Traditional treatment of infectious diseases is based on compounds that kill or inhibit growth of bacteria. A major concern with this approach is the frequent development of resistance to antibiotics. The discovery of communication systems (quorum sensing systems) regulating bacterial virulence has afforded a novel opportunity to control infectious bacteria without interfering with growth. Compounds that can override communication signals have been found in the marine environment. Using Pseudomonas aeruginosa PAO1 as an example of an opportunistic human pathogen, we show that a synthetic derivate of natural furanone compounds can act as a potent antagonist of bacterial quorum sensing. We employed GeneChip â microarray technology to identify furanone target genes and to map the quorum sensing regulon. The transcriptome analysis showed that the furanone drug speci®c-ally targeted quorum sensing systems and inhibited virulence factor expression. Application of the drug to P.aeruginosa bio®lms increased bacterial susceptibility to tobramycin and SDS. In a mouse pulmonary infection model, the drug inhibited quorum sensing of the infecting bacteria and promoted their clearance by the mouse immune response.

Formation of biofilm is a survival strategy for bacteria and fungi to adapt to their living environment, especially in the hostile environment. Under the protection of biofilm, microbial cells in biofilm become tolerant and resistant to antibiotics and the immune responses, which increases the difficulties for the clinical treatment of biofilm infections. Clinical and laboratory investigations demonstrated a perspicuous correlation between biofilm infection and medical foreign bodies or indwelling devices. Clinical observations and experimental studies indicated clearly that antibiotic treatment alone is in most cases insufficient to eradicate biofilm infections. Therefore, to effectively treat biofilm infections with currently available antibiotics and evaluate the outcomes become important and urgent for clinicians. The review summarizes the latest progress in treatment of clinical biofilm infections and scientific investigations, discusses the diagnosis and treatment of different biofilm infections and introduces the promising laboratory progress, which may contribute to prevention or cure of biofilm infections. We conclude that, an efficient treatment of biofilm infections needs a well-established multidisciplinary collaboration, which includes removal of the infected foreign bodies, selection of biofilm-active, sensitive and well-penetrating antibiotics, systemic or topical antibiotic administration in high dosage and combinations, and administration of anti-quorum sensing or biofilm dispersal agents.

Bacteria survive in nature by forming biofilms on surfaces and probably most, if not all, bacteria (and fungi) are capable of forming biofilms. A biofilm is a structured consortium of bacteria embedded in a self-produced polymer matrix consisting of polysaccharide, protein and extracellular DNA. Bacterial biofilms are resistant to antibiotics, disinfectant chemicals and to phagocytosis and other components of the innate and adaptive inflammatory defense system of the body. It is known, for example, that persistence of staphylococcal infections related to foreign bodies is due to biofilm formation. Likewise, chronic Pseudomonas aeruginosa lung infections in cystic fibrosis patients are caused by biofilm growing mucoid strains. Gradients of nutrients and oxygen exist from the top to the bottom of biofilms and the bacterial cells located in nutrient poor areas have decreased metabolic activity and increased doubling times. These more or less dormant cells are therefore responsible for some of the tolerance to antibiotics. Biofilm growth is associated with an increased level of mutations. Bacteria in biofilms communicate by means of molecules, which activates certain genes responsible for production of virulence factors and, to some extent, biofilm structure. This phenomenon is called quorum sensing and depends upon the concentration of the quorum sensing molecules in a certain niche, which depends on the number of the bacteria. Biofilms can be prevented by antibiotic prophylaxis or early aggressive antibiotic therapy and they can be treated by chronic suppressive antibiotic therapy. Promising strategies may include the use of compounds which can dissolve the biofilm matrix and quorum sensing inhibitors, which increases biofilm susceptibility to antibiotics and phagocytosis.

Synthetic furanone compounds inhibited bacterial quorum-sensing in P. aeruginosa and exhibited favourable therapeutic effects on P. aeruginosa lung infection.

