Michael Givskov

Phenotypes of Non-Attached Pseudomonas aeruginosa Aggregates Resemble Surface Attached Biofilm

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.

Quorum Sensing Regulation in Aeromonas hydrophila

We present detailed results on the C4-HSL-mediated quorum sensing (QS) regulatory system of the opportunistic Gram-negative bacterium Aeromonas hydrophila. This bacterium contains a particularly simple QS system that allows for a detailed modeling of kinetics. In a model system (i.e., the Escherichia coli monitor strain MH205), the C4-HSL production of A. hydrophila is interrupted by fusion of gfp(ASV). In the present in vitro study, we measure the response of the QS regulatory ahyRI locus in the monitor strain to predetermined concentrations of C4-HSL signal molecules. A minimal kinetic model describes the data well. It can be solved analytically, providing substantial insight into the QS mechanism: at high concentrations of signal molecules, a slow decay of the activated regulator sets the timescale for the QS regulation loop. Slow saturation ensures that, in an A. hydrophila cell, the QS system is activated only by signal molecules produced by other A. hydrophila cells. Separate information on the ahyR and ahyI loci can be extracted, thus allowing the probe to be used in identifying the target when testing QS inhibitors.

Virulence of mucoid Pseudomonas aeruginosa strains in cystic fibrosis: New sequential infection model

Macrolides long-term therapy has demonstrated beneficial effects on pulmonary status in CF patients, but there is a lack of literature on microbiological variations possibly associated with it. We studied lung function (FEV1%), nutritional status (BMI) and sputum culture in 65 CF patients (32 males, mean age 29.11 years), in whom long-term Azithromycin therapy (250–500mg/die, 3/7 days a week, for at least 1 year) was performed. Moreover we compared, for sputum cultures results, 10 patients (Group A: 4 males, mean age 23.5 years, range 12.5−44, Sputum culture: S.a.oxa S in 7/10, S.a. oxa R in 1/10, S.a.+ P.a. in 2/10) out of these series, with 10 CF patients (Group B) matched for age and sputum culture, in whom long-term Azithromycin therapy was never performed. In 44/65 patients (68%) mean BMI improved (T0: 19.64; T12: 19.83); in 41/64 patients (63%) mean FEV1% increased (T0: 56.17%; T12: 60.21%); in 20/65 patients sputum bacteria showed new multiresistance antibiotic pattern and in 14/65 (21.5%) sputum cultures exhibited small colony variant (SCV) as new bacterial phenotypes: S.a. in 9 pts, P.a. in 6 pts and mucoid P.a. in 1 pt. The sputum culture comparison between Group A and Group B showed no difference on new multiresistence antibiotic bacteria developement, but 2 SCV S.a. and 1 SCV P.a. in Group A and no SCV bacteria in Group B were isolated. In conclusion our data, according to international leterature, demonstrate that long-term Macrolides therapy have a positive impact on respiratory function and nutritional status in CF, but we hypothesize that this therapy could be associated with SCV bacteria development in CF sputa.