Antimicrobial Ionic Liquids: Ante-Mortem Mechanisms of Pathogenic EPEC and MRSA Examined by FTIR Spectroscopy.

Ionic liquids (ILs) have gained considerable attention due to their versatile and designable properties. ILs show great potential as antibacterial agents, but understanding the mechanism of attack on bacterial cells is essential to ensure the optimal design of IL-based biocides. The final aim is to achieve maximum efficacy while minimising toxicity and preventing resistance development in target organisms. In this study, we examined a dose-response analysis of ILs' antimicrobial activity against two pathogenic bacteria with different Gram types in terms of molecular responses on a cellular level using Fourier-transform infrared (FTIR) spectroscopy. In total, 18 ILs with different antimicrobial active motifs were evaluated on the Gram-negative enteropathogenic Escherichia coli (EPEC) and Gram-positive methicillin-resistant Staphylococcus aureus (MRSA). The results showed that most ILs impact bacterial proteins with increasing concentration but have a minimal effect on cellular membranes. Dose-response spectral analysis revealed a distinct ante-mortem response against certain ILs for MRSA but not for EPEC. We found that at sub-lethal concentrations, MRSA actively changed their membrane composition to counteract the damaging effect induced by the ILs. This suggests a new adaptive mechanism of Gram-positive bacteria against ILs and demonstrates the need for a better understanding before using such substances as novel antimicrobials.

Antimicrobial and Virucidal Potential of Morpholinium-Based Ionic Liquids.

Witnessed by the ongoing spread of antimicrobial resistant bacteria as well as the recent global pandemic of the SARS-CoV-2 virus, the development of new disinfection strategies is of great importance, and novel substance classes as effective antimicrobials and virucides are urgently needed. Ionic liquids (ILs), low-melting salts, have been already recognized as efficient antimicrobial agents with prospects for antiviral potential. In this study, we examined the antiviral activity of 12 morpholinium based herbicidal ionic liquids with a tripartite test system, including enzyme inhibition tests, virucidal activity determination against five model viruses and activity against five bacterial species. The antimicrobial and enzymatic tests confirmed that the inhibiting activity of ILs corresponds with the number of long alkyl side chains and that [Dec2Mor]+ based ILs are promising candidates as novel antimicrobials. The virucidal tests showed that ILs antiviral activity depends on the type and structure of the virus, revealing enveloped Phi6 phage as highly susceptible to the ILs action, while the non-enveloped phages PRD1 and MS2 proved completely resistant to ionic liquids. Furthermore, a comparison of results obtained for P100 and P001 phages demonstrated for the first time that the susceptibility of viruses to ionic liquids can be dependent on differences in the phage tail structure.

A Novel Method for Sampling and Long-Term Monitoring of Microbes That Uses Stickers of Plain Paper.

Detection of pathogens is crucial in food production areas. While it is well established, swabbing as a state-of-the-art sampling method offers several drawbacks with respect to yield, standardization, overall handling, and long-term monitoring. This led us to develop and evaluate a method that is easier to use at a lower cost and that should be at least as sensitive. After evaluating sundry promising materials, we tested text-marking paper stickers for their suitability to take up and release Listeria monocytogenes with their nonsticky paper side over a 14-day time period using quantitative PCR. The recovery rate was similar to that in previous studies using conventional swabs, and we also confirmed the feasibility of pooling besides resilience to cleansing and disinfection. In a proof-of-concept experiment that sampled several locations, such as door handles, the occurrences of L. monocytogenes and Escherichia coli were determined. The results suggest that the presented sticker system might offer a promising cost-effective alternative sampling system with improved handling characteristics.IMPORTANCE As a ubiquitous bacterium, Listeria monocytogenes has a propensity to enter food production areas inadvertently via fomites such as door handles and switches. While the bacterium might not be in direct contact with the food products, knowing the microbial status of the surroundings is essential for risk assessment. Our investigation into a novel quantitative PCR (qPCR)-based sampling system with the highest sensitivity and ability to monitor over long periods of time, yet based on paper, proved to be cost-effective and reasonably convenient to handle.

Evidence of a reverse side-chain effect of tris(pentafluoroethyl)trifluorophosphate [FAP]-based ionic liquids against pathogenic bacteria.

