Characterization of a communityacquired- MRSA USA300 isolate from a river sample in Austria and whole genome sequence based comparison to a diverse collection of USA300 isolates

The increasing emergence of multi-resistant bacteria in healthcare settings, in the community and
in the environment represents a major health threat worldwide. In 2016, we started a pilot project to
investigate antimicrobial resistance in surface water. Bacteria were enriched, cultivated on selective
chromogenic media and species identification was carried out by MALDI-TOF analysis. From a river in
southern Austria a methicillin resistant Staphylococcus aureus (MRSA) was isolated. Whole genome
sequence analysis identified the isolate as ST8, spa type t008, SCCmecIV, PVL and ACME positive,
which are main features of CA-MRSA USA300. Whole genome based cgMLST of the water isolate and
comparison to 18 clinical MRSA USA300 isolates from the Austrian national reference laboratory for
coagulase positive staphylococci originating from 2004, 2005 and 2016 and sequences of 146 USA300
isolates arbitrarily retrieved from the Sequence Read Archive revealed a close relatedness to a clinical
isolate from Austria. The presence of a CA-MRSA USA300 isolate in an aquatic environment might
pose a public health risk by serving as a potential source of infection or a source for emergence of new
pathogenic MRSA clones.
Methicillin resistant strains of Staphylococcus aureus (MRSA) are the leading cause of nosocomial infections1.
Manifestations vary from minor skin infection to fatal disease. MRSA have an outstanding ability to acquire antibiotic
resistance genes leading to resistance to multiple antibiotic classes2. Since the late 1960s hospital acquired
MRSA (HA-MRSA) became endemic in hospitals worldwide3. In the 1990s new clones affecting also healthy
individuals in the community, featuring increased virulence as well as the power to spread easily, arose4. This
so called community acquired MRSA (CA-MRSA) became prevalent worldwide with predominant clones in
certain geographic areas. Clonal Complex (CC) 1 (USA400) and CC8 (USA300) are the major lineages in the
United States, CC80 is the predominant lineage in Europe, CC59 and CC30 appear in Asia and the southwest
Pacific5,6. CA-MRSA is now epidemic in the United States mainly due to dissemination of the USA300 clone
which belongs to multi locus sequence type (MLST) 8/SCCmec IV and harbours the lukS-lukF genes, encoding
the Panton-Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME) cluster7. While virulence
of these strains enhances dissemination, the potential to acquire resistance to multiple antibiotic classes
hinders treatment of MRSA infections1. CA-MRSA clones are not restricted to a geographical region. The ability of geographically predominant CA-MRSA clones to spread worldwide and to cause infections on other continents
has been demonstrated2. In several European countries, where ST80 still represents the prevalent CA-MRSA
clone, also infections due to the North American USA300 clone have been reported8–11. Consequently the identification
of new potential infection sources is essential for infection control.
Antimicrobial resistance (AMR) is an increasing global problem and the fact that resistant pathogens are not
restricted to clinical settings but can be increasingly found in the environment is alarming12–14. In aquatic systems,
horizontal gene transfer (HGT) of resistance genes between bacteria leads to the evolution of AMR bacteria,
which in turn find back into clinical settings. AMR dissemination of resistant strains occur through direct contact
of humans to the AMR bacteria in the aquatic system or from contact to resistant environmental bacteria which
pass resistance genes to human or animal pathogens15. So called high risk clones (HRCs), which are characterized
by enhanced virulence and multiple antibiotic resistances, pose a serious public health risk16. Aanensen et al.8
identified three key elements to tackle the public health threat caused by HRCs: genetic population structure and
identification of HRCs, assessment of risks posed by virulence and resistance determinants and risk management
by implementation of prevention and control strategies.
The Austrian Agency for Health and Food Safety, the major Austrian organization responsible for consumer
protection and public health, started a pilot project analysing surface water samples from rivers and bathing sites
with the aim to identify possible public health risks due to AMR HRCs in 201617. Environmental screening for
HRCs and high discriminatory typing identified a CA-USA300 isolate in a river water sample in Austria in 2016.
In this report we describe a detailed characterization of this USA300 MRSA isolate and its phylogenetic relatedness
to clinical CA-MRSA USA300 isolates.