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Journal of Antimicrobial Chemotherapy (2000) 46, 807–810 ... aInstitute of Infectious Diseases and Public Health, University of Ancona, c/o Azienda ..... European Journal of. Clinical Microbiology and Infectious Diseases 18, 292–5. 4. Ling ...

JAC

Journal of Antimicrobial Chemotherapy (2000) 46, 807–810

Comparative activities of polycationic peptides and clinically used antimicrobial agents against multidrug-resistant nosocomial isolates of Acinetobacter baumannii A. Giacomettia*, O. Cirionia, M. S. Del Pretea, F. Barchiesia, A. Mataloni Paggia, E. Petrellib and G. Scalisea 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 The in vitro activity of buforin II, cecropin P1, indolicidin, magainin II and ranalexin, alone and in combination with eight clinically used antimicrobial agents was investigated against 12 multidrugresistant strains of Acinetobacter baumannii isolated from immunocompromised patients. Antimicrobial activities were measured by MIC, MBC and viable count. The peptides had a varied range of inhibitory values: overall, the organisms were more susceptible to buforin II (MIC range 0.25–16 mg/L), cecropin P1 (0.50–32 mg/L) and magainin II (0.50–16 mg/L). Synergy occurred when magainin II was combined with β-lactam antibiotics.

Introduction Acinetobacter spp. are emerging as a major cause of nosocomial infections or in outbreaks of cross-infection, particularly in intensive care units, where antimicrobial use is greatest and the host is most susceptible.1–3 Acinetobacter baumannii is the most frequently isolated Acinetobacter spp. in human infections. It is ubiquitous and can be isolated from soil, water, human skin and the environment. It is resistant to many of the antibiotics currently used, and resulting infections are often difficult to treat. Particularly it is frequently resistant to most of the β-lactams and aminoglycosides.4,5 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 Gramnegative 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.

Materials and methods Organisms 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 Sigma–Aldrich (Milan, Italy).

*Corresponding author. Tel: 39-071-5963467; Fax: 39-071-5963468; E-mail: [email protected]

807 © 2000 The British Society for Antimicrobial Chemotherapy

A. Giacometti et al. inoculum of 5  105 cfu/mL. Polypropylene 96-well plates (Sigma–Aldrich) 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 10–6 dilutions and plating 10 L 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

The in vitro activity of various antibiotics was also evaluated: chloramphenicol, netilmicin and ofloxacin (all from Sigma–Aldrich), co-amoxiclav (SmithKline Beecham, Milan, Italy), aztreonam (Menarini, Firenze, Italy), ceftazidime (Glaxo–Wellcome, Verona, Italy), meropenem (Zeneca, Rome, Italy) and piperacillin (Wyeth–Lederle, Aprilia, Italy). The range of concentrations of polycationic peptides tested was 0.125–64 mg/L, and of the other antimicrobial agents was 0.25–256 mg/L.

MIC and MBC determinations The MIC of polycationic peptides was determined using a microbroth dilution method with Mueller–Hinton (MH) broth (Becton Dickinson Italia, Milan, Italy) and an initial

Table I. Antimicrobial susceptibility of Acinetobacter baumannii: number of strains inhibited MIC (mg/L) Agent

0.25

0.50

1

2

4

8

16

BFII CP1 IND MGII RNL AMC ATM CAF CAZ MEM NET OFX PIP

1

2 2

2 3

2

3

2 1 3 2 2

3 2 3 2 3

2 3 2 2 2 3

1 1 2 2 3 4 3 3 3 2 2 7

1

1 1

2 1 1

1 2 1

1 1 2 2

32

64

1 2

1

2 2 5 4 3 3 3 1

1 2 3 2 4 2 3

128

2 2 2 2

256

256

2

3

4

6

128

256

3 5

2 2

5 2 2

2 5

4

8

MBC (mg/L) Agent BFII CP1 IND MGII RNL AMC ATM CAF CAZ MEM NET OFX PIP

0.25

0.50

1

2

4

8

16

32

64

1

1 2

2 2

1 2 4 2 3

1 1

2

2 3 3 3 7

1 1 2

2

3 2 2 4

2 2 4

1 6 2

2 2 5 4

2 2 3 4

256

13

1

1 1 2

2 1 2 2

1

Abbreviations: BFII, buforin II; CP1, cecropin P1; IND, indolicidin; MGII, magainin II; RNL, ranalexin; AMC, co-amoxiclav; ATM, aztreonam; CAF, chloramphenicol; CAZ, ceftazidime; MEM, meropenem; NET, netilmicin; OFX, ofloxacin; PIP, piperacillin

808

809

BFII, buforin II; CP1, cecropin P1; IND, indolicidin; MGII, magainin II; RNL, ranalexin; AMC, co-amoxicclav; ATM, aztreonam; CAF, chloramphenicol; CAZ, ceftazidime; MEM, meropenem; NET, netilmicin; OFX, ofloxacin; PIP, piperacillin.

