4 Material und Umwelt
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AbstractThis work addresses the critical need for multifunctional materials and substrate‐independent high‐precision surface modification techniques that are essential for advancing microdevices and sensing elements. To overcome existing limitations, the versatility of mussel‐inspired materials (MIMs) is combined with state‐of‐the‐art multiphoton direct laser writing (DLW) microfabrication. In this way, 2D and 3D MIM microstructures of complex designs are demonstrated with sub‐micron to micron resolution and extensive post‐functionalization capabilities. This study includes polydopamine (PDA), mussel‐inspired linear, and dendritic polyglycerols (MI‐lPG and MI‐dPG), allowing their direct microstructure on the substrate of choice with the option to tailor the patterned topography and morphology in a controllable manner. The functionality potential of MIMs is demonstrated by successfully immobilizing and detecting single‐stranded DNA on MIM micropattern and nanoarray surfaces. In addition, easy modification of MIM microstructure with silver nanoparticles without the need of any reducing agent is shown. The methodology developed here enables the integration of MIMs in advanced applications where precise surface functionalization is essential.
Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements.
In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern.
Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria.
At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy.
The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined.
The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion.
The overarching goal of this project is to develop a predictive model for efflux-mediated antimicrobial tolerance in bacterial multicellular assemblies. Our central hypostasis is that efflux pump activity causes emergent antibiotic tolerance of multicellular bacterial populations, through the interplay of efflux mediated spatial interactions and efflux-linked persistence. To test this hypothesis, we will use a combination of microscopy, microbial killing assays, computational modelling, and data analysis, integrating information from 3 types of multicellular assembly: colonies, cell-to-cell interactions in a monolayer microfluidic device, and 3D flow chamber biofilms. Building on our preliminary observations, we will experimentally characterize the link between colony structure and spatial patterns of efflux gene expression in strains that differ in their levels of efflux. We will develop a mathematical model to test whether local growth inhibition of neighbors due to effluxing cells, coupled with local environment-dependent regulation of efflux, can account qualitatively for these results. By including persister cell formation in our model we will predict, and measure, the emergent function of antimicrobial tolerance in our colonies. To fully understand how tolerance emerges from the interplay between efflux-mediated spatial interactions and efflux-linked persister cell formation, we need quantitative measurements at the single cell level. To this end, we will use a microfluidic setup with cells growing in a monolayer to qualify in detail the dependence of efflux expression and persister cell formation on nutrient conditions, the correlation between efflux and persister formation, and the spatial range of efflux-mediated neighbour growth inhibition. To predict and quantitatively understand the emergent multicellular function of tolerance, we will perform individual-based modelling of biofilm growth, using as input the parameters measured on the single-cell level with our microfluidics experiments. Our simulations will predict biofilm spatial structure development, patterns of efflux and persister formation and, ultimately, tolerance to antimicrobial challenge. These predictions will be directly tested in flow-cell biofilm experiments. We are currently generating acrAB-tolC knockout-strain, without efflux activity, and a strain with an inducible acrAB-tolC efflux pump. To distinguish the different strains under the microscope, they were labeled with genes encoding for different fluorescent proteins. All strains are currently characterized in terms of growth, minimum inhibitory concentration of different antimicrobial substances, colony morphology, and biofilm formation ability. On the theoretical side, we are currently working on modeling the system at various scales and degree of detail, ranging from coarse-grained continuum models to stochastic, individual-based models. Some exploratory work was doe to test existing software for individual-based modelling that may be adapted for our purpose. Furthermore, we are in the process of developing more coarse-grained models. This work involves some physiological modelling and literature search, focusing on working mechanisms of efflux pumps and kinetic models for import and export of antibiotics.
Bacteria are ubiquitous and colonize all types of surfaces, including those in close proximity to humans, such as skin, food, and everyday objects. This raises the question of whether their presence represents a problem to be mitigated or a potential source of benefit to be harnessed, thereby stimulating scientific inquiry into the role of surface-associated bacteria in diverse domains ranging fromhuman health to industrial biotechnology. Aim: The objective of this project is to explore the impact of modifying surface topography on bacterial adhesion behavior. By manipulating the physical characteristics of the substrate, the attachment and detachment dynamics of bacteria can potentially be modified, leading to novel strategies for controlling bacterial colonization in various applications, such as medical devices. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were tested on LIPSS-covered Fused Silica samples.
