1 Institute of Microbiology, 2 Department of Biomedical Sciences and Technology, Udine Medical School, University of Udine, p.zza S.M. Misericordia 1, 33100 Udine; 3 National Laboratory CIB, AREA Science Park, Padriciano-Trieste; 4 Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy
Received 20 January 2003, returned 24 February 2003; revised 11 June 2003; accepted 18 June 2003
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Abstract |
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Methods: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains. The killing mechanism was investigated further by means of a two-colour fluorescent permeabilization assay, and by scanning electron microscopy (SEM).
Results: The Bacteroides fragilis group, Clostridium reference strains and most clinical isolates were inhibited in vitro by 12 µM (3.26.4 mg/L) SMAP-29, and killed by 1.5- to 2-fold higher peptide concentrations. The anaerobic bacterial cells were 90%100% permeabilized within 2 h of exposure to bactericidal concentrations of the peptide. The SEM images of bacteria exposed to SMAP-29 provide morphological evidence that the envelope is an important target of the bactericidal activity of this peptide. These results are consistent with earlier studies indicating that SMAP-29 kills aerobic bacteria with a membranolytic mechanism, and suggest that both aerobic and anaerobic bacteria share surface features that are targeted by this peptide.
Conclusions: These studies demonstrate that the spectrum of antibacterial activity of SMAP-29 includes the B. fragilis group and Clostridium species, and encourage further investigations of the therapeutic potential of this peptide.
Keywords: antimicrobial peptide, cathelicidin, anaerobic bacteria, membrane permeabilization
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Introduction |
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Several species of anaerobic bacteria are isolated frequently from human clinical specimens and may be responsible for serious infections. In particular, Bacteroides fragilis group strains among Gram-negative organisms, and Clostridium perfringens, Clostridium difficile and other Clostridium species among Gram-positive anaerobes may cause serious infections, i.e. mixed chronic/abscess processes, bacteraemia, post-operative wound infections and toxin-associated syndromes.2 The rapid spread of antibiotic-resistant clinical isolates has impaired effective treatment of these anaerobic infections, by decreasing the efficacy of several classes of molecules currently used in therapy, such as ß-lactams, tetracyclines, macrolides and clindamycin.3 Even metronidazole, which was long considered a fully active anti-anaerobe antibiotic, has become less effective following the emergence of less-susceptible B. fragilis isolates.4
Several novel compounds are currently under investigation for their therapeutic potential. Among these are the cationic peptides of the innate immune system, which represent a wide natural host-defence mechanism. A large number of these molecules have been isolated from animals, plants and bacteria in the last two decades.5,6 Their in vitro and in vivo effects against several groups of pathogenic aerobic bacteria and fungi have been determined,6,7 revealing potent activity against these organisms. A limited number of investigations have revealed their effects on anaerobic bacteria.812
In this study, the in vitro activity of SMAP-29,13 a high-potency and broad-spectrum7,14 antimicrobial peptide belonging to the cathelicidin family,15 was evaluated against reference and clinical anaerobic bacteria belonging to the B. fragilis group, C. difficile and C. perfringens species, including strains displaying antibiotic resistance mechanisms. The mechanism of antimicrobial activity was investigated using a two-colour fluorescence permeabilization assay and scanning electron microscopy (SEM).
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Materials and methods |
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A total of eight reference anaerobic strains were used in the assays: B. fragilis NCTC 9343, Bacteroides distasonis NCTC 11152, Bacteroides ovatus NCTC 11153, Bacteroides thetaiotaomicron NCTC 10582, Bacteroides eggerthii NCTC 11155, Bacteroides vulgatus NCTC 11154, C. difficile ATCC 10463 and C. perfringens ATCC 13124. In addition, 100 clinical isolates (70 from the B. fragilis group and 30 Clostridium spp.) isolated in the last 5 years by the Udine General Hospital and by the University Hospital were tested. Clostridium spp. isolates included toxigenic C. difficile strains.16 All the isolates were subcultured from frozen stock cultures, stored at 80°C in 10% glycerol Brucella broth (Oxoid, Milan, Italy) vials, onto freshly made Brucella agar base (Oxoid) plates, supplemented with 5 mg/L of haemin (Sigma-Aldrich, Milan, Italy), 1 mg/L of vitamin K1 (Sigma-Aldrich) and 5% defibrinated sheep blood (Oxoid). Plates were incubated in anaerobic jars (Anaerogen system, Oxoid) at 37°C for 4872 h, and checked for purity. Colonies were picked up from plates and resuspended in WilkinsChalgren broth (Oxoid), supplemented with 5 mg/L haemin and 1 mg/L vitamin K1, for use in the antibiotic susceptibility assays.
