In 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.

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.

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.

Foods with health-promoting effects beyond nutritional values have been gaining increasing research focus in recent years, although not much has been published on this subject in relation to bacterial infections. With respect to treatment, a novel antimicrobial strategy, which is expected to transcend problems with selective pressures for antibiotic resistance, is to interrupt bacterial communication, also known as quorum sensing (QS), by means of signal antagonists, the so-called QS inhibitors (QSIs). Furthermore, QSI agents offer a potential solution to the deficiencies associated with use of traditional antibiotics to treat infections caused by bacterial biofilms and multidrug-resistant bacteria. Several QSIs of natural origin have been identified, and in this study, several common food products and plants were extracted and screened for QSI activity in an attempt to isolate and characterize previously unknown QSI compounds active against the common opportunistic pathogen Pseudomonas aeruginosa . Several extracts displayed activity, but horseradish exhibited the highest activity. Chromatographic separation led to the isolation of a potent QSI compound that was identified by liquid chromatography-diode array detector-mass spectrometry (LC-DAD-MS) and nuclear magnetic resonance (NMR) spectroscopy as iberin—an isothiocyanate produced by many members of the Brassicaceae family. Real-time PCR (RT-PCR) and DNA microarray studies showed that iberin specifically blocks expression of QS-regulated genes in P. aeruginosa .

Quorum sensing (QS)-deficient Pseudomonas aeruginosa biofilms formed in vitro are more susceptible to tobramycin than QS-proficient P. aeruginosa biofilms, and combination treatment with a QS inhibitor (QSI) and tobramycin shows synergistic effects on the killing of in vitro biofilms. We extended these results to an in vivo P. aeruginosa foreign-body biofilm model. The effect of treatment initiated prophylactically was compared with treatment initiated 11 days post-insertion. Silicone tube implants pre-colonized with wild-type P. aeruginosa were inserted into the peritoneal cavity of BALB/c mice. Mice were treated with intraperitoneal or subcutaneous injections of the QSIs furanone C-30, ajoene or horseradish juice extract in combination with tobramycin. Mice were euthanized on day 1, 2, 3 or 14 post-infection for the estimation of quantitative bacteriology, histopathology and cytokine measurements. Combination treatment of P. aeruginosa resulted in a significantly lower cfu per implant as compared with the placebo groups for all QSIs tested. For early-initiated treatment, a significant difference in clearing was also observed between the combination group and the single-treatment groups, and between the placebo group and the single-treatment groups. In one case a significant difference in clearing was found between the two single-treatment groups. Synergistic antimicrobial efficacy could be achieved when treating mice with both a QSI and tobramycin, resulting in an increased clearance of P. aeruginosa in a foreign-body infection model. Our study highlights the important prospects in developing an early combinatory treatment strategy for chronic infections.