Filtern
Dokumenttyp
- Vortrag (20) (entfernen)
Sprache
- Englisch (20)
Referierte Publikation
- nein (20)
Schlagworte
- Biofilms (20) (entfernen)
Organisationseinheit der BAM
Eingeladener Vortrag
- nein (20)
Near Ambient Pressure XPS opens up a new world of possibilities for measurements with XPS. While there are examples where NAP-XPS has been used to study electrochemical processes and heterogeneous catalysis, little attention has been paid to its potential use in biological materials. Until now, bacteria have only been characterised with conventional XPS, which requires tedious sample preparation usually involving freeze drying, a treatment that may degrade biological sample constituents. By studying biological samples in their native wet states, new insight about composition, absorption and transport of drugs through cell membranes and extracellular polymeric substance (EPS) layers can be obtained. Both artificial model-films of exopolysaccharides and biofilms of Escherichia Coli have been characterised at pressures ranging from ultra-high vacuum to 15 mbar by using SPECS’ EnviroESCA NAP-XPS instrument and conventional XPS. By applying antimicrobials to model biofilms, some of which are known to be resistant towards the antimicrobial in question, the distribution of antimicrobials in biofilms has been studied. Capabilities and limitations of the approach will be discussed.
A drawback for X-ray photoelectron spectroscopy is that the measurements must be performed under ultra-high vacuum, which limits the type of samples which can be studied. However, by applying a differentially pumped aperture positioned close to the surface, even wet samples can be measured at near ambient pressure while the energy analyser is still under ultra-high vacuum, as illustrated below. Successful XPS-measurements with pressure up to 30 mbar have been reported using this approach, which opens up a new world of possibilities for ambient pressure measurements with XPS.
While there are examples where NAP-XPS has been used to study electrochemical processes and heterogeneous catalysis, little attention has been paid to its potential use in biological materials. Until now, bacteria have only been characterised with conventional XPS, which requires tedious sample preparation usually involving freeze drying, a treatment that may degrade biological sample constituents. By studying biological samples in their native wet states, new insight about composition, absorption and transport of drugs through cell membranes and extracellular polymeric substance (EPS) layers can be obtained.
Both artificial model-films of exopolysaccharides and biofilms of Escherichia Coli have been characterised at pressures ranging from ultra-high vacuum to 15 mbar by using SPECS’ EnviroESCA NAP-XPS instrument and conventional XPS. By applying antimicrobials to model biofilms, some of which are known to be resistant towards the antimicrobial in question, the distribution of antimicrobials in biofilms has been studied. Measurement capabilities and limitations of the approach will be discussed.
The XPS information depth of approximately 10 nm is in the same size range as the outer membrane of the gram-negative bacteria, which makes XPS a suitable tool for determining the elemental composition of the bacterial surface and monitor changes caused by outer stress like dehydration or exposure to antimicrobials. However, bacteria are inherently in a hydrated state, and therefore only compatible to ultra-high vacuum after extensive sample preparation, which may degrade the sample constituents. This issue is addressed by the development of near-ambient pressure (NAP)-XPS, which enables bacteria and biofilms to be characterised in their native wet state. Artificial biofilms, bacteria and biofilms of Escherichia coli have been characterised with the laboratory NAP-XPS instrument EnviroESCA from SPECS GmbH, at pressures ranging from high vacuum to 12 mbar, and in both humid and dry environment. By studying biological samples in their native wet state, new insight about composition and transport of drugs through cell membranes and the extracellular polymeric substance (EPS) of biofilms can be obtained. In this contribution, the latest progress on biofilm characterisation by NAP-XPS will be presented, and measurement capabilities and limitations will be discussed.
A presentation held for the seminar "Ausgewählte analytische Methoden der Physik" hosted by Prof. Birgit Kanngießer at TU Berlin. The first part focus on depth-dependent XPS-measurements (XPS, synchrotron HAXPES) to obtain a concentration profile of iodine in an artificial biofilm. In the second part, NAP-XPS measurements of various bacterial samples are presented.
The proposed ISO Technical Report provides a description of a variety of physical methods of analytical chemistry by which bacteria and biofilms can be analysed. The state of the art, sample requirements and strengths associated with each method are identified. Presented at the DIN-meeting for NA 062-08-16 AA „Chemische Oberflächenanalyse und Rastersondenmikroskopie“
X-ray photoelectron spectroscopy (XPS) provides elemental and chemical information from the outermost ~10 nm of the sample surface. This is in the same order of magnitude as the thickness of the outer bacterial membrane of gram-negative bacteria, as well as outer membrane molecules as exopolysaccharides and lipopolysaccharides, commonly attached to the cell surface. With the development of near-ambient pressure (NAP)-XPS, bacteria can be analysed with minimal sample preparation.