Increased interest in ionic liquids (ILs) is due to their designable and tunable unique physicochemical properties, which are utilized for a wide variety of chemical and biotechnological applications. ILs containing the tris(pentafluoroethyl)trifluorophosphate ([FAP]) anion have been shown to have excellent hydrolytic, electrochemical and thermal stability and have been successfully used in various applications. In the present study the influence of the cation on the toxicity of the [FAP] anion was investigated. Due to the properties of [FAP] ILs, the IL-toxicity of seven cations with [FAP] compared to [Cl] was examined by determination of minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) on six Gram-positive and six Gram-negative clinically-relevant bacteria. For the first time, to our knowledge, the results provide evidence for a decrease in toxicity with increasing alkyl side-chain length, indicating that the combination of both ions is responsible for this 'reverse side-chain effect'. These findings could portend development of new non-toxic ILs as green alternatives to conventional organic solvents.

Virucidal or Not Virucidal? That Is the Question-Predictability of Ionic Liquid's Virucidal Potential in Biological Test Systems.

For three decades now, ionic liquids (ILs), organic salts comprising only ions, have emerged as a new class of pharmaceuticals. Although recognition of the antimicrobial effects of ILs is growing rapidly, there is almost nothing known about their possible virucidal activities. This probably reflects the paucity of understanding virus inactivation. In this study, we performed a systematic analysis to determine the effect of specific structural motifs of ILs on three different biological test systems (viruses, bacteria and enzymes). Overall, the effects of 27 different ILs on two non-enveloped and one enveloped virus (P100, MS2 and Phi6), two Gram negative and one Gram positive bacteria (E. coli, P. syringae and L. monocytogenes) and one enzyme (Taq DNA polymerase) were investigated. Results show that while some ILs were virucidal, no clear structure activity relationships (SARs) could be identified for the non-enveloped viruses P100 and MS2. However, for the first time, a correlation has been demonstrated between the effects of ILs on enveloped viruses, bacteria and enzyme inhibition. These identified SARs serve as a sound starting point for further studies.

Hydrophobic ionic liquids for quantitative bacterial cell lysis with subsequent DNA quantification.

DNA is one of the most frequently analyzed molecules in the life sciences. In this article we describe a simple and fast protocol for quantitative DNA isolation from bacteria based on hydrophobic ionic liquid supported cell lysis at elevated temperatures (120-150 °C) for subsequent PCR-based analysis. From a set of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide was identified as the most suitable for quantitative cell lysis and DNA extraction because of limited quantitative PCR inhibition by the aqueous eluate as well as no detectable DNA uptake. The newly developed method was able to efficiently lyse Gram-negative bacterial cells, whereas Gram-positive cells were protected by their thick cell wall. The performance of the final protocol resulted in quantitative DNA extraction efficiencies for Gram-negative bacteria similar to those obtained with a commercial kit, whereas the number of handling steps, and especially the time required, was dramatically reduced. Graphical Abstract After careful evaluation of five hydrophobic ionic liquids, 1-butyl-1-methylpyrrolidinium bis(trifluoromethylsulfonyl)imide ([BMPyr + ][Ntf 2- ]) was identified as the most suitable ionic liquid for quantitative cell lysis and DNA extraction. When used for Gram-negative bacteria, the protocol presented is simple and very fast and achieves DNA extraction efficiencies similar to those obtained with a commercial kit. ddH 2 O double-distilled water, qPCR quantitative PCR.

Predictability of ionic liquid toxicity from a SAR study on different systematic levels of pathogenic bacteria.

Ionic liquids (ILs), a new class of solvents with unique and tunable physicochemical properties, were initially envisioned as working alternatives to traditional organic solvents. However, they have now proven to have a wide range of alternative chemical and biochemical applications. Due to their increasing use, environmental and toxicity concerns are growing, but resolutions are hindered by the sheer number of possible variants. In order to assess and possibly predict IL-toxicity, a structure-activity relationship (SAR) approach was adopted using defined structural motifs. These included varied cationic alkyl side-chain lengths, cation lipophilicity and diverse anion effects. The predictive powers of such SARs in respect of antibacterial effects were compared using a total of 28 ILs on six Gram-negative and six Gram-positive pathogenic bacteria. Endpoints were minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC). Results indicate that while certain limited IL-toxicity responses in bacteria can be predicted from SARs, they caution that predictions cannot be generalized across species. This study demonstrates the complex species-specific nature of IL-toxicity and the current limitations of SAR predictability.

Evaluation of the performance of quantitative detection of the Listeria monocytogenes prfA locus with droplet digital PCR.

