Sample collection
Scorpion specimens were collected from different locations of Kuhdasht city since Mar. 21, 2018 to Mar. 21, 2019. Since scorpions are mainly night-active and their cuticles glow under ultraviolet (UV) light, scorpions were searched by a UV lamp at night. The observed specimens were taken from the tail area by scorpion pliers, placed in a separate sterile plastic container, and transferred to the laboratory. The sampling date, the collector name, weather conditions and environmental factors at the time of sampling, station name, geographical location (GPS), and altitude were recorded for each sample.
Identification of scorpions
The scorpions were identified by a stereomicroscope (Olympus SZ-CTV) using the identification keys of Farzanpay (Farzanpay 1987), Dehghani (Dehghani 2006), Navidpour (Navidpour et al. 2011), and Kovarik (Kovařík 2007) and then studied phenometrically. The patterns of morphological differences between the studied scorpions specimens in the registered stations of Kuhdasht city (Table 1) based on measurable traits were examined. The studied populations were separated from each other in the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) tree (Figure 1). The applied techniques used were based on standardized methods.
Staphylococcus aureus isolation
In the laboratory, normal saline swabs were gently mopped on the surface of the body and telson of scorpion samples and cultured on Blood Agar and Nutrient Agar media. The bacteria were purified, stained by Gram staining, and then S. aureus was initially identified using biochemical tests, namely catalase, coagulase, blood agar hemolysis, DNase production, and mannitol salt agar (Ghaznavi-Rad and Ekrami 2018). All the employed equipment was sterile. This step was performed separately for each sample.
DNA extraction
The boiling method was used to extract DNA. First, two to three colonies of the bacterium were transferred to a microtube containing 300 µl of DNase/RNase-Free distilled water. Then, the lids of the microtubes were closed completely with parafilm and placed in boiling water for 15 min. Next, the suspension was centrifuged (10000 g, 5 min), the DNA-containing supernatant was transferred to clean sterile microtubes, and kept at −20 °C for further experiments (Kazemnia et al. 2014).
Molecular identification of bacteria
PCR method was used for accurate and final identification of S. aureus isolates. At this stage, the isolates were identified using specific primers, namely 5'-GCGATTGATGGTGATACGGTT-3' (forward) and 5'-AGCCAAGCCTTGACGAACTAAAGC-3' (reverse). The final volume (25µl) of the reaction included 3 μl of DNA template, 0.7 μl of each primer (10 pM), 0.5 μl of dNTPs (25 μM), 0.2 μl of Smart TaqDNA polymerase, 1 μl of MgCl2 (2 mM), 2.5 μl of 10X Taq buffer (Amersham, Pharmacia Biotech), and 16.4 μl of distilled water. Finally, all PCR products were electrophoresed on the gel (Brakstad et al. 1992). Sequencing was performed for all 16 PCR products. These sequences were recorded with accession numbers MZ816748 to MZ816763 in GenBank.
Detection of toxin genes
The Sea, Seb, Sec, Sed, See, Pvl, Tsst1, Eta, and Etb toxin genes in S. aureus were identified by the PCR method with primers used in previous studies (Japoni-Nejad et al. 2013; Leke et al. 2017). To detect MRSA isolates by the genotypic method, the mecA gene was examined in all isolates by PCR. The final volume (25 µl) of the reaction was similar to the previous step. Finally, all PCR products were electrophoresed on the gel.
Antibiotic resistance pattern and identification of MRSA isolates
The antibiotic resistance pattern in isolates was determined on Müller-Hinton agar medium by the disk diffusion method (Kirby-Bauer) according to the Clinical and Laboratory Standards Institute (CLSI). Antibiotics used were penicillin (10 units), cefoxitin (30 μg), gentamicin (10 μg), tobramycin (10 μg), azithromycin (15 μg), tetracycline (30 μg), ciprofloxacin (30 μg), clindamycin (2 μg), trimethoprim (5 μg), and rifampin (5 μg) (Weinstein et al. 2019). The phenotypes of MRSA isolates were detected by the disk diffusion method using the cefoxitin disk (30 μg).
Drawing a phylogenetic tree and Statistical Analysis
The MVSP (Multivariate Statistical Package) software was used to draw a phylogenetic tree and classify scorpions. Statistical analyses were done by SPSS (Statistical Package for the Social Sciences) software at a significance level of 0.05.