For a chronic infection to be established, bacteria must be able to cope with hostile conditions such as low iron levels, oxidative stress, and clearance by the host defense, as well as antibiotic treatment. It is generally accepted that biofilm formation facilitates tolerance to these adverse conditions. However, microscopic investigations of samples isolated from sites of chronic infections seem to suggest that some bacteria do not need to be attached to surfaces in order to establish chronic infections. In this study we employed scanning electron microscopy, confocal laser scanning microscopy, RT-PCR as well as traditional culturing techniques to study the properties of Pseudomonas aeruginosa aggregates. We found that non-attached aggregates from stationary-phase cultures have comparable growth rates to surface attached biofilms. The growth rate estimations indicated that, independently of age, both aggregates and flow-cell biofilm had the same slow growth rate as a stationary phase shaking cultures. Internal structures of the aggregates matrix components and their capacity to survive otherwise lethal treatments with antibiotics (referred to as tolerance) and resistance to phagocytes were also found to be strikingly similar to flow-cell biofilms. Our data indicate that the tolerance of both biofilms and non-attached aggregates towards antibiotics is reversible by physical disruption. We provide evidence that the antibiotic tolerance is likely to be dependent on both the physiological states of the aggregates and particular matrix components. Bacterial surface-attachment and subsequent biofilm formation are considered hallmarks of the capacity of microbes to cause persistent infections. We have observed non-attached aggregates in the lungs of cystic fibrosis patients; otitis media; soft tissue fillers and non-healing wounds, and we propose that aggregated cells exhibit enhanced survival in the hostile host environment, compared with non-aggregated bacterial populations.
During infection, Pseudomonas aeruginosa employs bacterial communication (quorum sensing [QS]) to coordinate the expression of tissue-damaging factors. QS-controlled gene expression plays a pivotal role in the virulence of P. aeruginosa, and QS-deficient mutants cause less severe infections in animal infection models. Treatment of cystic fibrosis (CF) patients chronically infected with P. aeruginosa with the macrolide antibiotic azithromycin (AZM) has been demonstrated to improve the clinical outcome. Several studies indicate that AZM may accomplish its beneficial action in CF patients by impeding QS, thereby reducing the pathogenicity of P. aeruginosa. This led us to investigate whether QS inhibition is a common feature of antibiotics. We present the results of a screening of 12 antibiotics for their QS-inhibitory activities using a previously described QS inhibitor selector 1 strain. Three of the antibiotics tested, AZM, ceftazidime (CFT), and ciprofloxacin (CPR), were very active in the assay and were further examined for their effects on QS-regulated virulence factor production in P. aeruginosa. The effects of the three antibiotics administered at subinhibitory concentrations were investigated by use of DNA microarrays. Consistent results from the virulence factor assays, reverse transcription-PCR, and the DNA microarrays support the finding that AZM, CFT, and CPR decrease the expression of a range of QS-regulated virulence factors. The data suggest that the underlying mechanism may be mediated by changes in membrane permeability, thereby influencing the flux of N-3-oxo-dodecanoyl-Lhomoserine lactone.
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