This study details a thorough analysis of leaky and waveguide modes in biperiodic diffractive nanostructures. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a highly sensitive refractive index biosensing platform that can resolve 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes.
Biocides are used for a wide range of purposes, including disinfectants or preservatives. They play a major role in the prevention of microbial infections in healthcare and animal husbandry. The use of biocides often leads to the discharge of active biocidal substances into wastewater streams, causing the exposure of wastewater microbial communities to subinhibitory concentrations. In turn, it is known that wastewater treatment plants (WWTP) are hotspots for antibiotic resistant bacteria. Since similar mechanisms confer resistance to biocides and antibiotics, exposure to biocides can result in co-selection of antibiotic resistant bacteria in WWTP Here, we want to investigate co-selection processes of antibiotic resistance in natural WWTP microbial communities upon biocide exposure. Microbial communities were sampled at the WWTP Ruhleben in Berlin and characterized regarding their susceptibility against different clinically relevant antibiotics. To investigate the link between biocide exposure and antibiotic resistance, changes in the susceptibility level after exposure to environmentally relevant concentrations of the commonly used biocide didecyldimethylammonium chloride (DDAC) will be determined by enumerating resistant and non-resistant E. coli on selective plates with and without antibiotics and DDAC. In case of antibiotics, clinical breakpoint concentrations according to EUCAST will be used to discriminate between susceptible and resistant strains. In case of DDAC (and biocides in general), clinical breakpoints do not exist. Therefore, we determined a cut-off concentration at which the majority of naturally-occurring E. coli strains cannot grow anymore based on (I) the MIC (minimal inhibitory concentration) distribution, and (II) by plating wastewater communities onto selective indicator agar plates loaded with increasing DDAC concentration. Additionally, antibiotic cross-resistance will be determined by spotting single colonies, isolated from DDAC-selective plates onto antibiotic plates. The results of our experiments will help to determine selective concentrations and to estimate the risk of antibiotic co-selection and cross-resistance in microbial WWTP communities upon biocide exposure.
Consequences of tolerance to disinfectants on the evolution of antibiotic resistance in E. coli
(2023)
Biocides are used as disinfectants and preservatives; one important active substance in biocides is benzalkonium chloride (BAC). BAC-tolerant bacterial strains can survive short treatments with high concentrations of BAC. BAC tolerance and resistance have been linked to antibiotic resistance. Here, the selection dynamics between a BAC-tolerant Escherichia coli strain and a sensitive wild type were investigated under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the BAC-tolerant strain was selected over the wild type at all ciprofloxacin concentrations investigated, with a minimum selection concentration (MSC) of 1/10th of the minimum inhibitory concentration (MIC) of the wild type. Furthermore, the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin was assessed by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance
to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. The importance of these results is highlighted by the fact that concentrations of ciprofloxacin well above the calculated MSC can be found in environmental samples such as hospital wastewaters and livestock slurry. In turn, BAC is used as a disinfectant in the same settings. Thus, the selection of BAC-tolerant strains at sub-inhibitory concentrations of ciprofloxacin can contribute to the stabilization and spread of BAC-tolerance in natural populations. The prevalence of such strains can impair the effects of BAC disinfections.
Ratiometric green–red fluorescent nanosensors for fluorometrically monitoring pH in the acidic range were designed from 80 nm-sized polystyrene (PS) and silica (SiO2) nanoparticles (NPs), red emissive reference dyes, and a green emissive naphthalimide pH probe, analytically and spectroscopically characterized, and compared regarding their sensing performance in aqueous dispersion and in cellular uptake studies. Preparation of these optical probes, which are excitable by 405 nm laser or LED light sources, involved the encapsulation of the pH-inert red-fuorescent dye Nile Red (NR) in the core of self-made carboxylated PSNPs by a simple swelling procedure and the fabrication of rhodamine B (RhB)-stained SiO2-NPs from a silane derivative of pH-insensitive RhB. Subsequently, the custom-made naphthalimide pH probe, that utilizes a protonation-controlled photoinduced electron transfer process, was covalently attached to the carboxylic acid groups at the surface of both types of NPs. Fluorescence microscopy studies with the molecular and nanoscale optical probes and A549 lung cancer cells confirmed the cellular uptake of all probes and their penetration into acidic cell compartments, i.e., the lysosomes, indicated by the switching ON of the green naphthalimide fluorescence. This underlines their suitability for intracellular pH sensing, with the SiO2-based nanosensor revealing the best performance regarding uptake speed and stability.
