a Departments of Microbiology and Immunology, d Parasitology , e Medical Technology and f Pediatrics, College of Medicine, National Cheng Kung University, Tainan; b Department of Chemistry, National Taiwan University; c Department of Medical Research, Veteran General Hospital, Taipei, Taiwan, Republic of China
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Abstract |
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Introduction |
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In our previous studies, we found that carboxyfullerene could inhibit Escherichia coli-induced meningitis by decreasing the damage caused by infiltrating neutrophils on the bloodbrain barrier,13 but not by direct inhibition of the growth of E. coli.14 Moreover, we also demonstrated that group A streptococcal infection could be inhibited by carboxyfullerene, and that carboxyfullerene not only enhanced the bactericidal activity of infiltrating neutrophils, but also inhibited the growth of Streptococcus pyogenes A-20.15 Since carboxyfullerene had differential effects on E. coli and S. pyogenes A-20, the efficacy of carboxyfullerene on different Gram-positive and Gram-negative bacteria was studied further. In this study, we demonstrate that carboxyfullerene has bactericidal activity on Gram-positive staphylococci and streptococci, and that its lethal action was achieved by insertion into the cell walls of bacteria and disruption of their structure.
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Materials and methods |
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The water-soluble carboxylic acid C60 derivative with C3 symmetry, an effective free-radical scavenger, was synthesized as described previously.9 In this study, C63(COOH)6 (referred to as C3) dissolved in phosphate-buffered saline (PBS; 10 mg/mL) was used throughout.
Bacteria
Sixteen clinical isolates from National Cheng Kung University Hospital consisting of two oxacillin-resistant Staphylococcus aureus, two Staphylococcus epidermidis, three group B streptococci, four vancomycin-resistant Enterococcus faecalis (nos 002005), three vancomycin-susceptible E. faecalis (nos 474, 487, 496) and two Streptococcus pneumoniae (serotypes 6 and 14) were used. As well as the strains mentioned above, S. pyogenes clinical isolate A-20 and its isogenic mutant S. pyogenes SW507, which was deficient in the speB gene, and another S. pyogenes NZ-131 and its speB- isogenic mutant, S. pyogenes SW510, were also used in this study.16 In our previous studies, the in vivo protective effect of carboxyfullerene on S. pyogenes SW507-induced infection was more effective than the S. pyogenes A-20-induced one (unpublished observation), so the in vitro effect of carboxyfullerene on these bacteria was studied further. Furthermore, Gram-negative bacteria, including E. coli and Pseudomonas aeruginosa, which were obtained from the Culture Collection and Research Center (Food Industry Research & Development Institute, Hsinchu, Taiwan), and a clinical isolate of each of Salmonella typhi and Klebsiella pneumoniae were used for comparison.
Growth curves
The growth curves for bacteria after treatment with carboxyfullerene were determined. Staphylococcus spp. and Streptococcus spp., except S. pneumoniae, were cultured in brainheart infusion broth and TSBY broth [tryptic soy broth (Difco Laboratories, Detroit, MI, USA) containing yeast extract 0.5% (w/v)], at 37°C for 12 h, respectively. S. pneumoniae was cultured in ToddHewitt broth (Difco Laboratories) at 37°C for 12 h in 5% CO2 in air. Gramnegative bacteria, including E. coli, P. aeruginosa, S. typhi and K. pneumoniae, were cultured in LuriaBertani (LB) broth [1% (w/v) NaCl, 1% (w/v) tryptone, 0.5% (w/v) yeast extract]. After 12 h, the bacteria were subcultured (1/50 volume) into fresh broth for a further 12 h. During subculture, different concentrations (5200 mg/L) of carboxyfullerene were added to the bacterial suspensions, and the growth curves of bacteria at different times were determined using a spectrophotometer (Beckman Instruments, Somerset, NJ, USA) by measuring the optical density at 600 nm. For quantification of viable bacteria, bacterial suspensions collected at different times were diluted 105-107 and 0.1 mL plated on to blood or LB agar plates. The number of colony-forming units (cfu) of bacteria was counted and was expressed as cfu/mL. These experiments were repeated three times.
MICs and MBCs
MICs and minimal bactericidal concentrations (MBCs) were determined according to NCCLS methodology.17 Bacteria were cultured for 12 h and then subcultured into fresh broth; the concentration of bacteria was adjusted to 5 x 105 cfu/mL in 2 mL broth. Different concentrations (5500 mg/L) of carboxyfullerene were added to the bacterial suspension, which was incubated overnight. The minimal concentration of carboxyfullerene in non-turbid tubes represented the MIC. Non-turbid tubes were subcultured (0.1 mL) on to agar plates and incubated overnight. The surviving cfu/mL of bacteria were calculated from the number of colonies grown. The minimal concentration of carboxyfullerene on agar plates, where the number of cfu/mL was <0.1% of the initial concentration of bacteria, was taken to represent the MBC.
Direct bactericidal assay
As well as the MBC described above, direct bactericidal activity of carboxyfullerene after a short period (<3 h incubation) was determined. High concentrations of S. pyogenes A-20 (1 x 109 cfu/mL) were obtained from exponential phase cultures and incubated with various concentrations of carboxyfullerene (10100 mg/L) in PBS. At various intervals, the concentration of bacteria was determined by plating on to blood agar (Becton Dickinson, Cockeysville, MD, USA) overnight. To avoid the carry-over effect of carboxyfullerene remnant in culture medium, the suspension of bacteria collected at various intervals was diluted with PBS at least 10 times; therefore, the lower limit of detection for 0.1 mL samples was 1 x 102 cfu/mL. PBS was used to prevent bacteria from continued growth, but it will also cause a slight decrease in bacterial viability during the 3 h incubation. These experiments were repeated three times. The result was expressed as the mean cfu ± s.d./mL.