Fast and reliable pathogen detection is an important issue for human health. Since conventional microbiological methods are rather slow, there is growing interest in detection and quantification using molecular methods. The droplet digital polymerase chain reaction (ddPCR) is a relatively new PCR method for absolute and accurate quantification without external standards. Using the Listeria monocytogenes specific prfA assay, we focused on the questions of whether the assay was directly transferable to ddPCR and whether ddPCR was suitable for samples derived from heterogeneous matrices, such as foodstuffs that often included inhibitors and a non-target bacterial background flora. Although the prfA assay showed suboptimal cluster formation, use of ddPCR for quantification of L. monocytogenes from pure bacterial cultures, artificially contaminated cheese, and naturally contaminated foodstuff was satisfactory over a relatively broad dynamic range. Moreover, results demonstrated the outstanding detection limit of one copy. However, while poorer DNA quality, such as resulting from longer storage, can impair ddPCR, internal amplification control (IAC) of prfA by ddPCR, that is integrated in the genome of L. monocytogenes ΔprfA, showed even slightly better quantification over a broader dynamic range. Graphical Abstract Evaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA.

Antimicrobial effects of short chained imidazolium-based ionic liquids­influence of anion chaotropicity.

Ionic liquids (ILs), a new solvent class composed solely of ions, have already found their way into numerous chemical and biochemical applications. Due to their unique properties and wide application range, research utilizing this new technology for biotechnological applications is steadily increasing. However, progress is hampered by lack of toxicological data, especially concerning IL anions and their general underlying toxicity mechanisms. The present study investigated for the first time the influence of the chaotropicity of the anion for nine imidazole based ILs on their antimicrobial behavior. The results indicate that for ILs with small cations ([C(n)mim](+) with n=2 and 4), the chaotropicity of the anion is a major factor regarding antimicrobial behavior, while for [C6mim](+) based ILs a surfactant-like behavior was identified that explains their high toxicity. It could also be shown that with increasing anion chaotropicity the surfactant-like behavior of the cation is strengthened. Identification of chaotropicity as an underlying mode of antimicrobial action of ILs presents a new point of adjustment for future design with regard to their toxicological behavior.

Quantification of Gram-positive bacteria: adaptation and evaluation of a preparation strategy using high amounts of clinical tissue.

BACKGROUND: A preparation method for quantification of bacteria in tissues is obligatory to reduce tissue mass, concentrate the target, purify, remove inhibitory substances and to achieve constant target recovery rates. No preparation method has been available until now for a high mass of tissue applicable for routine use and analytical veterinary diagnostics. RESULTS: This study describes an easy-to-use tissue preparation protocol to quantify Gram-positive bacteria from a large volume of tissue matrix. A previously published sample preparation method (Matrix-Lysis) from food science was successfully adapted for clinical use on tissues from pigs, including cerebrum, spinal cord, lung, liver, ileum, colon, caecum, kidney and muscle tissue. This tissue preparation method now permits quantification of pathogens from 5 g of organic matrix, which is a 20-200 fold increase by weight compared to other methods. It is based on solubilization of the sample matrix with either a chaotrope plus detergent or divalent salts as solubilization agents. The method was designed as a modular system, offering the possibility to change lysis buffers, according to tissue solubilization characteristics and the intended detection method (molecular or culture). Using Listeria monocytogenes as model organism, viable cell quantification or DNA extraction and quantitative real-time PCR were performed after Matrix-Lysis to determine recovery rates and detection limit (LOD). The adapted Matrix-Lysis protocol resulted in high recovery rates (mean value: 76% ± 39%) for all tested organs, except kidney, and recovery was constant over 5 log scales for all tested buffer systems. The LOD for Matrix-Lysis with subsequent plate count method (PCM) was as low as 1 CFU/5 g, while for qPCR based detection the LOD was 102 bacterial cell equivalents (BCE)/5 g for two buffer systems. CONCLUSIONS: This tissue preparation is inexpensive and can be easily used for routine and analytical veterinary diagnostics. Inoculation studies or hazard assessments can profit from this tissue preparation method and it is anticipated that this study will be a valuable source for further research on tissue preparation strategies.

Corrigendum: Decoding neuropathic pain: Can we predict fluctuations of propagation speed in stimulated peripheral nerve?

[This corrects the article DOI: 10.3389/fncom.2022.899584.].

Bacterial Resistance Toward Antimicrobial Ionic Liquids Mediated by Multidrug Efflux Pumps.

