Azithromycin in Pseudomonas aeruginosa Biofilms: Bactericidal Activity and Selection of nfxB Mutants

Abstract

Azithromycin (AZM) has shown promising results in the treatment of Pseudomonas aeruginosa chronic lung infections such as those occurring in cystic fibrosis (CF) patients. We evaluated the effect of hypermutation and alginate hyperproduction on the bactericidal activity and resistance development to AZM in P. aeruginosa biofilms. Strains PAO1, its mucA mutant (PAOMA), and their respective mutS-deficient hypermutable derivatives (PAOMS and PAOMSA) were used. Biofilms were incubated with several AZM concentrations for 1, 2, 4, or 7 days, and the numbers of viable cells were determined. During the first 2 days, AZM showed bactericidal activity for all the strains, but in extended AZM incubation for strain PAOMS and especially strain PAOMSA, a marked increased in the number of viable cells was observed, particularly at 4 g/ml. Biofilms formed by the lineages recovered from the 7-day experiments showed enhanced AZM resistance. Furthermore, most of the independent lineages studied, including those obtained from biofilms treated with AZM concentrations as low as 0.5 g/ml, showed MexCD-OprJ hyperexpression and mutations in nfxB. The role of nfxB mutation in AZM resistance was further confirmed through the characterization of nfxB and mexD knockout mutants. Results from this work show that, although AZM exhibits bactericidal activity against P. aeruginosa biofilms, resistant mutants are readily selected and that, furthermore, they frequently show cross-resistance to other unrelated antipseudomonal agents such as ciprofloxacin or cefepime but hypersusceptibility to others such as imipenem or tobramycin. Therefore, these results should help guide the selection of appropriate antipseudomonal therapies in CF patients under AZM maintenance treatment.The establishment of Pseudomonas aeruginosa chronic respiratory infection is mediated by a complex adaptive process that includes physiological changes, mainly represented by the transition from a planktonic to a biofilm mode of growth (3) and by the selection of an important number of adaptive mutations required for long-term persistence (42). The biofilm mode of growth is one of the most important factors in the persistence of chronic lung infections due to its increased resistance to the host defense mechanisms, including mechanical clearance and clearance mediated by complement, antibodies, or phagocytes, and to its inherent resistance to antibiotics (14).A common feature of P. aeruginosa chronic lung infections, including those occurring in patients suffering from cystic fibrosis (CF), bronchiectasias, or chronic obstructive pulmonary disease, is the very high prevalence (30 to 60% of patients) of hypermutable (or mutator) strains deficient in the DNA mismatch repair system in contrast to what is observed in acute infections (Ͻ1%) (2,8,23,31,32). The presence of hypermutable strains has been found to be linked to the high antibiotic resistance rates of P. aeruginosa strains isolated from patients with chronic lung infections (23, 32). It has also been shown by in vitro and in...

The Pseudomonas aeruginosa CreBC Two-Component System Plays a Major Role in the Response to β-Lactams, Fitness, Biofilm Growth, and Global Regulation

“…cDNA synthesis, fragmentation, labeling, and hybridization were performed according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. An expression analysis was performed as described previously (15,32,33). Only transcripts showing Ն2-fold increases or decreases were considered to be differentially expressed.…”

Overexpression of AmpC and Efflux Pumps in Pseudomonas aeruginosa Isolates from Bloodstream Infections: Prevalence and Impact on Resistance in a Spanish Multicenter Study

“…Mean values (Ϯ standard deviations) of mRNA levels obtained in three independent duplicate experiments were considered. Previously obtained PAO1 mutants overexpressing these mechanisms were used as controls (22,23,29).…”

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

“…The overexpression of MexCDOprJ has deleterious consequences for bacterial fitness in infections, reducing characteristics such as siderophore, protease, and phenazine production; cytotoxicity; motility; and, very significantly, virulence (220,234). In spite of these clear disadvantages, nfxB mutants are readily selected following exposure to fluoroquinolones, azithromycin, and even biocides like triclosan both in vitro and during clinical therapy (37,100,168). Another regulator is MexZ, which belongs to the TetR family and normally represses the transcription of the mexXY genes by binding to the mexZmexX intergenic region (157,264).…”