Histamine Promotes Pseudomonas aeruginosa Biofilm Formation and Renders P. aeruginosa Biofilms More Resistant to Gentamicin and Azithromycin

Abstract

Introduction: Pseudomonas aeruginosa biofilms contribute to the persistent presence of this bacterium in the cystic fibrosis airways. P. aeruginosa produces histamine in vitro and expresses histamine receptors. We investigated whether histamine regulated P. aeruginosa biofilm formation in vitro and contributed to bacterial virulence in Galleria mellonella.

Methods: P. aeruginosa biofilms were measured by staining bacteria adhered on polystyrene with crystal violet. Histamine concentrations were measured by ELISA. G. mellonella survival upon inoculation with P. aeruginosa was measured in the absence or presence of histamine.

Results: The concentration of histamine in the BHI broth was 140 ng/mL (1.3 μ<sc>m</sc>). Addition to the broth of diamine oxidase (DAO), an enzyme that catabolizes histamine, reduced by ∼3-fold the concentration of histamine and by 2-fold PAO1 strain biofilms. Addition of histamine (10-9<sc>m</sc>-10-4<sc>m</sc>) to the LB medium augmented P. aeruginosa biofilms. Maximum effects were observed with concentrations of 10-5<sc>m</sc> and 10-8<sc>m</sc> for the mucoid NH57388A strain and the PAO1 strain, respectively. DAO reduced mucoid NH57388A biofilms induced by histamine (10-4<sc>m</sc>) added to the LB medium. Addition of histamine to 48 h formed biofilms reduced anti-biofilm activities of gentamicin and azithromycin. Inoculation of G. mellonella with the PAO1 strain led to augmented histamine concentration in the haemolymph. Inoculation of histamine (10-8<sc>m</sc>) reduced the survival rate of G. mellonella infected with the PAO1 strain.

Conclusion: Histamine produced during periods of infection may augment P. aeruginosa virulence by promoting the biofilm mode of life of this bacterium.

Keywords: Antibiotic resistance; Biofilms; Histamine; P. aeruginosa.

Fig. 1.

Effect of DAO on P. aeruginosa biofilm formation. a The PAO1 strain in BHI broth was added to polystyrene plates in the absence (−) or presence of DAO. After 18 h, biofilms were quantified as described in the Materials and Methods section. The data represent mean values ± SEM of 14 independent experiments. **p < 0.01 as determined by a one-way ANOVA with post hoc Bonferroni test. Ns, not significant. b The concentration of histamine in the BHI broth incubated without (−) or with DAO (10 mU/mL) for 18 h at 37°C was measured by ELISA. c Experiments similar to the ones described in (a) were performed using BHI broth-containing sodium pyruvate (10 mm). The data represent mean values ± SEM of 5 independent experiments. ***p < 0.01 as determined by a paired Student’s t test. d Experiments similar to the ones described in (a) were performed using heat inactivated DAO (10 mU/mL). The data represent mean values ± SEM of 11 independent experiments. **p < 0.01 as determined by one-way ANOVA with post hoc Bonferroni test. Ns, not significant. e The mucoid NH57388A strain in BHI broth was added to polystyrene plates in the absence (−) or presence of DAO. Biofilms were quantified as described in (a). The data represent mean values ± SEM of 8–13 independent experiments. Ns, not significant as determined by one-way ANOVA with post hoc Bonferroni test. f The mucoid NH57388A in LB medium was added to polystyrene plates in the absence (−) or presence of DAO (10 mU/mL), histamine (His) (10⁻⁴m) or a combination of DAO (10 mU/mL) and histamine (His) (10⁻⁴m). After 18 h, biofilms were quantified as described in (a). The data represent mean values ± SEM of 6 independent experiments. **p < 0.01 as determined by one-way ANOVA with post hoc Bonferroni test. Ns, not significant. g The PAO1 strain (left panel) or the mucoid strain (right panel) were incubated in BHI broth in the absence (control) or presence of DAO (10 mU/mL). After each hour, turbidimetry was read to record bacterial growth. A representative experiment is shown.

Fig. 2.

Histamine promotes biofilm formation by PAO1 and mucoid NH57388A strains. a The mucoid NH57388A strain in LB medium was incubated in polystyrene wells without (−) or with histamine (His) (10⁻⁸m–10⁻⁴m). After 18 h, biofilms were quantified as described in Fig. 1a. The data represent mean values ± SEM of 7 independent experiments. **p < 0.01 as determined by a one-way ANOVA with post hoc Bonferroni test. b Similar experiments to the one described in (a) were carried out with the PAO1 strain. The data represent mean values ± SEM of 6 independent experiments. **p < 0.01 as determined by a one-way ANOVA with post hoc Bonferroni test. c The mucoid NH57388A strain (left panel) or the PAO1 strain (right panel) were incubated in LB medium with histamine (His) (10⁻⁷m–10⁻⁴m). After each hour, turbidimetry at 600 nm was read. The OD values versus time of culture are plotted. A representative experiment is shown. Ns, not significant.

Fig. 3.

Effect of gentamicin and azithromycin on P. aeruginosa biofilms. a A schematic representation of the experimental setting. b Forty-eight hours mature mucoid NH57388A biofilms were incubated for 24 h in the absence of any effector (−), or in the presence of histamine (His) (10⁻⁴m), gentamicin (Gen) (5 µg/mL, 10 µg/mL, 20 µg/mL or 100 µg/mL), azithromycin (Azi) (50 µg/mL, 150 µg/mL, 300 µg/mL), or a combination of gentamicin (5 µg/mL, 10 µg/mL, 20 µg/mL or 100 µg/mL) and histamine (10⁻⁴m), or a combination of azithromycin (50 µg/mL, 150 µg/mL, 300 µg/mL) and histamine (10⁻⁴m). Thereafter, biofilms were quantified as described in the legends to Fig. 1a. c Similar experiments, as described in (b), were performed with the PAO1 strain. The data represent mean values ± SEM of 5–8 independent experiments. *p < 0.05, **p < 0.01 as determined by a paired Student’s t test. Ns, not significant.

Fig. 4.

Histamine produced in G. mellonella in response to P. aeruginosa infection contributes to pathogenicity. a The haemolymph of six 18 h surviving larvae inoculated with either PBS or the PAO1 strain (2 CFU) were collected and the concentration of histamine in the pooled cell-free supernatants was measured using an ELISA. The data represent mean values ± SD of triplicates. b (left panel) G. mellonella were inoculated, through the proleg, with 2 CFU of the PAO1 strain (dashed line) or with 2 CFU of the PAO1 strain and histamine (10⁻⁸m) (solid line). b (right panel), similar experiment to the one described in (b) was carried out using 5 CFU of the PAO1 strain. Larval survival (Kaplan-Meier survival analysis) was recorded after 24 h–48 h. *p < 0.05 as determined by a Mantel-Cox test.