Detection of Staphylococcus aureus and their toxin genes inhabit on the scorpions surface

The transmission of infectious agents by arthropods is of particular importance. Every year, many people are bitten by scorpions around the world. Staphylococcus aureus is of the most important infectious bacteria. This study aimed to investigate the distribution of S. aureus in scorpion specimens and the presence of some toxin genes in these species. The fauna of scorpions in the Kuhdasht region was studied for one year. Then, S. aureus was identified on the body surface of scorpions by biochemical and molecular methods, and the presence of Sea, Seb, Sec, Sed, See, Pvl, Tsst1, Eta, Etb, and mecA genes was examined by the PCR method. The pattern of antibiotic resistance was determined by the use disk diffusion method. MRSA isolates were identified using genotypic and phenotypic methods. Of 75 studied scorpion specimens, Hottentotta saulcyi was the most abundant species. Sixteen (21.3%) isolates of S. aureus were identified from all samples. The highest and lowest antibiotic resistance levels belonged to penicillin and clindamycin, respectively. MRSA was observed in 50% of the isolates. Thirteen out of 16 isolates possessed at least one of the toxin genes. Due to the presence of S. aureus on the body surface of scorpions, it should always be expected that an infection may occur after the bite. Moreover, the presence of toxin genes in the studied isolates showed that infection with these bacteria would seriously threaten one’s health.


Introduction
Arthropods are a large branch of invertebrates that encompass organisms such as Gnathostomata, Arachnids, Crustaceans, etc. A group of arthropods are harmless to humans and also play an important role in the biological cycle. Some groups are harmful to humans and can cause numerous injuries through bites, stings, or other toxic and inflammatory secretions, in addition to transmitting various pathogens (Leitner et al. 2015). Some arthropods can only injure the site of the bite, and in some cases, bacterial contamination of the mouthpieces of these arthropods can cause secondary infections. Many pathogens are transmitted by arthropods. Examples include plague, Crimean Congo haemorrhagic Fever, and Dengue Fever, which are transmitted by fleas, ticks, and Aedes mosquitoes, respectively (Organization 2014).
Scorpion bites are one of the most important health and medical problems and, in addition to the resultant anxiety and concern; the problem incurs high treatment costs and poses many people to the risk of death every year. Therefore, the development of infection after arthropod bites is a special medically important issue (Ahmadi et al. 2020).
Staphylococcus aureus is a gram-positive and facultative anaerobic cocci, which is medically the most important species in the genus Staphylococcus. This bacterial strain can cause a wide range of simple skin infections (such as pimples, boils, scabs, styes, and abscesses) to dangerous diseases, such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome, and septicemia. S. aureus is one of the five most common causes of nosocomial infections, in particular postoperative wound infections (Khan et al. 2017;Lee et al. 2018). Toxins of this bacterium play Communicated by Erko Stackebrandt. an important role in its pathogenesis. Recent studies, for instance, have shown an association between PVL (Panton-Valentine Leukocidin) and methicillin-resistant strains of S. aureus, or the toxic shock syndrome toxin-1 gene produces a protein that causes systemic disease (Hennekinne et al. 2012;Oliveira et al. 2018;Kim 2019). The Exfoliative toxin is another important toxin of this bacterium that can cause complications such as staphylococcal scaly skin syndrome. Staphylococcal enterotoxins are a class of superantigens and are also involved in food poisoning (Hennekinne et al. 2012;Oliveira et al. 2018;Kim 2019). On the other hand, infections caused by methicillin-resistant S. aureus (MRSA) have turned into a global concern. The presence of the mecA gene is the cause of drug resistance in MRSA (Goering et al. 2019). Therefore, it is of paramount importance to identify these important isolates in scorpions. The prevalence of S. aureus in scorpions and the identification of pathogenic genes in isolated bacteria have not been studied up to now.
Annually, about 50,000 scorpion stings occur in Iran. According to the registered statistics, more than 170 cases of scorpion sting occur in the city of Kuhdasht every year. One of the provinces of Iran, where the frequency of scorpion stings is high, is Ilam, which is adjacent to the city of Kuhdasht. On the other hand, due to the high frequency of scorpion stings in the city of Kuhdasht, this city was selected for the study. This study aimed to investigate the fauna of scorpions in Kuhdasht city, the distribution of S. aureus on the surface of scorpions, the frequency of Sea, Seb,Sec,Sed,See,Pvl,Tsst1,Eta,Etb, and mecA genes in S. aureus isolates, and the frequency of MRSA in these isolates.

