Methicillin-resistant
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).
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.
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).
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).
The presence of
The DNA sequence-based methods,
Data were analysed with the free online software MedCal’c (
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).
Distribution of presumptive CA-MRSA isolates among clinical specimens.
Specimen | Results value is number (%) |
||
---|---|---|---|
2010 | 2014 | 2015 | |
(n=16) | (n=20) | (n=25) | |
Skin and soft tissue | 12 (75) | 13 (65) | 13 (52) |
Aspirate tracheae | 1 (6.3) | 4 (20) | 4 (16) |
Knee joint puncture | - | 1 (5) | 1 (4) |
Urine | 1 (6.3) | 1 (5) | 2 (8) |
Nose swab | - | 1 (5) | 1 (4) |
Blood culture | - | - | 1 (4) |
Bone | - | - | 1 (4) |
Faeces | - | - | 1 (4) |
Nasopharynx | - | - | 1 (4) |
Ear swab | 2 (12.4) | - | - |
Presumptive CA-MRSA strains isolated during 2014 were associated with 46 different
Distribution of CA-MRSA
Most frequent
2010* |
2014 |
2017 |
|||||||
---|---|---|---|---|---|---|---|---|---|
Rank | Frequency % (number) | MLSTa | Frequency % (number) | MLSTa | Frequency (number) | % MLSTa | |||
1 | t015 | 21.8 (20) | ST45 | t011 | 17.1 (21) | ST398b | t011 | 17.0 (31) | ST398b |
2 | t011 | 13.0 (12) | ST398b | 9.8 (12) | ST97 | t034 | 6.6 (12) | ST398b | |
3 | 6.5 (6) | ST45 | t002 | 8.1 (10) | ST5 | t127 | 5.5 (10) | ST1 | |
4 | t091 | 6.5 (6) | ST7 | t127 | 6.5 (8) | ST1 | 5.5 (10) | ST45 | |
5 | t008 | 5.4 (5) | ST8 | t034 | 4.9 (6) | ST398b | t002 | 4.9 (9) | ST5 |
6 | t002 | 4.3 (4) | ST5 | 4.9 (6) | ST5 | t015 | 4.4 (8) | ST45 | |
7 | t005 | 4.3 (4) | ST22 | t015 | 4.1 (5) | ST45 | 3.8 (7) | ST97 | |
8 | t026 | 4.3 (4) | ST45 | t050 | 3.3 (4) | ST45 | 3.3 (6) | ST130 | |
9 | t127 | 3.3 (3) | ST1 | t008 | 2.4 (3) | ST8 | t091 | 2.2 (4) | ST7 |
10 | t020 | 2.2 (2) | ST22 | t2164 | 2.4 (3) | ST5 | t223 | 2.2 (4) | ST22 |
>10 | 28.4 (26) | ST1, ST5, | t026, t032, | 36.5 (45) | ST5, ST7, | 44.6 (81) | ST1, ST5, | ||
t034, t041, | ST7, ST8, | t091, t095, | ST8, ST15, | t008, t010, | ST8, ST22, | ||||
t105, t108, | ST22, ST30, | t116, t189, | ST22, ST30, | t021, t022, t044, | ST30, ST45, | ||||
t116, t174, | ST45, ST72, | t216, t223, | ST45, ST59, | t050, |
ST59, ST80, | ||||
t355, t595, | ST88, ST130, | t230, t267, | ST88, ST97, | t122, t131, t177, | ST88, ST97, | ||||
t737, t791, | ST152/377, | t331, t355, | ST152/377, | t216, t304, t316, | ST152/377, | ||||
ST225, | t448, t622, | ST130, ST188, | t331, t334, t447, | ST130, | |||||
ST228, ST398 | t693, |
ST398, ST627, | t448, t583, t589, | ST188, | |||||
t808, |
ST1774, | t595, t685, t786, | ST247, | ||||||
t1081, t1094, | ST3138 | ST398, | |||||||
t1509, t1911, | t1192, t1340, | t1344, t1451, t1510, | ST913, | ||||||
t1451, t1689, | t1523, t1689, | ST3138 | |||||||
t1842, t1943, | t1943, t2032, t2135, | ||||||||
t2289, t2576, t2970, | |||||||||
t5500, t7268, | t3002, t3213, |
||||||||
t14168, | t3380, t3673, t3992, | ||||||||
t14540, | t4272, t4335, t4960, | ||||||||
t14541, | t5168, t5510, |
||||||||
t14542, | |||||||||
t14546** | t15607**, |
||||||||
t15609**, t15610**, | |||||||||
t15628**, t15629**, | |||||||||
t15773**, t15823** |
Legend: *Previously published (9); **New
Cluster analysis was performed using the
The distribution of the most prevalent STs varied in different geographic regions (Figure 2). In 2014 12
The distribution of presumptive CA-MRSA clones circulating in Slovenia for 2010, 2014 and 2015.
