Molecular investigation of methicillin-resistant staphylococcus aureus isolates from blood : USA 600 emerges as the major type

Introduction: The widespread emergence of methicillin-resistant Staphylococcus aureus is turning into a real worry in public health. The goals of the present study were to identify resistance and virulence encoding genes and molecular characteristics of methicillin-resistant S. aureus bloodstream isolates. Methodology: A cross-sectional study was conducted on 84 S. aureus bloodstream isolates during a 10-month period. To evaluate antibiotic susceptibility of the isolates, we used Kirby-Bauer disk diffusion method. In addition, the prevalence of antimicrobial resistance and toxins genes was assessed using polymerase chain reaction. Isolates were typed according to polymorphisms seven housekeeping genes by MLST. Results: All the isolates were resistant to methicillin. The most prevalent resistance gene was mecA gene (100%) followed by tetM (57.1%), aac (6 ́)-Ie/aph (2 ̋) (53.6%), ant (4 ́)-Ia (46.4%), ermA (45.2%), msrA (35.7%), msrB (33.3%), aph (3 ́)-IIIa (33.3%), ermB (31%), ermC (16.7%), and mupA (14.3%) genes. The presence of toxin encoding genes tst, pvl, eta, and etb were detected in 25%, 14.3%, 3.6% and 3.6%, respectively. The isolates were classified into five different sequence types: ST45 (29.8%), ST239 (27.4%), ST858 (21.4%), ST22 (17.8%), and ST59 (3.6%). All the high-level mupirocin-resistant (HLMUPR) strains belonged to ST239, while the low-level mupirocin resistant (LLMUPR) strains belonged to ST22 (13%) and ST239 (6%). Conclusions: To the best of our knowledge, the present study is the first report of ST59 in MRSA bloodstream isolates in Iran. Our data demonstrated the need for thorough epidemiological monitoring to detect emergence and dissemination of MDRMRSA types in our hospitals.


Introduction
Methicillin-resistant Staphylococcus aureus (MRSA), which is a major global cause of infection in both community and hospital, accounts for a broad range of serious human infections ranging from skin infections to sever infections, including bacteremia [1].According to the literature, compared with noninvasive MRSA infections, higher mortality rate is reported in cases with invasive infections due to MRSA [2].Although different STs have been found to predominate in a variety of geographical regions, one of the most prevalent sequence types (STs) is USA600 or ST45.The first USA600 strain was reported in some European countries such as Germany and the Netherlands.Since then, numerous surveillance studies have revealed a steady increase in the incidence of this type in the United States and Canada [2,3].According to the published data, reported mortality rates among patients with MRSA bloodstream infections (BSIs) were found to be 20 to 30%.Recent studies have reported mortality rates of 60% for patients with MRSA BSIs caused by the USA600, suggesting remarkable ability of this pathogen to produce virulence factors [2][3][4].The increasing prevalence of MRSA infections and its ability to spread in hospitals and health care facilities is becoming a major challenge for infection control.During the past years, the widespread use of antibiotics has led to the emergence and dissemination of invasive MRSA strains that systematically harbor multiresistance genes and cause failure treatment [5][6][7].In addition to mecA gene, the emergence of co-existence of genes encoding resistance to other antibiotics is the new concern, along with the particular concern of invasive bloodstream infections [8][9][10][11].A major concern with managing MRSA infections in patients with blood stream infection is the lack of awareness about the molecular types and antibiotic resistance pattern of this bacterium either phenotypically or genotypically [1].Molecular typing methods have been developed to track MRSA isolates among which Multi-Locus Sequence Typing (MLST) technique, with its high discriminatory abilities, has been introduced as an effective method for typing MRSA strains [12,13].With regards to the lack of sufficient information about molecular characterization of MRSA strains isolated from blood infection, the present study was conducted to understand phenotypic and genotypic resistance pattern and to identify STs of MRSA obtained from patients with bacteremia in Tehran, Iran.

Study design and population
In the current cross-sectional study, we investigated 84 MRSA isolates, recovered over a 10-month period from November 2016 to August 2017, from patients referring to 12 medical centers located in different areas of Tehran (capital city of Iran).The frequency of MRSA isolated from studied hospitals in Tehran was as following; 12 isolates (14.3%) from hospital A, 12 isolates from hospital C (14.3%), 11 isolates from hospital G (13.1%), 10 isolates from hospital I (11.9%), 9 isolates from hospital E (10.7%), 8 isolates (9.5%) from hospital B, 6 isolates (7.1%) from hospital D, 5 isolates (5.9%) from hospital H, 4 isolates (4.8%) from hospital J, 3 isolates (3.6%) from hospital F, 3 isolates (3.6%) from hospital K and one isolate (1.2%) from hospital L. One isolate per patient was included in the study and duplicate samples were excluded.The study was approved by the Ethics Committee at Shahid Beheshti University of Medical Sciences, Tehran, Iran (IR.SBMU.MSP.REC.1396.2).Blood cultures obtained from patients with suspected sepsis so as to order to identify S. aureus were processed using standard microbiological procedures including colony morphology on blood agar, Gram-stain microscopy, bhemolysis on 5% sheep blood agar, growth on mannitol salt agar, and giving positive results for catalase, coagulase, and DNase tests.Isolates were confirmed as S. aureus via detecting the presence of the femA and nucA genes (encoding peptidyl transferase and thermostable nuclease, respectively) using PCR [5,14].

