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Understanding Stimulation of Conjugal Gene Transfer by Nonantibiotic Compounds: How Far Are We?
(2024)
A myriad of nonantibiotic compounds is released into the environment, some of which may contribute to the dissemination of antimicrobial resistance by stimulating conjugation. Here, we analyzed a collection of studies to (i) identify patterns of transfer stimulation across groups and concentrations of chemicals, (ii) evaluate the strength of evidence for the proposed mechanisms behind conjugal stimulation, and (iii) examine the plausibility of alternative mechanisms. We show that stimulatory nonantibiotic compounds act at concentrations from 1/1000 to 1/10 of the minimal inhibitory concentration for the donor strain but that stimulation is always modest (less than 8-fold). The main proposed mechanisms for stimulation via the reactive oxygen species/SOS cascade and/or an increase in cell membrane permeability are not unequivocally supported by the literature. However, we identify the reactive oxygen species/SOS cascade as the most likely mechanism. This remains to be confirmed by firm molecular evidence. Such evidence and more standardized and high-throughput conjugation assays are needed to create technologies and solutions to limit the stimulation of conjugal gene transfer and contribute to mitigating global antibiotic resistance.
Microbiologically influenced corrosion (MIC) is a crucial issue for industry and infrastructure. Biofilms are known to form on different kinds of surfaces such as metal, concrete, and medical equipment. However, in some cases the effect of microorganisms on the material can be negative for the consistency and integrity of the material. Thus, to overcome the issues raised by MIC on a system, different physical, chemical, and biological strategies have been considered; all having their own advantages, limitations, and sometimes even unwanted disadvantages. Among all the methods, biocide treatments and antifouling coatings are more common for controlling MIC, though they face some challenges. They lack specificity for MIC microorganisms, leading to cross-resistance and requiring higher concentrations. Moreover, they pose environmental risks and harm non-target organisms. Hence, the demand for eco-friendly, long-term solutions is increasing as regulations tighten. Recently, attentions have been directed to the application of nanomaterials to mitigate or control MIC due to their significant antimicrobial efficiency and their potential for lower environmental risk compared to the conventional biocides or coatings. Use of nanomaterials to inhibit MIC is very new and there is a lack of literature review on this topic. To address this issue, we present a review of the nanomaterials examined as a biocide or in a form of a coating on a surface to mitigate MIC. This review will help consolidate the existing knowledge and research on the use of nanomaterials for MIC mitigation. It will further contribute to a better understanding of the potential applications and challenges associated with using nanomaterials for MIC prevention and control.
Bakterielle Biofilme stellen in medizinischen und industriellen Bereichen ein ernsthaftes Problem dar. Eine der größten gesellschaftlichen Herausforderungen liegt in der zunehmenden Resistenz von Bakterien gegen Biozide, die bei antimikrobiellen Behandlungen eingesetzt werden, z.B. durch übermäßigen Einsatz in Medizin, Industrie und Landwirtschaft oder durch Reinigung und Desinfektion in Privathaushalten. Daher sind neue effiziente bakterienabweisende Strategien, die den Einsatz von Bioziden vermeiden, dringend erforderlich.
Ein vielversprechender Weg zur Erzielung bakterienabweisender Oberflächen liegt in der berührungslosen und aseptischen großflächigen Laserbearbeitung von technischen Oberflächen. Maßgeschneiderte Oberflächentexturen, ermöglicht durch verschiedene Laserbearbeitungsstrategien, die zu topographischen Skalen im Bereich von Nanometern bis Mikrometern führen, können eine Lösung für diese Herausforderung darstellen.
In dem Vortrag wird ein Überblick über den aktuellen Stand der Technik bei der subtraktiven Texturierung von Laseroberflächen zur Kontrolle der Biofilmbildung bei verschiedenen Bakterienstämmen und in unterschiedlichen Umgebungen gegeben. Auf der Grundlage spezifischer Eigenschaften von Bakterien und laserbearbeiteten Oberflächen werden die Herausforderungen antimikrobieller Oberflächendesigns erörtert und zukünftige Richtungen aufgezeigt.
