In Vitro Activity of the Histatin Derivative P-113 against Multidrug-Resistant Pathogens Responsible for Pneumonia in Immunocompromised Patients

Organisms.

Clinical isolates of P. aeruginosa, methicillin-resistant S. aureus, and S. maltophilia (20 strains for each species) cultured from sputum, bronchoalveolar lavage, or blood derived from immunocompromised patients with pneumonia were tested. All isolates were considered responsible for nosocomially acquired infections since the positive specimen cultures were obtained more than 48 h after admission and there was no evidence of infection at the time of admission. The isolates were obtained from distinct patients coming from central Italy with unrelated sources of infection and admitted to the Hospital Umberto I, Ancona, Italy, from January 2001 to December 2003. Identification of the strains was performed according to standard procedures. The identification was confirmed by means of the API 20 systems (bioMérieux Italia, Italy). The different API codes and susceptibility patterns stated the independence of all isolates. S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 were used as quality control strains.

Synthetic peptides and antimicrobial agents.

P-113 (AKRHHGYKRKFH-NH₂) was synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry according to the following procedure. (i) Five- and 15-min deprotection steps were performed with 20% piperidine in the mixture N,N-dimethylformamide (DMF)-N-methyl-2-pyrrolidone (NMP)(1:1 [vol/vol]) in the presence of 1% Triton. (ii) The coupling reactions were carried out with the protected amino acid diluted in a mixture DMF-NMP (1:1 [vol/vol]) in the presence of 1% Triton, using N,N′-diisopropylocarbodiimide (DIC) as the coupling reagent in the presence of 1-hydroxybenzotriazole (HOBt) (Fmoc-AA-DIC-HOBt at 1:1:1) for 1 h. The completeness of each coupling reaction was monitored by the chloranil test (4, 7). The protected peptidyl resin was treated with a mixture of 95% trifluoroacetic acid, 2.5% water, and 2.5% triisopropylsilane (TIS) for 1 h. After cleavage, the solid support was removed by filtration, and the filtrate was concentrated under reduced pressure. The cleaved peptide was precipitated with diethyl ether and lyophilized. P-113 was purified by solid-phase extraction (SPE) on Kromasil sorbent (C₈, 5 μm, 100 Å) by the protocol described in reference 12. The resulting fractions with purity greater than 97 to 98% were tested by high-performance liquid chromatography. The peptide was analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The peptide was solubilized in phosphate-buffered saline (pH 7.2), yielding a 1-mg/ml stock solution. Solutions of drugs were made fresh on the day of assay or stored at −80°C in the dark for short periods.

For comparison, the in vitro activities of the following antibiotics were evaluated: amikacin, vancomycin, and colistin sulfate from Sigma-Aldrich; clarithromycin from Abbott, Rome, Italy; ciprofloxacin from Bayer, Milan, Italy; ceftazidime from GlaxoSmithKline, Verona, Italy; imipenem from Merck, Sharp & Dohme, Milan, Italy; and piperacillin-tazobactam from Wyeth-Lederle, Aprilia, Italy. Laboratory standard powders were diluted in accordance with the manufacturers' recommendations, yielding a 1-mg/ml stock solution. Stock solutions of these antimicrobial drugs were stored at −80°C until they were used. The concentration range assayed for each antibiotic was 0.125 to 128 μg/ml.

MIC and MBC determinations.

The MIC was determined by a broth microdilution method with Mueller-Hinton (MH) broth (Becton Dickinson Italia, Milan, Italy) and an initial inoculum of 5 × 10⁵ CFU/ml, according to the procedures outlined by the National Committee for Clinical Laboratory Standards (15). Polystyrene 96-well plates (Becton Dickinson and Co., Franklin Lakes, N.J.) were incubated for 18 h at 37°C in air. When peptides were tested, since they have a tendency to precipitate, plates were shaken throughout the study. The MIC was taken as the lowest drug concentration at which observable growth was inhibited. The minimal bactericidal concentration (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.

All organisms were grown at 37°C in MH broth. Aliquots of exponentially growing bacteria were resuspended in fresh MH broth at approximately 10⁷ cells/ml and separately exposed to each peptide at two times the MIC for 0, 2, 5, 10, 20, 30, 40, 50, and 60 min at 37°C. After these times, samples were serially diluted and plated onto MH agar plates to obtain viable colonies. The limit of detection for this method was approximately 10 CFU/ml.

