This study provides the first report of the frequency of SEs and TSST-1 encoding genes among human CoNS isolates in Iran. Some investigators have reported lack of enterotoxin genes associated with human and veterinary CoNS isolates [
27,
28]. The
sec gene was the most common classic enterotoxin-encoding gene among all enterotoxin genes, which was detected in 48.4% of the isolates obtained from wound cultures. The frequency followed by
seb and
see, harbored by 27% and 12% of the isolates, respectively. While the
sea, sed, seh and
sem genes were rarely found (2.2%, 3.2%, 4.4% and 5.4%, respectively). The low prevalence of some SEs genes including
sem in this study could be due to the theory that indicated enterotoxin-like (SEl) toxin genes are more abundant among commensal strains as compared to pathogenic ones [
29]. Other studies also showed significantly higher rates of
sec in CoNS isolated from coalho cheese, goat milk, industry meat and newborns, which ranged from 46.8% to 78% [
5,
30‐
32]. Andrade et al. (2019) researched the occurrence of
sea,
seb,
sec,
sed,
see,
seg,
seh,
sei and
sej genes in strains of
Staphylococcus coagulase-positive (CoPS) and negative isolated from coalho cheese, and detected the presence of the following genes:
seh (53.2%) in CoPS strains and
sec (46.8%) in CoNS strains [
30]. Lyra et al. (2013), also investigated SEs-encoding genes (
sea,
sec,
sed,
see,
seg,
seh,
sei) in 44 strains of CoNS isolated from goat milk and found the presence of the
sec in 55.6% of the strains and
sea gene was found only in one strain [
5]. These results indicate that the occurrence of the SEs genes isolated from coalho cheese and goat milk were not very diverse. Pinheiro et al. reported the presence of
sea,
seb,
sec,
sed, see,
seg,
seh and
sei genes among
S. epidermidis and S
. haemolyticus isolated from blood cultures, while
sei,
seg and
sea were the most frequent genes in both species [
33]. It can relate to the fact that nosocomial isolates may be better equipped with virulence factors obtained by facilitated transfer through selective pressure. This is in contrast with other similar works which have been indicated the occurrence of
sea gene in the higher frequency compared to other enterotoxin genes. Veras et al. evaluated the potential of CoNS and CoPS strains associated with outbreaks of
staphylococcal food poisoning in Brazil and reported that enterotoxin genes were observed amongst 70% of the CoNS isolates. 38% found to harbor
sea, 29% amplified only
seb, and the concomitant presence of the
sea and
seb genes were reported in 24% of the isolates. Genes for
sec and
sed (either alone or concomitantly) were found infrequently [
34]. In another study, the
sea and
seb genes have also described as the most common SEs gene among the
staphylococcal species causing bovine mastitis [
35]. These differences between studies may be related to geographic origin of the isolates, number and genetic structure of each isolate. In the current study, all of the strains producing of the enterotoxin SEA were positive for both
sea and
seb. The concomitant presence of these two genes in the same bacterium is explained by the fact that they occupy the same chromosome locus [
36]. 17(46%) and 11 (61%) of toxigenic
S. epidermidis and
S. haemolyticus strains showed two or more the SEs genes in association that
seb +
sec were detected as the most frequent. Other studies have also reported the concomitant presence of SEs genes in these organisms. Cunha et al. (2006) revealed the simultaneous presence of the
seb and
sec genes in 11%,
sea/seb and
sea/seb/sec in 5% of the CoNS obtained from newborns hospitalized in the Neonatal Unit of the Hospital of the Botucatu Medical School [
32]. In another work, the concomitant incidence
sea and
seb genes have observed in 20% of the
S. epidermidis and
S. haemolyticus strains recovered from blood cultures [
33]. The
tsst gene was identified in 25% of CoNS species including 28.6% of
S. epidermidis, 21.6% of
S. haemolyticus and 20% of
S. saprophyticus strains. In contrary with our work, other studies have found
tsst gene in any of CoNS species isolated from newborns and cows with bovine mastitis [
32,
37]. But this gene has been detected in 87% of
S. aureus isolated from wound and blood cultures and in 15.5% of
S. aureus obtained from bovine mastitis milk [
38,
39]. Our findings demonstrated a high percentage of toxigenic strains among
S. epidermidis (75.5%,
n = 37), followed by
S. haemolyticus (48.6%,
n = 18). This is in accordance with other findings (Pinheiro et al. and Cunha et al.), which
S. epidermidis was described as the CONS with the highest potential to produce of enterotoxins among identified isolates [
33,
40]. The current study indicated a higher incidence of SEs genes among strains recovered from positive blood cultures(42.3%), so that 55% and 38% of all
S. haemolyticus and
S. epidermidis strains contained at least one enterotoxin gene, predominating followed by urine (37.2%), catheter (13.5%) and wound (6.7%) sources. This was in agreement with a similar survey carried out by Pinheiro et al. who exhibited a high prevalence of SEs genes in isolated
S. epidermidis and
S. haemolyticus strains from blood cultures with frequencies of 95.3% and 79.8%, respectively [
33]. This is likely due to the fact that genes encoding some virulence factors were up-regulated in human blood over time. The coordinated expression of diverse virulence factors during infections (e.g., expression of adhesins early during colonization versus production of toxins late in infection to facilitate tissue spread) hints at the existence of multiple regulatory elements that respond to a variety of different environmental signals [
41‐
43]. Considering the antimicrobial susceptibility patterns, the highest resistance rates were determined for cefoxitin in 53.8% and trimethoprim-sulfamethoxazole in 46.2% of the isolates. The highest frequency of