Antimicrobial agents
SMAP-29 is a cathelicidin-derived peptide deduced from sheep myeloid mRNA, as previously reported.13 The peptide used in this study was synthesized chemically as a 28 residue peptide (RGLRRLGRKIAHGVKKYGPTVLRIIRIA), amidated at the C terminus, as indicated by the presence of C-terminal glycine, a common amidation signal in cathelicidin peptides.15 The correct peptide was obtained in >60% yield, and with a measured mass of 3198.0 ± 0.3 compared with a calculated mass of 3197.99 Da, and was homogeneous after preparative purification, as confirmed by mass spectrometry and analytical RP-HPLC.
Standard laboratory powders of amoxicillin, cefoxitin, clindamycin and tetracycline (Sigma Aldrich) were reconstituted according to NCCLS indications.17
Antimicrobial testing
Microdilution assays to establish MIC and MBC values of SMAP-29, as well as timekill assays to determine the kinetics of inactivation of selected strains by the peptide, were performed as recommended by the NCCLS17 and by reference protocols.18 However, in order to prevent dehydration of samples and to optimize viable cell counting, the final broth volume of each well was increased to 300 µL instead of 100 µL. Supplemented WilkinsChalgren broth was implemented in the assays using B. fragilis and Clostridium strains. For each strain, experiments were performed in duplicate.
Aliquots of bacterial suspensions were dispensed into microtitre plate wells; different amounts of SMAP-29 were added to each well, in order to obtain a serial concentration range (0.832 mg/L) (0.25 µM to 10 µM), except for growth control wells, which contained an equal amount of bacterial suspension and PBS instead of SMAP-29. Negative controls contained SMAP-29 at the highest concentration used without bacterial suspension in WilkinsChalgren broth. Microtitre plates were incubated at 37°C in anaerobic jars (Anaerogen system, Oxoid) for 2448 h. MICs were determined by visual reading of well turbidity, whereas MBCs were evaluated from the same test by viable counting assay: MBC was defined as the lowest concentration of peptide that killed 99.9% of the test inoculum.18
The killing kinetics of selected reference strains (B. fragilis NCTC 9343, C. perfringens ATCC 13124, C. difficile ATCC 10463) were performed on the basis of standard protocols.18 The experiments were performed in duplicate for each strain. Bacterial suspensions were obtained during the late logarithmic phase from WilkinsChalgren broth cultures and adjusted to 106107 cfu/mL. Aliquots were dispensed into microtitre plate wells, and SMAP-29 was added to the following final concentrations: 0.5 x MIC, 1 x MIC, 2 x MIC. At different time points (0, 2, 4, 6, 8 h), aliquots of samples were collected, briefly spun at 5200g three times (centrifuge mod.5810, Eppendorf, Milan, Italy), rinsed in PBS (to avoid antibiotic carry-over), serially diluted if needed to ensure accurate colony counting and cultured anaerobically on Brucella blood agar plates at 37°C for 2448 h. Data were analysed and expressed as log10 cfu/mL over time.
The antibiotic susceptibility of reference and clinical strains belonging to the B. fragilis group to amoxicillin, cefoxitin, clindamycin and tetracycline was determined by a broth microdilution method.17 Breakpoints for susceptibility, intermediate susceptibility and resistance of B. fragilis group isolates against antimicrobial agents other than SMAP-29 were based on NCCLS indications.17
Membrane permeabilization assay
The ability of SMAP-29 to permeabilize anaerobic bacterial membranes was tested using a two-colour fluorescence assay of bacterial viability (LIVE/DEAD BacLight Bacterial Viability Kit, Molecular Probes, Eugene, OR, USA). The method utilizes mixtures of a green fluorescent nucleic acid stain (SYTO 9) and propidium iodide (PI), a red fluorescent nucleic acid stain. As indicated by the manufacturer, SYTO 9 labels all bacteria in a population with intact membranes. Conversely, propidium iodide only penetrates damaged bacterial membranes. The presence of both SYTO 9 and PI, as revealed by a decrease in the SYTO 9 fluorescence with respect to intact cells, denotes membrane damage. This method has been reported previously.19,20
A 1:1 (v/v) mixture of the two dyes was used to monitor bacterial populations exposed to SMAP-29 during the killing-curve experiments. Aliquots of bacterial suspensions were taken, using the same peptide concentrations and interval time points as for the killing curves, washed three times with PBS and finally suspended in the same buffer. Final dilutions were performed to obtain an appropriate density for microscopic counts: 5.0 x 1056.5 x 105 cells/mL, which was estimated to be the ideal concentration range required for counting a mean value of 100 cells/optical field (total magnification: 650x)(Axioskop, Zeiss, Germany). Samples were exposed to a standard mixture of the two dyes, as indicated by the manufacturers protocol. A total amount of 10 µL of each sample was fixed on slides (fluorescence three-well slides, PBI, Milan, Italy) and examined under UV light (Axioskop, Zeiss). For each time point of observation, at least 25 different optical fields were considered, and a mean of 100 bacteria/optical field were counted. Values are expressed as mean percentage of red fluorescent bacteria versus mean percentage value of total amount of fluorescent cells, derived from multiple optical field counts and from at least duplicate experimental data sets.
