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- ItemDiscrimination between pathogenic and non-pathogenic E. coli strains by means of Raman microspectroscopy(Berlin ; Heidelberg : Springer, 2020) Lorenz B.; Ali N.; Bocklitz T.; Rösch P.; Popp J.Bacteria can be harmless commensals, beneficial probiotics, or harmful pathogens. Therefore, mankind is challenged to detect and identify bacteria in order to prevent or treat bacterial infections. Examples are identification of species for treatment of infection in clinics and E. coli cell counting for water quality monitoring. Finally, in some instances, the pathogenicity of a species is of interest. The main strategies to investigate pathogenicity are detection of target genes which encode virulence factors. Another strategy could be based on phenotypic identification. Raman spectroscopy is a promising phenotypic method, which offers high sensitivities and specificities for the identification of bacteria species. In this study, we evaluated whether Raman microspectroscopy could be used to determine the pathogenicity of E. coli strains. We used Raman spectra of seven non-pathogenic and seven pathogenic E. coli strains to train a PCA-SVM model. Then, the obtained model was tested by identifying the pathogenicity of three additional E. coli strains. The pathogenicity of these three strains could be correctly identified with a mean sensitivity of 77%, which is suitable for a fast screening of pathogenicity of single bacterial cells. [Figure not available: see fulltext.]. © 2020, The Author(s).
- ItemTowards Bacteria Counting in DI Water of Several Microliters or Growing Suspension Using Impedance Biochips(Basel : MDPI, 2020) Kiani, Mahdi; Tannert, Astrid; Du, Nan; HĂ¼bner, Uwe; Skorupa, Ilona; BĂ¼rger, Danilo; Zhao, Xianyue; Blaschke, Daniel; Rebohle, Lars; Cherkouk, Charaf; Neugebauer, Ute; Schmidt, Oliver G.; Schmidt, HeidemarieWe counted bacterial cells of E. coli strain K12 in several-microliter DI water or in several-microliter PBS in the low optical density (OD) range (OD = 0.05–1.08) in contact with the surface of Si-based impedance biochips with ring electrodes by impedance measurements. The multiparameter fit of the impedance data allowed calibration of the impedance data with the concentration cb of the E. coli cells in the range of cb = 0.06 to 1.26 Ă— 109 cells/mL. The results showed that for E. coli in DI water and in PBS, the modelled impedance parameters depend linearly on the concentration of cells in the range of cb = 0.06 to 1.26 Ă— 109 cells/mL, whereas the OD, which was independently measured with a spectrophotometer, was only linearly dependent on the concentration of the E. coli cells in the range of cb = 0.06 to 0.50 Ă— 109 cells/mL.
- ItemIn vitro assembly of plasmid DNA for direct cloning in Lactiplantibacillus plantarum WCSF1(San Francisco, California, US : PLOS, 2023) Blanch-Asensio, Marc; Dey, Sourik; Sankaran, ShrikrishnanLactobacilli are gram-positive bacteria that are growing in importance for the healthcare industry and genetically engineering them as living therapeutics is highly sought after. However, progress in this field is hindered since most strains are difficult to genetically manipulate, partly due to their complex and thick cell walls limiting our capability to transform them with exogenous DNA. To overcome this, large amounts of DNA (>1 μg) are normally required to successfully transform these bacteria. An intermediate host, like E. coli, is often used to amplify recombinant DNA to such amounts although this approach poses unwanted drawbacks such as an increase in plasmid size, different methylation patterns and the limitation of introducing only genes compatible with the intermediate host. In this work, we have developed a direct cloning method based on in-vitro assembly and PCR amplification to yield recombinant DNA in significant quantities for successful transformation in L. plantarum WCFS1. The advantage of this method is demonstrated in terms of shorter experimental duration and the possibility to introduce a gene incompatible with E. coli into L. plantarum WCFS1.
- ItemReduce and refine: Plasma treated water vs conventional disinfectants for conveyor-belt cleaning in sustainable food-production lines(Melville, NY : American Inst. of Physics, 2021) Weihe, Thomas; Schnabel, Uta; Winter, Hauke; Möller, Timon; Stachowiak, Jörg; Neumann, Sabine; SchlĂ¼ter, Oliver; Ehlbeck, JörgSustainable and microbiologically secure foodstuff production lines are of increasing scientific interest and are in the focus of recent research programs. Additionally, they are of great importance for the production industry due to the prevention of food-borne illnesses caused by pathogens such as Salmonella sp., Listeria monocytogenes, or Escherichia coli. These pathogens are responsible for production losses, loss of customer acceptance, and severe food-borne illnesses. A pathogenic threat is frequently combated with sanitizing steps of the production lines. For conveyor band cleaning, this study compares the cleaning abilities of nitric acid (HNO3) and plasma treated water (PTW), which have been sprayed via a commercially available nozzle on two different polymeric surfaces (polysiloxane and polyurethane). Additionally, the cleaning agents HNO3 and PTW have been characterized through their pH and their conductivity. These findings have been underpinned by experiments that focus on a possible influence of nozzle abrasion, such as brass and stainless-steel nanoparticles, on the antimicrobial potential of PTW and HNO3. Adversely acting effects like an enhanced abrasion of conveyer band materials due to PTW or HNO3 treatment have been checked by using light microscopic micrographs and topographic scans in high-resolution mode. Based on the presented results of the experiments, the suitability of an in-place sanitation step in foodstuff production lines has been demonstrated on a laboratory scale.
