Meticillin-resistant Staphylococcus aureus isolated from Iranian hospitals : virulence factors and antibiotic resistance properties

Staphylococcus aureus is an important opportunistic pathogen responsible for a variety of diseases. Indiscriminate prescription of antibiotics caused severe antibiotic resistance especially against commonly used drugs. Th e present investigation was carried out to study the distribution of Panton-Valentine Leukocidin gene, SCCmec types and antibiotic resistance properties of meticillin-resistant Staphylococcus aureus isolated from Iranian hospitals. A total of 132 clinical specimens were collected from two major Iranian hospitals. Samples were cultured and their positive results were subjected to several PCR methods. Th e patterns of antibiotic resistance were studied using the disk diff usion method. We found that 66 out of 132 samples (50) were positive for Staphylococcus aureus. Th e most commonly infected samples were superfi cial and surgical wounds (66.12). Th e incidence of mecA, tetK, ermA, ermC, tetM, aacA-D, linA, msrA, vatA, vatC and vatB antibiotic resistance genes were 80.30, 34.84, 30.30, 25.75, 24.24, 19.69, 7.57, 7.57, 6.06, 3.03 and 1.51, respectively. Totally, 40.90 of isolates harbored the Panton-Valentine Leukocidin gene. Of 53 mec positive strains, the distribution of SCCmec V, SCCmec III, SCCmec IVa, SCCmec IVc and SCCmec IVb were 28 (52.83), 13 (24.52), 6 (11.32), 4 (7.54) and 2 (3.77), respectively. All isolates were resistant to penicillin, cephalothin, cefazoline and ceftriaxone. Th e high levels of Staphylococcus aureus resistance against commonly used antibiotics as well as high presence of SCCmec types of meticillin-resistant virulent strains of Staphylococcus aureus suggest that infections with these strains require more advanced hospital care with emerging demand for novel


INTRODUCTION
Th e increasing prevalence of bacterial resistance to commonly used antibiotics, may result in an insuffi cient array of substances to combat some bacterial infections and it is more important in hospitals.Staphylococcus aureus (S. aureus) has long been recognized as a major pathogen of hospital acquired infections.Th e bacterium can colonize individuals both in the community and hospital settings [1].Infections caused by this bacterium are treated mainly with meticillin but in recent years, increasing numbers of meticillin resistant S. aureus (MRSA) strains have been reported worldwide from patients with community-acquired infections [2][3][4].Globally, fi fteen to forty fi ve percent of S. aureus strains isolated from hospital infections were meticillin resistant [5,6].S. aureus is the most common cause of skin and soft-tissue infections (such as impetigo, furunculosis, superfi cial and surgical wounds and abscess), as well as systemic infections (such as pneumonia, urinary tract infections-UTIs and endocarditis) [7][8][9][10][11][12][13].
Th e pathogenicity of S. aureus depends on various bacterial surface components and extracellular proteins.Th e frequent recovery of staphylococcal isolates that produce leukocidal toxins from patients with deep skin soft tissue infections, particularly furunculosis, cutaneous abscesses, severe necrotizing pneumonia, and even UTIs, suggests that the Panton-Valentine leukocidin (PVL) is a virulence factor that has a major role in pathogenicity [7][8][9][10][11][12][13][14].PVL-positive strains are associated with skin diseases, accounting for 96 of the cases [15,16].PVL has also been associated with severe infections, including pneumonia [11], purpura fulminans [17] and osteomyelitis [18].Th is toxigenic gene has also been isolated from the cases of Lemierre' s syndrome [19], Fournier' s  gangrene [20], community-acquired necrotizing and hemorrhagic pulmonary infections aff ecting previously healthy children and young adults [14,21].
SCCmec elements are currently classifi ed into types I, II, III, IV and V according to the nature of the mec and ccr gene complexes, and are further classifi ed into subtypes according to diff erences in their J region DNA [22][23][24].Complete identifi cation of bacterial genetic background and the SCCmec element is very important for molecular typing of MRSA [9,25].
PVL is mostly associated with community-acquired MRSA infections and distinguishable from nosocomial MRSA by no multi-drug resistance and carriage of the type IV staphylococcal chromosome cassette element (SCCmec type IV) [26,27].Th e changing trend of MRSA epidemiology, showed the use of PVL locus detection as a marker of MRSA isolates, alongside with non multi resistant pattern and SCCmec type IV or V [28].
Th ere were no previously published data about the distribution of PVL and SCCmec types among multi-drug resistant strains of S. aureus isolated from Iranian hospitals.According to the indiscriminate and excessive prescribing of antibiotics in Iran, it is necessary to study the distribution of PVL gene, SCCmec types and antibiotic resistance properties of MRSA isolated from various types of hospital' s infections.

