In vitro activity and killing effect of temporin A on nosocomial isolates of Enterococcus faecalis and interactions with clinically used antibiotics

Andrea Giacometti1, Oscar Cirioni1, Wojciech Kamysz2, Giuseppina D'Amato1, Carmela Silvestri1, Maria Simona Del Prete1, Alberto Licci1, Jerzy Lukasiak2 and Giorgio Scalise1

1 Institute of Infectious Diseases and Public Health, Università Politecnica delle Marche, Ancona, Italy; 2 Faculty of Pharmacy, Medical University of Gdansk, Gdansk, Poland


* Corresponding author. Tel: +39-071-5963715; Fax: +39-071-5963468; Email: anconacmi{at}interfree.it

Received 30 June 2004; returned 11 September 2004; revised 28 September 2004; accepted 19 November 2004


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objective: To study the in vitro activity of temporin A, a basic, highly hydrophobic, antimicrobial peptide amide derived from the skin of the European red frog Rana temporaria, alone and in combination with co-amoxiclav, imipenem, ciprofloxacin, linezolid and vancomycin, against 42 nosocomial isolates of Enterococcus faecalis. Fourteen of these were resistant to vancomycin.

Methods: Antimicrobial activity of temporin A was measured by MIC, MBC and time–kill studies and by the chequerboard titration method.

Results: All isolates were inhibited at concentrations of 1 to 16 mg/L. Combination studies carried out with E. faecalis ATCC 29212 and ATCC 51299 demonstrated synergy only when the peptide was combined with co-amoxiclav and imipenem.

Conclusions: Our findings show that temporin A is active against E. faecalis and that its activity could be enhanced when it is combined with other antimicrobial agents.

Keywords: antimicrobial peptides , Gram-positive infections , susceptibility , antibiotic combinations , enterococci


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Enterococci are often involved in nosocomial infections and outbreaks often originate in ICUs. In the last decade they have demonstrated an increasing frequency of multidrug resistance, including high-level resistance to aminoglycosides, penicillins, chloramphenicol, tetracycline and glycopeptides. In particular, glycopeptide resistance among enterococci has become increasingly common in many European countries and in North America.1,2 Most enterococcal infections are caused by Enterococcus faecalis, which causes 80–90% of human enterococcal infections and is more likely to be resistant even to antibiotics of last resort.2 The problem of the emergence of resistance demands an increased effort to search for antimicrobial compounds with new mechanisms of action.1,2

Antimicrobial cationic peptides are a new class of antibiotic with a unique mechanism of action. They are part of an innate immune system that is widely distributed in nature and that has been found in many different organisms including animal, plant and bacterial species.3,4

Temporins are a family of linear 10–13 residue cationic peptides isolated from the skin of the European red frog Rana temporaria.5 They are among the smallest antimicrobial peptides, amidated at the C terminus. Those containing one basic residue, either lysine or arginine, in the sequence (net charge +2) were found to be active specifically against Gram-positive bacteria and Candida albicans. Temporin A is a basic, highly hydrophobic, antimicrobial peptide amide (FLPLIGRVLSGIL-NH2) that has variable antibiotic activity against a broad spectrum of microorganisms. There are currently different hypotheses concerning the mechanism of action by which temporins kill organisms: insertion into the hydrophobic core of the cell membrane, interaction with anionic heads and hydrocarbon tails of bacterial phospholipids, binding to DNA or altering enzyme activities.5,6

The aim of this study was to evaluate the in vitro activity of temporin A and its bactericidal effect over time for a large number of E. faecalis hospital isolates, including vancomycin-resistant (VR) strains, as well as to investigate its in vitro interaction with five clinically used antibiotics.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Organisms

The quality control strains E. faecalis ATCC 29212 and ATCC 51299 and 42 nosocomial isolates of E. faecalis were tested. Fourteen isolates exhibited resistance to vancomycin. The isolates were obtained from patients coming from Central Italy with unrelated sources of infection and admitted to the Hospital Umberto I, Ancona, Italy, from January 2000 to December 2003. The strains were cultured from surgical wound infections (23 strains), urine (13) and blood (6).

