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A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

ABSTRACT: In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by Pseudomonas aeruginosa within the airway plays an important role in contributing to resistance to treatment. An in vitro biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies employed clinical cystic fibrosis (CF) isolates (n = 3) and reference nonmucoid and mucoid multidrug-resistant (MDR) CF isolates (n = 7). Bacterial growth and biofilm development and disruption were studied using cell viability assays and image analysis with scanning electron and confocal laser scanning microscopy. Pseudomonal growth in AS medium was associated with increased ATP production (P < 0.05) and the formation (at 48 h) of discrete (>10-?m) microcolonies. In conventional growth medium, colistin retained an ability to inhibit growth of planktonic bacteria, although the MIC was increased (0.1 to 0.4 ?g/ml) in AS medium compared to Mueller-Hinton (MH) medium. In contrast, in an established-biofilm model in AS medium, the efficacy of colistin was decreased. OligoG CF-5/20 (?2%) treatment, however, induced dose-dependent biofilm disruption (P < 0.05) and led to colistin retaining its antimicrobial activity (P < 0.05). While circular dichroism indicated that OligoG CF-5/20 did not change the orientation of the alginate carboxyl groups, mass spectrometry demonstrated that the oligomers induced dose-dependent (>0.2%; P < 0.05) reductions in pseudomonal quorum-sensing signaling. These findings reinforce the potential clinical significance of microcolony formation in the CF lung and highlight a novel approach to treat MDR pseudomonal infections.

SUBMITTER: Pritchard MF

PROVIDER: S-EPMC5571347 | biostudies-literature | 2017 Sep

REPOSITORIES: biostudies-literature

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A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

Pritchard Manon F MF Powell Lydia C LC Jack Alison A AA Powell Kate K Beck Konrad K Florance Hannah H Forton Julian J Rye Philip D PD Dessen Arne A Hill Katja E KE Thomas David W DW

Antimicrobial agents and chemotherapy 20170824 9

In chronic respiratory disease, the formation of dense, 3-dimensional "microcolonies" by <i>Pseudomonas aeruginosa</i> within the airway plays an important role in contributing to resistance to treatment. An <i>in vitro</i> biofilm model of pseudomonal microcolony formation using artificial-sputum (AS) medium was established to study the effects of low-molecular-weight alginate oligomers (OligoG CF-5/20) on pseudomonal growth, microcolony formation, and the efficacy of colistin. The studies empl ...[more]

PMID: 28630204

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Project description:Alginate oligosaccharide (AOS), prepared from depolymerised alginate, a natural polysaccharide occurring in the cell walls of brown algae, provides beneficial effects for intestinal health. However, the underlying mechanisms by which AOS supplementation maintains the intestinal integrity of weaned pigs remain obscure. Here, we aimed to determine how AOS modulates the intestinal integrity of weaned pigs. Twenty-four weaned pigs were assigned to two treatments: a control group (basal diet) and an AOS group (the basal diet supplemented with 100 mg kg-1 AOS). On day 15, eight pigs per treatment were randomly selected and sacrificed for serum and intestinal samples. We observed that AOS supplementation enhanced the intestinal integrity, as evidenced by the increased (P < 0.05) intestinal occludin protein abundance. Compared to the control group, AOS ingestion both elevated (P < 0.05) the jejunal and ileal catalase activity and decreased (P < 0.05) the duodenal and jejunal tumour necrosis factor-α concentration and mast cell tryptase expression. Furthermore, AOS down-regulated (P < 0.05) the duodenal toll-like receptor 4 (TLR4) and its down-stream signals, myeloid differentiation factor 88 (MyD88), interleukin-1 receptor-associated kinase 1 (IRAK1) and tumour necrosis factor receptor-associated factor 6 (TRAF6) mRNA levels, as well as jejunal nucleotide-binding oligomerisation domain protein 1 (NOD1) and its adaptor molecule, receptor-interacting serine/threonine-protein kinase 2 (RIPK2), mRNA levels. Additionally, phospho-nuclear factor-κB (p-NF-κB) p65 protein abundance in the duodenum and jejunum was down-regulated (P < 0.05) following AOS supplementation. According to the above results, the enhanced intestinal integrity in AOS-supplemented pigs appears to be associated with the elevated antioxidant capacity and the reduced mast cell degranulation, as well as the inhibited pro-inflammatory cytokines production via inhibiting the TLR4/NF-κB and NOD1/NF-κB signalling pathways.

