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Uninvited and welcomed guests in museums – biological management of clothes moths and woodworms
(2020)
In museums and historic houses clothes, moths (Tineola bisselliella, Tinea pellionella) and the furniture beetle (Anobium punctatum) are the most economically important pests on textiles or wooden artifacts, respectively. Their management is essential to protect cultural ethnological heritage and natural history collections for future generations.
Pest management strategies have changed over time. Today, intensive knowledge on pest biology and overall material science are key cornerstones in IPM concepts - also for the Museum environment. The important first steps for sustainable pest management are risk assessment, early pest detection and identification of pathways of infestation. These steps are followed by physical and biological means of control, which have lately gained more importance than applying biocides. Several potentially effective biological enemies of clothes moths and woodworm have been known for a long time, but their promotion for pest control in Museums and historic houses is just beginning.
This short review summarizes current concepts of pest life cycle interruption by applying good quarantine and very specific biological measures. The lessons learned from recent faunistic surveys, life-history studies as well as behavioral observations of parasitoids and predators of clothes moths and woodworm may supplement the pest management tool box. The need for further research in this field is addressed.
Uninvited and Welcomed Guests in Museums – Biological management of
clothes moths and woodworms
(2019)
In museums and historic houses clothes, moths (Tineola bisselliella, Tinea pellionella) and the furniture beetle (Anobium punctatum) are the most economically important pests on textiles or wooden artifacts, respectively. Their management is essential to protect cultural ethnological heritage and natural history collections for future generations.
Pest management strategies have changed over time. Today, intensive knowledge on pest biology and overall material science are key cornerstones in IPM concepts - also for the museum environment. The important first steps for sustainable pest management are risk assessment, early pest detection and identification of pathways of infestation. These steps are followed by physical and biological means of control, which have lately gained more importance than applying biocides.
Several potentially effective biological enemies of clothes moths and woodworm have been known for a long time, but their promotion for pest control in museums and historic houses is just beginning.
This short review summarizes current concepts of pest life cycle interruption by applying good quarantine and very specific biological measures. The lessons learned from recent faunistic surveys, life-history studies as well as behavioral observations of parasitoids and predators of clothes moths and woodworm may supplement the pest management tool box. The need for further research in this field is addressed.
Antimicrobial touch surfaces have been introduced in healthcare settings with the aim of supporting 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 may drive the evolution and spread of antimicrobial resistance. This review highlights studies that indicate risks associated with resistance on antimicrobial surfaces by different processes, including evolution by de-novo mutation and horizontal gene transfer, and species sorting of inherently resistant bacteria dispersed on to antimicrobial surfaces. The review focuses on antimicrobial surfaces made of copper, silver and antimicrobial peptides because of the practical application of copper and silver, and the promising characteristics of antimicrobial peptides. The available data point to a potential for resistance selection and a subsequent increase in resistant strains via cross-resistance and co-resistance conferred by metal and antibiotic resistance traits. However, translational studies describing the development of resistance to antimicrobial touch surfaces in healthcare-related environments are rare, and will be needed to assess whether and how antimicrobial surfaces lead to resistance selection in These settings. Such studies will need to consider numerous variables, including the antimicrobial concentrations present in coatings, the occurrence of biofilms on surfaces, and the humidity relevant to dry-surface environments. On-site tests on the efficacy of antimicrobial Coatings should routinely evaluate the risk of selection associated with their use.
Introduction: Biofilms are regarded as a common cause of chronic infections on medical devices. Preventive and therapeutic strategies against biofilm infections commonly involve applications of multiple antimicrobial substances: antimicrobial coatings on the implanted biomaterials in combination with systemically administered antibiotics. While this practice of combination therapy harbours the risk of developing cross-resistance, it might also provide the possibility to implement specific antimicrobial-antibiotic combinations (AACs) that can slow down the selection of antibiotic resistant strains.
Hypothesis and aims: Specific AACs can exert combinatorial effects on the growth of susceptible and antibiotic-resistant Pseudomonas aeruginosa that either suppress or increase their individual effects.
Our aim is to identify AACs with antagonistic or synergistic effects on pseudomonal biofilms and to understand their impact on selection of resistant strains. Specifically, we want to identify AACs that select for and against antibiotic resistance during biofilm formation.
Methodology: We screened for AACs that cause antagonistic or synergistic effects on planktonic P. aeruginosa.
To study the effect of antimicrobial-antibiotic exposure on resistance selection in bacterial biofilms, we will grow resistant and sensitive strains on PDMS surfaces with and without antimicrobial coatings and expose them to antibiotics.
Results: Several combinations with synergistic or antagonistic interaction on the growth rate of P. aeruginosa were detected. We observed a strong antagonism when combining the antimicrobial substance chlorhexidine with the carbapenem drug meropenem. A meropenem-resistant mutant showed a selection advantage in low concentrations of chlorhexidine combined with a sub-inhibitory concentration of meropenem over the wild-type. No antagonistic effect was observed for the same combination when E. coli was exposed to chlorhexidine and meropenem, suggesting a non-chemical basis for the observed effect on P. aeruginosa.
Conclusion: Gaining a better understanding about resistance selection during biofilm formation on biomedical surfaces will enable us to mitigate against biofilm-associated antimicrobial resistance.
Background Bacterial biofilms are regarded as the most common cause of chronic infections and are often associated with medical devices, such as implants and catheters. Bacteria growing in biofilms produce a protective, extracellular matrix, which enables them to tolerate much higher antimicrobial concentrations than free-living bacteria and survive long enough to acquire antimicrobial resistance. Preventive and therapeutic strategies against biofilm infections in clinical settings commonly involve the application of multiple antimicrobials: biocidal coatings on the biomaterials and systemically administered antibiotics. This frequent practice harbors the risk of the development of cross-resistance via shared resistance mechanisms between antimicrobials used in material coatings and administered antibiotics.
Aim Our goal is to determine how bacteria adapt to antimicrobials during biofilm formation on surfaces coated with antimicrobials and how population dynamics within biofilms affect the transmission of resistance mutations. Specifically, we want to identify antimicrobial-antibiotic-combinations that select for and against antibiotic resistance in biofilms by following the population dynamics of resistant and susceptible strains in competition assays on a single cell level.
