Understanding bacterial biofilms in patients with cystic fibrosis: current and innovative approaches to potential therapies

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1 Journal of Cystic Fibrosis 1 (2002) Review Understanding bacterial biofilms in patients with cystic fibrosis: current and innovative approaches to potential therapies Niels Høiby* Department of Clinical Microbiology and Danish Cystic Fibrosis Centre in Copenhagen, Rigshospitalet, University of Copenhagen, Blegdamsvej, Copenhagen, Denmark Abstract Chronic P. aeruginosa infection is characterized by production of mucoid alginate and formation of microcolonies (biofilm) as seen in the lungs of cystic fibrosis patients. Oxygen radicals produced by the inflammatory response polymorphonuclear leucocytes induces the alginate production. The biofilm mode of growth is the survival strategy of environmental bacteria and alginate biofilms are also protected against antibiotics and against the immune response in the lungs of the patient. Quorum sensing is important for early and mature biofilm formation and also for the severity of the infection. The new knowledge of the mechanisms involved in biofilm formation opens up new possibilities for therapeutic intervention strategies involving e.g. inhibitors of quorum sensing European Cystic Fibrosis Society. Published by Elsevier Science B.V. All rights reserved. Keywords: Bacteria; Biofilms; Cystic fibrosis; Pseudomonas aeruginosa 1. Introduction The chronic P. aeruginosa lung infection in cystic fibrosis (CF) is a biofilm (Fig. 1) w1x characterized by 1. the mucoid phenotype producing abundance of alginate in vitro and in the patients w1x, 2. microcolonies surrounded by alginate in sputum and in post-mortem investigations w2,1x, 3. the bacteria stay inside the mucus and grow under anaerobic conditions using nitrate as electron acceptor w3x, 4. the bacteria are not located on the epithelial cells w3x, but they induce an endobronchiolitis without spreading to the blood or to other organs w2x, 5. high levels of antibodies against alginate and other P. aeruginosa antigens w1x, 6. resistance against the patients defence mechanisms and against antibiotic treatment w1x. *Department of Clinical Microbiology 9301, Rigshospitalet, Juliane Maries Vej 22, DK-2100 Copenhagen, Denmark. Tel.: q ; fax: q address: hoiby@inet.uni2.dk (N. Høiby). Oxygen radicals produced by the inflammatory response polymorphonuclear leucocytes (PMNs) induces mutations leading to the alginate production which is so characteristic for P. aeruginosa biofilm infection in CF w4x. Quorum sensing is also involved in mature biofilm formation in vitro and in vivo w5x. The biofilm mode of growth is the survival strategy of environmental bacteria like P. aeruginosa and alginate biofilms are also protected against antibiotics and against the immune response in the lungs of the patient w1x. The tissue damage is due to immune complex mediated chronic inflammation dominated by PMNs leading to released leukocyte proteases w1x. 2. Nature of P. aeruginosa biofilms and its resistance to antibiotics Bacteria growing in biofilms are much more resistant to antibiotics compared to planktonic growing cells of the same isolate: minimal inhibitory concentration (MIC) and minimal bactericidal concentration can be fold greater in old biofilms whereas young biofilms are less resistant. The same sensitivity as in the planktonic bacteria is found if the bacteria from the /02/$ - see front matter 2002 European Cystic Fibrosis Society. Published by Elsevier Science B.V. All rights reserved. PII: S Ž

2 250 N. Høiby / Journal of Cystic Fibrosis 1 (2002) Fig. 1. Development of P. aeruginosa biofilm in the bronchioles of CF patients w1 5,21,42 46x. Planctonic P. aeruginosa cells (PA) adhere reversibly to the gel phase of airway lining fluid using the pole of their cells and the flagella and pili. Irreversible attachment occurs to the axis of the bacterial cells which divide and form clusters and an early biofilm. This step involves type 4 pili and the lasir encoded quorum sensing system which major product is the signal molecule C12-HSL (N-(3-oxododecanoyl)-homoserine lactone. The production of alginate involves mutations in the muca gene of PA induced by oxygen radicals (OR) released together with elastase (ELA) (yellow clouds) from activated PMN. Autolysing PMNs release DNA. Maturation of the biofilm with formation of water channels, pores and tower-like and mushroom-like structures involves the rhlr encoded quorum sensing system which major product is the signal molecule C4-HSL (N-butanoylhomoserine lactone). Pieces of biofilms and also planctonic PA cells are released from the surface of mature biofilms. Most of the PA biofilm is present in sputum and not located on the epithelial cells of the respiratory airways and the biofilm is depleted of oxygen (ANAEROBIC). The tissue damage is due to the inflammatory reaction aggravated by the antibody response to the components of the biofilm (immune complex mediated tissue damage). resistant biofilm are liberated and reinvestigated. The ordinary techniques used in the laboratory to determine antibiotic susceptibility of planktonic growing bacteria cannot predict the possibility of eradication of the bacteria growing in biofilms w6x. The resistance against antibiotics of biofilm growing bacteria can be due to several factors such as slow growth, reduced oxygen concentrations at the base of the biofilm, maybe a penetration barrier based on binding of e.g. the positively charged aminoglycosides to the negatively charged alginate polymers, the presence of b-lactamase from the bacteria, which cleaves andyor traps b-lactam-antibiotics and overexpression of efflux pumps w3,7 11x. The increased resistance of the biofilm growing bacteria means that antibacterial therapy usually fails with respect to eradication of the bacteria in the biofilm, although the ordinary susceptibility tests in the laboratory demonstrate sensitivity to the antibiotics used. However, the antibiotic treatment regularly leads to temporary clinical improvement of the patient and lung

