In vitro activity of citropin 1.1 alone and in combination with clinically used antimicrobial agents against Rhodococcus equi

Andrea Giacometti1,*, Oscar Cirioni1, Wojciech Kamysz2, Carmela Silvestri1, Maria Simona Del Prete1, Alberto Licci1, Giuseppina D'Amato1, 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


* Correspondence address. Institute of Infectious Diseases and Public Health, c/o Ospedale Regionale, via Conca 71, Ancona I-60020, Italy. Tel: +39-071-5963715; Fax: +39-071-5963468; E-mail: anconacmi{at}interfree.it

Received 9 November 2004; returned 8 February 2005; revised 21 May 2005; accepted 6 June 2005


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Objectives: The aim of this study was to investigate the in vitro activity of citropin 1.1, an antimicrobial peptide derived from the Australian tree frog Litoria citropa, alone and in combination with ampicillin, ceftriaxone, doxycycline, netilmicin, ciprofloxacin, rifampicin, linezolid, vancomycin, clarithromycin and imipenem against 12 nosocomial isolates of Rhodococcus equi.

Methods: Antimicrobial activity of citropin 1.1 was measured by MIC, MBC, time–kill studies and chequerboard titration method.

Results: All isolates were inhibited at concentrations of citropin 1.1 between 2 and 8 mg/L. Combination studies demonstrated synergy only when the peptide was combined with clarithromycin, doxycycline and rifampicin.

Conclusions: Our findings show that citropin 1.1 is active against R. equi and that its activity could be enhanced when it is combined with hydrophobic antibiotics.

Keywords: Gram-positive cocci , antimicrobial peptides , synergy , antibiotics


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Rhodococcus equi, an aerobic, facultative intracellular, Gram-positive, acid-fast coccobacillus, is a well-known pathogen in domestic animals, especially horses, which causes suppurative bronchopneumonia with a high mortality rate in young foals.1,2 R. equi has also emerged as a significant opportunistic pathogen in immunocompromised hosts, especially those infected with the HIV. Clinical reports have shown that, despite antibiotic therapy, frequent relapses occur during the course of the disease.1,2 Although combined therapy with erythromycin and rifampicin has dramatically improved the survival rate in foals, this treatment regimen is not without problems for the recently reported emergence of rifampicin resistance in R. equi.13

In the last few years many positively charged polypeptides have been isolated from a wide range of animal, plant and bacterial species.4 The dual hydrophobic and hydrophilic nature of these molecules is important for their initial interaction with the bacterial membrane, which may allow entry of several substrates inside the cell.5

The citropins are antimicrobial peptides isolated from the dorsal glands of Australian tree frogs. They possess a broad spectrum of biological activity and some are specific to certain pathogens. Citropin 1.1 is a small peptide (16 residues) produced by both the dorsal and submental glands of the Australian green tree frog Litoria citropa. It has a significant antimicrobial, anticancer and nitric oxide synthetase activities.6

The aim of the present study was to evaluate the in vitro activity of citropin 1.1 and its bactericidal effect for several R. equi strains, as well as to investigate its in vitro interaction with 10 clinically used antibiotics.


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

Twelve nosocomial isolates of R. equi were tested. The isolates were obtained from distinct patients from Central Italy with unrelated sources of infection and admitted to the Hospital Umberto I, Ancona Italy, from January 1990 to December 2003. The strains were identified according to the following criteria: Gram-positive coccobacillary non-spore-forming non-motile organisms, weak or partial acid fastness, mucoid colonies, salmon-pink/coral coloured colonies, inability to ferment carbohydrates, inability to hydrolyse casein, hypoxanthine, xanthine, tyrosine and gelatin, catalase positive, urease positive, oxidase negative, nitrate reduction positive, and alkaline phosphatase positive. The identification was confirmed by means of the API Strep systems (bioMérieux Italia, Italy). The different API codes and susceptibility patterns stated the independence of all isolates.

