Tobramycin at subinhibitory concentration inhibits the RhlI/R quorum sensing system in a Pseudomonas aeruginosa environmental isolate

Abstract

Background: Antibiotics are not only small molecules with therapeutic activity in killing or inhibiting microbial growth, but can also act as signaling molecules affecting gene expression in bacterial communities. A few studies have demonstrated the effect of tobramycin as a signal molecule on gene expression at the transcriptional level and its effect on bacterial physiology and virulence. These have shown that subinhibitory concentrations (SICs) of tobramycin induce biofilm formation and enhance the capabilities of P. aeruginosa to colonize specific environments.

Methods: Environmental P. aeruginosa strain PUPa3 was grown in the presence of different concentrations of tobramycin and it was determined at which highest concentration SIC, growth, total protein levels and translation efficiency were not affected. At SIC it was then established if phenotypes related to cell-cell signaling known as quorum sensing were altered.

Results: In this study it was determined whether tobramycin sensing/response at SICs was affecting the two independent AHL QS systems in an environmental P. aeruginosa strain. It is reasonable to assume that P. aeruginosa encounters tobramycin in nature since it is produced by niche mate Streptomyces tenebrarius. It was established that SICs of tobramycin inhibited the RhlI/R system by reducing levels of C4-HSL production. This effect was not due to a decrease of rhlI transcription and required tobramycin-ribosome interaction.

Conclusions: Tobramycin signaling in P. aeruginosa occurs and different strains can have a different response. Understanding the tobramycin response by an environmental P. aeruginosa will highlight possible inter-species signalling taking place in nature and can possible also have important implications in the mode of utilization for human use of this very important antibiotic.

Figure 1

Effect of subinhibitory concentration of tobramycin on swarming motility in P. aeruginosa PUPa3. a) Swarming of P. aeruginosa PUPa3 on 0.5% LB agar plates at 37°C after 24 hrs. WT swarms (A), but in presence of SIC of tobramycin (0.05 μg/mL) cannot swarm (B). Swarming was not present in the rhlI mutant (C) nor in double mutant lasI/rhlI (D). b) Swarming of P. aeruginosa PUPa3 on 0.5% LB agar plates at 37°C after 40 hrs. Normal swarming occurs in WT (A), no swarming is present in WT on plate supplemented with SIC of tobramycin (B) and swarming was restored upon the addition of 2 μM exogenous synthetic C4-HSL (C). Swarming in the rhlI mutant was also restored upon the addition of 2 μM exogenous synthetic C4-HSL (D), but not upon the addition of 2 μM exogenous synthetic 3-oxo-C12-HSL (E).

Figure 2

Effect of subinhibitory concentration of tobramycin on biofilm growth. Biofilm formation was reduced when PUPa3 was grown under subinhibitory concentration of tobramycin (Tob). Biofilm formation was rescued by adding 2 μM exogenous C4-HSL (Tob + C4HSL).

Figure 3

Pyocyanin production in PUPa3 in early stationary growth phase. All strains were grown in LB media. Presence of SIC of tobramycin in wild type significantly decreases levels of pyocyanin production (P < 0.05). Values shown in graph are means from three independent biological experiments, normalized to culture density (OD₆₀₀). WT, is strain PUPa3; Tob, is strain PUPa3 grown in the presence of SIC of tobramycin (0.05 μg/mL); Tob C4HSL, is strain PUPa3 grown in the presence of SIC of tobramycin (0.05 μg/mL) and 2 μM of C4-HSL; rhlI, is the rhlI knock-out mutant of strain PUPa3; rhlI C4HSL, is the rhlI knock-out mutant of strain PUPa3 grown in the presence of exogenously added 2 μM of C4-HSL.

Figure 4

Proteolytic activities determined in the appropriate indicator LB plates. The P. aeruginosa lasI mutant (A) showed four-fold reduced proteolytic activity compared to WT (B). WT shows no difference in proteolytic activity in the presence of 0.05 μg/mL SIC of tobramycin (C).

Figure 5

lasI and rhlI gene promoter activities and quantitative determination of 3-oxo-C12-HSL. a)lasI gene promoter activities measured via β-galactosidase assay in PUPa3 in different stages of growth. The white colorless bars represents the control culture P. aeruginosa PUPa3 with the vector pMP220; light grey represents P. aeruginosa PUPa3 harboring pLASI; dark grey, represents P. aeruginosa PUPa3 harboring pLASI with addition of SIC of tobramycin (0.05 μg/mL). b) rhlI gene promoter activities measured via β-galactosidase assay in PUPa3 in different stages of growth. The white colorless bars represents the control culture P. aeruginosa PUPa3 with the vector pMP220; light grey represents P. aeruginosa PUPa3 harboring pRHLI; dark grey, represents P. aeruginosa PUPa3 harboring pRHLI with addition of SIC of tobramycin (0.05 μg/mL). The β-galactosidase activities were performed at different stages of growth: assay 1 was performed during log phase, assay 2 during early stationary phase, assay 3 and assay 4 during stationary phase. Each assay was performed in biological triplicates. Error bars represent the standard deviations calculated from at least three separate experiments.

Figure 6

C4-HSL and 3-oxo-C12-HSL quantification. a) C4-HSL quantification using two E. coli luxCDABE specific C4-HSL biosensors harbored in pSB406 and pSB536. Bioluminescence induced by AHL extracts of PUPa3 corresponding to 7 × 10⁹ cells grown in presence of SIC of tobramycin (0.05 μg/mL) at 37°C was defined as the percentage relative to the same strain without antibiotic (white bar). Each assay was an independent experiment measured in triplicates. +/- represents extract from PUPa3 grown in presence or absence of SIC of tobramycin added to biosensor culture. b) 3-oxo-C12-HSL quantification: the columns represent the 3-oxo-C12-HSL quantification determined by using the biosensor P. putida SM17 (pRSAL220). E represents 5 ml of biosensor culture supplemented with 5 μl ethyl-acetate, +/- addition of 5 μl of AHL extract of PUPa3 (equivalent to 9.2 × 10⁹ cells) grown in presence or absence of SIC of tobramycin (0.05 μg/mL).

Figure 7

Swarming of P. aeruginosa PUPa3 on 0.5% LB agar plates at 37°C after 24 hrs. In the presence of SIC tobramycin (0.05 μg/mL) no swarming is present in WT (B), but the swarming was restored with the expression of RmtA (C), RmtC (D) or NpmA (E) methyltransferases as in LB plates inoculated with WT (A).