Modeling Polygenic Antibiotic Resistance Evolution in Biofilms

The recalcitrance of biofilms to antimicrobials is a multi-factorial phenomenon, including genetic, physical, and physiological changes. Individually, they often cannot account for biofilm recalcitrance. However, their combination can increase the minimal inhibitory concentration of antibiotics needed to kill bacterial cells by three orders of magnitude, explaining bacterial survival under otherwise lethal drug treatment. The relative contributions of these factors depend on the specific antibiotics, bacterial strain, as well as environmental and growth conditions. An emerging population genetic property—increased biofilm genetic diversity—further enhances biofilm recalcitrance. Here, we develop a polygenic model of biofilm recalcitrance accounting for multiple phenotypic mechanisms proposed to explain biofilm recalcitrance. The model can be used to generate predictions about the emergence of resistance—its timing and population genetic consequences. We use the model to simulate various treatments and experimental setups. Our simulations predict that the evolution of resistance is impaired in biofilms at low antimicrobial concentrations while it is facilitated at higher concentrations. In scenarios that allow bacteria exchange between planktonic and biofilm compartments, the evolution of resistance is further facilitated compared to scenarios without exchange. We compare these predictions to published experimental observations

Anodic biofilms as the interphase for electroactive bacterial growth on carbon veil

© 2016 American Vacuum Society. The structure and activity of electrochemically active biofilms (EABs) are usually investigated on flat electrodes. However, real world applications such as wastewater treatment and bioelectrosynthesis require tridimensional electrodes to increase surface area and facilitate EAB attachment. The structure and activity of thick EABs grown on high surface area electrodes are difficult to characterize with electrochemical and microscopy methods. Here, the authors adopt a stacked electrode configuration to simulate the high surface and the tridimensional structure of an electrode for large-scale EAB applications. Each layer of the stacked electrode is independently characterized using confocal laser scanning microscopy (CLSM) and digital image processing. Shewanella oneidensis MR-1 biofilm on stacked carbon veil electrodes is grown under constant oxidative potentials (0, +200, and +400mV versus Ag/AgCl) until a stable current output is obtained. The textural, aerial, and volumetric parameters extracted from CLSM images allow tracking of the evolution of morphological properties within the stacked electrodes. The electrode layers facing the bulk liquid show higher biovolumes compared with the inner layer of the stack. The electrochemical performance of S. oneidensis MR-1 is directly linked to the overall biofilm volume as well as connectivity between cell clusters

An in-vitro evaluation of C-di-GMP mediated biofilm dispersal as a potential strategy to restore antimicrobial efficacy