Antimicrobial agents

Citropin 1.1 (GLFDVIKKVASVIGGL-NH2) was synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase method and was purified by HPLC on a Knauer K501 two-pump system with a Kromasil C8 column 10 x 250 mm (5 µm particle diameter, 100 Å pore size) with a flow rate of 5 mL/min and gradient 20–50% A/90 min (A = 0.1% TFA in acetonitrile, B = 0.1% aqueous TFA), absorbance at 226 nm. The resulting fractions with purity greater than 97–98% were tested by HPLC (Kromasil C8 column, 4.6 x 150 mm). The peptide was analysed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). Ampicillin, ceftriaxone, doxycycline, netilmicin, rifampicin and vancomycin (all from Sigma-Aldrich, Milan, Italy), clarithromycin (Abbott, Rome, Italy), ciprofloxacin (Bayer, Milan, Italy), imipenem (Merck, Sharp & Dohme, Milan, Italy), and linezolid (Pharmacia & Upjohn, Kalamazoo, MI, USA) were tested as control agents.

MIC and MBC determinations

Laboratory standard powders were diluted in accordance with manufacturers' recommendations. Solutions of drugs were made fresh on the day of assay or stored at –80°C in the dark for no longer than 2 weeks. The concentration range assayed for citropin 1.1 was 0.125–64 mg/L, and 0.250–256 mg/L for the other antimicrobial agents.

MICs and MBCs were assayed according to the procedures outlined by the NCCLS.7 Experiments were performed in triplicate. Values are presented as the median of each triplicate.

Bacterial killing assay

All clinical isolates were used to study the in vitro killing effect of citropin 1.1. Aliquots of exponentially growing bacteria were resuspended in fresh MH broth at ~107 cells/mL and exposed to citropin 1.1 at 2 x MIC for 0, 5, 10, 15, 20, 25, 30, 40, 50 and 60 min at 37°C. After these times, 0.1 mL samples were serially diluted in 10 mM sodium HEPES buffer (pH 7.2) to minimize the carryover effect and plated onto MH agar plates to obtain viable colonies. The limit of detection for this method was ~10 cfu/mL.

Synergy studies

In interaction studies, six strains of R. equi 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 citropin 1.1 and 0.25–256 mg/L for clinically used antibiotics. The fractionary 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 Experiments were performed in triplicate. FIC values are presented as the median of each triplicate.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
All isolates were inhibited by citropin 1.1 at concentrations between 2 and 8 mg/L. Interestingly, it showed MBC ranges of 2–16 mg/L. Comparative evaluation of other antimicrobial agents did not show important differences in their activity. In fact, all antimicrobial agents were highly active against at least 90% of R. equi isolates, with exhibited MICs less or similar to those of citropin 1.1. However, most of the control agents showed MBCs much higher than their MICs. The results are summarized in Table 1. Killing by citropin 1.1 was shown to be very rapid: its activity on bacterial isolates was complete after 20 min exposure period at a concentration of 2 x MIC.


View this table:
[in this window]
[in a new window]
 
Table 1. MICs and MBCs of citropin 1.1 and other clinically used antibiotics for 12 R. equi clinical isolates

 
In interaction studies synergy was observed only for combinations between citropin 1.1 and rifampicin, doxycycline or clarithromycin. For each of the six strains FIC indices were <0.5. Actually, median values of 0.385, 0.385 and 0.312 were observed by testing citropin 1.1 combined with rifampicin, doxycycline and clarithromycin, respectively. In contrast, the other experiments with the other antibiotics gave values between 1.167 and 1.833.

Polymorphonuclear cells and monocytes/macrophages represent the first defence line against invading microorganisms, but although R. equi can be susceptible to neutrophil-mediated killing, it is able to resist innate macrophage defences and establishes residence within the intracellular environment of that phagocyte.13 The ability to replicate within the macrophage is associated with virulence, and correlates in animals with the possession of a large plasmid and expression of the plasmid-encoded and surface-expressed lipoprotein. Despite the good in vitro activities of traditional antibiotics, therapy is often partially effective and relapses occur during the course of the disease.2,3 For this reason, new strategies based on novel molecular targeting agents are warranted to further improve the long-term outcome of R. equi infection treatment. It is well known that the major killing mechanism of the neutrophils is based on the cationic peptides associated with azurophilic granules of these dedicated antimicrobial phagocytes.4,9 Accordingly, it may be useful to investigate the therapeutic strategy of combining clinically used antibiotics with peptides against bacteria able to infect macrophages.

