Increased susceptibility to azithromycin of Pseudomonas aeruginosa biofilms using RPMI 1640 testing media

Abstract

Azithromycin (AZM) is efficient for treatment of chronic Pseudomonas aeruginosa biofilm lung infections, despite of resistance in conventional susceptibility testing. It has been shown that planktonic P. aeruginosa are more susceptible to AZM when tested in RPMI 1640 medium. The aim of the study was to test the susceptibility to AZM of P. aeruginosa biofilms in LB vs RPMI 1640 media. We investigated the effect of AZM on planktonic and biofilms of (WT) P. aeruginosa (PAO1), the hypermutable (ΔmutS) and the antibiotic-resistant phenotype(ΔnfxB) mutants. The effect of AZM on young and mature biofilms was investigated in the modified Calgary Biofilm Device by estimation of the minimal biofilm inhibitory concentration (MBIC). The AZM MBIC₉₀ in LB/RPMI1640 on young biofilms treated for 24 h was 16/4 μg/mL for PAO1, 32/8 μg/mL for ΔmutS, and 256/16 μg/mL for ΔnfxB, while in mature biofilms was 256/2 μg/mL for PAO1 and ΔmutS and 16/1 μg/mL for ΔnfxB. The effect of AZM was improved when the treatment was prolonged to 72 h, supporting the intracellular accumulation of AZM. An increased susceptibility of P. aeruginosa biofilms to AZM was observed in RPMI 1640 than in LB medium. Our results might improve susceptibility testing and dosing of AZM for treatment of biofilm infections.

Keywords: Pseudomonas aeruginosa; RPMI‐1640; azithromycin; biofilm.

Fig. 1

Planktonic growth curves in LB and RPMI 1640 media. The graph shows the absorbance of planktonic cultures of PAO1 (wt1 and wt2), PAOΔmutS, and PAOΔnfxB. In order to get the logarithmic curves, the initial values, that were recorded as 0, were counted as 0.000001. Statistical significance was determined using a two‐way ANOVA and Sidak's test for multiple comparisons. Six replicates for each condition were performed. ns, nonsignificant, *** p≤0.001.

Fig. 2

MIC AZM on LB (A) and RPMI‐1640 (B) of Pseudomonas aeruginosa PAO1ΔmutS. MIC ciprofloxacin on LB (C) and RPMI 1640 (D) of P. aeruginosa PAOΔnfxB.

Fig. 3

Planktonic growth curves of PAO1‐wt1 (A & B) and wt2 (E & F), PAOΔmutS (C & D), and PAOΔnfxB (G & H) with different azithromycin concentrations in LB medium (left) and RPMI 1640 medium (right). Statistical significance was determined using a two‐way ANOVA and Sidak's test for multiple comparisons. Six replicates for each condition were performed.

Fig. 4

Biofilm formation on modified Calgary Device of the Pseudomonas aeruginosa strains during 72 h incubation. The chart shows the absorbance of the CV that stained the biofilm biomass on the pegs. The formation was measured in PAO1 (wt1 and wt2), PAOΔmutS, and PAOΔnfxB. CV staining means for arbitrary units (OD). The bacteria were cultured in LB medium without antibiotic. Statistical significance was determined using a two‐way ANOVA and Sidak's test for multiple comparisons. Mean with SEM. Six replicates for each condition were performed. **p≤0.01; ***p≤ 0.001.