The Prevalence of antimicrobial resistance in Staphylococcus aureus and coagulase-negative Staphylococci strains isolated from inpatients CURRENT STATUS: UNDER REVIEW

Objectives Globally nosocomial infection is a significant problem. Methicillin resistance Staphylococcus (MRSA) are major causes of nosocomial infections. Aim of this study was to determine the prevalence and antimicrobial susceptibility of MRSA and MRCoNS in the southwest of Iran. This cross-sectional study was conducted on 221 non-duplicated staphylococci isolates collected from teaching hospital in Shiraz. The prevalence of MRSA and MRCoNS in clinical samples was identified with conventional microbiological tests. After identification, all of the isolates were subjected to antimicrobial susceptibility test and PCR to identify the presence of femA , mecA and pvl genes. Result 168 (76%) S. aureus and 53 (24%) CoNS were detected. 70 (41.7%) MRSA among 168 S. aureus , and 26 (15.48%) MRCoNS among 53 CoNS were examined. Chloramphenicol (65%) and Gentamicin (23%) were the most active antibacterial agents against MRSA and MRCoNS, whereas these isolates were susceptible to SXT (100%). The frequency of pvl gene among S. aureus strains was 3.57%. There is need for developing the antibiotic policy and limiting the use of powerful antibiotics. stool and sputum with initial diagnosis of S. aureus . Bacterial isolates were identified by Gram stain, colony characteristics and conventional biochemical tests, namely catalase and coagulase (slide and tube) reaction; growth on mannitol salt agar and. S. aureus ATCC 25923 was used as susceptibility control strains for this study.


Introduction
Staphylococcus aureus is a remarkably successful pathogen in the healthcare and community setting (1). It is capable of persistence as a pathogen, because of its frequent resistance to multiple antimicrobial agents and its virulence determinants such as enzymes and toxins (2). In recent decades, methicillin resistant S. aureus (MRSA) has been a major agent of health care-and community-associated infections manifesting from minor skin diseases to life-threatening infections in many countries (3). Coagulase-negative staphylococci (CoNS) are part of the human normal flora; especially, the skin is also associated with severe infections such as bacteremia and septicemia, particularly in patients with indwelling devices or immunocompromised patients (4). CoNSs consist of a variety of Staphylococcus spp. and are usually resistant to most ß-lactams antibiotics including methicillin (5). Methicillin-resistant CoNS (MRCoNS) is growing worldwide and has a major role in clinical infections (6). The main mechanism of resistance to methicillin in staphylococci is the expression of mecA gene coding penicillin binding protein 2a (PBP2a), a transpeptidase with low affinity for β-lactams which confers resistance to methicillin and other β-lactam antibiotics in staphylococci harboring the gene (8). mecA is carried on a mobile genetic element called the Staphylococcal Chromosome Cassette mec (SCCmec) (9). So far, 12 SCCmec types described that HA-MRSA strains carry SCCmec types I, II and III, while CA-MRSA strains have SCCmec types IV and V (10). In recent years, treatment of MRSA has been one of the major problems in hospitals because these bacteria are multiply resistant and are susceptible only to glycopeptide antibiotics such as vancomycin and investigational drugs (11). Panton-Valentine Leukocidin (PVL or lukS/F) is a bicomponent pore-forming toxin causing tissue necrosis and lysis of phagocytic leukocytes that is associated with deep-seated abscess, multiple lesions and multiple antibiotic resistances (12). PVL presents in the majority of community associated MRSA isolates and rarely presents in hospital isolates (13). Epidemiological data suggest that high virulence of community-acquired MRSA is associated with PVL genes (14). Both endemic and epidemic MRSA infections occur globally as infected and colonized patients in hospitals (15). In our hospitals, the incidence rate of MRSA has been increased in clinical isolates and the prevalence of MRCoNs is also rising. Although many studies have been done on prevalence and antibiotic susceptibility of Staphylococcus, many of these studies have focused only on MRSA, not on MRCoNS which are similarly important. Therefore, this study aimed to determine the prevalence and antimicrobial susceptibility of MRSA and MRCoNS in teaching hospital in the southwest of Iran.

