%) and 2010 (n=8, 8.7%), but this result was not 
statistically significant (Table 4). 
Associations of PVL positive MRSA with infections were 
reported in all study years. In 2010 PVL were detected 
in five presumptive CA-MRSA strains among 16 clinical 
specimens. All PVL positive strains were isolated from skin 
and soft tissue infections (n=5, 31.2%) and belonged to spa 
types t002 (n=1), t008 (n=1), t355 (n=1), t791 (n=1) and 
t4365 (n=1). Among 20 clinical specimens, six PVL positive 
presumptive CA-MRSA strains were isolated in 2014 from 
skin and soft tissue infections, belonging to spa types t002 
(n=4), t050 (n=1) and t2164 (n=1). In 2015 four PVL positive 
out of 25 presumptive CA-MRSA strains were isolated from 
skin and soft tissue infections (n=3, 12%) and faeces (n=1, 
4%). These PVL positive strains belonged to spa types t002 
(n=1), t010 (n=1), t127 (n=1) and t131 (n=1).
3.4 SCCmec Types Among All Presumptive CA-MRSA
The most frequently confirmed type was SCCmec IV (2014, 
n=62, 50.1%; 2015, n=88, 48.4%) and SCCmec V (2014, 
n=42, 34.4%; 2015, n=66, 36.3%). SCCmec I was detected 
in 1.6% (n=2) isolates in 2014, and in 1.6% (n=3) in 2015, 
while SCCmec II was detected in 5.7% (n=7) isolates in 
Table 4. Virulence factors of Slovenian presumptive CA-MRSA isolates from 2010, 2014 and 2015.
Legend: *Data already shown by Dermota et al. 2015; sea staphylococcal enterotoxin gene type A, seb staphylococcal enterotoxin gene 
type B, sec staphylococcal enterotoxin gene type C, sed staphylococcal enterotoxin gene type D, see staphylococcal enterotoxin gene 
type E, locus egc locus enterotoxin gene cluster, PVL Panton-Valentine leukocidin, tst toxic shock syndrome toxin gene, eta exfoliative 
toxin type A, etb exfoliative toxin type B, etd exfoliative toxin type D
sea
seb
sec
sed
see
Locus egc
PVL
tst
eta
etb
etd
0.15
0.77
 < 0.0001
0.92
-
0.11
0.63
0.02 
0.61
-
-
2.5 (3)
1.6 (2)
10.7 (13)
14.7 (18)
 0
43.4 (53)
10.7 (13)
1.6 (2)
0.8 (1)
 0
0
6.5 (6)
2.2 (2)
35.8 (33)
15.2 (14)
0
54.3 (50)
8.7 (8)
8.7 (8)
0
0
0
0.61
0.60
0.01
0.15
-
0.03
0.31
0.33
0.79
-
0.39
8.2 (15)
3.3 (6)
22.5 (41)
9.3 (17)
0
40.7 (74)
5.5 (10)
12.6 (23)
0.5 (1)
0
1.6 (3)
Virulence factor
p-value 
(Chi-square test)
2010*
(n=92)
2014
(n=122)
2015
(n=182)
Year 2015 vs. 2010Year 2014 vs. 2010% (number) of presumptive CA-MRSA isolates 
2014, and in 1.1% (n=2) in 2015. 5.7% (n=7) isolates in 2014 
and 3.2% (n=6) isolates in 2015 were non-typable. SCCmec 
XI was detected in 1.6% (n=2) isolates in 2014, and in 8.2% 
(n=15) in 2015.
4 DISCUSSION
Several changes were observed in the clonal distribution 
and virulence properties of presumptive CA-MRSA strains 
during the years 2014 and 2015 in comparison to 2010 
(13). Similar to as in 2010, clones ST5, ST45 and ST398 
were most common during 2014 and 2015 study, but with 
different rankings.
In 2014 and 2015, the first major clone (2014, 28 strains, 
22.9%; 2015, 50 strains, 27.5%) was related to a pig-
associated clone, ST398, while in 2010 ST398 was found in 
15.2% (n=14) and was the second most common clone. In 
2010, most strains belonging to ST398 were predominantly 
found in rural areas of north eastern and southern Slovenia 
(regions D and E), where livestock breeding is an important 
agricultural activity. In 2014 and 2015, the density of 
ST398 was also confirmed in north eastern and southern 
Slovenia (regions D and E), but ST398 was also detected 
in non-agricultural regions, namely A, C, F and G (Figure 
3). Since LA-MRSA has spread throughout Europe, the 
number of colonized people and infections is increasing 
(4, 7). The main reservoirs of LA-MRSA ST398 are pigs, 
poultry, cattle, and companion animals, and close contact 
with animals is a risk factor for LA-MRSA carriage (4, 7). 