Pseudomonas aeruginosa is an opportunistic pathogen of considerable medical importance, owing to its pronounced antibiotic tolerance and association with cystic fibrosis and other life-threatening diseases. The aim of this study was to highlight the genes responsible for P. aeruginosa biofilm tolerance to antibiotics and thereby identify potential new targets for the development of drugs against biofilm-related infections. By developing a novel screening approach and utilizing a public P. aeruginosa transposon insertion library, several biofilm-relevant genes were identified. The Pf phage gene (PA0720) and flagellin gene (fliC) conferred biofilm-specific tolerance to gentamicin. Compared with the reference biofilms, the biofilms formed by PA0720 and fliC mutants were completely eliminated with a 4-fold-lower gentamicin concentration. Furthermore, the mreC, pprB, coxC, and PA3785 genes were demonstrated to play major roles in enhancing biofilm tolerance to gentamicin. The analysis of biofilm-relevant genes performed in this study provides important novel insights into the understanding of P. aeruginosa antibiotic tolerance, which will facilitate the detection of antibiotic resistance and the development of antibiofilm strategies against P. aeruginosa.
Surfaces of metallic copper and copper alloys effectively inactivate microorganisms and viruses. However, the exact inactivation mode is still under debate. Main factors are assumed to include direct contact with the metallic surface, influx of Cu(I)/Cu(II) ions and the generation of reactive oxygen species (ROS). Laser-induced periodic surface structures (LIPSS) are frequently reported to act antibacterial, mainly by prevention of bacterial adhesion due to a limited number of possible adhesion points or by increasing the overall surface of intrinsically antibacterial materials. In time-kill experiments with E. coli and S. aureus we analyzed the impact of LIPSS on the toxicity of metallic copper and brass. We also conducted ROS accumulation assays and conclude that the application of LIPSS is not generally straight forward to obtain or improve antibacterial surfaces. Thus, the antibacterial effects of LIPPS.
Bacterial biofilms pose serious problems in medical and industrial settings. One of the major societal challenges lies in the increasing resistance of bacteria against biocides used in antimicrobial treatments, e.g., via overabundant use in medicine, industry, and agriculture or cleaning and disinfection in private households. Hence, new efficient bacteria-repellent strategies avoiding the use of biocides are strongly desired. One promising route to achieve bacteria-repellent surfaces lies in the contactless and aseptic large-area laser-processing of technical surfaces. Tailored surface textures, enabled by different laser-processing strategies that result in topographic scales ranging from nanometers to micrometers may provide a solution to this challenge. This article presents a current state-of-the-art review of laser-surface subtractive texturing approaches for controlling the biofilm formation for different bacterial strains and in different environments. Based on specific properties of bacteria and laser-processed surfaces, the challenges of anti-microbial surface designs are discussed, and future directions will be outlined.
Excessive discharge of quaternary ammoniumdisinfectants such as benzalkonium chloride (BAC) into aquatic systems can trigger several physiological responses in environmental microorganisms. In this study, we isolated a less-susceptible strain of Aeromonas hydrophila to BAC, designated as INISA09, froma wastewater treatment plant in Costa Rica. We characterized its phenotypic response upon exposure to three dierent concentrations of BAC and characterizedmechanisms related to its resistance using genomic and proteomic approaches. The genome of the strain, mapped against 52 dierent sequenced A. hydrophila strains, consists of approximately 4.6Mb with 4,273 genes. We found a massive genome rearrangement and thousands of missense mutations compared to the reference strain A. hydrophila ATCC 7966.
