New antimicrobials in the management of cystic fibrosis

The chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) is a biofilm1 characterized by (i) the mucoid phenotype producing an abundance of alginate in vitro and in the patients, (ii) microcolonies surrounded by alginate in sputum and in post-mortem investigations and bacteria staying on the surface of the airways as an endobronchiolitis without spreading to the blood or to other organs, (iii) high levels of antibodies against alginate and other P. aeruginosa antigens, and (iv) resistance to the patients' defence mechanisms and to antibiotic treatment.1 Oxygen radicals produced by the inflammatory response (polmorphonuclear leucocytes; PMNs) induce mutations leading to the alginate production that is so characteristic of P. aeruginosa biofilm infection in CF.2 Quorum sensing is also involved in mature biofilm formation in vitro and in vivo.3 The biofilm mode of growth is the survival strategy of environmental bacteria such as P. aeruginosa, and alginate biofilms are also protected against antibiotics and against the immune response in the lungs of the patient.1 The tissue damage is due to immune complex-mediated chronic inflammation dominated by PMNs leading to release of leucocyte proteases.1 Bacteria growing in biofilms are much more resistant to antibiotics compared with planktonic-growing cells of the same isolate: MIC and minimal bactericidal concentration (MBC) can be 100- to 1000-fold greater in old biofilms whereas young biofilms are less resistant. The same sensitivity as in planktonic bacteria is found if the bacteria from the resistant biofilm are liberated and re-investigated. Standard laboratory techniques used to determine antibiotic susceptibility of planktonic-growing bacteria cannot predict the possibility of eradication of bacteria growing in biofilms.4 The resistance to antibiotics of biofilm-growing bacteria could 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 β-lactamase from the bacteria, which cleaves and/or traps β-lactam-antibiotics, and overexpression of efflux pumps.5–9 The increased resistance of biofilm-growing bacteria means that antibacterial therapy usually fails to eradicate the bacteria in the biofilm, although the standard laboratory susceptibility tests demonstrate sensitivity to the antibiotics used. On the other hand, antibiotic treatment regularly leads to temporary clinical improvement of the patient and lung function in parallel with a decrease in the number of (planktonic) bacteria (cfu) in sputum.10 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.11 The number of P. aeruginosa cfu in sputum may be as high as 108–1010 per mL. This implies that mutations occur in sputum. A high frequency (36%) of hypermutable P. aeruginosa has been found in CF lung infection.12 By means of population analysis Giwercman et al.13 have shown that at the onset of therapy the majority of P. aeruginosa in sputum were sensitive to the β-lactam antibiotics used for treatment, whereas a minority were already resistant. This minority was identical (by typing) 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.13 The resistance was shown to be caused by partially derepressed production of chromosomal β-lactamase that could be further induced.14 Furthermore, free β-lactamase activity, probably exported in liberated microvesicles,7 could be detected in CF sputum and the concentration increased during the course of piperacillin, ceftazidime, cefsulodin and imipenem therapy.14 Aztreonam therapy led to the opposite result because the β-lactamase activity decreased and aztreonam was able to mask β-lactamase activity by acting as an inhibitor.14 Double β-lactam therapy with aztreonam and e.g. piperacillin, ceftazidime or imipenem may therefore be a future approach. Development of resistance to ciprofloxacin is regularly seen,15 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 selective pressure in patients receiving colistin by daily inhalation.11,16 The high frequency of hypermutable P. aeruginosa in CF,12 the high level of resistance to ciprofloxacin due to several different mutations15 and the induction of mutations by means of oxygen radicals from PMNs leading to alginate production,2 combined with the antioxidant imbalance in the CF lung,17 have led us to suggest that it is the chronic inflammation dominated by PMNs that induces a high level of mutation 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-acetylcysteine18 may be a new way to delay development of conventional resistance to antibiotics in P. aeruginosa in CF patients. A logical consequence of 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.11 Thus, it is possible to prevent or at least delay the onset of chronic P. aeruginosa infection in most CF patients by early aggressive therapy of intermittent colonization with oral ciprofloxacin in combination with colistin inhalation. Inhalation of tobramycin has also been shown to be effective.11 This early, aggressive therapeutic approach is widely accepted and has been published by a European CF consensus conference.11 Further improvement of this approach may possibly be achieved by the addition of macrolides (see below) or N-acetylcysteine (see above), since early alginate production characterizes therapeutic failures. 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 β-lactam antibiotic. This principle is called ‘maintenance chemotherapy' (= chronic suppressive therapy = elective therapy).11 The principle is 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 during these intervals. This treatment strategy has significantly improved the survival of CF patients.11 Maintenance therapy with oral ciprofloxacin every 3 months as well as frequent (three to four times a year) non-elective intravenous treatment of acute exacerbations of chronic P. aruginosa infection are also efficient.11 Maintenance treatment with high doses of inhaled tobramycin on–off every other month is a new and promising approach, which is effective and convenient for the patients.19 The principles of maintenance therapy are widely accepted and have been published by a European CF consensus conference.11 The mechanism of action of the antibiotics in 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 (piperacillin + tobramycin).20 Furthermore, sub-inhibitory concentrations of antibiotics have been shown to suppress the production of exoproducts such as proteases and phospholipase C and alginate of P. aeruginosa, and colistin binds to P. aeruginosa lipopolysaccharide.21–23 According to these results, therefore, the decrease in cfu of planktonic bacteria and to some degree of biofilm bacteria and 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.11 Further improvement of this approach will focus on eradication of the biofilm (see below). 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. One promising new group of agents is peptide antibiotics, some of which are highly active against P. aeruginosa and which hopefully may be developed for inhalation therapy.24 Another new therapeutic approach may emerge from the synergy described between some β-lactam antibiotics and specific phospholipids that increase the permeability of the P. aeruginosa outer membrane by binding both Ca2+ and Mg2+.25 Aminoglycosides are bound to the alginate of the biofilm, but since alginate lyases can facilitate the penetration of these antibiotics by dissolving the alginate of the biofilm26 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, 10–20 V, 20 mA over a distance of 2.5 mm).27 This combination kills the bacteria in the biofilm although neither the antibiotic nor the electric current alone is 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.23 In CF patients with chronic P. aeruginosa infection similar effects have been reported in a recent controlled study using azithromycin 250 mg/day for 3 months.28 The efficacy of macrolides in spite of their lack of bacteriostatic or bactericidal effect against P. aeruginosa is probably due to an anti-inflammatory activity and a sub-MIC effect, which inhibit the production of proteins such as the exoproteases of P. aeruginosa and inhibit and destroy the biofilm matrix.23 Part of the reason for this sub-MIC effect seems to be that azithromycin and maybe other macrolides inhibit the quorum sensing of P. aeruginosa,29 which is necessary for maturation of the biofilm and which turns on the genes that regulate the production of many virulence factors of these bacteria. Other promising agents that inhibit quorum sensing have been detected in nature, e.g. the halogenated furanones, which are produced by Delisea pulchra, an alga that is relatively free of bacterial colonization on its surface.30 The hope is, therefore, that such compounds may also prevent or destroy P. aeruginosa biofilms in the lungs of CF patients.