Our data demonstrate that citropin 1.1 is active against R. equi and show a rapid bactericidal effect. These findings confirm the activity of naturally derived peptides against this organism, as already described by our group.10 Combination studies showed that it exhibited positive interaction with hydrophobic antibiotics. Previous reports demonstrated that polycationic peptides present properties of synergy with lipophilic agents such as rifampicin, macrolides, fusidic acid and novobiocin. It is a complex mechanism that probably involves the peptide-induced entrance of large lipophilic molecules into the cell. The cationic peptides, by triggering the activity of bacterial murein hydrolases, may cause degradation of the peptidoglycan and have direct membrane-permeabilizing activity with maximal entry of hydrophobic compounds. Several cationic antimicrobial peptides are known to interact with bacterial membranes, making the outer protective shield more permeable.4,9 It is possible that the synergic interaction is a result of a combined effect of increased access to the intracellular target for clarithromycin, doxycycline or rifampicin, and secondary effects of the peptides themselves. A hypothesis including increased uptake and accessibility to the target, combined with drugs acting on a common pathway, can explain the observed synergy for hydrophobic antibiotics. In fact, other mechanisms may be involved in this interaction: it has been demonstrated that cationic peptides by disintegrating the biological membranes yield to uncoupling of the oxidative respiration.4

In conclusion, our data suggest that citropin 1.1, alone or in combination with conventional antibiotics, may be a promising agent for the management of R. equi infections.


    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. Beaman BL, Saubolle MA, Wallace RJ. Nocardia, Rhodococcus, Streptomyces, Oerskovia, and other aerobic actinomycetes of medical importance. In: Murray PR, Baron EJ, Pfaller MA et al., eds. Manual of Clinical Microbiology, 6th edn. Washington, DC: American Society for Microbiology, 1995; 379–99.

2. Hodalus MK. Pathogenesis and virulence of Rhodococcus equi. Vet Microbiol 1997; 56: 257–68.[CrossRef][ISI][Medline]

3. Asoh N, Watanabe H, Fines-Guyon M et al. Emergence of rifampicin-resistant Rhodococcus equi with several types of mutations in the rpoB gene among AIDS patients in northern Thailand. Antimicrob Agents Chemother 2003; 41: 2337–40.

4. Hancock REW, Scott MG. The role of antimicrobial peptides in animal defences. Proc Natl Acad Sci USA 2000; 97: 8856–61.[Abstract/Free Full Text]

5. Vaara M, Porro M. Group of peptides that act synergistically with hydrophobic antibiotics against Gram-negative enteric bacteria. Antimicrob Agents Chemother 1996; 40: 1801–5.[Abstract]

6. Wabnitz PA, Bowie JH, Wallace JC et al. The antibiotic citropin peptides from the Australian tre frog Litoria citropa. Structure determination using electrospray mass spectrometry. Rapid Commun Mass Spectrom 1999; 13: 1724–32.[CrossRef][ISI][Medline]

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

8. Odds FC. Synergy, antagonism and what the chequerboard puts between them. J Antimicrob Chemother 2003; 52: 1.[CrossRef][ISI][Medline]

9. Cudic M, Otvos L Jr. Intracellular targets of antibacterial peptides. Curr Drug Targets 2002; 3: 101–6.[ISI][Medline]

10. Giacometti A, Cirioni O, Ancarani F et al. In vitro activity of polycationic peptides alone and in combination with clinically used antimicrobial agents against Rhodococcus equi. Antimicrob Agents Chemother 1999; 43: 2093–6.[Abstract/Free Full Text]





This Article
Abstract
Full Text (PDF)
All Versions of this Article:
56/2/410    most recent
dki236v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Disclaimer
Request Permissions
Google Scholar
Articles by Giacometti, A.
Articles by Scalise, G.
PubMed
PubMed Citation
Articles by Giacometti, A.
Articles by Scalise, G.