Epidemiology of Methicillin-Resistant Staphylococcus aureus in Slovakia, 2020 – Emergence of an Epidemic USA300 Clone in Community and Hospitals

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of health care-associated infections. Additionally, over the decades, the spread of community-associated (CA-MRSA) clones has become a serious problem. The aim of this study was to gain data on the current epidemiology of MRSA in Slovakia. Between January 2020 and March 2020, single-patient MRSA isolates (invasive and/or colonizing) were collected in Slovakia from hospitalized inpatients (16 hospitals) or outpatients (77 cities). Isolates were characterized via antimicrobial susceptibility testing, spa typing, SCCmec typing, the detection of mecA/mecC, genes coding for Panton-Valentine leukocidin (PVL), and the arcA gene (part of the arginine catabolic mobile element [ACME]). Out of 412 isolates, 167 and 245 originated from hospitalized patients and outpatients, respectively. Inpatients were most likely older (P < 0.001) and carried a strain exhibiting multiple resistance (P = 0.015). Isolates were frequently resistant to erythromycin (n = 320), clindamycin (n = 268), and ciprofloxacin/norfloxacin (n = 261). 55 isolates were resistant to oxacillin/cefoxitin only. By clonal structure, CC5-MRSA-II (n = 106; spa types t003, t014), CC22-MRSA-IV (n = 75; t032), and CC8-MRSA-IV (n = 65; t008) were the most frequent. We identified PVL in 72 isolates (17.48%; 17/412), with the majority belonging to CC8-MRSA-IV (n = 55; arcA+; t008, t622; the USA300 CA-MRSA clone) and CC5-MRSA-IV (n = 13; t311, t323). To the best of our knowledge, this is the first study on the epidemiology of MRSA in Slovakia. The presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV was found, as was, importantly, the emergence of the global epidemic USA300 CA-MRSA clone. The extensive spread of USA300 among inpatients and outpatients across the Slovakian regions warrants further investigation. IMPORTANCE The epidemiology of MRSA is characterized by the rise and fall of epidemic clones. Understanding the spread, as well as the evolution of successful MRSA clones, depends on the knowledge of global MRSA epidemiology. However, basic knowledge about MRSA epidemiology is still fragmented or completely missing in some parts of the world. This is the first study of MRSA epidemiology in Slovakia to identify the presence of the epidemic HA-MRSA clones CC5-MRSA-II and CC22-MRSA-IV and, importantly and unexpectedly, the emergence of the global epidemic USA300 CA-MRSA clone in the Slovakian community and hospitals. So far, USA300 has failed to spread in Europe, and this study documents an extensive spread of this epidemic clone in a European country for the first time.

view of MRSA as a nosocomial pathogen (3,4). Community-associated MRSA (CA-MRSA) strains have become common in the last two decades. CA-MRSA strains are frequently associated with increased virulence and genetic factors, such as Panton-Valentine leukocidin (PVL). Some CA-MRSA lineages also possess arginine catabolic mobile elements (ACME) that are responsible for increased survival on human skin. The distinctive feature of CA-MRSA is its capacity to cause severe infection in young and healthy people, compared to healthcare-associated MRSA (HA-MRSA), which affects patients of advanced age as well as those with comorbidities (4).
The population structure of MRSA is characterized by the presence of epidemic clones. The selective pressure of antibiotics, along with the subsequent acquisition of resistance, is the main contributing factor to the expansion of epidemic clones, such as the global expansion of ST5-MRSA-II (5) or ST8-MRSA-IV (USA300 [CC8]) (6).
Slovakia has been a participating country in the European monitoring of the resistance prevalence among bacterial isolates that cause invasive infections European Antimicrobial Resistance Surveillance Network (EARS-Net) since 2011. According to EARS-NET data, the frequency of MRSA in Slovakia is relatively high, ranging between 25% to 30% of invasive staphylococcal isolates between 2016 and 2020 (7). However, little is known about the population structure of MRSA clones circulating in the country. Only one isolate from Slovakia was investigated in the study by Rolo et al., which focused on community-associated S. aureus across Europe (8). Following our previous study of MRSA epidemiology in the Czech Republic (9), we aimed to collect and characterize the representative number of MRSA isolates circulating in the community and hospitals in Slovakia. We want to use the acquired data to fill the gap in Slovak MRSA epidemiology and compare it with the situation in the Czech Republic as well as other neighboring countries.