Project description:Biofilms play an important role in the pathogenesis of group A streptococcus (GAS), a Gram-positive pathogen responsible for a wide range of infections and with a significant public health impact. Although most GAS serotypes are able to form biofilms, there is a large amount of heterogeneity between individual strains in biofilm formation, as measured by standard crystal violet assays. It is generally accepted that biofilm formation includes the initial adhesion of bacterial cells to a surface followed by microcolony formation, biofilm maturation, and extensive production of extracellular matrix that links together proliferating cells and provides a scaffold for the three-dimensional (3D) biofilm structure. However, our studies show that for GAS strain JS95, microcolony formation is not an essential step in static biofilm formation, and instead, biofilm can be effectively formed from slow-growing or nonreplicating late-exponential- or early-stationary-phase planktonic cells via sedimentation and fixation of GAS chains. In addition, we show that the GAS capsule specifically contributes to the alternative sedimentation-initiated biofilms. Microcolony-independent sedimentation biofilms are similar in morphology and 3D structure to biofilms initiated by actively dividing planktonic bacteria. We conclude that GAS can form biofilms by an alternate noncanonical mechanism that does not require transition from microcolony formation to biofilm maturation and which may be obscured by biofilm phenotypes that arise via the classical biofilm maturation processes.IMPORTANCE The static biofilm assay is a common tool for easy biomass quantification of biofilm-forming bacteria. However, Streptococcus pyogenes biofilm formation as measured by the static assay is strain dependent and yields heterogeneous results for different strains of the same serotype. In this study, we show that two independent mechanisms, for which the protective capsule contributes opposing functions, may contribute to static biofilm formation. We propose that separation of these mechanisms for biofilm formation might uncover previously unappreciated biofilm phenotypes that may otherwise be masked in the classic static assay.

Project description:BackgroundThe choice of antibiotic, and the use of single or combined therapy are controversial areas in the treatment of respiratory infection due to Pseudomonas aeruginosa in cystic fibrosis (CF). Advantages of combination therapy include wider range of modes of action, possible synergy and reduction of resistant organisms; advantages of monotherapy include lower cost, ease of administration and reduction of drug-related toxicity. Current evidence does not provide a clear answer and the use of intravenous antibiotic therapy in CF requires further evaluation. This is an update of a previously published review.ObjectivesTo assess the effectiveness of single compared to combination intravenous anti-pseudomonal antibiotic therapy for treating people with CF.Search methodsWe searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register, comprising references identified from comprehensive electronic database searches and handsearches of relevant journals and abstract books of conference proceedings. Most recent search of the Group's Trials Register: 07 October 2020. We also searched online trials registries on 16 November 2020.Selection criteriaRandomised controlled trials (RCTs) comparing a single intravenous anti-pseudomonal antibiotic with a combination of that antibiotic plus a second anti-pseudomonal antibiotic in people with CF.Data collection and analysisTwo authors independently assessed trial quality and extracted data. We assessed the certainty of the data using GRADE.Main resultsWe identified 59 trials, of which we included eight trials (356 participants) comparing a single anti-pseudomonal agent to a combination of the same antibiotic and one other. There was a wide variation in the individual antibiotics used in each trial. In total, the trials included seven comparisons of a beta-lactam antibiotic (penicillin-related or third generation cephalosporin) with a beta-lactam-aminoglycoside combination and three comparisons of an aminoglycoside with a beta-lactam-aminoglycoside combination. There was considerable heterogeneity amongst these trials, leading to difficulties in performing the review and interpreting the results. These results should be interpreted cautiously. Six of the included trials were published between 1977 and 1988; these were single-centre trials with flaws in the randomisation process and small sample size. Overall, the methodological quality was poor and the certainty of the evidence ranged from low to moderate. The review did not find any differences between monotherapy and combination therapy in either the short term or in the long term for the outcomes of different lung function measures, bacteriological outcome measures, need for additional treatment, adverse effects, quality of life or symptom scores.Authors' conclusionsThe results of this review are inconclusive. The review raises important methodological issues. There is a need for an RCT which needs to be well-designed in terms of adequate randomisation allocation, blinding, power and long-term follow-up. Results need to be standardised to a consistent method of reporting, in order to validate the pooling of results from multiple trials.

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A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

Publications

A Low-Molecular-Weight Alginate Oligosaccharide Disrupts Pseudomonal Microcolony Formation and Enhances Antibiotic Effectiveness.

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