a Institute of Infectious Diseases and Public Health, University of Ancona, c/o Azienda Ospedaliera Umberto I, Piazza Cappelli 1, Ancona I-60121; b Department of Infectious Diseases, San Salvatore Hospital, Pesaro, Italy
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Abstract |
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Introduction |
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One way to overcome the problems of the emergence of resistance is to use new antimicrobial compounds and/or combination therapy. In the last few years many polypeptides have been isolated from a wide range of animal, plant and bacterial species.4 Recent reports hypothesize that these compounds cross the outer membrane of Gram-negative bacteria via the self-promoted uptake pathway. The initial step in this process should be a high-affinity binding of the peptide to surface lipopolysaccharide (LPS), causing the displacement of divalent cations that stabilize adjacent LPS molecules.6 Additionally, this event may lead to self-promoted uptake of the destabilizing compound across the outer membrane and subsequent channel formation in the cytoplasmic membrane, resulting in cell death. Finally, it has been shown that the peptides may act by inserting into the cytoplasmic membrane and triggering the activity of bacterial murein hydrolases, resulting in damage or degradation of the peptidoglycan and lysis of the cell.7
In this study we investigated the in vitro activity of buforin II, cecropin P1, indolicidin, magainin II and ranalexin alone and in combination with eight clinically used antimicrobial agents against 12 multidrug-resistant nosocomial isolates of A. baumannii.
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Materials and methods |
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The quality control strain A. baumannii ATCC 19606 and 12 nosocomial isolates of A. baumannii were tested. They were isolated from distinct patients with unrelated sources of infection over a 5 year period.
Antimicrobial agents
Buforin II, cecropin P1, indolicidin, magainin II and ranalexin were obtained from SigmaAldrich (Milan, Italy). The in vitro activity of various antibiotics was also evaluated: chloramphenicol, netilmicin and ofloxacin (all from SigmaAldrich), co-amoxiclav (SmithKline Beecham, Milan, Italy), aztreonam (Menarini, Firenze, Italy), ceftazidime (GlaxoWellcome, Verona, Italy), meropenem (Zeneca, Rome, Italy) and piperacillin (WyethLederle, Aprilia, Italy). The range of concentrations of polycationic peptides tested was 0.12564 mg/L, and of the other antimicrobial agents was 0.25256 mg/L.
MIC and MBC determinations
The MIC of polycationic peptides was determined using a microbroth dilution method with MuellerHinton (MH) broth (Becton Dickinson Italia, Milan, Italy) and an initial inoculum of 5 x 105 cfu/mL. Polypropylene 96-well plates (SigmaAldrich) were incubated for 18 h at 37°C in air. The MIC was defined as the lowest peptide concentration that reduced growth by more than 50% of that in the control well.8 The number of viable organisms in each well was determined by performing 106 dilutions and plating 10 µL of each dilution on to MH agar plates and incubating overnight. The MBC was determined by plating out the contents of the wells showing no visible growth of bacteria on to MH agar plates and incubating at 37°C for 18 h. The MBC was defined as the lowest concentration of each drug that prevented any residual colony formation.8 Experiments were performed in triplicate.
The MIC of the other antibiotics was determined by a microbroth dilution method according to the procedures outlined by the National Committee for Clinical Laboratory Standards.9 Polystyrene 96-well plates (Becton Dickinson and Co., Franklin Lakes, NJ, USA) were incubated for 18 h at 35°C in air. The MIC was taken as the lowest drug concentration at which observable growth was inhibited. The MBC was taken as the lowest concentration of each drug that resulted in more than 99.9% reduction of the initial inoculum. Experiments were performed in triplicate.
Bacterial killing assay
To study the in vitro killing effect of the peptides the control strain ATCC 19606 and two representative strains of A. baumanii, Ab-03.96 and Ab-02.98, were selected. The former was the most susceptible to all the peptides, the second the least susceptible. Aliquots of exponentially growing bacteria were resuspended in fresh MH broth at c. 107 cells/mL and exposed to each peptide at 4 x MIC for 0, 5, 10, 15, 20, 25, 30, 40, 50 or 60 min at 37°C. After these times samples were diluted serially in 10 mM sodium HEPES buffer (pH 7.2) to minimize the carryover effect and plated on to MH agar plates to obtain viable colonies.
Synergy studies
In interaction studies, the three strains mentioned above were used to test the antibiotic combinations by a chequerboard titration method using 96-well polypropylene microtitre plates. Chloramphenicol, co-amoxiclav, aztreonam, netilmicin, ofloxacin, piperacillin, ceftazidime and meropenem were tested in combination with each peptide at a temperature of 35°C. The ranges of drug dilutions used were 0.12564 mg/L for peptides and 0.25256 mg/L for clinically used antibiotics. The fractional inhibitory concentration (FIC) index for combinations of two antimicrobials was calculated according to the equation: FIC index = FICA + FICB = A/MICA + B/MICB, where A and B are the MICs of drug A and drug B in the combination, MICA and MICB are the MICs of drug A and drug B alone, and FICA and FICB are the FICs of drug A and drug B. The FIC indexes were interpreted as follows: 0.5, synergy; >0.5 to 1.0, addition; >1.0 to <4.0, indifference; and
4.0, antagonism.
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Results and discussion |
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Combination studies showed that magainin II acted synergically with ß-lactams. The mechanism of this positive interaction remains largely unknown, even though it might be caused by increased access of the ß-lactam antibiotics to the cytoplasmic membrane following breakdown of peptidoglycan by magainin II.
Magainins are produced by the African clawed frog Xenopus laevis. They are -helical ionophores that dissipate ion gradients in cell membranes, causing lysis.10 The helical, amphiphilic structure is responsible for their affinity for membranes. It has been shown that an increase in their concentration caused the artificial lipid bilayer thickness to decrease, suggesting adsorption within the head-group region of the lipid bilayer. Moreover, magainin II is non-haemolytic and this property may result from a peptidecholesterol interaction in mammalian membranes that inhibits the formation of peptide structure capable of lysis. The powerful antibacterial activity and the synergic interactions demonstrated between magainin II and ß-lactam antibiotics against a selected panel of an important contemporary multidrug-resistant Gram-negative pathogen make the cationic peptides potentially valuable as an adjuvant for antimicrobial chemotherapy. Nevertheless, very few in vivo studies of cationic peptide action have been published and there are unanswered concerns about in vivo efficacy and unknown toxicities. Future research towards these objectives based on animal models is needed.
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Notes |
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References |
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Hancock, R. E. W. & Chapple, D. S. (1999). Peptide antibiotics. Antimicrobial Agents and Chemotherapy 43, 131723.
7 . Hancock, R. E. W. (1997). Antibacterial peptides and the outer membranes of gram-negative bacilli. Journal of Medical Microbiology 46, 13.[ISI][Medline]
8 . Hancock, R. E. W. (1998). Hancock Laboratory: Methods. Department of Microbiology and Immunology, University of British Columbia, British Columbia, Canada. [Online.] http://www.interchg.ubc.ca/bobh/MIC.htm. [29 September 1998, last date accessed by author.]
9 . National Committee for Clinical Laboratory Standards. (1993). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A3. NCCLS, Villanova, PA.
10 . Zasloff, M. (1987). Magainins, a class of antimicrobial peptides from Xenopus skin: isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proceedings of the National Academy of Sciences of the USA 84, 544953.[Abstract]
Received 27 March 2000; returned 12 June 2000; revised 23 June 2000; accepted 5 July 2000