Methodology To study the effect of antimicrobial-antibiotic exposure on resistance development and population dynamics on bacterial biofilms in a multidrug environment, we will grow Pseudomonas aeruginosa on glass surfaces with and without antimicrobial coatings and expose them to antibiotics. First, we will screen in vitro for combinations of antibiotics and antimicrobials that select for and against antibiotic resistance. Second, effective combinations will be chosen for in-depth investigations during bacterial adhesion and of mature biofilms of resistant and susceptible genotypes. Third, based on the outcome of the screen and the obtained mechanistic understanding we will choose a clinical example in which we study the relevance of our findings in biofilms grown in vivo.
Relevance Studying the biointerfacial interactions between bacterial biofilms and medical devices in terms of population dynamics as well as on single cell level during multidrug selection will help us understand how drug resistance develops and spreads in persistent biofilm infections. Based on our findings we aim to provide clinical recommendations for improved administration of antibiotics/antimicrobials in combination with medical device materials in order to mitigate against biofilm associated antimicrobial resistance.
Selection of resistance in bacteria grown on antimicrobial surfaces in a multidrug environment
(2018)
Bacterial biofilms are regarded as the most common cause of chronic infections and are often associated with medical devices, such as implants and catheters. Bacteria growing in biofilms produce a protective, extracellular matrix, which enables them to tolerate much higher antimicrobial concentrations than free-living bacteria and survive long enough to acquire antimicrobial resistance. Preventive and therapeutic strategies against biofilm infections in clinical settings commonly involve the application of multiple antimicrobials: antimicrobial coatings on the biomaterials and systemically administered antibiotics. This frequent practice harbors the risk of the development of cross-resistance via shared resistance mechanisms between antimicrobials used in material coatings and administered antibiotics.
Our goal is to determine how population dynamics within biofilms affect the transmission of resistance mutations. Specifically, we want to identify antimicrobial-antibiotic-combinations that select for and against antibiotic resistance in biofilms by following the population dynamics of resistant and susceptible strains in competition assays. Gaining a better understanding about resistance development and spread in persistent biofilm infections will enable us to provide clinical recommendations for improved administration of antibiotics/antimicrobials in combination with medical device materials to mitigate against biofilm associated antimicrobial resistance.
Here, we will discuss our first findings on the effects of combinations of the carbapenem drug meropenem and various antimicrobials.
Background Bacterial biofilms are regarded as the most common cause of chronic infections and are often associated with medical devices, such as implants and catheters. Bacteria growing in biofilms produce a protective, extracellular matrix, which enables them to tolerate much higher antimicrobial concentrations than free-living bacteria and survive long enough to acquire antimicrobial resistance. Preventive and therapeutic strategies against biofilm infections in clinical settings commonly involve the application of multiple antimicrobials: biocidal coatings on the biomaterials and systemically administered antibiotics. This frequent practice harbors the risk of the development of cross-resistance via shared resistance mechanisms between antimicrobials used in material coatings and administered antibiotics.
Aim Our goal is to determine how bacteria adapt to antimicrobials during biofilm formation on surfaces coated with antimicrobials, how antimicrobial resistance mutations are acquired and evolve within mature biofilms, and how population dynamics within biofilms affect the transmission of resistance mutations. Specifically, we want to identify antimicrobial-antibiotic-combinations that select for and against antibiotic resistance in biofilms by following the population dynamics of resistant and susceptible strains in competition assays.
Methodology We will grow biofilms of Pseudomonas aeruginosa on glass surfaces with and without antimicrobial coatings and expose them to antibiotics. Then we will track their physiological properties, evolutionary adaptations, and population dynamics. First, we will screen in vitro for combinations of antibiotics and antimicrobials that select for and against antibiotic resistance. Second, effective combinations will be chosen for in-depth investigations during bacterial adhesion and of mature biofilms of resistant and susceptible genotypes. Third, based on the outcome of the screen and the obtained mechanistic understanding we will choose a clinical example in which we study the relevance of our findings in biofilms grown in vivo.
Relevance Studying the biointerfacial interactions between bacterial biofilms and medical devices in terms of population dynamics as well as on single cell level during multidrug selection will help us understand how drug resistance develops and spreads in persistent biofilm infections. Based on our findings we aim to provide clinical recommendations for improved administration of antibiotics/antimicrobials in combination with medical device materials in order to mitigate against biofilm associated antimicrobial resistance.
Environmental sustainability and –stability of Materials concerning the Migration of pollutants
(2018)
MaUS is an acronym for ”Material und Umweltsimulationen“. Plastics are in the focus of environmental politics due to their long-term behaviour and therefore to their persistence. Not only that they appear as visible contaminants in the sea and on the beach, but their unknown behaviour concerning their additives as well as the related transformation products are anxious. Therefore, we wish to establish a certified reference method to provide a method for testing plastics.
Aim of this project is the development of fast motion standard reference methods for testing plastics regarding to their environmental compatibility. To establish these testing methods, we use polystyrene (PS) and polypropylene (PP) with environmental relevant brominated flame retardants, known for their persistent bioaccumulative and toxic (PBT) properties. In case of PS the material contains 1 wt% of 1,2,5,6,9,10-hexabromocyclododecan (HBCD) and in case of PP 0.1 wt% bromodiphenylether (BDE-209), which is known as a substance of very high concern (SVHC). Furthermore, we use polycarbonate (PC), which is still used as material in baby flasks and releases Bisphenol A (BPA), an estrogenic active substance.
As an additional material PTFE is used for its importance as a source for two ubiquitous environmental substances (PFOS and PFOA), whose toxicological effects are still incompletely known.
The focus in this current work is set on the transfer of potential pollutants out of applied materials mentioned above into environmental compartments like water or soil. Here an accelerated aging concept should be developed to shortened time consuming natural processes. For these resulting simulations we use a programmable weathering chamber with dry and wet periods and with high and low temperatures. These programmes run for several weeks and according to a defined sampling schedule we take water samples, run a clean-up procedure by SPE (Molecular imprinted polymers (MiPs) resp. polymer-based cartridges (Waters Oasis HLB)) and analyse them by HPLC-UV resp. LC-MS/MS. Of most interest in case of flame retardants are photocatalytic transformation products. Therefore, we conduct a non-target-screening resp. a suspected target-screening by LC-MS/MS and HRMS.
