Polyamine effects on antibiotic susceptibility in bacteria.
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Journal: 2007/August - Antimicrobial Agents and Chemotherapy
ISSN: 0066-4804
Abstract:
Biogenic polyamines (e.g., spermidine and spermine) are a group of essential polycationic compounds found in all living cells. The effects of spermine and spermidine on antibiotic susceptibility were examined with gram-negative Escherichia coli and Salmonella enterica serovar Typhimurium bacteria and clinical isolates of Pseudomonas aeruginosa and with gram-positive Staphylococcus aureus bacteria, including methicillin-resistant S. aureus (MRSA). Exogenous spermine exerted a dose-dependent inhibition effect on the growth of E. coli, S. enterica serovar Typhimurium, and S. aureus but not P. aeruginosa, as depicted by MIC and growth curve measurements. While the MICs of polymyxin and ciprofloxacin were in general increased by exogenous spermine and spermidine in P. aeruginosa, this adverse effect was not observed in enteric bacteria and S. aureus. It was found that spermine and spermidine can decrease the MICs of beta-lactam antibiotics in all strains as well as other types of antibiotics in a strain-dependent manner. Significantly, the MICs of oxacillin for MRSA Mu50 and N315 were decreased more than 200-fold in the presence of spermine, and this effect of spermine was retained when assessed in the presence of divalent ions (magnesium or calcium; 3 mM) or sodium chloride (150 mM). The effect of spermine on the sensitization of P. aeruginosa and MRSA to antibiotics was further demonstrated by population analysis and time-killing assays. The results of checkerboard assays with E. coli and S. aureus indicated a strong synergistic effect of spermine in combination with beta-lactams and chloramphenicol. The decreased MICs of beta-lactams implied that the possible blockage of outer membrane porins by exogenous spermine or spermidine did not play a crucial role in most cases. In contrast, only the MIC of imipenem against P. aeruginosa was increased by exogenous spermine and spermidine, and this resistance effect was abolished in a mutant strain devoid of the outer membrane porin OprD. In E. coli, the MICs of carbenicillin, chloramphenicol, and tetracycline were decreased in two acrA mutants devoid of a major efflux pump, AcrAB. However, retention of the spermine effect on antibiotic susceptibility in two acrA mutants of E. coli suggested that the AcrAB efflux pump was not the target for a synergistic effect by spermine and antibiotics and ruled out the hypothesis of spermine serving as an efflux pump inhibitor in this organism. In summary, this interesting finding of the effect of spermine on antibiotic susceptibility provides the basis for a new potential approach against drug-resistant pathogens by use of existing beta-lactam antibiotics.
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Antimicrob Agents Chemother 51(6): 2070-2077
PMID: 17438056

Polyamine Effects on Antibiotic Susceptibility in Bacteria<sup><a href="#fn1" rid="fn1" class=" fn">▿</a></sup>

Department of Biology, Georgia State University, Atlanta, Georgia
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 2006 Nov 23; Revised 2007 Feb 2; Accepted 2007 Apr 5.
Copyright © 2007, American Society for Microbiology

Abstract

Biogenic polyamines (e.g., spermidine and spermine) are a group of essential polycationic compounds found in all living cells. The effects of spermine and spermidine on antibiotic susceptibility were examined with gram-negative Escherichia coli and Salmonella enterica serovar Typhimurium bacteria and clinical isolates of Pseudomonas aeruginosa and with gram-positive Staphylococcus aureus bacteria, including methicillin-resistant S. aureus (MRSA). Exogenous spermine exerted a dose-dependent inhibition effect on the growth of E. coli, S. enterica serovar Typhimurium, and S. aureus but not P. aeruginosa, as depicted by MIC and growth curve measurements. While the MICs of polymyxin and ciprofloxacin were in general increased by exogenous spermine and spermidine in P. aeruginosa, this adverse effect was not observed in enteric bacteria and S. aureus. It was found that spermine and spermidine can decrease the MICs of β-lactam antibiotics in all strains as well as other types of antibiotics in a strain-dependent manner. Significantly, the MICs of oxacillin for MRSA Mu50 and N315 were decreased more than 200-fold in the presence of spermine, and this effect of spermine was retained when assessed in the presence of divalent ions (magnesium or calcium; 3 mM) or sodium chloride (150 mM). The effect of spermine on the sensitization of P. aeruginosa and MRSA to antibiotics was further demonstrated by population analysis and time-killing assays. The results of checkerboard assays with E. coli and S. aureus indicated a strong synergistic effect of spermine in combination with β-lactams and chloramphenicol. The decreased MICs of β-lactams implied that the possible blockage of outer membrane porins by exogenous spermine or spermidine did not play a crucial role in most cases. In contrast, only the MIC of imipenem against P. aeruginosa was increased by exogenous spermine and spermidine, and this resistance effect was abolished in a mutant strain devoid of the outer membrane porin OprD. In E. coli, the MICs of carbenicillin, chloramphenicol, and tetracycline were decreased in two acrA mutants devoid of a major efflux pump, AcrAB. However, retention of the spermine effect on antibiotic susceptibility in two acrA mutants of E. coli suggested that the AcrAB efflux pump was not the target for a synergistic effect by spermine and antibiotics and ruled out the hypothesis of spermine serving as an efflux pump inhibitor in this organism. In summary, this interesting finding of the effect of spermine on antibiotic susceptibility provides the basis for a new potential approach against drug-resistant pathogens by use of existing β-lactam antibiotics.

