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Surface characterisation of Escherichia coli under various conditions by near-ambient pressure XPS
(2018)
Bacteria are inherently in a hydrated state and therefore not compatible to ultra-high vacuum techniques such as XPS without prior sample preparation involving freeze drying or fast freezing.
This has changed with the development of near-ambient pressure (NAP)-XPS, which makes it possible to characterise the bacterial surface with minimal sample preparation. This paper presents NAP-XPS measurements of Escherichia coli under various NAP conditions: at 11 mbar in a humid environment, at 2 mbar after drying in the chamber, pre-dried at 4 mbar, and at 1 mbar after overnight pumping at 10^−4 mbar. The high-resolution spectra of carbon, nitrogen, and oxygen are presented and found to be in general agreement with XPS measurements from freeze-dried and fast-frozen bacteria. However, it was found that the amount of carbon components associated with polysaccharides increases relative to aliphatic carbon during drying and increases further after overnight pumping. This implies that drying has an impact on the bacterial surface.
Antimicrobial resistance is a major threat to human health. The prevalence of multi-drug resistant (MDR) bacteria is predicted to increase in the future requiring robust control strategies. One cornerstone to prevent the spread of MDR bacteria in clinical settings is the application of disinfectants to improve hygiene standards. However, bacteria can evolve resistance to disinfectants, which in turn can confer cross-resistance to antibiotics. Additionally, clonal bacterial populations can display phenotypic heterogeneity with respect to the tolerance of antibiotic stress leading to a prolonged survival of a sub-population; this phenomenon is termed persistence. Persistence to antibiotics is an evolvable trait and can serve as a stepping stone for the evolution of genetically encoded resistance. Until now, there is a lack of systematic studies that investigate if bacterial populations establish persister subpopulations that tolerate disinfectant stress longer than the majority of the population. Our results in E. coli indicate that persistence is a bacterial survival strategy against benzalkonium chloride, a widely used disinfectant.
In future experiments, we will investigate the evolution of persistence in the face of fluctuating exposure to disinfectants and whether persistence facilitates resistance against disinfectants. Lastly, we will test how tolerance and resistance against disinfectants affects susceptibility against antibiotics.
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.
Microbial modelling of sulfate-reducing bacteria (SRB) and methanogenic archaea (ME) using iron
(2019)
Sulfate reducing bacteria (SRB) and methanogenic archaea (MA) are commonly found in the oil and gas environments. The formation of hydrogen sulfide (HS-) is particularly concerning for the petroleum industry due to its corrosiveness. However, the activities of SRB are limited to the concentration of sulfate present in the environment, whereas methanogens can utilize substrates such as H¬2 for methanogenesis. MA is commonly found in sulfate-free environments, such as deep sediments, and are known to form interspecies electron transfer relationships with SRB. Recently, SRB and MA capable of microbiologically influenced corrosion (MIC) by using elemental iron as a direct electron source (EMIC) have gained increased attention. On the iron surface, EMIC-SRB can outcompete EMIC-MA in the presence of sulfate, but this changes as sulfate depletes. The formation of FeS on the metal surface can be further utilized by MA for methanogenesis as it provides a conductive path. However, the possible kinetics involved of the overall process are currently unknown.
We obtained a co-culture of EMIC-SRB and EMIC-MA to investigate the growth rates and electrical potential changes under different environmental conditions, including changes in pH, temperature and salinity. Results indicate that under neutral conditions and using iron as the sole substrate, methane production (up to 5 mM) starts after sulfate was depleted. Electrochemical measurements will be conducted on the co-culture under different conditions to determine the changes in the electrical potential in correlation with the sulfate and methane concentration. Fluorescence and electron microscope images of the biofilm structure will be used to visualize cell distribution and morphology. This study embarks the first step of understanding the relationship between EMIC-SRB and EMIC-MA. Such knowledge is important for the field of microbial electrophysiology and can be further explored for industrial applications.
