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.

Don't Shut the Stable Door after the Phage Has Bolted-The Importance of Bacteriophage Inactivation in Food Environments.

In recent years, a new potential measure against foodborne pathogenic bacteria was rediscovered-bacteriophages. However, despite all their advantages, in connection to their widespread application in the food industry, negative consequences such as an uncontrolled phage spread as well as a development of phage resistant bacteria can occur. These problems are mostly a result of long-term persistence of phages in the food production environment. As this topic has been neglected so far, this article reviews the current knowledge regarding the effectiveness of disinfectant strategies for phage inactivation and removal. For this purpose, the main commercial phage products, as well as their application fields are first discussed in terms of applicable inactivation strategies and legal regulations. Secondly, an overview of the effectiveness of disinfectants for bacteriophage inactivation in general and commercial phages in particular is given. Finally, this review outlines a possible strategy for users of commercial phage products in order to improve the effectiveness of phage inactivation and removal after application.

Essential role of polymerases for assay performance - Impact of polymerase replacement in a well-established assay.

The quantitative real-time polymerase chain reaction (qPCR) is one of the most commonly molecular methods used today. It is central to numerous assays that have since been developed and described around its optimization. The Listeria monocytogenes prfA qPCR assay has been studied in great detail and due to its comprehensive knowledge, excellent performance (sensitivity of one single copy), and internal amplification control, it represents a suitable test platform for qPCR examinations. In this study, we compared ten different polymerases (or ready-to-use mastermixes) as possible (economic) alternatives to our gold standard Platinum Taq polymerase. We sought to determine the reproducibility of these assays under modified conditions, which are realistic because published assays are frequently used with substituted polymerases. Surprisingly, there was no amplification at all with some of the tested polymerases, even although the internal amplification control worked well. Since adaptation of the thermal profile and of MgCl2 concentration could restore amplification, simple replacement of the polymerase can destroy a well-established assay leading up to >106-fold less analytical sensitivity. Further, validation using Poisson and PCR-Stop analyses revealed limits to some assay-polymerase combinations and emphasize the importance of validation.

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.

PCR-Stop analysis as a new tool for qPCR assay validation.

Progressively more qPCR assays have been developed in recent years in numerous fields of application. These assays are routinely validated using calibration curves, but essential validation per se such as Poisson analysis is frequently neglected. However, validation is crucial for determination of resolution and quantitative and qualitative limits. The new test method PCR-Stop analysis presented in this work investigates assay performance during initial qPCR cycles. PCRs with one to five pre-runs are performed while the subsequent main qPCR runs reflect pre-run replication rates. Ideally, DNA doubles according to pre-runs, there is no variation between replicates and qPCR starts immediately at the first cycle with its average efficiency. This study shows two exemplary qPCR assays, both with suitable calibration curves and efficiencies. We demonstrated thereby the benefits of PCR-Stop analysis revealing quantitative and qualitative resolution of both assays, the limits of one of those assays and thus avoiding misinterpretations in qPCR analysis. Furthermore, data displayed that a well performing assay starts indeed with its average efficiency.

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.

Virucidal Influence of Ionic Liquids on Phages P100 and MS2.

An increasing number of publications describe the potential of ionic liquids (ILs) as novel antimicrobials, antibacterial coatings and even as active pharmaceutical ingredients. Nevertheless, a major research area, notably their impact on viruses, has so far been neglected. Consequently the aim of this study was to examine the effects of ILs on the infectivity of viruses. A systematic analysis to investigate the effects of defined structural elements of ILs on virus activity was performed using 55 ILs. All structure activity relationships (SARs) were tested on the human norovirus surrogate phage MS2 and phage P100 representing non-enveloped DNA viruses. Results demonstrate that IL SAR conclusions, established for prokaryotes and eukaryotes, are not readily applicable to the examined phages. A virus-type-dependent IL influence was also apparent. Overall, four ILs, covering different structural elements, were found to reduce phage P100 infectivity by ≥4 log10 units, indicating a virucidal effect, whereas the highest reduction for phage MS2 was about 3 log10 units. Results indicate that future applications of ILs as virucidal agents will require development of novel SARs and the obtained results serve as a good starting point for future studies.

Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

Viruses shape a diversity of ecosystems by modulating their microbial, eukaryotic, or plant host metabolism. The complexity of virus-host interaction networks is progressively fathomed by novel metagenomic approaches. By using a novel metagenomic method, we explored the virome in mammalian cell cultures and clinical samples to identify an extensive pool of mobile genetic elements in all of these ecosystems. Despite aseptic treatment, cell cultures harbored extensive and diverse phage populations with a high abundance of as yet unknown and uncharacterized viruses (viral dark matter). Unknown phages also predominated in the oropharynx and urine of healthy individuals and patients infected with cytomegalovirus despite demonstration of active cytomegalovirus replication. The novelty of viral sequences correlated primarily with the individual evaluated, whereas relative abundance of encoded protein functions was associated with the ecologic niches probed. Together, these observations demonstrate the extensive presence of viral dark matter in human and artificial ecosystems.-Thannesberger, J., Hellinger, H.-J., Klymiuk, I., Kastner, M.-T., Rieder, F. J. J., Schneider, M., Fister, S., Lion, T., Kosulin, K., Laengle, J., Bergmann, M., Rattei, T., Steininger, C. Viruses comprise an extensive pool of mobile genetic elements in eukaryote cell cultures and human clinical samples.

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.

A Systematic Investigation of Parameters Influencing Droplet Rain in the Listeria monocytogenes prfA Assay - Reduction of Ambiguous Results in ddPCR.

The droplet digital polymerase chain reaction (ddPCR) determines DNA amounts based upon the pattern of positive and negative droplets, according to Poisson distribution, without the use of external standards. However, division into positive and negative droplets is often not clear because a part of the droplets has intermediate fluorescence values, appearing as "rain" in the plot. Despite the droplet rain, absolute quantification with ddPCR is possible, as shown previously for the prfA assay in quantifying Listeria monocytogenes. Nevertheless, reducing the rain, and thus ambiguous results, promotes the accuracy and credibility of ddPCR. In this study, we extensively investigated chemical and physical parameters for optimizing the prfA assay for ddPCR. While differences in the concentration of all chemicals and the dye, quencher and supplier of the probe did not alter the droplet pattern, changes in the PCR cycling program, such as prolonged times and increased cycle numbers, improved the assay.

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.

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.