10.2478/sjph-2020-0030 Zdr Varst. 2020;59(4):236-244
243
In recent years, LA-MRSA carriage was also reported in 
humans without any animal contact (7-9). MRSA ST398 has 
also been introduced into the health care setting, mainly 
in areas with a high density of livestock farming (5). 
The second major clone (2010, 9 strains, 9.8%; 2014, 21 
strains, 17.2%; 2015, 17 strains, 9.3%) was related to the 
ST5. Despite the difference in percentages, no significance 
in ST5 distribution was observed.
The third major clone (2010, 35 strains, 38.0%; 2014, 19 
strains, 15.5%; 2015, 29 isolates, 15.9%) was related to the 
ST45. Spa type t015, SCCmec type IV decreased from 17.4% 
(n=16) in 2010 to 3.3% (n=4) in 2014 and 2.7% (n=5) in 2015. 
Spa type t728, SCCmec type IV also decreased from 6.5% 
(n=6) in 2010 to 0.8% (n=1) in 2014 and 5.5% (n=10) in 2015. 
Isolates with spa type t728 were found in 2010 in only one 
region in Slovenia, E, but in 2015 they were also found in 
two other regions, A and D. 
European clone ST80 and Balkan clone ST152/377, which 
are circulating in Europe and our neighbouring countries 
(Italy, Austria, Croatia), are rare in Slovenia (24, 25). In 
2010, PVL gene was detected in eight (8.7%) strains that 
belonged to ST5, ST7, ST8, ST22, ST72, ST88, ST152/377 
and ST772. In 2014 and 2015, 23 (7.6%) PVL positive strains 
were associated with ST1, ST5, ST22, ST80 and ST152/377. 
PVL positive ST5 (t002) significantly increased from 1.3% 
(n=2) in 2010 to 7.4% (n=9) in 2014 (p-value=0.0466). PVL 
positive MRSA strains were also found in 2014 and 2015 in 
10 clinical samples (nine samples from wounds, one from 
faeces), but in Slovenia PVL positive clones are surprisingly 
rarely distributed (25). 
mecC positive MRSA strains were found in 1.1% in 2010 (14), 
while in 2014 and 2015 we observed an increase to 1.6% 
and 8.2%, respectively. mecC positive MRSA, associated 
with ST130 and ST3138, were observed in 2014 in the rural 
area of Southern Slovenia (region E), but in 2015 mecC 
MRSA were distributed throughout Slovenia, also in non-
rural regions A, C, G and F (Figure 3). Most mecC positive 
MRSA were detected in asymptomatic carriers; in two 
patients, mecC were detected from clinical specimens 
(sputum, blood culture) (spa type t1048, t15608). In the 
literature, mecC MRSA have mainly been reported in 
humans from screening specimens, as well as from clinical 
specimens, and the range of infections caused by mecC-
carrying MRSA is the same as seen in other S. aureus, 
including life-threatening diseases such as bacteraemia (7, 
10, 26). 
Our study has the following limitations. The first is 
that the definition of presumptive CA-MRSA is based on 
the susceptibility pattern. As we have written in the 
manuscript, in Slovenia no systematic surveillance of 
MRSA isolates is performed. To the best of our knowledge 
the HA-MRSA strains in our country are resistant to beta-
lactam antibiotics, fluoroquinolones, macrolides and 
lincosamides (27). Because of the emerging CA-MRSA in 
Europe, in 2006 a definition for presumptive CA-MRSA 
as a screening tool, the same as in other countries, was 
set based on the susceptibility pattern. To compare 
the data from 2014 and 2015 with that from 2010, the 
same definition for presumptive CA-MRSA was used. The 
authors are aware, however, that our definition is not 
reliable without epidemiological data, as in 2010, due 
to incomplete epidemiological data of health care risk 
factors. The authors are also aware that our definition 
based on susceptibility pattern does not cover all CA-MRSA 
strains (e.g. more resistant CA-MRSA) and also includes 
more sensitive HA-MRSA strains. Another limitation of our 
study is that most isolates were recovered from screening 
specimens at mucocutaneous site.  
In this work we confirmed the changes in clonal distribution 
of presumptive CA-MRSA in Slovenia. The most frequent 
sequence type during the 2014/2015 study period was 
ST398, a pig-associated clone, which is mostly distributed 
among asymptomatic carriers. While previously ST398 and 
mecC positive strains were geographically limited, they 
have spread throughout the country since 2010. 
Future surveillance studies of molecular epidemiology 
with improved molecular methods and whole genome 
sequencing (WGS) of systematically collected MRSA are 
very important in Slovenia in order to monitor changes in 
clonal distribution.
CONFLICT OF INTEREST 
The authors declare that no conflicts of interest exist in 
relation to this research.
FUNDING
The study was financed by internal funding sources.
ETHICAL APPROVAL
Not required.
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244
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