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.

ABSTRACTIn relation to emerging multiresistant bacteria, development of antimicrobials and new treatment strategies of infections should be expected to become a high-priority research area. Quorum sensing (QS), a communication system used by pathogenic bacteria likePseudomonas aeruginosato synchronize the expression of specific genes involved in pathogenicity, is a possible drug target. Previousin vitroandin vivostudies revealed a significant inhibition ofP. aeruginosaQS by crude garlic extract. By bioassay-guided fractionation of garlic extracts, we determined the primary QS inhibitor present in garlic to be ajoene, a sulfur-containing compound with potential as an antipathogenic drug. By comprehensivein vitroandin vivostudies, the effect of synthetic ajoene towardP. aeruginosawas elucidated. DNA microarray studies of ajoene-treatedP. aeruginosacultures revealed a concentration-dependent attenuation of a few but central QS-controlled virulence factors, including rhamnolipid. Furthermore, ajoene treatment ofin vitrobiofilms demonstrated a clear synergistic, antimicrobial effect with tobramycin on biofilm killing and a cease in lytic necrosis of polymorphonuclear leukocytes. Furthermore, in a mouse model of pulmonary infection, a significant clearing of infectingP. aeruginosawas detected in ajoene-treated mice compared to a nontreated control group. This study adds to the list of examples demonstrating the potential of QS-interfering compounds in the treatment of bacterial infections.

Polymorphonuclear neutrophilic leukocytes (PMNs) play a central role in innate immunity, where they dominate the response to infections, in particular in the cystic fibrosis lung. PMNs are phagocytic cells that produce a wide range of antimicrobial agents aimed at killing invading bacteria. However, the opportunistic pathogen Pseudomonas aeruginosa can evade destruction by PMNs and thus cause persistent infections. In this study, we show that biofilm cells of P. aeruginosa recognize the presence of attracted PMNs and direct this information to their fellow bacteria through the quorum sensing (QS) signalling system. The bacteria respond to the presence of PMNs by upregulating synthesis of a number of QS-controlled virulence determinants including rhamnolipids, all of which are able to cripple and eliminate cells of the host defence. Our in vitro and in vivo analyses support a 'launch a shield' model by which rhamnolipids surround the biofilm bacteria and on contact eliminate incoming PMNs. Our data strengthen the view that cross-kingdom communication plays a key role in P. aeruginosa recognition and evasion of the host defence.

No abstract

Many of the virulence factors produced by the opportunistic human pathogen Pseudomonas aeruginosa are quorum-sensing (QS) regulated. Among these are rhamnolipids, which have been shown to cause lysis of several cellular components of the human immune system, e.g. monocytederived macrophages and polymorphonuclear leukocytes (PMNs). We have previously shown that rhamnolipids produced by P. aeruginosa cause necrotic death of PMNs in vitro. This raises the possibility that rhamnolipids may function as a 'biofilm shield' in vivo, which contributes significantly to the increased tolerance of P. aeruginosa biofilms to PMNs. In the present study, we demonstrate the importance of the production of rhamnolipids in the establishment and persistence of P. aeruginosa infections, using an in vitro biofilm system, an intraperitoneal foreign-body model and a pulmonary model of P. aeruginosa infections in mice. Our experimental data showed that a P. aeruginosa strain unable to produce any detectable rhamnolipids, due to an inactivating mutation in the single QS-controlled rhlA gene, did not induce necrosis of PMNs in vitro and exhibited increased clearance compared with its wild-type counterpart in vivo. Conclusively, the results support our model that rhamnolipids are key protective agents of P. aeruginosa against PMNs. KeywordsRhamnolipid; Pseudomonas aeruginosa; mouse models; biofilm; PMN Pseudomonas aeruginosa is an opportunistic human pathogen causing serious infections in immuno-compromised individuals, and is the most frequent Gram-negative, bacterial * Contributed equally to this work. † Current address: