Introduction their use. So far, few anti-biofilm agents

Introduction

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common causes of infection in the world (1). Glycopeptides, such as vancomycin, are considered the first line of treatment (2). However, vancomycin susceptibility among clinical studies has decreased (3,4). Alternatives to treating the MRSA infection are restricted. Moreover, resistance to new antibacterial agents such as, linezolid and daptomycin, have emerged (5).

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Biofilms are microbial sessile communities described by the bacterium that are adhered to abiotic or biotic surfaces or to each other. Biofilms are surrounded by a polymer matrix and show an altered phenotype compared to planktonic cells (6). Biofilm cells are recognized to be 10–1,000 times more resistant to antimicrobial agents compared to planktonic cells (7). This may be a result of a reduced penetration of antibiotics, a declined growth rate of the biofilm cells, and/or a decreased metabolism of bacterial cells in biofilms. The most important problem is biofilm formation by S. aureus, which is the cause of chronic infections; these infections are resistant to most currently accessible antibiotics (8). Recently, the development of new antibacterial agents have been limited and their antimicrobial activity causes selective pressure with antimicrobial resistance as a predictable result of their use. So far, few anti-biofilm agents are presented for clinical use (9). Therefore, new agent’s guides to fight biofilm-related infections are immediately needed. Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are known as catalytic domains of the bacteriophage-derived endolysin LysK and were formerly described to demonstrate lytic activity against MRSA. Results showed that concentrations of ?1 ?g/mL of the purified engineered chimeric CHAP-amidase protein have considerable antibacterial activity against MRSA (10). Knowledge of the ability of phage lysin to lyse the staphylococcal biofilms has been restricted. Investigation of CHAP-amidase has revealed that have (something is missing) anti-staphylococcal biofilm activities (11, 12). The aim of this study was to test the CHAP-amidase effects on biofilm growth formation of MRSA.

 

Methodology

Clinical strains

In this study, 48 isolates of MRSA were recovered from skin and soft tissue infection. All isolates were recognized by microbiological methods including colony morphology, Gram staining, catalase activity, DNAse tests, tube coagulase test, and mannitol fermentation. Methicillin resistance was confirmed using a cefoxitin disk (30 ?g) and 1?g oxacillin disk as recommended by Clinical and Laboratory Standards Institute (CLSI) on Mueller-Hinton agar plates (13). Polymerase chain reaction (PCR) was used to verify the presence of the mecA gene (14).

Biofilm formation assay

MRSA isolates were grown overnight at 37? in liquid Luria Bertani. The culture was diluted in 1:100 medium and 200µl of bacterial suspension was used to inoculate sterile 96-well microplates. After 24h at 37?, the wells were washed 3 times with 300µl of distilled water, dried and stained with 300µl of 2% crystal violet solution in water for 45min. Study of the biofilm formation was performed in 96-well cell culture plates (SPL Lifescience, Korea) by adding 200µl of acetic acid-ethanol (5: 95, Vol/Vol) to solubilize the dye. 100µl from each well was transferred to new 96-well microplates. Optical density (OD) was determined at 590 nm. The absorbance was recorded by the microplate reader. The biofilm formation was divided into three categories in this study: the strains with OD590 OD590 0.70 were defined as biofilm formers of the weak level, moderate level, and strong level, respectively, based on the ODs. All assays were performed in triplicate. The un-inoculated medium was used as a control. The mean OD590 value from the control wells was subtracted from the mean OD590 of tested wells (14).

Bacterial culture, subcloning, and protein expression

The E. coli strain BL21 (DE3) (Invitrogen, Carlsbad, CA) was grown at 37°C in Luria-Bertani (LB) medium. The LB medium was then supplemented with ampicillin (100?g/mL) for plasmid selection. The subcloning of the CHAP-amidase-encoding sequence into plasmid and transformation of pEX and pET-22b (Novagen, USA) vectors into bacterial cells were followed by standard restriction enzyme digestion and sequence analysis, which were used to verify the cloning procedure (10). Protein expression was performed in BL21 (DE3) strain. In brief, the recombinant pET-22b plasmid was transformed into bacterial cells and the cells were cultured in LB medium containing 100?g/mL of ampicillin. The culture was further incubated at 37°C and protein expression was induced by the addition of isopropyl ?-D-1-thiogalactopyranoside (IPTG) to the final concentration of 1mM at logarithmic phase (corresponding to 0.5–0.6 OD590). Cells were harvested after 4h and protein expression was evaluated by SDS-PAGE (sodium-dodecyl sulfate polyacrylamide gel electrophoresis). The recombinant expression cultures of BL21 were harvested by centrifugation and the pellets were lysed via sonication. The protein was purified by using the modified nickel-chromatography Ni-NTA purification system. The concentration of purified protein was determined by spectrophotometry using the Bradford assay.

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