Population diversity of Listeria monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010.

It has been possible to determine the genotype diversity of Listeria monocytogenes in the actual cheese lots of acid curd cheese that caused a multinational outbreak between 2009 and 2010. Following product recall in January 2010 all lots were investigated. A total of 422 L. monocytogenes isolates were characterized by genotyping. In a first approach the PCR serogroups were defined by multiplex-PCR assays. Subsequently, the isolates were subtyped by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). Sequence types were assigned by submitting the DNA sequences to the Listeria MLST database at the Institute Pasteur. The serogroup PCR resulted in a homogeneous 1/2a - 3a (genetic linage II) cluster. The generated PFGE patterns divided the strains into two clusters (type 1 and 2) diverging at a homogeneity level of 74%. PFGE-type 2 was predominant, accounting for 98.3% (n = 415/422) of the isolates and was isolated during the whole period of acid curd cheese processing (01.12.2009-13.01.2010). 1.7% of all tested L. monocytogenes isolates (n = 7/422) belonged to PFGE-type 1 and were isolated from 28% of all cheese lots (n = 5/18) produced between the time span of 08.12.2009 to 13.01.2010. Furthermore, PFGE-type 1 and 2 showed the same PFGE patterns as the human outbreak strains (clone 1 and clone 2).

The challenge to quantify Listeria monocytogenes--a model leading to new aspects in molecular biological food pathogen detection.

In this work, we discuss the latest insights concerning advantages and disadvantages and the nature of microbiological and molecular methods for quantitative food pathogen detection. The assessment of molecular methods must be brought on a basis that considers the nature of molecular methods and their underlying mechanism. A potential approach to setting up the development, validation and structure of an analytical chain is presented based on quantitative real-time PCR (qPCR). This is analysed exemplary on the basis of recent work using the model organism Listeria monocytogenes. Several prerequisites for successful quantitative detection of this pathogen will be discussed. In particular, sample preparation, controls for all methodical steps and the validation of the core assay qPCR are addressed, which constitute the basis for a reliable analytical detection chain for molecular biological pathogen detection from food. Microbiological methods are analysed based on growth of the single cell, which is the fundament of these traditional methods.

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. METHODS AND RESULTS: The combined enrichment/real-time PCR method amplifying the prfA locus was performed according to [Rossmanith et al.(2006) Res Microbiol, 157, 763-771]. Qualitative and quantitative examination of the samples was performed according to the standard method ISO-11290. Comparison of the combined enrichment/real-time PCR method with ISO-11290 resulted in 100% relative accuracy, 100% relative sensitivity and 100% relative specificity. CONCLUSIONS: A previously published study describing the validation of the method, including samples after storage at -80 degrees C, resulted in lower performance values. In contrast, the samples were stored at +4 degrees C in this study. The results of this study indicate an effect of storage, thus masking the true performance of the method. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of this study are discussed together with the previously published data to demonstrate the excellent qualities of this rapid (< or = 30 h) method when applied to fresh specimens stored at +4 degrees C.

The use of chromogenic bacteria as coloured substitutes for pathogens: a simple strategy during design and development of a new method for sample pretreatment.

AIMS: In the present study, chromogenic (red) bacteria were used to simulate actual target bacteria during set-up and optimization of an isolation process of bacteria, designed for food samples. Isolation of bacteria from food in the context of molecular biological detection of food pathogens is a multistep process. Development of such a separation method requires continuous monitoring of the location of the presumable targets in the sample tubes. Therefore, red-coloured pigmented bacteria were used as substitutes for the actual target bacteria, during the establishment of a new sample preparation technique. METHODS AND RESULTS: The chromogenic bacteria Micrococcus roseus and Serratia marcescens were confirmed to withstand the physical (e.g. centrifugal forces) and chemical (e.g. lysis buffer composition) conditions required during establishment of the new technique. Furthermore, the suitability of these model bacteria to substitute for the actual target pathogens (Salmonella enterica subsp. enterica serovar Typhimurium and Listeria monocytogenes) was assured by testing the physical properties of the model bacteria with respect to the proposed separation methods. CONCLUSION: Visibility of the pigmented bacteria within the complex sample matrices served to allocate bacterial content during the various steps necessary for finalization of the method protocol. The presumptive bacterial targets can be allocated simply by visualization of their bright red colour silhouetted against the background sample matrix. SIGNIFICANCE AND IMPACT OF STUDY: The use of pigmented bacteria as substitutes for actual colourless target bacteria during design and development of a bacterial isolation method is a simple and inexpensive application. It saves a huge amount of time and resources, as the proof of principle of new methods is possible in rapid succession.

Determination of the sedimentation coefficient distribution by least-squares boundary modeling.

A new method is presented for the calculation of apparent sedimentation coefficient distributions g*(s) for the size-distribution analysis of polymers in sedimentation velocity experiments. Direct linear least-squares boundary modeling by a superposition of sedimentation profiles of ideal nondiffusing particles is employed. It can be combined with algebraic noise decomposition techniques for the application to interference optical ultracentrifuge data at low loading concentrations with significant systematic noise components. Because of the use of direct boundary modeling, residuals are available for assessment of the quality of the fits and the consistency of the g*(s) distribution with the experimental data. The method can be combined with regularization techniques based on F statistics, such as used in the program CONTIN, or alternatively, the increment of s values can be adjusted empirically. The method is simple, has advantageous statistical properties, and reveals precise sedimentation coefficients. The new least-squares ls-g*(s) exhibits a very high robustness and resolution if data acquired over a large time interval are analyzed. This can result in a high resolution for large particles, and for samples with a high degree of heterogeneity. Because the method does not require a high frequency of scans, it can also be easily used in experiments with the absorbance optical scanning system. Published 2000 John Wiley & Sons, Inc.