Pseudomonal Biofilm Research Articles

Novel Inhibitors of the Pseudomonas aeruginosa Virulence Factor LasB: a Potential Therapeutic Approach for the Attenuation of Virulence Mechanisms in Pseudomonal Infection

Pseudomonas elastase (LasB), a metalloprotease virulence factor, is known to play a pivotal role in pseudomonal infection. LasB is secreted at the site of infection, where it exerts a proteolytic action that spans from broad tissue destruction to subtle action on components of the host immune system. The former enhances invasiveness by liberating nutrients for continued growth, while the latter exerts an immunomodulatory effect, manipulating the normal immune response. In addition to the extracellular effects of secreted LasB, it also acts within the bacterial cell to trigger the intracellular pathway that initiates growth as a bacterial biofilm. The key role of LasB in pseudomonal virulence makes it a potential target for the development of an inhibitor as an antimicrobial agent. The concept of inhibition of virulence is a recently established antimicrobial strategy, and such agents have been termed "second-generation" antibiotics. This approach holds promise in that it seeks to attenuate virulence processes without bactericidal action and, hence, without selection pressure for the emergence of resistant strains. A potent inhibitor of LasB, N-mercaptoacetyl-Phe-Tyr-amide (K(i) = 41 nM) has been developed, and its ability to block these virulence processes has been assessed. It has been demonstrated that thes compound can completely block the action of LasB on protein targets that are instrumental in biofilm formation and immunomodulation. The novel LasB inhibitor has also been employed in bacterial-cell-based assays, to reduce the growth of pseudomonal biofilms, and to eradicate biofilm completely when used in combination with conventional antibiotics.

Structural features of tympanostomy tube biofilm formation in ciprofloxacin-resistant Pseudomonas otorrhea

Bacterial biofilm formation has been implicated in the high rate of persistent otorrhea after tympanostomy tube insertion. In this study, we evaluated Pseudomonal biofilm formation from intractable post tympanostomy tube otorrhea in children. Twelve patients (seven males, five females) with unilateral post tympanostomy tube P. aeruginosa otorrhea were evaluated prospectively. All patients were treated with ciprofloxacin otic drops but the otorrhea failed to resolve. Ear discharge for culture was collected from the external auditory canal using a swab. The tympanostomy tubes were removed and collected for evaluation of biofilm formation using a scanning electron microscopy. In all cases, ciprofloxacin-resistant P. aeruginosa was the only organism grown. The surface of the silicone tube contained undulations or microfissures throughout. The thick biofilms present on most tube surfaces were densities with no intervening spaces, consistent with biofilms. Biofilms can be directly observed by scanning electron microscopy. Therefore, our results demonstrate that bacterial aggregates called biofilms, that are resistant to treatment by antibiotics, can be detected by standard culture techniques, and may play a major etiologic role in posttympanostomy otorrhea.

In vitro resistance to bacterial biofilm formation on coated fluoroplastic tympanostomy tubes

Bacterial biofilm formation has been implicated in persistent posttympanostomy otorrhea and irreversible tube contamination. The use of a tympanostomy tube with a resistance to biofilm formation by the most common organisms associated with persistent infection may decrease the incidence of chronic otorrhea and the need for tube removal. In this investigation, scanning electron microscopy was used to compare a phosphorylcholine-coated fluoroplastic tympanostomy tube to plain fluoroplastic and silver oxide-impregnated fluoroplastic for resistance to biofilm formation after in vitro incubation with Staphylococcus aureus or Pseudomonas aeruginosa. Only a biofilm from Pseudomonas formed on the untreated fluoroplastic tubes, whereas the silver oxide-impregnated tubes developed biofilms from both S aureus and P aeruginosa. In contrast, the coated fluoroplastic tube showed resistance to both staphylococcal and pseudomonal biofilm adhesion. This is the first study to demonstrate the effect of a surface treatment of fluoroplastic as a method to inhibit biofilm formation by both S aureus and P aeruginosa. This reinforces our previous studies showing that surface-adherence properties such as charge or slickness or both may be more beneficial than antibacterial treatments in preventing film adhesion. (Otolaryngol Head Neck Surg 2000;123:246–51.)

Experimental pneumonia with Pseudomonas aeruginosa in immunosuppressed guinea pigs as a model for biofilm-associated infection

To establish a model where the role of bacterial biofilms in chronic pneumonia with Pseudomonas aeruginosa could be investigated, hydrocortisone-treated guinea pigs were given P. aeruginosa, strain 2126 by inhalation which were used throughout this study, in planktonic form. In these animals, the bacteria were recovered only from the lungs more than 4 weeks after infection. The persistence in bacterial colonization in the lungs coincided with the formation of glanulomatous lesions that surrounded spherical grains consisting of outer shell and inner bacterial colonies. The outer shell of grain was stained with ruthenium red and was presumed to be polyanionic and therefore to be a biofilm-like material. In normal animals without hydrocortisone-treatment, the number of neutrophils recovered from bronchoalveolar lavage fluid increased significantly from 3 hours after infection and subsequently the inhaled bacteria were eliminated from the lungs by day 3 of infection. This early influx of neutrophils into the lungs tended to be suppressed by treatment with hydrocortisone. The formation of grains did not take place in the lungs of normal animals, indicating the significant role of grain-formation in the initiation and the prolongation of bacterial colonization in the lungs. P. aeruginosa, strain 2126, incubated in saline formed thick biofilms on the surface of teflon piece. Levofloxacin (LVFX), a quinolone antibacterial, exhibited killing activity against the bacteria in in vitro-forming biofilms at MIC. In contrast, gentamicin (GM), an aminoglycosid antibiotic, and ceftazidime (CAZ), a beta-lactams antibiotic, showed no such killing activity at MIC. Treatment of this model with oral LVFX achieved complete eradication of the bacteria, whereas subcutaneous injection of GM or CAZ was hardly effective. The pharmacokinetic study on these antibacterials revealed that the doses used in this study were sufficient to obtain the pulmonary levels of these drugs far above MIC even in GM and CAZ. These data indicate that the outer shell of grains, a characteristic finding in the pulmonary lesions of this model, may be one of the forms of pseudomonal biofilms and that this model represents the significant role of biofilm mode of growth of P. aeruginosa in persistence in pulmonary colonization.