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.

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.

A Fast and Easy ATP-Based Approach Enables MIC Testing for Non-resuscitating VBNC Pathogens.

Many bacteria enter the viable but non-culturable (VBNC) state to maximize resources and increase their tolerance to harmful conditions to cope with environmental stress, which has been described for a plethora of important human and foodborne pathogens. VBNC pathogens can potentially present a serious risk to human health as they are invisible to routine microbiological culture-based methods. Of high importance is the increased tolerance to antibiotics or disinfectant measures while in the VBNC state. The greatest remaining challenge for such investigations is the lack of an appropriate, cost-effective multi-species screening method due to experimental constraints. In this study, we investigated if de novo ATP production of cells in the VBNC state is a suitable indicator for overall cell viability that can be utilized to determine the minimum ATP inhibitory concentration (MAIC) of antibiotics and other antimicrobials. To validate this approach, heat-stress time-kill experiments were performed with both culturable and VBNC cells. We developed a comprehensive experimental setup and demonstrated the applicability of this VBNC-MIC assay for testing the tolerance of 12 strains of 4 important bacterial species (Escherichia coli, Bacillus cereus, Pseudomonas aeruginosa, and Listeria monocytogenes) in the VBNC state to eight important antimicrobials including four different antibiotics. We confirmed that bacteria in the VBNC state were resistant to all tested antibiotics (ampicillin, imipenem, ciprofloxacin, and gentamicin) and additionally insensitive to disinfectants (benzalkonium chloride and trioctylmethylammonium chloride) and preservatives (bronopol and sodium azide). These data emphasize the need for further research regarding the characteristics of bacterial pathogens in the VBNC state and present the advantages and high-throughput capabilities of ATP determinations to investigate tolerance of VBNC pathogens to antimicrobials. The presented method should be helpful in order to identify appropriate countermeasures, treatments, or disinfectants when confronted with bacterial pathogens in the VBNC state.

FTIR Spectroscopy Suggests a Revised Mode of Action for the Cationic Side-Chain Effect of Ionic Liquids.

Over the past decades, ionic liquids (ILs) have gained considerable attention from the scientific community because of their versatile and designable properties. As a result, there are numerous IL applications, not only in organic synthesis, catalysis, or extraction but also as active pharmaceutical ingredients or novel antimicrobials. While considerable effort has been put into developing quantitative structure-activity relationship (QSAR) models for IL toxicity prediction, little is known about their actual mode of action. In this study, Fourier transform infrared (FTIR) spectroscopy is used to monitor IL induced molecular responses directly at the cellular level. Investigation of the well-known cationic alkyl side-chain effect (increasing side-chain length leads to increasing toxicity) of imidazolium- and ammonium-based ILs on two bacterial pathogens, enteropathogenic  Escherichia coli (EPEC) and methicillin-resistant Staphylococcus aureus (MRSA), surprisingly revealed two distinct modes of action. Contrary to prior models, it was only for [TMC16A][Cl], where a molecular response in the membrane was found, while ILs with shorter side-chain lengths predominantly affected bacterial proteins. The results of this study highlight the importance of further direct investigations of the impact of ILs at the cellular level to improve toxicity prediction and assess the usefulness of spectroscopic methods, such as FTIR spectroscopy at achieving this goal.

Induction of the viable but non-culturable state in bacterial pathogens by household cleaners and inorganic salts.

Effective monitoring of microbial pathogens is essential for a successful preventive food safety and hygiene strategy. However, as most monitoring strategies are growth-based, these tests fail to detect pathogenic bacteria that have entered the viable but non-culturable (VBNC) state. The present study reports the induction of the VBNC state in five human pathogens by commercially available household cleaners in combination with inorganic salts. We determined that non-ionic surfactants, a common ingredient in household cleaners, can induce the VBNC state, when combined with salts. A screening study with 630 surfactant/salt combinations indicates a correlation between the hydrophobicity of the surfactant and VBNC induction in L. monocytogenes, E. coli, S. enterica serovar Typhimurium, S. aureus and toxin-producing enteropathogenic E. coli. Cells that were exposed to combinations of surfactants and salts for 5 min and up to 1 h lost their culturability on standard growth media while retaining their ATP production, fermentation of sugars and membrane integrity, which suggests intact and active metabolism. Screening also revealed major differences between Gram-negative and Gram-positive bacteria; the latter being more susceptible to VBNC induction. Combinations of such detergents and salts are found in many different environments and reflect realistic conditions in industrial and domestic surroundings. VBNC cells present in industrial environments, food-processing plants and even our daily routine represent a serious health risk due to possible resuscitation, unknown spreading, production of toxins and especially their invisibility to routine detection methods, which rely on culturability of cells and fail to detect VBNC pathogens.

Influence of Environmental Factors on Phage-Bacteria Interaction and on the Efficacy and Infectivity of Phage P100.

When using bacteriophages to control food-borne bacteria in food production plants and processed food, it is crucial to consider that environmental conditions influence their stability. These conditions can also affect the physiological state of bacteria and consequently host-virus interaction and the effectiveness of the phage ability to reduce bacteria numbers. In this study we investigated the stability, binding, and replication capability of phage P100 and its efficacy to control Listeria monocytogenes under conditions typically encountered in dairy plants. The influences of SDS, Lutensol AO 7, salt, smear water, and different temperatures were investigated. Results indicate that phage P100 is stable and able to bind to the host under most conditions tested. Replication was dependent upon the growth of L. monocytogenes and efficacy was higher when bacterial growth was reduced by certain environmental conditions. In long-term experiments at different temperatures phages were initially able to reduce bacteria up to seven log10 units after 2 weeks at 4°C. However, thereafter, re-growth and development of phage-resistant L. monocytogenes isolates were encountered.