Virulence factors and antibiotic resistance properties of the Staphylococcus epidermidis strains isolated from hospital infections in Ahvaz, Iran

Background Resistant Staphylococcus epidermidis strains are considered to be one of the major causes of human clinical infections in hospitals. The present investigation was done to study the pattern of antibiotic resistance and the prevalence of virulence and antibiotic resistance genes amongst the S. epidermidis strains isolated from human hospital infections. Methods One hundred hospital infectious samples were collected and S. epidermidis strains were identified using culture and biochemical tests. Isolated strains were subjected to disk diffusion and PCR. Results Forty-six out of 100 hospital infectious samples (46%) were positive for S. epidermidis. S. epidermidis strains harbored the highest prevalence of resistance against penicillin (95.65%), tetracycline (91.30%), erythromycin (82.60%), cefazolin (78.26%), and trimethoprim-sulfamethoxazole (73.91%). All S. epidermidis strains had resistance against at least three different types of antibiotics, while the prevalence of resistance against more than seven types of antibiotics was 17.39%. AacA-D (69.56%), tetK (56.52%), mecA (45.65%), msrA (39.13%), and tetM (39.13%) were most commonly detected antibiotic resistance genes. The prevalence of vatC (4.34%), ermA (8.69%), vatA (8.69%), vatB (13.04%), ermC (13.04%), and linA (10.86%) were lower than other detected antibiotic resistance genes. ClfA (32.60%), agrIII (17.39%), and etB (13.04%) were the most commonly detected virulence factors. Conclusions The presence of virulent and multi-drug resistance S. epidermidis strains showed an important public health issue in hospitals.


Background
Hospital infections are considered as a major issue all around the world. Bacteria have the highest impact on the occurrence of hospital infections [1][2][3][4][5][6][7]. Staphylococcus spp. are commensal bacteria of human skin and have been isolated from diverse clinical sources such as urinary tract infections (UTIs), respiratory tract infections (RTIs), wound infections (WIs), soft tissue infections, blood infections, and endocarditis [3,[8][9][10]. It has been suggested that Staphylococcus epidermidis (S. epidermidis) is one of the most important species of this group.
It is a Gram-positive, non-spore forming, nonmotile, facultative anaerobic, and catalase-positive and coagulasenegative bacterium responsible for different types of hospital and nosocomial infections. Indwelling medical devices are considered a major vector of S. epidermidis in hospitalized patients [11,12]. S. epidermidis results in approximately 13% of prosthetic valve endocarditis infections, with a high rate of intracardiac abscess formation (38%) and mortality (24%) [13].
Some potential virulence factors including toxic shock syndrome toxin-1 (TSST-1 encoded by tst), exfoliative toxins A and B (eta and etb), clumping factor (clfA), and types I, II, and III of the accessory gene regulator (agr) are responsible for virulence characters of the S. epidermidis strains isolated from human clinical infections [10]. The Xregion gene of Staphylococcus strains has a high degree of importance in the occurrence of diseases, and it may have a variation rate (or clock speed) that provides suitable discrimination for outbreak investigation [10]. The IgG-binding region is responsible for causing host specificity and various immunological responses against Staphylococcus strains. The X-region and IgG-binding region have been detected in various types of staphylococcal infections [10].
Scarce researches have been conducted on epidemiological and molecular aspects of the S. epidermidis strains in hospital infections in Iran. Thus, the current research was done to study the prevalence rate, distribution of virulence factors, and antimicrobial resistance properties of S. epidermidis strains isolated from various types of human clinical infections collected from Ahvaz city, Iran.