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 (
The distribution and prevalence of ST398 clone and
Two out of 11 tested virulence genes were not detected in the studied strain collection (
Frequencies of virulence associated genes and
2010* |
2014 |
2017 |
|||||||
---|---|---|---|---|---|---|---|---|---|
Rank | Number of isolates | Frequency % | Number of isolates | Frequency % | Number of isolates | Frequency % | |||
6 | 6.5 | t008 (4), t041 (1), t2032 (1) | 3 | 2.5 | t008 (2), t003 (1) | 15 | 8.2 | t002 (5), t021 (1), t122 (1), t127 (1), t304 (3), t976 (2), t4335 (1), t12135 (1) | |
2 | 2.2 | t002 (1), t026 (1) | 2 | 1.6 | t216 (1), t1340 (1) | 6 | 3.3 | t216 (3), t976 (2), t2289 (1) | |
33 | 35.8 | t002 (1), t015 (18), t026 (3), t116 (1), t728 (5), t737 (1), t791 (1), t1079 (1), t1231 (1), t13070 (1) | 13 | 10.7 | t015 (2), t026 (1), t050 (1), t095 (1), t116 (2), t230 (1), t331 (3), t622 (1), t14540 (1) | 41 | 22.5 | 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) | |
14 | 15.2 | t002 (2), t003 (1), t008 (4), t015 (2), t020 (1), t846 (1), t1094 (1), t1179 (1), t2032 (1) | 18 | 14.7 | t002 (9), t003 (3), t015 (1), t026 (1), t1192 (1), t2164 (2), t14541 (1) | 17 | 9.3 | t002 (4), t003 (2), t008 (2), t010 (2), t011 (1), t050 (1), t067 (1), t359 (1), t447 (1), t2032 (1), t15607 (1) | |
none | 0 | - | none | 0 | - | none | 0 | - | |
Locus |
50 | 54.3 | 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) | 53 | 43.4 | 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) | 74 | 40.7 | 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) |
PVL | 8 | 8.7 | t002 (2), t005 (1), t008 (1), t091 (1), t355 (1), t791 (1), t4335 (1) | 13 | 10.7 | t002 (9), t050 (1), t127 (1), t2164 (1), t355 (1) | 10 | 5.5 | t002 (4), t010 (1), t044 (1), t067 (1), t127 (1), t131 (1), t595 (1) |
8 | 8.7 | t026 (1), t105 (1), t728 (5), t1111 (1) | 2 | 1.6 | t015 (1), t728 (1) | 23 | 12.6 | t002 (4), t015 (1), t122 (1), t223 (3), t359 (1), t685 (1), t728 (10), t4335 (1), t15610 (1) | |
none | - | - | 1 | 0.8 | t015 (1) | 1 | 0.5 | t991 (1) | |
none | - | - | none | - | - | none | - | - | |
none | - | - | none | - | - | 3 | 1.6 | t044 (1), t131 (1), t991 (1) |
Legend: *Data already shown by Dermota et al. 2015;
Virulence factors of Slovenian presumptive CA-MRSA isolates from 2010, 2014 and 2015.
2010* (n=92) | 2014 (n=122) | 2015 (n=182) | p-value (Chi-square test) | ||
---|---|---|---|---|---|
Virulence factor | |||||
% (number) of presumptive CA-MRSA isolates | Year 2014 vs. 2010 | Year 2015 vs. 2010 | |||
6.5 (6) | 2.5 (3) | 8.2 (15) | 0.15 | 0.61 | |
2.2 (2) | 1.6 (2) | 3.3 (6) | 0.77 | 0.60 | |
35.8 (33) | 10.7 (13) | 22.5 (41) | < 0.0001 | 0.01 | |
15.2 (14) | 14.7 (18) | 9.3 (17) | 0.92 | 0.15 | |
0 | 0 | 0 | - | - | |
Locus |
54.3 (50) | 43.4 (53) | 40.7 (74) | 0.11 | 0.03 |
PVL | 8.7 (8) | 10.7 (13) | 5.5 (10) | 0.63 | 0.31 |
8.7 (8) | 1.6 (2) | 12.6 (23) | 0.02 | 0.33 | |
0 | 0.8 (1) | 0.5 (1) | 0.61 | 0.79 | |
0 | 0 | 0 | - | - | |
0 | 0 | 1.6 (3) | - | 0.39 |
Legend: *Data already shown by Dermota et al. 2015;
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
The most frequently confirmed type was SCC
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).
In recent years, LA-MRSA carriage was also reported in humans without any animal contact (7, 8, 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.
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).
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
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.