Sodium nitrite blocks the activity of aminoglycosides against Pseudomonas aeruginosa biofilms

Abstract

Sodium nitrite has broad antimicrobial activity at pH 6.5, including the ability to prevent biofilm growth by Pseudomonas aeruginosa on the surfaces of airway epithelial cells. Because of its antimicrobial activity, nitrite is being investigated as an inhaled agent for chronic P. aeruginosa airway infections in cystic fibrosis patients. However, the interaction between nitrite and commonly used aminoglycosides is unknown. This paper investigates the interaction between nitrite and tobramycin in liquid culture, abiotic biofilms, and a biotic biofilm model simulating the conditions in the cystic fibrosis airway. The addition of nitrite prevented killing by aminoglycosides in liquid culture, with dose dependence between 1.5 and 15 mM. The effect was not blocked by the nitric oxide scavenger CPTIO or dependent on efflux pump activity. Nitrite shifted the biofilm minimal bactericidal concentration (MBC-biofilm) from 256 μg/ml to >1,024 μg/ml in an abiotic biofilm model. In a biotic biofilm model, the addition of 50 mM nitrite decreased the antibiofilm activity of tobramycin by up to 1.2 log. Respiratory chain inhibition recapitulated the inhibition of aminoglycoside activity by nitrite, suggesting a potential mechanism of inhibition of energy-dependent aminoglycoside uptake. In summary, sodium nitrite induces resistance to both gentamicin and tobramycin in P. aeruginosa grown in liquid culture, as an abiotic biofilm, or as a biotic biofilm.

FIG 1

In a modified time-kill assay done in LB aerobic culture, the addition of 1.5 mM to 15 mM nitrite blocked the bactericidal activity of the aminoglycosides tobramycin (A) and gentamicin (B). Brackets indicate differences with P values of <0.05 by one-way ANOVA followed by Tukey testing (n = 4 for panel A and 3 for panel B). (C) Nitrite increases the MBC-biofilm for gentamicin. At 48 h, there was robust biofilm growth as seen by crystal violet staining (inset). Biofilms were treated for 2 h with gentamicin ± 75 mM nitrite. For control biofilms, the MBC-biofilm was 256 μg/ml, while for nitrite-treated biofilms, the MBC-biofilm was >1,024 μg/ml (n = 4; data from a representative assay are shown).

FIG 2

In a modified time-kill assay done in LB aerobic culture, the addition of 400 μM KCN (A), 5 mM NaN₃ (B), or 50 μM CCCP (C) blocked the bactericidal activity of tobramycin. Brackets indicate differences with P values of <0.05 by one-way ANOVA followed by Tukey testing (n = 3 or 4).

FIG 3

(A) In liquid culture, induction of resistance to tobramycin is not blocked by addition of the nitric oxide scavenger CPTIO. (B) Induction of resistance to tobramycin by nitrite is not blocked by the addition of the efflux pump inhibitor PAβN to a log-phase aerobic culture. Concentrations used were as follows: tobramycin, 2.5 μg/ml; nitrite, 15 mM; CPTIO, 1.5 mM; and PAβN, 20 μg/ml. Brackets indicate differences that are not significant (n.s) by one-way ANOVA (n = 6 for panel A and 3 for panel B). The addition of the nitric oxide scavenger PTIO or the efflux pump inhibitor PAβN did not block the induction of resistance in abiotic PAO1 biofilms grown on microtiter dishes. (C) CFU counts from 24-h biofilms treated for 2 h and then incubated with 1% Triton to recover attached bacteria (n = 4). Differences were analyzed by two-way ANOVA followed by multiple-comparison testing (P < 0.05 for differences between the control and other conditions; differences were nonsignificant for comparisons between nitrite and PTIO or PaβN). Similar results were obtained using 48-h-old biofilms in an MBC-biofilm assay. (D) Representative MBC-biofilm plate with control growth inhibited at 256 μg/ml, while the MBC-biofilm for the other conditions was >1,024 μg/ml (n = 3; a representative image is shown). To confirm that efflux pump upregulation is not required for nitrite-induced gentamicin resistance, ΔMexY (E) and ΔMexXY (F) strains were grown as biofilms on microtiter dishes for 24 h before being treated with gentamicin and 75 mM nitrite for 2 h. Attached bacteria were recovered in 1% Triton and counted. P values were <0.05 by two-way ANOVA for differences between control and nitrite treatments.

FIG 4

Nitrite induces resistance to aminoglycosides within biofilms. PAO1 biofilms were grown on the surface of CFBE-wt cells for 6 h and then treated with nitrite (A) or nitrite and tobramycin (B) for 90 min. Brackets indicate differences with P values of <0.05 by one-way ANOVA followed by pairwise comparison testing. (C) The zone of inhibition is unchanged in biofilm bacteria exposed to nitrite compared to unexposed bacteria (n = 3; representative images are shown).