Introduction

Staphylococcus aureus (S. aureus) is known as a normal human skin flora and continues to be an important pathogen, particularly in nosocomial infections (1-3).The most multidrug resistant type is methicillin-resistant S.aureus (MRSA), a leading cause of life threatening infections (3-5). The increasing trend of antibiotic resistance, along with the capacity of biofilm formation on medical devices and tissue may cause the additional antibiotic resistance and further treatment fails(6). Biofilms are the population of microorganisms with the ability to grow on biotic and abiotic surfaces by the production of polysaccharide intercellular adhesion (PIA) encoded by ica ADBC(6-8). While some antibiotics are commonly used to treat staphylococcal infections, they are ineffective on them, but these therapeutic antibiotics may be capable of impressing the biofilm formation in sub-minimal inhibitory concentrations and further elimination or development of infections depends on reducing or inducing effect of these levels. The exposure of bacteria to low levels of antibiotic concentrations may take place for different reasons such as bioavailability reduction of antibiotics in systemic circulation(9), systemic absorption of topically applied antibiotics, and application of antibiotics as a growth promoters(10) may lead to find subinhibitory concentrations of these antibiotics. In some cases, even the existence of the biofilm may decrease the penetration of antibiotics to the internal milieu of biofilms and are consequently exposed to the lower levels of antibiotics due to diffusion gradients(11, 12).In addition, the wide range of  antibiotics with low doses , as growth promoters in agriculture, put the organisms in exposure to low dose of drugs(10). Therefore, studying the effect of clinical antibiotics prescribed routinely by physicians for staphylococcal infections, on the biofilm formation seems necessary to improve the treatment process. The current study aimed at determined to the inducing and reducing effects of common antibiotics in sub-minimal inhibitory concentrations, on MRSA biofilm formation.

Materials and methods

Bacterial isolates

Twenty-eight clinical isolates of S.aureus were obtained from the microbial bank of Antimicrobial Resistance Research Center (ARRC) of Iran University of Medical Sciences, Tehran, Iran. All strains were collected from inpatients with burn injuries and conserved in tryptic soy broth (TSB) with 20% glycerol. An MRSA ATCC 33591 got along with the rest. Staphylococcus aureus ATCC 25922 was used as the quality control. The strains were recultured from TSB on blood agar and incubated for 18 hours, for further analyses.

Antibiotics

Ten types of antibiotics from different classes routinely used in treatment of staphylococcal infections were selected, including cefazolin, rifampicin, vancomycin, oxacillin, clindamycin, cotrimoxazole, minocycline, linezolid, azithromycin, and clarithromycin. It should be noted that various types of antimicrobial agents are used against staphylococcal infections. In the current study, the tested antibiotics were selected from the most therapeutic options especially in Iran. The disks and powders of the antibiotics were purchased from MAST (UK) and Sigma Aldrich (4), respectively.

Identification tests

Isolates were confirmed by their Gram-positive, catalase positive, DNase positive reaction, mannitol fermentation activity, and coagulase positive results (13).

Detection of MRSA strains 

Disk diffusion test to evaluate the resistance to oxacillin and cefoxitin was done as described in Clinical and Laboratory Standards Institute (CLSI) guidelines(14), focused on cefoxitin, which gives more accurate result than oxacillin.

Detection of biofilm producing Staphylococcus aureus

Qualitative detection of biofilm-producer strains was performed by culturing 28 strains on Congo red agar (CRA)(15). This method is based on the morphological characteristics of biofilm-producer and non-producer bacteria cultured on Congo red medium, composed of brain heart infusion broth (BHI) 37g/L, sucrose 50 g/L, agar No.1 10 g/L, and Congo red 0.8 g/L. Congo red stain was prepared as a concentrated aqueous solution and sterilized at 121˚C for 15 minutes. Then, it was added to the rest of autoclaved medium. The strains were inoculated in streaks and incubated at 35°C for 24 and 48 hours, aerobically. Black colonies with a dry crystalline consistency indicated biofilm production, while the non-biofilm-producing strains formed red colonies(16). The quantitative detection of biofilm production of each strain, carried out by microtiter plate biofilm assay, described below.

Determination of minimum inhibitory concentration value

MIC assays were performed against 10 antimicrobial agents in Mueller-Hinton broth(MHB) according to CLSI guidelines for broth microdilution susceptibility test(14).Strains were incubated at 37°C for 20 to 24 hours, and susceptibility of the isolates was determined according to the standard CLSI breakpoints. The MIC is determined as the lowest concentration of an antimicrobial agent at which the bacterial growth is completely inhibited.

Microtiter plate biofilm assay, as a quantitative biofilm production assay

The impact of 10 antibiotics was assessed on biofilm formation in subinhibitory concentrations as previously described (2,17,18). Briefly, aliquots (100 µL) of antibiotic solutions (512 µg/mL) were added in triplicate to100 µL tryptic soy broth (TSB) supplemented with 1% glucose in the first well of a 96-well tissue culture microtiter plate (Falcon). The serial dilutions were provided with the concentrations ranged from 0.06to 128 µg/ml(serial dilution: 0.06, 0.125, 0.5, 1, 2, 4, 8, 16, 32, 64, 128 µg/mL) and wells containing uninoculated TSB+glucose 1% served as a negative controls. Aliquots of inocula (100 µLeach, ca. 10⁴ to 10⁵ CFU/ml) were added to the wells. Positive controls were prepared by mixing 100 µL of inocula and 100 µLofTSB+glucose1%without antibiotics. The plates were covered and incubated at 37°C for 24 hours.