The World Health Organization (WHO) has deemed antimicrobial resistance (AMR) in bacteria to be a critical threat to society (World Health Organization 2016, World Health Organization 2017). AMR and specifically multi-drug resistant (MDR) bacteria represent an increasingly serious public health concern worldwide. The need for novel therapeutic approaches to prevent and/or eradicate microbial infections is urgent. AMR can evolve due to mutations that reduce antibiotic susceptibility or due to activation of various innate mechanisms of tolerance, for example, biofilm formation. Bacteria exhibit a complex life-cycle, whereby they can switch from a planktonic, free-living form, to a densely packed biofilm form. Biofilms are an aggregated, sessile, often surface attached form of bacterial mode of growth. Biofilms provide complex 3D structures to bacterial communities, allowing them to interact with each other and respond to changes in their environment differently than their planktonic counterparts (Costerton, Geesey et al. 1978). 60-80% of infections are biofilm-related, once biofilms attach and mature bacteria can exhibit 10 to 1000 times less susceptibility to antimicrobials (Høiby, Bjarnsholt et al. 2010). The use of dispersal agents has been suggested as a potential treatment strategy against microbial biofilms (Banin, Brady et al. 2006, Frederiksen, Pressler et al. 2006, Landini, Antoniani et al. 2010, Barraud, J. Kelso et al. 2015). This strategy consists of manipulating biofilm dispersal to induce changes in biofilm matrix and metabolic states of biofilm cells. These changes are believed to enhance antimicrobial efficacy, increasing antimicrobial killing efficiency, shortening duration of treatment and reducing the risk of emergence of resistance. Investigating the effectiveness of biofilm dispersal as part of an antimicrobial treatment strategy is challenging, and the consequences and the underlying mechanisms of such strategy remain unclear. So far, no dispersal agents have yet been used extensively in a clinical setting (Fleming and Rumbaugh 2017). The present research aims at providing knowledge about how c-di-GMP mediated biofilm dispersal will impact antimicrobial synergy treatment against bacterial biofilms and whether it will result in the emergence of resistant/tolerant variant populations. To achieve these objectives, a strain of Pseudomonas aeruginosa, PAO1/pBAD-yhjH which carries an inducible phosphodiesterase (PDE) enzyme allowing for controlled biofilm dispersal, was utilized (Chua, Tan et al. 2013). A strain PAO1/pJN105 carrying an empty vector plasmid was used as a control. Using antimicrobial agents, alone or in combination, under static in-vitro conditions, c-di-GMP mediated biofilm dispersal was shown to enhance drug synergy against P. aeruginosa biofilms (Chapter 2), without increasing the risk of releasing resistant/tolerant variants (Chapter 3). The potential of biofilm dispersal strategy was also assessed against an evolved AMR strain (Chapter 4). A colistin-resistant derivative of PAO1/pBAD-yhjH was generated and characterized: Significant differences in morphology, motility, and virulence factor production were identified between the parent PAO1/pBAD-yhjH strain and its colistin resistant derivative. Whole genome sequencing and analysis revealed point mutations in phoQ and lpxC, two genes involved in the biosynthesis of lipid-A located in the bacterial membranes and the biological target of colistin. These mutations resulted in upregulated expression of the arn operon which is known to lead to the modification of Lipid-A. The colistin-resistant derivative also showed enhanced susceptibility to carbapenems and glycopeptides, suggesting that the development of colistin resistance resulted in a trade-off in sensitivity to other classes of antimicrobials. This was further explored by testing biofilms for sensitivity to a range of antimicrobial combinations. It was observed that colistin plus vancomycin demonstrated a synergistic effect only against the dispersed biofilms of the colistin resistant strain. The results of this work indicate the potential of c-di-GMP mediated biofilm dispersal as a potential strategy for efficient treatment of biofilm-associated infections.Doctor of Philosoph

In Vitro Evaluation of Biofilm Dispersal as a Therapeutic Strategy To Restore Antimicrobial Efficacy

As a proof-of-concept study, the direct impact of biofilm dispersal on the in vitro efficacy of imipenem and tobramycin was evaluated against 3-day-old biofilms of Pseudomonas aeruginosa. Arabinose induction of biofilm dispersal via activation of the phosphodiesterase YhjH in the P. aeruginosa engineered strain PAO1/pBAD-yhjH resulted in increased antimicrobial efficacy and synergy of the imipenem-tobramycin combination. These results support the use of biofilm dispersal to enhance antimicrobial efficacy in the treatment of biofilm-associated infections, representing a promising therapeutic strategy.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Modeling Polygenic Antibiotic Resistance Evolution in Biofilms

The recalcitrance of biofilms to antimicrobials is a multi-factorial phenomenon, including genetic, physical, and physiological changes. Individually, they often cannot account for biofilm recalcitrance. However, their combination can increase the minimal inhibitory concentration of antibiotics needed to kill bacterial cells by three orders of magnitude, explaining bacterial survival under otherwise lethal drug treatment. The relative contributions of these factors depend on the specific antibiotics, bacterial strain, as well as environmental and growth conditions. An emerging population genetic property—increased biofilm genetic diversity—further enhances biofilm recalcitrance. Here, we develop a polygenic model of biofilm recalcitrance accounting for multiple phenotypic mechanisms proposed to explain biofilm recalcitrance. The model can be used to generate predictions about the emergence of resistance—its timing and population genetic consequences. We use the model to simulate various treatments and experimental setups. Our simulations predict that the evolution of resistance is impaired in biofilms at low antimicrobial concentrations while it is facilitated at higher concentrations. In scenarios that allow bacteria exchange between planktonic and biofilm compartments, the evolution of resistance is further facilitated compared to scenarios without exchange. We compare these predictions to published experimental observations.ISSN:1664-302

