Antimicrob Agents Chemother 50(5): 1623-1627

Polyamines Increase Antibiotic Susceptibility in <em>Pseudomonas aeruginosa</em>

Department of Biology, Georgia State University, Atlanta, Georgia 30303

^{Corresponding author. Mailing address: Department of Biology, Georgia State University, 24 Peachtree Center Avenue, Atlanta, GA 30303. Phone: (404) 651-2531. Fax: (404) 651-2509. E-mail:}ude.usg.etagnal@ldcoib.

Received 2005 Jun 14; Revised 2005 Jul 27; Accepted 2006 Feb 1.

Abstract

Pseudomonas aeruginosa is an opportunistic human pathogen. Treatment is complicated by frequent acquired resistance to antipseudomonal therapies. Polyamines (cadaverine, putrescine, spermidine, and spermine) are ubiquitous polycationic compounds essential for all living organisms. In a dose-dependent manner, polyamines increased the susceptibility of P. aeruginosa to 14 β-lactam antibiotics, chloramphenicol, nalidixic acid, and trimethoprim as demonstrated by a reduction in MIC of up to 64-fold. This effect was partially antagonized (25 to 50%) by the presence of 10 mM of Mg^{or Ca}^{. In contrast, the effects of the outer membrane permeabilizers, polymyxin B nonapeptide and EDTA, were completely abolished by 3 mM Mg}^{or Ca}^{. Changes on the outer membrane barrier by these compounds were assessed by activity measurements of periplasmic β-lactamase. The results showed that while EDTA and polymyxin B serve as outer membrane disorganizing agents as expected, exogenous spermidine and spermine did not exhibit any apparent effect on outer membrane permeability or rupture. In summary, these results strongly suggest that the increased antibiotic susceptibility by polyamines is exerted by a mechanism that differs from that of EDTA and polymyxin B. Polyamines might be potentially useful in antipseudomonal therapies by increasing the effectiveness of certain β-lactam antibiotics.}

Abstract

Pseudomonas aeruginosa is a gram-negative human pathogenic bacterium responsible for severe nosocomial infections, life-threatening infections in immunocompromised persons, and chronic infections in cystic fibrosis patients (6, 23, 29, 30). Antimicrobial treatment is often difficult because of development of resistant strains (12). While tremendous efforts have been devoted to deciphering molecular details of resistance mechanisms, little has been done to identify methods for increasing antibiotic susceptibility to available drugs. Antibiotic susceptibility enhancement was first reported in the late 1950s (21, 31) and was related to outer membrane permeabilization of gram-negative bacteria by cationic and chelating agents. The outer membrane of gram-negative bacteria consists of an asymmetric double layer of polyanionic lipopolysaccharide (LPS) molecules (outer leaflet) and glycerophospholipids (inner leaflet). LPS molecules are electrostatically linked by divalent cations (e.g., Mg^{and Ca}^{), forming a “tiled-roof” structure with strong integrity that functions as an effective permeability barrier against hydrophobic antibiotics, detergents, dyes, and macromolecules (}20, 25, 27). However, this structure can be weakened by removing divalent ions or replacing them with other cationic agents. This results in an increase of outer membrane permeability and sensitizes the bacteria to hydrophobic antibiotics, detergents, or dyes (27). Several such compounds (e.g., EDTA, polymyxin B nonapeptide [PMBN], lysine polymers, and protamine) have been reported to sensitize gram-negative bacteria to antimicrobial agents in this manner (27).

Vaara and coworkers have extensively investigated cationic agents that increase outer membrane permeability (20, 25, 27). PMBN is the one of the best-characterized cationic outer membrane permeabilizers and sensitizes enteric bacteria to hydrophobic antibiotics. However, PMBN is extremely nephrotoxic, and thus, its use in clinical applications is markedly reduced (27).

Natural polyamines, including cadaverine, putrescine, spermidine, and spermine, are a group of ubiquitous cationic compounds found in all living organisms. Spermine is present in eukaryotic cells whereas the others are present in both prokaryotic and eukaryotic cells. Polyamines are essential for optimal cell growth and viability, and intracellular concentrations of polyamines are at millimolar levels in both prokaryotic and eukaryotic cells (7). In prokaryotic cells, polyamines have been reported as potential regulatory molecules in DNA replication, transcription, translation, and enzyme activities (8). A recent report suggested that in Escherichia coli, polyamines enhance the expression of a set of regulatory genes at the level of translation and subsequently stimulate the transcription of hundreds of genes required for optimal cell growth and viability (33). Other reports suggest a role for polyamines in the protection of cells from external toxic conditions, such as oxidative stress (9, 26), radiation (10), acidic pH (22, 24), and other toxic agents (2, 16). Polyamines are also involved in control of membrane permeability by blocking outer membrane porin channels (e.g., OmpF and OmpC) in E. coli (3). In contrast, the synthetic polyamine analogues naphthylacetylspermine and methoctramine were reported to increase the outer membrane permeability by disruption of LPS integrity, resulting in increased susceptibility of E. coli to hydrophobic antibiotics (32). An early study conducted by Vaara and Vaara concluded that cadaverine, spermidine, and spermine at submillimolar concentrations had neither bactericidal nor sensitizing activity to antibiotics in E. coli (28).

We previously studied arginine metabolisms and polyamine utilization in P. aeruginosa PAO1 (14) to explore the physiological roles of polyamines in this organism. We reported that exogenous natural polyamines can enhance the susceptibility of P. aeruginosa PAO1 to multiple antibiotics, including β-lactams, chloramphenicol, nalidixic acid, and trimethoprim, but not to erythromycin, novobiocin, and fusidic acid. We also presented data in support of the notion that the mechanism of antibiotic susceptibility by polyamines is fundamentally different from that associated with EDTA or PMBN.

Acknowledgments

This work was supported by National Science Foundation grants 0316005 and 0415608.

We are grateful to David Y. Graham for critical review of the manuscript.

Acknowledgments

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