New update on molecular diversity of clinical Staphylococcus aureus isolates in Iran: antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing

Staphylococcus aureus is often considered as a potential pathogen and resistant to a wide range of antibiotics. The pathogenicity of this bacterium is due to the presence of multiple virulence factors and the ability to form biofilm. SCCmec types I, II and III are mainly attributed to HA-MRSA, while SCCmec types IV and V have usually been reported in CA-MRSA infections. In this study, we performed a cross-sectional study to determine the antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing of clinical S. aureus isolates in Iran. S. aureus isolates were identified using microbiological standard methods and antibiotic susceptibility tests were performed as described by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Inducible resistance phenotype and biofilm formation were determined using D-test and tissue culture plate methods, respectively. Multiplex-PCRs were performed to detect adhesion and virulence factors, antibiotic resistance genes, biofilm formation and SCCmec typing by specific primers. Among 143 clinical samples, 67.8% were identified as MRSA. All isolates were susceptible to vancomycin. The prevalence of cMLSB, iMLSB and MS phenotypes were 61.1%, 22.2% and 14.8%, respectively. The TCP method revealed that 71.3% of isolates were able to form biofilm. The predominant virulence and inducible resistance genes in both MRSA and MSSA isolates were related to sea and ermC respectively. SCCmec type III was the predominant type. Data show the high prevalence rates of virulence elements among S. aureus isolates, especially MRSA strains. This result might be attributed to antibiotic pressure, facilitating clonal selection.


Introduction
Staphylococcus aureus (S. aureus) is one of the most common causes of healthcare and community-acquired infections and so responsible for a wide variety of illnesses, from soft and skin tissue infections (SSTIs) to life-threatening infections such as septicemia, toxic shock, hospital-and community-acquired pneumonia (HAP and CAP) and endocarditis [1]. S. aureus clinical isolates often promote infections by expressing various exotoxins such as heat-stable staphylococcal enterotoxins (SEs), staphylokinase (SAK), toxic shock syndrome toxin-1 (TSST-1), capsular polysaccharides, lipase, exfoliative toxins (ETA and ETB), hemolysins (α, β, γ, δ) and leukocidins (Panton-Valentine leukocidin; PVL, LukE/D) [2]. From the clinical point of view, indwelling medical devices or catheter-related infections such as central venous catheters (CVC), are at risk of S. aureus-related infection. The ability to form a stable biofilm is one of the most crucial factors in S. aureus pathogenicity and biofilm-associated S. aureus infections are often resistant to antibiotic therapy and innate host immune system [3]. Biofilm formation requires polysaccharide intercellular adhesin (PIA), which is encoded and regulated by the intercellular adhesion (icaADCB) operon. This operon includes an N-acetylglucosamine transferase (icaA and icaB), a predicted exporter (icaC), and a deacetylase (icaD) [1,4].
Multidrug-resistant S. aureus (MDRSA), is becoming a serious global concern, as a common cause of nosocomialand community-acquired infections [5]. In recent years, methicillin-resistant S. aureus (MRSA), which is now the most common MDR, has emerged with the acquisition of Staphylococcal Cassette Chromosome mec (SCCmec) elements, which carry a mecA gene that encodes a penicillinbinding protein (PBP2a or PBP2′) with a low affinity to β-lactams [6]. MRSA is spread worldwide and is common cause of health care (HAIs)-and community-acquired (CAIs) infections. SCCmec determinants are classified into various types based on the combination of ccr and mec genes complexes, which includes 5 and 8 mec and ccr classes, respectively. To date, at least 13 types of SCCmec elements have been recognized and all SCCmec types have individual characteristics. In general, SCCmec type I, II, and III are distributed in the hospital-associated MRSA (HA-MRSA) and type IV and V are present in the community-acquired MRSA (CA-MRSA) [7].
The Mupirocin-a topical ointment that broadly used for SSTIs and nasal decolonization of MRSA-is effective on the isoleucyl-tRNA synthetase (IleRS) which is encoded by ileS gene, interfering with protein synthesis. According to the minimal inhibitory concentration (MIC), two mupirocinresistant phenotypes have been identified; MIC 8-256 µg/ ml (low-level resistant-LLR or LMR) and MIC ≥ 512 µg/ ml (high-level resistant-HLR or HMR). A point mutation in ileS-1 gene (mupL) led to LLR isolates, while HLR is usually mediated by a conjugate plasmid-borne ileS-2 (mupA) gene which encodes a new IleRS that is not bound by mupirocin [8].
The aim of this study was a new update on molecular diversity of antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing of clinical S. aureus isolates in Iran.

