Tolerance to the antimicrobial peptide colistin in Pseudomonas aeruginosa biofilms is linked to metabolically active cells, and depends on the pmr and mexAB‐oprM genes

Abstract

SummaryBacteria living as biofilm are frequently reported to exhibit inherent tolerance to antimicrobial compounds, and might therefore contribute to the persistence of infections. Antimicrobial peptides are attracting increasing interest as new potential antimicrobial therapeutics; however, little is known about potential mechanisms, which might contribute to resistance or tolerance development towards these compounds in biofilms. Here we provide evidence that a spatially distinct subpopulation of metabolically active cells in Pseudomonas aeruginosa biofilms is able to develop tolerance to the antimicrobial peptide colistin. On the contrary, biofilm cells exhibiting low metabolic activity were killed by colistin. We demonstrate that the subpopulation of metabolically active cells is able to adapt to colistin by inducing a specific adaptation mechanism mediated by the pmr operon, as well as an unspecific adaptation mechanism mediated by the mexAB-oprM genes. Mutants defective in either pmr-mediated lipopolysaccharide modification or in mexAB-oprM-mediated antimicrobial efflux were not able to develop a tolerant subpopulation in biofilms. In contrast to the observed pattern of colistin-mediated killing in biofilms, conventional antimicrobial compounds such as ciprofloxacin and tetracycline were found to specifically kill the subpopulation of metabolically active biofilm cells, whereas the subpopulation exhibiting low metabolic activity survived the treatment. Consequently, targeting the two physiologically distinct subpopulations by combined antimicrobial treatment with either ciprofloxacin and colistin or tetracycline and colistin almost completely eradicated all biofilm cells.

New trends in peptide-based anti-biofilm strategies: a review of recent achievements and bioinformatic approaches

“…Interestingly, in S. aureus, the regulatory system GraRS, which is involved in upregulation of biofilm production, has been reported to mediate the resistance of the planktonic cells to AMPs (Herbert et al 2007). In cystic fibrosis, where P. aeruginosa biofilms cause pneumonia, results show that colistin kills the stalk subpopulation (a deeper layer with low metabolic activity) preferentially, whereas the metabolically active cap-forming subpopulation in the upper layer becomes colistin resistant due to the up-regulation of the pmr and mexAB-oprM genes (Haagensen et al 2007;Pamp et al 2008).…”

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…Reduced diffusion in combination with high cell densities results in steep chemical gradients from the outer surface towards the central core of an in vitro biofilm [26,32]. This causes a very heterogenic growth pattern throughout in vitro biofilms, which in turn influences regional tolerance towards different types of antibiotic treatments [33,34]. Tobramycin tolerance in P. aeruginosa has also been shown to be influenced by QS-systems [19].…”

“…Using continuous flow-cell conditions and Confocal Laser Scanning Microscopy (CLSM), we are closer to understanding the processes involved in the initial attachment of cells to surfaces in vitro [46][47][48]. Combining molecular techniques with CLSM to construct and visualise knock-out strains of bacteria, has shown the contribution of motility and QS to biofilm development [30,33,46,47,49].…”

Adaptive and Mutational Resistance: Role of Porins and Efflux Pumps in Drug Resistance

“…The exposure of biofilms to antibiotics also contributes to the development of adaptive resistance; in some cases, the molecular mechanisms involved differ from those of planktonic cells. For example, a challenge of P. aeruginosa biofilms with colistin triggers the adaptation of the upper layers of the biofilm through the overexpression of the lipopolysaccharide (LPS) modification (arn) operon as well as the efflux pump mexAB-oprM (194), both of which were proposed to be necessary for acquired resistance to colistin. The deeper layers of the biofilm, however, lacked the capacity to develop this response and remained sensitive to colistin.…”