Antibiotic susceptibility testing were carried out using the Kirby-Bauer disk diffusion technique according to Clinical and Laboratory Standard Institute 2013 guidelines,17 Applied antibiotics (Mast, England) including cefoxitin 30 μg, tetracycline (TE) 10 μg, gentamycin (GM) 10 μg, erythromycin (E) 15 μg, vancomycin (VAN) 30 μg, amikacin (AK) 30 μg, imipenem (IMP) 10 μg, linezolid (LZ) 10 μg, tigecycline (TG) 15 μg, ciprofloxacin (CIP) 5 μg, clindamycin (CD) 2 μg, tobramycin (TO) 10 μg, rifampin (RF) 5 μg, and trimethoprim/sulfamethoxazole (TMP/SMX) 1.25/23.75 μg. Methicillin resistance was examined using cefoxitin disk and confirmed by the mecA-specific PCR.

Total DNA was extracted using the DNA extraction Kit (BioFlux Co., Japan), according to the manufacturer’s instructions. Quality of extracted DNA was assessed spectrophotometrically by the Nanodrop ND-1000 (Nanodrop Technologies, Inc., Wilmington, DE, USA). Following the DNA extraction, PCR targeting of nuc gene was used for confirmation of S. aureus strains. PCR was carried out in a final volume of a 20 μL reaction mixture containing 10 μL of 2X Taq premix Mastermix (Parstous Biotech Co., Iran), 5 μL of sterile double distilled water, 1 μL of forward primer, 1 μL of reverse primer, and 3 μL of DNA sample. The DNA of samples, as well as the DNA of positive control (S. aureus ATCC 25423) and a negative control (S. epidermidis), were amplified by an initial denaturation step for 5 minutes at 94 °C followed by 35 cycles of 94 °C for 60 seconds, 50 °C for 60 seconds, and 72 °C for 1 minute and a final extension step at 72 °C for 10 minutes in a Bio-Rad Thermal Cycler (Bio-Rad Laboratories, Inc., USA). The products PCR were subjected to 1.5% agarose gel electrophoresis.

The PCR assay targeting mecA gene coding methicillin resistance was performed for all isolates. Primer sequences included: forward (5'- GTAGAAATGACTGAACGTCCGATAA-3') and reverse (5'- CCAATTCCACATTGTTTCGGTCTAA-3'). S. aureus ATCC 25923 was included as the positive control. PCR was carried out in a final volume of a 20 μL. The initial denaturation was at 92 °C for 5 minutes. Denaturation at 94 °C for 60 seconds, annealing at 56 °C for 1 minute, and extension at 72 °C for 1 minute was maintained for 35 cycles.

Each isolate was tested by seven multiplex PCRs according to the method of Holtfreter for the genes: (i) sea, seh, and seo; (ii) sed, etd, and eta; (iii) see, seb, and sem; (iv) sen, seq, and sej; (v), ser, seu, and sep; (vi) sel, sei, and TSST; and (vii) seg, sek, and sec. Primers sequences are provided in Table 2. The reaction mixture for the PCR assay was 20 μL, and was prepared as previously described. DNA denaturation at 95 °C for 5 minutes was followed by 35 cycles of amplification (95 °C for 30 seconds, 55 °C for 30 seconds, and 72 °C for 60 seconds), ending with a final extension at 72 °C for 10 minutes.18,19

One sample of each enterotoxin, as well as mecA PCR products (amplicons), was sequenced by Bioneer Co., Korea mediated by Takapouzist Co., Iran and the data were analyzed using the Chromas software.

Data was analyzed using SPSS Statistics (SPSS Inc. Released 2007. SPSS for Windows, Version 11.0. Chicago, SPSS Inc). Descriptive statistical methods were used to determine the frequency, percentage, and mean. Chi-square test was used to compare the qualitative results, and independent t-test to compare quantitative data. A p-value ≤ 0.050 was considered significant in comparative data.

Results

Among the 200 specimens, the total number of samples from males was 105 (52.5%) and 95 from females (47.5%). The distribution of isolates amoung the samples is shown in Table 2.

The results of antibiotic resistance of MRSA and MSSA isolates have been presented in Table 3. Chi-squared analysis of the presence of the virulence and resistance genes showed preferential distribution to MRSA in comparison with MSSA isolates (p ≤ 0.050). There was a significant relationship between the pathogenicity of MSSA and MRSA and the presence of antibiotics resistance agents.

In our study, MRSA isolates were more prevalent in males (54.0%) than females (46.0%), whereas MSSA isolates were more prevalent in females (59.0%) than males (41.0%) with no significant statistical difference (p= 0.109). There was no significant association of MRSA and MSSA with age (p= 0.126).

The distribution of SAg genes among MRSA and MSSA strains is presented in Table 4. Among MRSA isolates, 92 (92.0%) harbored SAg genes and the most frequent toxin gene was sea (45.0%), followed by sec (39.0%). Among MSSA isolates, 89 (89.0%) harbored SAg genes and the most prevalent genes were sea 42 (42.0%), sek 38 (38.0%), sec 35 (35.0%), and TSST-1, which were harbored by 10.0% of strains. None of the investigated isolates carried the eta gene. The etd gene in MRSA was detected only in two strains that belonged to puncture samples. In MSSA, etd was detected in two strains isolated from blood and puncture samples. The association of SAg genes with MRSA and MSSA isolates showed a higher prevalence of enterotoxin seq, seg, and sei in MRSA than MSSA with significant difference (p = 0.014), (p = 0.001) and (p = 0.009), respectively [Table 4]. There were no significant difference among MRSA and MSSA with other SAg (p > 0.050).

