Characterization of Staphylococcus aureus Isolated From Wound Infectious in Diabetes Clinic of Hazrat Fatemeh Zahra (SA) Hospital

Staphylococcus aureus has long been recognized as an important pathogen of hospital acquired infections (1). Due to the expansion of the drug resistance pattern and importance of antibiotic resistance, the study of these microbial strains is one of the major challenges which should be taken into consideration (2). Methicillin resistance in S. aureus is mediated by a penicillin binding protein (PBP2a) which is encoded by the mecA gene (2-4). According to previous studies, 50% to 90% of S. aureus strains isolated from hospital infections were resistant to methicillin (5,6). Staphylococcus aureus is the most common cause of skin and soft-tissue infections like impetigo, furunculosis, superficial and surgical wounds and abscess (7-13). PIA is a polysaccharide composed of β1-6-linked N-acetyl glucosamine with partially deacetylate residues, which encloses the human cells or medical tools and protects the microorganism against both host immune system and antibiotic treatment (6). In this study, we investigated effective MSCRAMM-encoding genes, during biofilm formation on Congo Red Agar (CRA) medium and polystyrene plates, via phenotypic and genotypic screening of methicillin resistant S. aureus strains isolated from the patients with nosocomial infections in Hazrat Fatemeh Zahra (SA) hospital, Isfahan, Iran.

Phenotypic Assay on Slime Production and PCR Amplification The slime production assay was performed by cultivation of the S. aureus strains on CRA plates as described in different studies. No slime producing strains produced pinkish red, smooth colonies with a darkening at the center. Slime producing isolates grow as black colonies and non-slime producers constitute red colonies. These biofilm-producing isolates were selected to determine antibiotic resistance pattern (12,16). The bacterial genomic DNA of S. aureus strains were extracted with a plasmid Minikit (QIAGEN, Germany) according to the protocol (18)(19)(20)(21)(22). The biofilm genes were determined by specific primers listed in Table 1 (23)(24)(25)(26).

Results
In this study, we investigated the S. aureus strains isolated from wound infections from the patients in diabetes clinic of Hazrat Fatemeh Zahra (SA) hospital. The antibiotic resistance pattern of isolates was determined by disk diffusion agar method. The results showed that 95 isolates out of 132 samples were methicillin-resistant S. aureus (MRSA) (27). All the 95 isolates produced characteristic black colonies on CRA, and were categorized as slime producers. All of the 95 MRSA isolates produced biofilm in different levels. High rate of resistance was observed to methicillin (85.2%), erythromycin (73.8%), ciprofloxacin (86.1%), and penicillin (86.6%). On the contrary, the low rate of resistance was seen to vancomycin (0%) and nitrofurantoin (12%). The phenotypic method showed that 48.3% of the isolates were highly able to produce biofilm, 29.1% were intermediate biofilm producers, and 10.6% of the isolates were low biofilm producers (27).
Frequency of S. aureus isolates were detected in different patients most notably in the age range of 61-70 years old. This study demonstrated that icaC (73.2%) and icaD (49.2%) genes had the highest frequency among Staphylococcus aureus Isolated From Wound Infectious the genes involved in biofilm formation. The frequency of other genes involved in biofilm formation is shown in Figure 1.

Discussion
The mechanism of biofilm formation in S. aureus is weakly understood. However, in fact these genes are associated with both slime production and biofilm formation according to Ammendolia (24,25).
However, the present study showed different expression levels of the genes found in 95 biofilm forming strains. Moreover, all of the strains harboring icaA (35.3%), icaD (49.2%), icaB (28.1%), and icaC (73.2%) produced slime on CRA, micro plate titration, and genotypic methods. Thus, no association was found between icaA and icaD genes. Slime production as detected with CRA and micro plate titration is similar to that in the study of Rohde in 2001 (28). Statistical analysis on biofilm formation and frequency of genes showed that there was a significant relationship between biofilm formation by the phenotypic methods and presence of icaD and icaA genes (P value <0.05).
The prevalence of clfA, clfB, cna, and eno genes in the tested strains were comparable with those found in S. aureus strains isolated from different niches and hosts in various studies. Although we found that the prevalence of biofilm formation was high in human infections, the prevalence of fnbA, fnbB, and other genes was much higher in the study of Vandecasteele et al in 2003 (25). According to our results, the frequency of clfA was 42.5%, which is comparable to Peacock et al study in 2002. They also illustrated that the expression level of the clfA gene in the S. aureus strains increased 24 hours after growth. Statistical analysis verified a significant relationship between phenotypic and genotypic frequency of the genes (P value= 0.0015) (29). PCR test results showed that nine MSCRAMM-encoding genes like clfA, clfB, fib, and fnbA were detected in all of the strains isolated. Moreover, it  (19).

Conclusions
This study mainly tried to investigate the expression of 12 genes involved in biofilm formation in MRSA isolated from wound infections in diabetes clinic of Hazrat Fatemeh Zahra (SA) hospital. According to the study results, the expression of these genes significantly increased. In addition, high prevalence of antibiotic resistance pattern was one of the important reasons for the development of drug resistance in patients.

Conflict of Interests
None.