Environmental sustainability and –stability of materials concerning the migration of pollutants
(2019)
In addition to previously reported results on the accelerated weathering of polystyrene samples (PS) containing 1 wt. % hexabromocyclododecane (HBCD), we present the first results of our investigations of polypropylene samples (PP) containing 0.1 wt. % BDE-209. All studied polymer samples were exposed to a defined weathering schedule in a climate chamber in accordance to regulation EN ISO 4892-3:2006. For the determination of BDE-209 in the collected raining water the samples were prepared in accordance to a validated protocol. Before the analyses each sample was spiked with isotopically labeled BDE-209. Subsequently the samples were extracted with isooctane. The obtained extracts were concentrated, and the resulting solutions were analyzed by GC/MS. Additionally, the total bromine content was monitored for the weathered and untreated samples using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and X-ray fluorescence analysis (XRF) as a non-destructive and rapid method. In general, the resulting data from the accelerated weathering will be compared to those from the natural weathering experiments. Here, the surfaces of the test pieces were analyzed by LA-ICP-MS and XRF as well. Moreover, soil bed tests were conducted in a well characterized model soil. This soil (boulder-clay, sand with 12 % loam, particle size in total 0.2-4 mm) is filled in a free-draining basin inside of an air-conditioned cellar. In this manner, TOC, water capacity and humidity are recorded parameters. To induce a leaching process from the samples by the raining water, the target water content is calculated to 8%. The actual humidity is measured by a tensiometer, assuring the duration of the raining period. A defined humidity is a fundamental parameter for a biological activity in the soil. The test polymers were placed up to the half in the soil. Microbial activity of the soil is monitored by a reference polymer (polyurethane) and should induce the release of HBCD and BDE-209 out of the test materials. These released analytes will be captured by passive samplers (silicone tubes) placed in a distinct distance to the polymer samples in the soil. The soil bed experiments are complementary to the weathering experiments due to the biological activity in the soil.
The evolutionary success of termites has been driven largely by a complex communication system operated by a rich set of exocrine glands. As many as 20 different exocrine organs are known in termites. While some of these organs are relatively well known, only anecdotal observations exist for others. One of the exocrine organs that has received negligible attention so far is the labral gland. In this study, we examined the structure and ultrastructure of the Labrum in soldiers of 28 termite species. We confirm that the labral gland is present in all termite species, and comprises two secretory regions located on the ventral side of the labrum and the dorso-apical part of the hypopharynx. The Labrum of Neoisoptera has a hyaline tip, which was secondarily lost in Nasutitermitinae, Microcerotermes and species with snapping soldiers. The epithelium of the gland generally consists of class 1 secretory cells, with an addition of class 3 secretory cells in some species. A common feature of the secretory cells is the abundance of smooth endoplasmic reticulum, an organelle known to produce lipidic and often volatile secretions. Our observations suggest that the labral gland is involved in communication rather than defence as previously suggested. Our study is the first to provide a comprehensive picture of the structure of the labral gland in soldiers across all Termite taxa.
Wood protection technology in the marine environment has changed over the last decades and will continue to do so. New active ingredients, newer formulations, and novel wood-based materials including physically- and chemically-modified wood, together with increasing concerns over environmental impacts of wood preservatives, urgently demand a major revision of EN 275 “Wood preservatives – Determination of the Protective Effectiveness against Marine Borers”, dated from 1992. This IRG document reports on the technical work in CEN TC 38 regarding the revision of this standard. A Task Group within WG 24 of CEN TC 38 was formed consisting of experts from different field of competence (e.g. wood preservatives industry, wood scientists, marine biologists, archaeologists and cultural heritage conservators). Starting by e-mail correspondence in 2014, and continuing with four physical meetings (Berlin 2x, Florence, Venice) with experts from Germany, Italy, Sweden, and UK were held so far. Significant items for revision in EN 275 were identified as: number of replicates, duration of the test, dimension of specimens, number of test sites, number of reference species, reference material including reference preservative, re-immersion of specimens after non-destructive periodical evaluation for longer periods of time vs higher number of replicates for successive destructive examinations without re-immersion, utilization of X- ray apparatus and specific software to ease evaluation, etc.
Furthermore, the task group is working on a standardized lab test for time-saving evaluation of different wood qualities for their potential to resist attack by limnorids. The suitability of this lab test will be determined by round robin tests as soon as safe face-to-face collaboration permits. The outcome will be published as a CEN TR (Technical Report) document, with a view to eventual adoption within the revised standard.
In nature, the cellular environment of DNA includes not only water and ions, but also other components and co-solutes, which can exert both stabilizing and destabilizing effects on particular oligonucleotide conformations. Among them, ectoine, known as an important osmoprotectant organic co-solute in a broad range of pharmaceutical products, turns out to be of particular relevance. In this article, we study the influence of ectoine on a short single-stranded DNA fragment and on double-stranded helical B-DNA in aqueous solution by means of atomistic molecular dynamics (MD) simulations in combination with molecular theories of solution. Our results demonstrate a conformation-dependent binding behavior of ectoine, which favors the unfolded state of DNA by a combination of electrostatic and dispersion interactions. In conjunction with the Kirkwood–Buff theory, we introduce a simple Framework to compute the influence of ectoine on the DNA melting temperature. Our findings reveal a significant linear decrease of the melting temperature with increasing ectoine concentration, which is found to be in qualitative agreement with results from denaturation experiments. The outcomes of our Computer simulations provide a detailed mechanistic rationale for the surprising destabilizing influence of ectoine on distinct DNA structures.
The development of the microbiologically influenced corrosion ( MIC ) -specific inductively coupled plasma-time of flight-mass spectrometry ( ICP-ToF-MS ) analytical method presented here, in combination with the investigation of steel-MIC interactions, contributes significantly to progress in instrumental MIC analysis. For this, a MIC-specific staining procedure was developed, which ensures the analysis of intact cells. It allows the analysis of archaea at a single cell level, which is extremely scarce compared to other well-characterized organisms. The detection method revealed elemental selectivity for the corrosive methanogenic strain Methanobacterium -affiliated IM1. Hence, the possible uptake of individual elements from different steel samples was investigated and results showed the cells responded at a single-cell level to the different types of supplemented elements and displayed the abilities to uptake chromium, vanadium, titanium, cobalt, and molybdenum from solid metal surfaces. The methods developed and information obtained will be used in the future to elucidate underlying mechanisms, compliment well-developed methods, such as SEM-EDS, and develop novel material protection concepts.
