Genetic analysis of methicillin‐susceptible Staphylococcus aureus clinical isolates: High prevalence of multidrug‐resistant ST239 with strong biofilm‐production ability

Abstract Background The distributions of methicillin‐susceptible Staphylococcus aureus (MSSA) are divers geographically with different genetic backgrounds. Data related to molecular characteristics of MSSA compare to methicillin‐resistant Staphylococcus aureus (MRSA) is sparse. Methods In this cross‐sectional study, antimicrobial susceptibility testing, virulence genes analysis, biofilm formation, accessory gene regulator (agr) typing, and multilocus sequence typing (MLST) characterized on 75 MSSA isolates. Results Multidrug‐resistance MSSA was found to be 84%. Forty‐eight (64%) isolates were toxinogenic with 34 and 14 isolates carrying pvl and tst representing 45.3% and 18.7%. The most common SE genes were sed (20%), sec (16%), and sea (16%). Fifty‐five (73.3%) isolates were confirmed as biofilm producer with a markedly high prevalence of fnbA (93.3%), fnbB (86.7%), icaA (65.3%), icaD (53.3%), can (24%), ebp (10.7%), and bap (1.3%). A total of 3 agr types (I, 73.3%; III, 16%; II, 10.7%) and 4 clonal complexes (CCs) and sequence types (STs), namely CC8/ST293 (45.3%), CC/ST22 (28%), CC/ST30 (16%), and CC/ST5 (10.7%) were detected in this study. All the high and low‐level mupirocin resistance strains belonged to ST239 and ST22 strains, respectively. All the fusidic acid‐resistant isolates carried fusC and belonged to ST30. Conclusions These findings indicated that ST239 with strong biofilm production ability is the most common type in MSSA strains isolated from patients. It seems that the antimicrobial resistance profiles, toxin, and biofilm formation were closely associated with specific STs. Further studies are required to identify and control of these clonal lineages in our area.


| INTRODUC TI ON
Staphylococcus aureus as a major human pathogen in both hospitals and community settings is responsible for a variety of diseases ranging from skin and soft-tissue infections (SSTIs), gastrointestinal disorders, and pneumonia to serious and life-threatening diseases. [1][2][3] Some of researchers from Asia reported a dramatic increase in the annual rate of visits for staphylococcal infections to healthcare settings which consequently represents a special concern related to the rate of mortality and morbidity. 4,5 However, it is well established that several infections are caused by methicillin-susceptible S aureus (MSSA). 6,7 According to the evidence, variable rates of staphylococcal infections caused by MSSA have been reported in previously published data from Iran, ranged from 23.1% to 76.4%. 8 Nowadays, infection associated with MSSA is a major public health crisis representing a priority for healthcare settings. 2,3,8 Compelling evidence has indicated that virulence genes may play an important role in serious infections related to MSSA, and it is further exacerbated by widening resistance to currently available antibacterial agents. 9,10 Emerging simultaneous resistance to multiple antibacterial agents among MSSA strains has significantly limited the availability of chemotherapeutic agents for the treatment of staphylococcal infections and leads to deterioration of the disease. [11][12][13][14] S aureus produces virulence determinants including adhesions (collagen-binding protein, clumping factor, fibronectin-binding protein, and elastin-binding protein), toxins (toxic shock syndrome toxin-1, panton-valentine leukocidin, exfoliative), enterotoxins (SEs), staphylokinase hemolysin, and lipase which are related to the severity of the infection. [15][16][17] Several investigators noted concerns about biofilm formability on biotic and abiotic surfaces especially medical devices, by MSSA strains. 8,18,19 Furthermore, biofilm formability can play a key role in the development of resistance, unsuccessful eradication of infection, and resistance to host immune response. 8 MSSA isolates usually present fast dissemination and high genetic variability which makes their epidemiological understanding more complex. [2][3][4]6,10,15 Based on the evidence, MSSA isolates could be a reservoir for MRSA clones and are essential for controlling the potential emergence of new epidemic MRSA clones. 6,20 Therefore, gaining adequate knowledge about genotypic characteristics, understanding of the resistance and virulence pattern and the ability of biofilm formation is helpful to prevent and control the spread of MSSA strains. According to the published data characteristics of MSSA strains in Iran remain poorly understood. the current study was performed to investigate the resistance pattern, biofilm-forming ability, and the presence of virulence factors, biofilm, and adhesion genes. Multilocus sequence typing (MLST), accessory gene regulator (agr) typing were used to characterize the genotype of the MSSA strains.
Healthcare-associated MSSA (HA-MSSA) infection was defined if the positive culture of MSSA was obtained on or after the third day of admission to a hospital. 21 The Ethics Committee of the Shahid Beheshti University of Medical Sciences in Tehran, Iran approved this research (IR. SBMU. MSP.REC. 1398. 818). All isolates were confirmed as S aureus by using standard microbiological methods as well as polymerase chain reaction (PCR) assay for the presence of the nuc gene as described previously. 22 The S aureus isolates susceptible to cefoxitin disc (30 µg, Mast Co., UK) and negative for the presence of mecA gene by PCR were considered as MSSA strains. 20,22

| DNA isolation and resistance, virulence and biofilm genes analysis by PCR-based assays
Genomic DNA was isolated using the phenol-chloroform extraction method with a slight modification including adding 5 μL of 5 mg/mL of lysostaphin (Sigma-Aldrich) to cell suspension and incubation at 37°C for 30 minutes to 1 hour. 24 The quantity of DNA was adjusted approximately to 100 ng/μL which evaluated by a NanoDrop-2000 spectrophotometer (Thermo Fisher Scientific). All of the isolates shock syndrome toxin (tst) genes by PCR assay with oligonucleotide primers as previously described. [24][25][26][27] We also used PCR assay to assess biofilm by the presence of icaA, icaB, icaC, icaD, can, ebp, fnbB, fnbA, and bap genes. 19,23

