Mechanical properties of the superficial biofilm layer determine the architecture of biofilms

© 2016 The Royal Society of Chemistry. Cells in biofilms sense and interact with their environment through the extracellular matrix. The physicochemical properties of the matrix, particularly at the biofilm-environment interface, determine how cells respond to changing conditions. In this study we describe the application of atomic force microscopy and confocal imaging to probe in situ the mechanical properties of these interfacial regions and to elucidate how key matrix components can contribute to the physical sensing by the cells. We describe how the Young's modulus of microcolonies differs according to the size and morphology of microcolonies, as well as the flow rate. The Young's modulus increased as a function of microcolony diameter, which was correlated with the production of the polysaccharide Psl at later stages of maturation for hemispherical or mushroom shaped microcolonies. The Young's modulus of the periphery of the biofilm colony was however independent of the hydrodynamic shear. The morphology of the microcolonies also influenced interfacial or peripheral stiffness. Microcolonies with a diffuse morphology had a lower Young's modulus than isolated, circular ones and this phenomenon was due to a deficiency of Psl. In this way, changes in the specific polysaccharide components imbue the biofilm with distinct physical properties that may modulate the way in which bacteria perceive or respond to their environment. Further, the physical properties of the polysaccharides are closely linked to the specific architectures formed by the developing biofilm

Weak acids as an alternative anti-microbial therapy

Weak acids such as acetic acid and N-acetyl cysteine (NAC) at pH less than their pKa can effectively eradicate biofilms due to their ability to penetrate the biofilm matrix and the cell membrane. However, the optimum conditions for their activity against drug resistant strains, and safety, need to be understood for their application to treat infections or to inactivate biofilms on hard surfaces. Here, we investigate the efficacy and optimum conditions at which weak acids can eradicate biofilms. We compared the efficacy of various mono and triprotic weak acids such as N-acetyl cysteine (NAC), acetic acid, formic acid and citric acid, in eradicating biofilms. We found that monoprotic weak acids/acid drugs can kill mucoid P. aeruginosa mucA biofilm bacteria provided the pH is less than their pKa, demonstrating that the extracellular biofilm matrix does not protect the bacteria from the activity of the weak acids. Triprotic acids, such as citric acid, kill biofilm bacteria at pH ​< ​pKa1. However, at a pH between pKa1 and pKa2, citric acid is effective in killing the bacteria at the core of biofilm microcolonies but does not kill the bacteria on the periphery. The efficacy of a monoprotic weak acid (NAC) and triprotic weak acid (citric acid) were tested on biofilms formed by Klebsiella pneumoniae KP1, Pseudomonas putida OUS82, Staphylococcus aureus 15981, P. aeruginosa DK1-NH57388A, a mucoid cystic fibrosis isolate and P. aeruginosa PA_D25, an antibiotic resistant strain. We showed that weak acids have a broad spectrum of activity against a wide range of bacteria, including antibiotic resistant bacteria. Further, we showed that a weak acid drug, NAC, can kill bacteria without being toxic to human cells, if its pH is maintained close to its pKa. Thus weak acids/weak acid drugs target antibiotic resistant bacteria and eradicate the persister cells in biofilms which are tolerant to other conventional methods of biofilm eradication

Female Sexual Polymorphism and Fecundity Consequences of Male Mating Harassment in the Wild

Genetic and phenotypic variation in female response towards male mating attempts has been found in several laboratory studies, demonstrating sexually antagonistic co-evolution driven by mating costs on female fitness. Theoretical models suggest that the type and degree of genetic variation in female resistance could affect the evolutionary outcome of sexually antagonistic mating interactions, resulting in either rapid development of reproductive isolation and speciation or genetic clustering and female sexual polymorphisms. However, evidence for genetic variation of this kind in natural populations of non-model organisms is very limited. Likewise, we lack knowledge on female fecundity-consequences of matings and the degree of male mating harassment in natural settings. Here we present such data from natural populations of a colour polymorphic damselfly. Using a novel experimental technique of colour dusting males in the field, we show that heritable female colour morphs differ in their propensity to accept male mating attempts. These morphs also differ in their degree of resistance towards male mating attempts, the number of realized matings and in their fecundity-tolerance to matings and mating attempts. These results show that there may be genetic variation in both resistance and tolerance to male mating attempts (fitness consequences of matings) in natural populations, similar to the situation in plant-pathogen resistance systems. Male mating harassment could promote the maintenance of a sexual mating polymorphism in females, one of few empirical examples of sympatric genetic clusters maintained by sexual conflict

Exploring the equity of GP practice prescribing rates for selected coronary heart disease drugs: a multiple regression analysis with proxies of healthcare need

