Genotype distribution of methicillin-susceptible Staphylococcus aureus clinical isolates in Iran: high multiresistant clonal complex 8

Objective Compared to methicillin-resistant Staphylococcus aureus (MRSA), there have been few studies focused on the molecular characterization of methicillin-susceptible Staphylococcus aureus (MSSA). In this cross-sectional study, 85 MSSA isolates were characterized by antimicrobial susceptibility testing, virulence genes analysis, accessory gene regulator (agr) typing, and S. aureus protein A locus (spa) typing. Results In present study, 9 different clonal complexes namely CC8-MSSA-t037 (22.4%), CC8-MSSA-t008 (11.8%), CC7-MSSA-t091 and CC30-MSSA-t021 (each 9.4%), CC8-MSSA-t037 (8.3%), CC398-MSSA-t034 (7.1%), CC22-MSSA-t005 (5.9%), CC5-MSSA-t002 and CC15-MSSA-t084 (each 4.7%), CC22-MSSA-t790 and CC59-MSSA-t437 (each 3.5%), CC22-MSSA-t1869, CC5-MSSA-t045, and CC45-MSSA-t015 (each 2.3%), CC30-MSSA-t318 and CC15-MSSA-t491 (each 1.2%) were found. agr types detected in tested strains were mainly type I (76.5%), II (12.9%), and III (10.6%). Of 85 MSSA examined isolates, 48 (56.5%) isolates were toxinogenic with 27 producing pvl (31.8%) and 21 tst (24.7%). The findings of the study show a high genetic diversity in MSSA strains warranting continued surveillance to provide critical insights into control and treatment of MSSA infections.


Introduction
Staphylococcus aureus is a common hospital-and community-acquired pathogen [1]. It is responsible for a multitude of human infections ranging from minor skin and soft tissue infections to serious and life-threatening conditions [2]. Although the epidemiology of S. aureus strain diversity appears to differ by geographic region, there has been a dramatic increase in the prevalence of S. aureus strains associated with human infections around the world and this appears to be especially true for methicillin-susceptible Staphylococcus aureus (MSSA) [3][4][5]. MSSA is a challenge for health-care settings and is becoming a public health concern [1, 6]. Compelling evidence suggests virulence genes may play an important role in serious MSSA infections, which are further exacerbated by the widespread circulation and emergence of drug-resistant strains [3]. Antimicrobial resistance is a barrier to successful control of S. aureus infections [7,8]. Knowledge of genetic variability, clonal relatedness, and dissemination of staphylococcal infections may help to provide crucial insight for implementation of infection control programs, rational use of antibiotics, and better understand the evolution of these species [9,10].
Although there is information about characteristics of MSSA strains in Iran, limited attention has been given to clonal diversity, and virulence gene prevalence among strains. To address these data limitations, the current study was performed to investigate the genetic background of MSSA strains isolated from patients.

Bacterial isolates
Eighty-five MSSA isolates were obtained from hospitalized patients at four hospitals affiliated to Shahid Beheshti University of Medical Sciences during an 9-month collection period from March 2019 to November 2019. This study protocol was approved by the Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran (IR. SBMU. MSP.REC. 1398. 774). Furthermore, we confirmed S. aureus isolates phenotypically by using standard microbiological techniques. Polymerase chain reaction (PCR) assay targeting the S. aureus-specific nuc gene was applied to verify the isolates [11,12]. The S. aureus isolates susceptible to cefoxitin disc (30 µg, Mast Co., UK) in a disc diffusion assay using established methods (CLSI 2018) and negative for the presence of mecA gene by PCR were considered as MSSA strains [12].

Evaluation of antimicrobial activities
In present study, Kirby-Bauer disk diffusion method was applied to determine the antimicrobial susceptibility of isolates based on the clinical and laboratory standards institute (CLSI) criteria (CLSI 2018). The antimicrobial agents included penicillin, teicoplanin, gentamicin, kanamycin, amikacin, tobramycin, clindamycin, erythromycin, tetracycline, linezolid, rifampicin, mupirocin, ciprofloxacin, quinupristin-dalfopristin, and trimethoprim-sulfamethoxazole (Mast Co., UK). The minimal inhibitory concentrations (MIC) values of vancomycin was evaluated by broth microdilution method. Susceptibility test was quality controlled by using S. aureus ATCC 25923, ATCC 43300, and ATCC 29213 strains.

DNA isolation and screening of the key virulence related genes
Genomic DNA was isolated using the phenol-chloroform extraction method. All of the isolates were screened for virulence encoding genes namely: exfoliative toxin genes (eta, and etb), Panton-Valentine leukotoxin gene (pvl), and toxic shock syndrome toxin (tsst-1) by PCR assay [12][13][14]. The S. aureus ATCC49775 and toxin positive S. aureus strains obtained from our previous were used as reference strains [14]. The S. aureus strain ATCC 25923 were also used as negative control.

Molecular typing methods
Multiplex PCR was performed for agr type detection using primer set comprising a common forward primer (Pan) and reverse primers (agr1, agr2, agr3, and agr4) specific to each agr group [15]. agr types were identified by comparing the banding patterns of isolates to RN6390 (agr type I), RN6607 (agr type II), RN8465 (agr type III), RN4550 (agr type IV), and RN6911 (negative control), as reference strains. PCR amplification was used for spa typing as described previously [16]. In this method polymorphic X region of spa gene amplified by PCR with forward (5′-AGA CGA TCC TTC GGT GAG C-3′) and reverse (5′-GCT TTT GCA ATG TCA TTT ACTG-3′) primers. The purified PCR products were sequenced and then edited. The Ridom SpaServer database (http://www.spase rver.ridom .de) was applied to determine the spa type of each isolate. Each set of PCR reactions include a spa-type t008 isolate from our previous study as positive control sample (14), and a reaction mixture containing no template DNA as a control for possible false positive results.
According to the evidence, the agr genotypes are strictly associated with the clonal lineages [34,35]. In the current study, the agr type I, as the most predominant type (76.4%), was associated with CC8, CC22, CC7, CC45, CC398, and CC59 isolates.

Limitations
Our research had some limitations. Firstly, present work lacks detailed clinical information about the patients, Secondly, our samples were not collected consecutively. Thirdly, whole genome sequencing technique was not applied in the present work due to some of technical limitations.