Results
The present investigation was done to assess the antibiotic resistance properties and distribution of virulence genes amongst the S. epidermidis strains isolated from different types of hospital infectious samples. Forty-six out of 100 hospital infectious samples (46%) were positive for S. epidermidis. Table 2 represents the antibiotic resistance pattern of the S. epidermidis strains isolated from hospital infectious samples. S. epidermidis strains harbored the highest prevalence of resistance against penicillin (95.65%), tetracycline (91.30%), erythromycin (82.60%), cefazolin (78.26%), and trimethoprim-sulfamethoxazole (73.91%) antibiotic agents. Reversely, S. epidermidis strains harbored the lowest prevalence of resistance against nitrofurantoin (34.78%) and mupirocin (50%) antibiotic agents. The prevalence of resistance against ciprofloxacin, clindamycin, azithromycin, and rifampin antibiotic agents were 69.56%, 65.21%, 60.86%, and 60.86%, respectively. Figure 1 represents the prevalence of multi-drug resistant S. epidermidis strains isolated from hospital infectious samples. Multidrug-resistant S. epidermidis strains were determined as those who had at least simultaneous resistance against three or more than three types of antibiotics. All S. epidermidis strains had resistance against at least three different types of antibiotics, while the prevalence of resistance against more than seven types of antibiotics was 17.39%. Table 3 represents the distribution of antibiotic resistance genes amongst the S. epidermidis strains isolated from hospital infectious samples. We found that aacA-D (69.56%), tetK (56.52%), mecA (45.65%), msrA (39.13%), and tetM (39.13%) were most commonly detected antibiotic resistance genes amongst the S. epidermidis strains. The prevalence of vatC (4.34%), ermA (8.69%), vatA (8.69%), vatB (13.04%), ermC (13.04%), and linA (10.86%) were lower than other detected antibiotic resistance genes. The prevalence of msrA and msrB antibiotic resistance genes were 36.13% and 26.08%, respectively.

Discussion
S. epidermidis is a common commensal bacterium of the human skin and mucosa. While S. epidermidis has long been considered nonpathogenic, it is now recognized as a relevant opportunistic pathogen [11,12,14]. Most S. epidermidis-related not only are associated with intravascular devices (prosthetic heart valves, shunts, etc.) but also commonly occur in prosthetic joints, catheters, and large wounds. However, recently published data revealed the high prevalence of S. epidermidis in the cases of human clinical infections [11,12,14]. Additionally, nosocomial S. epidermidis isolates were characterized by their pronounced resistance against many commonly used antibiotics [11,12,14]. The present study was done to assess the antibiotic resistance pattern and distribution of antibiotic resistance and virulence genes amongst the S. epidermidis strains isolated from different types of human clinical infections. Findings showed that 46% of human clinical infection samples were positive for S. epidermidis strains. Relatively high prevalence of S. epidermidis in the human clinical infections of the present study is may be due to the ubiquitous presence of the bacterium in the hospital environment, its ability to biofilm formation, and finally failure to observe sanitary and disinfection principles in hospitals in Ahwaz city, Iran. Because of the ubiquitous prevalence of S. epidermidis as a commensal bacterium, it is often difficult for a clinician to decide whether an isolate represents the causative agent of an infection or an unspecific culture contamination.
Amongst all virulence markers found in the S. epidermidis strains, genes encoding clumping factor (clfA), IgG-binding region, toxic shock syndrome toxin (tst), exfoliative toxins (eta and etb), accessory gene regulator (agr), and X-region were recognized as the most important markers in occurrence of infectious diseases caused by S. epidermidis [10]. Eftekhar    genes, clfB (78.60%) and etb (2.90%) had the highest and lowest prevalence, respectively. The prevalence of tsst-1 gene amongst the S. epidermidis strains of our research was low (4.34%). Similar findings have also been reported from Iran (11.60%) [32], Sweden (22.00%) [33], Malaysia (0.50%) [34], and Colombia (10.00%) [35]. Tsst-1 gene is a pyrogenic toxin that encodes a 21.9 KDa extracellular toxin causing toxic shock syndrome (TSS). It is known as a severe acute disease distinguished by symptoms such as fever, rash, hypotension, and dysfunction of multiorgan systems. In addition, TSS secretion into the human blood may raise the rate of neonatal TSS-like exanthematous disease, Kawasaki syndrome, and sudden infant death syndrome [32]. Regarding the other detected genes, the eta gene was presented in 6.52% of strains. The prevalence of etb gene was 13.04%. The incidence rate of the eta and etb in the present study was higher than that reported in other investigations conducted on Iran (0.68%) [32], Colombia (3.00%) [35], and Malaysia (0%) [34]. A higher prevalence of eta gene was reported in studies conducted in Czech (10.00%) [36] and Turkey (19.20%) [37]. It was detected that the prevalence of the etb gene differs amongst numerous investigations, ranging from 0% in Colombia [35] and Malaysia [34] to 9.20% in Turkey [37]. Ghasemian et al. [38] reported the high prevalence of the clfA and clfB genes (100%). The incidence of the clfA gene in the bacterial strains of our research was relatively high (32.60%). A higher prevalence of this gene was reported from Brazil [39] and China [40]. Another important detected gene amongst the S. epidermidis strains was agr. The prevalence of agrI, agrII, and agrIII virulence genes amongst the S. epidermidis strains were 8.69%, 10.86%, and 17.39%, respectively. Agr virulence gene was also predominant amongst the S. epidermidis strains isolated from clinical samples recovered from China [40], USA [41], and Iran [42]. The accessory gene regulator (agr) locus influences the expression of many virulence genes in the S. epidermidis. Four allelic groups of agr, which generally inhibit the regulatory activity of each other, have been identified within the species. Interference in virulence gene expression caused by different agr groups has been suggested to be a mechanism for isolating bacterial populations and a fundamental basis for subdividing the species [43]. It encodes a twocomponent signal transduction system that leads to downregulation of surface proteins and upregulation of secreted proteins during in vitro growth. A role for agr in virulence has been demonstrated by the attenuated virulence of agr mutants in different animal infection models [43].