Synergy studies.

In interaction studies, 18 strains (six for each species) were used to test the antibiotic combinations by a checkerboard titration method using 96-well polypropylene microtiter plates. The ranges of drug dilutions used were 0.125 to 64 μg/ml for P-113 and 0.25 to 256 μg/ml for clinically used antibiotics. The fractionary inhibitory concentration (FIC) index for combinations of two antimicrobials was calculated according to the equation FIC index = FIC_A + FIC_B = A/MIC_A + B/MIC_B, where A and B are the MICs of drugs A and B in the combination, MIC_A and MIC_B are the MICs of drug A and drug B alone, and FIC_A and FIC_B are the FICs of drug A and drug B. The FIC indices were interpreted as follows: <0.5, synergy; 0.5 to 4.0, indifferent; and >4.0, antagonism (6).

MIC and MBC results.

All gram-negative organisms were inhibited by P-113 at concentrations of 0.5 to 8 μg/ml. In particular, S. maltophilia was more susceptible than P. aeruginosa, for which the MIC ranges were 0.5 to 4 and 1 to 8 μg/ml, respectively. In contrast, against the S. aureus clinical isolates, the peptide exhibited a MIC range of 2 to 16 μg/ml.

Overall, high rates of resistance to the clinically used antibiotics were demonstrated. The results are summarized in Table 1.

Bacterial killing assay.

Killing by P-113 was shown to be very rapid: its activity against the gram-negative organisms was complete after a 10-min exposure period at a concentration of two times the MIC. On the other hand, the peptide exhibited a slower bactericidal effect on S. aureus. Figure 1 summarizes the results obtained from the quality control strains S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 and the multiresistant clinical isolate S. maltophilia 02.02. These results were similar for all other strains.

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FIG. 1.

Time-kill kinetics of P-113 against the quality control strains S. aureus ATCC 43300 and P. aeruginosa ATCC 27853 and the multiresistant clinical isolate S. maltophilia 02.02. The results were similar for all other strains.

Synergy studies.

In the combination studies, relevant differences were detected between the two control strains. In detail, when S. aureus ATCC 43300 was tested, synergy was never observed, even if indifference was demonstrated between P-113 and clarithromycin. In contrast, FIC indices of 0.312 were observed by testing P-113 combined with ceftazidime, tazobactam-piperacillin, and imipenem against P. aeruginosa ATCC 27853, while the combination between the two peptides P-113 and colistin showed an indifferent effect. The results are summarized in Table 2.

Our data demonstrate that P-113 has a powerful bactericidal effect on multiresistant clinical isolates of P. aeruginosa and S. maltophilia: against these pathogens, its activity is comparable to that of colistin sulfate, a well-characterized antimicrobial peptide. Nevertheless, different from colistin, P-113 exhibited good activity against methicillin-resistant strains of S. aureus too. Although the differences in the antimicrobial spectra between the two peptides cannot be explained on the basis of current knowledge, our results point out two potential therapeutic uses of P-113.

First, data from synergy studies suggest that it could be usefully administered in combinations with beta-lactam antibiotics to treat severe gram-negative infections. As mentioned above, the cationic peptides allow maximal entry of several substrates inside the cell: the synergistic interaction with beta-lactam antibiotics could be due to their increased passage through the outer bacterial membrane. On the other hand, peptides and beta-lactams may have a common target: it had been hypothesized that cationic peptides might render bacteria nonviable by activating their autolytic wall enzymes, such as muramidases (8).

Second, like colistin, P-113 could have potential for use in inhalant antibacterial treatment because of its strong activity against the most important multiresistant pathogens responsible for severe respiratory infections in immunocompromised patients. Since the worldwide emergence of antimicrobial resistance is an increasing problem in human medicine, the antimicrobial peptides are promising compounds: several studies have shown that peptide-resistant mutants emerge rarely because of their mechanism of action, which primarily involves the chemical and physical structure of the biological membrane. For this reason, they represent a conserved theme in host antimicrobial defenses throughout nature. Today the increasing amount of data concerning the antibacterial activity of polycationic peptides makes these agents potentially valuable as an adjuvant for antimicrobial chemotherapy.