a

0.187 0.250 0.312 0.250 0.375 1.5 1.0 1.5 1.0 1.0 1.0 1.0 1.25 1.5 1.25 1.5 0.750 1.0 0.750 1.5 1.25 2.0 1.5 1.0 1.0 1.25 1.25 1.0 0.750 1.5 1.25 2.0 1.25 1.25 1.25 1.25 1.0 1.25 1.5 1.25 0.312 0.312 0.187 0.312 0.375 1.5 1.0 1.5 1.0 1.25 1.0 1.0 1.0 1.5 1.25 1.25 1.5 1.0 1.25 0.750 1.0 1.5 1.25 2.0 1.5 1.5 1.25 0.750 1.0 1.0 1.0 1.25 1.5 0.750 1.25 1.0 0.750 1.0 1.25 1.5 0.187 0.312 0.187 0.250 0.312 1.5 1.0 1.5 1.0 0.750 1.5 1.25 1.0 1.5 1.0 2.0 1.0 1.0 1.25 0.750 1.0 1.5 1.25 1.25 AMC ATM CAZ PIP MEM CAF NET OFX

1.25 1.25 1.25 1.0 0.750 1.25 1.25 2.0

MGII IND BFII RNL MGII IND CP1 BFII RNL MGII IND CP1 BFII

The A. baumannii isolates were more susceptible to buforin II (MIC range 0.25–16 mg/L), magainin II (range 0.50–16 mg/L) and cecropin P1 (range 0.50–32 mg/L) and less susceptible to indolicidin (range 2–64 mg/L) and ranalexin (range 2–64 mg/L) (Table I). In vitro viable counts did not show important differences between the control strain ATCC 19606 and the two representative strains, Ab-03.96 and Ab-02.98. Killing by buforin II was shown to be the most rapid: its activity was complete after a 10–15 min exposure period. Killing by cecropin P1, magainin II and

Agenta

Results and discussion

A. baumannii 03-96

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 0.125–64 mg/L for peptides 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 to 1.0, addition; 1.0 to 4.0, indifference; and 4.0, antagonism.

Acinetobacter baumannii ATCC 19606

Synergy studies

FIC indexes

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  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.

Table II. Results of interaction studies between polycationic peptides and other drugs

Bacterial killing assay

CP1

A. baumannii 02-98

RNL

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.

1.0 1.0 1.25 1.25 1.5 1.0 1.0 1.5

Acinetobacter baumannii susceptibility

A. Giacometti et al. indolicidin was complete after a 15–20 min exposure period, while killing by ranalexin was complete after a 20–25 min exposure period. In the combination studies synergy was never observed, with the exception of the combinations between magainin II and β-lactam antibiotics. FIC indexes in the range 0.187– 0.375 were observed by testing magainin II combined with each β-lactam, while the other experiments gave values between 0.750 and 2.0 (Table II). This study emphasizes the importance of the search for alternative antibacterial agents. Buforin II, cecropin P1 and magainin II were highly active against these multiresistant organisms and showed a rapid bactericidal effect. Interestingly, these observations are in agreement with recent reports that showed that killing by peptides was very rapid and resulted in log orders of cell death within minutes of peptide addition.6 Combination studies showed that magainin II acted synergically with β-lactams. The mechanism of this positive interaction remains largely unknown, even though it might be caused by increased access of the β-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 headgroup region of the lipid bilayer. Moreover, magainin II is non-haemolytic and this property may result from a peptide–cholesterol 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 β-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.

References 1. Bergogne-Bérézin, E. & Towner, K. J. (1996). Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clinical Microbiology Reviews 9, 148–65. 2. Siau, H., Yuen, K. Y., Ho, P. L., Wong, S. S. & Woo, P. C. (1999). Acinetobacter bacteremia in Hong Kong: prospective study and review. Clinical Infectious Diseases 28, 26–30. 3. Ruiz, J., Nunez, M. L., Perez, J., Simarro, E., Martinez-Campos, L. & Gomez, J. (1999). Evolution of resistance among clinical isolates of Acinetobacter over a 6-year period. European Journal of Clinical Microbiology and Infectious Diseases 18, 292–5. 4. Ling, J. M., Ng, T. K., Cheng A. F. & Norrby, S. R. (1996). Susceptibilities to 23 antimicrobial agents and β-lactamase production of blood culture isolates of Acinetobacter species in Hong Kong. Scandinavian Journal of Infectious Diseases Supplementum 101, 21–5. 5. Fass, R. J., Barnishan, J., Solomon, M. C. & Ayers, L. W. (1996). In vitro activities of quinolones, beta-lactams, tobramycin, and trimethoprim-sulfamethoxazole against nonfermentative gramnegative bacilli. Antimicrobial Agents and Chemotherapy 40, 1412–8. 6. Hancock, R. E. W. & Chapple, D. S. (1999). Peptide antibiotics. Antimicrobial Agents and Chemotherapy 43, 1317–23. 7. Hancock, R. E. W. (1997). Antibacterial peptides and the outer membranes of gram-negative bacilli. Journal of Medical Microbiology 46, 1–3. 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, 5449–53. Received 27 March 2000; returned 12 June 2000; revised 23 June 2000; accepted 5 July 2000

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