Samples and Staphylococcus identifi cation
From February to May 2013, a total of 132 clinical samples from individuals suff ering from various types of infections including blood (n=13), UTIs (n=16), respiratory infections and lung abscesses (n=24), superfi cial and surgical wounds (n=62) and abscesses (n=17) were collected from 2 major hospitals of Iran (Baqiyatallah and Peyambaran Hospitals, Tehran, Iran).All samples were transported to the Microbiology and Infectious Diseases Research Center of the Islamic Azad University of Shahrekord in a cooler with ice-packs.All samples were directly cultured into 7 sheep blood agar (Merck, Darmstadt, Germany) and incubated aerobically at 37°C for 48 h.After incubation, suspicious colonies were examined by using techniques appropriate for diagnosing Staphylococcus spp.(microscopical morphology, catalase and coagulase production).Studied colonies were cultured on Tryptic Soy Broth (TSB) (Merck, Darmstadt, Germany) and Tryptic Soy Agar (TSA) (Merck, Darmstadt, Germany).After growth, staphylococci were identifi ed on the basis of colony characteristics, Gram staining, pigment production, hemolytic and the following biochemical reactions: catalyses activity, coagulated test (rabbit plasma), Oxidase test, glucose O/F test, resistance to bacitracin (0.04 U), mannitol fermentation on Mannitol Salt Agar (MSA) (Merck, Darmstadt, Germany), urease activity, nitrate reduction, novobiocin resistance, phosphatase, deoxyribonuclease (DNase) test and carbohydrate (xylose, sucrose, trehalose and maltose, fructose, lactose, mannose) fermentation tests [29].

DNA extraction and Staphylococcus confi rmation
A typical colony of the biochemically identifi ed S. aureus was cultivated in 1 mL TSB for 24 h at 37°C.Chromosomal DNA was extracted from the typical colonies using the DNA Genomic Purifi cation Kit (Fermentas, Germany) according to the manufacturer' s instructions.Presence of S. aureus in each DNA samples was confi rmed using the Banada et al. [31] method.Th e PCR reaction mix consisted of 1 X PCR buff er (10 mM Tris-HCl, pH 8.3, 50 mM KCl and 0.001 (w/v) gelatin) with 4 mM MgCl2, 250 mM of each nucleotide (deoxynucleoside triphosphate), 0.5 mM of each primer (forward and reverse), 4 ng of the molecular beacon and 4 U of Taq DNA polymerase (Fermentas, Germany).

PCR detection of mecA and PVL genes
Two pairs of primers were used for amplifi cation of mecA and PVL genes of the S. aureus strains [32,33].Th e PCR reactions were performed in a total volume of 25 μL, including 1.5 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 Triton X-100, 200 μM dNTPs each (Fermentas, Germany), 2.5 μL PCR buff er (10X), 25 pmoL of each primer, 1.5 U of Taq DNA polymerase (Fermentas, Germany) and 5 μL (40-260 ng/μL) of the extracted DNA template of the Staphylococcus isolates.Th e two sets of primer pairs were used in each reaction mixture.Th e DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) were used in all PCR reactions for DNA amplifi cations.Th e thermal cycler was adjusted as follows: 94°C for 10 min, followed by 10 cycles of 94°C for 1 min, 55°C for 1 min, and 72°C for 1.5 min, and 25 cycles of 94°C for 1 min, 52°C for 1 min, and 72°C for 1.5 min, followed by fi nal extension at 72°C for 7 min; the PCR products were stored in the thermal cycler at 4°C until they were collected.

Antibiotic resistance genes amplifi cation
Th e presences of tetK, tetM, ermA, ermC, aacA-D, linA, msrA, vatA, vatC and vatB genes were analyzed using the Kumar et al. [34] technique.List of primers are shown in Table 1.Th e PCR reactions were performed in a total volume of 25 μL, including 2 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 Triton X-100, 150 μM dNTPs each (Fermentas, Germany), 2.5 μL PCR buff er (10X), 25 pmoL of each primers, 2 U of Taq DNA polymerase (Fermentas, Germany), and 4 μL (40-260 ng/μL) of the extracted DNA template of the Staphylococcus isolates.Th e four set of primer pairs were used in each reaction mixture.Th e thermal cycler was adjusted as follows: 94°C 5 min, 30 cycles of 1 min at 95°C for the denaturation step and 1 min at 55°C for the annealing-extension step and followed by fi nal extension at 72°C for 90 seconds.

