Spa Diversity and Genetic Characterization of t127 Methicillin-resistant Staphylococcus Aureus in a Tertiary Greek Hospital

Introduction: Methicillin-resistant Staphylococcus aureus (MRSA) causes severe community and hospital acquired infections. Identication of staphylococcal cassette chromosome mec (SCCmec), multilocus-sequence typing, and sequencing of S. aureus protein A (spa) gene are used for MRSA typing. The aim was to investigate the spa types of MRSA isolates in a tertiary hospital in Greece and analyse the whole genome sequences of two t127 MRSA isolates. Methods: Totally, 39 MRSA isolates collected during July 2019 to June 2020 in “Georgios Gennimatas” General Hospital of Thessaloniki, Greece, were included in the study. Identication and antimicrobial susceptibility testing were performed using VITEK II automated system, and spa typing was performed. A minimum spanning tree was used to display the spa type frequencies and the genetic distances among them. Two t127-MRSA isolates (IM-MRSA and PD-MRSA) were selected for WGS. Results: Twenty-two different spa types were detected, with t002, t003, and t422 being the most frequent (5/39, 12.8% each), followed by t1994 (4/39, 10.3%). The isolates presented high genetic diversity and, taking into account the time between hospital admission and sampling, intrahospital spread did not occur. Even the two t127 isolates were assigned to different sequence types, ST9-XII-t127 and ST1-IVa-t127. Plasmids and genes conferring antimicrobial resistance and virulence were also identied. Conclusions: Various spa types were identied and together with the information about the time between hospital admission and sampling supports polyclonal MRSA spread in the hospital excluding a nosocomial infection. WGS provides a more detailed analysis distinguishing even the isolates belonging to the same spa type. The current study provides an insight into the spa types of in a tertiary hospital in and together with the information of time interval between admission and sampling suggests polyclonal introductions in the various wards. The extensive resistance to most antimicrobial classes needs further attention since it is associated with high antimicrobial consumption. A more detailed genetic characterization is gained when WGS is applied, as shown by the results of the analysis of the two t127 MRSA isolates, which showed that although they the same spa type, they were different strains. Molecular surveillance studies


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a gram-positive bacterium causing community-acquired and nosocomial infections worldwide (Tong et al. 2015). According to the latest epidemiological report of the European Centre for Disease Prevention and Control (ECDC), the prevalence of MRSA in Greece is among the highest in Europe (European Centre for Disease Prevention and Control. 2020).
Various methods are used for MRSA typing, such as the identi cation of staphylococcal cassette chromosome mec (SCCmec), the multilocus-sequence typing (MLST), and the sequencing of the highly polymorphic repeat region of S. aureus protein A (spa) gene (Enright et al. 2000;Bosch et al. 2016; Kaya et al. 2018). The detection of spa-clusters could be used as a rapid tool for the study of MRSA epidemiology in a hospital and for separation between relapse and re-infection (Satta et al. 2013).
Previous studies in Greece showed that spa types t044, t003 and t037 are the predominant types among MRSA isolated from clinical sources (Kachrimanidou et al. 2014;Nikolaras et al. 2019). In Europe, the most prevalent are the spa-types t032, t008 and t067, while t011, t108 and t034 predominate among livestock-associated (LA)-MRSA (Asadollahi et al. 2018). LA-MRSA were rst detected in 2003 in pigs (Voss et al. 2005), while later, they have been isolated also in patients with no previous contact with livestock (Larsen et al. 2017). Knowledge of MRSA epidemiology is of great importance in organising and implementing infection control measures, especially in hospital settings (Leekha et al. 2020 The aim of the present study was to evaluate the antimicrobial resistance patterns and the distribution and prevalence of spa types of MRSA isolated from patients hospitalized in various wards of a tertiary hospital in Greece, and to further characterize the whole genome sequences of two isolates belonging to spa type t127, which is often associated with LA-MRSA.