EnviroESCA is a laboratory based NAP-XPS instrument, equipped with a monochromated Al Kα radiation source and a differentially pumped energy analyser connected to an exchangeable sample environment. It allows for measurements in various gas-atmospheres, including water vapor, which makes it possible to characterise bacteria and other biological samples close to their natural, hydrated state. Artificial model-biofilms of exopolysaccharides, planktonic Pseudomonas Fluorescens and biofilms of Escherichia Coli have been characterised in hydrated and dried state.
High-resolution XPS-spectra from carbon, oxygen, nitrogen and phosphorous can be assigned to carbohydrates, lipids and proteins in general agreement with literature. Especially the carbon 1s peak is of interest. A series of measurements of an E. coli biofilm from 11 mbar in humid environment to 1 mbar air reveal changes in the C1s peak, which suggests that the bacterial surface undergo substantial Change.
Bacterial samples are typically freeze dried or cryo-prepared prior to XPS analysis to allow for measurements in ultra-high vacuum (UHV). The sample environment in the near-ambient pressure (NAP) XPS instrument EnviroESCA allows for measurements in up to 15 mbar water vapor, thus, sample preparation is no longer restricted to UHV-compatible techniques. For instance, biofilms grown in medium can be transferred directly from the medium to the measurements chamber, maintaining a humid environment throughout the measurements. Considering the complexity of bacterial samples, sample preparation must be carefully considered in order to obtain meaningful and reproducible results.
In this talk, various strategies for sample preparation of bacteria and biofilms for NAP-XPS measurements will be discussed. Model systems of planktonic bacteria, artificial biofilms resembling the exopolysaccharide matrix and biofilms have been characterised in various conditions. The stability and homogeneity of the samples was assessed by monitoring the C1s core level peak at different sample locations. The quality of the XPS-spectra is also influenced by the gas environment, which will be exemplified by core level spectra of P. Fluorescens acquired in air, water vapor and ultra-high vacuum.
Bacterial biofilms have the capacity to develop and thrive in virtually all circumstances and surfaces, even in the most challenging environmental conditions. The pervasive and recalcitrant existence of biofilms renders them to be a significant safety risk and economical encumbrance in a wide array of industries and technologies, and therefore is a priority area of research.
It is essential to develop an improved understanding of the mechanisms implicated during biofilm formation, such as in the case of the diffusion of bacterial-secreted extracellular electron transporters, which are purported to play an important role during biocorrosion by exoelectrogenic bacteria. Hence, a fundamental understanding of electron transfer mechanisms between bacteria and extracellular electron acceptors will contribute insight to our understanding of charge transport and chemistry at the biofilm – external insoluble electron acceptor interface.
In the present work, Attenuated Total Reflection - Fourier transform-infrared (ATR-FTIR) spectroscopy has been coupled to electrochemical techniques for the nondestructive, in situ spectro-electrochemical monitoring of biofilms in real-time. Shewanella sp. have been selected for this investigation due to their adaptable exoelectrogenic respiratory capacities and their notable ability to reduce metals via several different mechanisms of extracellular electron transfer mechanisms, including self-secreted flavin shuttles. Gold-thin film model substrates have been used due to their inert nature and for their ability to permit precise manipulation of the substrate surface polarization. Additionally, hydrogels comprised of calcium cross-linked alginate have been used to mimic the architectural features of extracellular polymeric substances which are integral to a bacterial biofilm, to allow the study of electron-transporting flavin molecules in an artificial biofilm. The results will demonstrate patterns of diffusion, akin to how flavins would diffuse in a naturally occurring biofilm, and how polarization affects this process. Furthermore, insight will be gained on how the redox behavior of flavins can influence the development and evolution of a biofilm.
This interdisciplinary approach should shed light on bacterial electron transfer mechanisms which could contribute towards emerging technologies which seek to better understand such mechanisms for novel antifouling strategies, renewable energies, and bioremediation.
Antimicrobial surfaces have broad use in multiple settings including touch surfaces in hospitals, implanted devices, or consumer products. Their aim is to support existing hygiene procedures, and to help combat the increasing threat of antimicrobial resistance. However, concerns have been raised over the potential selection pressure exerted by such surfaces, which might drive the evolution and spread of antimicrobial resistance. In my presentation, I will highlight the risks and knowledge gaps associated with resistance on antimicrobial surfaces by different processes including evolution by de novo mutations and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed onto antimicrobial surfaces. The latter process has the potential to select for antibiotic resistance via cross-resistance between traits that confer resistance to both the antimicrobial surface coating and antibiotics. Conditions in which antibiotics and antimicrobial coatings are present simultaneously (e.g. implants) will lead to more complex interactions that can either result in the selection for or against antibiotic resistance. We mapped these interactions between several antimicrobials and antibiotics on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological (i.e. synergy and antagonism) and evolutionary (i.e. cross-resistance and collateral sensitivity) combination effects. Understanding these interactions opens the door to tailor therapeutic interventions to select against resistance. In additions, we need new methods and translational studies that investigate resistance development to antimicrobial surfaces under realistic conditions. Therefore, I will present recent developments in our lab on the development of such a method based on existing efficacy standards.
