Patrick Mester

Broad range evaluation of the matrix solubilization (matrix lysis) strategy for direct enumeration of foodborne pathogens by nucleic acids technologies.

A previously published rapid (<5 h) proof-of-concept protocol for the concentration of the foodborne pathogen Listeria monocytogenes from milk, based on the solubilization of the food matrix, was further evaluated. The original protocol was modified to detect gram-negative and other gram-positive bacteria and to broaden the range of food matrices by using Lutensol instead of sodium dodecyl sulfate as the main detergent in the buffer. A new protocol using a protease and sucrose buffer was established for the analysis of meat and fish. Matrix lysis was used for dairy products, ice cream, milk, fish, meat, eggs, and blood. Solubilization of the foodstuffs resulted in bacterial pellets of reasonable size for further quantification. Using L. monocytogenes, Staphylococccus aureus, Bacillus cereus, Escherichia coli, and Salmonella Typhimurium as model organisms, microscopic analysis of the remaining bacterial pellets revealed that the recovered bacteria remained physically intact, albeit their viability was compromised. In model experiments using free DNA, the free target DNA was reduced by 5 log units. The compatibility of matrix lysis with subsequent real-time PCR analysis has been demonstrated with salmon, chicken, egg, ice cream, cheese, and blood samples that were artificially contaminated with L. monocytogenes, S. aureus, and Salmonella Typhimurium. These experiments resulted in an average recovery of 48.7% (relative standard error, 83.4%) of the inoculated bacterial cells with the real-time PCR assay. The average detection limit of the method was 7.3 CFU/ml for all examined foodstuffs and bacterial target organisms.

Use of ionic liquid-based extraction for recovery of salmonella typhimurium and Listeria monocytogenes from food matrices.

Methods for rapid separation (<5 h) and concentration of bacteria based on solubilization of complex food matrices have been developed recently to facilitate rapid molecular detection methods. However, a major disadvantage of these protocols is the resulting lack of viability of the microorganisms under study due to extensive use of chemicals and enzymes, which can inhibit subsequent quantitative microbiological analyses. In this study, a new class of organic salts, ionic liquids, were used for solubilization of various foodstuffs, with subsequent molecular and microbiological quantification methods. This approach was applied to gram-positive Listeria monocytogenes and gram-negative Salmonella enterica serovar Typhimurium. By introducing the ionic liquid 1-ethyl-3-methylimidazolium thiocyanate into an existing food solubilization protocol, both molecular and microbiological quantification methods could be used subsequently without losing performance or prolonging the analysis. These experiments resulted in an average recovery of 87% of inoculated bacterial cells with real-time PCR, 85% recovery on nonselective agar plates, and 43% on selective medium. These results illustrate the feasibility of applying ionic liquids in sample pretreatment steps for rapid detection and quantification of bacterial pathogens.

Antimicrobial effects of short chained imidazolium-based ionic liquids—Influence 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.

Demonstration of the effective performance of a combined enrichment /real-time PCR method targeting the prfA gene of Listeria monocytogenes by testing fresh naturally contaminated acid curd cheese

Aims:  A rapid real‐time PCR‐based method for the detection of Listeria monocytogenes was applied to the examination of 44 Quargel cheese samples from a recent outbreak in Austria and compared to the standard method according to ISO‐16140.

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

ABSTRACT 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.

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.

Biased spectroscopic protein quantification in the presence of ionic liquids

AbstractOwing to their unique physicochemical properties, ionic liquids have gained much recognition as solvents or co-solvents in a wide variety of biochemical applications. In the context of protein analytics, four similar 1-alkyl-3-methylimidazolium-based ionic liquids have been analysed for their applicability as co-solvents. Spectroscopic bovine serum albumin (BSA) quantification experiments in the presence of ionic liquids were performed and for two ionic liquids a concentration-dependent effect has been found that can lead to biased protein quantification. It could be shown that the biased spectroscopic analysis of the tested ionic liquids is dependent on the length of the alkyl side chain (>C4) of the 1-alkyl-3-methylimidazolium-based cation, and the chaotropicity of the anion. Once accounted for and properly calibrated when using spectroscopic methods, these effects can be avoided thus facilitating correct protein quantification in the presence of ionic liquids. FigureConcentration dependent but non-linear shift of UV/Vis spectra of ionic liquids

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

AbstractFast 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 AbstractEvaluating the absolute quantification potential of ddPCR targeting Listeria monocytogenes prfA

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.

Cloning and Characterisation of a Δ-prfA Listeria monocytogenes Strain Containing an Artificial Single Copy Genomic Internal Amplification Control (IAC) for Use as Internal Sample Process Control (ISPC)

Conventional internal amplification controls (IAC) are DNA-based controls which monitor the amplification reaction of real-time PCR in food pathogen detection. Food pathogen detection using real-time PCR, however, includes necessarily sample preparation and DNA isolation/purification. This modular structure leads to an analytical chain. To cover the whole analytical chain, the concept of the IAC has to be extended to internal sample process controls (ISPCs) which include supporting pre-analytical steps. One concept for such ISPCs is the use of recombinant bacterial cells comprising a deleted target and an artificial competitive target instead, which are derived from the actual target strain. In this work, we present an ISPC for the molecular detection of Listeria monocytogenes. A Δ-prfA L. monocytogenes EGDe strain was cloned with a pPL2 phage insertion vector to include a single copy artificial DNA target, resulting in a fluorescence signal not interfering with the respective signal of the L. monocytogenes EGDe wild-type strain during real-time PCR. The recombinant strain was confirmed and characterized with conventional and real-time PCR including sequencing. Microbiological examination revealed a distinct phenotype pattern on selective plate media which enables discrimination of Δ-prfA L. monocytogenes EGDe from wild-type L. monocytogenes EGDe and Listeria innocua. The ISPC was applied in an examination of artificially contaminated ultra high temperature-treated milk to demonstrate its analytical suitability. The resulting corrected recovery values of the ISPC as obtained by the whole molecular quantification procedure correspond to the respective values determined for the actual target strain (P ≤ 0.05).