Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics

Abstract

Pseudomonas aeruginosa is one of the six bacterial pathogens, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp., which are commonly associated with antimicrobial resistance, and denoted by their acronym ESKAPE. P. aeruginosa is also recognized as an important cause of chronic infections due to its ability to form biofilms, where the bacteria are present in aggregates encased in a self-produced extracellular matrix and are difficult or impossible to eradicate with antibiotic treatment. P. aeruginosa causes chronic infections in the lungs of patients with cystic fibrosis and chronic obstructive lung disease, as well as chronic urinary tract infections in patients with permanent bladder catheter, and ventilator-associated pneumonia in intubated patients, and is also an important pathogen in chronic wounds. Antibiotic treatment cannot eradicate these biofilm infections due to their intrinsic antibiotic tolerance and the development of mutational antibiotic resistance. The tolerance of biofilms to antibiotics is multifactorial involving physical, physiological, and genetic determinants, whereas the antibiotic resistance of bacteria in biofilms is caused by mutations and driven by the repeated exposure of the bacteria to high levels of antibiotics. In this review, both the antimicrobial tolerance and the development of resistance to antibiotics in P. aeruginosa biofilms are discussed. Possible therapeutic approaches based on the understanding of the mechanisms involved in the tolerance and resistances of biofilms to antibiotics are also addressed.

Keywords: Pseudomonas aeruginosa; antibiotic; biofilm; resistance; tolerance.

Figure 1

Mechanisms involved in biofilm-associated tolerance against different classes of antibiotics. Beta-lactams are represented in gray ellipses, fluoroquinolones in green, aminoglycosides in blue and antimicrobial peptides (colistin) in orange. Mechanisms which confer tolerance to several classes of antibiotics are listed in the area where the ellipses overlap. Mechanisms which are specific to an antibiotic class are listed in the respective ellipse area.

Figure 2

The effect of ciprofloxacin, colistin and the combination of the two antibiotics on P. aeruginosa PAO1 biofilm subpopulations. The images show vertical sections through flow-chamber-grown P. aeruginosa biofilm microcolonies that were treated with either no antibiotic as control (A), ciprofloxacin (B), colistin (C), or ciprofloxacin and colistin (D). Live bacterial cells appear green due to expression of gfp, whereas dead bacterial cells appear red due to staining with propidium iodide (Pamp et al., 2009). Figure is adapted from Cytometry Part A 75:90–103 with permission from the John Wiley and Sons.

Figure 3

The development of nfxB mutants in 72 h-old PAO1 flow-cell biofilm during treatment with low-dose ciprofloxacin. Biofilms of PAO1 mCherry-P_CD-gfp (mCherry integrated at the attB site; mini-Tn7::P_CD-gfp inserted upstream of the glmS gene) were grown in three independent channels of flow-cell chambers for 72 h and were then treated with 0.2 μg/ml CIP for a total of 96 h. Imaging by CLSM was done every 24 h. Red color represents wild-type cells due to constitutive expression of mCherry, whereas green color shows nfxB mutants due to expression of the P_CD-gfp reporter which is upregulated specifically in these mutants. The images show orthogonal 3D biofilm views (left panel) or perspective view (right panel) with overlay of red and green channel fluorescence (Zaborskyte et al., 2016). Figure is reproduced from Antimicrobial Agents and Chemotherapy, 61, Issue 3, e02292–16 with permission from the American Society of Microbiology.