JAC Advance Access originally published online on April 24, 2006
Journal of Antimicrobial Chemotherapy 2006 58(1):95-100; doi:10.1093/jac/dkl145
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Studies on the antimicrobial activity of cecropin A–melittin hybrid peptides in colistin-resistant clinical isolates of Acinetobacter baumannii
1 Service of Infectious Diseases, Hospitales Universitarios Virgen del Rocío Avda. Manuel Siurot s/n, 41013 Sevilla, Spain; 2 Service of Emergency and Critical Care, Hospitales Universitarios Virgen del Rocío Avda. Manuel Siurot s/n, 41013 Sevilla, Spain 3 Centro de Investigaciones Biológicas (CSIC), C/ Ramiro de Maeztu 9 28040 Madrid, Spain 4 Departament of Experimental and Health Sciences, Universitat Pompeu Fabra Dr Aiguader 80, 08003 Barcelona, Spain 5 Service of Microbiology, Hospitales Universitarios Virgen del Rocío Avda. Manuel Siurot s/n, 41013 Sevilla, Spain 6 Department of Microbiology, School of Medicine University of Sevilla, Apdo. 914, 41080 Sevilla, Spain
*Corresponding author. Tel/Fax: +34-955012376; E-mail: jeronimopachon{at}telefonica.net
Received 27 October 2005; returned 17 February 2006; revised 9 March 2006; accepted 27 March 2006
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Objectives: Acinetobacter baumannii has successfully developed resistance against all common antibiotics, including colistin, one of the last active drugs against this pathogen. We have tested whether the differences in lethal mechanism between polymyxin B and the cecropin A–melittin hybrid peptide CA(1-8)M(1-18), shown previously with a colistin-susceptible strain, can be exploited as a new chemotherapeutic alternative against colistin-resistant clinical isolates. Furthermore, the effect of capsule on the bactericidal activity of cecropin A–melittin analogues (CAMs) was tested.
Methods: MICs and MBCs of the four CAMs were determined for 13 clinical isolates. The bactericidal acti-vity of the antimicrobial peptides was measured using time–kill curves. The presence or absence of capsule was determined using Indian ink stain.
Results: The MIC ranges of CA(1-8)M(1-18) and three of its shortened analogues, namely CA(1-7)M(2-9), its N
-terminal octanoylated analogue and CA(1-7)M(5-9), for A. baumannii strains were 2–8, 2–4, 2–8 and 4–4 mg/L, respectively. MBCs differed by a factor of two at the most. All of the cecropin A–melittin peptides showed bactericidal activity in time–kill curves against four A. baumannii strains. The bactericidal activity of CAMs was not affected by the presence of capsule.
Conclusions: These results indicate that this class of peptides has a fast microbicidal effect on the colistin-resistant A. baumannii isolates, regardless of considerable structural variation among the four peptides and varying colistin MIC for the strains included in the study. Overall, the cecropin A–melittin peptides appear to be a promising alternative to overcome polymyxin resistance in A. baumannii.
Keywords: colistin resistance , A. baumannii , polymyxin , bactericidal activity , capsule
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Introduction |
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Acinetobacter baumannii is a Gram-negative bacillus that acts as an opportunistic pathogen, causing severe nosocomial infections with high mortality rates.1–3 Furthermore, A. baumannii has shown an outstanding capacity to develop resistance against the common antibiotics, including carbapenems and other extended-spectrum ß-lactams, tetracyclines, fluoroquinolones and aminoglycosides, through a variety of mechanisms.4–6 This has led to an impressive reduction in the available active antibiotics, including imipenem, which was until recently the gold standard for the treatment of A. baumannii infections.7,8 This systematic reduction during the past years has left polymyxins as the only alternative treatment.9 The use of colistin as monotherapy against A. baumannii has shown success with sepsis,10,11 with meningitis and ventriculitis,9,12–15 and in blood clearance in a rabbit model of endocarditis.16 Recently, reports of sporadic outbreaks of colistin-resistant A. baumannii5,17,18 have prompted the search for new antimicrobial agents, including eukaryotic antibiotic peptides.19–22
Antimicrobial peptides are key components of the innate immunity in all multicellular organisms.23–26 Expectations about their use as new powerful antibacterial agents have been raised on the basis of their mechanism of action, which involves the permeation of the bacterial membranes and thus possible reduced likelihood of emergence of resistance.27–30 A significant number of antimicrobial peptides have been tested in vitro against different species of the genus Acinetobacter.20,22,31,32 More recently, one of the diastereomers of the synthetic peptide K4L7 was successful in the treatment of an experimental murine bacteraemia caused by Acinetobacter.33 Some studies have also addressed the activity of these peptides against multiresistant Acinetobacter isolates.19,20,22,32 Many of these belong to the group of cecropin A–melittin peptides, formed by hybridization of the N-terminal sequences of cecropin A and melittin, with an improved microbicidal spectrum relative to the parental peptides.34 In a previous work,22 we demonstrated that the lethal mechanisms in Acinetobacter for these peptides and polymyxin B differed in their interaction with the inner membrane of Acinetobacter. Also, in a strain resistant to polymyxin B and colistin, other antimicrobial peptides such as rBPI21 and cecropin P1 showed an MIC lower than that of polymyxin B and were active in bactericidal assays.5
The aim of this study was to determine the activity of four selected cecropin A–melittin analogues (CAMs) against colistin-resistant clinical isolates of A. baumannii, as a first step towards the in vivo use of these peptides as an alternative to polymyxins.