Draft Genome Sequence of the Model Naphthalene-Utilizing Organism Pseudomonas putida OUS82

Pseudomonas putida OUS82 was isolated from petrol- and oil-contaminated soil in 1992, and ever since, it has been used as a model organism to study the microbial assimilation of naphthalene and phenanthrene. Here, we report the 6.7-Mb draft genome sequence of P. putida OUS82 and analyze its featured pathways for biodegradation.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

Biofilm development of an opportunistic model bacterium analysed at high spatiotemporal resolution in the framework of a precise flow cell

Life of bacteria is governed by the physical dimensions of life in microscales, which is dominated by fast diffusion and flow at low Reynolds numbers. Microbial biofilms are structurally and functionally heterogeneous and their development is suggested to be interactively related to their microenvironments. In this study, we were guided by the challenging requirements of precise tools and engineered procedures to achieve reproducible experiments at high spatial and temporal resolutions. Here, we developed a robust precise engineering approach allowing for the quantification of real-time, high-content imaging of biofilm behaviour under well-controlled flow conditions. Through the merging of engineering and microbial ecology, we present a rigorous methodology to quantify biofilm development at resolutions of single micrometre and single minute, using a newly developed flow cell. We designed and fabricated a high-precision flow cell to create defined and reproducible flow conditions. We applied high-content confocal laser scanning microscopy and developed image quantification using a model biofilm of a defined opportunistic strain, Pseudomonas putida OUS82. We observed complex patterns in the early events of biofilm formation, which were followed by total dispersal. These patterns were closely related to the flow conditions. These biofilm behavioural phenomena were found to be highly reproducible, despite the heterogeneous nature of biofilm.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio

A new therapeutic avenue for bronchiectasis : dry powder inhaler of ciprofloxacin nanoplex exhibits superior ex vivo mucus permeability and antibacterial efficacy to its native ciprofloxacin counterpart

Non-cystic fibrosis bronchiectasis (NCFB) characterized by permanent bronchial dilatation and recurrent infections has been clinically managed by long-term intermittent inhaled antibiotic therapy among other treatments. Herein we investigated dry powder inhaler (DPI) formulation of ciprofloxacin (CIP) nanoplex with mannitol/lactose as the excipient for NCFB therapy. The DPI of CIP nanoplex was evaluated against DPI of native CIP in terms of their (1) dissolution characteristics in artificial sputum medium, (2) ex vivo mucus permeability in sputum from NCFB and healthy individuals, (3) antibacterial efficacy in the presence of sputum against clinical Pseudomonas aeruginosa strains (planktonic and biofilm), and (4) cytotoxicity towards human lung epithelial cells. Despite their similarly fast dissolution rates in sputum, the DPI of CIP nanoplex exhibited superior mucus permeability to the native CIP (5-7 times higher) attributed to its built-in ability to generate highly supersaturated CIP concentration in the sputum. The superior mucus permeability led to the CIP nanoplex's higher antibacterial efficacy (>3 log10 CFU/mL). The DPI of CIP nanoplex exhibited similar cytotoxicity towards the lung epithelial cells as the native CIP indicating its low risk of toxicity. These results established the promising potential of DPI of CIP nanoplex as a new therapeutic avenue for NCFB.MOH (Min. of Health, S’pore)Accepted versio

Sex Steroids Induce Membrane Stress Responses and Virulence Properties in Pseudomonas aeruginosa (vol 11, e01774-20, 2020)

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