Microbiologically influenced corrosion (MIC) is a highly unpredictable process dictated by the environment, microorganisms, and the respective electron source. Interaction pathways between cells and the metal surface remain unclear. The development of this novel single cell-inductively coupled plasma-time of flight-mass spectrometry analytical method and a MIC-specific staining procedure facilitate the investigation of steel-MIC interactions. With this it is possible to analyze the multi-elemental fingerprint of individual cells. The detection method revealed elemental selectivity for the corrosive methanogenic archaeal strain Methanobacterium-affiliated IM1. The interface between material and environmental analysis thus receives special attention, e.g., when considering MIC on solid steel. Hence, the possible uptake of individual elements from different steel samples is investigated. Results showed the cells responded at a single-cell level to the different types of supplemented elements and displayed the abilities to interact with chromium, vanadium, titanium, cobalt, and molybdenum from solid metal surfaces. The information obtained will be used in the future to elucidate underlying mechanisms and develop possible material protection concepts, thus combining modern methods of analytical sciences with materials research.
Deciphering microbiological influenced corrosion processes on steel with single cell-ICP-ToF-MS
(2022)
Microbiologically influenced corrosion (MIC) is a highly unpredictable process dictated by the environment, microorganisms, and the respective electron source. Interaction pathways between cells and the metal surface remain unclear. The development of this novel single cell-inductively coupled plasma-time of flight-mass spectrometry analytical method and a MIC-specific staining procedure facilitate the investigation of steel-MIC interactions. With this it is possible to analyze the multi-elemental fingerprint of individual cells. The detection method revealed elemental selectivity for the corrosive methanogenic archaeal strain Methanobacterium-affiliated IM1. The interface between material and environmental analysis thus receives special attention, e.g., when considering MIC on solid steel. Hence, the possible uptake of individual elements from different steel samples is investigated. Results showed the cells responded at a single-cell level to the different types of supplemented elements and displayed the abilities to interact with chromium, vanadium, titanium, cobalt, and molybdenum from solid metal surfaces. The information obtained will be used in the future to elucidate underlying mechanisms and develop possible material protection concepts, thus combining modern methods of analytical sciences with materials research.
References.
Microbial communities in the immediate environment of socialized invertebrates can help to suppress pathogens, in part by synthesizing bioactive natural products. Here we characterized the core microbiomes of three termite species (genus Coptotermes) and their nest material to gain more insight into the diversity of termite-associated bacteria.
Sampling a healthy termite colony over time implicated a consolidated and highly stable microbiome, pointing toward the fact that beneficial bacterial phyla play a major role in termite fitness. In contrast, there was a significant shift in the composition of the core microbiome in one nest during a fungal infection, affecting the abundance of wellcharacterized Streptomyces species (phylum Actinobacteria) as well as less-studied bacterial phyla such as Acidobacteria. High-throughput cultivation in microplates was implemented to isolate and identify these less-studied bacterial phylogenetic group.
Amplicon sequencing confirmed that our method maintained the bacterial diversity of the environmental samples, enabling the isolation of novel Acidobacteriaceae and expanding the list of cultivated species to include two strains that may define new species within the genera Terracidiphilus and Acidobacterium.
Infections with multi-resistant Gram negative pathogens are a major threat to our health system. In order to serve the needs in antibiotics development we selected untapped bioresources and implemented high throughput approaches suitable for the discovery of strains producing antibiotics with anti-Gram negative activity.
Our approaches rely on the hypothesis that Termite associated bacteria are likely to produce potent antibiotics to defend their hosts against entomopathogenic microorganisms. Termite nests and guts harbor suitable, highly diverse microbiomes in which bacterial taxa are present known to potentially produce natural compounds. In
a first step the diversity of Coptotermes species nest microbiomes was assessed carefully by using 16S rDNA amplicon sequencing on the Illumina MiSeq platform and nest material was selected to retrieve viable cells by using Nycodenz density gradient centrifugation. In order to analyze the diversity of the culturable termite nest microbiome, bacterial cells were either distributed in 384-well plates
(approach 1) or encapsulated in small spheric agarose beads by an high throughput microfluidics technique (approach 2). Cultures obtained from approach 1 were scaled-up in 96-well Duetz-systems for characterization of diversity and for rapid supernatant screening using the bioluminescence-labeled E. coli pFU166. The generated
droplets of approach 2 simultaneously received a small population of GFP-tagged Gram negative screening cells and were sorted for low fluorescence using FACS. After elimination of redundancy we performed a fast scale-up of active strains.
Implementation of this pipeline allows us to prioritize antibiotics producing strains in a ultra-high throughput fashion and by cultivation of broad diversity in our approches.
The growth rate of single bacterial cells is continuously disturbed by random fluctuations in biosynthesis rates and by deterministic cell-cycle events, such as division, genome duplication, and septum formation.
It is not understood whether, and how, bacteria reject these growth-rate disturbances. Here, we quantified growth and constitutive protein expression dynamics of single Bacillus subtilis cells as a function of cell-cycle progression. We found that, even though growth at the population level is exponential, close inspection of the cell cycle of thousands of single Bacillus subtilis cells reveals systematic deviations from exponential growth.
Newborn cells display varying growth rates that depend on their size. When they divide, growth-rate Variation has decreased, and growth rates have become birth size independent. Thus, cells indeed compensate for growth-rate disturbances and achieve growth-rate homeostasis. Protein synthesis and growth of single cells displayed correlated, biphasic dynamics from cell birth to division. During a first phase of variable duration, the absolute rates were approximately constant and cells behaved as sizers. In the second phase, rates increased, and growth behavior exhibited characteristics of a timer strategy. These findings demonstrate that, just like size homeostasis, growth-rate homeostasis is an inherent property of single cells that is achieved by cell-cycle-dependent rate adjustments of biosynthesis and growth.
Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations which can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfection, knowledge concerning persistence to disinfectants and its link to resistance evolution is currently lacking. Here, we show that E. coli displays persistence against a widely used disinfectant benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance. BAC tolerance is associated with reduced cell surface charge and mutations in the novel tolerance locus lpxM. Moreover, the fitness cost incurred by BAC tolerance turned into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings provide a mechanistic underpinning for the faithful application of disinfectants to prevent multi-drug-resistance evolution and to steward the efficacy of biocides and antibiotics.
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.
Biocides used as disinfectants are important to prevent the transmission of pathogens, especially during the current antibiotic resistance crisis. This crisis is exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic Treatment and facilitate resistance evolution. Here, we show that E. coli displays persistence against a widely used disinfectant, benzalkonium chloride (BAC). Periodic, persister-mediated failure of disinfection rapidly selects for BAC tolerance, which is associated with reduced cell Surface charge and mutations in the lpxM locus, encoding an enzyme for lipid A biosynthesis.
Moreover, the fitness cost incurred by BAC tolerance turns into a fitness benefit in the presence of antibiotics, suggesting a selective advantage of BAC-tolerant mutants in antibiotic environments. Our findings highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
Introduction: Effective disinfection is crucial to maintain hygiene and to prevent the spread of infections. Phenotypic heterogeneity in disinfection survival (i.e. tolerance) may result in failure of disinfection, which in turn may foster the evolution of resistance to both disinfectants and antibiotics. However, the consequences of phenotypic heterogeneity for disinfection outcome and resistance evolution are not well understood. Goal: This study investigates the impact of phenotypic heterogeneity on the survival and evolution of Escherichia coli during disinfection with six commonly used substances. Furthermore, the consequences of evolved disinfectant tolerance for antibiotic resistance evolution are studied. Materials & Methods: The extent of population heterogeneity during disinfection is derived by determining time-kill kinetics and analysis with mathematical modelling. The link between population heterogeneity and evolvability of disinfectant tolerance was assessed by laboratory evolution experiments under periodic disinfection. The ability of disinfectant tolerant strains to evolve antibiotic resistance is assessed by serial transfer experiments with increasing concentrations of different antibiotics and by whole genome sequencing. Results: Multi-modal time-kill kinetics in three of the six disinfectants suggest the presence of disinfectant-tolerant subpopulations (i.e. persister cells). Importantly, the ability and extent to evolve population-wide tolerance under periodic disinfection is related with the presence of persister cells and the level of phenotypic heterogeneity during disinfection. Interestingly, the probability of high-level resistance evolution to certain antibiotics is attenuated in disinfectant tolerant strains as compared to the sensitive ancestor. Whole-genome sequencing reveals epistatic interactions between disinfectant tolerance and antibiotic resistance mutations, preventing access to canonical evolutionary paths to resistance. Summary: Our findings suggest that phenotypic heterogeneity can facilitate disinfection survival and the evolution of population wide tolerance, which can impact future antibiotic resistance evolution.
The exposure to antimicrobial substances drives the evolution of antimicrobial resistance. Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work at BAM we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics. Within our group, I mainly focus on the following two questions: How does phenotypic heterogeneity in bacteria affect the ability to survive treatment with biocides? And what are the consequences of phenotypic heterogeneity for the evolution of resistance to biocides and antibiotics?
I will share published and unpublished results which demonstrate that phenotypic heterogeneity can enable the survival of biocide treatment and, through this, facilitate the evolution of AMR. On the other hand, we find that adaptation to a biocide can unexpectedly impair the ability to evolve resistance against an antibiotic.
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 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.
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.
Phenotypic heterogeneity in disinfection: sources and consequences for antimicrobial resistance
(2022)
A summary of projects here at BAM which investigate the influence of phenotypic heterogeneity on the outcome of disinfection and the influence on antimicrobial resistance. This presentation was given in the Theory Seminar of the Quantitative and Theoretical Biology group of Prof. Oliver Ebenhöh at HHU Düsseldorf
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.
The exposure to antimicrobial substances drives the evolution of antimicrobial resistance (AMR). Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics.
Here, I present results which demonstrate that phenotypic heterogeneity in E. coli, namely the formation of tolerant persister cells, can enable the survival of disinfection and consequentially facilitate the evolution of AMR. Using experimental evolution that selects for tolerance, we find that mutations in lipid A biosynthesis arise following periodic treatment with benzalkonium chloride (BAC), a widely used disinfectant. BAC tolerance is associated with additional, diverse mutations as well as changes in the susceptibility to antibiotics from different classes and changes in motility and biofilm formation, suggesting extensive regulatory re-wiring in the evolved clones. Remarkably, we find that fitness costs (growth rate reduction) incurred by BAC tolerance are alleviated in the presence of antibiotics. These findings demonstrate the complexity underlying the adaptation to antimicrobials and highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
The exposure to antimicrobial substances drives the evolution of antimicrobial resistance (AMR). Biocides are antimicrobials used as disinfectants, antiseptics and preservatives. They find application on a large scale in the industrial and medical sector, but also in private households. In terms of mass, the worldwide use of biocides exceeds that of antibiotics. Thus, despite their important role in preventing the spread of pathogens, due to their ubiquity, biocides are suspected to be drivers of the antimicrobial resistance crisis. In our work we try to understand how biocides contribute to the emergence of AMR, what the underlying adaptation principles and mechanisms are and how they compare to those found for antibiotics.
Here, I present results which demonstrate that phenotypic heterogeneity in E. coli, namely the formation of tolerant persister cells, can enable the survival of disinfection and consequentially facilitate the evolution of AMR. Using experimental evolution that selects for tolerance, we find that mutations in lipid A biosynthesis arise following periodic treatment with benzalkonium chloride (BAC), a widely used disinfectant. BAC tolerance is associated with additional, diverse mutations as well as changes in the susceptibility to antibiotics from different classes and changes in motility and biofilm formation, suggesting extensive regulatory re-wiring in the evolved clones. Remarkably, we find that fitness costs (growth rate reduction) incurred by BAC tolerance are alleviated in the presence of antibiotics. These findings demonstrate the complexity underlying the adaptation to antimicrobials and highlight the links between persistence to disinfectants and resistance evolution to antimicrobials.