Background There is a small, but growing body of literature highlighting inequities in GP practice prescribing rates for many drug therapies. The aim of this paper is to further explore the equity of prescribing for five major CHD drug groups and to explain the amount of variation in GP practice prescribing rates that can be explained by a range of healthcare needs indicators (HCNIs). Methods The study involved a cross-sectional secondary analysis in four primary care trusts (PCTs 1–4) in the North West of England, including 132 GP practices. Prescribing rates (average daily quantities per registered patient aged over 35 years) and HCNIs were developed for all GP practices. Analysis was undertaken using multiple linear regression. Results Between 22–25% of the variation in prescribing rates for statins, beta-blockers and bendrofluazide was explained in the multiple regression models. Slightly more variation was explained for ACE inhibitors (31.6%) and considerably more for aspirin (51.2%). Prescribing rates were positively associated with CHD hospital diagnoses and procedures for all drug groups other than ACE inhibitors. The proportion of patients aged 55–74 years was positively related to all prescribing rates other than aspirin, where they were positively related to the proportion of patients aged >75 years. However, prescribing rates for statins and ACE inhibitors were negatively associated with the proportion of patients aged >75 years in addition to the proportion of patients from minority ethnic groups. Prescribing rates for aspirin, bendrofluazide and all CHD drugs combined were negatively associated with deprivation. Conclusion Although around 25–50% of the variation in prescribing rates was explained by HCNIs, this varied markedly between PCTs and drug groups. Prescribing rates were generally characterised by both positive and negative associations with HCNIs, suggesting possible inequities in prescribing rates on the basis of ethnicity, deprivation and the proportion of patients aged over 75 years (for statins and ACE inhibitors, but not for aspirin)

Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

Background: Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome- wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods: We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results: As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions: Plant-RRBS offers high-throughput and broad, genome- dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations

Postcopulatory sexual selection

The female reproductive tract is where competition between the sperm of different males takes place, aided and abetted by the female herself. Intense postcopulatory sexual selection fosters inter-sexual conflict and drives rapid evolutionary change to generate a startling diversity of morphological, behavioural and physiological adaptations. We identify three main issues that should be resolved to advance our understanding of postcopulatory sexual selection. We need to determine the genetic basis of different male fertility traits and female traits that mediate sperm selection; identify the genes or genomic regions that control these traits; and establish the coevolutionary trajectory of sexes

Differential Dynamics of Transposable Elements during Long-Term Diploidization of Nicotiana Section Repandae (Solanaceae) Allopolyploid Genomes

PubMed ID: 23185607This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Selecting Indicator Portfolios for Marine Species and Food Webs: A Puget Sound Case Study

Ecosystem-based management (EBM) has emerged as a promising approach for maintaining the benefits humans want and need from the ocean, yet concrete approaches for implementing EBM remain scarce. A key challenge lies in the development of indicators that can provide useful information on ecosystem status and trends, and assess progress towards management goals. In this paper, we describe a generalized framework for the methodical and transparent selection of ecosystem indicators. We apply the framework to the second largest estuary in the United States – Puget Sound, Washington – where one of the most advanced EBM processes is currently underway. Rather than introduce a new method, this paper integrates a variety of familiar approaches into one step-by-step approach that will lead to more consistent and reliable reporting on ecosystem condition. Importantly, we demonstrate how a framework linking indicators to policy goals, as well as a clearly defined indicator evaluation and scoring process, can result in a portfolio of useful and complementary indicators based on the needs of different users (e.g., policy makers and scientists). Although the set of indicators described in this paper is specific to marine species and food webs, we provide a general approach that could be applied to any set of management objectives or ecological system

Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis

© 2017 The Authors Despite aggressive antibiotic therapy, bronchopulmonary colonization by Pseudomonas aeruginosa causes persistent morbidity and mortality in cystic fibrosis (CF). Chronic P. aeruginosa infection in the CF lung is associated with structured, antibiotic-tolerant bacterial aggregates known as biofilms. We have demonstrated the effects of non-bactericidal, low-dose nitric oxide (NO), a signaling molecule that induces biofilm dispersal, as a novel adjunctive therapy for P. aeruginosa biofilm infection in CF in an ex vivo model and a proof-of-concept double-blind clinical trial. Submicromolar NO concentrations alone caused disruption of biofilms within ex vivo CF sputum and a statistically significant decrease in ex vivo biofilm tolerance to tobramycin and tobramycin combined with ceftazidime. In the 12-patient randomized clinical trial, 10 ppm NO inhalation caused significant reduction in P. aeruginosa biofilm aggregates compared with placebo across 7 days of treatment. Our results suggest a benefit of using low-dose NO as adjunctive therapy to enhance the efficacy of antibiotics used to treat acute P. aeruginosa exacerbations in CF. Strategies to induce the disruption of biofilms have the potential to overcome biofilm-associated antibiotic tolerance in CF and other biofilm-related diseases

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber