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 (2019) [16], but do not agree with Darban-Sarokhalil et al (2016) [30]. In a study directed by Kateete et al (2011), 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 (2007) 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, (2019) [33], Mir et al (2019) [16] and Pournajaf et al (2014) [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 (2019) [33], VAN is still the best option in the treatment of patients with MRSA infection. In compare 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 (2014) [34] and Antonov et al (2015) [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 (2019) [33] and McNeil et al (2011) [36] studies. Solgi et al (2019) [33] declare that low-level MUP resistance may be occurred due to another responsible genes such as; mupL/D/W/O/T. Contrary to our study, Mir et al (2019) [16], showed that 85.6% of the isolates were resistant to MUP. On the other hand, Chen et al (2012) reported high frequency of MUP-resistant MRSA isolates in burn centers [37]. This topical bacteriostatic antimicrobial 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 decolonization of nasal carriage, burn, diabetic foot, bedsores and other skin lesions could be related to 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 (2019) [33], Khodabandeh et al (2019) [14] and Gupta et al (2009) [38], but have conflict with Seifi et al (2012) [39], Adhikari et al (2017) [11], Ruiz-Ripa et al (2019) [40] and Deotale et al (2010) [10] studies. These conflicts may be related to 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 (2019) [33], and Gupta et al (2009) [38], the frequency of cMLSB phenotype was higher than iMLSB, but in another study, the frequency of iMLSB phenotype showed to be higher than cMLSB. Therefore, notice of regional frequency of MLSB resistant isolates is very important for microbiology laboratories to choose to perform D-test regularly. In concordance with Khodabandeh et al (2019) [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 contradicts with the study conducted by Ghanbari et al (2016) [41] and Saribas et al (2006) [42]. This discrepancy could be due to genetic variation and spread of single clone in our area. Distribution of AMEs genes in our samples were as fallows; aph(3')-I (33%), aph(3')-IIIa (62.8%), aac(6′)/aph(2′′) (24.7%) and ant(4')-Ia (85.6%) in the MRSA isolates and aph(3')-I (36.9%), aph(3')-IIIa (45.6%), aac(6′)/aph(2′′) (13%) and ant(4')-Ia (26.1%) in MSSA. M-PCR showed that 8.2% (n = 8/97), 12.4% (n = 12/97), and 33% (n = 32/97) of MRSA isolates carried simultaneously aph(3')-IIIa/aac(6′)/aph(2′′), aph(3')-I/aph(3')-IIIa, and aph(3')-IIIa/ant(4')-Ia genes, respectively. Only 2.1% (n = 2/97) isolates were positive for all AMEs tested genes. These results are inconsistent with the studies of Khosravi et al (2017) [43] and Goudarzi et al (2020) [44]. In agreement with our study, the ant(4')-Ia was the most prevalent gene in Yadegar et al (2009) [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 resistance 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 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 phenotype, 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 (2018) [15] and Gowrishankar et al (2016) [28]. One reason for this discrepancy is source of the samples (food and pharyngitis samples vs our clinical samples). Contrary to our study, Ghasemian et al (2015) [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 (2018) 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 was not found in MSSA isolates. In a study performed by Mehrotra et al (2000) [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 source of the sample (anterior nasal swabs vs clinic samples). Sabouni et al (2014) [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 (2019) [16] showed that the frequency of hla, hlb, hld, hlg, tst and pvl genes was 92.8%, 34.7%, 89.8%, 11.9%, 10.7%, and 0.5% respectively. In line with our study, hla gene had the highest frequency among isolates (94.4% for MRSA and 89.8% for MSSA). Exfoliative toxin A and B were detected in the 11.3% and 6.2% of MRSA isolates, respectively. In the MSSA strains, the etA gene was present in 4.3% and etB in 6.5% of isolates. These data are similar to the results reported by Sila et al (2009) [48]. Also to support Sabouni et al (2014) 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 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 MSCRAMMs-encoding 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 (2019) 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 (2019) 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 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 (2019) [16] and Mkrtchyan et al (2017) [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 (2013) [51] and Teare et al (2010) [52], the prevalence of pvl gene was 14.6% and 2% respectively. Surprisingly, in concordance with Mir et al (2019) [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 (2013) [53] and Ghanbari et al (2016) [41], SCCmec typing did not show 100% type ability and had poor discriminatory power, as 6.4% of MRSA strains were nontypable. 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 (2016) [54], Ghanbari et al (2016) [41] and Moosavian et al (2017) [55], the most common SCCmec type was type III. However Jamshidi et al (2019) [56] and Boye et al (2007) [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.