Bacterial isolates
Thirty-nine MRSA isolates collected during one-year period (July 2019 to June 2020) from 39 patients (18 males, 46%) hospitalized in various wards of "Georgios Gennimatas" General Hospital of Thessaloniki in Greece, were included in the study. The median age of the patients was 59 years (range 0.25 -91 years). The isolates were recovered from wound (18, 46.2%), ear swab ( ve, 12.8%), blood, throat and nasal swabs (four, 10.3% each), central intravenous catheter (two, 5.1%), urine and eye swab samples (one, 2.5%, each) ( Table 1). Eight isolates (20.5%) were taken through testing for colonization, while 29 (79.5%) were collected from infection sites. The distinction between infection and colonization was performed using previously described criteria (Geladari et al. 2017). The time between patients' admission to the hospital and sampling was estimated in order to associate or not with nosocomial infection (>48 or <48 hours, respectively) ( Table 1).

Microbiological methods
All samples were cultured in blood agar, and strain identi cation and antimicrobial susceptibility testing were performed using the GP ID and AST-P659 cards in VITEK II automated system, respectively (BioMérieux, Marcyl'Étoile, France). The minimum inhibitory concentration (MIC) was interpreted according to the Clinical and Laboratory Standards Institute (CLSI) breakpoints reported in January 2019 (CLSI. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-Fifth Informational Supplement M100-S29. CLSI. Wayne). MRSA isolates were de ned as resistant to oxacillin (Lee et al. 2018). Cefoxitin-screen test in VITEK 2 was used as a surrogate marker for the detection of mecA gene (John et al. 2009).
DNA extraction -Spa typing DNA was extracted using the DNA extraction kit (Qiagen, Hilden, Germany). The spa gene was ampli ed and sequenced. Typing was performed through the Rindom Spa server (spaserver.ridom.de). A minimum spanning tree (MST) was generated using the spa-clustering method of the spa-typing plugin of BioNumerics v.7.1 software (Applied Maths, Sint-Martens-Latem, Belgium), which is connected to the SeqNet/Ridom Spa Server (https://www.spaserver.ridom.de/).

Whole genome sequencing -Bioinformatic analysis
Since t127 is often related with LA-MRSA, two t127 isolates [one collected from a patient hospitalized in internal medicine (IM-MRSA), and a second one from a patient hospitalized in the pediatric ward (PD-MRSA)] were selected for WGS analysis. WGS was performed on Ion Torrent PGM Platform (Life Technologies Corporation, Grand Island, NY, USA). All procedures were conducted according to manufacturer's guidelines. PCR products were loaded on Ion-316 TM chip kit V2BC. The Ion PGM Hi-Q (200) chemistry (Ion PGM Hi-Q Sequencing kit, A25592) was applied.
The consensus sequence was taken using S. aureus strain WHC09 (GenBank accession number CP077755) as reference sequence. MLST analysis was performed using the web-based MLST-DTU tool (

Spa typing
Twenty-two different spa types were detected, with t002, t003, and t422 being the most frequent (5/39, 12.8% each), followed by t1994 (4/39, 10.3%), t127 and t328 (2/39, 5.1% each) ( Table 1). Different spa types were detected in the various wards, except the paediatric ward where t1994 (3/4, 75%) predominated. Most of the samples were taken <48h after admission; this was the case also for the t1994 isolates, suggesting that they were not associated with nosocomial infection (Table 1, Figure 1). Analysis of whole genome sequences IM-MRSA and PD-MRSA isolates were assigned to ST9 and ST1, respectively. They were carrying SCCmec elements belonging to XII and IVa types, respectively.
The antimicrobial resistance and virulence genes, and the plasmids carried by the two isolates are seen in Table   2. Speci cally, both had antimicrobial resistance genes for β-lactams (mecA and blaZ), aminoglycosides Regarding plasmid content, IM-MRSA carried a plasmid replicon type rep22 (of the pGSA11 group), while PD-MRSA transferred plasmid replicon types rep7a (of the pGSA2 group, which includes the repC cassette), rep7c, rep10 (of the pGSA3 group which includes the ermC gene) and rep5 along with rep16 (of the pGSA22 group which includes the blaZ gene) ( Table 2). Table 2 Genetic characteristics of two t127 MRSA strains of the study.