RESULTS
Patient data. In total, 412 single-patient MRSA isolates were collected in Slovakia, and these came from 167 inpatients hospitalized in 16 hospitals and 245 outpatients attending health care facilities in 77 cities (Fig. 1).
The isolates originated from the following types of samples: nose swab (n = 187), wound swab (n = 114), throat swab (n = 54), genitourinary tract samples (n = 14), ear swab (n = 10), lower airway samples (n = 10), blood cultures (n = 7), catheters (n = 4), eye swab (n = 4), and others (n = 8). In 165 (40.05%; 165/412) and 243 (58.98%; 243/412) of the cases, the isolates Emergence of USA300 CA-MRSA in Slovakia Microbiology Spectrum were considered to be the cause of infection and asymptomatic carriage, respectively. For four isolates, assignment to an invasive or colonizing group was not possible. The median age of patients was 45 years (range of 1 day to 98 years), and 199 (48.30%; 199/412) of the patients were men. For men and women, there was no statistically significant difference regarding the age, region, infection/colonization status, origin of the isolate, clonal lineage, or PVL positivity of the isolate. The inpatients (median age of 67.49 years) and patients with MRSA infections (59.36 years) were significantly older than were the outpatients (27.47 years; P , 0.001) and the patients with asymptomatic colonization (32.64 years; P = 0.002). For the associations of various isolate characteristics, see Fig. 2.
Clonal structure. Among the 412 isolates, 66 spa types were identified. 28 spa types were present among inpatients, and 53 spa types were present among outpatients. Isolates carried SCCmec cassette type I (n = 41), type II (n = 106), type IV (n = 248), and type V (n = 9). For 404 isolates, the MLST clonal complexes (CC) derived from the spa types and the SCCmec types were available. A total of 14 MRSA clonal lineages were identified. Six major clonal lineages, each representing more than 40 isolates, accounted for 94.17% (388/412) of the isolates ( Table 2).
The CC5-MRSA-II was the most common MRSA lineage (25.73%; 106/412). It was present in all regions and dominating in both inpatients (32.34%; 54/167) and outpatients (21.22%; 52/245), with no statistical difference between invasive and colonizing strains (P = 1.000). The majority of isolates belonged to spa types t014 (n = 55) and t003 (n = 42), which are representatives of the Rhine-Hesse MRSA clone (ST225-MRSA-II). Patients infected or colonized by CC5-MRSA-II (median age of 70.8 years) were significantly older (P , 0.001), compared to patients with other lineages, with the exception of CC22-MRSA-IV (P = 1.000) (for a boxplot comparing the age distribution of the patients among MRSA clones, see the Supplemental Material). The CC5-MRSA-II isolates were resistant to four or more categories of antibiotics (P , 0.001). The typical resistance profile (97.17%; 103/106) was the combination of resistance to antistaphylococcal beta-lactams, macrolides, fluoroquinolones, and lincosamides.
The second most common lineage was CC22-MRSA-IV (18.20%; 75/412), which is known also as an EMRSA-15 or Barnim clone. All isolates carried SCCmec subtype IVh, and the most frequent spa type was t032 (n = 65), whereas other spa types were rare. CC22-MRSA-IV was associated more with inpatients (47 of 75 isolates; P , 0.001) and older age (median age of 67.28 years, P , 0.001), compared to other lineages, with the exception of CC5-MRSA-II. All of the isolates were resistant to fluoroquinolones. In 60 (80.00%; 60/75) isolates, a combined resistance to macrolides and lincosamides was detected.
Eight lineages were represented by four or fewer isolates (Table 2). One MRSA (t7603; CC130) isolate carrying the mecC gene was found.

DISCUSSION
The Slovakia MRSA invasive infection prevalence of 24.81% (134/540) is considerably higher, compared to neighboring countries, such as the Czech Republic (9.28%; 194/2,089), Austria (4.40%; 125/2,843), and Poland (13.84%; 187/1,351) (7), but no data on the characteristics of MRSA strains are available from this region. To fill this knowledge gap, we collected 412 MRSA isolates, including inpatient and outpatient MRSA cases, covering all regions of Slovakia.
Interestingly, the third most frequently detected lineage in this study was CC8-MRSA-IV (15.78%; 65/412), with characteristic features of the US epidemic CA-MRSA clone USA300, that is, SCCmec subtype IVa with genes coding for PVL and arcA (15) in the majority of isolates (83.33%; 54/65). The onset and subsequent spread of the USA300 epidemic in America in 2005 (16) were followed by several studies in other countries that were set up to monitor the spread of CA-MRSA and/or the USA300 clone, in particular. In Europe, USA300 was found to be the most frequent (18%; 9/51) among 51 imported CA-MRSA cases that were reported between 2011 and 2016 in 7 countries (17). Other studies detecting the sporadic presence of USA 300 MRSA isolates in Europe concluded this to be associated with multiple Emergence of USA300 CA-MRSA in Slovakia Microbiology Spectrum introductions of USA300 from abroad, with only a limited local spread (6,(18)(19)(20)(21). Originally, USA300 was frequently found in inpatients without any obvious risk factors for MRSA acquisition, and it often caused invasive infection and exhibited limited antimicrobial resistance (4). Later, the strain acquired additional resistance genes (6) and started to be found more frequently in health care facilities (22). Correspondingly, the results of our study found that USA300 isolates were significantly more often the cause of infection, rather than mere colonization, but they were frequently resistant to erythromycin and norfloxacin/ciprofloxacin. Additionally, they were derived from both outpatient and inpatients. Importantly, the spread of this clone in the USA led to an increase in the number of MRSA infections (23), and severe forms of Staphylococcal infections, such as necrotizing fasciitis and pneumonia, became more common (4,24). The burden of USA300 on the Slovak health care system as well as the factors driving the spread of USA300 in Slovakia are currently unknown. Further studies will be needed to assess the risks for Slovak patients and possible dissemination into neighboring countries. PVL positivity (a marker of CA-MRSA) among all isolates was significantly associated with infection, younger age, and an outpatient setting, pointing to the higher virulence of these strains, as reported previously (4). Independently of PVL positivity, CA-MRSA characteristics, such as a younger age and an outpatient setting, were associated with CC5-MRSA-I (t010, t045), CC5-MRSA-IV (t311, t179, t3235), and CC45-MRSA-IV (t015, t026, t330, t671, t1231), but, of these, only the 11 CC5-MRSA-IV (9Â t311; 2Â t3235) isolates were positive for PVL. These lineages, found in between 9.95% (41/412) and 13.11% (54/412) of isolates, are rarely, if ever, found in hospital-based studies in the Czech Republic, Poland, and Austria (9,11,13,14,(25)(26)(27), stressing that focusing only on hospital epidemiology could significantly underestimate the presence of strains that are circulating in the community and causing significant disease burden.
Conclusions. To the best of our knowledge, this is the first study on the molecular epidemiology of MRSA in Slovakia. This study confirmed the presence of the epidemic clones CC5-MRSA-II and CC22-MRSA-IV that were reported from neighboring countries and identified a high prevalence of the global epidemic CA-MRSA clone, namely, USA300 (CC8-MRSA-IV; PVL1; arcA1), among inpatients and outpatients. Taking into account the data from other European countries, the high prevalence of the USA300 clone in Slovakia is highly unexpected and warrants further investigation to understand the possible risk of its dissemination.