The effective elimination of foodborne pathogens through cleaning and disinfection measures is of great importance to the food processing industry. As food producers rely heavily on disinfectants to control pathogenic bacteria in their facilities, the increasing spread of tolerant, often even multidrug resistant, strains is of particular concern. In addition to efforts to prevent or at least reduce development and spread of strains resistant to disinfectants and sanitizers, there is an urgent need for new and effective antimicrobials. One new class of promising antimicrobials is ionic liquids (ILs), which have been reported to be effective against resistant strains as they interact with bacterial cells in multiple ways, but investigations of their effectivity against MDR bacteria or specific defense mechanisms are still limited. This study investigates the role of multidrug efflux pumps of the Resistance Nodulation-Division family (RND) on the resistance of bacterial pathogens Escherichia coli and Salmonella enterica serovar Typhimurium toward 10 antimicrobial active ILs. Results reveal that, while known structure-activity relationships (SARs), such as the side-chain effect, were found for all strains, antimicrobial ILs with one elongated alkyl side chain were significantly affected by the RND efflux pump, highlighting the importance of efflux pumps for future IL toxicity studies. In case of antimicrobial ILs with multiple side chains and different cationic head groups, two ILs were identified that were highly active against all investigated strains with little to no effect of the efflux pump. The results obtained in this study for RND efflux pumps can serve as a starting point for identifying and designing antimicrobial ILs as effective biocides against MDR bacteria.

Reoptimization of parameterized problems.

Parameterized complexity allows us to analyze the time complexity of problems with respect to a natural parameter depending on the problem. Reoptimization looks for solutions or approximations for problem instances when given solutions to neighboring instances. We combine both techniques, in order to better classify the complexity of problems in the parameterized setting. Specifically, we see that some problems in the class of compositional problems, which do not have polynomial kernels under standard complexity-theoretic assumptions, do have polynomial kernels under the reoptimization model for some local modifications. We also observe that, for some other local modifications, these same problems do not have polynomial kernels unless NP ⊆ coNP / poly . We find examples of compositional problems, whose reoptimization versions do not have polynomial kernels under any of the considered local modifications. Finally, in another negative result, we prove that the reoptimization version of Connected Vertex Cover does not have a polynomial kernel unless Set Cover has a polynomial compression. In a different direction, looking at problems with polynomial kernels, we find that the reoptimization version of Vertex Cover has a polynomial kernel of size 2 k + 1 using crown decompositions only, which improves the size of the kernel achievable with this technique in the classic problem.

Decoding Neuropathic Pain: Can We Predict Fluctuations of Propagation Speed in Stimulated Peripheral Nerve?

To understand neural encoding of neuropathic pain, evoked and resting activity of peripheral human C-fibers are studied via microneurography experiments. Before different spiking patterns can be analyzed, spike sorting is necessary to distinguish the activity of particular fibers of a recorded bundle. Due to single-electrode measurements and high noise contamination, standard methods based on spike shapes are insufficient and need to be enhanced with additional information. Such information can be derived from the activity-dependent slowing of the fiber propagation speed, which in turn can be assessed by introducing continuous "background" 0.125-0.25 Hz electrical stimulation and recording the corresponding responses from the fibers. Each fiber's speed propagation remains almost constant in the absence of spontaneous firing or additional stimulation. This way, the responses to the "background stimulation" can be sorted by fiber. In this article, we model the changes in the propagation speed resulting from the history of fiber activity with polynomial regression. This is done to assess the feasibility of using the developed models to enhance the spike shape-based sorting. In addition to human microneurography data, we use animal in-vitro recordings with a similar stimulation protocol as higher signal-to-noise ratio data example for the models.

Proof of concept for recombinant cellular controls in quantitative molecular pathogen detection.

In this study, we present the concept of internal sample process controls (ISPCs) to monitor the efficiency of an analytical chain using sample preparation and quantitative PCR (qPCR). A recombinant Listeria monocytogenes ΔprfA (targeted deletion) strain containing a competitive artificial single-copy genomic target was applied to naturally contaminated samples to demonstrate its analytical suitability as an ISPC.

Mechanisms of degradation of DNA standards for calibration function during storage.

