1. Background
Staphylococcus aureus (S. aureus) isolates are ubiquitous pathogens that cause a wide spectrum of clinical signs, mild and status to systemic and even fatal infections (1). Staphylococcus aureus infections can cause by healthcare or community settings and usually occur in afflict hospitalized and/or immunocompromised individuals (2). MRSA isolates can resist a wide range of antibiotics, which make the treatment of infections much more difficult.
MRSA isolates are resistant to beta-lactam antibiotics via a new Penicillin Binding Protein2a (PBP2a) that confer significantly reduced affinity to beta-lactams (3). The mecA gene encoding this PBP is located in SCCmec elements (4). Moreover, MRSA isolates acquired from nosocomial are named HA-MRSA with Staphylococcal Cassette Chromosome mec (SCCmec) types I, II and III (5), while those acquired from community are called CA- MRSA that harbor SCCmec types IV and V. On the other hand, the accessory gene regulator (agr) genes play a vital role in the S. aureus pathogenesis and therefore, the appreciation of the relationship between these genes and clinical signs may be useful for these genes. Furthermore, S. aureus isolates can colonize on skin surface and epithelium of the body via a number of surface-attached and secreted proteins (6-8). Thus, colonized individuals may be at risk of endogenous infections of S. aureus entering into the sterile sites of the body via wounds or indwelling medical devices (9). Biofilm formation plays a vital role in chronic and persistent infections caused by S. aureus. Biofilm formation takes place through either the icaADBC (via synthesis of a polysaccharide named PIA) genes, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) or both (10). The icaAD genes encode the enzymes assembling the PIA. Enzymes named sortases are responsible for attaching these components covalently to the peptidoglycan (11) and thus these components contribute to the biofilm formation (12). MSCRAMMs play an indispensable role in triggering of endovascular, bone and joint and prosthetic-device infections (13). These molecules interact with proteins such as collagen (mostly with Cna), fibronectin (mainly via Fn, via FnbAB), fibrinogen (Fib, with ClfAB and Fib) and thus evade the immune system responses and therefore, progress the infection (10, 14, 15). Clumping Factor A (ClfA) protein, for instance, binds to fibrinogen (Fg) and is also responsible for clumping of S. aureus in blood plasma, through which culminating in arthritis and endocarditis (16). On this base, detection of various genes related to the pathogenesis and antibiotic resistance is an important cue to insight the capabilities of clinical isolates in different specific sites of infections.
2. Objectives
The current study aimed to characterize isolates of S. aureus collected from wound samples.
3. Materials and Methods
3.1. Bacterial Isolates
A total of 15 S. aureus wound isolates collected from hospitalized patients were evaluated, inpatients were included (n = 15) and all the outpatients (data not available) were excluded, from July 2012 to January 2013 in Tehran, Iran. Afterwards, the isolates were identified with conventional tests including catalase, coagulases, acid production from mannitol on mannitol salt agar and DNase tests.
3.2. Genomic DNA Extraction
The total genomic DNA was extracted through the preparation of a suspension of bacterial isolates in 200 µL of TE buffer and then with lysostaphin (including 200 µL and 20 µL of lysostaphin [2 µg/mL, Sigma]). The total DNA was isolated according to Straubinger method (17).
3.3. DNA Amplification
DNA was amplified with specific primers previously published to detect mecA gene and SCCmec types, agr genes and several biofilm related genes among the wound isolates (18).
The annealing temperature was 55°C (30 seconds) for mecA gene and 51°C (one minute) for SCCmec types, according to Zhang study (19). The specific primers for mecA gene and SCCmec types are shown in Table 1. To observe the PCR products by electrophoresis, 5 µL of each product was blended with 1 µL of each gel red and loading buffer dyes, and were run in 1% agarose gel electrophoresis and was observed by transluminator UV.