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In Pseudomonas aeruginosa the production of multiple virulence factors depends on cell-to-cell communication through the integration of N-acylhomoserine lactone (AHL)-and 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS)-dependent signalling. Mutation of genes encoding the efflux protein MexI and the porin OpmD from the MexGHI-OpmD pump resulted in the inability to produce N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) and PQS and a marked reduction in N-butanoyl-L-homoserine lactone levels. Both pump mutants were impaired in growth and exhibited enhanced rather than reduced antibiotic resistance. Provision of exogenous PQS improved growth and restored AHL and virulence factor production as well as antibiotic susceptibility, indicating that the pump mutants retained their capacity to respond to PQS. RT-PCR analysis indicated that expression of the PQS biosynthetic genes, phnA and pqsA, was inhibited when the mutants reached stationary phase, suggesting that the pleiotropic phenotype observed may be due to intracellular accumulation of a toxic PQS precursor. To explore this hypothesis, double mexI phnA (unable to produce anthranilate, the precursor of PQS) and mexI pqsA mutants were constructed; the improved growth of the former suggested that the toxic compound is likely to be anthranilate or a metabolite of it. Mutations in mexI and opmD also resulted in the attenuation of virulence in rat and plant infection models. In plants, addition of PQS restored the virulence of mexI and opmD mutants. Collectively, these results demonstrate an essential function for the MexGHI-OpmD pump in facilitating cell-to-cell communication, antibiotic susceptibility and promoting virulence and growth in P. aeruginosa. INTRODUCTIONPseudomonas aeruginosa is an ubiquitous Gram-negative c-proteobacterium capable of causing disease in humans, animals and plants (Cao et al., 2001a;Lyczak et al., 2002). P. aeruginosa produces a vast array of extracellular virulence factors, the majority of which are regulated both in a cell population density-dependent manner, via cell-to-cell communication or 'quorum sensing' Withers et al., 2001;Bassler, 2002;Smith & Iglewski, 2003), and in a growth phase-dependent manner (Diggle et al., 2002(Diggle et al., , 2003. P. aeruginosa possesses two N-acylhomoserine lactone (AHL)-dependent quorum sensing systems (reviewed by Winzer & Williams, 2001; Cámara et al., 2002). The las system comprises the N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL) synthase, LasI and its cognate response regulator LasR, whilst the rhl system involves the N-butanoyl-L-homoserine lactone (C4-HSL) synthase and the response regulator RhlR (Winzer & Williams, 2001; Cámara et al., 2002). The las system regulates the production of extracellular virulence determinants such as elastase, the LasA protease, alkaline protease and exotoxin A (Winzer & Williams, 2001; Cámara et al., 2002). It also plays a role in controlling the expression of the xcpP and xcpR genes involved in the regulation of the Abbreviations: AHL, N-acylhomos...

The consequences of O-acetylated alginate-producing Pseudomonas aeruginosa biofilms in the lungs of chronically infected cystic fibrosis (CF) patients are tolerance to both antibiotic treatments and effects on the innate and the adaptive defense mechanisms. In clinical trials, azithromycin (AZM) has been shown to improve the lung function of CF patients. The present study was conducted in accordance with previous in vitro studies suggesting that the effect of AZM may be the inhibition of alginate production, blockage of quorum sensing (QS), and increased sensitivity to hydrogen peroxide and the complement system. Moreover, we show that AZM may affect the polymerization of P. aeruginosa alginate by the incomplete precipitation of polymerized alginate and high levels of readily dialyzable uronic acids. In addition, we find that mucoid bacteria in the stationary growth phase became sensitive to AZM, whereas cells in the exponential phase did not. Interestingly, AZM-treated P. aeruginosa lasI mutants appeared to be particularly resistant to serum, whereas bacteria with a functional QS system did not. We show in a CF mouse model of chronic P. aeruginosa lung infection that AZM treatment results in the suppression of QS-regulated virulence factors, significantly improves the clearance of P. aeruginosa alginate biofilms, and reduces the severity of the lung pathology compared to that in control mice. We conclude that AZM attenuates the virulence of P. aeruginosa, impairs its ability to form fully polymerized alginate biofilms, and increases its sensitivity to complement and stationary-phase killing, which may explain the clinical efficacy of AZM.Mucoid Pseudomonas aeruginosa plays an important role in chronic lung infections among patients with CF due to its ability to form alginate-producing biofilms, which enable the bacteria to resist treatments with antibiotics and which affect the actions of the cells of the immune system (23). Macrolides such as azithromycin (AZM) are normally used against grampositive bacteria, whereas many gram-negative rods, such as Pseudomonas spp., are inherently resistant to macrolides (56). However, several clinical studies from Japan (36, 39, 65) have highlighted the beneficial effects of different macrolides, including AZM, in the treatment of patients with diffuse panbronchiolitis, a disease which is very similar to CF. The first indication that AZM therapy could also benefit the lung function in CF patients was given by Jaffe et al. (27) and was subsequently confirmed by other investigators (9,16,58,74). The mechanisms of action macrolides are multiple and may include an anti-inflammatory effect (36, 40), inhibition of a key enzyme in the alginate synthesis pathway (47), modulation of the production of quorum-sensing (QS) bacterial virulence factors (33,44,69), and/or inhibition of protein synthesis after prolonged exposure (67, 68).In P. aeruginosa, the production of several virulence factors is regulated by cell-to-cell communication, based on the lasand rhl-encoded QS systems (6), w...

The time course of activity of colistin and imipenem against mucoid and nonmucoid Pseudomonas aeruginosa growing in a biofilm showed that compared with those for planktonic bacteria, the kinetics of colistin and imipenem retained the concentration-and time-dependent killing, respectively, but higher doses of antibiotics and longer dosing periods were required for biofilm eradication. Biofilms of mucoid P. aeruginosa were more difficult to eradicate than nonmucoid biofilms.