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.
Wild bees are important pollinators for agricultural crops and solitary species such as Osmia bicornis are particularly suitable for pollination management. Wild bees share floral resources with managed honey bees and may be exposed to emerging infectious diseases. Although studies have explored the prevalence of pathogens in solitary wild bee species, data regarding the impact of pathogens on solitary bee health are lacking.
We carried out experiments examining whether the solitary bee species O. bicornis is susceptible to infection with the emerging pathogen The results obtained indicate that N. ceranae may be able to infect O. bicornis but its impact on host fitness is negligible: survival rates did not differ between Control and inoculated bees, although male survival was marginally lower after infection. To explore the possible field-relevance of our findings, we collected wild bees near an infected and a non-infected hive and showed that N. ceranae was shared between managed and wild bees, although only the in presence of infected honey bees.
The findings of the present study show that O. bicornis is susceptible to pathogen spillover and could act as a potential reservoir host for N. ceranae in pollinator networks. Additional studies on this species incorporating sublethal effects, multiple infections and other interacting stressors are warranted.
In recent years, the fabrication of laser-generated surface structures on metals such as titanium surfaces have gained remarkable interests, being technologically relevant for applications in optics, medicine, fluid transport, tribology, and wetting of surfaces.
The morphology of these structures, and so their chemistry, is influenced by the different laser processing parameters such as the laser fluence, wavelength, pulse repetition rate, laser light polarization type and direction, angle of incidence, and the effective number of laser pulses per beam spot area.
However, the characterization of the different surface structures can be difficult because of constraints regarding the analytical information from both depth and the topographic artifacts which may limit the lateral and depth resolution of elemental distributions as well as their proper quantification. A promising technique to investigate these structures even at the nano-scale is Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS), a very surface sensitive technique that at the same time allows to perform depth-profiling, imaging and 3D-reconstruction of selected ion-sputter fragment distributions on the surface.
In this study we combine chemical analyses such as Energy Dispersive X-ray spectroscopy (EDX) and high-resolution scanning electron microscopy (SEM) analyses with ToF-SIMS to fully characterize the evolution of various types of laser-generated micro- and nanostructures formed on Ti and Ti alloys at different laser fluence levels, effective number of pulses and at different pulse repetition rates (1 – 400 kHz), following irradiation by near-infrared ultrashort laser pulses (925 fs, 1030 nm) in air environment or under argon gas flow.
We show how this combined surface analytical approach allows to evaluate alteration in the surface chemistry of the laser-generated surface structures depending on the laser processing parameters and the ambient environment.
Inter-pulse accumulation of heat could affect the chemical and morphological properties of the laser processed material surface. Hence, the laser pulse repetition rate may restrict the processing parameters for specific laser-induced surface structures. In this study, the evolution of various types of laser-induced micro- and nanostructures at various laser fluence levels, effective number of pulses and at different pulse repetition rates (1 – 400 kHz) are studied for common metals/alloys (e.g. steel or titanium alloy) irradiated by near-infrared ultrashort laser pulses (925 fs, 1030 nm) in air environment. The processed surfaces were characterized by optical and scanning electron microscopy (OM, SEM), energy dispersive X-ray spectroscopy (EDX) as well as time of flight secondary ion mass spectrometry (TOF-SIMS). The results show that not only the surface morphology could change at different laser pulse repetition rates and comparable laser fluence levels and effective number of pulses, but also the surface chemistry is altered. Consequences for medical applications are outlined.
Biofilm formation in industrial or medical settings is usually unwanted and leads to serious health problems and high costs. Inhibition of initial bacterial adhesion prevents biofilm formation and is, therefore, a major mechanism of antimicrobial action of surfaces. Surface topography largely influences the interaction between bacteria and surfaces which makes topography an ideal base for antifouling strategies and eco-friendly alternatives to chemical surface modifications. Femtosecond laser-processing was used to fabricate sub-micrometric surface structures on silicon and stainless steel for the development of antifouling topographies on technical materials.
Social insects nesting in soil environments are in constant contact with entomopathogens and have evolved disease resistance mechanisms within a colony to prevent the occurrence and spread of infectious diseases. Among these mechanisms: mutual grooming reduces the cuticular load of pathogens, and burial of cadavers and cannibalism can prevent pathogens from replicating within the group. We explored how the rate and type of collective behavioural response is determined by stepwise infection dynamics operating at the level of the individual. Specifically, we infected the eastern subterranean termite Reticulitermes flavipes with different types of infectious particle and infection route of the entomopathogenic fungus Metarhizium anisopliae and recorded behavioural responses of nestmates to individuals at different times during the progression of infections. As expected, termites groomed conidia-exposed individuals significantly more than controls. Interestingly, grooming was significantly elevated after fungal germination than before, suggesting that pathogen growth cues act as strong stimulators of allogrooming. Conidia-exposed termites were cannibalized, but only after they became visibly ill. By contrast, termites did not groom blastospore-injected individuals more than controls at any time-point following infection. Instead, we found that blastospore-injected individuals were continually cannibalized at a low-level following injection with either viable or heat-killed blastospores, with a marked increase in cannibalism after termites injected with viable blastospores became visibly ill and were close to death. Together, these findings point to the importance of host condition as a cue for social hygienic behavior, and that the host itself appears to emit essential sickness cues that act as targets for its own sacrifice. This demonstrates that termites have independently evolved to both identify and destructively respond to sickness.
The termites are a derived eusocial lineage of otherwise non-social cockroaches. Understanding the proximal and ultimate drivers of this major evolutionary transition represents an important goal in biology. One outstanding question concerns the evolution of termite immunity, which is thought to have undergone broad-sweeping adaptations in order to enhance group-level immune protection. To understand the evolutionary origins of termite immunity, we conducted qualitative and quantitative transcriptome analyses along a gradient of sociality. Firstly, we aimed to identify large-scale genetic shifts in immune traits linked to eusociality by comparing immune gene repertoires in solitary and subsocial cockroaches and across a range of eusocial termite lineages. Secondly, we compared the responses of a solitary cockroach, a subsocial wood-roach and different castes of a lower termite species to a non-specific immune challenge, in order to understand how sociality may have influenced the evolution of immune gene regulation. Firstly, we found that termites have a broadly representative repertoire of canonical immune genes as compared to gregarious cockroaches and subsocial wood-roaches. Secondly, with respect to immune challenge, the solitary cockroach and the subsocial wood-roach displayed a similarly comprehensive induced response, while the termite response was considerably dampened by comparison and strongly influenced by caste; with reproductives displaying a generally higher constitutive level of immune-gene expression compared to sterile castes. In summary we did not find termite eusociality to be associated with significant changes in immune gene diversity, but rather to be linked with significant modifications to the regulation of immunity following the origin of division of labour.