The content of the wells were aspirated and the microtiter plates were washed twice with sterile normal saline to eliminate unbound cells, and then, dried. To fix the attached cells, 190 µL of methanol (99%) was added to each well, left for 20 minutes, and then, decanted and dried. Afterward, the adherent cells were stained for 5 minutes with 190 µL of Gram’s crystal violet. The wells were then, rinsed with tap water 3 times to rinse the excess stain off. Plates were air-dried at room temperature. To quantitate the biofilm biomass, the dye bound to the attached cells were released by adding 190 µL of glacial acetic acid 33% v/v to the wells, and the plates were placed in ELISA (the enzyme-linked immunosorbent assay) reader after shaking gently for 30 seconds to measure the absorbance at 630 nm wavelength. The percentage of biofilm inhibition is expressed as [(OD₆₃₀ value at0µg/mL of antibiotic −[ OD₆₃₀ value in the presence of an antibiotic) / OD₆₃₀ value at 0 µg/mL of antibiotic ] × 100 (18). The positive values showed inhibitory effect, and negative values represent inducing effect of antibiotics on biofilm formation according to the formula.

Statistical analysis

The significance of differences between the mean of optical densities of groups, with and without each antibiotic (12 groups; the serial dilutionranged from 0.06to128 µg/mL) were examined by one-way analysis of variance (ANOVA), followed by the Dunnett multiple comparison test. A P-value of

Results

Strain selection

Two conditions were considered to select the appropriate strains for further studies: the ability to produce biofilm, and showing resistance to various antibiotics. Among the 28 strains of S.aureus isolated from clinical samples, 5strains were qualified on the basis of biofilm production by the Congo red agar (CRA) method ;the strains with black colonies were considered as biofilm-producer (figure 1), and high resistance to antibiotics used in the current study.

The strains disqualified for different reasons such as low biofilm-forming ability or high sensitivity to the mentioned antibiotics. One MSSA and 3 MRSA strains were detected by the disk diffusion method for resistance to oxacillin and cefoxitin. MRSA ATCC 33591 was used along with these 4clinical strains.

MIC determination

MIC values of 5 isolates against cefazolin, rifampicin, vancomycin, oxacillin, clindamycin, cotrimoxazole, minocycline, linezolid, azithromycin, and clarithromycin are shown in Table 1.

Table 1. MICs of the Antibiotics Tested in the Current Study

MIC, minimuminhibitory concentration; CAZ, cefazolin; AZM, azithromycin; VAN, vancomycin; OXA, oxacillin; MIN, minocycline; RIF, rifampin; CLI, clindamycin; TMP/SMX, trimethoprim-sulfamethoxazole; LZD, linezolid; CLR, clarithromycin

 Of the studied isolates, all the 5isolates were resistant to azithromycin, clarithromycin, minocycline, clindamycin and linezolid, and 1strain (S.aureus 595) was resistant to rifampicin, whereas all the strains were susceptible to vancomycin. Four of the five isolates, were resistant to cefazolin, oxacillin, and cotrimoxazole. These resistance patterns can help to ignore the direct impact of antibacterial activity of agents on the growth, and subsequently, the biofilm production of strains.

Influence of sub-MICs on biofilm formation

By means of the microtiter plate biofilm assay, isolates were incubated in the presence of antimicrobial concentrations ranging from 1× to 1/1024 MIC (these concentrations are below the MICs of the strains accounted for sub-minimal inhibitory concentration).These MIC ratios, which refer to continuous dilutions of antibiotics, are different for each strains depending on their MIC, and it should be noted that for the strains, which showed the MIC of over 128 µg/mL, the ratios of MIC were defined by considering the least sum of MIC(128 µg/mL). The percentages of biofilm inhibition or induction are shown in Table 2.

Table 2. Inhibition of Biofilm Formation of Staphylococcus aureus Isolates at Six Sub-MICs of the Tested Antibiotics

^a*: P<0.05

^b: The ratio of MIC mentioned in the table depends on the MIC of each strain. Some concentrations were excluded because they were not in the range of the tested area (0.06to 128 µg/mL).

^c:Asthe range of testing concentrations was started from 0.06 µg/mL, and the sub-MICs of 4 strains for rifampin were less than this concentration, they were not included in this table.

ATCC, American type culture collection

Since most of the significant effects (P<0.05) were observed in the range of 1/2 to 1/64 MIC, this range was considered in the mentioned table and other concentrations were not shown. The lowest effect was observed when linezolid was applied in all 5 isolates in the presence of at least 2subinhibitory concentrations, which had significant inhibiting effect on biofilm formation(P<0.05). Afterward, cefazolin (in 4strains) and clarithromycin, oxacillin, clindamycin, and minocycline had significant reducing effects on the biofilm formation of 3isolates, followed by cotrimoxazole (2strains) and vancomycin (1strain).

In contrast, some antibiotics showed inducing effects on biofilm formation. For instance, azithromycin and vancomycin had great inducing impacts on the biofilm growth of 3 strains, in the presence of at least 1subinhibitory concentration, followed by oxacillin and cotrimoxazole in 2strains, and 1 strain, respectively, when minocycline and clindamycin were used. The number of strains, which induced, reduced, and affected by the antibiotics is shown in Table 3.

Table3. The Effect of Sub-minimal Inhibitory Concentrations of Antibiotics on the Level of Staphylococcus aureus Biofilm Formation

Table 3 is a simplified form of Table2; for more information, refer to the previous table.