Conclusions
The present investigation is the first report of the phenotypic and genotypic analysis of antibiotic resistance in the S. epidermidis strains isolated from human hospital infectious samples in Iran. The total prevalence of S. epidermidis strains in hospital infectious samples was 46%. Considerable prevalence of resistance against penicillin, tetracycline, erythromycin, cefazolin, and trimethoprim-sulfamethoxazole and high distribution of aacA-D, tetK, mecA, and tetM antibiotic resistance genes may pose a potential public health threat. Additionally, clfA, agrIII, and etB were the most commonly detected virulence factors. A high prevalence of multidrug resistant S. epidermidis in the human clinical infectious samples is another important finding of the present study. Moreover, the presence of antibiotic resistance genes and also virulence factors in some S. epidermidis strains should be considered as a serious health hazard. Further researches are required to understand additional epidemiological aspects such as the exact relations between antibiotic resistance genes and virulence factors of the S. epidermidis strains in hospital infectious samples.

Bacterial isolation
S. epidermidis was identified by conventional bacteriological tests. The sample was enriched in a tryptic soy broth, and grown on mannitol salt agar, and then catalase, tube coagulase and urease tests, and carbohydrate fermentation were performed. S. epidermidis is catalasepositive, coagulase-negative, urease-positive, unable to ferment D-mannitol and D-trehalose, and able to ferment D-mannose and D-maltose [44,45].

Antibiotic resistance pattern
Patterns of antimicrobial resistance of the S. epidermidis strains were studied using the Kirby-Bauer method. A simple disk diffusion technique on the Mueller-Hinton agar (Merck, Germany) medium was used for this purpose. susceptibility of S. epidermidis isolates was tested against several types of antibiotic agents including penicillin (10 μg/disk), cefazolin (30 μg/disk), clindamycin (2 μg/disk), mupirocin (30 μg/disk), azithromycin (15 μg/ disk), erythromycin (15 μg/disk), tetracycline (30 μg/disk), ciprofloxacin (5 μg/disk), trimethoprim-sulfamethoxazole (25 μg/disk), nitrofurantoin (300 μg/disk), and rifampin (5 μg/disk) (Oxoid, UK). The instructions of the Clinical and Laboratory Standards Institute were used for this purpose [46]. The plates containing the disks were allowed to stand for at least 30 min before incubated at 37°C for 24 h. The diameter of the zone of inhibition produced by each antibiotic disc was measured and interpreted using the CLSI zone diameter interpretative standards [46]. S. epidermidis ATCC 12228 was used as a quality control organism in antimicrobial susceptibility determination.
DNA extraction and amplification of virulence factors and antibiotic resistance genes S. epidermidis isolates were sub-cultured on TSB media (Merck, Germany) and further incubated for 48 h at 37°C. Genomic DNA was extracted from bacterial colonies using the DNA extraction kit (Fermentas, Germany) according to the manufacturer's instruction. Table 1 represents the list of primers and PCR conditions used for the amplification of virulence factors and antibiotic resistance genes [47]. A programmable DNA thermo-cycler (Eppendorf Mastercycler 5330, Eppendorf-Nethel-Hinz GmbH, Hamburg, Germany) was used in all PCR reactions.

Statistical analysis
Statistical analysis was done using the SPSS 21.0 statistical software (SPSS Inc., Chicago, IL, USA). Chi-square test and Fisher's exact two-tailed test were used to assess any significant relationship between the prevalence of S. epidermidis strains, virulence factors, and their antibiotic resistance properties. P value < 0.05 was considered as statistical significant level.
Abbreviation CLSI: Clinical and Laboratory Standards Institute