@2024 Afarand., IRAN

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ISSN: 2252-0805    The Horizon of Medical Sciences 2016;22(3):201-207

Background
Nosocomial pathogens are among the important factors that can interfere in the recovery of hospitalized patients [1].
Previous Studies
... [2-9] improper treatment of infections caused by negative coagulase staphylococci and sometimes inappropriate or excessive use of a wide range of antibiotics have led to widespread resistances in this group including the resistance to methicillin in the bacteria [10]. ... [11-15]. PCR (Polymerase Chain Reaction) is one of the most sensitive and accurate molecular methods that can be used for tracing different genes in a DNA molecule [16, 17]. … [18-20]. By eliminating the DNA extraction phase, PCR implementation can get more quickly when studying a large number of samples [21].
Aim(s)
The study aimed to compare two methods of PCR using extracted DNA with the extraction kit and direct PCR in determining the genes resistant to methicillin in negative coagulase staphylococci.
Research type
This is a cross-sectional, descriptive study.
Research society, place and time
This study was carried out in 2015 on 135 samples of Staphylococcus epidermidis and 88 samples of Staphylococcus saprophyticus.
Sampling method and number
Out of 839 samples, 135 samples of Staphylococcus epidermidis and 88 samples of Staphylococcus saprophyticus were taken from the blood, wounds, catheter and urine of patients hospitalized in Zahedan`s medical centers. The obtained samples were transferred to the microbiology laboratory of Zahedan University of Medical Sciences using BHI environment (Merck; Germany). To determine Streptococci species, samples were cultured on blood agar environment (Merck; Germany) with 5% of sheep blood and the species of negative coagulase staphylococci were identified using microbial detection methods such as Gram staining, biochemical tests such as catalase, coagulase, a survey of DNase production, cultivation on mannitol salt agar environment and examining the novobiocin sensitivity. According to CLSI (Clinical and Laboratory Standards Institute), the strains of Staphylococcus epidermidis were differentiated from Staphylococcus saprophyticus using disks of 30-mg novobiocin, 10-mg bacitracin and 100-unit polymyxin B. In order to differentiate between Staphylococcus hominis and Staphylococcus epidermidis, fosfomycin and deferoksamine antibiotics were used, and the samples with diameters greater than 30 mm for fosfomycin antibiotic as well as the ones with diameters greater than 20 mm for deferoksamine, were considered Staphylococcus epidermidis.
Used devices&materials
After separation of bacteria species, the samples were stored at -20° C until DNA isolation. The standard strain of Staphylococcus aureus ATCC33591 was used as the positive control as was the standard strain of Staphylococcus aureus ATCC25923 for the negative control. In order to extract the DNA, first, stored isolates were cultured at -20° C in blood agar environment and were incubated at 37 ° C for 24 hours. Then a colony of each cultured isolate was inoculated in 5 micro liters of Luria Brittany broth medium and incubated for 20 hours at 37 ° C. The tubes were then removed from the incubator and 1.5 micro liters of the medium was added to 1.5-mL micro-tubes. The stages of DNA extraction were done using the PR881614 extraction kit (Sinagene; Iran) according to the manufacturer's procedure. To examine the quality of extracted DNAs, first through dilution of the extracted DNAs, their OD (optical density) were studied and 5 micro liters of DNA was electrophoresed on 1% agarose gel (Figure 1). The resulted DNAs were stored at -20° C until PCR test. In the direct PCR method, instead of DNA extraction and performing its multiple stages, the new colony of bacteria was used as 24-hour cultures. Freshness of the colonies and not keeping them for doing PCR should be considered. Conducting PCR using extracted DNA and direct PCR: to identify mecA and 16srDNA genes, the primers of these genes were employed (Table 1). In order to carry out the PCR reaction, the final volume of 25 micro liters was used containing 1 microliter of template DNA, 1 microliter of each primer at 10 pico-molar concentration, 12.5 micro liters of the master mix (Ampliqon; Germany) and 10.5 micro liters of deionized distilled water. PCR mixture without the template DNA was deployed as the positive control [14]. Then the PCR reaction was performed for mecA gene using a thermocycler machine (BioRad; USA), including initial denaturation at 95 ° C for 5 minutes, 35 cycles of denaturation at 94 ° C for 2 minutes, primer connection phase at 57 ° C for 2 minutes and replication of target segment at 72 ° C for one minute. The PCR program for 16srDNA gene was similar to mecA gene. PCR products were stored in a refrigerator at +4° C for electrophoresis. For conducting direct PCR reaction, instead of using template DNA and going through different stages of extraction, one of the newly-cultured bacterial colonies was taken with a microbiological loop and was inoculated in the prepared mixture. For better solution of the colony, the final mixture was vortexed for 10 seconds. To prepare the final mixture, the final volume was brought to 25 micro liters according to the first method, and instead of 1 micro liter of each primer, 3 micro liters of each primer were added and the reaction was done through the same temperature cycle. For the electrophoresis of PCR product, 8 micro liters of it were electrophoresed in 2% agarose gel and the final result was examined using the gel documentation system, the CCD-Tab1 model (Kiagene; Iran).
Findings by Text
In both used methods, 16srDNA and mecA genes with a bond length of 420 and 310 were reproduced successfully relating to the gene of identifying Staphylococcus and methicillin-resistant gene, respectively. Besides, the quality of observed bonds was quite similar (Figures 2 and 3). Out of 135 samples of Staphylococcus epidermidis, 119 cases (88.1%) were extracted through PCR method using the DNA extracted by the kit and 117 cases (86.6%) had the mecA gene using direct PCR. Out of 88 samples of Staphylococcus saprophyticus, 71 cases (80.6%) were extracted by PCR using extracted DNA and 68 cases (77.2%) had the mecA gene through direct PCR.
Main comparison to the similar studies
... [22-31]. In a study on diphtheria toxins using direct PCR, Nakao et al. demonstrated that this method has acceptable sensitivity for diagnosing diphtheria toxin and clinical samples can be evaluated in terms of the presence of tox gene through this method [32]. In a study, Fod Vagan et al. used direct PCR method to evaluate Escherichia coli bacterium and the obtained results in this study indicate the accordance of this method with the DNA extraction-based method while the obtained bonds in both methods are almost equal and reliable [33]. Inglis et al. investigated the molecular tracing of Campylobacter species using direct PCR. [34]. ... [35-37].
Suggestions
Considering the lack of enough time to do further experiments, it seems that performing the direct PCR can be better and more tested through further studies.
Limitations
Cross-sectional nature of this study, insufficient time to execute additional tests, and not doing this experiment with other bacterial species are the limitations of this study.
Conclusions
To save time and money, direct PCR can be used for detection of negative coagulase staphylococcus resistant to methicillin.
Acknowledgments
The authors of this article are grateful to the officials of the research center for infectious-tropical diseases of Zahedan Medical Sciences University and colleagues of microbiology laboratory in Zahedan Medical University for helping them accomplishing this research.
Conflict of interest
Non-declared
Ethical Permissions
This study was approved by ethics and scientific research committees of Zahedan University of Medical Sciences in 2014.
Funding Sources
This study was financially supported by the Research and Technology Deputy of Zahedan University of Medical Sciences, using the funds of research projects.

TABLES and CHARTS

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