The global rise of antibiotic resistance has made the proper use of disinfectants more important than ever. Their application in clinical l settings is an integral part of antibiotics stewardship by preventing the occurrence and spread of infections. However, improper use of disinfectants also harbours the risk for the evolution of tolerance and resistance to disinfectants, but also to antibiotics. It is therefore crucial to understand whether and how bacteria can survive chemical disinfection and which conditions facilitate the evolution of tolerance and resistance.
Here, we study the heterogeneity in the response of isogenic E. coli populations exposed to different levels of commonly used disinfectants. At concentrations below the minimal inhibitory concentration (MIC), we find that certain disinfectants induce prolonged lag times in individual cells, a phenotype that has been associated with persistence against antibiotics. At concentrations above the MIC, we find heterogeneous killing for a range of the tested substances. Interestingly, for the three cationic surfactants that were tested, we find kill kinetics revealing the presence of a tolerant subpopulation that can withstand disinfection longer than most of the population. We will present results from an ongoing evolution experiment in which we test the potential for evolution of population-wide tolerance and resistance through intermittent exposure to lethal doses of a cationic surfactant.
Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.
This study details a theoretical analysis of leaky and waveguide modes in biperiodic all-dielectric holograms. By tuning diffraction orders and subsequently confining local density of optical states at two distinct resonance wavelengths, we present a new class of highly sensitive refractive index biosensing platforms that are capable of resolving 35.5 to 41.3 nm/RIU of spectral shift for two separate biological analytes.
The survey of Canadian shale sites showed a dominance of halophilic microorganisms, including Halomonas (HA). Nitrate-amended incubations of the field samples under high salinity (14.6% NaCl), revealed a dominance of HA (>72%) and an accumulation of nitrite. Nitrite accumulation directly inhibited the growth of SRB, thereby decreasing their souring and corrosion risks. However, accumulated nitrite may also contribute to iron corrosion, which will be tested by using different concentrations of nitrate as an electron acceptor to HA. Different salinities are further tested on HA strains supplemented with iron coupons to determine their effects on iron corrosion rates. HA incubated with separate cultures of corrosive methanogen and SRB were tested to determine whether a positive or adverse effect will occur between them. Lastly, analyses of iron coupons will be conducted using TOF-SIMS, FIB-SEM and EDS for corrosion product characterization
BIOCIDE
(2022)
Quaternary ammonium compounds (QACs) are widely used as active agents in disinfectants, antiseptics, and preservatives. Despite being in use since the 1940s, there remain multiple open questions regarding their detailed mode-of-action and the mechanisms, including phenotypic heterogeneity, that can make bacteria less susceptible to QACs. To facilitate studies on resistance mechanisms towards QACs, we synthesized a fluorescent quaternary ammonium compound, namely N-dodecyl-N,N-dimethyl-[2-[(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]ethyl]azanium-iodide (NBD-DDA). NBD-DDA is readily detected by flow cytometry and fluorescence microscopy with standard GFP/FITC-settings, making it suitable for molecular and single-cell studies. As a proof-of-concept, NBD-DDA was then used to investigate resistance mechanisms which can be heterogeneous among individual bacterial cells. Our results reveal that the antimicrobial activity of NBD-DDA against Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa is comparable to that of benzalkonium chloride (BAC), a widely used QAC, and benzyl-dimethyl-dodecylammonium chloride (BAC12), a mono-constituent BAC with alkyl-chain length of 12 and high structural similarity to NBD-DDA. Characteristic time-kill kinetics and increased tolerance of a BAC tolerant E. coli strain against NBD-DDA suggest that the mode of action of NBD-DDA is similar to that of BAC. As revealed by confocal laser scanning microscopy (CLSM), NBD-DDA is preferentially localized to the cell envelope of E. coli, which is a primary target of BAC and other QACs. Leveraging these findings and NBD-DDA‘s fluorescent properties, we show that reduced cellular accumulation is responsible for the evolved BAC tolerance in the BAC tolerant E. coli strain and that NBD-DDA is subject to efflux mediated by TolC. Overall, NBD-DDA’s antimicrobial activity, its fluorescent properties, and its ease of detection render it a powerful tool to study resistance mechanisms of QACs in bacteria and highlight its potential to gain detailed insights into its mode-of-action.