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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 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.
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.
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.