sea gene responsible for the synthesis of the SEA was detected among strains that exhibited phenotypic resistance to chloramphenicol, erythromycin, cefoxitin and trimethoprim-sulfamethoxazole antibiotics. The
sec gene among strains that acquired resistant to gentamicin and
sed in strains with phenotypic resistance to doxycycline, levofloxacin, novobiocin were the most abundant identified SEs genes. Clindamycin and rifampicin resistant strains showed higher frequencies of CoNS containing the
seh gene in association with other SEs genes. In addition, in this regard, the largest frequency of
see gene was found among isolates showing phenotypically resistance toward cefoxitin and doxycycline antibiotics and 18% of all CoNS isolates harbored
see gene and exhibited resistance to trimethoprim-sulfamethoxazole, clindamycin, levofloxacin and novobiocin antibiotics concurrently. Regarding the results of statistical analysis in CoNS isolates observed a significant association between the incidence of SEs genes and resistance to antimicrobial agents. Schroeder et al, and Motamedi et al. described the role of the occurrence infective determinant-associated genes in the development of resistance to the antimicrobial agents and fatal
Staphylococcal infections [
44,
45]. 11 (12%) Out of 91 isolates were found to be resistant to levofloxacin, which the most dominant detected CoNS species was
S. epidermidis (46%), followed by
S. haemolyticus (36%) and
S. saprophyticus (18%). The most remarkable percentage of the resistant isolates were found in the blood infections (64%), followed by catheter-associated urinary tract infections (18%). 45% of the identified isolates that exhibited phenotypic resistance to levofloxacin were carrying the SEs and TSST
1 genes that
seb +
sec were the most frequent, and the majority of toxigenic strains of CoNS were shown to belong to
S. haemolyticus. In this regard, our finding is similar to another study in which the proportion of resistance to levofloxacin ranged from 0% in methicillin-resistant
S. haemolyticus (MRSH) to 7.7% in methicillin-resistant
S. epidermidis (MRSE) strains [
46]. These findings confirm which levofloxacin is a fluoroquinolone that exerts a potent effect against MRSH, MRSE and CoNS strains. It should be noted that, the access very little available literature on any information concerning the
gyrA,
gyrB,
grlA, and
grlB genes in
staphylococci isolates was available. Among the phenotypically-resistant strains to levofloxacin, 5 (45.4%) out of 11 isolates determined to be positive for carrying of levofloxacin-conferring genes. Notably, the combinations of resistance genes were conserved in these strains. Amongst the positive isolates, 6 (54.5%) were positive for four genes, 3 (27.2%) were positive for three genes and 1 (9%) harbored two genes. In levofloxacin-resistant
S. haemolyticus strains the
grlA +
grlB and
gyrB genes were responsible for inducing resistance to levofloxacin. In a survey carried out by Osman et al.,
gyrA,
gyrB and
grlA genes in
S. haemolyticus strains were absent and 66.6% and 33.33% of MSSA (methicillin susceptible
Staphylococcus aureus) isolates carried
gyrA and
gyrB genes, which in contrary to our results the prevalence percentage of these genes were reported with high incidence, 50%, 75% and 50%, respectively [
1]. According to published results by Osman et al., the prevalence of
gyrA and
grlA genes among various
staphylococcus species in accordance with our results were with high incidence (63% and 70.4%, respectively) and
gyrB gene in contrary with our findings was identified with incidence low, 26% [
47]. Fluoroquinolones (FQs) are categorized as effective antibiotics against a wide variety of organisms and having a role in the chemotherapy and postexposure prophylaxis for organisms, that could be used in biologic warfare. A extreme resistance could be attributed to the extensive use of FQs which can led to the development of drug resistance among
staphylococcus isolates and other bacterial species [
47]. Regarding to the prevalence proportion of insertion sequences, IS256 and IS257 were identified with the highest frequency among
S. haemolyticus and
S. epidermidis strains with the frequencies of 58.6%, 41.4% and 38.2%, 54.4%, respectively. Studies analyzing the prevalence rate of IS256 among
S. epidermidis strains have been reported diverging results from 46.7% to 81% [
48‐
50]. Previous reports have demonstrated that IS256 is significantly associated with multi-resistant, biofilm-forming
S. epidermidis isolates resident in the hospital setting [
51]. It was also indicated association of the IS256 with the genomes of aminoglycoside-resistant
staphylococci and
enterococci isolates [
52,
53]. In addition, in this study statistically found significant association between the incidence of IS256 and resistance to gentamicin as well as between the presence of IS257 and resistance to cefoxitin (
p value 0.033 and 0.004, respectively). The IS257 is a mobile genetic element, which associated with genes mediating biofilm formation and genes conferring resistance to beta-lactamase, aminoglycosides and tetracycline antibiotics [
54,
55]. The IS256 can be used as a potential molecular marker to discriminate invasive strains from commensal strains of
S. epidermidis [
56]. Montanaro et al. demonstrated a dramatic correlation between the presence of IS256 and resistance to gentamicin [
57]. Considering the antimicrobial susceptibility patterns, strains harboring IS256 and IS257 were the most frequent among those with resistance to cefoxitin (53% and 76%, respectively). It was also found that IS256 may influence the expression of pathogenesis-related genes [
50]. In current research IS256 was the most common among isolates harboring
sea also statistically found significant relationship between production of SEA and the occurrence of IS256.