In Vitro Activity of Aurein 1.2 Alone and in Combination with Antibiotics against Gram-Positive Nosocomial Cocci

Abstract

This study was performed to evaluate the in vitro activity of the amphibian peptide aurein 1.2 and to investigate its interaction with six antibiotics against nosocomial gram-positive cocci. All isolates were inhibited at concentrations of 1 to 16 mg/liter. Synergy was demonstrated when aurein 1.2 was combined with clarithromycin and minocycline.

Recent interest in the search for alternative therapeutics is growing because of the drug resistance problem with traditional antibiotics (2, 4). Antimicrobial peptides have attracted much attention because of their favorable properties, such as rapid killing, wide spectrum, and rare development of drug resistance (8-10). Amphibian skin is one of the most generous sources of these peptides (1, 12). These molecules are produced and stored in dermal structures called granular glands, which release their contents on the skin of the frog upon adrenergic stimulation or injury.

A number of different types of molecules have been identified from the glandular skin secretions of Australian anurans of the Litoria genus. Recently, the primary structures and activity spectra of 17 novel peptides, named aureins, that are present in the secretions from the dorsal granular glands of the green and golden bell frog, Litoria aurea (13), have been reported. These peptides show broad-spectrum antibiotic and anticancer activities.

Aurein 1.2 is an amphipathic and α-helical peptide; with only 13 amino acid residues, it is one of the smallest amphibian peptides so far reported (13, 14). The aim of the present study was to evaluate the in vitro activities of aurein 1.2 and its bactericidal effects for a large number of gram-positive cocci, including streptococci, methicillin-resistant staphylococci, and vancomycin-resistant enterococci, causing nosocomial infections, as well as to investigate its in vitro interactions with six clinically used antibiotics.

Organisms.

Methicillin-susceptible (MS) Staphylococcus aureus ATCC 29213, methicillin-resistant (MR) S. aureus ATCC 43300, vancomycin-susceptible (VS) Enterococcus faecalis ATCC 29212, vancomycin-resistant (VR) E. faecalis ATCC 51299, and Streptococcus pyogenes ATCC 19615 were used as quality control strains. Twenty nosocomial isolates of each species, except for VR E. faecalis (12 strains), were tested.

Antimicrobial agents.

Aurein 1.2 was synthesized by 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase chemistry by the Faculty of Pharmacy, Medical University of Gdańsk, Gdańsk, Poland (6). The peptide was purified by high-performance liquid chromatography (HPLC) on a Knauer K501 two-pump system and analyzed by analytical HPLC and matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry.

Vancomycin and minocycline (Sigma-Aldrich, Milan, Italy), amoxicillin-clavulanate (GlaxoSmithKline, Verona, Italy), imipenem (Merck, Sharp, & Dohme, Milan, Italy), clarithromycin (Abbott, Rome, Italy), and linezolid (Pharmacia & Upjohn, Kalamazoo, MI) were tested as control agents.

MIC and MBC determinations.

Laboratory standard powders were diluted in accordance with manufacturers' recommendations. MICs and minimal bactericidal concentrations (MBCs) were determined according to the procedures outlined by the Clinical and Laboratory Standards Institute (3). Experiments were performed in triplicate.

Bacterial killing assay.

The ATCC control strains were used to study the in vitro killing effect of aurein 1.2. Aliquots of exponentially growing bacteria were resuspended in fresh Mueller-Hinton broth at approximately 10⁷ cells/ml and exposed to aurein 1.2 at concentrations equivalent to twice and four times the MIC for 0, 5, 10, 15, 20, 25, 30, 40, 50, and 60 min at 37°C. After these times, samples were first serially diluted in 10 mM sodium HEPES buffer (pH 7.2) to minimize the carryover effect and then plated onto Mueller-Hinton agar plates to obtain viable colonies (7).

Synergy studies and cytotoxicity assay.

Six strains of each species were tested by a checkerboard titration method. The fractional inhibitory concentration (FIC) indexes were interpreted as follows: <0.5, synergy; 0.5 to 4.0, indifferent; >4.0, antagonism (5). In addition, time-kill synergy studies were performed at recommended subinhibitory concentrations (one-fourth and one-half the MIC). Synergy or antagonism was defined as a ≥100-fold increase or decrease, and indifference was defined as a <10-fold increase or decrease, in colony counts at 24 h by the combination compared to the most active single agent, and the number of surviving organisms in the presence of the combination had to be 100 CFU/ml below the starting inoculum.

For the cytotoxicity assay, A-549 cells from a human lung carcinoma (BioWhittaker Inc., Walkersville, MD) were cultured in 25-cm² tissue culture flasks. The cytotoxicity of aurein 1.2 at the MBC was determined by the CellTiter 96 AQ cell proliferation assay (Promega Corp., Lyon, France).

All isolates were inhibited by aurein 1.2 at concentrations of 1 to 16 mg/liter. In detail, for the control strains S. aureus ATCC 29213, S. aureus ATCC 43300, E. faecalis ATCC 29212, E. faecalis ATCC 51299, and S. pyogenes ATCC 19615, the peptide exhibited MICs of 8, 8, 8, 16, and 4 mg/liter and MBCs of 16, 16, 32, 32, and 8 mg/liter, respectively. Overall, high rates of resistance to the clinically used antibiotics, except for linezolid, were demonstrated for multiresistant strains. The results are summarized in Table 1. As shown in Table 1, the good activity of aurein 1.2 was confirmed by the fact that the MBCs (range, 2 to 64 mg/liter) were comparable to those of other antibiotics.