Detection of SCCmec types
Th e SCCmec types were detected using the Zhang et al. [12] method.Lists of primers are shown in Table 2. Th e PCR reactions were performed in a total volume of 25 μL, including 2 mM MgCl 2 , 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1 Triton X-100, 250 μM dNTPs each (Fermentas, Germany), 2.5 μL PCR buff er (10X), 25 pmoL of each primer, 2 U of Taq DNA polymerase (Fermentas, Germany), and 5 μL (40-260 ng/μL) of the extracted DNA template of the Staphylococcus isolates.Th e thermal cycler was adjusted as follows: beginning with an initial denaturation step at 94°C for 5 min followed by 10 cycles of 94°C for 45 s, 65°C for 45 s, and 72°C for 1.5 min and another 25 cycles of 94°C for 45 s, 55°C for 45 s, and 72°C for 1.5 min, ending with a fi nal extension step at 72°C for 10 min.
In order to confi rm the PCR results, the sequencing method was used.For this reason, PCR products of some positive samples were purifi ed with High pure PCR product purifi cation kit (Roche Applied Science, Germany) according to manufacturer' s recommendations.Single DNA strands were sequenced with ABI 3730 XL device and Sanger sequencing method (Macrogen, Korea).Result of the sequence of each gene was aligned with the gene sequences recorded in the GenBank database located at NCBI.

Gel electrophoresis
Fifteen microliters of PCR products were resolved on a 1.5 agarose gel containing 0.5 mg/mL of ethidium bromide in Tris-borate-EDTA buff er at 90 V for 1 h, also using suitable molecular weight markers.Th e products were examined under ultraviolet illumination.

Statistical analysis
Th e results were transferred to a Microsoft Excel spreadsheet (Microsoft Corp., Redmond, WA) for analysis.Statistical analysis was performed using SPSS/16.0 software (SPSS Inc., Chicago, IL) for signifi cant relationship between incidences of PVL gene and SCCmec types of S. aureus isolated from clinical samples.A χ 2 test and Fisher' s exact 2-tailed test analysis

Ethical considerations
Th e present study was accepted by the ethical committees of the Baqiyatallah and Peyambaran Hospitals, Tehran, Iran and Microbiology and Infectious Diseases Center of the Islamic Azad University of Shahrekord Branch, Iran.Written informed consent was obtained from all of the study patients or their parents.

RESULTS
Of 132 clinical samples, 66 were positive for S. aureus.Superfi cial and surgical wounds had the highest incidence of S. aureus (66.12), while blood samples had the lowest incidence (15.38) (Table 3).
Th ere were no positive results for the PVL gene in the S. aureus strains of blood and urinary system.V phonotype (52.83) and III phonotype (24.52) had the highest incidence of SCCmectypes (Table 6).
Th e incidence of IVa, IVc and IVb SCCmec types were 11.32, 7.54 and 3.77, respectively.Th ere were no positive results for I, II and IVd types.All of the S. aureus isolates were resistant to penicillin, cefalotin, cefazoline and ceftriaxone (Table 7).

DISCUSSION
In our research we found high incidence of multi-drug resistant S. aureus strains in patients' clinical samples.All of the examined blood, urine, respiratory secretions, superfi cial and surgical wounds and abscesses of the patients were infected with these strains.Unfortunately, these results showed that the Iranian hospitals' environments were so infected.Also, our results showed that antibiotics were used in a highly irregular manner in Iranian hospitals.Th ese two fi ndings may lead to the emergence of resistant staphylococcal diseases which can infect patients and even healthy people in hospitals.Similar results have been reported previously by some authors [8][9][10][11][12][13].
In our study, microorganisms showed lowest resistance to imipenem.Totally, 6.06 of the S. aureus isolates of our study were resistant to imipenem.Fifty seven percent of MRSA strains of the Boyce et al. [41] were imipenem resistant which was entirely higher than our results.Similar studies have been reported by Totsuka et al. [42] and Yamazaki et al. [43].We have observed no bacterial resistance to vancomycin and nitrofurantoin antibiotics in our study.Resistance against vancomycin have been showed previously [44][45][46].Current recommendations showed that antibiotics may trigger release of PVL and progression to bad clinical complications.Furthermore, due to the inappropriate prescription, it was not surprising that our study found that resistance to penicillin, cefalotin, cefazoline, ceftriaxone, azithromycin and tetracycline were 100, 100, 100, 100, 62.12 and 57.57, respectively.
Th e Staphylococcus strains of our study had the highest incidence in superfi cial and surgical wounds (66.12).Signifi cant diff erences (p<0.05) were observed for the incidence of bacteria between the superfi cial and surgical wounds and blood and urine samples.Similar results have been reported previously [47][48][49].Th e main reason for this fi nding is the fact that the S. aureus strains represent the major micro fl ora of a healthy skin.Also, these strains are ubiquitous