Sample collection
Scorpion specimens were collected from different locations in Kuhdasht city from 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, 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), 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. 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 (10,000 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).

Detection of toxin genes and mecA gene
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.

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.

Identification of scorpions and phylogenetic tree
A total of 75 scorpions were collected from the registered stations in Kuhdasht city. The caught scorpions were examined for 17 measurable traits (Table 1). Phenometric studies included Hottentotta saulcyi (42), Scorpio maurus (16), Hemiscorpius lepturus (10), Compsobuthus matthiesseni (5), Androctonus crassicauda (1), and Orthochirus iranus (1) species, respectively. H. saulcyi (56%) was the least abundant and A. crassicauda and O. iranus (1.33%) had the highest abundance. UPGMA results showed the presence of two branches in the tree (Fig. 1). In the smaller branch, H. saulcyi was isolated from the other species. This branch is divided into two sub-branches. Paye-Astan station was separated from other populations of this species. The second sub-branch was divided into two groups, the smaller group consisting of Qebleh and Southern 2) were placed in a separate group. The larger branch consisted of two sub-branches. The smaller subdivision was subdivided into two groups, where Scorpio maurus of Paye-Astan station (No. 4) was separated from the rest and formed a smaller group. In the larger group, H. lepturus in the Garab station (No. 5) was separated from the others, and S. maurus in the Garab, Qebleh and Keshmahoor stations was placed in the larger subgroup. This subgroup was subdivided into two parts, and Garab station was separated from the other two stations. The larger subdivision was also divided into two groups. The larger group was divided into two subgroups, where the O. iranus formed a smaller subgroup in the Northern Kuhdasht station and was separated from the populations of C. matthiesseni. The larger subgroup was divided into two parts, with a smaller population in Southern Kuhdasht. The larger section was divided into two sub-sections, the smaller subsection consisting of the Paye-Astan station and the larger sub-section comprising Garab and Keshmahoor stations. The smaller group consisted of two subgroups, of which H. lepturus in Keshmahoor was a smaller subgroup. The larger subgroup was subdivided into two parts, where S. maurus was the smaller part in the Northern Kuhdasht station, and H. lepturus of Paye-Astan and S. maurus of Southern Kuhdasht stations formed the larger part. The average characteristics of the samples collected at Tange Ab Barik and Garab were very close.

Staphylococcus aureus isolation and molecular detection
Based on molecular and biochemical characteristics, 16 (21.3%) isolates of S. aureus were identified out of 75 studied scorpion specimens. The detected isolates were, respectively, S. maurus (7,

Antibiotic resistance pattern and identification of MRSA isolates
Examination of the antibiotic resistance pattern of the studied isolates showed that the highest and lowest levels of antibiotic resistance belonged to penicillin and clindamycin, respectively. Of the 16 studied isolates, two isolates were resistant to all tested antibiotics. Considering that 9 isolates were resistant to cefoxitin, 9 isolates were probably MRSA ( Table 2).

Detection of toxin and mecA genes
Of the 16 isolates of S. aureus, three (18.75%) isolates had none of the toxin genes studied, while the other isolates possessed at least one of the toxin genes. On the other hand, sed, sec, and pvl genes were observed in none of the isolates. With a frequency of 50%, the mecA gene was identified in two samples of H. saulcyi, one sample of O. iranus, and five samples of S. maurus. In other words, 8 MRSA isolates were identified genotypically. Since eight out of nine isolates were considered MRSA by the phenotypic method and had a positive PCR test of the mecA gene, one false positive sample was detected in the phenotypic method. The results indicated that the two species, A. crassicauda and H. lepturus, contained none of the studied toxin genes (Table 3).