Antimicrobial agents

Temporin A was synthesized manually by the solid phase method using the Fmoc/But procedure (Faculty of Pharmacy, Medical University of Gdansk, Poland) and was purified by reversed-phase (Vydac C-18, 10 x 250 mm) high-pressure liquid chromatography (HPLC) on a Knauer K501 two-pump system. The product was analysed by HPLC, chemical analysis, and matrix-assisted laser-desorption ionization mass spectrometry (MALDI-TOF). Temporin A was dissolved in distilled H2O at 20 times the required maximum concentration. Successively, serial dilutions of the peptide were prepared in 0.01% acetic acid containing 0.2% bovine serum albumin in polypropylene tubes.

In addition, vancomycin (Sigma–Aldrich, Milan, Italy), co-amoxiclav (GlaxoSmithKline, Verona, Italy), ciprofloxacin (Bayer, Milan, Italy), linezolid (Pharmacia & Upjohn, Kalamazoo, MI, USA) and imipenem (Merck, Sharp & Dohme, Milan, Italy) were tested as control agents.

MIC and MBC determinations

Laboratory standard powders were diluted in accordance with the manufacturers' recommendations. Solutions of drugs were made fresh on the day of assay or stored at –80 °C in the dark for up to 2 weeks.

MICs were assayed at 5 x 105 cfu/mL on Mueller–Hinton (MH) broth by the microbroth dilution method according to the procedures outlined by the National Committee for Clinical Laboratory Standards.7 Polypropylene 96-well plates (Sigma–Aldrich) were incubated for 18 h at 37 °C in air and, since several peptides 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 MBC was taken as the lowest concentration of each drug that resulted in more than 99.9% reduction of the initial inoculum. Experiments were carried out in triplicate.

Bacterial killing assay

The control strains E. faecalis ATCC 29212 and ATCC 51299 were used to study the in vitro killing effect of temporin A. Exponentially growing bacteria were resuspended in fresh MH broth at approximately 107 cells/mL and exposed to temporin A at 2x MIC for 0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min at 37 °C. After these times, samples were serially diluted in 10 mM of sodium HEPES buffer (pH 7.2) to minimize the carryover effect and plated onto MH agar plates to obtain viable colonies.

Synergy studies

The control strains E. faecalis ATCC 29212 and ATCC 51299 were used to test the antibiotic combinations by a chequerboard titration method using 96-well polypropylene microtitre plates. The ranges of drug dilutions used were: 0.125–64 mg/L for temporin A and 0.25–256 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; >0.5–4.0, no interaction; and >4.0, antagonism.8


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All isolates were inhibited by temporin A at concentrations of 1–16 mg/L. In particular, both the vancomycin-susceptible (VS) and the vancomycin-resistant strains showed MIC50 of 4 mg/L and MIC90 of 8 mg/L. For the control strains ATCC 29212 and ATCC 51299, the peptide exhibited MICs of 2 and 4 mg/L, and MBCs of 4 and 8 mg/L, respectively. High rates of resistance to ß-lactams and ciprofloxacin were demonstrated by VRE, whereas all isolates were susceptible to linezolid. The results are summarized in Table 1.


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Table 1. MICs and MBCs of temporin A and five clinically used antibiotics for 42 clinical isolates of Enterococcus faecalis

 
Killing by temporin A was shown to be very rapid: its activity on the control strains was complete after a 20–25 min exposure period at a concentration of 2x MIC.

In the combination studies, no relevant difference was detected between the two control strains. Synergy was never observed, with the exception of the combinations between temporin A and ß-lactams. Actually, FIC indexes of 0.312 were observed by testing temporin A combined with co-amoxiclav and imipenem, whereas the other experiments with ciprofloxacin, vancomycin and linezolid gave values between 0.750 and 2.0 (Table 2).


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Table 2. Results of interaction studies between temporin A and other drugs

 
This study emphasizes the importance of the search for alternative antimicrobial agents with activity against nosocomial isolates of E. faecalis. Since standard therapies for enterococcal infections are based on combinations of antimicrobial agents, there is great interest in combining drugs to improve the spectrum of their activity.1,2 In this study, in vitro experiments with temporin A were carried out to determine its bactericidal activity and whether synergy, antagonism, or indifference would be the predominant response when the combinations with other antibiotics used clinically were tested against E. faecalis.

