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Organisationseinheit der BAM
- 4 Material und Umwelt (138) (entfernen)
Background. Disinfection is an important mitigation strategy to control and prevent the spread of infections. Incomplete or incorrect usage of disinfection may promote evolution of resistance against disinfectants and antibiotics. Ideally, disinfection reduces the number of surviving bacteria and the chance for resistance evolution. Resistance describes the ability to grow in previously inhibitory concentrations of an antimicrobial, whereas tolerance is associated with enhanced survival of lethal doses. Individual bacteria from the same population can display considerable heterogeneity in their ability to survive treatment (i.e. tolerance) with antimicrobials, which can result in unexpected treatment failure.
Objective. In this study, we investigated six active substances of disinfectants, preservatives, and antiseptics against a population of E. coli to identify the presence of a tolerant subpopulation.
Methods. We performed time-kill experiments and analyzed the data with a mathematical model to statistically infer whether the data is best explained by the presence of a tolerant subpopulation.
Results. The analysis identified bimodal kill kinetics for benzalkonium chloride, didecyldimethylammonium chloride, and isopropanol. In contrast, kill kinetics by chlorhexidine, glutaraldehyde, and hydrogen peroxide were best explained by unimodal kill kinetics. These findings have implications for the risk of disinfection failure. In addition, we are currently performing adaptive laboratory evolution (ALE) experiments with the different disinfectants to investigate the potential consequences of tolerant sub-populations for the evolution of antimicrobial resistance and tolerance.
The amount of plant-available phosphorus (P) in soil strongly influences the yield of plants in agriculture. Therefore, various simple chemical extraction methods have been developed to estimate the plant-available P pools in soil. More recently, several experiments with the DGT technique have shown that it has a much better correlation to plant-available P in soils than standard chemical extraction methods (e.g. calcium-acetate-lactate (CAL), Colwell, Olsen, water) when soils with different characteristics are considered. However, the DGT technique cannot give information on the plant-available P species in the soil. Therefore, we combined DGT with solution 31P nuclear magnetic resonance (NMR) spectroscopy. This was achieved by using a modified DGT device in which the diffusive layer had a larger pore size, the binding layer incorporated an adsorption material with a higher capacity, and the device had a larger exposure area. The spectroscopic investigation was undertaken after elution of the deployed DGT binding layer in a NaOH solution. Adsorption tests using solutions of known organic P compounds showed that a sufficient amount of these compounds could be adsorbed on the binding layer in order for them to be analyzed by solution 31P NMR spectroscopy. Furthermore, various intermediates of the hydrolysis of trimetaphosphate in soil could be also analyzed over time.
Disinfectants are important to prevent the transmission of pathogens, especially in the face of the current antibiotic resistance crisis. The crisis is further exacerbated by phenotypically tolerant persister subpopulations that can survive transient antibiotic treatment and facilitate resistance evolution. Despite the transient nature of disinfectant application, persistence to disinfectants and its role for the evolution of tolerance and cross-resistance to antibiotics has not been studied. Our work shows that E. coli displays persistence against several widely used disinfectants, including benzalkonium chloride (BAC), didecyldimethylammoniumchlorid (DDAC) and isopropanol. The molecular mechanism of BAC persistence is triggered in stationary phase and affected by several antibiotic persister genes (hipA, tisB, tolC, relA, spoT). Experimental evolution and population dynamic modeling show that repeated failure of disinfection due to persisters rapidly selects for BAC tolerance underpinned by reduced cell surface charge due to mutations in genes related to lipid A acylation (lpxML). Furthermore, evolved BAC tolerance affects the susceptibility to antibiotics, leading to positive selection of disinfectant tolerant strains at environmentally relevant antibiotic concentrations and variations in evolvability of antibiotic resistance due to epistatic effects. These results highlight the need for faithful application of disinfectants to steward their efficacy and the efficacy of antibiotics. A better understanding of the bacterial response to disinfectants is crucial to understand and avert the ongoing antimicrobial resistance crisis.
Fungal biofilms on materials: describing and modelling growth of the black fungus Knufia petricola
(2023)
Fungi that grow as biofilms are associated with clinical settings as well as various cases of material fouling and material damage. Black fungi as biofilm formers have been rarely studied so far. Their conspicuous dark pigmentation, EPS production, adhesion capabilities and adaptations to stresses allow black fungi to develop biofilms on materials under harsh conditions. For example, rock-inhabiting black fungi withstand sun irradiation and dehydration and are therefore ubiquitous on arid surfaces like solar panels and marble monuments.
