RESULTS:

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis patients. P. aeruginosa colonizes the lungs by forming biofilm microcolonies throughout the lung. Quorum sensing (QS) renders the biofilm bacteria highly tolerant to otherwise lethal doses of antibiotics and protects against the bactericidal activity of polymorphonuclear leukocytes (PMNs). It has been previously demonstrated that QS is inhibited by garlic extract. In this study the synergistic effects of garlic and tobramycin and PMNs activities have been evaluated. P. aeruginosa was grown in vitro in continuous-culture once-through flow chambers with and without garlic extract. The garlic-treated biofilms were susceptible to both tobramycin and PMN grazing. Furthermore the PMNs showed an increase in respiratory burst activation when incubated with the garlic-treated biofilm. Garlic extract was administered as treatment for a mouse pulmonary infection model. Mice were treated with garlic extract or placebo for 7 days with the initial 2 days being prophylactic before P. aeruginosa was instilled in the left lung of the mice. Bacteriology mortality histopathology and cytokine production were used as indicators. The garlic treatment initially provoked a higher degree of inflammation and significantly improved clearing of the infecting bacteria. The results indicate that a QS-inhibitory extract of garlic renders P. aeruginosa sensitive to tobramycin respiratory burst and phagocytosis by PMNs as well as leading to an improved outcome of pulmonary infections.

The opportunistic human pathogen Pseudomonas aeruginosa is the predominant micro-organism of chronic lung infections in cystic fibrosis (CF) patients. P. aeruginosa colonizes the CF lungs by forming biofilm structures in the alveoli. In the biofilm mode of growth the bacteria are highly tolerant to otherwise lethal doses of antibiotics and are protected from bactericidal activity of polymorphonuclear leukocytes (PMNs). P. aeruginosa controls the expression of many of its virulence factors by means of a cell–cell communication system termed quorum sensing (QS). In the present report it is demonstrated that biofilm bacteria in which QS is blocked either by mutation or by administration of QS inhibitory drugs are sensitive to treatment with tobramycin and H2O2 and are readily phagocytosed by PMNs in contrast to bacteria with functional QS systems. In contrast to the wild-type QS-deficient biofilms led to an immediate respiratory-burst activation of the PMNs in vitro. In vivo QS-deficient mutants provoked a higher degree of inflammation. It is suggested that quorum signals and QS-inhibitory drugs play direct and opposite roles in this process. Consequently the faster and highly efficient clearance of QS-deficient bacteria in vivo is probably a two-sided phenomenon: down regulation of virulence and activation of the innate immune system. These data also suggest that a combination of the action of PMNs and QS inhibitors along with conventional antibiotics would eliminate the biofilm-forming bacteria before a chronic infection is established.

The pathogenesis of Pseudomonas aeruginosa is at least partially attributable to its ability to synthesize and secrete the siderophore pyoverdin and the two zinc metalloproteases elastase and LasA and its ability to form biofilms in which bacterial cells are embedded in an alginate matrix. In the present study a lysophospholipid 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate [also called monopalmitoylphosphatidic acid (MPPA)] which accumulates in inflammatory exudates was shown to inhibit the extracellular accumulation of P. aeruginosa PAO1 alginate elastase LasA protease and the siderophore pyoverdin. MPPA also inhibited biofilm formation. The inhibitory effects of MPPA occur independently of rpoS expression and without affecting the accumulation of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl-L-homoserine lactone and may be due at least in part to the ability of MPPA to bind divalent cations.

Pseudomonas aeruginosa and Burkholderia cepacia are capable of forming mixed biofilms in the lungs of cystic fibrosis patients. Both bacteria employ quorum-sensing systems which rely on N-acylhomoserine lactone (AHL) signal molecules to co-ordinate expression of virulence factors with the formation of biofilms. As both bacteria utilize the same class of signal molecules the authors investigated whether communication between the species occurs. To address this issue novel Gfp-based biosensors for non-destructive in situ detection of AHLs were constructed and characterized. These sensors were used to visualize AHL-mediated communication in mixed biofilms which were cultivated either in artificial flow chambers or in alginate beads in mouse lung tissue. In both model systems B. cepacia was capable of perceiving the AHL signals produced by P. aeruginosa while the latter strain did not respond to the molecules produced by B. cepacia. Measurements of extracellular proteolytic activities of defined quorum-sensing mutants grown in media complemented with AHL extracts prepared from culture supernatants of various wild-type and mutant strains supported the view of unidirectional signalling between the two strains.

The structural organization of four microbial communities was analysed by a novel computer program COMSTAT which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria Pseudomonas putida P. aureofaciens P. fluorescens and P. aeruginosa tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure – mean thickness roughness substratum coverage and surface to volume ratio – showed that the four Pseudomonas strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum and formed flat uniform biofilms. P. aureofaciens resembled P. aeruginosa but had a stronger tendency to form micro-colonies. Finally the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0·03 mM 0·1 mM or 0·5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover biofilm roughness increased with lower citrate concentrations whereas surface to volume ratio increased with higher citrate concentrations.

The pathogenesis of Pseudomonas aeruginosa is associated with expression of virulence factors many of which are controlled by two N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Escherichia coli strains equipped with a luxR-based monitor system expressing green fluorescent protein (GFP) in the presence of exogenous AHL molecules were used to detect the production of AHLs from P. aeruginosa in vivo. Mice were challenged intratracheally with alginate beads containing P. aeruginosa and E. coli and killed on different days after the challenge. By means of confocal scanning laser microscopy GFP-expressing E. coli bacteria could be detected in the lung tissues indicating production and excretion of AHL molecules in vivo by the infecting P. aeruginosa. AHL signals were detected mainly in lung tissues exhibiting severe pathological changes. These findings support the view that expression of AHL molecules by P. aeruginosa during infection coincides with its pathogenesis.