Chest
Volume 125, Issue 2, Supplement, February 2004, Pages 62S-69S
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Effects of Subinhibitory Concentrations of Macrolide Antibiotics on Pseudomonas aeruginosa

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Biofilm-forming bacteria such as Staphylococcus, Haemophilus, and Pseudomonas species resist phagocytosis by host immune cells and the actions of antimicrobial agents. In susceptible individuals, such as patients with cystic fibrosis (CF) or diffuse panbronchiolitis (DPB), strains of Pseudomonas aeruginosa produce a number of virulence determinants that permit colonization and infection of the respiratory tract. P aeruginosa strains isolated from CF and DPB patients typically have a mucoid colony morphology. This is due to the overproduction of alginate, an exopolysaccharide capsule that is composed of D-mannuronic and L-guluronic acids. In addition, the P aeruginosa type IV pilus mediates cell surface translocation by a process known as twitching motility. Both alginate production and twitching motility contribute to the virulence of P aeruginosa, as does the formation of biofilms. Biofilms bind cells and organic and inorganic materials to each other, and to a variety of substrata. Their tightly formed structure reduces antimicrobial activity, promotes bacterial adhesion to lung epithelia, and prevents bacterial dehydration. Prior work has suggested that macrolides have therapeutic value in patients with DPB and CF. We hypothesized that the improved clinical status of these patients was due, in part, to macrolides inhibiting the production of P aeruginosa virulence determinants. Traditionally, macrolides have not been considered to exhibit antipseudomonal activity, as their mean inhibitory concentration (MIC) values for clinical and environmental strains of the microbe range from 50 to 550 μg/mL. In this study, we found that sub-MIC levels of clarithromycin substantially inhibited twitching motility. In addition, the incubation of biofilm-grown P aeruginosa with clarithromycin altered the structure and architecture of the biofilm. Investigating the potential nonribosomal effects of macrolides on opportunistic pathogens such as P aeruginosa and elucidating the molecular mechanisms that underlie the inhibition of twitching motility may lead to more effective treatments of pulmonary infections in patients with CF and DPB.

Section snippets

Initiating Infection: Adhesin-Receptor Interaction

The type IV pili of P aeruginosa are polar, polymer filaments composed of a single protein subunit, PilA, or pilin. Pilin contains a highly conserved hydrophobic amino terminal region, which forms its helical core, and hydrophilic outer proteins with variable domains adapted to different functions. By virtue of its helical structure, pili rotation likely supplies the force needed for extension and retraction of the pilus strand. Microscopy studies by Bradley,9 Semmler et al,10 and Skerker and

Twitching Motility: An Essential Component of Virulence

Video images of piliated P aeruginosa colonies illustrate the fact that twitching motility along smooth surfaces promotes rapid colony expansion.1011 At the interstitial surface between the agar and the underlying plastic or glass of a Petri dish, a series of concentric rings develop, giving the appearance of a halo (Fig 2).10 Nonpiliated P aeruginosa variants or strains that harbor mutations in genes affecting pilus function fail to carry out twitching motility (Fig 2).10 Semmler and colleagues

Twitching Motility and Biofilm Formation

In the experiments of O'Toole and Kolter,17 phase contrast microscopy was used to visualize the edges of P aeruginosa microcolonies with intact twitching motility. They were described as irregular and as the result of functional type IV pili. In contrast, type IV pili-deficient strains demonstrated smooth-edged colonies. These investigators proposed that flagellar-mediated motility is required to bring P aeruginosa within proximity of a surface, and is necessary to overcome the repulsive forces

The Effects of Macrolides on Pilus Formation and Assembly

Two main mechanisms by which macrolides might act in P aeruginosa infections are to modify the inflammatory response to infection and by directly inhibiting the activity or production of P aeruginosa virulence determinants (eg, secreted virulence factors, motility, quorum sensing, and biofilm production). While the exact mode of action has yet to be fully elucidated, it has been postulated as secondary to binding of the 50S ribosomal subunit, which interferes with the elongation of polypeptide

The Effects of Macrolides on Biofilm Formation

We hypothesized that if macrolides interfere with cell surface assembly of type IV pili and the function of twitching motility, then these agents should affect some step in biofilm formation. We incubated P aeruginosa for variable periods of time in the presence or absence of sub-MIC levels of clarithromycin. We then applied microtiter assays to quantify biofilm formation. Unbound planktonic bacteria were first removed, and the surface-bound biofilms were stained with crystal violet to

Conclusion

A number of hypotheses have been offered to explain the antipseudomonal mechanism of activity of macrolides, including the innate structural aspects of 14-membered and 15-membered macrolides, the inhibition of protein synthesis that is known to contribute to the assembly, transport, and secretion of bacterial exoproducts, and the macrolide-mediated alterations in lipopolysaccharide and outer membrane proteins of the pathogen that facilitate macrolide entry and intracellular accumulation.

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CME Questions

The American College of Chest Physicians designates this continuing medical education activity for 1 credit hour in category 1 of the Physician's Recognition Award of the American Medical Association. To obtain credit, please complete the question form at http://www.chestnet.org. Credit can be obtained ONLY through our online process.

  • 1.

    All of the following contribute to the virulence of P aeruginosa except:

    • A.

      Decreased production of alginate

    • B.

      Twiching motility

    • C.

      Formation of biofilms

    • D.

References (25)

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    Infect Immun

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  • DE Bradley

    A function ofPseudomonas aeruginosaPAO polar pili: twitching motility

    Can J Microbiol

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    Learning objectives:

    • 1.

      To understand the determinants of Pseudomonas aeruginosa virulence.

    • 2.

      To understand the adhesin-receptor interaction as the primary step in the establishment of infection.

    • 3.

      To understand the importance of twitching motility for P aeruginosa colonization.

    • 4.

      To understand the effects of macrolides on twitching motility.

    • 5.

      To understand the effects of macrolides on biofilm formation.

    Dr. Wozniak, Ms. Keyser, or the department(s) with which they are affiliated have received something of value (ie, any item, payment, or service valued in excess of $750.00) from Abbott Laboratories related directly or indirectly to the subject of this submission.

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