Scanning electron microscopy
Late log phase cultures of B. fragilis NCTC 9343 and C. perfringens ATCC 13124 strains were resuspended at 1 x 1085 x 108 cfu/mL in 0.1 M Ca2+- and Mg2+-free PBS (pH 7.4) and exposed to SMAP-29 at the MIC value. Control samples were run in the same microwell plate, in the presence of PBS only. Samples were incubated in anaerobic jars at 37°C for 4 h; afterwards, bacterial suspensions were spun briefly at 5200g at room temperature, washed in PBS and finally resuspended in 0.5 mL of the same buffer. Cells were fixed with an equal volume of 5% glutaraldehyde (Sigma) at 4°C for 2 h, and filtered through Isopore filters (0.2 µm pore size; Millipore, Bedford, MA, USA). Filters were washed extensively with PBS, dehydrated with absolute ethanol, treated by gold coating and mounted on plates for SEM observation; samples were examined by a Leica Stereoscan (Deerfield, IL, USA) electron scanning microscope.
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Results |
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The in vitro activity of the antimicrobial peptide SMAP-29 against selected anaerobic species was evaluated using eight reference strains. These included Gram-negative (Bacteroides spp.) and Gram-positive spore-forming species (Clostridium spp.). Both the inhibitory (MIC) and the bactericidal (MBC) concentrations were determined. As reported in Table 1, SMAP-29 displayed antimicrobial activity against all the reference strains, with no appreciable differences between Gram-negative and -positive species. The MIC values ranged between 2.4 and 6.4 mg/L (0.752.0 µM). The MBCs were 1.5 to 2 x MICs, consistent with a bactericidal mechanism of action for SMAP-29. It is also important to note that the values are similar, or only slightly higher than those reported for a variety of aerobic bacteria, confirming the potency and broad spectrum of activity of SMAP-29.14
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The MICs at which 50% and 90% of 100 clinical isolates were inhibited by SMAP-29 (MIC50, MIC90), and the distribution of MICs for these organisms, are summarized in Table 2. MICs of 3.212.8 mg/L (14 µM) were determined for all Gram-negative spp. The B. fragilis isolates tested had been screened previously for tetracycline and macrolide resistance genes carried by chromosomal conjugal elements.21 MICs of SMAP-29 against Clostridium spp. were in the range 1.69.6 mg/L (0.53 µM), except for one C. perfringens strain isolated from a blood culture that required higher peptide concentrations (MIC of 19.2 mg/L) (6 µM) (Table 2).
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Of the 70 B. fragilis group isolates tested, 49 (70%) were found to be amoxicillin resistant, 28 (40%) clindamycin resistant, five (7%) cefoxitin resistant and 52 (74.3%) tetracycline resistant at breakpoint concentrations. The peptide efficiently inhibited both antibiotic-susceptible and -resistant B. fragilis group isolates (Table 3).
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Figure 1 shows the killing kinetics for SMAP-29 against three reference strains: B. fragilis NCTC 9343, C. perfringens ATCC 13124 and C. difficile ATCC 10463 (Figure 1). Bacterial viability was determined as cfu counts. In all cases, SMAP-29 was found to inhibit the bacterial growth at 0.5 x MIC, and was bactericidal at higher concentrations ( 1 x MIC) in a dose- and time-dependent manner. Particularly, at 2 x MIC,
corresponding to the MBC for all the species analysed, the peptide caused one and two log decreases of B. fragilis and C. difficile after 2 h incubation, respectively, with >105 organisms/mL killed at 8 h for C. difficile (Figure 1). Slower killing kinetics were observed for C. perfringens ATCC 13124, with a maximum colony decrease of about 3.5 log after 8 h incubation with SMAP-29 at 2 x MIC.