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Bacterial strains
A total of 13 clinical pan-resistant A. baumannii strains, with resistance to cefotaxime, ceftazidime, imipenem, amoxicillin, amikacin, piperacillin/tazobactam, doxycycline and colistin, were collected from clinical isolates at the Virgen del Rocío University Hospitals, Sevilla, Spain.
A. baumannii was identified by Gram stain appearance, colony morphology, motility, cytochrome oxidase reaction and growth at 44°C, as well as by the semiautomated MicroScan Walk Away method (Dade-Behring, West Sacramento, CA, USA). All the strains were confirmed as A. baumannii by amplified ribosomal rRNA gene restriction analysis (ARDRA).35 The isolates were stored at –70°C until required.
Antimicrobials and other reagents
Reagents of the highest purity available were purchased from Sigma (Madrid, Spain) or Merck (Darmstadt, Germany).
The peptides used in this study included four CAMs, named according to the parental peptides (CA and M for cecropin A and melittin, respectively), followed by the stretch of their sequence included in the corresponding hybrid. Four peptides were assayed: CA(1-8)M(1-18) [KWKLFKKIGIGAVLKVLTTGLPALIS-NH2] (MW = 2793.78), with 26 amino acids,36 and three shortened analogues, namely CA(1-7)M(2-9) [KWKLFKKIGAVLKVL-NH2] (MW = 1770.19),37 its N-terminal octanoylated analogue [Oct-KWKLFKKIGAVLKVL-NH2] (MW = 1898.5)38 and CA(1-7)M(5-9) [KWKLFKKVLKVL-NH2] (MW = 1544.07), the shortest analogue. CAMs were synthesized by solid phase chemical synthesis, using Fmoc standard protocols, except for the octanoylated analogue, which was synthesized as described previously.38 CAMs were purified by reverse phase HPLC, characterized by MALDI–TOF mass spectrometry and quantified by tryptophan fluorescence and amino acid analysis.38 Purity was higher than 98%.
Standard laboratory powder of colistin (C-4461; Sigma, St Louis, MO, USA) was used and prepared according to the guidelines of the CLSI.39
Capsule stain
A capsule stain technique was used to confirm the presence of capsule in the A. baumannii strains.40 Indian ink was used to provide a dark background against which the shapes of unstained cells are clearly visible. Crystal violet was used to stain the bacteria. Because the capsule does not stain with either of the two dyes, the capsule appears as a transparent halo between the purple colour of the bacteria and the dark background.