- ItemAutomated and rapid identification of multidrug resistant Escherichia coli against the lead drugs of acylureidopenicillins, cephalosporins, and fluoroquinolones using specific Raman marker bands(Weinheim : Wiley-VCH-Verl., 2020) Götz, Theresa; Dahms, Marcel; Kirchhoff, Johanna; Beleites, Claudia; Glaser, Uwe; Bohnert, JĂ¼rgen A.; Pletz, Mathias W.; Popp, JĂ¼rgen; Schlattmann, Peter; Neugebauer, UteA Raman-based, strain-independent, semi-automated method is presented that allows the rapid (<3 hours) determination of antibiotic susceptibility of bacterial pathogens isolated from clinical samples. Applying a priori knowledge about the mode of action of the respective antibiotic, we identified characteristic Raman marker bands in the spectrum and calculated batch-wise weighted sum scores from standardized Raman intensity differences between spectra of antibiotic exposed and nonexposed samples of the same strains. The lead substances for three relevant antibiotic classes (fluoroquinolone ciprofloxacin, third-generation cephalosporin cefotaxime, ureidopenicillin piperacillin) against multidrug-resistant Gram-negative bacteria (MRGN) revealed a high sensitivity and specificity for the susceptibility testing of two Escherichia coli laboratory strains and 12 clinical isolates. The method benefits from the parallel incubation of control and treated samples, which reduces the variance due to alterations in cultivation conditions and the standardization of differences between batches leading to long-term comparability of Raman measurements. © 2020 The Authors. Journal of Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
- ItemOptimized polymer-based glucose release in microtiter plates for small-scale E. coli fed-batch cultivations(London : BioMed Central, 2020) Keil, Timm; Dittrich, Barbara; Lattermann, Clemens; BĂ¼chs, JochenBackground: Small-scale cultivation vessels, which allow fed-batch operation mode, become more and more important for fast and reliable early process development. Recently, the polymer-based feeding system was introduced to allow fed-batch conditions in microtiter plates. Maximum glucose release rates of 0.35 mg/h per well (48-well-plate) at 37 °C can be achieved with these plates, depending on the media properties. The fed-batch cultivation of fluorescent protein-expressing E. coli at oxygen transfer rate levels of 5 mmol/L/h proved to be superior compared to simple batch cultivations. However, literature suggests that higher glucose release rates than achieved with the currently available fed-batch microtiter plate are beneficial, especially for fast-growing microorganisms. During the fed-batch phase of the cultivation, a resulting oxygen transfer rate level of 28 mmol/L/h should be achieved. Results: Customization of the polymer matrix enabled a considerable increase in the glucose release rate of more than 250% to up to 0.90 mg/h per well. Therefore, the molecular weight of the prepolymer and the addition of a hydrophilic PDMS-PEG copolymer allowed for the individual adjustment of a targeted glucose release rate. The newly developed polymer matrix was additionally invariant to medium properties like the osmotic concentration or the pH-value. The glucose release rate of the optimized matrix was constant in various synthetic and complex media. Fed-batch cultivations of E. coli in microtiter plates with the optimized matrix revealed elevated oxygen transfer rates during the fed-batch phase of approximately 28 mmol/L/h. However, these increased glucose release rates resulted in a prolonged initial batch phase and oxygen limitations. The newly developed polymer-based feeding system provides options to manufacture individual feed rates in a range from 0.24-0.90 mg/h per well. Conclusions: The optimized polymer-based fed-batch microtiter plate allows higher reproducibility of fed-batch experiments since cultivation media properties have almost no influence on the release rate. The adjustment of individual feeding rates in a wide range supports the early process development for slow, average and fast-growing microorganisms in microtiter plates. The study underlines the importance of a detailed understanding of the metabolic behavior (through online monitoring techniques) to identify optimal feed rates. © 2020 The Author(s).