| Isolates | SCCmec | agr | clfAB | fnbA | fnbB | eno | cna | ebps | bbp | Antibiotic Resistance (Resistance) |
|---|---|---|---|---|---|---|---|---|---|---|
| 1 | III | III | + | + | + | + | + | - | - | CIP, E, GM, T, Amx, CD |
| 2 | V | I | + | - | + | + | - | - | - | Amx, T |
| 3 | III | I | + | + | - | + | + | - | - | CIP, E, T, Amx, CD |
| 4 | III | IV | + | + | - | + | + | - | - | CIP, E, GM, T, Amx, CD |
| 5 | III | I | + | + | + | + | + | + | - | CIP, E, GM, T, Amx, CD |
| 6 | III | I | + | + | + | - | + | - | - | CIP, E, GM, T, Amx, CD |
| 7 | III | I | + | - | - | + | - | - | - | CIP, E, GM, T, Amx, CD, SXT |
| 8 | V | II | + | + | - | + | - | - | - | CIP, E, T, Amx |
a Abbreviations: Amx, amoxicillin; CD, clindamycin; CIP, ciprofloxacin; E, erythromycin; GM, gentamicin; T, tetracycline.
b The frequencies of clfAB, fnbAB, fib, eno, cna, ebps and bbp genes were 100%, 100%, 65% (n= 13), 55% (n = 11), 70% (n = 14), 70% (n = 14), 55% (n = 11), 0% and 0%, respectively.
3.4. Analysis of Data
The SPSS software and Chi-square were employed to analyze the collected data.
4. Results
4.1. Bacterial Isolates
Nine S. aureus isolates were collected from males and eleven from females. The isolates were collected from intensive care unit (45%, n = 9), outpatients included in the study (25%, n = 5), emergency (25%, n = 5) and infectious diseases (5%, n = 1).
4.2. The Antibiotic Susceptibility Test Pattern
The majority of the isolates were resistant to amoxicillin (80%, n = 16), tetracycline (45%, n = 9) and erythromycin (35%, n = 7), but all were susceptible to vancomycin and linezolid. Resistance to ciprofloxacin, trimethoprim-sulfamethoxazole, clindamycin and gentamicin were 30% (n = 6), 15% (n = 3), 20% (n = 4) and 20% (n = 4), respectively.
4.3. Methicillin Resistance
Eight (40%) isolates were methicillin resistant, S. aureus (MRSA) with oxacillin, and the mecA gene was detected in these isolates. Two MRSA were resistant to all the antibiotics, except to linezolid and vancomycin. Moreover, three isolates were only susceptible to SXT, in addition to vancomycin and linezolid. The AST pattern of MRSA is exhibited in Table 1.
4.4. The SCCmec types
The majority of (90%, n = 18) MRSA harbored SCCmec type III, and two isolates harbored type V. MRSA wound isolates with SCCmec type III were resistant to a wider spectrum of the antibiotics.
4.5. The agr Specific Groups
The majority (70%, n = 14) of the isolates belonged to agr I, followed by agr II (15%, n = 3), agr IV (10%, n = 2) and agr III (5%, n = 1).
4.6. The Frequency of Genes Encoding the MSCRAMMs
The frequencies of clfAB, fnbAB, fib, eno, cna, ebps and bbp genes were 100%, 100%, 65% (n = 13), 55% (n = 11), 70% (n = 14), 70% (n = 14), 55% (n = 11), 0% and 0%, respectively. The relationship between these genes and methicillin resistance or other characteristics of S. aureus from wound samples was not confirmed.
5. Discussion
In the current study, the majority of the isolates (90%) were resistant to amoxicillin. This occurs because of plasmid carried beta- lactamases that are capable of transmitting to new isolates more rapidly. Moreover, MRSA isolates had a wider spectrum of antibiotic resistance. All the isolates were susceptible to vancomycin and linezolid; likewise the majority (75%) were susceptible to SXT. These results exhibit that three of the aforementioned antibiotics remain the last resorts to remove these isolates. Several previous studies indicated that vancomycin (glycopeptides) is the last drug to control MRSA. In 2012 and 2013, the prevalence of MRSA in Namazi and Faghihi hospitals of Shiraz were 146 (42.3%) and 199 (57.7%), respectively (20). In the study by Rahimi in 2009, 88% of the collected isolates were MRSA (21). In the study by Ghasemian et al., the inducible clindamycin and methicillin resistance were not high (22). Results of a survey performed by Adebayo were similar to those of the current study, in which he showed that all the isolates were susceptible to vancomycin and linezolid, and higher antibiotic resistance was detected among MRSA strains (23). Similarly, Nitishkumar observed that all the tested isolates were susceptible to vancomycin and the antibiotic resistance was significantly higher in MRSA than MSSA strains (24). These results show that vancomycin and linezolid antibiotics remain among few effective antibiotics to remedy MRSA infections.