Temporin A was equally active against both VSE and VRE clinical isolates. Time–killing studies have shown a rapid bactericidal effect, even if the inactivation of E. faecalis appears to be slower than that observed for Gram-negative bacilli.9 One can hypothesize that the much thicker cell wall of the Gram-positive organism, formed by multilayer peptidoglycan and teichoic acid, could slow the approach of the peptide to the cytoplasmic membrane resulting in slower killing activity.

Combination studies showed that temporin A acted synergically with co-amoxiclav and imipenem. The mechanism of this positive interaction remains largely unknown, even though it might be due to the contemporaneous effect of temporin A and the ß-lactams on peptidoglycan. In addition, some peptides are thought to inhibit synthesis of DNA, RNA and cellular proteins and to be able to insert themselves into the cytoplasmic membrane triggering the activity of bacterial murein hydrolases and leading to degradation of the peptidoglycan with lysis of the cell.3,4 In addition, temporin A might increase the access of the ß-lactams to the cytoplasmic membrane following breakdown of peptidoglycan while, on the other hand, it is possible that the ß-lactams create new sites in the biological membranes for peptide entry.

In conclusion, temporin A has been shown here to have powerful in vitro activity against both VS and VR E. faecalis isolates and synergic interactions with ß-lactam antibiotics. These characteristics make this peptide potentially valuable for the future as an adjuvant for antimicrobial therapy for enterococcal infections, although a recent report described a weak activity of temporin A against VR strains of Enterococcus faecium.10 Nevertheless, the clinical applications of cationic peptides are hard to interpret due to the lack of pharmacokinetic and safety studies with these compounds: they are currently being tested as such in humans for a number of indications, but today very few in vivo pharmacokinetic studies have been published and there are unanswered concerns about their in vivo efficacy and toxicity. Future research towards these objectives and based on animal models may resolve these issues.


    Acknowledgements
 
This work was supported by the Italian Ministry of Education, University and Research (PRIN 2003).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Cormican, M. G. & Jones, R. N. (1996). Emerging resistance to antimicrobial agents in Gram-positive bacteria. Enterococci, staphylococci and nonpneumococcal streptococci. Drugs 51, S6–S12.

2 . Diekema, D. J., BootsMiller, B. J., Vaughn, T. E. et al. (2004). Antimicrobial resistance trends and outbreak frequency in United States hospitals. Clinical Infectious Diseases 38, 78–85.[CrossRef][ISI][Medline]

3 . Hancock, R. E. W. (2000). Cationic antimicrobial peptides: towards clinical applications. Expert Opinion on Investigational Drugs 9, 1723–9.[ISI][Medline]

4 . Hancock, R. E. W. & Chapple, D. S. (1999). Peptide antibiotics. Antimicrobial Agents and Chemotherapy 43, 1317–23.[Free Full Text]

5 . Simmaco, M., Mignogna, G., Canofeni, S. et al. (1996). Temporins, antimicrobial peptides from the European red frog Rana temporaria. European Journal of Biochemistry 242, 788–92.[Abstract]

6 . Zhao, H., Rinaldi, A. C., Di Giulio, A. et al. (2002). Interactions of the antimicrobial peptides temporins with model biomembranes. Comparison of temporins B and L. Biochemistry 41, 4425–36.[CrossRef][ISI][Medline]

7 . National Committee for Clinical Laboratory Standards. (2003). Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically: Approved Standard M7-A6. NCCLS, Villanova, PA, USA.

8 . Odds, F. C. (2003). Synergy, antagonism and what the chequerboard puts between them. Journal of Antimicrobial Chemotherapy 52, 1.[ISI][Medline]

9 . Cirioni, O., Giacometti, A., Ghiselli, R. et al. (2004). Potential therapeutic role of histatins derivative P-113D in experimental rat models of Pseudomonas sepsis. Journal of Infectious Diseases 190, 356–64.[CrossRef][ISI][Medline]

10 . Harjunpaa, I., Kuusela, P., Smoluch, M. T. et al. (1999). Comparison of synthesis and antibacterial activity of temporin A. FEBS Letters 449, 187–90.[CrossRef][ISI][Medline]





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