To understand and control their ability to colonise and deteriorate materials, one should assess and model black fungi’s growth patterns. But so far, no mathematical model has been developed to describe their growth. Knufia petricola A95, representing rock-inhabiting fungi from Chaetothyriales, is genetically amenable and can serve as a model for biofilm studies in black fungi. The primary objective of this project is to develop a growth model for K. petricola A95 which will enable to define and predict material colonisation of black fungi.
Dedicated experimental work with K. petricola will allow the quantitative assessment of the impact of environmental conditions (e.g. pH, nutrients, etc.) on the growth behaviour at the biofilm and single cells level. Data which will be used to validate and develop an individual-based model (based on the iDynoMICS modelling platform) that explains how fungal biofilms form, colonise materials, and cause deterioration.
Thus far, research has been conducted on the impact of different concentrations and sources of major elements (e.g. C, N, …), as well as trace elements (e.g. Cu, Mg, …), on the colony shape and biomass of Knufia petricola A95 biofilms. To study the behaviour of single cells, the length of the cell cycle in different growth media has been determined via the combined use of microfluidic devices and confocal microscopy.
DHN (1,8-dihydroxynaphthalene) melanin is produced by Ascomycetes via slightly differing synthetic routes. Polyketide synthases release YWA1, AT4HN or T4HN. YWA1 and AT4HN are deacetylated by ‘yellowish-green’ hydrolases, and T4HN is converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. The melanogenic genes are tightly, partially or not clustered in the genomes, and are often regulated in a spatial and/or temporal fashion. By contrast, microcolonial fungi/black yeasts – a polyphyletic group of Ascomycetes dwelling in hostile habitats such as bare rock surfaces – feature constitutive DHN melanogenesis. Here, we report on the DHN melanogenic genes of Knufia petricola (Eurotiomycetes/Chaetothyriales).
Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, which are hindered by slow growth, lack of sexual cycles and transformation difficulties, CRISPR/Cas9-based genetic tools were implemented (Erdmann et al. 2022, Front Fungal Biol). Now efficient targeted mutagenesis of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Still more progress on assigning functions to yet unknown genes can be expected if a forward genetics approach is available. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis of K. petricola. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase (AcTPase) and by this the regulatable transposition of the resistance cassette-containing Ds transposon is desired. However, endogenous promoters for nitrate assimilation and galactose catabolism - often used in fungi for regulatable gene expression - are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in oligotrophic fungi. Therefore, the metabolism-independent Tet-on system was combined with the AcTPase coding sequence and subsequently transformed into Ds-carrying K. petricola strains. In total, four auxotrophic Ac|Ds starter strains containing the Ds transposon at different position of ade2 or ura3 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA-lacking media resulted in prototrophic colonies (revertants) for some but not all Ac|Ds strains. Currently, the transposition events in the obtained revertants are studied to validate the procedure. First amplicon sequencing of excision sites revealed footprint patterns, proving the transposon jumped.
Cryomyces antarcticus – a cryptoendolithic black fungus endemic to Antarctica – is taxonomically classified in phylum Ascomycota, class Dothideomycetes incertae sedis. C. antarcticus has shown high capability to survive extreme environmental conditions like those found in space (ionizing radiation, vacuum, microgravity), thus fueling fundamental astrobiological questions like “searching for life beyond Earth” (Onofri et al. 2020, Extremophiles Astrobiol Model). Its extraordinary resilience has been attributed to the presence of thick, highly melanized cell walls, which may contain both DHN and DOPA melanins (Pacelli et al. 2020, Appl Microbiol Biotechnol). To better understand the contribution of DHN melanin to the overall resilience of C. antarcticus, we initially adopted chemicals e.g., tricyclazole to inhibit the DHN melanin synthetic pathway; however, these studies gave inconclusive results. Eventually, we decided to generate melanin-deficient mutants by genetic engineering. Using the genetic toolkit developed for the black fungus Knufia petricola (Voigt et al. 2020, Sci Rep; Erdmann et al. 2022, Front Fungal Biol), we designed a strategy for mutating the key enzyme (polyketide synthase)-encoding gene capks1 by transient delivery of Cas9 and capks1-specific sgRNA from AMA-containing plasmids and PCR-generated donor DNA i.e., resistance cassettes flanked by ~75-bp-long sequences homologous to capks1. For this, the melanin-PKS encoding ortholog was identified in the C. antarcticus CBS 116301 genome (mycocosm.jgi.doe.gov) and used to design primers for re-sequencing of the capks1 locus in the strain CCFEE 515. Transformation of C. antarcticus is challenging because of its very slow growth; we expect that 4-6 months are needed from obtaining enough biomass for cell wall lysis until transferring putatively resistant transformants for genotyping. Important parameters were evaluated: protoplasts can be generated, and they survive the transformation procedure, and suitable concentrations of selective agents have been identified. Nowadays, we are waiting for the first C. antarcticus mutants considered to be deficient in DHN melanogenesis.