In vitro activity of the cecropin A–melittin peptides against A. baumannii
MICs and MBCs of colistin and the four CAMs were determined in duplicate for the A. baumannii strains by the broth microdilution method.39 Mueller–Hinton II Broth Cation Adjusted (MHBCA; Becton Dickinson, Cockeysville, MD, USA) was used as the growth medium and an initial inoculum of 5 x 105 cfu/mL at exponential growth phase was used.39 MIC was defined as the lowest concentration of antimicrobial agent that completely inhibits growth of the organism in the microdilution wells as detected by the unaided eye. MBC was defined as the lowest concentration of drug that resulted in >99.9% reduction of the initial inoculum. Initial inoculum and bacterial growth were determined by serial dilutions in physiological serum and subculture in Columbia III agar with 5% sheep blood and incubation at 37°C for 20 h. The concentrations of antimicrobials ranged from 0.06 to 128 mg/L. Due to the absence of a standard control strain for synthetic peptides, Pseudomonas aeruginosa ATCC 27853, A. baumannii ATCC 19606, Escherichia coli ATCC 25922 and Ac157, a pan-resistant A. baumannii strain, previously tested with CAMs and generously provided by Professor M. López-Brea (Microbiology Department, La Princesa University Hospital, Madrid, Spain), were used as control strains. A strain with an MIC 
2 mg/L was considered susceptible to colistin and one with an MIC 
4 mg/L was considered resistant.41,42
Time–killing curves
The bactericidal activity of the CAMs was measured in 4 of the 13 strains using the time–killing method. The 208628 and 201630 strains were used, because they showed different MICs of colistin (64 and 8 mg/L, respectively) and MICs of the CAMs in the low range. The 183280 strain, with rough (183280R) and smooth (183280S) varieties, was selected to evaluate the influence of a morphological characteristic on the antimicrobial activity.
Aliquots of 20 mL of MHBCA and an initial inoculum of 5 x 105 cfu/mL of each A. baumannii strain were prepared for the time–killing curves. CAM concentrations of 1-, 2- and 4-fold the MIC for each strain were employed. Bacterial growth was measured at 0, 2, 4, 8 and 24 h after incubation, plating 10-fold dilutions on Columbia III agar with 5% sheep blood and incubating at 37°C for 20 h. Tubes with bacterial inoculum but without the antimicrobial were used as growth controls, and tubes without bacterial inoculum and antimicrobial were used as sterility controls. An antibiotic was considered bactericidal when a reduction of 3 log10 cfu/mL compared with the initial inoculum concentration was achieved.43,44
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Capsule stain
The A. baumannii smooth variety (183280S) and the other two strains (208628 and 201630) showed a transparent halo around a purple cell confirming the presence of capsule. In contrast the rough variety (183280R) did not show the transparent halo, confirming the absence of capsule.
In vitro activity of colistin and the cecropin A–melittin peptides against A. baumannii
MICs and MBCs of colistin and the four CAMs are shown in Table 1. CA(1-7)M(2-9) was the CAM that showed the lowest MIC50 (2 mg/L), with an MIC range of 2–4 mg/L, and CA(1-8)M(1-18) showed the highest MIC90 (8 mg/L), with an MIC range of 2–8 mg/L for the 13 A. baumannii tested strains (Table 1).
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MICs and MBCs of peptides for the selected strains (208628, 201630, 183280R and 183280S) are shown in Table 2. In general, the CA(1-7)M(2-9) and Oct-CA(1-7)M(2-9) peptides showed the lowest MICs and MBCs for these strains.
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Time–killing curves
All of the CAMs showed bactericidal activity at any of the used concentrations in the time–killing curves against the four selected A. baumannii strains (Figure 1). This activity was higher and longer in time when an increasing concentration of CAM in relation to the MIC was used. Thus, in general regrowth occurred at MIC and 2x MIC of the four peptides for the 208628 and 201630 strains. Also, there was regrowth at MIC of all peptides, except CA(1-7)M(5-9), for the 183280S and 183280R strains. CA(1-8)M(1-18) showed the highest bactericidal activity against the four tested strains. As a general rule, the results of time–killing curves showed rather good correlation with those for MBC, with exceptions for CA(1-7)M(2-9) against strains 208628 and 201630, for Oct-CA(1-7)M(2-9) against strain 208628 and for CA(1-7)M(5-9) against 201630 when assayed at their respective MBC. The bactericidal activities for the CAMs against the 183280S and 183280R strains were very similar.