- ItemArgon Humidification Exacerbates Antimicrobial and Anti-MRSA kINPen Plasma Activity(Basel : MDPI, 2023) Clemen, Ramona; Singer, Debora; Skowski, Henry; Bekeschus, SanderGas plasma is a medical technology with antimicrobial properties. Its main mode of action is oxidative damage via reactive species production. The clinical efficacy of gas plasma-reduced bacterial burden has been shown to be hampered in some cases. Since the reactive species profile produced by gas plasma jets, such as the kINPen used in this study, are thought to determine antimicrobial efficacy, we screened an array of feed gas settings in different types of bacteria. Antimicrobial analysis was performed by single-cell analysis using flow cytometry. We identified humidified feed gas to mediate significantly greater toxicity compared to dry argon and many other gas plasma conditions. The results were confirmed by inhibition zone analysis on gas-plasma-treated microbial lawns grown on agar plates. Our results may have vital implications for clinical wound management and potentially enhance antimicrobial efficacy of medical gas plasma therapy in patient treatment.
- ItemEfficacy of A Poly(MeOEGMA) Brush on the Prevention of Escherichia coli Biofilm Formation and Susceptibility(Basel : MDPI, 2020) Alves, PatrĂcia; Gomes, Luciana Calheiros; RodrĂguez-Emmenegger, Cesar; MergulhĂ£o, Filipe JosĂ©Urinary tract infections are one of the most common hospital-acquired infections, and they are often associated with biofilm formation in indwelling medical devices such as catheters and stents. This study aims to investigate the antibiofilm performance of a polymer brush—poly[oligo(ethylene glycol) methyl ether methacrylate], poly(MeOEGMA)—and evaluate its effect on the antimicrobial susceptibility of Escherichia coli biofilms formed on that surface. Biofilms were formed in a parallel plate flow chamber (PPFC) for 24 h under the hydrodynamic conditions prevailing in urinary catheters and stents and challenged with ampicillin. Results obtained with the brush were compared to those obtained with two control surfaces, polydimethylsiloxane (PDMS) and glass. The polymer brush reduced by 57% the surface area covered by E. coli after 24 h, as well as the number of total adhered cells. The antibiotic treatment potentiated cell death and removal, and the total cell number was reduced by 88%. Biofilms adapted their architecture, and cell morphology changed to a more elongated form during that period. This work suggests that the poly(MeOEGMA) brush has potential to prevent bacterial adhesion in urinary tract devices like ureteral stents and catheters, as well as in eradicating biofilms developed in these biomedical devices. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
- ItemEngineered living hydrogels for robust biocatalysis in pure organic solvents(Maryland Heights, MO : Cell Press, 2022) Gao, Liang; Feng, Lilin; Sauer, Daniel F.; Wittwer, Malte; Hu, Yong; Schiffels, Johannes; Li, XinEngineered living hydrogels that can protect cells from harsh environments have achieved preliminary successes in biomedicine and environmental remediation. However, their biocatalytic applications in pure organic solvents have not been explored. Here, living hydrogels were engineered by integrating genetically modified Escherichia coli cells into alginate hydrogels for robust biocatalysis in pure organic solvents. The biocompatible hydrogels could not only support cell growth and diminish cell escape but could also act as protective matrices to improve organic solvent tolerance, thereby prolonging catalytic activity of whole-cell biocatalysts. Moreover, the influence of hydrogel microenvironments on biocatalytic efficiency was thoroughly investigated. Importantly, the versatility of engineered living hydrogels paves the way to achieve robust biocatalytic efficiency in a variety of pure organic co-solvents. Overall, we are able to engineer living hydrogels for regio-selective synthesis in pure organic solvents, which may be particularly useful for the innovation of living hydrogels in biocatalysis.
- ItemEffect of Chlorine Dioxide Treatment on Human Pathogens on Iceberg Lettuce(Basel : MDPI AG, 2021) Hassenberg, Karin; Praeger, Ulrike; Herppich, Werner B.In the vegetable processing industry, the application of chlorine dioxide (ClO2) as a disinfectant solved in washing water to eliminate undesirable microorganisms harmful to consumers’ health and the shelf life of produce has been discussed for years. The disinfection efficacy depends on various factors, e.g., the location of microorganisms and the organic load of the washing water. The present study analyzed the sanitation efficacy of various concentrations of water-solved ClO2 (cClO2: 20 and 30 mg L−1) on Escherichia coli (1.1 Ă— 104 cfu mL−1), Salmonella enterica (2.0 Ă— 104 cfu mL−1) and Listeria monocytogenes (1.7 Ă— 105 cfu mL−1) loads, located on the leaf surface of iceberg lettuce assigned for fresh-cut salads. In addition, it examined the potential of ClO2 to prevent the cross-contamination of these microbes in lettuce washing water containing a chemical oxygen demand (COD) content of 350 mg L−1 after practice-relevant washing times of 1 and 2 min. On iceberg leaves, washing with 30 mg L−1 ClO2 pronouncedly (1 log) reduced loads of E. coli and S. enterica, while it only insignificantly (<0.5 Ă— log) diminished the loads of L. monocytogenes, irrespective of the ClO2 concentration used. Although the sanitation efficacy of ClO2 washing was only limited, the addition of ClO2 to the washing water avoided cross-contamination even at high organic loads. Thus, the application of ClO2 to the lettuce washing water can improve product quality and consumer safety.