Transmission electron microscopic examination
The bacteria collected from the exponential phase were cultured with carboxyfullerene (50 mg/L) for 30 min at 37°C, then washed several times with PBS. Subsequently, the bacteria were fixed with 1.2% glutaldehyde in PBS containing 1 g/L of ruthenium red for the staining of the capsule at 4°C for 1 h.18 After several washes, the bacteria were osmicated in 1% osmium tetraoxide (Electron Microscopy Science, Washington, PA, USA) for 1 h, and then were dehydrated in a graded series of ethanol, cleared in propylene oxide and flat-embedded in EPON plastic [30% (w/w) dodecenyl succinic anhydride, 51% (w/w) EM bed-812, 18% (w/w) dadic methyl anhydride, 1% (w/w) 2,4,6-tri(dimethylaminomethyl) phenol] (Electron Microscopy Science). Thin sections (80 µm) were examined using an electron microscope (JEOL JEM-1200EX) at 75 kV.
Anti-carboxyfullerene antibody binding assay
The anti-carboxyfullerene antibody binding assay was performed using protein G/ASepharose beads coated with anti-carboxyfullerene antibodies, which were generated by immunizing BALB/c mice with 50 µg carboxyfullerene in Freund's complete adjuvant (Sigma Chemical Co., St Louis, MO, USA) five times.19 In order to remove antibacterial antibodies present in sera, the sera from naive and carboxyfullerene-primed mice were pre-absorbed with S. aureus, S. pyogenes, E. coli or P. aeruginosa. The pre-absorbed sera were incubated at 4°C overnight with Sepharose beads containing protein A and protein G (Amersham Pharmacia Biotech, Uppsala, Sweden), which are known to have high affinity for the Fc portion of immunoglobulin G.20,21 Bacteria collected from the exponential phase were cultured with carboxyfullerene (50 mg/L) for 30 min at 37°C, then washed several times with PBS. The treated bacteria were incubated with Sepharose beads that had conjugated with anti-carboxyfullerene antibodies for 1 h at room temperature, and non-binding bacteria were washed off with PBS containing 0.5% (v/v) Tween-20. The bacteria bound to Sepharose beads were visualized using Gram's stain.
Membrane perturbation assay
The membrane perturbing assay was performed using propidium iodide (PI) fluorescence detection by flow cytometry.22 The bacteria (2 x 107 cfu/mL) collected from the exponential phase were cultured with different concentrations (5200 mg/L) of carboxyfullerene at 37°C for various times. At various intervals, the bacteria were collected and stained with PI (400 mg/L) for 10 min, and dye penetration was measured by the presence of fluorescence emission at 560 nm.
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Results |
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Discussion |
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How carboxyfullerene kills staphylococci and streptococci is intriguing and worth further investigation. Carboxyfullerene can insert into the cell walls of staphylococci and streptococci within 30 min, as demonstrated by our anti-carboxyfullerene antibody binding assay. This only occurred in Gram-positive and not Gram-negative bacteria. The differential effects of carboxyfullerene on Gram-positive and Gram-negative bacteria might be a result of the fact that there are different components in Grampositive and Gram-negative cell walls. The cell wall of Gram-positive bacteria consists of many layers of peptidoglycan and teichoic acid, which is absent in Gram-negative bacteria, whereas that of Gram-negative bacteria contains one or very few layers of peptidoglycan, but its outer membrane consists of lipoprotein, lipopolysaccharides and phospholipids.29 Once carboxyfullerene is intercalated into the Gram-positive bacterial cell wall, it might disrupt the structure of the cell wall and cause bacterial death, as suggested by the membrane perturbation assay. Huang et al.12 reported that carboxyfullerene can interact with lipid bilayers; the binding efficiency between water-soluble fullerenes and microbes might determine its activity. Mashino et al.30 reported that cationic ammonium fullerene derivatives, C60-bis(N,N-dimethylpyrrolidinium iodide) and C60-bis(N-methylpiperazinium iodide) can suppress the growth of E. coli, indicating the importance of the side chain.
The MICs of carboxyfullerene in Gram-positive cocci were 550 mg/L. This dosage is lower than the LD50 (250 mg/L) in mammalian cells such as lymphocytes, neutrophils, RAW 264.7 and A549 cells (unpublished observations). The LD50 of fullerenol in mice by intraperitoneal injection is 1000 mg/kg.31 Addition of mouse sera to as much as 5% did not affect the bactericidal effect of carboxyfullerene (unpublished observations). Carboxyfullerene inhibited group A streptococcal infection by modulating the function of neutrophils and suppressing the growth of S. pyogenes A-20 directly in vivo.15 In this study, we demonstrated that carboxyfullerene inhibits the growth of Gram-positive cocci through intercalation into the cell walls, disrupting the structure and causing death. This suggests that carboxyfullerene could be considered as a new antimicrobial agent against Gram-positive cocci.
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Acknowledgements |
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Notes |
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References |
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Received 18 July 2001; returned 22 October 2001; revised 29 November 2001; accepted 20 December 2001