Abstract

Biogenic polyamines (e.g., putrescine, spermidine, and spermine) are essential cationic compounds in all living organisms (1, 11). Inside the cells, these positively charged molecules are mostly associated with acidic macromolecules, such as genomic DNA and rRNA. The involvement of polyamine in numerous cellular functions has been extensively studied in eukaryotes and bacteria (3, 11, 13-15).

Recently, we reported that exogenous polyamine can affect the antibiotic susceptibility of Pseudomonas aeruginosa PAO1 in two opposite ways (11, 18, 19). Specifically, exogenous polyamine increased the MICs of cationic peptides, aminoglycosides, and quinolone antibiotics. At the genetic level, exogenous polyamine induced the expression of the oprH-phoPQ operon and the pmrHFIJKLM operon, both of which participate in the resistance mechanisms for polymyxin B and other cationic peptide antibiotics (25, 26). On the other hand, the MICs of β-lactams and chloramphenicol were decreased by the presence of exogenous polyamine. While the possible molecular mechanism for this observed sensitization effect is not known, we have ruled out the possibility of polyamine effects on the expression or specific activity of β-lactamase and on the disruption of outer membrane permeability (18).

In Escherichia coli, polyamine was reported to block the flow of certain β-lactams through the OmpF and OmpC porins, which serve as channels for the entrance of these antibiotics through the outer membrane (5). Similarly, the outer membrane porin OprD of P. aeruginosa was reported as the channel for a specific type of β-lactam antibiotics including imipenem and meropenem (22, 33). Once inside the cells, these antibiotics can still be extruded by the operation of efflux pumps, e.g., the AcrAB complex of E. coli (20, 31). An increased level of efflux pump activity is one of the major mechanisms for multidrug resistance in clinical isolates (32). Potentially, this adverse effect of induced efflux can be overcome by efflux pump inhibitors, which are compounds that physically block the pumps (16, 23).

Without an outer membrane, gram-positive bacteria are highly subject to the antibacterial activities of β-lactams. However, the emergence and spread of strains resistant to β-lactams have been a major problem in clinical treatments, e.g., methicillin-resistant Staphylococcus aureus (MRSA) in the health care setting. Even more worryingly, MRSA is now emerging in community clinical treatments. Vancomycin and teicoplanin have been the last line of antibiotics against MRSA, but overuse has led to the emergence of vancomycin-intermediate and vancomycin-resistant S. aureus (VISA and VRSA, respectively). The treatment options for these infections are severely compromised, and thus, new antimicrobial remedies against MRSA, VISA, and VRSA are urgently required (4, 7, 17, 28).

In this study, we extended the initial observation of polyamine effects on antibiotic susceptibility to clinical isolates of P. aeruginosa and strains of E. coli, Salmonella enterica serovar Typhimurium, and S. aureus, including MRSA and VISA. The possible interaction of polyamine with the outer membrane porin OprD and its subsequent effect on the efficacies of imipenem and other β-lactams were characterized in an oprD knockout mutant of P. aeruginosa PAO1. We also analyzed the potential effect of spermine on drug efflux by employing the AcrA mutant strains of E. coli. In addition, the results indicated that exogenous spermine enhances killing of these bacteria by β-lactams with a strong synergistic effect.

Acknowledgments

This work was supported by National Science Foundation grant 0415608.

We are grateful to Phang C. Tai, Parjit Kaur, James Versalovick, and Mark Smeltzer for providing bacterial strains.

Acknowledgments

Footnotes

Published ahead of print on 16 April 2007.

Footnotes

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