Study design and identification of the isolates
This cross-sectional study was performed from March 2016 to March 2017 in Nemazee teaching hospitals in Shiraz. A total of 221 isolates were collected from different clinical specimens such as the blood, urine, stool and sputum with initial diagnosis of S. aureus. Bacterial isolates were identified by Gram stain, colony characteristics and conventional biochemical tests, namely catalase and coagulase (slide and tube) reaction; growth on mannitol salt agar and. S. aureus ATCC 25923 was used as susceptibility control strains for this study.

DNA extraction and polymerase chain reaction (PCR) assay
Whole DNAs of methicillin resistance staphylococci were extracted, using boiling method and used as PCR templates. Detection of the presence of the mecA, femA and pvl genes was confirmed by PCR assay with specific primers (13,17). PCR amplifications were performed on a T100™ thermal cycler (Bio-Rad, Hercules, CA, USA). The cycling condition was set up as follows: initial denaturation at 96°C for 3 min; followed by 35 cycles of 30 s at 96°C, annealing for 1 min at 55°C and 2 min at 72°C; and an extension for 10 min at 72°C. PCR products were loaded into the wells of agarose gel (1.5%) carefully and electrophoresed at 75 V for 90 min. Staining was performed with safe stain load dye (CinnaGen Co., Iran) and then observed under the UV trans-illuminator.

Statistical analysis
Statistical analyses of the parameters were conducted using SPSS for Windows statistical software 122 (55.2%) were from male and female patients, respectively. Among MRCoNS isolates, the frequency of S. epidermidis and S. saprophyticus was 76.9% and 23.1%, respectively. Also, in both male and female patients, the frequency of S. epidermidis (75.5%) was more than S. saprophyticus (15.24). the isolates were collected from different wards of hospitals; MRSA and MRCoNS were isolated more commonly from internal 19 (27.1%) and clinical treatment 10 (38.5%) wards. The frequency of MRSA and MRCoNS in different wards is shown in Table 1. The most common sites of bacterial isolation in MRSA and MRCoNS were from bloodstream infections (40%), as presented in Table 2.

A: Trimethoprim/Sulfamethoxazole
The prevalence of Panton-Valentine Leukocidin (PVL) toxin in the MRSA isolates by detection of pvl gene was 6 (3.57%). The PCR results of the confirmatory (femA and mecA) and the pvl genes.

Discussion
Many strains of Staphylococcus aureus have been colonized on the surface of humans and animals' bodies, and some of these bacteria are pathogenic (18). The prevalence of MRSA is now increasing worldwide and this bacterium has become a major concern. Different Staphylococcus species have the ability to transmit the genes vertically. Today, coagulase-negative staphylococci (CoNS) are among the main human pathogens (19).  (26) and, as in this study, demonstrated the importance of rapid detection of CoNS. Among the samples examined in this study, 6 (3.57%) had pvl gene. The occurrence of pvl gene in previous stud ies has been reported from 2 to 35% (26,27). In compari son, the obtained results for pvl gene in the present study were near the lower limit obtained from similar studies. Chantratita in Thailand estimated this frequency at 16% in hospital patients (27). Kong in Taiwan reported the prevalence of this gene in about 13% (28). Odhiano estimated the prevalence of pvl in a hospital in Tehran to be 20% (29). Motamedi reported this prevalence in Ahwaz to be around 3%(30).
In conclusion, the results of the current study showed the prevalence of MRSA and MRCoNS strains resistance against widely used antimicrobial agents. The regular follow-up and continuous monitoring of antibiotic resistance profiles of hospital pathogens, especially staphylococci, will also be useful for selecting an appropriate antibiotic, knowing the changing trends of antibiotic susceptibility pattern, and developing hospital antibiotic policy.

Limitations
The present study had some limitations. First, this was a single-center study; therefore,