The results of the basic local alignment search tool (BLAST) of intended genes product indicated that it has the same DNA sequences and all PCR assay results were confirmed.

Discussion

In this study, the frequency of MRSA among S. aureus isolates was 50.0%. Our results are in agreement with another study from Shiraz that reported a MRSA prevalence of 42.3%.20 Among the 100 confirmed MRSA isolates, 46 (46.0%) and 54 (54.0%) were isolated from females and males, respectively, which is similar to previous findings in Shiraz, Iran.20 In addition, higher numbers of MRSA was observed in patients aged 45–60 and 15–30 years old, which is consistent with the results from a Malaysian study.21 Studies on the prevalence of MRSA isolates in clinical samples are inconsistent. Some studies, such as our results, indicated a higher prevalence of MRSA in blood (40.0%) and very low rates (5.0%) in pus and pharyngeal swabs samples. In the study performed by Alfatemi et al,20 most cases of MRSA (39.79%) were obtained from sputum and the fewest number (2.05%) were gathered from eye and nose samples. A higher percentage of MRSA was found in blood and urine than other specimens was similar to other studies.21,22

Most MRSA isolates were multiple-drug resistant. Resistance to TE and GM was higher in MRSA isolates than MSSA isolates, which in conflict with Asadollahi et al,23 study. Although some investigations in the recent years have reported increasing number of VAN-resistant MRSA isolates, the results of the current study showed that VAN is still the main antibiotic of choice for treatment of serious and threatening infections caused by MRSA and MSSA isolates. Our results also demonstrated that all isolates were susceptible to VAN, which is in agreement with another study from Iran.23

Studies on the prevalence of enterotoxin gene in MRSA and MSSA isolates are inconsistent. We observed a significant association among the blood samples and high prevalence of enterotoxin in blood (p = 0.001) were observed, while there was no significant relationship between SAg with other samples (p > 0.050). The frequency of the sea gene in MRSA isolates from various specimens in our study was 45.0%. This figure is similar to studies performed in Tehran (32.07%),24 Gorgan (58.8%),25 and Shiraz (27.39%),20 Iran, but higher than that of a study from Germany (12%),17 Ilam (17%),23 Iran, and Korea (17.5%).26 Report of sea (35.4%) gene prevalence in China was consistent with our result.19 In our study, the frequency of the seb gene was 34.0%, which was inconsistent with other studies in Iran (Gorgan 61.3%,25 Ilam 2%,23 and Tehran 73.58%)27 and studies from other countries
(China 5%,28 Canada 15.78%).29 These differences could be associated with geographical differences, number of samples, and year. The frequency rates of sed gene in studies performed in Tehran,28 Shiraz,20 Korea,30 and Columbia,31 were 3.77%, 2.05%, 2.9%, and 7%, respectively.

In this study, 90.5% of isolates carried at least one type of enterotoxin. This result was inconsistent with the results obtained by Asadollahi et al,23 and in accordance with Alfatemi et al.20 These discrepancies might be due to the different study locations.
There was no significant correlation between enterotoxins of MRSA and MSSA strains observed, except for seq, seg, and sei, which had a statistically significant difference p = 0.014, p = 0.001 and p = 0.009, respectively.

The frequency of TSST-1 gene in MRSA strains in our study was 12.0%, which were obtained from blood (8%) and nasal septal (4%). Our results were consistent with a study from Shiraz (11.64%)20 and Germany (9.14%),17 and conflicting with those from Ilam (46%)23 and several other countries (Korea 72.2%, Czech 50%, and 0–78% in the US). In our study, none of the investigated isolates harbored the eta gene. The frequency rates of eta gene in different studies from Iran were very low (Shiraz and Ilam were 0.68% and 1%, respectively).20,23 This was also the finding in other countries including Germany, Turkey, and Colombia31 (2%, 19.2%, and 3%, respectively). Furthermore, in our study, the frequency rate of etd gene in all strains of S. aureus was 2.0%, while the etd gene was not found in any of the isolates tested by Wu et al.19 The frequency of MRSA enterotoxin genes in different countries and even within a country in different cities or hospitals, have not been determined to be the same. This could be due to differences in the geographical features of each country, the hospital wards where the specimens collected from, number of samples, patients, and health conditions.

One of the strengths of our study compared to similar studies is the number of clinical samples taken from different locations and also the application of seven multiplex PCR complex for detection more than of 20 Staphylococcus toxin genes. Our study is limited by not using advanced typing methods such as pulsed-field gel electrophoresis or multilocus sequence typing for epidemiological interpretation.

Conclusions

Our isolates exhibited a high rate of resistance to CIP and TMP/SMX. There is a significant relationship between virulence genes and antibiotic resistance patterns. Genetic investigations of clinical MRSA and MSSA could provide a global and comprehensive aspect of risk prediction, which can be suitable for short-term and long-term health care policies.

Disclosure

The authors declared no conflicts of interest. The authors would like to acknowledge the Vice Chancellor of Hamadan University of Medical Sciences for funding of the current study.

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