Cephalosporin-3'-Diazeniumdiolate NO Donor Prodrug PYRRO-C3D Enhances Azithromycin Susceptibility of Nontypeable Haemophilus influenzae Biofilms

Abstract

PYRRO-C3D is a cephalosporin-3-diazeniumdiolate nitric oxide (NO) donor prodrug designed to selectively deliver NO to bacterial infection sites. The objective of this study was to assess the activity of PYRRO-C3D against nontypeable Haemophilus influenzae (NTHi) biofilms and examine the role of NO in reducing biofilm-associated antibiotic tolerance. The activity of PYRRO-C3D on in vitro NTHi biofilms was assessed through CFU enumeration and confocal microscopy. NO release measurements were performed using an ISO-NO probe. NTHi biofilms grown on primary ciliated respiratory epithelia at an air-liquid interface were used to investigate the effects of PYRRO-C3D in the presence of host tissue. Label-free liquid chromatography-mass spectrometry (LC/MS) proteomic analyses were performed to identify differentially expressed proteins following NO treatment. PYRRO-C3D specifically released NO in the presence of NTHi, while no evidence of spontaneous NO release was observed when the compound was exposed to primary epithelial cells. NTHi lacking β-lactamase activity failed to trigger NO release. Treatment significantly increased the susceptibility of in vitro NTHi biofilms to azithromycin, causing a log fold reduction (10-fold reduction or 1-log-unit reduction) in viability (P < 0.05) relative to azithromycin alone. The response was more pronounced for biofilms grown on primary respiratory epithelia, where a 2-log-unit reduction was observed (P < 0.01). Label-free proteomics showed that NO increased expression of 16 proteins involved in metabolic and transcriptional/translational functions. NO release from PYRRO-C3D enhances the efficacy of azithromycin against NTHi biofilms, putatively via modulation of NTHi metabolic activity. Adjunctive therapy with NO mediated through PYRRO-C3D represents a promising approach for reducing biofilm-associated antibiotic tolerance.

Keywords: Haemophilus influenzae; antibiotic resistance; biofilms; nitric oxide; proteomics.

FIG 1

(a) Structure of PYRRO-C3D and NO release mechanism following β-lactam ring cleavage by β-lactamase. (b) NO release from PYRRO-C3D (50 μM in PBS) following activation with β-lactamase (penicillinase).

FIG 2

PYRRO-C3D elicits a direct antibacterial effect on planktonic NTHi. (a) Planktonic NTHi growth in the presence of PYRRO-C3D measured by absorbance (OD₆₀₀; n = 4). (b) Viability of 72-h in vitro NTHi biofilm following 2-h treatment with PYRRO-C3D as measured by CFU enumeration (n = 4). (c) NO release from 50 μM PYRRO-C3D in the presence of planktonic NTHi. The signal was recorded over 15 min before quenching with the β-lactamase inhibitor clavulanate (n = 2).

FIG 3

PYRRO-C3D increases NTHi in vitro biofilm susceptibility toward azithromycin treatment. (a and b) NTHi biofilms grown in vitro for 72 h and treated with 50 μM PYRRO-C3D and 4 mg/ml azithromycin (Azithro), both individually and in combination, for 2 h were assessed for viability through CFU enumeration (a) and COMSTAT analysis (b) of live stained bacteria (n = 5). (c and d) Viability of the supernatant population following treatment measured by CFU enumeration (c) and maximum biofilm thickness measured by confocal microscopy (d). Values that were statistically significantly different are indicated by bars and asterisks as follows: *, P ≤ 0.05; **, P < 0.01; ***, P ≤ 0.001. Values were not statistically significantly different (ns) are also indicated.

FIG 4

PYRRO-C3D treatment increases NTHi biofilm density. Confocal images of NTHi biofilms treated with 50 μM PYRRO-C3D and 4 mg/ml azithromycin, both individually and in combination, for 2 h were processed using COMSTAT software to calculate the average diffusion distance between live bacteria within biofilms. *, P ≤ 0.05.

FIG 5

Response of NTHi biofilms to PYRRO-C3D is NO mediated. (a and b) Viability of 72-h in vitro NTHi biofilms following 2 h treatment with 50 μM DEA/NO, cephaloram, and PYRRO-C3D (a) and 50 μM cPTIO and clavulanate (Clav) (b), both individually and in combination with 4 mg/ml azithromycin for 2 h. Viability was measured by CFU enumeration. *, P ≤ 0.05.

FIG 6

NO release from PYRRO-C3D is dependent on NTHi β-lactamase production. Viability of 72-h in vitro biofilms formed by β-lactamase-producing (HI6) and non-β-lactamase-producing (HI5) NTHi isolates following treatment with 50 μM PYRRO-C3D and 4 mg/ml azithromycin, both individually and in combination for 2 h, as assessed by CFU enumeration (n = 5). **, P ≤ 0.01; ns, not significant.

FIG 7

PYRRO-C3D treatment increases azithromycin susceptibility of NTHi biofilms grown on primary respiratory epithelial cells. (a) Measurement of NO release from 50 μM PYRRO-C3D in the presence of primary respiratory epithelial cells isolated from the air-liquid interface (ALI) before and after activation with 10 U of β-lactamase (β-lact) (penicillinase). (b) Viability of 72-h NTHi biofilms grown at the ALI on primary respiratory epithelial cells following 2-h treatment with 50 μM PYRRO-C3D and 4 mg/ml azithromycin, both alone and in combination, as assessed by CFU enumeration (n = 5). *, P ≤ 0.05; **, P ≤ 0.01. (c) SEM image of a 72-h NTHi biofilm formed at an ALI on primary respiratory epithelial cells. Magnification of ×5,000.