Bacterial adhesion on surfaces of medical, water and food applications may lead to infections, water or food spoilage and human illness. In comparison to traditional static and macro flow chamber assays for biofilm formation studies, microfluidic chips allow in situ monitoring of biofilm formation under various flow regimes, have better environment control and smaller sample requirements.
In this work, a novel microfluidic platform is developed to investigate biofilm adhesion under precisely controlled bacteria concentration, temperature, and flow conditions. This platform central unit is a single-inlet microfluidic flow cell with a 5 mm wide chamber designed and tested to achieve ultra-homogenous flow in the central area of chamber. Within this area, defined microstructures are integrated that will disturb the homogeneity of the flow, thus changing bacterial adhesion pattern.
Here we present the monitoring of bacterial biofilm formation in a microfluidic chip equipped with a microstructure known as micro-trap. This feature is based on a 3D bacteria trap designed by Di Giacomo et al. and successfully used to sequester motile bacteria.
At first, fluorescent particles similar in size to Escherichia coli (E. coli) are used to simulate bacteria flow inside the flow cell and at the micro-trap. The turbulences induced by the trap are analyzed by imaging and particle tracking velocimetry (PTV). Secondly, the model strain E. coli TG1, ideal and well described for biofilm studies, is used to analyze biofilm formation in the micro-trap. Therefore, a stable fluorescent strain E. coli TG1-MRE-Tn7-141 is constructed by using Tn7 transposon mutagenesis according to the method described by Schlechter et al. Sequestering of E. coli cells within the micro-trap was followed using epifluorescence microscopy.
The novel microfluidic platform shows great potential for assessment of bacterial adhesion under various flow regimes. The performance of structural feature with respect to the generation of turbulences that promote or reduce bacterial adhesion can be systematically examined.
The combination of flow analysis and fluorescent strain injection into the microfluidic chip shows that the micro-trap is useful for capturing bacteria at defined positions and to study how flow conditions, especially micro-turbulences, can affect biofilm formation. It represents a powerful and versatile tool for studying the relation between topography and bacteria adhesion.
The overarching goal of this project is to develop a predictive model for efflux-mediated antimicrobial tolerance in bacterial multicellular assemblies. Our central hypostasis is that efflux pump activity causes emergent antibiotic tolerance of multicellular bacterial populations, through the interplay of efflux mediated spatial interactions and efflux-linked persistence. To test this hypothesis, we will use a combination of microscopy, microbial killing assays, computational modelling, and data analysis, integrating information from 3 types of multicellular assembly: colonies, cell-to-cell interactions in a monolayer microfluidic device, and 3D flow chamber biofilms. Building on our preliminary observations, we will experimentally characterize the link between colony structure and spatial patterns of efflux gene expression in strains that differ in their levels of efflux. We will develop a mathematical model to test whether local growth inhibition of neighbors due to effluxing cells, coupled with local environment-dependent regulation of efflux, can account qualitatively for these results. By including persister cell formation in our model we will predict, and measure, the emergent function of antimicrobial tolerance in our colonies. To fully understand how tolerance emerges from the interplay between efflux-mediated spatial interactions and efflux-linked persister cell formation, we need quantitative measurements at the single cell level. To this end, we will use a microfluidic setup with cells growing in a monolayer to qualify in detail the dependence of efflux expression and persister cell formation on nutrient conditions, the correlation between efflux and persister formation, and the spatial range of efflux-mediated neighbour growth inhibition. To predict and quantitatively understand the emergent multicellular function of tolerance, we will perform individual-based modelling of biofilm growth, using as input the parameters measured on the single-cell level with our microfluidics experiments. Our simulations will predict biofilm spatial structure development, patterns of efflux and persister formation and, ultimately, tolerance to antimicrobial challenge. These predictions will be directly tested in flow-cell biofilm experiments. We are currently generating acrAB-tolC knockout-strain, without efflux activity, and a strain with an inducible acrAB-tolC efflux pump. To distinguish the different strains under the microscope, they were labeled with genes encoding for different fluorescent proteins. All strains are currently characterized in terms of growth, minimum inhibitory concentration of different antimicrobial substances, colony morphology, and biofilm formation ability. On the theoretical side, we are currently working on modeling the system at various scales and degree of detail, ranging from coarse-grained continuum models to stochastic, individual-based models. Some exploratory work was doe to test existing software for individual-based modelling that may be adapted for our purpose. Furthermore, we are in the process of developing more coarse-grained models. This work involves some physiological modelling and literature search, focusing on working mechanisms of efflux pumps and kinetic models for import and export of antibiotics.