Clinical sampling and laboratory identification
A total of 143 non-duplicative clinical samples were collected from admission patients referred to teaching therapeutic hospitals (Shahid Beheshti & Ruhani Hospitals, Babol, Iran) in a period of 8 months from September 2019 to April 2020. The samples were transported to the microbiology laboratory in Brain-Heart Infusion Broth (Merck Co., Germany). Each sample was cultured on Mannitol Salt Agar (supplemented with 7.5% sodium chloride) (Merck Co., Germany) and incubated at 37 °C for 24 h. All S. aureus colonies were identified based on routine biochemical and microbiological standard tests [12].

Inducible resistance phenotype
The Inducible resistance phenotype was recognized using the double disk test including, Clindamycin (CD; 2 µg) and Erythromycin (ERY; 15 µg) disks applied 20 mm separately [10,11]. After an incubation time of 24 h at 35 °C, a flattening inhibition zone adjacent to the ERY disk representing an inducible type (D-shaped zone) of MLS B resistance (IR), whereas no-susceptibility to both ERY and CD was mentioned as a constitutive type (CR). The nonappearances of a D-shaped zone in ERY-resistant and CD-susceptible isolates were interpreted as the M/MS B efflux phenotype [14].

Quantitative biofilm production assay
In Brief, pure colonies were inoculated in 10 mL of 1% glucose-rich tryptic soy broth (TSBglu), incubated at 37 °C for 24 h in a stationary growth phase and diluted 1:100 with fresh medium. Each well of sterile 96 well-flat bottom polystyrene tissue culture microtiter plates (Falcon® 3046, Lincoln Park, NJ) was full with 200 µL aliquots of diluted cultures. Sterile TSBglu broth was used as a negative control. All plates were incubated at 37 °C for 24 h and then, substances of all wells were gradually removed by tapping the petri. The wells were washed three times with 0.3 mL of phosphate buffer saline (PBS, pH 7.2) to remove loosely attached and floating "planktonic" microorganisms. Biofilm formed by adherent "sessile" isolates in petri was immobile with sodium acetate (NaA) and stained with crystal violet (0.1% w/v). Extra dye was removed by washing with sterile deionized water and plates were kept for drying. Adherent S. aureus cells frequently formed biofilm on the sides of the wells and were regularly stained with crystal violet (CV; 1%). The investigation of biofilm formation was assessed by adding the 200 µL of 95% CH 3 −CH 2 −OH (ethanol) to decolorize the wells. The optical density (OD) of stained adherent isolates was measured with a micro ELISA autoreader (Bio-Tek Instruments, USA) at a wavelength of 570 nm (OD570 nm). Biofilm formation was recorded as follows: non-biofilm forming (A570 < 1); weak (1 < A570 < 2); ++, moderate (2 < A570 < 3); +++, strong (A570 > 3) [1,15].

Multiplex-polymerase chain reactions (M-PCRs)
M-PCRs reactions were performed for detection of virulence, resistance and biofilm corresponding genes. Chromosomal DNA was extracted from the pure colonies using the Bacterial Genomic DNA Extraction kit (TaKaRa Biotechnology Co., Ltd, Dalien, China). The DNA concentration and purity were evaluated using a Nanodrop ND-1000 spectrophotometer (Thermo Fisher Scientific, UK) and then kept at − 20 °C until further use. The details of the primers used in this study are shown in Table 1. The process of M-PCR reactions in the final volume of 25 µL was performed according to Table 2 in an Eppendorf MasterCycle Gradient Thermocycler (Eppendorf, Hamburg, Germany). M-PCRs products were electrophoresed in a 1% agarose/0.5 × TBE (45 mM-Tris-borate, 1 mM-EDTA) gel stained with 0.1 µL/mL Gel Red™ (Biotium, USA), then photographed under an UV trans-illuminator (Tanon, China).

Data analysis
SPSS version 18.0 for Windows (SPSS Inc., Chicago, USA) was used for statistical analysis. P ≤ 0.05 was considered as a statistical significance.

