Changing epidemiology of presumptive community-associated-methicillin-resistant Staphylococcus aureus in Slovenia in 2014-2015 compared to 2010

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 (13). MRSA infections in the community can also be caused by livestock-associated MRSA (LA-MRSA) (1, 4, 5, 6, 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, 5, 6, 7, 8, 9). In 2011 a novel divergent mecA gene homologue (mecA_LGA251), 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.

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. 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.