As far as we are aware, this is the first report of the efficacy of antibiotic combinations against P. aeruginosa grown under anaerobic conditions. Our findings also extend previous studies using MCBT for CF P. aeruginosa grown under aerobic planktonic conditions and in biofilm mode by incorporating colistin combinations into the protocols. The emergence of high levels of resistance to many antipseudomonal antibiotics has seen a resurgence of interest in nebulized colistin for CF lung infection.
The increased resistance of P. aeruginosa
to antibiotics in advanced CF lung disease has been attributed to genetic mechanisms including the emergence of hypermutable P. aeruginosa
), the generation of biofilms, and exposure of the organisms to an environment which is anaerobic, acidic, and nutrient depleted (13
). It is believed that P. aeruginosa
is able to grow under anaerobic conditions in CF lung sputum using NO3
, or nitrous oxide as a terminal electron acceptor. Anaerobic growth has been achieved in vitro by the addition of nitrates to conventional media (33
). However, in this study MHB II supported the anaerobic growth of all isolates to 108
within 48 h without supplementation. It is assumed that MHB II contains sufficient nitrates for anaerobic growth. The major ingredient of MHBII, beef extract, is the likely source.
The first-line antipseudomonal antibiotics, the beta-lactams, aminoglycosides, and fluoroquinolones, act along different pathways, but all are most effective against rapidly dividing bacteria. Theories that these agents would be less effective against P. aeruginosa
growing more slowly in the static, anaerobic mucus in the CF lung are supported by the results of this study. Various other processes are known to underpin the adverse effect of anaerobic respiration on the efficacy of antibiotics, e.g., the activity of aminoglycosides is dependent on internalization by specific transport mechanisms requiring oxidative phosphorylation (13
). In one recent study it was reported that addition of nitrate decreased susceptibilities of the organisms to antibiotics, suggesting that local oxygen deficiencies and the presence of nitrate contribute to the reduced susceptibilities (3
There is only limited literature addressing the susceptibilities of P. aeruginosa
grown anaerobically to antimicrobial agents. Davey and colleagues reported that the minimal bactericidal concentration of ciprofloxacin for P. aeruginosa
under anaerobic conditions was only slightly raised, while that for gentamicin was raised eightfold (7
). In the current study all antibiotics were found to be less effective under anaerobic and biofilm conditions than under aerobic conditions. Colistin and meropenem were the most effective single agents under all conditions; at the other extreme amikacin was ineffective against all isolates under all conditions. Interestingly, there was no clear correlation between the class of drug or mode of growth and bactericidal activity. Colistin, meropenem, and ciprofloxacin were bactericidal against more isolates under anaerobic conditions than in biofilm mode, whereas tobramycin, ticarcillin-clavulanate, and ceftazidime were more effective under biofilm than anaerobic conditions. Resistance to colistin has rarely been reported even in the face of the selection pressure of a daily dose by inhalation (8
). Although the efficacy of colistin was markedly diminished when tested under anaerobic conditions, it remained the most effective single agent. Meropenem had bactericidal activity against more isolates than high-dose tobramycin under planktonic aerobic conditions. These two agents, however, were equally effective under anaerobic conditions, and high-dose tobramycin was bactericidal for more isolates under biofilm conditions. Notably, three of the four most effective dual combinations under all conditions (colistin with ciprofloxacin or cotrimoxazole or azithromycin) do not involve parenteral administration or the need for hospitalization and are therefore potentially useful for out-of-hospital maintenance.
It was interesting that the proportion of P. aeruginosa isolates susceptible to single and combination antibiotics under anaerobic conditions was not significantly different from that in the biofilm mode of growth. Since colistin was effective against all isolates as a single agent, it was not surprising that colistin combinations were the most effective under all conditions. Aside from colistin, combinations containing meropenem were the most effective under aerobic as well as anaerobic and biofilm conditions.
Biofilm-specific resistance is now accepted as a complex physiological process. It has been reported that P. aeruginosa
responds to anaerobic conditions by increased production of alginate, which poses a physical barrier to antibiotics (33
). The adsorption of positively charged antibiotics, such as the aminoglycosides, to the negatively charged alginate polymers also retards penetration (12
). Hypoxic conditions within the deeper layers of the biofilm and the overexpression of efflux pumps combined with the slower growth rates in anaerobic and biofilm cultures are also likely to contribute to resistance. It has been reported that antibiotic-resistant phenotypic variants of P. aeruginosa
with an enhanced ability to form biofilms arise at high frequency both in vitro and in the lungs of CF patients (9
). There is also evidence that the bacteria residing within biofilms employ other genetic mechanisms to resist the action of antibiotics. Mah and colleagues recently proposed a model wherein periplasmic glucans bind to and sequester antibiotics including tobramycin (18
). Tolerance to antibiotics in biofilm cultures has also been attributed to the presence of “persister” cells (30
), although the underlying mechanisms are unknown. Our data also provide further evidence that the use of antagonistic combinations may contribute to the persistence of infection.
There is evidence to suggest that the apparent activity of macrolides against P. aeruginosa
in vivo may relate to its anti-inflammatory activity and/or modulation of the production of alginate or other virulence factors including elastase and rhamnolipid by quorum-sensing systems. These quorum-sensing systems are activated under stationary phase and under anaerobic conditions (16
). The effect may also be due to a sub-MIC effect that interferes with the biofilm matrix. Macrolides paired with other agents have shown synergistic activity against multidrug-resistant, planktonically grown P. aeruginosa
). Surprisingly, in this study, antibiotic combinations containing azithromycin proved to be more effective against aerobic, planktonically grown isolates than against the corresponding isolates grown under anaerobic conditions or in biofilms. Furthermore, among anaerobically or biofilm-grown isolates, antagonism was observed in up to 19% of combinations containing azithromycin. This finding may have implications for the widespread use of azithromycin in the CF population (29
Increased evidence indicating that the CF lung contains P. aeruginosa living in both planktonic and biofilm mode in conditions varying from aerobic through hypoxic to anaerobic has important implications for selection of optimal antibiotic combinations, particularly since the relative size of each population is not known. There is evidence from this study that antibiotic combinations bactericidal under aerobic or anaerobic planktonic conditions in vitro are not necessarily bactericidal against organisms growing in biofilm mode and may even be antagonistic or vice versa. It is also possible that anaerobic cultures and biofilms generated in vitro do not accurately reflect the hostile environment of the CF lung and/or that the concentrations of antibiotics used in our study may not reflect the concentrations found in CF airway mucus. Eradication of P. aeruginosa from the chronically infected CF lung is rarely achieved and may not be a realistic goal using current antimicrobials. Nonetheless, selections of antibiotic combinations that are commonly bactericidal against clinical isolates grown under aerobic planktonic, anaerobic planktonic, and biofilm conditions may prove to be more effective in reducing bacterial load. Clinical trials will be needed to determine whether this approach leads to improvements in lung function and thus quality and longevity of life for CF patients.