Results suggest a reduction in immune gene repertoires in termites and possible complementary expression between termite castes. With comparative genomics we will investigate the evolution of gene families related to immunity, try to understand where reductions and expansions take place and relate these changes to shifts in sociality and ecology. The role of TEs in expansions and contractions of immune gene families will be investigated. For these analyses, we propose to generate high quality, highly contiguous genomes of species from different levels of sociality, covering all major termite families. With comparative transcriptomics we will investigate the expression of immune genes in different castes. Via network analyses we will identify pathways indicated in differential immunity between castes and between species of different sociality levels. We will investigate how these pathways have been rewired along the transitions to higher levels of sociality and how, intra-specifically, they change between castes.
A broad suite of immune adaptations have evolved in social insects which hold close parallels with the immune systems of multicellular individuals. However, comparatively little is known about the evolutionary origins of immunity in social insects. We tackle this by identifying immune genes from 18 cockroach and termite species, spanning a gradient of social lifestyles. Termites have undergone contractions of major immune gene families during the early origin of the group, particularly in antimicrobial effector and receptor proteins, followed by later re-expansions in some lineages. In a comparative gene expression analysis, we find that reproductive individuals of a termite invest more in innate immune regulation than other castes. When colonies encounter immune-challenged nestmates, gene expression responses are weak in reproductives but this pattern is reversed when colony members are immune-challenged individually, with reproductives eliciting a greater response to treatment than other castes. Finally, responses to immune challenge were more comprehensive in both subsocial and solitary cockroaches compared to termites, indicating a reduced overall ability to respond to infection in termites. Our study indicates that the emergence of termite sociality was associated with the evolution of a tapered yet caste-adapted immune system.
Zearalenone (ZEN) and its sulfate and glucoside conjugates have been detected in (a broad variety of) food and feed commodities1. Both conjugated derivatives are formed as part of fungal or plant secondary metabolism and thus, belong to the group of modified mycotoxins2. After consumption of contaminated foodstuff, the conjugates can be hydrolyzed by human intestinal microbiota leading to liberation of ZEN that implies an underestimation of the true ZEN exposure. In order to include ZEN conjugates in routine analysis, as well as for toxicological investigation reliable standards are needed.
The objective of the present study was to develop a simple and economic method for biosynthesis of ZEN conjugates. Preceding experiments on the biotransformation of ZEN by Rhizopus and Aspergillus species showed a mixed metabolite formation3. Therefore, these known ZEN conjugating fungal strains were screened for their potential to selectively synthesize the ZEN derivatives ZEN-14-sulfate (Z14S), ZEN-14-glucoside (Z14G) and ZEN-16-glucoside (Z16G). The screening was conducted by adding ZEN to liquid fungal cultures. Cultivation conditions and ZEN incubation time were varied. All media samples were analyzed for metabolite formation by HPLC-MS/MS. Z14S was exclusively formed by A. oryzae. Under optimized conditions a specific biosynthesis of Z14G by R. oryzae and Z16G by R. oligosporus was achieved. After liquid-liquid-extraction and preparative chromatographic cleanup 1H-NMR purities of ≥ 73% for Z14S, ≥ 82% for Z14G and ≥ 50% for Z16G were obtained. In addition, a consecutive biosynthesis was developed by first using Fusarium graminearum for ZEN biosynthesis on rice based liquid medium. After inactivation of Fusarium the subsequent conjugation reaction was conducted utilizing Aspergillus and Rhizopus species under the various optimized conditions.
In this study an easy and cost-efficient biosynthesis for Z14S, Z14G and Z16G was developed. The developed biosynthesis could be also used for other metabolites like ZEL conjugates. Our results of the in vitro screening indicate also the formation of a ZEL-glucoside and α ZEL-sulfate as major metabolites by R. oryzae. In sum, under optimized cultivation conditions fungi can be easily utilized for a targeted and stereospecific synthesis of ZEN conjugates.
Coastal oceans receive large amounts of anthropogenic fixed nitrogen (N), most of which is denitrified in the sediment before reaching the open ocean. Sandy sediments, which are common in coastal regions, seem to play an important role in catalysing this N‐loss. Permeable sediments are characterized by advective porewater transport, which supplies high fluxes of organic matter into the sediment, but also leads to fluctuations in oxygen and nitrate concentrations. Little is known about how the denitrifying communities in these sediments are adapted to such fluctuations. Our combined results indicate that denitrification in eutrophied sandy sediments from the world's largest tidal flat system, the Wadden Sea, is carried out by different groups of microorganisms. This segregation leads to the formation of N2O which is advectively transported to the overlying waters and thereby emitted to the atmosphere. At the same time, the production of N2O within the sediment supports a subset of Flavobacteriia which appear to be specialized on N2O reduction. If the mechanisms shown here are active in other coastal zones, then denitrification in eutrophied sandy sediments may substantially contribute to current marine N2O emissions.
The feasibility of microbial hydrogen consumption to mitigate the hydrogen embrittlement (HE) under different cathodic potentials was evaluated using the Devanathan-Stachurski electrochemical test and the hydrogen permeation efficiency h. The hydrogen permeation efficiency h in the presence of strain GA-1 was lower than that in sterile medium. The cathodic potential inhibited the adherence of strain GA-1 to AISI 4135 steel surface, thereby reducing the hydrogen consumption of strain GA-1. The adherent GA-1 cells were capable of consuming ‘cathodic hydrogen’ and reducing the proportions of absorbed hydrogen, indicating that it is theoretically possible to control HE by hydrogen-consuming microbes.
