Nitrite modulates bacterial antibiotic susceptibility and biofilm formation in association with airway epithelial cells

Abstract

Pseudomonas aeruginosa is the major pathogenic bacteria in cystic fibrosis and other forms of bronchiectasis. Growth in antibiotic-resistant biofilms contributes to the virulence of this organism. Sodium nitrite has antimicrobial properties and has been tolerated as a nebulized compound at high concentrations in human subjects with pulmonary hypertension; however, its effects have not been evaluated on biotic biofilms or in combination with other clinically useful antibiotics. We grew P. aeruginosa on the apical surface of primary human airway epithelial cells to test the efficacy of sodium nitrite against biotic biofilms. Nitrite alone prevented 99% of biofilm growth. We then identified significant cooperative interactions between nitrite and polymyxins. For P. aeruginosa growing on primary CF airway cells, combining nitrite and colistimethate resulted in an additional log of bacterial inhibition compared to treating with either agent alone. Nitrite and colistimethate additively inhibited oxygen consumption by P. aeruginosa. Surprisingly, whereas the antimicrobial effects of nitrite in planktonic, aerated cultures are nitric oxide (NO) dependent, antimicrobial effects under other growth conditions are not. The inhibitory effect of nitrite on bacterial oxygen consumption and biofilm growth did not require NO as an intermediate as chemically scavenging NO did not block growth inhibition. These data suggest an NO-radical independent nitrosative or oxidative inhibition of respiration. The combination of nebulized sodium nitrite and colistimethate may provide a novel therapy for chronic P. aeruginosa airway infections, because sodium nitrite, unlike other antibiotic respiratory chain "poisons," can be safely nebulized at high concentration in humans.

Keywords: Biofilm; Colistimethate; Colistin; Polymyxin; Pseudomonas aeruginosa; Sodium nitrite.

Conflict of interest statement

Conflict of interest: Dr. Gladwin is listed as a co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases. Dr. Gladwin consults with Mast-Aires Pharmaceuticals on the development of a phase II proof of concept trial using inhaled nitrite for pulmonary arterial hypertension.

Copyright © 2014 Elsevier Inc. All rights reserved.

Figures

Figure 1. Nitrite prevents P. aeruginosa biofilm formation on airway epithelial cells

To test if nitrite prevented biotic biofilm formation, Pseudomonas aeruginosa biofilms were grown on CFBE-wt cells seeded onto coverslips and imaged by live cell microscopy after 6 hours of growth. DAPI (blue) staining shows the epithelial cell nuclei. (A) Untreated biofilms showed abundant growth of (GFP)-P. aeruginosa in characteristic clusters. (B) Biofilms treated with 15mM showed very few bacteria present. Volumetric projections of control (C) vs 15 mM (D) treated biofilms demonstrate the decreased bacterial biomass with 15 mM nitrite exposure. COMSTAT biomass quantification showed an 80% reduction in biomass (numbers on panels). (E) A static co-culture model was used to establish a dose-response relationship for nitrite in biofilm prevention. Increasing concentrations of sodium nitrite were added to epithelial - P. aeruginosa co-cultures following an attachment period. Bacterial colony forming units (CFU) were counted 5 hours later. All concentrations caused a statistically significant decrease in bacteria (one-way ANOVA followed by Tukey test). (F) Transepithelial electrical resistance measurements were used to assess epithelial integrity in the presence of nitrite. Resistance was not significantly different from control at 15mM and 50 mM nitrite (one-way ANOVA followed by Tukey test, p>0.05).

Figure 2. Nitrite sensitizes P. aeruginosa to polymyxins

(A) The static co-culture model was used to determine the efficacy of colistin sulfate at disrupting established P. aeruginosa biotic biofilms. P. aeruginosa was added to the apical surface of CFBE-wt cells. After 6 hours of maturation, the biofilms were treated with colistin sulfate for 90 minutes and the number of bacteria was enumerated with a CFU dilution assay. 20μg/ml colistin sulfate caused a 3-log reduction in CFUs. Planktonic time-kill assays were used to determine the interaction between nitrite and polymyxins. Subinhibitory concentrations of (B) colistin sulfate (1.5μg/ml) and (C) colistimethate (70μg/ml) showed increased antimicrobial activity against P. aeruginosa strain PA14 when combined with subinhibitory concentrations of acidified sodium nitrite. p<0.05 for polymyxin vs polymyxin + nitrite by two-way ANOVA. (D) Representative cultures showed reduced bacterial growth (turbidity) when both CMS and nitrite are present.

Figure 3. Nitrite and colistimethate additively prevent biotic biofilm formation on primary CF human airway epithelial cells

PA01 biofilms were grown on primary CF HBE cells. After attachment, developing biofilms were treated with 300mM nitrite, colistimethate 20μg/ml or a combination of the agents, p-values indicated from two-way ANOVA.

Figure 4. Increased bacterial killing with nitrite and polymyxins is not due to increased intracellular availability of nitrite

(A) Bacteria were grown in the presence of 15 mM nitrite for 5.5 hours, rinsed and exposed to colistimethate in liquid aerobic culture for 60 minutes. Nitrite pretreatment sensitized P. aeruginosa to colistimethate in planktonic culture. (B) Planktonic cultures were exposed to polymyxin b (PMB), polymyxin b nonapeptide (PMBN) and 12 mM nitrite for 5.5 hours. No significant difference was seen by one-way ANOVA between PMBN treatment with or without nitrite. PMB + nitrite was significantly lower than PMB or nitrite alone by one-way ANOVA followed by Tukey test.

Figure 5. Inhibition of respiration by nitrite and polymyxins

A) Inhibiting respiration with 0.4mM KCN or 5mM NaN3 increases polymyxin susceptibility of P. aeruginosa grown in aerobic LB culture. Colistin sulfate (COL) was used at 1.5μg/ml while colistimethate (CMS) was used at 20μg/ml (both sub inhibitory doses in PA14 under these conditions). (B) Representative tracings of bacterial oxygen consumption measured using a Clark-type electrode in LB with increasing concentrations of nitrite. (C) At low concentrations, both nitrite and CMS inhibit oxygen consumption by 20%. The combination of CMS and nitrite additively blocked oxygen uptake. Brackets show p values <0.05 from one-way ANOVA followed by Tukey test. Data shown as mean +/- SD. (D) Increased killing by nitrite and CMS in combination is not blocked by the addition of the scavenger CPTIO in planktonic culture. (E) Representative tracing of oxygen uptake from a Clark-type electrode. 15mM nitrite decreases oxygen consumption by 50%. Pretreatment with ImM CPTIO or 15μM hemoglobin did not prevent nitrite induced oxygen consumption blockade.

Figure 6. Biofilm cytostasis is nitric oxide independent

(A) Nitrite inhibited growth of PA14 in planktonic culture. (B) To confirm that nitrite induced cytostasis in planktonic cultures is nitric oxide dependent, log-phase oxygenated cultures were grown with nitrite and the nitric oxide scavengers CPTIO or oxyhemoglobin. Growth inhibition was blocked bythe addition of either agent. (C) To determine if the prevention of biofilm growth by nitrite requires nitric oxide, static epithelial- P. aeruginosa co-cultures were treated with nitrite and the nitric oxide scavenger CPTIO. Growth inhibition was unchanged by the addition of 1mM CPTIO.