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The four CAMs were active at micromolar concentrations against the set of A. baumannii strains tested, regardless of their respective MICs of colistin. The MIC and MBC for a given isolate and peptide were similar or with only one dilution difference. Similar null or very scarce variation between these two parameters was also reported for peptides rBPI121 and cecropin P1 tested against another polymyxin-resistant A. baumannii.5 The killing curves showed a fast decrease in the number of viable bacteria and, in more than 50% of cases, reached the maximal killing activity at 4x MIC, with a decrease of more than 4 log10 cfu/mL even after 24 h of incubation. This demonstrated a remarkable bactericidal behaviour, in agreement with previous results obtained in multidrug-resistant strains of A. baumannii with these analogues19,22,32 or other antimicrobial peptides.31,45 In some cases, this activity was observed at concentrations higher than MBC. This discordance between MBC and bactericidal activity evaluated by time–killing curves has been also observed in other studies with colistin.46 Time–killing curves showed that the bactericidal effect of CAMs was higher and longer-acting when the concentrations increased, which suggests that, similar to colistin, the most important pharmacokinetic/pharmacodynamic parameter in its activity is the relation Cmax/MIC.16,47
No differences between the 183280R and 183280S strains in MIC, MBC and time–killing curve results were found. Morphological differences among strains depend on the presence or absence of the capsule, which offers protection against phagocytosis, and allows differentiation in serological types of some species such as Streptococcus pneumoniae.48 However, the presence of capsule in the genus Acinetobacter is not taxonomically discriminating or a virulence element49 and the present work also shows that it is not a resistance element against the CAMs. Interestingly, the presence of capsule in Klebsiella pneumoniae50 or A-layer in Aeromonas salmonicida51 acts as a shield against a wide variety of antimicrobial peptides.
Cecropin A–melittin hybrid peptides have been extensively studied as bactericidal agents,34 and some of them have been tested against Acinetobacter.19,20,22 In the present work the four CAMs displayed very similar bactericidal activities against A. baumannii. Remarkably, in a previous work on the pseudomonicidal activities of a large set of CAMs, a lower activity was associated with peptides with shorter sequences,52 a trend not confirmed in the reduced set of CAMs tested in this study. The N-terminally modified Oct-CA(1-7)M(2-9) showed that acylation did not improve the activity against Acinetobacter, in agreement with our previous results against E. coli.38 On the other hand, this same analogue has shown an improved leishmanicidal effect, and acylation of other antimicrobial peptides has increased their antimicrobial and antiendotoxic activities relative to the non-acylated analogue.45,53 A more detailed study on acylation will be required in order to unveil the parameters involved in this discrepant behaviour.
Whether the cecropin A–melittin peptides will be a good clinical alternative against the increasing threat of a widespread dissemination of pan-resistant A. baumannii, including colistin, is a subject for further studies. The potentiality of these peptides as an antimicrobial template relies on the large number of modifications available to improve their activity and tolerability. In fact, recently, a diastereomer of an artificial antimicrobial peptide was tested successfully in a murine bacteraemia model.33 In addition, the design of these peptides as cecropin A–melittin hybrids has very favourable effects on cytotoxicity (usually monitored as haemolytic activity), since the cluster of basic residues 21–24 associated with this activity in melittin is avoided. In vitro assays of haemolytic activity for CA(1-8)M(1-18) and CA(1-7)M(2-9) showed a full lack of effect at concentrations below 200 µM.37,54 In our hands, CA(1-7)M(5-9), the shortest analogue, followed a similar trend; in contrast, Oct-CA(1-7)M(2-9), the only N-acylated peptide in this study, at 10 µM induced 45% of the haemolysis levels produced by 0.1% Triton X-100 (L. Rivas, data not shown). Furthermore, several cecropin A–melittin peptides have been safely tested in vivo in mice,55,56 rabbit57 and dog58 models, in the latter case, with the mildly cytotoxic Oct-CA(1-7)M(2-9) analogue also assayed in this work.
Furthermore cecropin A–melittin peptides have shown a very low level of induction of resistance, including those mechanisms triggered by the antibiotic peptide itself.59 Besides, other facets of combination therapy, such as their synergy with ß-lactams,32 may further reduce the threat of induction of resistance against these peptides, a point that, though still controversial, has been advocated by some authors.59–61
To investigate these peptides further, we are currently testing the tolerability and the bactericidal activity of these CAMs in an animal model of colistin-resistant Acinetobacter sepsis.
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No declarations were made by the authors of this paper.
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This work was supported by research grants from the Consejería de Salud de la Junta de Andalucia (41/02 to J. P.), Spain, by the Spanish Network for the Research in Infectious Diseases (ISCIII, C03/14 to J. P. and L. R.), Spain, by the Fondo de Investigación Sanitaria, Spain (FIS PI04/0827, PI04/0624 and PI04/0885 to L. R., J. P. and D. A., respectively) and by the CICyT (BIO2002-04091-C03-01 and BIO2003-09056-CO2-O2 to D. A. and L. R., respectively).