Establishment of molecular diagnostics offering quantitative technology is directly associated with real-time polymerase chain reaction (PCR). This rapid, accurate and sensitive method requires careful execution, including reliable calibration standards. The storage of such standards is crucial to prevent nucleic acid decay and to ensure stable results using real-time PCR. In this study, a broad investigation of possible causes of DNA degradation during storage was performed, including GC-content of the fragments, long-term storage, rapid freeze-and-thaw experiments, genomic DNA and short DNA fragments of different species, the influence of shear stress and the effect of nuclease remaining after DNA isolation. Several known chemical DNA degradation mechanisms have been matched with the experimental data through a process of elimination. Protocols for practical application, as well as a theoretical model describing the underlying mechanisms of deviation of real-time PCR results due to decay of standard DNA, have been developed. Primary amines in the buffer composition, which enhance depurination of the DNA helix, and shear stress due to ice crystal formation, could be identified as major sources of interaction. This results in degradation of the standard DNA, as well as in the probability of occurrence of mismatches affecting real-time PCR performance.

Sample Preparation for qPCR Detection of Listeria from Food.

Quantitative PCR, if performed properly, is a highly sensitive and robust tool. Nevertheless, its application to the particular case of pathogen detection from foodstuffs necessitates special requirements for reliable results. Firstly, a robust analytical chain, involving sample preparation and DNA isolation with purification, is necessary to ensure optimal performance. Secondly, for reliable quantification of Listeria monocytogenes from food, reproducible controls for all steps of the analytical chain are needed, which can give quantitative information about the performance of each individual step of the detection chain. Ideally, each individual sample should include a so-called internal sample process control (ISPC) which passes through all steps of the analytical chain and is phenotypically similar to the target organism (in this case L. monocytogenes).This chapter describes the modular and rapid (3 h) sample preparation method "matrix lysis" for the quantification of L. monocytogenes from food and gives detailed information regarding the application of an ISPC based on the example of the L. monocytogenes Δ-prfA/+IAC strain.

qPCR Validation on the Basis of the Listeria monocytogenes prfA Assay.

Quantitative real-time polymerase chain reaction (qPCR) is one of the most used molecular methods. There are numerous qPCR assays on the market, some of them for pathogen detection, and the development of new assays still continues. However, what methods are suitable for assay performance validation and which information do they provide? For conclusions based on qPCR data, it is essential to know which capacities and limitations an assay has. This chapter gives an overview of methods for qPCR assay performance validation and the respective insights and how to combine them. Most of those validation methods have been published in connection with the prfA assay, which specifically detects Listeria monocytogenes. Thereby, it could be demonstrated that this assay reliably quantifies even a single copy of the prfA gene and is thus suitable for detection of Listeria monocytogenes.

A new long-term sampling approach to viruses on surfaces.

The importance of virus disease outbreaks and its prevention is of growing public concern but our understanding of virus transmission routes is limited by adequate sampling strategies. While conventional swabbing methods provide merely a microbial snapshot, an ideal sampling strategy would allow reliable collection of viral genomic data over longer time periods. This study has evaluated a new, paper-based sticker approach for collection of reliable viral genomic data over longer time periods up to 14 days and after implementation of different hygiene measures. In contrast to swabbing methods, which sample viral load present on a surface at a given time, the paper-based stickers are attached to the surface area of interest and collect viruses that would have otherwise been transferred onto that surface. The major advantage of one-side adhesive stickers is that they are permanently attachable to a variety of surfaces. Initial results demonstrate that stickers permit stable recovery characteristics, even at low virus titers. Stickers also allow reliable virus detection after implementation of routine hygiene measures and over longer periods up to 14 days. Overall, results for this new sticker approach for virus genomic data collection are encouraging, but further studies are required to confirm anticipated benefits over a range of virus types.

How to Evaluate Non-Growing Cells-Current Strategies for Determining Antimicrobial Resistance of VBNC Bacteria.

Thanks to the achievements in sanitation, hygiene practices, and antibiotics, we have considerably improved in our ongoing battle against pathogenic bacteria. However, with our increasing knowledge about the complex bacterial lifestyles and cycles and their plethora of defense mechanisms, it is clear that the fight is far from over. One of these resistance mechanisms that has received increasing attention is the ability to enter a dormancy state termed viable but non-culturable (VBNC). Bacteria that enter the VBNC state, either through unfavorable environmental conditions or through potentially lethal stress, lose their ability to grow on standard enrichment media, but show a drastically increased tolerance against antimicrobials including antibiotics. The inability to utilize traditional culture-based methods represents a considerable experimental hurdle to investigate their increased antimicrobial resistance and impedes the development and evaluation of effective treatments or interventions against bacteria in the VBNC state. Although experimental approaches were developed to detect and quantify VBNCs, only a few have been utilized for antimicrobial resistance screening and this review aims to provide an overview of possible methodological approaches.