3 N. Høiby / Journal of Cystic Fibrosis 1 (2002) function in parallel to the decrease of the number of (planktonic) bacteria (CFU) in sputum w12,13x. 3. Traditional resistance mechanisms in P. aeruginosa biofilms The development of resistance to antibiotics occurs frequently in CF due to the intensive selective pressure provided by the large amount of antibiotics used in these patients w14x. Mucoid and non-mucoid variants of the same strain is frequently present in sputum simultaneously but the non-mucoid variants are more resistant to antibiotics possibly reflecting a higher antibiotic selection pressure out-side the alginate biofilm w15x. The number of P. aeruginosa CFU in sputum may be as 8 10 high as yml. This implies that mutations occur in sputum. Actually, high frequency (36%) of hypermutable P. aeruginosa has been found in CF lung infection w16x. By means of population analysis of the P. aeruginosa majority and minority populations as regard to resistance to b-lactam antibiotics Giwercman et al. w17x have shown, that at the onset of therapy the majority of P. aeruginosa CFU in sputum was sensitive to the antibiotics used for treatment, whereas a minority of CFU already was resistant. The clonality of this minority was identical to the sensitive majority population. During therapy, however, the resistant minority population took over and became predominant as a consequence of the selective pressure imposed by the antibiotic therapy w17x. In these studies the conventional mechanism of resistance to b-lactam antibiotics was shown to be partially due to depressed production of chromosomal b-lactamase which could be further induced w18x. Moreover, free b-lactamase activity, probably exported in liberated microvesicles w9x, could be detected in CF sputum and the level increased to high concentrations during the course of piperacillin, ceftazidime, cefsulodin and imipenem therapy w18x. Aztreonam therapy led to the opposite result because the b-lactamase activity decreased and aztreonam was able to mask b-lactamase activity by acting as an inhibitor w18x. Double b-lactam therapy with aztreonam and e.g. piperacillin, ceftazidime or meropenem is therefore a rational approach. Development of resistance to ciprofloxacin is regularly seen w19x, and resistance to aminoglycosides is also a problem in CF patients with chronic P. aeruginosa infection, whereas resistance to colistin is seldom seen in spite of a daily selective pressure in patients receiving colistin by inhalation w14,20x. The high frequency of hypermutable P. aeruginosa in CF w16x, the high level of resistance to ciprofloxacin due to several different mutations w19x and the induction of mutations by means of oxygen radicals from PMNs leading to alginate production w4x and the antioxidant imbalance in the CF lung w21x have lead us to suggest, that it is the chronic inflammation dominated by PMNs which induce high level of mutations in P. aeruginosa in CF lungs and the resistant mutants are then selected by the heavy use of antibiotics. If this is true, antioxidant therapy with N- acetylcystein w22x may be a new way to delay development of conventional resistance to antibiotics in P. aeruginosa in CF patients. 4. Therapeutic strategies for P. aeruginosa biofilms A logical consequence of the resistant biofilms is to aim at improved prophylaxis and early aggressive therapy before the biofilm is fully established and before the inflammatory reaction around the biofilm is recruited, leading to tissue damage and clinical symptoms w14x. In this way it is possible to prevent or at least delay the onset of the chronic P. aeruginosa infection in most CF patients by early aggressive therapy of the intermittent colonization with oral ciprofloxacin in combination with colistin inhalation. Inhalation of tobramycin or gentamicin has also been shown to be effective w14,23x. This early, aggressive therapeutic approach is widely accepted and has been published by a European CF consensus conference w12 14x. Further improvement of this approach may possibly be achieved by addition of macrolides or N-acetylcystein, since early alginate production characterizes therapeutic failures. It has recently been shown that extracellular DNA is present in P. aeruginosa biofilms and is essential for the early development of the biofilm w24x. Furthermore, bovine DNase I can completely inhibit the early development of P. aeruginosa biofilm and, consequently, early prophylactic use of DNase I in CF may therefore prevent the establishment of chronic lung infection with these bacteria w24x. Another logical consequence of the resistant biofilms is to suppress the number and activity of the P. aeruginosa bacteria in the lungs of CF patients using intravenous tobramycin in combination with a b-lactam antibiotic. This principle is called maintenance chemotherapy (chronic suppressive therapy or elective therapy) w14x. This principle is therefore to restore lung function repeatedly by regular 2 week courses of intensive intravenous treatment every 3 months in the CF centre or at home and, in addition, daily inhalations of colistin between and during the courses of intravenous antibiotics and sometimes also by giving oral ciprofloxacin between these courses. This treatment strategy has significantly improved the survival of CF patients w14x. Maintenance therapy with oral ciprofloxacin every 3 months is also efficient w14x. Frequent (3 4 times a year) non-elective intravenous treatment of acute exacerbations of the chronic P. aeruginosa infection may also be efficient w14x. Maintenance treatment with high doses of inhaled tobramycin on off every other months is another effective approach, which is convenient for

