Up-dating the Cholodny method using PET films to sample microbial communities in soil

The aim of this work was to investigate the use of PET (polyethylene terephtalate) films as a modern development of Cholodny’s glass slides, to enable microscopy and molecular-based analysis of soil communities where spatial detail at the scale of microbial habitats is essential to understand microbial associations and interactions in this complex environment. Methods. Classical microbiological methods; attachment assay; surface tension measurements; molecular techniques: DNA extraction, PCR; confocal laser scanning microscopy (CLSM); micro- focus X-ray computed tomography (μCT). Results. We first show, using the model soil and rhizosphere bacteria Pseudomonas fluorescens SBW25 and P. putida KT2440, that bacteria are able to attach and detach from PET films, and that pre-conditioning with a filtered soil suspension improved the levels of attachment. Bacteria attached to the films were viable and could develop substantial biofilms. PET films buried in soil were rapidly colonised by microorganisms which could be investigated by CLSM and recovered onto agar plates. Secondly, we demonstrate that μCT can be used to non-destructively visualise soil aggregate contact points and pore spaces across the surface of PET films buried in soil. Conclusions. PET films are a successful development of Cholodny’s glass slides and can be used to sample soil communities in which bacterial adherence, growth, biofilm and community development can be investigated. The use of these films with μCT imaging in soil will enable a better understanding of soil micro-habitats and the spatially-explicit nature of microbial interactions in this complex environment

Examining c-di-GMP and possible quorum sensing regulation in Pseudomonas fluorescens SBW25:links between intra and inter-cellular regulation benefits community cooperative activities such as biofilm formation

Bacterial success in colonizing complex environments requires individual response to micro-scale conditions as well as community-level cooperation to produce large-scale structures such as biofilms. Connecting individual and community responses could be achieved by linking the intracellular sensory and regulatory systems mediated by bis-(3′-5′)-cyclic dimeric guanosine monophosphate (c-di-GMP) and other compounds of individuals with intercellular quorum sensing (QS) regulation controlling populations. There is growing evidence to suggest that biofilm formation by many pseudomonads is regulated by both intra and intercellular systems, though in the case of the model Pseudomonas fluorescens SBW25 Wrinkly Spreader in which mutations increasing c-di-GMP levels result in the production of a robust cellulose-based air-liquid interface biofilm, no evidence for the involvement of QS regulation has been reported. However, our recent review of the P. fluorescens SBW25 genome has identified a potential QS regulatory pathway and other QS–associated genes linked to c-di-GMP homeostasis, and QS signal molecules have also been identified in culture supernatants. These findings suggest a possible link between c-di-GMP and QS regulation in P. fluorescens SBW25 which might allow a more sophisticated and responsive control of cellulose production and biofilm formation when colonising the soil and plant-associated environments P. fluorescens SBW25 normally inhabits.Анализ ц-ди-ГМФ и возможного чувства кворума у Pseudomonas fluorescens SBW 25: связь между внутри и межклеточной регуляцией способствует кооперативному поведению в сообществе и формированию биоплёнкиУспешность бактериальной колонизации сложных экониш требует индивидуального ответа на изменения условий на микроуровне равно как и кооперации на уровне сообщества для продукции таких крупно масштабных структур как биоплёнки. Координация индивидуальных ответ ов и ответов сообщества может быть достигнута путем связывания внутриклеточных сенсорных и регуляторных систем, опосредуемых бис-(3',5')-циклическим димерным гуанозинмонофосфатом (ц-ди-ГМФ) и другими соединениями индивидуумов с межклеточной регуляцией - чувством кворума (ЧК), контролирующем популяци ю. Накапливается всё больше доказательств того, что формирование биопленки многими псевдомонадами регулируется как внутри клеточными, так и меж клеточными регуляторными системами, хотя в случае модельной Pseudomonas fluorescens SBW25 Wrinkly Spreader, у которой мутации, повышающ ие уровни ц-ди-ГМФ, приводят к созданию прочной целлюлозной биоплёнки на границе раздела фаз воздух-жидкость, не было обнаружено ни ка кого свидетельства вовлечения кворум-зависимой регуляции. Однако наш недавний обзор генома P. fluorescens SBW25 выявил потенциальный ЧК-зависимый регуляторный пу ть и другие ЧК-зависимые гены, связанные с гомеостазом ц-ди-ГМФ, а молекулы ЧК-сигналинга были идентифицированы в культуре. Эти данные свидетельствуют о возможной связи между ц-ди-ГМФ-регуляцией и ЧК у P. fluorescens SBW25, что позволяет более сложный и гибкий контроль над продукцией целлюлозы и образовани ем биопленки при колонизации почв и экониш, aссоциированных с растениям и, - естественными средами обитания P. fluorescens SBW25

eDNA inactivation and biofilm inhibition by the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl)

The choice of effective biocides used for routine hospital practice should consider the role of disinfectants in the maintenance and development of local resistome and how they might affect antibiotic resistance gene transfer within the hospital microbial population. Currently, there is little understanding of how different biocides contribute to eDNA release that may contribute to gene transfer and subsequent environmental retention. Here, we investigated how different biocides affect the release of eDNA from mature biofilms of two opportunistic model strains Pseudomonas aeruginosa ATCC 27853 (PA) and Staphylococcus aureus ATCC 25923 (SA) and contribute to the hospital resistome in the form of surface and water contaminants and dust particles. The effect of four groups of biocides, alcohols, hydrogen peroxide, quaternary ammonium compounds, and the polymeric biocide polyhexamethylene guanidine hydrochloride (PHMG-Cl), was evaluated using PA and SA biofilms. Most biocides, except for PHMG-Cl and 70% ethanol, caused substantial eDNA release, and PHMG-Cl was found to block biofilm development when used at concentrations of 0.5% and 0.1%. This might be associated with the formation of DNA–PHMG-Cl complexes as PHMG-Cl is predicted to bind to AT base pairs by molecular docking assays. PHMG-Cl was found to bind high-molecular DNA and plasmid DNA and continued to inactivate DNA on surfaces even after 4 weeks. PHMG-Cl also effectively inactivated biofilm-associated antibiotic resistance gene eDNA released by a pan-drug-resistant Klebsiella strain, which demonstrates the potential of a polymeric biocide as a new surface-active agent to combat the spread of antibiotic resistance in hospital settings

Robust symbiotic microbial communities in space research

Naturally occurring symbiotic microbial communities (SMK) are the most robust assemblages for a multipurpose use in keeping humans healthy and soil fertile. Especially, safe and reliable SMK are needed for producing probiotics and ferments valuable for health problems prophylaxis. This is true for long-term expeditions, outposts, extraterrestrial permanently-manned bases where humans are exposed to adverse environmental factors, weakening the immune system. The kombucha beverage has been used in human society within millennia as a probiotic drink which is produced by naturally occurring mixed populations of living microorganisms. Here, we discuss the potential of the kombucha culture for outposts in far future missions