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1 Cisneros JM and Pachón J. (2003) Acinetobacter baumannii: a nosocomial pathogen difficult to control. Enferm Infecc Microbiol Clin 21:221–3.[Medline]
2 Cisneros JM, Reyes MJ, Pachón J, et al. (1996) Bacteremia due to Acinetobacter baumannii: epidemiology, clinical findings, and prognostic features. Clin Infect Dis 22:1026–32.[Web of Science][Medline]
3 Seifert H, Strate A, Pulverer G. (1995) Nosocomial bacteremia due to Acinetobacter baumannii. Clinical features, epidemiology, and predictors of mortality. Medicine (Baltimore) 74:340–9.[CrossRef][Medline]
4
Ribera A, Fernández-Cuenca F, Beceiro A, et al. (2004) Antimicrobial susceptibility and mechanisms of resistance to quinolones and ß-lactams in Acinetobacter genospecies 3. Antimicrob Agents Chemother 48:1430–2.
5
Urban C, Mariano N, Rahal J, et al. (2001) Polymyxin B-resistant Acinetobacter baumannii clinical isolate susceptible to recombinant BPI21 and cecropin P1. Antimicrob Agents Chemother 45:994–5.
6 Van Looveren M and Goossens H. (2004) Antimicrobial resistance of Acinetobacter spp. in Europe. Clin Microbiol Infect 10:684–704.[CrossRef][Web of Science][Medline]
7 Gallego L, Canduela MJ, Sevillano E, et al. (2004) Carbapenemase detection in Acinetobacter baumannii clones resistant to imipenem. Enferm Infecc Microbiol Clin 22:262–6.[Medline]
8
Jain R and Danziger LH. (2004) Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians. Ann Pharmacother 38:1449–59.
9 Jiménez-Mejías ME, Becerril B, Márquez-Rivas FJ, et al. (2000) Successful treatment of multidrug-resistant Acinetobacter baumannii meningitis with intravenous colistin sulfomethate sodium. Eur J Clin Microbiol Infect Dis 19:970–1.[CrossRef][Web of Science][Medline]
10 Markou N, Apostolakos H, Koumoudiou C, et al. (2003) Intravenous colistin in the treatment of sepsis from multiresistant Gram-negative bacilli in critically ill patients. Crit Care 7:R78–83.[CrossRef][Web of Science][Medline]
11 Sarria JC, Angulo-Pernett F, Kimbrough RC, et al. (2004) Use of intravenous polymyxin B during continuous venovenous hemodialysis. Eur J Clin Microbiol Infect Dis 23:340–1.[CrossRef][Web of Science][Medline]
12 Fernández-Viladrich P, Corbella X, Corral L, et al. (1999) Successful treatment of ventriculitis due to carbapenem-resistant Acinetobacter baumannii with intraventricular colistin sulfomethate sodium. Clin Infect Dis 28:916–7.[Web of Science][Medline]
13 Jimenez-Mejias ME, Pichardo-Guerrero C, Marquez-Rivas FJ, et al. (2002) Cerebrospinal fluid penetration and pharmacokinetic/pharmacodynamic parameters of intravenously administered colistin in a case of multidrug-resistant Acinetobacter baumannii meningitis. Eur J Clin Microbiol Infect Dis 21:212–4.[CrossRef][Web of Science][Medline]
14 Siegman-Igra Y, Bar-Yosef S, Gorea A, et al. (1993) Nosocomial Acinetobacter meningitis secondary to invasive procedures: report of 25 cases and review. Clin Infect Dis 17:843–9.[Web of Science][Medline]
15
Vasen W, Desmery P, Ilutovich S, et al. (2000) Intrathecal use of colistin. J Clin Microbiol 38:3523.
16 Rodriguez-Hernandez MJ, Jimenez-Mejias ME, Pichardo C, et al. (2004) Colistin efficacy in an experimental model of Acinetobacter baumannii endocarditis. Clin Microbiol Infect 10:581–4.[CrossRef][Medline]
17 Manikal VM, Landman D, Saurina G, et al. (2000) Endemic carbapenem-resistant Acinetobacter species in Brooklyn, New York: citywide prevalence, interinstitutional spread, and relation to antibiotic usage. Clin Infect Dis 31:101–6.[CrossRef][Web of Science][Medline]
18
Henwood CJ, Gatward T, Warner M, et al. (2002) Antibiotic resistance among clinical isolates of Acinetobacter in the UK, and in vitro evaluation of tigecycline (GAR-936). J Antimicrob Chemother 49:479–87.
19 Alarcón T, López-Hernández S, Andreu D, et al. (2001) In vitro activity of CA(1-8)M(1-18), a synthetic cecropin A-melittin hybrid peptide, against multiresistant Acinetobacter baumannii strains. Rev Esp Quimioter 14:184–90.[Medline]
20 Giacometti A, Cirioni O, Kamysz W, et al. (2003) Comparative activities of cecropin A, melittin, and cecropin A-melittin peptide CA(1-7)M(2-9)NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii. Peptides 24:1315–8.[CrossRef][Medline]
21 Rivas L and Andreu D. (2003) Eukaryotic antibiotic peptides: a new alternative in clinical practice? Enferm Infecc Microbiol Clin 21:358–65.[Medline]
22
Saugar JM, Alarcón T, López-Hernández S, et al. (2002) Activities of polymyxin B and cecropin A-melittin peptide CA(1-8)M(1-18) against a multiresistant strain of Acinetobacter baumannii. Antimicrob Agents Chemother 46:875–8.
23 Bowdish DM, Davidson DJ, Hancock REW. (2005) A re-evaluation of the role of host defence peptides in mammalian immunity. Curr Protein Pept Sci 6:35–51.[CrossRef][Web of Science][Medline]
24 Jerala R and Porro M. (2004) Endotoxin neutralizing peptides. Curr Top Med Chem 4:1173–84.[CrossRef][Medline]
25
Yeaman MR and Yount NY. (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55:27–55.
26 Reddy KV, Yedery RD, Aranha C. (2004) Antimicrobial peptides: premises and promises. Int J Antimicrob Agents 24:536–47.[CrossRef][Web of Science][Medline]
27 Calandra T. (2001) Pathogenesis of septic shock: implications for prevention and treatment. J Chemother 13:173–80.
28 David SA, Awasthi SK, Balaram P. (2000) The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides. J Endotoxin Res 6:249–56.[CrossRef]
29 Guerra AN, Fisette PL, Pfeiffer ZA, et al. (2003) Purinergic receptor regulation of LPS-induced signaling and pathophysiology. J Endotoxin Res 9:256–63.[CrossRef]
30 Hancock REW and Lehrer R. (1998) Cationic peptides: a new source of antibiotics. Trends Biotechnol 16:82–8.[CrossRef][Web of Science][Medline]
31
Ge Y, MacDonald DL, Holroyd KJ, et al. (1999) In vitro antibacterial properties of pexiganan, an analog of magainin. Antimicrob Agents Chemother 43:782–8.
32
Giacometti A, Cirioni O, Del Prete MS, et al. (2000) Comparative activities of polycationic peptides and clinically used antimicrobial agents against multidrug-resistant nosocomial isolates of Acinetobacter baumannii. J Antimicrob Chemother 46:807–10.
33
Braunstein A, Papo N, Shai Y. (2004) In vitro activity and potency of an intravenously injected antimicrobial peptide and its DL amino acid analog in mice infected with bacteria. Antimicrob Agents Chemother 48:3127–9.
34 Rivas L and Andreu D. (2003) Cecropin-melittin hybrid peptides as versatile templates in the development of membrane active antibiotics agents. In Menestrina G and Dalla Serra M (Eds.). Pore-Forming Peptides and Protein Toxins. Reading, Berkshire: Harwood Academic Publishers pp. 215–59.
35 Vaneechoutte M, Dijkshoorn L, Tjernberg I, et al. (1995) Identification of Acinetobacter genomic species by amplified ribosomal DNA restriction analysis. J Clin Microbiol 33:11–5.[Abstract]
36
Boman HC, Boman IA, Andreu D, et al. (1989) Chemical synthesis and enzymic processing of precursor forms of cecropins A and B. J Biol Chem 264:5852–60.
37 Andreu D, Ubach J, Boman A, et al. (1992) Shortened cecropin A-melittin hybrids. Significant size reduction retains potent antibiotic activity. FEBS Lett 296:190–4.[CrossRef][Web of Science][Medline]
38
Chicharro C, Granata C, Lozano R, et al. (2001) N-terminal fatty acid substitution increases the leishmanicidal activity of CA(1-7)M(2-9), a cecropin-melittin hybrid peptide. Antimicrob Agents Chemother 45:2441–9.
39 Clinical and Laboratory Standards Institute. (2005) Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically—Sixth Edition. (CLSI, Wayne, PA).