Mussel-inspired multifunctional coating for bacterial infection prevention and osteogenic induction
(2021)
Bacterial infection and osteogenic integration are the two main problems that cause severe complications after surgeries. In this study, the antibacterial and osteogenic properties were simultaneously introduced in biomaterials, where copper nanoparticles (CuNPs) were generated by in situ reductions of Cu ions into a mussel-inspired hyperbranched polyglycerol (MI-hPG) coating via a simple dip-coating method. This hyperbranched polyglycerol with 10 % catechol groups’ modification presents excellent antifouling property, which could effectively reduce bacteria adhesion on the surface. In this work, polycaprolactone (PCL) electrospun fiber membrane was selected as the substrate, which is commonly used in biomedical implants in bone regeneration and cardiovascular stents because of its good biocompatibility and easy post-modification. The as-fabricated CuNPs-incorporated PCL membrane [PCL-(MI-hPG)-CuNPs] was confirmed with effective antibacterial performance via in vitro antibacterial tests against Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and multi-resistant E. coli. In addition, the in vitro results demonstrated that osteogenic property of PCL-(MI-hPG)-CuNPs was realized by upregulating the osteoblast-related gene expressions and protein activity. This study shows that antibacterial and osteogenic properties can be balanced in a surface coating by introducing CuNPs.
Without precaution, insects may cause serious damage to Museum collections. Quarantine of potentially infested objects can be logistically challenging. Anoxia under controlled nitrogen atmosphere is a most compatible but also time-consuming method to eradicate insect pests in all kinds of different materials. Treatment results are usually effected by duration, temperature, humidity and residual oxygen content. During a two-year research project, 34 relevant pest insect species of all developmental stages were tested in several different materials (wood, paper, wool) to monitor treatment success and to determine optimum treatment parameters. Duration of Treatment ranged from one to three weeks at temperatures of 20 - 27 °C.
As expected, results showed significant differences in mortality among tested species. Highest tolerance of hypoxic conditions was found in older larvae of Hylotrupes bajulus. However, this species is an unlikely museum pest. Anobiids and other wood boring beetles are more often an issue related to cultural heritage. Tested imbedding materials in general had no mortality lowering influence. A combination of three weeks exposure time at up to 0.5 % residual oxygen and at 24 °C and 50 % RH is recommended for infested artefacts.
Without precaution, insects may cause serious damage to museum collections. Quarantine of potentially infested objects can be logistically challenging. Anoxia under controlled Nitrogen atmosphere is a most gentle but also time-consuming method to eradicate insect pests in all kinds of different materials. Treatment results are usually affected by duration, temperature, humidity and residual oxygen content. During a two-year research project, 34 relevant pest insect species of all developmental stages were tested in different materials (wood, paper, wool) to monitor treatment success and to determine optimum treatment parameters. Duration of treatment ranged from one to three weeks at temperatures of 20–27 °C. As expected, results showed significant differences in mortality among tested species. Highest tolerance of hypoxic conditions was found in elder larvae of Hylotrupes bajulus. Although this species is an unlikely museum pest, it may serve as an overall most tolerant reference. Anobiids and other wood boring beetles are more often an issue related to cultural heritage. A combination of three weeks exposure time at maximum 0.5% residual oxygen and 24 °C, alternatively 1%
residual oxygen and 27 °C are recommended for infested artefacts. Imbedding materials in general had no influence on mortality. This study was funded by Deutsche Bundesstiftung Umwelt (DBU).
The compatible solute ectoine: protection mechanisms, strain development, and industrial production
(2020)
Bacteria, Archaea, and Eukarya can adapt to saline environments by accumulating compatible solutes in order to maintain an osmotic equilibrium. Compatible solutes are of diverse chemical structure (sugars, polyols, amino acid derivatives) and are beneficial for bacterial cells not only as osmoregulatory solutes but also as
protectants of proteins by mitigating detrimental effects of freezing, drying, and high temperatures. The aspartate derivative ectoine is a widespread compatible solute in Bacteria and possesses additional protective properties compared with other compatible solutes and stabilizes even whole cells against stresses such as ultraviolet radiation or cytotoxins. Here, it is our intention to go beyond a simple description of effects, but to depict the molecular interaction of ectoine with biomolecules, such as proteins, membranes, and DNA and explain the underlying principles. The stabilizing properties of ectoine attracted industry, which saw the potential to market ectoine
as a novel active component in health care products and cosmetics. In joint efforts of industry and research, a large-scale fermentation procedure has been developed with the halophilic bacterium Halomonas elongata used as a producer strain. The development and application of ectoine-excreting mutants from H. elongata (“leaky” mutants) allow for the annual production of ectoine on a scale of tons. The details of
the strain development and fermentation processes will be introduced.
The compatible solute ectoine is a versatile protectant synthesized by many prokaryotes. It is used as an osmolyte helping microorganisms to maintain an osmotic equilibrium. In addition, ectoine acts as a stabilizer and protects proteins, membranes and whole cells against detrimental effects such as freezing and thawing, drying and high temperatures. Its protective effect is explained by the preferential exclusion model, which postulates that ectoine does not directly interact with biomolecules but is excluded from their surface. Interestingly, details on the interaction of ectoine with DNA are still unknown. Therefore, we studied the influence of ectoine on DNA and the mechanisms by which ectoine protects DNA against ionizing radiation. To emulate biological conditions, we used a sample holder comprising a silicon chip with a Si3N4 membrane, which allows for electron irradiation of DNA in aqueous solution. Analysis by atomic force microscopy revealed that without ectoine, DNA was damaged by irradiation with a dose of 1,7 +/-0,3 Gy. With ectoine, DNA remained undamaged, even after irradiation with 15 Gy. Simulations with dsDNA and ectoine in water revealed a preferential binding of the zwitterionic ectoine to the negatively charged DNA. According to the simulations, binding of ectoine will destabilize dsDNA. Destabilizing is probably caused by the transition of B-DNA to A-DNA and will reduce the DNA melting temperature, which was experimentally proven. The preferential binding provides a stable ectoine shell around DNA, which allows ectoine to reduce OH-radicals and electrons near the DNA and thereby mitigating the damaging effect of ionizing radiation.