Multilocus Sequence Typing (MLST)
MLST was performed on isolates based on seven housekeeping genes (arcC, aroE, glpF, gmK, pta, tpiA, and yqiL) as described by Enright et al. [13] Sequence type (ST) of each isolate was determined by comparing the sequences at each locus with those of the known alleles in the S. aureus MLST database (http://www.mlst.net/).

Discussion
The present study reports on several important findings in relation to molecular typing of MRSA strains isolated from BSI, including detection of ST45 as the most predominant type and high prevalence of mupirocin resistance (33.3%), which was distributed among ST239 and ST22.Meanwhile, we reported ST59 the first time in Iran for although at a low frequency (3.6%).
In contrast with a study conducted in the Iran, which reported low frequency of ST45 (4%) in MRSA strains isolated from patients with bacteremia [1], in the present study, we observed a high frequency of the ST45 (29.8%) among MRSA strains isolated from BSIs.These findings may reflect a true increase in the prevalence of ST45 in Iran.The study showed that none of the ST45 isolates were susceptible to gentamycin and penicillin while 60%, 40%, and 8% were susceptible to trimethoprim-sulfamethoxazole, erythromycin, and clindamycin respectively.These findings are in contrast to those reported in Moore et al's who reported low frequency of resistance to gentamycin (25%) among ST45 strains while it was found that all the isolates were resistant to erythromycin and clindamycin [2].Variability in resistance and toxin encoding genes within ST45 strains were previously reported by several investigators [4,16].The resistance gene profiles of the isolates have shown that the overwhelming majority of these isolates carried aac(6΄)-Ie/aph(2˝)(80%), and ant(4΄)-Ia (72%).All the isolates were PVL negative.S. aureus ST45 was previously identified in Iran [1], Netherlands [17], Switzerland [18], and Croatia [19].This high prevalence of ST45 isolates in our study may be attributed to high transmissibility and rapidity of its spread within the community and/or increase in the volumes of international travel.
According to the literature, ST239 have evolved from ST8 and ST30 parents and accounts for at least 90% of hospital-acquired-MRSA in the South American, Europe, and some other Asian countries, including Kuwait, Saudi Arabia and Malaysia [3,16,20,21].In the present study, multi resistant ST239 was the second most common type identified (27.4%), although at a low level.Our previous study from Iran, carried out to identify molecular characteristics of MRSA isolated from blood samples, showed that ST239 was the most predominant type (43%).In line with the studies conducted by Abimanyu et al. in India [22] and Udo et al. in Kuwait [20], who demonstrated that HLMUPR-MRSA strains belonged to ST239 clone, our findings showed that 12 HLMUPR-MRSA strains (14.3%) and 5 HLMUPR-MRSA strains (5.9%) belonged to ST239.Among our ST239 isolates, 12 (14.3%), 3 (3.6%),and 2 isolates (2.4%) were positive for tst, eta and etb genes, respectively.S. aureus ST239, harboring tst, eta, and etb genes, has been detected Iran [1], Korea [23], Taiwan [3], and Kuwait [20], as well.
As our results showed, ST585, as a single-locus variant (SLV) of ST239, was the third most-common type among the MRSA strains isolated from BSI (21.4%).Low frequency of this type has previously been reported [1,24].In our previous research on MRSA strains isolated from BSI, ST585 was detected in 10.9% of tested isolates [1], which was lower than the rate found in the present study.Ohadian Moghadamet et al. [24], in Iran, identified molecular characterization of MRSA isolates obtained from clinical samples.They found that MRSA isolates belonged to two major clonal complexes (CC); CC8 (ST239, ST585, ST2732, ST1294) and CC30 (ST30, ST36, ST1163) and the presence of ST585 was reported in 11.1% of the tested MRSA isolates.With regard to ST858 antibiotic resistance to gentamicin, tetracycline, and ciprofloxacin pattern, our findings support the recent study conducted by Goudarzi et al. [1].
Another type found among our strains was ST59.In contrast to a previous study in Taiwan [3], which reported a rate of 17.2% for ST59 strains isolated from blood, in the present study, we found low frequency of ST59 (3.6%), which all were positive for pvl encoding genes.Although antimicrobial resistance pattern in ST59 isolates may vary, we found that all the isolates were resistant to ciprofloxacin, tetracycline, and gentamicin.These findings, with regards to resistance to tetracycline, resembled those of the previous reports by Wang et al. [3] in Taiwan and Coombs et al. in Western Australia [28].To the best of our knowledge, it is the first report of ST59 in MRSA strains isolated from patients with BSI in Iran.Given the wide dissemination of clonal complex 59 (CC59)/ST59 in the Asia Pacific region, it is guessed that transmission of these isolates may occur, due to the rate of transportation between these countries.

Conclusion
In conclusion, the present survey showed that MRSA isolated from blood has genetic diversity, including Five STs with different toxin and resistance gene profiles which should seriously be taken into consideration.ST59 was reported for the first time in Iran, indicating the emergence of the new ST in our region.Further and periodical studies are needed to delineate the genetic changes in MRSA isolated from blood.

Figure 1 .
Figure 1.Detection of resistance genes of MRSA strains isolated from BSI.

Table 1 .
Molecular characterization of MRSA strains isolated from BSI.