Iron is arguably the most essential metal in living organisms. For rock-inhabiting fungi its acquisition might be unconventional as they (1) tend to inhabit iron-deficient, oxygen-rich surfaces like marble monuments and solar panels and (2) produce the black, iron-adsorbing pigment melanin. We used a range of analytical methods, ongoing mineral dissolution experiments and gene deletion mutants of the model rock-inhabiting fungus Knufia petricola to figure out the mechanisms and substrate deteriorating effects of iron uptake by these organisms. To study both siderophore-mediated and reductive iron assimilation (RIA), genes like sidC, encoding a putative siderophore synthetase and ftr1 and fet3 encoding the subunits of an iron permease-oxidase were deleted.
At iron deficient conditions, growth of the wild type (WT) and ΔsidC mutant was similar, whereas growth of the Δftr1-fet3 mutant and the double mutant ΔsidC/Δftr1-fet3 was diminished and absent, respectively. We were not able to detect the siderophore of K. petricola and the WT and mutants were not able to grow at low concentrations of strong iron chelators. However, in a cross-feeding experiment, an overexpression strain of sidC allowed more growth of ΔsidC/Δftr1-fet3 on iron deficient medium than the WT, whereas the ΔsidC mutant could not do so at all. Compared to the WT, the sidC overexpression strain also withstood oxidative stress better and had a shorter lag time and higher growth rate. Combined, these results indicate that K. petricola relies more on RIA than siderophore-mediated uptake as it likely excretes low quantities of a primarily intracellular siderophore. Interestingly, Δftr1-fet3 had a higher iron content than the WT at iron deficient conditions. This difference disappeared upon deletion of melanin synthesis (Δpks1 vs. Δpks1/Δftr1-fet3): melanin-bound iron can likely not be used without RIA. K. petricola’s chelation incapacity implies a habitat free of competition for iron while offering us a mitigation strategy.
Biocides are used for a wide range of purposes, including disinfectants or preservatives. They play a major role in the prevention of microbial infections in healthcare and animal husbandry. The use of biocides often leads to the discharge of active biocidal substances into wastewater streams, causing the exposure of wastewater microbial communities to subinhibitory concentrations. In turn, it is known that wastewater treatment plants (WWTP) are hotspots for antibiotic resistant bacteria. Since similar mechanisms confer resistance to biocides and antibiotics, exposure to biocides can result in co-selection of antibiotic resistant bacteria in WWTP Here, we want to investigate co-selection processes of antibiotic resistance in natural WWTP microbial communities upon biocide exposure. Microbial communities were sampled at the WWTP Ruhleben in Berlin and characterized regarding their susceptibility against different clinically relevant antibiotics. To investigate the link between biocide exposure and antibiotic resistance, changes in the susceptibility level after exposure to environmentally relevant concentrations of the commonly used biocide didecyldimethylammonium chloride (DDAC) will be determined by enumerating resistant and non-resistant E. coli on selective plates with and without antibiotics and DDAC. In case of antibiotics, clinical breakpoint concentrations according to EUCAST will be used to discriminate between susceptible and resistant strains. In case of DDAC (and biocides in general), clinical breakpoints do not exist. Therefore, we determined a cut-off concentration at which the majority of naturally-occurring E. coli strains cannot grow anymore based on (I) the MIC (minimal inhibitory concentration) distribution, and (II) by plating wastewater communities onto selective indicator agar plates loaded with increasing DDAC concentration. Additionally, antibiotic cross-resistance will be determined by spotting single colonies, isolated from DDAC-selective plates onto antibiotic plates. The results of our experiments will help to determine selective concentrations and to estimate the risk of antibiotic co-selection and cross-resistance in microbial WWTP communities upon biocide exposure.
Bacteria are ubiquitous and colonize all types of surfaces, including those in close proximity to humans, such as skin, food, and everyday objects. This raises the question of whether their presence represents a problem to be mitigated or a potential source of benefit to be harnessed, thereby stimulating scientific inquiry into the role of surface-associated bacteria in diverse domains ranging fromhuman health to industrial biotechnology. Aim: The objective of this project is to explore the impact of modifying surface topography on bacterial adhesion behavior. By manipulating the physical characteristics of the substrate, the attachment and detachment dynamics of bacteria can potentially be modified, leading to novel strategies for controlling bacterial colonization in various applications, such as medical devices. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were tested on LIPSS-covered Fused Silica samples.