Consequences of tolerance to disinfectants on the evolution of antibiotic resistance in E. coli
(2023)
Biocides are used as disinfectants and preservatives; one important active substance in biocides is benzalkonium chloride (BAC). BAC-tolerant bacterial strains can survive short treatments with high concentrations of BAC. BAC tolerance and resistance have been linked to antibiotic resistance. Here, the selection dynamics between a BAC-tolerant Escherichia coli strain and a sensitive wild type were investigated under four conditions: in the absence of antibiotics and in the presence of three different sub-inhibitory concentrations of the antibiotic ciprofloxacin in liquid cultures. The wild type was selected over the BAC-tolerant strain in the absence of antibiotics, while the BAC-tolerant strain was selected over the wild type at all ciprofloxacin concentrations investigated, with a minimum selection concentration (MSC) of 1/10th of the minimum inhibitory concentration (MIC) of the wild type. Furthermore, the evolvability of resistance of the two strains to inhibitory concentrations of ciprofloxacin was assessed by performing a serial dilution evolution experiment with gradually increasing ciprofloxacin concentrations. The wild type had a higher probability to develop resistance
to ciprofloxacin than the tolerant strain. By the end of the evolution experiment both strains evolved to grow at the highest ciprofloxacin concentration investigated, which was 2048 ×MIC of the wild type. The importance of these results is highlighted by the fact that concentrations of ciprofloxacin well above the calculated MSC can be found in environmental samples such as hospital wastewaters and livestock slurry. In turn, BAC is used as a disinfectant in the same settings. Thus, the selection of BAC-tolerant strains at sub-inhibitory concentrations of ciprofloxacin can contribute to the stabilization and spread of BAC-tolerance in natural populations. The prevalence of such strains can impair the effects of BAC disinfections.
Bacterial biofilms pose serious problems in medical and industrial settings. One of the major societal challenges lies in the increasing resistance of bacteria against biocides used in antimicrobial treatments, e.g., via overabundant use in medicine, industry, and agriculture or cleaning and disinfection in private households. Hence, new efficient bacteria-repellent strategies avoiding the use of biocides are strongly desired. One promising route to achieve bacteria-repellent surfaces lies in the contactless and aseptic large-area laser-processing of technical surfaces. Tailored surface textures, enabled by different laser-processing strategies that result in topographic scales ranging from nanometers to micrometers may provide a solution to this challenge. This article presents a current state-of-the-art review of laser-surface subtractive texturing approaches for controlling the biofilm formation for different bacterial strains and in different environments. Based on specific properties of bacteria and laser-processed surfaces, the challenges of anti-microbial surface designs are discussed, and future directions will be outlined.
Bacterial biofilms can pose a serious health risk to humans and are less susceptible
to antibiotics and disinfection than planktonic bacteria. Here, a novel method
for biofilm eradication based on antimicrobial photodynamic therapy utilizing
a nanoparticle in conjunction with a BODIPY derivative as photosensitizer was
developed. Reactive oxygen species are generated upon illumination with
visible light and lead to a strong, controllable and persistent eradication of
both planktonic bacteria and biofilms. One of the biggest challenges in biofilm
eradication is the penetration of the antimicrobial agent into the biofilm and
its matrix. A biocompatible hydrophilic nanoparticle was utilized as a delivery
system for the hydrophobic BODIPY dye and enabled its accumulation within the
biofilm. This key feature of delivering the antimicrobial agent to the site of action
where it is activated resulted in effective eradication of all tested biofilms. Here,
3 bacterial species that commonly form clinically relevant pathogenic biofilms
were selected: Escherichia coli, Staphylococcus aureus and Streptococcus
mutans. The development of this antimicrobial photodynamic therapy tool for
biofilm eradication takes a promising step towards new methods for the much
needed treatment of pathogenic biofilms.