Cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase are shown lytic activity against methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to determine the anti-biofilm effect of CHAP-amidase on MRSA.
The chimeric CHAP-amidase was synthesized and subcloned into the pET-22b (+) expression vector, and the recombinant protein was expressed in E.coli BL21 (DE3) strain. Next, affinity-based purification yielded ~12 mg soluble protein per liter of E. coli culture. The results of the study showed that concentrations of ?1 ?g/mL of the purified CHAP-amidase have significant antibacterial activity against MRSA. The concentrations of ?2 ?g/mL of the purified CHAP-amidase have significant anti-biofilm activity against MRSA. Anti-biofilm activity of CHAP-amidase against MRSA revealed significant difference at 1?g/mL in comparison with 0.5 ?g/mL (P<0.001). A synergistic interaction between CHAP-amidase and vancomycin for inhibition of biofilm formation was detected by using checkerboard assay. Fractional inhibitory concentration (FIC) index was 0.375. Conclusion The results of this study showed that the anti-biofilm activity against MRSA would allow the use of CHAP-amidase to be a promising candidate in successfully eradicating biofilm formation. Keywords: Staphylococcus aureus, Methicillin-resistant, Endolysin, Biofilm formation. 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). 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. Lytic activity against MSA was shown by the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) and amidase that are known to be the catalytic domains of the bacteriophage-derived endolysin LysK. 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 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 < 0.38, 0.38 > OD590 < 0.70, and 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.
Disk diffusion assay
The purified Chimeric CHAP-amidase was diluted in saline Tris lysis buffer (STB), containing 150 mM NaCl and 10 mM Tris-HCl (pH 7.5), to the final concentrations of 10, 5, 2, 1 and 0.5 µg/mL. Afterwards, a sterile 6 mm filter paper disk was used per concentration of CHAP-amidase. The saturated disks were spotted onto a freshly spread MRSA culture, while the control disk was soaked in STB. The culture was incubated for 20h at 37°C.
Determination of the MIC and MBC of CHAP-amidase
The MICs of Chimeric CHAP-amidase against MRSA were determined by the microdilution method, using Mueller-Hinton broth (MHB). Chimeric CHAP-amidase was diluted in a 96-well microtiter plate to final concentrations ranging from 10, 5, 1, and 0.5 ?g/ml. A 100-?l aliquot of the bacterial suspension (106 CFU/ml) was inoculated and incubated at 37°C for 18h. The MIC was determined as the lowest concentration that completely inhibited bacterial growth. The minimal bactericidal concentration (MBC) was assessed as the extract concentration that gave significant MIC values after streaking the culture on Trypticase soy agar. Experiments were carried out in triplicate.
Growth inhibition by CHAP-amidase added to mid-logarithmic-phase cultures
Twenty-milliliter aliquots of MRSA at an optical density at 578 nm (OD578) of 0.1 were cultured in the basic medium at 37°C with shaking at 150 rpm until mid-logarithmic phase was reached (approximately 4h). At that time, CHAP-amidase was added at a final concentration of 1×, 2×, 4×, and 8× MIC. The OD of the cultures was measured at the indicated time intervals. The experiment was carried out in triplicate, and the results were presented as the mean OD ± the standard deviation.
Effect of CHAP-amidase on biofilm formation
Overnight cultures of biofilm producing MRSA isolates grown in Luria Bertani broth were diluted to 106 CFU/ml. 100-?l aliquot was transferred to a 96-well microtiter plate and 100?l of a concentration of CHAP-amidase 0.5?g/ml, 1?g/ml, 2?g/ml, 4 ?g/ml, and 8?g/ml, dissolved in Luria Bertani broth, was added. After incubation at 37°C for 24h without agitation, the bacterial growth (based on the OD600) was determined by using a microplate reader. A biofilm assay was carried out. All assays were performed in triplicate. As a control, the un-inoculated medium was used. The mean OD590 value from the control wells was subtracted from the mean OD590 of the tested wells.