We identified 15,762 missense mutations mainly associated with transport, antimicrobial resistance, and outer membrane proteins.
In addition, a quantitative proteomic analysis revealed a significant upregulation of several efflux pumps and the downregulation of porins when the strain was exposed to three BAC concentrations.Other genes related tomembrane fatty acid metabolism and redox metabolic reactions also showed an altered expression.
Our findings indicate that the response of A. hydrophila INISA09 to BAC primarily occurs at the envelop level, which is the primary target of BAC. Our study elucidates the mechanisms of antimicrobial susceptibility in aquatic environments against a widely used disinfectant and will help better understand howbacteria can adapt to biocide pollution. To our knowledge, this is the first study addressing the resistance to BAC in an environmental A. hydrophila isolate. We propose that this bacterial species could also serve as a new model to study antimicrobial pollution in aquatic environments.
Microbiologically influenced corrosion (MIC) is a phenomenon of increasing concern which affects various materials and sectors of society. MIC describes the effects, often negative, that a material can experience due to the presence of microorganisms. Unfortunately, although several research groups and industrial actors worldwide have already addressed MIC, discussions are fragmented, while information sharing and willingness to reach out to other disciplines is limited. A truly interdisciplinary approach, that would be logical for this material/biology/chemistry-related challenge, is rarely taken. In this review we highlight critical non-biological aspects of MIC that can sometimes be overlooked by microbiologists working on MIC but are highly relevant for an overall understanding of this phenomenon. Here, we identify gaps, methods and approaches to help solve MIC related challenges, with an emphasis on the MIC of metals. We also discuss the application of existing tools and approaches for managing MIC and propose ideas to promote an improved understanding of MIC. Furthermore, we highlight areas where the insights and expertise of microbiologists are needed to help progress this field.
Climate change is becoming one of the greatest challenges facing our society, particularly due to the continued use of fossil fuels. The steadily increasing demand for energy and the continuously growing world population will further intensify these challenges. The development of renewable energies is therefore of central importance. The 2020 EU Energy Roadmap aims to increase the share of renewable energies (gross energy consumption) to 55% by 2050.
Hydrogen (H2) has the highest potential to become the primary renewable energy source. It is envisioned that by 2050 up to 24% of the total energy demand of Europe is to be ensured by H2. However, a decisive disadvantage of the climate friendly alternatives is the massive containment demand, which needs to be highly secure, cost efficient and easily extractable.
Underground geological formations (UGF) represent a seemingly optimal alternative to meet the rapidly increasing storage demand. In this context, many studies are currently underway to determine the feasibility and risks of UGF. However, little or no consideration is being given to microbiology. Therefore, in this Research Topic we will focus on achieving a greater understanding of the impact microorganisms exert on UGF, with a particular emphasis on interdisciplinary studies.
As many subsurface microbial communities can use H2 as an electron donor, production of seemingly undesirable metabolic byproducts, such as hydrogen sulfide, methane, and acids, are also to be expected. However, the rate of the H2 conversion by the microorganisms, how their metabolic activities impact the UGF on a short-term and long-term scale, the extent of damages microorganisms exert on the infrastructure, or potential use of microorganisms to enhance UGF are just a few questions that require urgent research to assess the role of microorganisms in this new anthropogenic use of the subsurface environment. These and many questions can be addressed in this article collection. In particular, understanding microbial community changes and activity rates will help assess operational and environmental risks, develop mitigation strategies and provide new insights on life under extreme conditions (i.e., pressure, salinity).
In this Research Topic, the editorial team particularly welcomes Original Research, Hypothesis and Theory, Method, and Review manuscripts that deal with the latest advances in microbiology in formations that are planned or currently prepared for hydrogen storage, from both fundamental and practical points of view. The ultimate objective is to promote a deeper understanding into the sustainability of UGF and generate interdisciplinary research involving microbiologists, reservoir engineers, geologists, chemists, physicists.
