Regulation Of Virulence-relevant Genes By Sub-inhibitory Concentrations Of Antibiotics In Pseudomonas Aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen. It can cause seriously acute and chronic infections in human beings, and is the third leading pathogen in nosocomial infections. Although some antibiotics are effective against P. aeruginosa, it bears innate resistance to many of the clinical important drugs. There is strong need to develop new anti-Pseudomonad drugs.Antibiotics are chemical substances secreted by microorganisms. Their well-known function is killing or inhibition of other microorganisms, however, it is now known antibiotics also possess many other biological activities. Under sub-inhibitory concentrations antibiotics can act as chemical signals playing an important role in regulating gene expression. In this study, we analyzed the regulation of P. aeruginosa virulence factors by sub-inhibitory antibiotics. We constructed promoter-lux reporter fusions on a low-copy-number plasmid to monitor the expression of virulent factors under different conditions in real-time. The results obtained indicate that sub-inhibitory antibiotics such as Tc (1/4 MIC and 1/8 MIC) have strong effect on the expression of the 31 virulence-related genes. Among these genes, 21 were activated to different levels by the above mentioned antibiotics. The highest activation was about 10-fold compared to the control. In order to find out the pathways of observed regulation, a reporter strain that contains the phzA1 fusion plasmid, pKD-phzA1, was subject to transposon mutagenesis. Mutants that showed different regulation profiles compared to the wild type reporter strain were screened for, and 53 mutants were collected. Some of these mutants displayed no regulation by Tetracycline and some showed changed regulation by sub-inhibitory concentrations of Tetracycline. Using arbitrary PCR, the interrupted genes in these mutants were identified, some of them seem to be novel transcriptional regulators. And one of the regulation pathways appears to be through the quorum-sensing pathways. These results indicate that there are complex pathways that sub-inhibitory antibiotics exert their regulatory effects on the responsive genes, and virulence is clearly affected by low concentration antibiotics. Uncovering these pathways would certainly help understanding the action and resistance of antibiotic, and the developing of new ones.