MATERIALS AND METHODS
Ethics. Ethical approval and informed consent were not needed for this study because it was a laboratorybased surveillance study and because the isolates were acquired as a part of routine diagnostics. The patient data were anonymized, and the results of the study have no impact on the patient's care.
Study design. Between January 3 rd , 2020, and March 27 th , 2020, non-duplicated (single-patient) MRSA isolates were collected in Slovakia from hospitalized patients (16 hospitals) and outpatients attending outpatient hospital departments (including emergency departments), outpatient medical centers, and general practitioners (77 cities) (Fig. 1). Isolates were cultured from blood samples, samples that were collected from the site of an infection, or samples that were collected during screening for asymptomatic colonization upon the request of a physician. The MRSA screening (nose, throat, and skin swabs) was performed based on local guidelines or at the discretion of the treating physician.
The isolates were grouped as (i) invasive when isolated from a primarily sterile site (i.e., blood-culture or catheter) and/or when the isolation of MRSA from a particular site corresponded with the presence of clinical symptoms of the infection or as (ii) colonizing when the isolation of MRSA was not directly related to the patient's diagnosis.
The following anonymized data were collected: age, sex, clinical setting (inpatient or outpatient), geographic location of the health care facility, sample type, and patient diagnosis.
Identification of the isolates and antimicrobial susceptibility testing. The species identification of isolates was confirmed via MALDI-TOF MS Biotyper v 3.1 (Bruker Daltonics). Antimicrobial susceptibility was tested via the disc diffusion method with the breakpoints recommended by EUCAST v12.0 (28). Inducible clindamycin resistance was tested via D tests (28). Resistance to tigecycline was detected via disc diffusion and confirmed via the gradient diffusion test. The multiplicity of antimicrobial resistance was counted by using previously described antistaphylococcal drug categories (29).
Isolates were tested for the presence of the mecA gene and for genes coding Panton-Valentine leucocidin (PVL) via qPCR (30). In cases of mecA negative results, the presence of the mecC gene was tested via PCR (31). In addition, the CC8-MRSA-IV isolates were tested via PCR for the presence of arcA (15).

Emergence of USA300 CA-MRSA in Slovakia
Microbiology Spectrum Clonal analysis. The spa typing was performed as previously described (32), and the individual spa types were assigned by using the Ridom StaphType software package, version 2.2.1 (Ridom, Germany). They were clustered into spa clonal complexes by using the based upon repeat pattern (BURP) algorithm. The multilocus sequence typing STs and/or clonal complexes (CC) were either inferred by using the SpaServer database (http://spaserver.ridom.de) or derived from previously published studies.
SCCmec types I to VI were determined by using multiplex PCR (33), and the subtyping of the IVa to IVd and IVg to IVh types was performed (34,35).
Statistics. Differences between groups were evaluated by using the chi-square test for categorical variables. For small sample sizes, Fisher¨s exact test was used. A Kruskal-Wallis test and a post hoc Dunn's test with the Holm correction were used for the comparisons of continuous variables.
A P value of #0.05 was considered to be indicative of a statistically significant result. The analyses were conducted by using the R statistical libraries "stats" and "rstatix".
Isolates with incomplete epidemiological or demographic data as well as those originating from lineages that were represented by fewer than 10 isolates were excluded from the statistical analysis. Data for 384 out of 412 isolates were included.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, XLSX file, 0.2 MB.