TABLE 1.

MICs and MBCs of aurein 1.2 and other clinically used antibiotics for clinical isolates

Strain (no. of isolates) and agent	MIC (mg/liter)a			MBC (mg/liter)b
	Range	50%	90%	Range	50%	90%
MR S. aureus (20)
Aurein 1.2	1-16	4	8	2-32	8	16
AMX-CLVc	2-128	16	128	8-128	64	256
Minocycline	1-16	4	8	2-64	8	32
Clarithromycin	0.50-16	4	16	4-128	32	128
Imipenem	2-128	16	128	8-128	64	256
Linezolid	0.25-4	0.5	2	0.50-2	0.5	2
Vancomycin	0.12-4	1	2	0.50-4	1	2
MS S. aureus (20)
Aurein 1.2	1-16	4	8	2-16	8	16
AMX-CLV	0.25-8	2	4	0.5-32	8	16
Minocycline	0.50-8	4	8	2-32	8	32
Clarithromycin	1-16	2	8	2-64	16	64
Imipenem	0.25-4	1	4	0.5-32	4	16
Linezolid	0.12-2	0.5	2	0.50-2	0.5	2
Vancomycin	0.12-2	1	2	0.50-4	1	2
VS Enterococcus faecalis (20)
Aurein 1.2	2-32	8	16	2-32	16	32
AMX-CLV	1-64	8	16	4-256	16	64
Minocycline	1-32	8	16	4-128	16	64
Clarithromycin	4-128	8	32	8-256	16	128
Imipenem	1-64	4	16	4-256	16	64
Linezolid	0.50-4	1	2	0.5-4	2	4
Vancomycin	0.25-4	1	2	1-4	2	4
VR Enterococcus faecalis (12)
Aurein 1.2	2-32	8	16	2-64	16	32
AMX-CLV	4-128	16	32	8-256	64	128
Minocycline	2-64	16	32	8-64	16	64
Clarithromycin	8-128	16	64	16-256	32	128
Imipenem	4-64	16	32	8-256	32	128
Linezolid	0.50-4	1	2	1-4	4	4
Vancomycin	32-128	32	64	64-256	64	256
Streptococcus pyogenes (20)
Aurein 1.2	1-8	2	4	2-16	4	8
AMX-CLV	0.25-2	0.50	1	0.5-4	1	4
Minocycline	0.50-8	4	8	1-32	8	16
Clarithromycin	0.50-8	2	8	1-32	8	32
Imipenem	0.12-2	0.50	1	0.5-4	1	4
Linezolid	0.12-2	0.5	1	0.50-2	1	2
Vancomycin	0.25-2	0.5	1	0.50-2	1	2

^a50% and 90%, MICs at which 50 and 90% of isolates are inhibited, respectively.

^b50% and 90%, MBCs at which 50 and 90% of isolates are killed, respectively.

^cAMX-CLV, amoxicillin-clavulanate at a ratio of 7:1.

At a concentration equivalent to twice the MIC, the killing activities of aurein 1.2 against staphylococci, enterococci, and streptococci were complete after 30-, 40-, and 20-min exposure periods, respectively. At a concentration equivalent to four times the MIC, its bactericidal activities against staphylococci, enterococci, and streptococci were complete after 20, 30, and 15 min, respectively.

Synergy was observed only for minocycline or clarithromycin. When aurein 1.2 was combined with minocycline, we observed FIC indexes of 0.385, 0.312, and 0.458 for S. aureus, E. faecalis, and S. pyogenes, respectively. When the peptide was combined with clarithromycin, FIC indexes of 0.312, 0.385, and 0.458 were found for the same strains. In contrast, combinations with other antibiotics gave FIC indexes ranging from 0.917 to 1.833. These data were confirmed by the time-kill synergy studies (data not shown).

Cytotoxic effects were practically absent at the concentrations tested (2.4% cytotoxicity).

The data of this study showed that staphylococci and streptococci were susceptible to aurein 1.2, while enterococci showed less susceptibility. Interestingly, aurein 1.2 was demonstrated to be equally active against susceptible and multiresistant clinical isolates. Time-kill studies have shown a rapid bactericidal effect, even if the inactivation of E. faecalis appears to be slower than that observed for the other cocci.

Combination studies showed that aurein 1.2 acted synergistically with hydrophobic antibiotics. Interactions between peptides and macrolides or tetracycline have not yet been extensively studied. The antimicrobial activities of macrolides and tetracyclines result from their abilities to inhibit protein synthesis by binding to the transpeptidation site of the larger ribosomal subunit. Aurein 1.2 adopts conventional amphipathic α-helical structures, a feature commonly found in cationic peptides. Interaction occurs at the membrane surface with the charged, normally basic peptide adopting an α-helical conformation and attaching itself to charged, normally anionic sites on the lipid bilayers (1, 14). Aurein 1.2 is a membrane permeabilizer and disorganizing agent, and these characteristics can allow maximal entry of the hydrophobic substrates (11, 16, 17). Furthermore, it is possible that the positive interaction is a combined effect of increased access to the intracellular target for antibiotics and secondary effects of the peptides themselves (11, 15).

In conclusion, aurein 1.2 might be considered an attractive molecule for future strategies against antibiotic-resistant pathogens.