Glutathione-Disrupted Biofilms of Clinical Pseudomonas aeruginosa Strains Exhibit an Enhanced Antibiotic Effect and a Novel Biofilm Transcriptome

Abstract

bPseudomonas aeruginosa infections result in high morbidity and mortality rates for individuals with cystic fibrosis (CF), with premature death often occurring. These infections are complicated by the formation of biofilms in the sputum. Antibiotic therapy is stymied by antibiotic resistance of the biofilm matrix, making novel antibiofilm strategies highly desirable. Within P. aeruginosa biofilms, the redox factor pyocyanin enhances biofilm integrity by intercalating with extracellular DNA. The antioxidant glutathione (GSH) reacts with pyocyanin, disrupting intercalation. This study investigated GSH disruption by assaying the physiological effects of GSH and DNase I on biofilms of clinical CF isolates grown in CF artificial sputum medium (ASMDM؉). Confocal scanning laser microscopy showed that 2 mM GSH, alone or combined with DNase I, significantly disrupted immature (24-h) biofilms of Australian epidemic strain (AES) isogens AES-1R and AES-1M. GSH alone greatly disrupted mature (72-h) AES-1R biofilms, resulting in significant differential expression of 587 genes, as indicated by RNA-sequencing (RNA-seq) analysis. Upregulated systems included cyclic diguanylate and pyoverdine biosynthesis, the type VI secretion system, nitrate metabolism, and translational machinery. Biofilm disruption with GSH revealed a cellular physiology distinct from those of mature and dispersed biofilms. RNA-seq results were validated by biochemical and quantitative PCR assays. Biofilms of a range of CF isolates disrupted with GSH and DNase I were significantly more susceptible to ciprofloxacin, and increased antibiotic effectiveness was achieved by increasing the GSH concentration. This study demonstrated that GSH, alone or with DNase I, represents an effective antibiofilm treatment when combined with appropriate antibiotics, pending in vivo studies.

Identification of α-tocopherol as a bioactive component ofDicranopteris lineariswith disrupting property against preformed biofilm ofStaphylococcus aureus

“…In Pseudomonas aeruginosa biofilms, pyocyanin intercalates directly with extracellular DNA, to confer structural integrity to the biofilm. The glutathione reacts with pyocyanin, directly interfering with pyocyanin's ability to intercalate with extracellular DNA and thus, resulting in the disruption of biofilms (Klare et al 2016). Besides that, a potent thiol-containing antioxidant, N-acetyl cysteine (NAC) have been reported to decrease biofilm formation of Enterococcus faecalis, Escherichia coli and Klebsiella pneumoniae, and may reduce the production of extracellular polysaccharide matrix while promoting the disruption of mature biofilm (Silveira et al 2013;Dinicola et al 2014).…”

Pseudomonas aeruginosa Extracellular Secreted Molecules Have a Dominant Role in Biofilm Development and Bacterial Virulence in Cystic Fibrosis Lung Infections

“…GSH alone was shown to have a significant effect on disruption of mature 72-h-old biofilms of the epidemic isolate AES-1R grown in ASMDM+, while the combined treatment with GSH and DNase I of biofilms from a range of CF isolates showed greater disruption and significantly increased susceptibility to ciprofloxacin killing. GSH-treated biofilms were also shown by RNA-sequencing to display a transcriptomic profile that was distinctly different from those of both mature biofilms and dispersed cells, including those resulting from dispersal agents such as NO [65]. In contrast to dispersed cells, GSH-disrupted biofilm cells significantly upregulated cyclic-di-GMP synthesis genes (siaA and siaB), and there was no concomitant induction of flagellar biosynthesis genes.…”

“…To investigate this further, Klare et al grew the CF P. aeruginosaAES-1 isolate R (isolated at the acute stage of infection)in an artificial sputum media (ASMDM+) that mimics CF sputum, and found it formed robust biofilms in comparison to its isogenic counterpart AES-1M (isolated at chronic infection). AES-1M which produces about 15 times less pyocyanin than AES-1R, and the exogenous addition of pyocyanin to AES-1M cultures facilitated enhanced biofilm formation [65] (Figure 2).…”

“…These cell structures and bio-polymers initiate hydrogen bonding and ionic interactions by colliding with bio-molecules anchored on the bacterial cell surface to stabilise the biofilm matrix and also to its adjacent cells and thereby help bacterial cells to overcome the physico-chemical energy barrier and promote bacterial cell-to-cell interactions and biofilm formation [7,64]. Confocal laser scanning microscopy (CLSM) analysis revealed that the intercalation of pyocyanin with eDNA facilitates P. aeruginosa PA14 wild-type biofilm formation while the absence of pyocyanin significantly inhibits biofilm formation [65]. To investigate this further, Klare et al grew the CF P. aeruginosaAES-1 isolate R (isolated at the acute stage of infection)in an artificial sputum media (ASMDM+) that mimics CF sputum, and found it formed robust biofilms in comparison to its isogenic counterpart AES-1M (isolated at chronic infection).…”

“…Recent studies in the Manos laboratory by Klare et al have demonstrated the excellent utility of GSH in disrupting P. aeruginosa biofilms. It was demonstratedusing CLSM that GSH-mediated inhibition of pyocyanin-DNA binding modulates P. aeruginosa biofilm architecture, significantly decreases biofilm biomass, surface coverage and leads to a significant increase in the percentage of dead bacterial cells [65]. GSH alone was shown to have a significant effect on disruption of mature 72-h-old biofilms of the epidemic isolate AES-1R grown in ASMDM+, while the combined treatment with GSH and DNase I of biofilms from a range of CF isolates showed greater disruption and significantly increased susceptibility to ciprofloxacin killing.…”