The topics of interest include, but are not limited to:
• Microbial diversity in different underground hydrogen storage sites or formations currently being considered for hydrogen storage
• Mechanism and impact of microbial growth under high H2 pressure
• Potential role of microorganisms in the short-term and long-term storage of hydrogen
• Potential influences of microorganisms on the hydrogen storage infrastructure systems, e.g., microbiologically influenced corrosion, biofilm growth
• Hydrogen-solid-microorganism interactions, including the influence of microbial growth on UGF geological parameters
• Mechanism and modelling of microbial impact on hydrogen storage
UGF relevant for this Research Topic include porous media, salt caverns, deep aquifers, hard rock caverns and depleted oil/gas reservoirs.
Disinfectants are important to prevent the transmission of pathogens, especially in the face of the current antibiotic resistance crisis. The crisis is further exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfectant application, persistence to disinfectants and its role for the evolution of tolerance and cross-resistance to antibiotics has not been studied. Our work shows that E. coli displays persistence against several widely used disinfectants, including benzalkonium chloride (BAC), didecyldimethylammoniumchlorid (DDAC) and isopropanol. The molecular mechanism of BAC persistence is triggered in stationary phase and affected by several antibiotic persister genes (hipA, tisB, tolC, relA, spoT). Experimental evolution and population dynamic modeling show that repeated failure of disinfection due to persisters rapidly selects for BAC tolerance underpinned by reduced cell surface charge due to mutations in genes related to lipid A acylation (lpxML). Furthermore, evolved BAC tolerance affects the susceptibility to antibiotics, leading to positive selection of disinfectant tolerant strains at environmentally relevant antibiotic concentrations and variations in evolvability of antibiotic resistance due to epistatic effects. These results highlight the need for faithful application of disinfectants to steward their efficacy and the efficacy of antibiotics. A better understanding of the bacterial response to disinfectants is crucial to understand and avert the ongoing antimicrobial resistance crisis.
Bacterial biofilms can pose a serious health risk to humans and are less susceptible
to antibiotics and disinfection than planktonic bacteria. Here, a novel method
for biofilm eradication based on antimicrobial photodynamic therapy utilizing
a nanoparticle in conjunction with a BODIPY derivative as photosensitizer was
developed. Reactive oxygen species are generated upon illumination with
visible light and lead to a strong, controllable and persistent eradication of
both planktonic bacteria and biofilms. One of the biggest challenges in biofilm
eradication is the penetration of the antimicrobial agent into the biofilm and
its matrix. A biocompatible hydrophilic nanoparticle was utilized as a delivery
system for the hydrophobic BODIPY dye and enabled its accumulation within the
biofilm. This key feature of delivering the antimicrobial agent to the site of action
where it is activated resulted in effective eradication of all tested biofilms. Here,
3 bacterial species that commonly form clinically relevant pathogenic biofilms
were selected: Escherichia coli, Staphylococcus aureus and Streptococcus
mutans. The development of this antimicrobial photodynamic therapy tool for
biofilm eradication takes a promising step towards new methods for the much
needed treatment of pathogenic biofilms.
Our earlier study based on historical and scientific analysis showed that Iranians used a considerable range of materials in the sizing process. Unlike many nations who used limited sizing materials to improve the mechanical strength and to smoothen the paper surface, Iranians have used various materials for sizing process from Taimurid (15th century) to Safawid (16th century) and Qajar (19th century) periods. Further to our earlier study on the Sizing Materials used in Persian manuscripts and miniature paintings, scientific analysis was carried out to investigate their behaviour against the mould fungus Aspergillus flavus. In the first stage fourteen different sizing materials that was identified from Persian historical recipes were reconstructed. In the second stage fungicidal property of each sizing samples against Aspergillus flavus fungus were identified that will be presented in this paper.