4 252 N. Høiby / Journal of Cystic Fibrosis 1 (2002) the patients w25x. The principles of maintenance therapy is widely accepted and has been published by a European CF consensus conference w14x. The mechanism of action of the antibiotics in the chronic infection caused by biofilm growing P. aeruginosa is not entirely clear. Although the biofilm mode of growth is the characteristic feature of the infection, planktonic bacteria susceptible to antibiotics also occur plentifully in the lungs. In addition, in vitro studies have shown that the number of biofilm growing bacteria can be reduced to 20% by high doses of combinations of antibiotics (piperacillinqtobramycin) w26x. Furthermore, sub-mic concentrations of some antibiotics have been shown to suppress the production of exoproducts such as proteases and phospholipase C (see below) and alginate of P. aeruginosa and colistin binds to LPS of P. aeruginosa w27 29x. According to these results, therefore, the decrease of the CFU of planktonic bacteria and to some degree of biofilm bacteria and the inhibition of exoproducts will reduce the antigenic load in the lungs and therefore probably also the concentration of immune complexes. Accordingly, inflammatory parameters and lung function improve during antibiotic therapy and in this way it is possible to maintain the lung function of the patients for years w12 14x. Further improvement of this approach will focus on eradication of the biofilm. 5. Innovative approaches and potential therapies for P. aeruginosa biofilms New antibiotics and new ways of using old antibiotics as indicated above are, however, needed due to the development of resistance to those antibiotics currently used in CF patients and in order to try to eradicate the biofilm w30x. A new groups of promising agents is peptide antibiotics, some of which are highly active against P. aeruginosa and which hopefully may be developed for inhalation therapy w31x. Another new therapeutic approach may emerge from the synergism described between some b-lactam antibiotics and specific phospholipids, which increase the permeability of the P. aeruginosa outer membrane by binding both 2q 2q Ca and Mg w32x. Aminoglycosides are bound to the alginate of the biofilm, but since alginate lyases can facilitate the penetrations of these antibiotics by dissolving the alginate of the biofilm w33x a new therapeutic approach may consist of inhalation of alginate lyases and aminoglycosides. A new interesting principle used to kill the biofilm growing bacteria, at least in vitro, is a combination of antibiotics and electricity (DC, V, 20 ma over a distance of 2.5 mm) w34x. This combination kills the bacteria in the biofilm although neither the antibiotic nor the electric current alone are effective. Whether these promising new antibiotics and principles will be adaptable to chronic pulmonary infection by P. aeruginosa in CF patients is uncertain. Chronic suppressive therapy by means of long-term daily macrolide therapy has significantly reduced symptoms and inflammatory parameters and increased the survival of Japanese patients suffering from diffuse panbronchiolitis and chronic P. aeruginosa lung infection w29x. In CF patients with chronic P. aeruginosa infection a similar effect has been reported in a recent controlled study using azithromycin 250 mgyday for 3 months w35x. The efficacy of macrolides in spite of their lack of bacteriostatic or bacteriocidal effect against P. aeruginosa is probably due to an anti-inflammatory activity and a sub-mic effect which inhibits the production of proteins such as the exoproteases of P. aeruginosa and inhibit and destroy the biofilm matrix w29x. Some of the reason for this sub-mic effect seems to be that azithromycin and maybe other macrolides inhibit the quorum sensing of P. aeruginosa w36x which is necessary for maturation of the biofilm and which turn on the genes which regulate the production of many virulence factors of these bacteria. Other promising agents have been detected in nature which inhibit quorum sensing e.g. the halogenated furanones, which are produced by the seaweed algae, Delisea pulchra, and these algae are relatively free of surface colonization w37x. Such compounds may also prevent or destroy P. aeruginosa biofilms in the lungs of CF patients. 6. Anti-inflammatory therapy Based on the concept of inflammation-mediated tissue damage, several clinical trials of the use of anti-inflammatory drugs in CF patients have been carried out. Side effects developtoo frequently when systemic prednisone is given w38x. Oral non-steroid anti-inflammatory agents (ibuprofen, peroxicam) and inhaled budesonide have been shown to have some efficacy in maintenance of the lung function without serious side effects and are now used routinely in some CF centres w39 41x. References w1x Høiby N, Johansen HK, Moser C, Song ZJ, Ciofu O, Kharazmi A. Pseudomonas aeruginosa and the biofilm mode of growth. Microbes Infect. 2001;3:1 13. w2x Baltimore RS, Christie CDC, Smith GJW. Immunohistopathologic localization of Pseudomonas aeruginosa in lungs from patients with cystic fibrosis implications for the pathogenesis of progressive lung deterioration. ARRD 1989;140: w3x Worlitzsch D, Tarran R, Ulrich M, et al. 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