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Membrane permeabilization |
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To visualize the effects of SMAP-29 on anaerobes, scanning electrographs (at 800020 000x magnification) were obtained from the Gram-negative B. fragilis NCTC 9343 strain and the Gram-positive spore-forming C. perfringens ATCC 13124 strain, after treatment with the peptide (Figure 4). With both strains, control cells incubated in broth in the absence of the peptide exhibited a regular, smooth surface (Figures 4a, c), whereas cells incubated with SMAP-29 at concentrations corresponding to the MIC value revealed severe membrane damage consistent with disruption of the membrane integrity. In fact, B. fragilis cells exposed to 1 x MIC (6.4 mg/L) of SMAP-29 for 4 h, revealed large globular surface protrusions (Figure 4b), and C. perfringens cells exposed to the peptide for the same time length appeared wrinkled, with numerous small clefts regularly distributed on the bacterial cell surface (Figure 4d).
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Discussion |
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It is important to note that prior studies of the in vivo efficacy of SMAP-29 have shown that this peptide is well tolerated and efficient when inoculated in sheep lung in a model of acute pneumonia against the respiratory pathogen Mannheimia haemolytica.24 Furthermore, SMAP-29 has been shown to provide full protection to mice injected intraperitoneally with lethal concentrations of Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus and encapsulated E. coli.1 In this respect, it should be noted that B. fragilis group species are primarily involved in intra-abdominal abscess formation and in peritonitis.2 However, a low therapeutic index has been observed when the peptide was administered intravenously,1 and further structureactivity relationship studies are required to identify synthetic SMAP-29 analogues with reduced systemic toxicity.
Detailed studies of anti-anaerobe potential are only reported for a minority of other antimicrobial peptides. Pexiganan, a 22 amino acid analogue of the magainin peptides isolated from the skin of the African clawed frog, is one of the most extensively investigated. Pexiganan proved to be effective against a variety of Gram-negative and -positive anaerobic species at concentrations of 64 mg/L or less, and was most active (MIC90 8 mg/L) against Bacteroides spp. and Propionibacterium acnes.9 Cecropin-melittin analogues (CAMEL), containing portions of the amino acid sequences of the two peptides, were found to be active against anaerobic microorganisms, with an MIC90 of 4 mg/L against B. fragilis, fusobacteria, propionibacteria and peptostreptococci.10 In a structureactivity relationship study, 16 CAMEL analogues exhibited MIC90 in the range 232 mg/L against 60 clinical anaerobic strains. Interestingly, in the study by Oh et al.,11 the activity of the hybrid peptides was compared to the bactericidal activity of commonly used antibiotics, and found to be equal or superior to those of metronidazole, cefoxitin, ciprofloxacin and chloramphenicol, although inferior to those of imipenem, clindamycin and piperacillin. Among the cathelicidin peptides, the porcine protegrin PG-1 was reported to kill within 3060 min some reference strains of Gram-negative anaerobic periodontal pathogens, such as Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia, at concentrations in the range 550 mg/L.12 In a recent study, SMAP-29 has been shown to be active against an F. nucleatum reference strain, with an MBC value of 10 mg/L, but not against a P. gingivalis reference strain (MBC > 100 mg/L).8
Overall, these data support the therapeutic usefulness of SMAP-29 in topical applications, and encourage further investigations aimed to evaluate the suitability of SMAP-29, or of synthetic analogues of this peptide, for application in mixed and anaerobic human infections. The synergistic effects of natural antimicrobial peptides and ß-lactams, as shown by Darveau et al.,25 suggest the use of SMAP-29 in association with traditional antibiotic treatments.
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Acknowledgements |
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Footnotes |
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References |
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2 . Finegold, S. M. (1995). Anaerobic infections in humans: an overview. Anaerobe 1, 39.[ISI]
3 . Finegold, S. M., & Wexler, H. M. (1996). Present status of therapy for anaerobic infections. Clinical Infectious Diseases 23, Suppl. 1, 914.[ISI]
4 . Dublanchet, A., Caillon, J., Edmond, J. P. et al. (1986). Isolation of Bacteroides strains with reduced sensitivity to 5-nitroimidazoles. European Journal of Clinical Microbiology 5, 3467.
5 . Boman, H. G. (1998). Gene-encoded peptide antibiotics and the concept of innate immunity: an update review. Scandinavian Journal of Immunology 48, 1525.[CrossRef][ISI][Medline]
6 . Lehrer, R. I. & Ganz, T. (1999). Antimicrobial peptides in mammalian and insect host defense. Current Opinion in Immunology 11, 237.[CrossRef][ISI][Medline]
7
.
