Quorum sensing (QS) governs the production of virulence factors and the architecture and sodium dodecyl sulphate (SDS) resistance of biofilm‐grown Pseudomonas aeruginosa. P. aeruginosa QS requires two transcriptional activator proteins known as LasR and RhlR and their cognate autoinducers PAI‐1 (N‐(3‐oxododecanoyl)‐l‐homoserine lactone) and PAI‐2 (N‐butyryl‐l‐homoserine lactone) respectively. This study provides evidence of QS control of genes essential for relieving oxidative stress. Mutants devoid of one or both autoinducers were more sensitive to hydrogen peroxide and phenazine methosulphate, and some PAI mutant strains also demonstrated decreased expression of two superoxide dismutases (SODs), Mn‐SOD and Fe‐SOD, and the major catalase, KatA. The expression of sodA (encoding Mn‐SOD) was particularly dependent on PAI‐1, whereas the influence of autoinducers on Fe‐SOD and KatA levels was also apparent but not to the degree observed with Mn‐SOD. β‐Galactosidase reporter fusion results were in agreement with these findings. Also, the addition of both PAIs to suspensions of the PAI‐1/2‐deficient double mutant partially restored KatA activity, while the addition of PAI‐1 only was sufficient for full restoration of Mn‐SOD activity. In biofilm studies, catalase activity in wild‐type bacteria was significantly reduced relative to planktonic bacteria; catalase activity in the PAI mutants was reduced even further and consistent with relative differences observed between each strain grown planktonically. While wild‐type and mutant biofilms contained less catalase activity, they were more resistant to hydrogen peroxide treatment than their respective planktonic counterparts. Also, while catalase was implicated as an important factor in biofilm resistance to hydrogen peroxide insult, other unknown factors seemed potentially important, as PAI mutant biofilm sensitivity appeared not to be incrementally correlated to catalase levels.

Variations in biofilm formation by, and antibiotic resistance of, Pseudomonas aeruginosa PAO1 (wild type) and the quorum-sensing-deficient mutants PDO100 (δrhlI), JP1 (δlasI) and JP2 (δlasIδrhlI) were studied. For PAO1, the maximum-accumulation phase of biofilm formation began immediately and a plateau phase was reached after 24 h, whereas the quorum-sensing mutants showed 36–48 h lags before entering the maximum-accumulation phase. After 72 h, the cell density of the PAO1 biofilms was c. 0.8–1.2 log greater than for the mutants. On a unit protein basis, total polysaccharide production was similar for PAO1 and PDO100, whereas JP1 and JP2 biofilms accumulated only c. 36% of the PAO1 level after 72 h. Fluorescent micrographs revealed that the PAO1 biofilms were much thicker than those of the quorum-sensing-deficient mutants. In the case of the PAO1 and PDO100 biofilms, most cells were attached to the top of the biofilm layer, whereas the bottom layer consisted predominantly of polysaccharides. The JP1 and JP2 biofilms were closely packed with cells, and little polysaccharide was visible. Cells in PAO1 biofilms were little affected by kanamycin, even at 100 mg/L, whereas those in PDO100 biofilms were susceptible to the highest concentration of kanamycin (100 mg/L) but not to lower concentrations (10 and 50 mg/L). In contrast, cells in JP1 and JP2 biofilms were susceptible to kanamycin at all three concentrations.

The use of acridine orange to visualize and quantify spatial variations in growth rate within Klebsiella pneumoniae colonies and biofilm was investigated. Bacterial colonies supported on polycarbonate filter membranes were grown on R2A agar plates. Some colonies were sampled for cell enumeration, while others were cryoembedded, sectioned, and stained with the fluorescent nucleic acid stain acridine orange. Spatial patterns of fluorescent color and intensity with depth in the colony were quantified using confocal microscopy and image analysis of stained cross sections. Colonies sampled in the midexponential phase were thin (20 microns), had high average specific growth rates (> 1 h-1), and had all the cells stained bright orange. Colonies sampled after more than 24 h of growth were thick (> 200 microns) and were growing slowly (mu < 0.15 h-1). These older colonies were characterized by distinct bands of orange at the colony edges and a dark green center. Stained biofilm cross sections displayed a similar orange band at the biofilm-bulk fluid interface and a green interior. Colony-average specific growth rates, determined by calculating the local slope of the cell accumulation versus time data, were correlated with colony-average fluorescence intensities. There was no correlation between average specific growth rate and orange or green intensity individually, but growth rate did correlate with the orange:green intensity ratio (r2 = 0.57). The resulting regression was used to predict specific growth rate profiles within colonies. These profiles indicated that bacteria were growing rapidly near the air and agar interfaces and more slowly in the center of the colonies when thicker than about 30 microns. The dimension of the orange bands ranged from 10 to 30 microns, which may indicate the thickness of growing regions. The inherent variability associated with this technique suggests that it is best applied in single species systems and that the results should be regarded as qualitative in nature.

Antimicrobial photodynamic inactivation (PDI) was shown to be a promising treatment modality for microbial infections. This study explores the effect of chitosan, a polycationic biopolymer, in increasing the PDI efficacy against Gram-positive bacteria, including Staphylococcus aureus , Staphylococcus epidermidis , Streptococcus pyogenes , and methicillin-resistant S. aureus (MRSA), as well as the Gram-negative bacteria Pseudomonas aeruginosa and Acinetobacter baumannii . Chitosan at <0.1% was included in the antibacterial process either by coincubation with hematoporphyrin (Hp) and subjection to light exposure to induce the PDI effect or by addition after PDI and further incubation for 30 min. Under conditions in which Hp-PDI killed the microbe on a 2- to 4-log scale, treatment with chitosan at concentrations of as low as 0.025% for a further 30 min completely eradicated the bacteria (which were originally at ∼10 ⁸ CFU/ml). Similar results were also found with toluidine blue O (TBO)-mediated PDI in planktonic and biofilm cells. However, without PDI treatment, chitosan alone did not exert significant antimicrobial activity with 30 min of incubation, suggesting that the potentiated effect of chitosan worked after the bacterial damage induced by PDI. Further studies indicated that the potentiated PDI effect of chitosan was related to the level of PDI damage and the deacetylation level of the chitosan. These results indicate that the combination of PDI and chitosan is quite promising for eradicating microbial infections.