Discussion
The present study provides an insight into the distribution of MRSA isolates of various spa types in a tertiary hospital in Greece. Most of the isolates were collected from infection sites and few from colonization sites; however, colonization usually precedes infection (Love et al. 2020). It was shown that most isolates were resistant to several antimicrobial categories. The high resistance to fusidic acid (46.2%) exceeds by far that observed among MRSA globally (2.6%) (Hajikhani et al. 2021). The resistance rate to mupirocin (15.4%) was higher than the 3.1% revealed in a previous multicenter surveillance study (Kresken et al. 2004); this is of crucial importance since resistance to mupirocin could potentially diminish the e cacy of MRSA decolonizing strategies (Bes et al. 2021). For both antimicrobials (fusidic acid and mupirocin) further studies are needed to elucidate the genetic background of the increased resistance (whether it is due to mutation(s) and/or acquired resistance genes). In contrast, resistance to levo oxacin was lower than that reported in a recently published study ( In total, 22 different spa types were identi ed, and most of them (16/22, 72.7%) were represented as singletons, suggesting a non-clonal MRSA distribution (Table 1, Figure 1). An exception was the t1994 predominance in the paediatric ward during a four-months' time period; however, it seems that it was not associated with intrahospital infection since the time of sampling was <48h from the admission to the hospital. The high prevalence of t002 and t003 spa types seen in the present study, has been reported in recent studies (Engelthaler et  LA-MRSA lack the human evasion genes scn (staphylococcal complement inhibitor), chp (chemotaxis inhibitory protein) and sak (staphylokinase), due to loss of the related φSa3 phage (Price et al. 2012). IM-MRSA lacked these three genes, while PD-MRSA lacked only the chp gene. IM-MRSA was isolated from wound infection and was assigned to ST9-XII, which, due to loss of scn, chp and sak genes, is considered to have been evolved from human to animal host (Yu et al. 2021 Comparative studies are needed to con rm which mechanism(s) took place. The second t127 isolate, PD-MRSA, was isolated from an axillary abscess and belonged to ST1-IVa-t127, which is originally considered a human-associated lineage. ST1-t127 isolates have been reported as one of the most frequent types isolated from human samples (Monaco et al. 2013).

Conclusions
The current study provides an insight into the spa types of MRSA in a tertiary hospital in Greece and together with the information of time interval between admission and sampling suggests polyclonal introductions in the various wards. The extensive resistance to most antimicrobial classes needs further attention since it is associated with high antimicrobial consumption. A more detailed genetic characterization is gained when WGS is applied, as shown by the results of the analysis of the two t127 MRSA isolates, which showed that although they belonged to the same spa type, they were different strains. Molecular surveillance studies are of high priority in the hospitals since they can lead the design of guidelines and infection control measures that must be applied to reduce and prevent the spread of MRSA.

Genome Sequences
The whole genome sequences of IM-MRSA and PD-MRSA were submitted to European Nucleotide Archive (ENA) under the study PRJEB47007 and received the Accession numbers ERS7262952 and ERS7262953, respectively.

Funding
This work was nancially supported by the European Union′s Horizon 2020 project VEO (grant number 874735).

Con icts of interest
The authors have no con icts of interest to declare that are relevant to the content of this article.
Availability of data and material  Figure 1 Minimum spanning tree based on spa-typing results of MRSA strains depending on the department of isolation. Each spa type is represented by a single node. The size of the node depends proportionally on the number of strains within the spa type. The colored sections represent a different ward. The distance between nodes represents the genetic diversity of the isolates.