40
Richardson WP and Sadoff JC. (1977) Production of a capsule by Neisseria gonorrhoeae. Infect Immun 15:663–4.
41 Clinical and Laboratory Standards Institute. (2005) Performance Standards for Antimicrobial Susceptibility Testing: Fifteenth International Supplement. (CLSI, Wayne, PA, USA).
42 Anon. (2000) Recomendaciones del grupo MENSURA para la selección de antimicrobianos en el estudio de la sensibilidad y criterios para la interpretación del antibiograma. Rev Esp Quimioter 13:73–86.[Medline]
43 Lorian V. (1996) Antimicrobial combinations. In Lorian V (Ed.). Antibiotics in Laboratory Medicine (Williams and Wilkins, MD, USA) pp. 339–41.
44 National Committee for Clinical Laboratory Standards. (1999) Methods for Determining Bactericidal Activity of Antimicrobial Agents: Approved Standard M26-A. (NCCLS, Wayne, PA, USA).
45 Avrahami D and Shai Y. (2003) Bestowing antifungal and antibacterial activities by lipophilic acid conjugation to D,L-amino acid-containing antimicrobial peptides: a plausible mode of action. Biochemistry 42:14946–56.[CrossRef][Medline]
46
Montero A, Ariza J, Corbella X, et al. (2002) Efficacy of colistin versus ß-lactams, aminoglycosides, and rifampin as monotherapy in a mouse model of pneumonia caused by multiresistant Acinetobacter baumannii. Antimicrob Agents Chemother 46:1946–52.
47 Beringer P. (2001) The clinical use of colistin in patients with cystic fibrosis. Curr Opin Pulm Med 7:434–40.[CrossRef][Medline]
48 Musher DM. (2005) Streptococcus pneumoniae. In Mandell D, Bennett JE, Dolin R (Eds.). Principles and Practice of Infectious Diseases 6th edn (Elsevier, Philadelphia, USA) pp. 2392–411.
49
Garcia A, Solar H, Gonzalez C, et al. (2000) Effect of EDTA on the resistance of clinical isolates of Acinetobacter baumannii to the bactericidal activity of normal human serum. J Med Microbiol 49:1047–50.
50
Campos MA, Vargas MA, Regueiro V, et al. (2004) Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides. Infect Immun 72:7107–14.
51 Henry MA and Secombes CJ. (2000) The A-layer influences the susceptibility of Aeromonas salmonicida to antibacterial peptides. Fish Shellfish Immunol 10:637–42.[CrossRef][Medline]
52
Scott MG, Yan H, Hancock REW. (1999) Biological properties of structurally related -helical cationic antimicrobial peptides. Infect Immun 67:2005–9.
53
Wakabayashi H, Matsumoto H, Hashimoto K, et al. (1999) N-Acylated and D enantiomer derivatives of a nonamer core peptide of lactoferricin B showing improved antimicrobial activity. Antimicrob Agents Chemother 43:1267–9.
54 Boman HG, Wade D, Boman IA, et al. (1989) Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids. FEBS Lett 259:103–6.[CrossRef][Web of Science][Medline]
55 Gough M, Hancock REW, Kelly NM. (1996) Antiendotoxin activity of cationic peptide antimicrobial agents. Infect Immun 64:4922–7.[Abstract]
56
Friedrich C, Scott MG, Karunaratne N, et al. (1999) Salt-resistant alpha-helical cationic antimicrobial peptides. Antimicrob Agents Chemother 43:1542–8.
57 Nos-Barbera S, Portolés M, Morilla A, et al. (1997) Effect of hybrid peptides of cecropin A and melittin in an experimental model of bacterial keratitis. Cornea 16:101–6.[Medline]
58
Alberola J, Rodríguez A, Francino O, et al. (2004) Safety and efficacy of antimicrobial peptides against naturally acquired leishmaniasis. Antimicrob Agents Chemother 48:641–3.
59 McPhee JB, Lewenza S, Hancock REW. (2003) Cationic antimicrobial peptides activate a two-component regulatory system, PmrA-PmrB, that regulates resistance to polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol Microbiol 50:205–17.[CrossRef][Web of Science][Medline]
60 Devine DA and Hancock REW. (2002) Cationic peptides: distribution and mechanisms of resistance. Curr Pharm Des 8:703–14.[CrossRef][Medline]
61 Hancock RE. (2003) Concerns regarding resistance to self-proteins. Microbiology 49:3343–5.
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