In the present study, MRSAs with SCCmec type III were significantly more resistant to antibiotics, indicating that these isolates are acquired from hospital (4).Other previous studies reported results similar to those of the current study findings: The study of Japoni in South of Iran reported that the frequency of SCCmec type III among MRSA isolates was74.3% (25). Likewise, Fatholahzadeh reported the type III in 78% (26) of the studied isolates. In the study by Ghasemian et al., SCCmec type III was reported as the major SCCmec type 31 (22). In the study by Reiter, among patients with cystic fibrosis, all harbored SCCmec type III (27).
The agr groups can regulate the S. aureus pathogenesis. It was determined that the majority of the wound isolates belonged to agr I. In a previous study, it was determined that the majority of the isolates belonged to agr I (28).There was no relationship between antibiotic resistance and agr groups. In the survey by Mirzaee, 63 MRSA isolates were collected and twenty nine (46%) of the isolates were strong biofilm producers. Moreover, the icaD and icaC genes were detected in all isolates, and the prevalence of icaA and B were 63% and 51%, respectively (12).
All the isolates in the current study harbored clfAB gene. The clumping factors play a critical role in attachment and colonization of S. aureus in body surfaces, such as skin surface and epithelium. Similar to our present study, Raphael detected clfB in 91.8% of the isolates (29). In the previous studies, the prevalence of these genes was similarly high (30, 31). To the authors` best knowledge, there is scarce previous studies detecting clfAB gene prevalence in Iran. Moreover, in the survey by Atshan, all strains harbored clfA, B genes (32). A study showed that 69.7% of the isolates harbored clfA, B genes. However, Momtaz confirmed that nearly 20% of S. aureus isolates that caused mastitis contained clfA gene (33). These studies suggested that S. aureus strains from different infection sites in addition to the epidemiological differences may contain different frequencies or express the clumping factors, essential for colonization. Besides, it was observed that all the MRSA and MSSA strains harbored clfAB gene. In the current study the frequencies of fnbA and fnbB were 65% and 55%, respectively. The gene encoding fibrinogen binding protein (fib) was present in 70% of the wound isolates. A study by Bodén detected the fib gene in all S. aureus strains (34). Similarly, the authors’ previous study determined that all clindamycin inducible resistant and vancomycin intermediate S. aureus (VISA) isolates contained this gene (22). The prevalence of eno and cna genes were 70% and 55%, respectively, exhibiting the important role of these genes to colonize S. aureus. Since the laminin and collagen constitute most of the proteins in the skin tissue, the protein products of these two genes have numerous receptors to attach to S. aureus. Aydiner reported that 78.4% of S. aureus isolates harbored cna gene (35). In contrast, Arciola showed that the cna gene was present in 46% of isolates (36). ebps and bbp genes were detected in none of the isolates. These two genes contribute to the colonization of S. aureus isolates to catheters and medical device surfaces and then culminate in systemic infections such as osteomyelitis bloodstream and other infections. In the study by Paniagua in catheter associated S. aureus isolates, for example, the most frequent biofilm related genes included: clfA, clfB, cna, bbp, ebps and ica (65.6 %, n = 21) (37). Similar to the current study, Tang studied various sources and then detected the bbp gene in only one S. aureus isolate (38). Therefore, several factors affect the frequency of the genes encoding pathogenesis factors and antibiotic resistance in S. aureus isolates, including clinical sites, epidemiological differences, the period of study, the locations in which the studies are performed and other factors that there is not no sufficient knowledge about them. The limitations of the current study was the low number of wound samples, the lack of measurement of expression capability of surface protein genes by RT-PCR and low knowledge about the degree of wound infection among patients and also MIC of isolates to vancomycin.
All the isolates and the majority of them were susceptible to vancomycin/linezolid and co-trimoxazole, respectively. The prevalence of MRSA in wound samples was 40% .The majority of MRSA harbored SCCmec type III. Moreover, most of the isolates belonged to agr I. the majority of the isolates contained clfAB, fib and eno genes responsible for colonization. The difference of wound isolates with other clinical isolates was not significant.