Antimicrobials can exert specific physiological effects when used in combination that are different from those when applied alone. These effects include physiological effects (i.e. synergy, antagonism and suppression) as well as evolutionary effects on the selection of resistant strains (i.e. cross-resistance and collateral sensitivity). While combination effects have been extensively mapped for antibiotic-antibiotic combinations, the combination effects of antibiotics with antimicrobials used as biocides or antiseptics have not been systematically investigated.
Here, we investigated the physiological and evolutionary consequences of combinations of antibiotics (meropenem, gentamicin and ciprofloxacin) and substances used as biocides or antiseptics (octenidine, benzalkonium chloride, cetrimonium bromide, chlorhexidine, povidone-iodine, silver) on growth and selection of Pseudomonas aeruginosa. We find prevalent physiological combination effects with synergy occurring 6 times and antagonism occurring 10 times. The effects are specific to the antibiotic-biocide combination with meropenem showing a tendency for antagonism with biocides (6 of 7), while gentamicin has a tendency for synergy (5 of 7). A particular strong antagonism is apparent for the meropenem-chlorhexidine combination, for which we conducted an in-depth study on the underlying molecular mechanism using RNASeq. Moreover, we find widespread effects of the biocide-antibiotic combinations on selection of P. aeruginosa strains resistant to the antibiotics, including cross-resistance and collateral sensitivity.
In conclusion, antibiotics and biocides or antiseptics exert physiological and evolutionary combination effects on the pathogen P. aeruginosa. These effects have consequences for the efficacy of both types of substances and for the selection of antimicrobial resistant strains in clinical applications with combined exposure (e.g. wound care, coated biomaterials).
Antimicrobial resistance (AMR) is a global health problem with the environment being an important compartment for the evolution, selection and transmission of AMR. These processes are impacted by pollution with antibiotics. However, biocides used as disinfectants and material preservatives are major pollutants by far excceding the market for antibiotics in terms of mass. Our work shows that biocides have the potential to affect evolutionary processes towards AMR by increasing the rates of de-novo mutation and conjugation. These effects depend on the species and biocidal substance. Importantly, chlorhexidine and quaternary ammonium compounds (QACs) affect rates of mutation and conjugation at environmentally relevant concentrations in E. coli. Moreover, our results show a connection between the RpoS-mediated general stress and the RecA-linked SOS response with increased rates of mutation and conjugation, but not for all biocides. Furthermore, our work highlights the potential of biocides to contribute to selection and transmission of AMR. We show that the application of biocides, especially QAC disinfectants, leads to the rapid evolution of tolerance (i.e. increased survival) in adaptive laboratory evolution (ALE) experiments. The evolved tolerant strains have a selective advantage in the presence of environmentally-relevant concentrations of antibiotics, which could lead to the stabilization of biocide tolerance in environments where biocides and antibiotics co-occur (e.g. wastewater, animal stables). ALE experiments with biocide tolerant strains indicate a decreased evolvability of resistance to antibiotics. Taken together, our work shows the importance of assessing the contribution of biocides on evolution, selection and transmission of AMR in the environment.
BIOCIDE
(2022)
Biocides, including disinfectants and antimicrobial surfaces (AMCs), are important to prevent the spread of pathogens and antimicrobial resistant bacteria via surfaces. However, concerns have been raised about the evolution and selection of resistance against disinfectants and AMCs. In turn, resistance against disinfectants and AMCs can be associated to antibiotic resistance due to cross-resistance and co-resistance. We need to understand the mechanisms and risks of disinfectants and AMCs for resistance and cross-resistance evolution to optimize their application and safeguard their long-term efficacy. We used adaptive laboratory evolution (ALE) experiments based on repeated exposure of bacteria to disinfectants. Our results show that repeated disinfection of E. coli with benzalkonium chloride in suspension results in a 2000-fold increase in survival within 5 exposure cycles. Adaption is linked to the initial presence of persister cells highly tolerant to benzalkonium chloride. We used the same approach to develop standardizable ALE experiments to determine resistance evolution to AMCs. The results highlight rapid adaptation of E. coli and P. aeruginosa towards copper surfaces. Moreover, there are multiple situations in the clinic or in the environment in which biocides and antibiotics co-occur and in which combination effects can shape their antimicrobial activity or their selective effects. Our work with P. aeruginosa shows prevalent combination effects of biocides and antibiotics, ranging from synergy to antagonism and resulting in the selection for or against antibiotic resistant strains. The combination effects are dependent on the biofilm mode-of-growth, manifesting in apparent differences in the structural arrangement of antibiotic sensitive and resistant strains in biofilms exposed to combinations. Furthermore, biocides affect rates of mutation and horizontal gene transfer, thereby having a potential facilitating effect on resistance evolution. Taken together, our work shows that the role of biocides as potential drivers of resistance evolution and selection deserves further study and regulative action.