Glutaraldehyde is widely used as a disinfectant and preservative, but little is known about its effects on bacterial susceptibility to antibiotics and the selection of tolerant phenotypes. We found that short-term exposure to sub-inhibitory levels of glutaraldehyde makes E. coli resistant to high doses of bactericidal antibiotics from different classes. This tolerance is associated with delayed, heterogeneous regrowth dynamics and global transcriptome remodeling. We identified over 1200 differentially expressed genes, including those related to antibiotic efflux, metabolic processes, and the cell envelope. The cells entered a disrupted state likely due to the unspecific mode-of-action of glutaraldehyde. Despite this unregulated response, we identified several differentially expressed genes not previously associated with antibiotic tolerance or persistence that induce antibiotic tolerance when overexpressed alone. These findings highlight how the unspecific mode-of-action of disinfectants can make bacteria temporarily resistant to antibiotics. They have implications for settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments where microorganisms are exposed to these substances, such as sewage systems. A trade-off arises from overcoming the disrupted state as quickly as possible and maintaining antibiotic tolerance.
Glutaraldehyde is widely used as a disinfectant and preservative, but little is known about its effects on bacterial susceptibility to antibiotics and the selection of tolerant phenotypes. We found that short-term exposure to sub-inhibitory levels of glutaraldehyde makes E. coli resistant to high doses of bactericidal antibiotics from different classes. This tolerance is associated with delayed, heterogeneous regrowth dynamics and global transcriptome remodeling. We identified over 1200 differentially expressed genes, including those related to antibiotic efflux, metabolic processes, and the cell envelope. The cells entered a disrupted state likely due to the unspecific mode-of-action of glutaraldehyde. Despite this unregulated response, we identified several differentially expressed genes not previously associated with antibiotic tolerance or persistence that induce antibiotic tolerance when overexpressed alone. These findings highlight how the unspecific mode-of-action of disinfectants can make bacteria temporarily resistant to antibiotics. They have implications for settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments where microorganisms are exposed to these substances, such as sewage systems. A trade-off arises from overcoming the disrupted state as quickly as possible and maintaining antibiotic tolerance.
Glutaraldehyde (GTA) is commonly used to disinfect medical equipment, in animal husbandry and in hydraulic fracturing. Its wide use bears the risk that microorganisms in different environments are exposed to potentially non-lethal doses of glutaraldehyde. To date, little is known about the effects of glutaraldehyde on the susceptibility of bacteria to antibiotics and its role in the selection of tolerant phenotypes.
Objectives
• To determine the effect of glutaraldehyde exposure on the survival of E. coli, S. aureus and P. aeruginosa to antibiotics
• To find the mechanistic basis for antibiotic tolerance upon glutaraldehyde exposure
Materials & Methods
Four bacterial isolates were exposed to sub-inhibitory glutaraldehyde. Antibiotic tolerance was determined by time-kill assays. Regrowth dynamics (lag times) were determined with ScanLag. E. coli was further investigated, using RNAseq to identify genes and processes involved in antibiotic tolerance. Mutants of candidate genes were screened for their antibiotic tolerance and heterogeneous target gene expression under stressed and unstressed conditions.
Results
Short-term exposure to sub-inhibitory levels of glutaraldehyde induced tolerance to high doses of bactericidal antibiotics. Tolerance to antibiotics was associated with highly heterogeneous regrowth dynamics and global transcriptome remodeling. Differentially expressed genes represented diverse biological functions and cellular components, including antibiotic efflux, metabolic processes, and the cell envelope. The heterogeneous regrowth dynamics and the diversity of the differentially expressed genes are likely related to the unspecific mode-of-action of glutaraldehyde. Among the many differentially expressed genes, several genes were identified that were not previously associated with antibiotic tolerance or persistence, which, when overexpressed alone, increased antibiotic tolerance.
Conclusion
Our results highlight how the big advantage of a disinfectant, its unspecific mode-of-action, can induce transient tolerance to antibiotics in bacteria. These findings have implications for 1.) settings where disinfectants and antibiotics are used in proximity, such as hospitals and animal husbandry, and 2.) for the selection dynamics of tolerant bacteria in fluctuating environments because of the trade-off that arises from overcoming the lag phase as fast as possible and maintaining antibiotic tolerance.