Discussion
In the current study, a high prevalence of MRSA (67.3%) was found, especially in samples obtained from ICU and NICU wards. These data are in agreement with Mir et al. [16], but do not agree with Darban-Sarokhalil et al. [30].
In a study directed by Kateete et al., all isolates were found to be MRSA [31]. These conflicts could be attributed to the sample types (burn vs. other samples), year of study, geographic location (Uganda vs. Iran), level of hygiene, different protocols in infection control, irrational antibiotic administration, and laboratory method for determination of methicillin-resistant isolates. In line with our study, Guardabassi et al. indicated that the 30 µg Fox disk diffusion method is preferred to most of the other recommended tests such as; oxacillin disc diffusion and oxacillin screen agar tests and it is currently an accepted method for recognition of MRSA isolates by Clinical and Laboratory Standards Institute strategies [32].
The resistance rate in MRSA isolates is significantly higher than in the MSSA (P ≤ 0.05), which is consistent with the study of Solgi et al. [33], Mir et al. [16] and Pournajaf et al. [24]. Among antibiotics used for MRSA strains, CIP showed the least anti-staphylococcal activity and VAN was the most effective. According to the study performed by Solgi et al. [33], VAN is still the best option in the treatment of patients with MRSA infection. In comparison with other studies, there has been an increase in resistance to antimicrobial in MRSA isolates. The resistance rates in the MRSA straits were as follows: CIP (91.7%), GM (87.6%), SXT (84.5%), ERY (83.5%), TET (76.3%), RIF (62.8%), CD (55.7%) and VAN (0.0%). So, 78.3% of our isolates were considered MDR. This could be due to the continuous and empirical usage of broad-spectrum antimicrobials and the lack of an appropriate antibiotic treatment strategy. According to our data, although 14.4% of MRSA strains were resistant to MUP, it's still recommended as an option in the removal of MSSA nasal colonization. These data are in agreement with Chaturvedi et al. [34] and Antonov et al. [35] studies. Interestingly, among 14.4% MUP-resistant MRSA strains, only 8.2% were positive for ileS-2 gene. These data are consistent with Solgi et al. [33] and McNeil et al. [36] studies. Solgi et al. [33] declare that low-level MUP resistance may be occurred due to another responsible gene such as; mupL/D/W/O/T. Contrary to our study, Mir et al. [16], showed that 85.6% of the isolates were resistant to MUP. On the other hand, Chen et al. reported a high frequency of MUP-resistant MRSA isolates in burn centers [37]. This topical bacteriostatic antimicrobial is mainly used for prophylaxis against S. aureus nasal carriage and other skin diseases. Its target is the bacterial isoleucyl transfer ribonucleic acid synthetase. The long-term use of MUP, mostly for the decolonization of nasal carriage, burns, diabetic foot, bedsores and other skin lesions could be related to the development of resistance to MUP [33].
D-test revealed that the prevalence of cMLSB, iMLSB and MS resistance phenotypes were 61.1%, 22.2% and 14.8%, respectively. This data has also been described by Solgi et al. [33], Khodabandeh et al. [14] and Gupta et al.    [38], but have conflict with Seifi et al. [39], Adhikari et al. [11], Ruiz-Ripa et al. [40] and Deotale et al. [10] studies. These conflicts may be related to the year of study, topographical locations and surveillance strategies, as well as limitation in drug prescription. The rate of inducible resistance varies from hospital to hospital and even from patient to patient. In agreement with Solgi et al. [33], and Gupta et al. [38], the frequency of cMLS B phenotype was higher than iMLS B , but in another study, the frequency of iMLS B phenotype showed to be higher than cMLS B . Therefore, notice of regional frequency of MLS B resistant isolates is very important for microbiology laboratories to choose to perform D-test regularly. In concordance with Khodabandeh et al. [14], ermC was the predominant gene on both MRSA and MSSA isolates. The prevalence of ermA, ermB, ermC and ereA in the MRSA isolates were 21.6%, 16.5%, 44.3% and 9.3%, respectively. ereA gene was only found in the MRSA isolates which were collected from ICU. The combination of ermA/ ermB/ermC genes was detected in only two MRSA isolates collected from blood samples. So, 26.1%, 15.2% and 23% of MSSA isolates were positive for ermA, ermB and ermC, respectively. Our findings contradict with the study conducted by Ghanbari et al. [41] and Saribas et al. [42]. This discrepancy could be due to genetic variation and the spread of a single clone in our area. Distribution of AMEs genes in our samples were as follows: aph ( [43] and Goudarzi et al. [44]. In agreement with our study, the ant(4')-Ia was the most prevalent gene in Yadegar et al. [45]. As a result, according to other studies, the AMEs gene in the MRSA strains was higher than MSSA, which could be due to the ability of these strains to acquire resistant genetic elements. In our study, isolates collected from blood and wound from patients hospitalized in ICU had the highest ability in biofilm formation. With this regard, 71.3% of the isolates were able to form a biofilm, including strong (67.6%), moderate (20.6%) and weak (11.7%). In MRSA isolates, 84.1%, 80.9% and 58.3% had a strong, moderate and weak phenotypes, respectively. So, 84.1%, 19% and 41.6% of MSSA strains had strong, moderate and weak biofilm phenotype, respectively. These data are in contrast with Avila-Novoa et al. [15] and Gowrishankar et al. [28]. One reason for this discrepancy is the source of the samples (food and pharyngitis samples vs. our clinical samples). Contrary to our study, Ghasemian et al. [27] declare that the prevalence of icaA, icaB, icaC and icaD were 73% (n = 16), 63.6% (n = 14), 73% (n = 16) and 73% (n = 16), respectively. So, they showed that there was no significant difference between MRSA and MSSA strains for the presence of icaADBC operon. In the present study, biofilm formation in MRSA isolates was far greater than MSSA. In line with Kord et al. study, the highest and lowest ica gene was icaA and icaD, respectively [46].