Calculation of the FIC for each antibiotic
The checkerboard technique
The checkerboard technique was performed using the combinations of vancomycin and CHAP-amidase. Concentration ranges from 8xMIC to 1/4xMIC for vancomycin and from 8xMIC to 1/4xMIC for CHAP-amidase were prepared in 96-well Microtiter plates. The concentration ranges were prepared in separate plates and then joined into a single plate in order to have different combinations of the antibacterial agents in each well. The bacterial inoculum was adjusted to 5×105cfu/ml and distributed in all the wells. Two wells were reserved for positive and negative controls in each plate, respectively. After incubation at 37°C for 24h, the Fractional Inhibitory Concentration Index (FICI) was calculated using the formula.
FIC for antibiotic A = MIC of antibiotic A in combination/ MIC of antibiotic A alone
FIC for antibiotic B = MIC of antibiotic B in combination /MIC of antibiotic B alone
åFIC = FIC for antibiotic A + FIC for antibiotic B
The sum of FICI was then interpreted as follows: synergy if ?FICI ? 0.5, additive effect if 0.5 < ?FIC ? 2, indifference if 2 < ?FIC ? 4, and antagonism if ?FIC > 4.
The statistical analysis was performed with SPSS (version 19, Chicago, IL, USA). The chi-square test or Fisher’s exact test was used to compare proportions. A p value of < 0.05 was considered significant. Results The results of disk diffusion assay shown that 1?g/mL is the lowest concentration of CHAP-amidase that is able to inhibit the growth of MRSA. The data of the study emphasized that CHAP-amidase shows more potent activity against MRSA at 10, 5, and 2?g/mL concentration. CHAP-amidase showed a notable disrupting activity against biofilms of MRSA isolates (Figure 1, 2, 3). Synergistic effects were detected while combining vancomycin with the CHAP-amidase using the checkerboard assay. The synergistic interactions were confirmed by the calculation of ?FIC, which was equal to 0.375 (Figure 4). Discussion The results of this study showed that CHAP-amidase has good bactericidal activity against planktonic and biofilm MRSA. It shows to be a promising alternative therapeutic agent for treatment of MRSA. Lysins are a novel class of anti-infective obtained from bacteriophage. Lysins are highly evolved enzymes produced to cleave essential bonds in the bacterial cell wall peptidoglycan for phage progeny release. Lysins can reduce bacteria from mucosal surfaces and biofilms. Also, lysins can act synergistically with antibiotics by re-sensitizing bacteria to resist antibiotics. The advantages over antibiotics are their specificity for the pathogen without disturbing the normal flora, their low probability of bacterial resistance, and their capacity to eradicate colonizing pathogens on mucosal surfaces. The antibacterial activity of bacteriophage Lysin CF-301 was verified against S. aureus persister cells in exponential-phase and stationary-phase populations (15). The results of the study of Schuch et al. showed that CF-301 is highly effective at disrupting biofilms and killing biofilm bacteria (16). Zhou et al data showed that the bacteriophage endolysin (lysin) LysK?amidase killed all 66 methicillin-resistant staphylococcal strains and disrupted biofilms (17). They recommended that LysK?amidase has the possibility to be an therapeutic agent option for treatment of MRSA. Yang et al. reported that a novel chimeric lysine (ClyF) has good bactericidal activity against planktonic and biofilm MRSA both in vitro and in vivo, and thus signifies a valuable antibacterial to fight MDR S. aureus(18). The chimeric muralytic enzyme P128 showed effective anti-biofilm activity against S. aureus (19). The bacteriophage-derived CHAPK murein peptidase was demonstrated to fast lyse several strains of MRSA while disrupting and preventing the formation of a staphylococcal biofilm (20-22). Our data demonstrated the presence of a synergistic interaction between CHAP-amidase and vancomycin as a commonly used antibiotic to reduce MRSA biofilm formation. The analysis verified this synergistic effect against intermediate and strong biofilm producer of MRSA isolates with FIC Index of 0.375. A major issue of this study was CHAP-amidase activity against biofilm formation. We found that CHAP-amidase was able to completely inhibit biofilm formation at 2µg/ml and 2× MIC. For MRSA, a biofilm-inhibiting effect was already observed in the MIC range and higher, signifying that biofilm formation was already affected at higher concentrations of CHAP-amidase.