Travis, S. M., Anderson, N. N., Forsyth, W. R. et al. (2000). Bactericidal activity of mammalian cathelicidin-derived peptides. Infection and Immunity 68, 274855.
8
.
Guthmiller, J. M., Vargas, K. G., Srikantha, R. et al. (2001). Susceptibilities of oral bacteria and yeast to mammalian cathelicidins. Antimicrobial Agents and Chemotherapy 45, 321619.
9
.
Ge, Y., MacDonald, D. L., Holroyd, K. J. et al. (1999). In vitro antibacterial properties of pexiganan, and analog of magainin. Antimicrobial Agents and Chemotherapy 43, 7828.
10 . Edlund, C., Hedberg, M., Engstrom, A. et al. (1998). Antianaerobic activity of a cecropin-melittin peptide. Clinical Microbiology and Infection 4, 1815.[Medline]
11
.
Oh, H., Hedberg, M., Wade, D. et al. (2000). Activities of synthetic hybrid peptides against anaerobic bacteria: aspects of methodology and stability. Antimicrobial Agents and Chemotherapy 44, 6872.
12 . Miyasaki, K. T., Iofel, R., Oren, A. et al. (1998). Killing of Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia by protegrins. Journal of Periodontal Research 33, 918.[ISI][Medline]
13 . Bagella, L., Scocchi, M. & Zanetti, M. (1995). cDNA sequences of the three sheep myeloid cathelicidins. FEBS Letters 376, 2258.[CrossRef][ISI][Medline]
14 . Skerlavaj, B., Benincasa, M., Risso, A. et al. (1999). SMAP29: a potent antibacterial and antifungal peptide from sheep leukocytes. FEBS Letters 463, 5862.[CrossRef][ISI][Medline]
15 . Zanetti, M., Gennaro, R. & Romeo, D. (1995). Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Letters 374, 15.[CrossRef][ISI][Medline]
16 . Arzese, A., Trani, G., Riul, L. et al. (1995). Rapid polymerase chain reaction method for specific detection of toxigenic Clostridium difficile. European Journal of Clinical Microbiology and Infectious Diseases 14, 71619.[ISI][Medline]
17 . National Committee for Clinical Laboratory Standards. (2000). Methods for Antimicrobial Susceptibility Testing of Anaerobic BacteriaFifth Edition: Approved Standard M11-A5. NCCLS, Villanova, PA, USA.
18 . Hindler, J. (1992). Section 5: Antimicrobial susceptibility testing. In Clinical Microbiology Procedures Handbook (Isenberg, H. D., Ed.), pp. 133. American Society for Microbiology, Washington, D. C., USA.
19 . Bouls, L., Prevost, M., Barbeau, B. et al. (1997). LIVE/DEAD® BacLightTM: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. Journal of Microbiological Methods 37, 7786.[CrossRef]
20
.
Virta, M., Lineri, S., Kankaanpaa, P. et al. (1998). Determination of complement-mediated killing of bacteria by viability staining and bioluminescence. Applied Environmental Microbiology 64, 51519.
21 . Arzese, A. & Botta, G. A. (1996). Detection of tetracycline resistance factors in Gram-negative anaerobes of human origin: involvement of horizontal transfer by Bacteroides conjugative transposons. In Proceedings of the XII European Meeting on Bacterial Gene Transfer and Expression, Siena, I, 1996. Abstract p. 72. Università degli Studi di Siena, Siena, Italy.
22 . Gennaro, R. & Zanetti, M. (2000). Structural features and biological activities of the cathelicidin-derived antimicrobial petides. Biopolymers 55, 3149.[CrossRef][ISI][Medline]
23 . Botta, G. A., Arzese, A., Minisini, R. et al. (1994). Role of structural and extracellular virulence factors in gram-negative anaerobic bacteria. Clinical and Infectious Diseases 18, Suppl. 4, 2604.
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.
Brogden, K. A., Kalfa, V. C., Ackermann, M. R. et al. (2001) The ovine cathelicidin SMAP29 kills ovine respiratory pathogens in vitro and in an ovine model of pulmonary infection. Antimicrobial Agents and Chemotherapy 45, 3314.
25 . Darveau, R. P., Cunningham, M. D., Seachord, C. L. et al. (1991). Beta-lactam antibiotics potentiate magainin 2 antimicrobial activity in vitro and in vivo. Antimicrobial Agents and Chemotherapy 35, 11539.[ISI][Medline]
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