In our study, all MRSA isolates were harbored sea gene. The seb, sec and sed were detected in 17.5%, 5.2% and 3.1% of MRSA isolates. Sec and sed were not found in MSSA isolates. In a study performed by Mehrotra et al. [22], among 107 strains collected from nasal swabs from healthy humans, 19.6% (n = 21), 24.3% (n = 26), 5.6% (n = 6), 7.5% (n = 8) and 1.9% (n = 2) were positive for sea, tst, seb, sec, and sed, respectively. This contrast may be related to the source of the sample (anterior nasal swabs vs. clinic samples). Sabouni et al. [47] showed that of 133 S. aureus isolates 48% (n = 64) were MRSA. The frequency of virulence-encoded genes was 40.6%, 19.6%, 12.8%, 11.3%, 9%, 4.5% and 3% for sea, seb, tsst, eta, etb, sed and sec, respectively. In contrast with the present study, among MSSA isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates. In our samples, none of the MSSA isolates were positive for tsst-1 gene. Mir et al. [16] showed that the frequency of hla,  [48]. Also to support Sabouni et al. study [47], etA gene was higher among MRSA isolates. However, no significant relationship was observed in the presence of etB between MRSA and MSSA strains. The etA and etB genes are more common in samples collected from Skin and soft tissue lesions [47]. Bacterial adherent to the target cell is the primary stage of infection. At this stage, attachment of S. aureus is facilitated by microbial surface component recognizing adhesive matrix molecules (MSCRAMMs) including, fnbA and fnbB (encoding fibronectin-binding proteins A and B), fib (encoding fibrinogen-binding proteins), cna (encoding collagen-binding protein), clfA and clfB (encoding clumping factors A and B), and eno (encoding laminin-binding protein) [49]. The prevalence of the MSCRAMMsencoding genes in the MRSA isolates was as follows: eno (84.5%), fib (75.3%), clfB (66%), clfA (56.7%), cna (52.3%), fnbA (15.5%) and fnbB (13.4%). These data are similar to Mir et al. study [16]. As a result, the frequency of MSCRAMMs genes in MRSA strains was higher than in MSSA, which could be due to the high pathogenicity of the strains. The distribution of these genes in the samples collected from the ICU was much higher than the samples of other units. Also, the strains collected from the wound, sputum, blood, and BAL samples, had the highest frequency of these genes, respectively. Mir et al.
reported that various molecules such as collagen, fibrinogen, fibronectin and other factors are present in the burn wound [16]. S. aureus encodes many MSCRAMMs that precisely interact with host cells and it enables the microbe to colonize on the burn wounds. One of the most important virulence factors in S. aureus infections, particularly in the skin and soft-tissue infections is the Panton-Valentine Leucocidin (PVL). This cytotoxin has been known as a virulence factor related to tissue necrosis such as necrotizing pneumonia (NP). M-PCR showed that 6.3% (n = 9/143) of S. aureus carried pvl gene. In contrast with Mir et al. [16] and Mkrtchyan et al. [50] studies, 8% of MRSA strains were positive for pvl gene, all of which were CA-MRSA. In our isolates, 55.7% and 44.3% of mecA-positive strains were HA-MRSA and CA-MRSA, respectively. In various studies directed by Rodrigues et al. [51] and Teare et al. [52], the prevalence of pvl gene was 14.6% and 2% respectively. Surprisingly, in concordance with Mir et al. [16] only one MSSA isolate (2.3%) was positive for pvl gene. According to our result, the frequency rate of types I, II, III, IV, and V of SCCmec was 23.7%, 11.3%, 34%, 18.5% and 6.2%, respectively. In line with Mariem et al. [53] and Ghanbari et al. [41], SCCmec typing did not show 100% type ability and had poor discriminatory power, as 6.4% of MRSA strains were non typable. Overall, 75% (n = 6 of 8) of pvl-positive MRSA strains belonged to the SCCmec type IV and V. In agreement with Taherirad et al. [54], Ghanbari et al. [41] and Moosavian et al. [55], the most common SCCmec type was type III. However, Jamshidi et al. [56] and Boye et al. [29] reported type IV as the most predominant type. This contrast may be related to the patients included in the study, multiple sclerosis (MS) cases vs. various Staphylococcus infections and geographical locations.

Conclusions
We determined the high prevalence of virulence elements and raised rate of antimicrobial resistance in our samples. MRSA strains also have a high ability to form biofilm. In addition, SCCmec type III was recognized as the predominant type. These data recommend that efficient control procedures must be considered to prevent the transmission of MRSA isolates among patients in hospital units, especially in the ICU.