Discussion
Our study demonstrated that scorpions identified in these areas belonged to three families: Hemiscorpiidae, Scorpionidae, and Buthidae. In a study conducted by Nematoallahi et al. in Aligudarz city, Hottentotta saulcyi had the highest distribution in different stations, which is consistent with our study (Nematollahi et al. 2020). Akbari et al. conducted a study in Kohgiluyeh and Boyer Ahmad and Chaharmahal and Bakhtiari provinces and found that the Buthidae family had the highest species diversity with six genera. They caught only one genus, Hemiscorpius, from the Hemiscorpiidae family, which is in line with the present study due to almost similar geographical conditions to Lorestan province (Akbari et al. 2001). It can generally be concluded that scorpions of the Buthidae family are the most abundant scorpions in the region and H. saulcyi is the dominant species in these areas. It should be noted that H. saulcyi, which is one of the most dangerous scorpions in Iran, and Hemiscorpius lepturus, which is one of the most dangerous scorpions and causes the death of many scorpion bites in Iran, comprised, respectively, 55 and 13% of scorpion species caught in Kuhdasht city, which can be a warning to the health officials of the province.
More importantly, although S. maurus is less dangerous to humans than the other species caught in this study, health workers should take diagnostic tests for S. aureus in people bitten by scorpions as this study proved that this bacterium is present on the body of these scorpions. It has now been proven that many bacteria live on the surface or inside the body of arthropods, acting as carriers of numerous pathogens that cause important human and animal diseases. These carriers play an important role in the transmission of bacteria to other organisms, including mammals. S. aureus is one of the pathogenic bacteria that have been confirmed to be present in association with some arthropods (Fagan et al. 2003;Nayduch et al. 2013;Abraham et al. 2017). The presence of some genes enables this bacterium to become more pathogenic and even cause death in some cases. No study has so far investigated the distribution of this bacterium in scorpions and the presence of the Sea, Seb,Sec,Sed,See,See,Pvl,Tsst1,Eta,Etb, and mecA genes in these species, making it impossible to compare the data of this study with similar studies. Furthermore, our results can provide a new perspective on the complications after scorpion bites. In other words, post-bite complications caused by scorpion were not considered previously to be possibly related to infection caused by S. aureus and its produced toxins. Depending on the type of scorpion bites, the bacterium can easily enter the tissue if the scorpion's body surface or telson is infected with the bacterium. In this study, 16 (21.3%) out of 75 scorpion samples contained this bacterium, which shows the presence of bacteria on the scorpion's body surface and telson and the importance of the study of bacterial infections after scorpion bites. Unfortunately, 11 (68.75%) out of the 16 studied isolates carried at least one of the toxin genes.
On the other hand, one of the current problems faced by humans is the presence of antibiotic-resistant bacteria (Organization 2017). In this study, a relatively high pattern of antibiotic resistance was observed in the isolates, and two isolates were resistant to all tested antibiotics; therefore, infection by these bacteria after scorpion bites can be extremely dangerous for the infected person. The two isolates were isolated from S. maurus in regions with more human communities; hence these antibiotic-resistant isolates might have been transferred to animals in the surrounding environment by human activities. MRSA is currently one of the major therapeutic challenges. MRSA isolates are divided into three groups: healthcare-associated MRSA (HA-MRSA), community-associated MRSA (CA-MRSA), and livestock-associated MRSA (LA-MRSA) (Junnila et al. 2020). Rapid identification and treatment of MRSA infections are the most important procedures to prevent the spread of infection and reduce the risk of mortality in patients (Blot et al. 2002). This is because the mecA gene is reportedly not found in MRSA strains; hence the molecular methods that identify the mecA gene are the gold standard methods (Wallet et al. 1996;Brown et al. 2005).
In this study, therefore, the identification of MRSA isolates by phenotypic and genotypic methods was very important because the exact and definite frequency of these isolates was determined here. According to our results, a significant frequency of 50% was obtained for MRSA isolates in scorpion communities carrying S. aureus. The higher frequency of MRSA isolates in S. maurus, which were more closely related to human communities, could probably result from the acquisition of these bacteria from humans and animals in contact with humans. In other words, these isolates probably originate from CA-MRSA and LA-MRSA, which in turn can indicate the role of humans in changing the pattern of bacterial distribution in the environment.
The present study presents the first results in the world and our findings confirm the possibility of infection with A. aureus after the scorpion bite. This, therefore, alarms that the possibility of infection by A. aureus should be considered after the scorpion bite based on the appearance of the lesion, and the necessary procedures should be taken in this regard. Similar studies are also recommended in other parts of the world to better determine the distribution of this bacterium and the genes studied in scorpions.