Glutaraldehyde is widely used as a disinfectant and preservative, but little is known about its effects on bacterial susceptibility to antibiotics and the selection of tolerant phenotypes. We found that short-term exposure to sub-inhibitory levels of glutaraldehyde makes E. coli resistant to high doses of bactericidal antibiotics from different classes. This tolerance is associated with delayed, heterogeneous regrowth dynamics and global transcriptome remodeling. We identified over 1200 differentially expressed genes, including those related to antibiotic efflux, metabolic processes, and the cell envelope. The cells entered a disrupted state likely due to the unspecific mode-of-action of glutaraldehyde. Despite this unregulated response, we identified several differentially expressed genes not previously associated with antibiotic tolerance or persistence that induce antibiotic tolerance when overexpressed alone. These findings highlight how the unspecific mode-of-action of disinfectants can make bacteria temporarily resistant to antibiotics. They have implications for settings where disinfectants and antibiotics are used in close proximity, such as hospitals and animal husbandry, and for the selection dynamics of tolerant pheno- and genotypes in fluctuating environments where microorganisms are exposed to these substances, such as sewage systems. A trade-off arises from overcoming the disrupted state as quickly as possible and maintaining antibiotic tolerance.
Background. Disinfection is an important mitigation strategy to control and prevent the spread of infections. Incomplete or incorrect usage of disinfection may promote evolution of resistance against disinfectants and antibiotics. Ideally, disinfection reduces the number of surviving bacteria and the chance for resistance evolution. Resistance describes the ability to grow in previously inhibitory concentrations of an antimicrobial, whereas tolerance is associated with enhanced survival of lethal doses. Individual bacteria from the same population can display considerable heterogeneity in their ability to survive treatment (i.e. tolerance) with antimicrobials, which can result in unexpected treatment failure.
Objective. In this study, we investigated six active substances of disinfectants, preservatives, and antiseptics against a population of E. coli to identify the presence of a tolerant subpopulation.
Methods. We performed time-kill experiments and analyzed the data with a mathematical model to statistically infer whether the data is best explained by the presence of a tolerant subpopulation.
Results. The analysis identified bimodal kill kinetics for benzalkonium chloride, didecyldimethylammonium chloride, and isopropanol. In contrast, kill kinetics by chlorhexidine, glutaraldehyde, and hydrogen peroxide were best explained by unimodal kill kinetics. These findings have implications for the risk of disinfection failure. In addition, we are currently performing adaptive laboratory evolution (ALE) experiments with the different disinfectants to investigate the potential consequences of tolerant sub-populations for the evolution of antimicrobial resistance and tolerance.
Background. Disinfection is an important mitigation strategy to control and prevent the spread of infections. Incomplete or incorrect usage of disinfection may promote evolution of resistance against disinfectants and antibiotics. Ideally, disinfection reduces the number of surviving bacteria and the chance for resistance evolution. Resistance describes the ability to grow in previously inhibitory concentrations of an antimicrobial, whereas tolerance is associated with enhanced survival of lethal doses. Individual bacteria from the same population can display considerable heterogeneity in their ability to survive treatment (i.e. tolerance) with antimicrobials, which can result in unexpected treatment failure.
Objective. In this study, we investigated six active substances of disinfectants, preservatives, and antiseptics against a population of E. coli to identify the presence of a tolerant subpopulation.
Methods. We performed time-kill experiments and analyzed the data with a mathematical model to statistically infer whether the data is best explained by the presence of a tolerant subpopulation.
Results. The analysis identified bimodal kill kinetics for benzalkonium chloride, didecyldimethylammonium chloride, and isopropanol. In contrast, kill kinetics by chlorhexidine, glutaraldehyde, and hydrogen peroxide were best explained by unimodal kill kinetics. These findings have implications for the risk of disinfection failure. In addition, we are currently performing adaptive laboratory evolution (ALE) experiments with the different disinfectants to investigate the potential consequences of tolerant sub-populations for the evolution of antimicrobial resistance and tolerance.
Bei dem Vortrag werden die potentiellen mikrobiologischen Einflüsse im Falle der Lagerung von Wasserstofflagerung in unterirdischen geologischen Formationen vorgestellt, die Zusammenarbeit mit internen und auch externen Partnern. Der Fokus liegt dabei auf UGF; anaerobe Umgebungen, Sulfate reduzierenden Bakterien, methanogenen Archaea, weiterleitenden Systeme und die Kombination der Untersuchung abiotisch/biotische