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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.
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.
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.
SRB are environmentally and industrially important microorganisms. The disadvantage of their metabolic activity (e.g. sulfate reduction) results in the formation of toxic sulfide that leads to microbial influenced corrosion. SRB have been responsible for biocorrosion of ferrous metal. One of mitigation strategy is the use of biocides. However, it has been shown that various bacteria develop antimicrobial resistance due to excessive use of biocides. Thus, a deeper understanding of the evolution of biocide resistance of SRB is necessary. Three commonly used biocides, THPS, BAC, and GLUT were applied to investigate the susceptibility of Desulfovibrio alaskensis G20.The minimum inhibitory and bactericidal concentration and the killing kinetics of the three biocides was determined. These results will be used to conduct evolution experiments to determine the evolution of resistance towards biocides of SRBs. The outcome of this work can be helpful to improve the management of MIC treatments.