bacteria that are found anywhere, in hospital environment as well.Respiratory swabs had also high incidence of S. aureus bacteria (50).Totally, 40.9 of the S. aureus isolates of our study carried the PVL gene.Th is gene had the highest incidence in the superfi cial and surgical wounds in our study (46.34).Th e main factor contributing to its high incidence in the wounds is the fact that the PVL gene is a bicomponent cytotoxin that is preferentially linked to furuncles, cutaneous abscesses and severe necrotic skin infections.We found no signifi cant statistical diff erences for distribution of the PVL gene between the various types of infections.Th e PVL gene was found in 100 of cases with pneumonia, 80 of cutaneous abscesses, 100 of cases with furunculosis and 33.33 of fi nger pulp infections in a study of Ezzat et al. [50].Many other investigations showed the presence of PVL genes in the staphylococcal infections including Esan et al. [51](Nigeria) (18), Khosravi et al. [52] (Iran) (7.23) and Shallcross et al. [53] (United Kingdom) (11.3).Also, the prevalence of PVL gene is estimated to be some 2-35 among MRSA strains in hospital infections as previously reported [54,55].Th e PVL toxin' s ability to cause the death of polymorphonuclear cells including neutrophils, basophils and eosinophils has been known since its very discovery.
Th e V phenotype (52.83) and III phenotype (24.52) were the most commonly detected SCCmec types among the mec positive strains of S. aureus of our study.We found signifi cant diff erences (p<0.05) between the incidence of SCCmec V and SCCmec IVc, SCCmec IVb and SCCmec Iva types.Similar studies have been reported previously [23,25,56,57].D'Souza et al. [56] showed that a total of 97 mecA-positive strains were SCCmec III (25), 136 were SCCmec IV (34), and 162 were SCCmec V (41) which was concordant to our results.Th ey also showed that all of the SCCmec III strains, 73 of SCCmec IV and V strains and 72 of SCCmec IV and SCCmec V strains were multidrug resistant.Moussa et al. [8] reported that the most predominant SCCmec type among the examined isolates in Saudi Arabia were type V (42.5) followed by, SCCmec type III 39 (38.6) which was similar to our fi ndings.
Th e above mentioned data highlight huge diff erences in the prevalence of SCCmec types in diff erent studies, as well as diff erences in PVL gene, antibiotic resistance genes and antibiotic resistance patterns in the clinical samples.Th is could be related to diff erences in the type of sample (stool, blood, urine, meat, milk, respiratory swabs, wound materials and other clinical samples) tested, number of samples, method of sampling, experimental methodology, geographical area, antibiotic prescription preference among clinicians, antibiotic availability, and climate diff erences in the areas where the samples were collected, which would have diff ered between each study.

CONCLUSION
In conclusion, we identifi ed a large number of MRSA, PVL virulence genes and antibiotic resistance properties in the S. aureus strains of hospitalized infections.Our data indicated that MRSA strains are predominant in Iran.High PVL gene distribution was also found.Our data revealed that tetK, tetM, ermA, ermC, aacA-D, linA, msrA, vatA, vatC and vatB genes and resistance to penicillin, cefalotin, cefazoline, ceftriaxone, azithromycin and tetracycline were the most commonly detected characteristics of the MRSA strains isolated from hospitals infections.Th e high levels of Staphylococcus aureus resistance against commonly used antibiotics as well as high presence of SCCmec types of meticillin-resistant in virulent strains of Staphylococcus aureus represent that infections with these strains require higher levels of hospital cares with emerging demand for novel antibiotics.Hence, the clinicians' role in judicious usage of antibiotics is pivotal.

TABLE 1 .
Oligonucleotide primers for amplifi cation of antibiotic resistance genes in Staphylococcus aureus strains isolated from Iranian hospitals.

TABLE 2 .
Oligonucleotide primers for amplifi cation of SCCmec types in Staphylococcus aureus strains isolated from Iranian hospitals.

TABLE 3 .
Distribution of Staphylococcus aureus in various types of hospital infections in Iran.

TABLE 4 .
Distribution of antibiotic resistance genes in Staphylococcus aureus strains isolated from hospital infections in Iran.

TABLE 7 .
Distribution of antibiotic resistance pattern in Staphylococcus aureus strains isolated from hospital infections in Iran.

TABLE 6 .
Distribution of SCCmec types in Staphylococcus aureus strains isolated from hospital infections in Iran.

TABLE 5 .
Distribution of PVL gene in Staphylococcus aureus strains isolated from hospital infections in Iran.