| agr typing
Staphylococcus aureus isolates underwent agr typing to determine specificity groups. Multiplex PCR-based protocol was used for amplification of the hypervariable domain of agr locus. agr types I, II, III, and IV were expected to produce 441-bp, 575-bp, 323-bp, and 659-bp fragments, respectively. 28

| MLST
Staphylococcus aureus isolates were further characterized by MLST and by amplifying and sequencing seven housekeeping genes (pta, arcC, tpi, aroE, gmk, yqiL, and glpF). Sequence types (STs) were determined by the submission of the allelic profile to the online MLST database website (https://pubml st.org/).   Table 1 gives information about the characterization of MSSA strains obtained from clinical samples.

| D ISCUSS I ON
Infections caused by MSSA in both community and healthcare setting is increasing and has gained great attention in recent years. 2,8,16,19 The present research had several findings including diverse genetic backgrounds of MSSA isolates with a predominance of C8/ST239 associated with lineages commonly observed among MRSA suggesting that MRSA probably originated from MSSA clones. High biofilm formation ability in clonal lineages of MSSA was observed (73.3%).
A relatively high pvl positive MSSA strains were noted that may reflect the important role of these isolates as reservoirs for pvl positive MRSA clones.
Data relating to susceptibility testing revealed that 10.7% of MSSA isolates were mupirocin resistant. This finding was differing from the previous study from other regions such as Iran (3%) 13 and South Africa (3%). 14 There are several studies that exhibited a high prevalence of mupirocin resistance among MRSA isolates comparing MSSA isolates. Conversely, our reported rate was higher than those reported previously among MRSA strains obtained from Iran. 13 A recent systematic review and meta-analysis conducted by Dadashi et al 29 showed that the pooled prevalence of mupirocin resistance and HLMUPR MRSA clinical isolates was 13.8% and 8.1%, respectively, in different parts of world that is higher than our reported rate. The reasons for resistance to mupirocin in present study could be injudicious and widespread use, uncontrolled policies in the prescription of this antibiotic, easy access to this antibiotic without prescription, inexpensive drugs, and spreading of specific lineage in these area.
Furthermore, we also observed that 3 (4%) and 5 (6.7%) isolates were HLMUPR and LLMUPR, respectively. In the study performed by Abdulgader et al 14 in South Africa on 212 S aureus isolates, 1% and 3% of the studied MSSA isolates indicated high and low levels of resistance to mupirocin. The reasons for discrepancies in rates of resistance to mupirocin could be studied population, spreading of specific lineage, differences in infection control strategies and unrestricted policies in the prescription of this antibiotic. The current survey displayed that all HLMUPR-MSSA strains carried mupA gene. In the study conducted in South Africa, a similar result was observed. 14 In our study, neither mupA nor mupB was detected in the LLMUPR isolates which is consistent with previous studies. 13,14 In the current study, the prevalence of iMLSB was found to be 21.3%, which was higher than those reported in Nepal (4.95%), 30 Brazil (5.8%), 11 India (9.3%) 31 and was near to reported rate in Ethiopia (21.4%). 32 In line with several studies performed in other parts of the world, we found a higher prevalence of cMLSB phenotype compare to iMLSB phenotype (29.3% vs 21.3%). 11 phenotype among MSSA isolates compare to iMLSB phenotype.
(3.6% vs 5.8%). However, high rates of cMLSB among MSSA isolates were also noted in other studies conducted in Nepal (20.8%), 30 and India (13.38%). 31   According to the evidence, virulence factors, biofilm formation, and subsequently antibiotic resistance regulated by agr system, which is strongly linked with specific clonal lineages. In present work, agr type I was identified as the predominant type, followed by type III and type II as previously reported in some studies. 3,10 Several investigators were highlighted the key role of biofilm and adhesion-related genes in biofilm formation. 18,44 In our strain collection, 84.7% of strains were biofilm producers.
This finding was contrary to previously published data, that exhibited biofilm production was higher in MRSA strains as compared to the MSSA strains. 18 Recently, a systematic review and meta-analysis reported a 56.8% rate of biofilm formation among MSSA strains isolated from clinical samples in Iran. 8 The results showed that the most common biofilm-related genes were fnbA, Their results also displayed that CC8 was a predisposing factor for strong biofilm formation. In Malaysia, CC8 (53.3%), followed by CC1 (20%), CC22 (16.7%), and CC7 (10%) were the most common biofilm producers. 46 Differences in biofilm formation abilities in clonal lineages of MSSA may be attributed to unique combinations of surface-associated and regulatory genes. 18 As illustrated in The presence of ST30-MSSA, known as the Southwest Pacific clone, has been noted in Australia, the UK, Germany, Lebanon, Abu Dhabi, and Kuwait. 12,18,28,42 Our finding that pvl positive ST30 was one of the most successful and persistent clones in MSSA isolates Notably, resistance to fusidic acid encoded by fusB was also detected in three isolates that belonged to ST30. This result was consistent with those in previous reports from Shore et al 36 in Ireland, which reported resistance to fusidic acid encoded by fusC in fewer than half of the tested isolates.
We noted a low prevalence of ST5 (10.7%) with a high biofilm-forming ability (62.5%) in this study. Similar observations but with different prevalence rates have been previously reported from Iran, 2 Kuwait, 3 Russia, 10 Belgium, 20 and China. 47