Dagmar Schoder

Survey on the Listeria contamination of ready-to-eat food products and household environments in Vienna, Austria.

Qualitative and quantitative contamination of ready‐to‐eat food‐stuffs with the pathogen Listeria monocytogenes was studied in 1586 samples collected from 103 supermarkets (n = 946) and 61 households (n = 640) in Vienna, Austria. Seventeen groups of ready‐to‐eat foods were classified into three risk categories for contamination (CP1–CP3). Three to four samples were randomly collected at the retail level from each CP. Regarding the households, the sampling procedure was started with food items of CP1, and if not available, was continued with sampling of food items of CP2 and finally of CP3. Additionally, 184 environmental samples (swabs from the kitchen area, dust samples from the vacuum cleaner) and faecal samples (household members and pet animals) were included. One‐hundred and twenty‐four (13.1%) and 45 (4.8%) samples out of 946 food samples collected from food retailers tested positive for Listeria spp. and L. monocytogenes, respectively, with five smoked fish samples exceeding the tolerated limit of 100 CFU/g food. Food‐stuffs associated with the highest risk of contamination were twice as frequently contaminated with L. monocytogenes as food‐stuffs associated with a medium risk of contamination. Products showing the highest contamination rate were fish and seafood (19.4%), followed by raw meat sausages (6.3%), soft cheese (5.5%) and cooked meat products/patés (4.5%). The overall contamination rate of foods collected at the household level was more than two times lower. Only 5.6% and 1.7% of 640 food‐stuffs analysed tested positive for Listeria spp. and L. monocytogenes, respectively. However, CP1 foods were rarely collected. Pulsed‐field gel electrophoresis (PFGE) typing of the collected L. monocytogenes isolates revealed a high degree of diversity between the isolates, with some exceptions. PFGE typing of isolates harvested from green‐veined cheese revealed a match among strains, although the manufacturer seemed to be distinguishable. Typing of household strains revealed an epidemiological link within one family. In this case, food‐stuffs and the kitchen environment were contaminated by an indistinguishable isolate. In addition, the same isolate was collected from a pooled faecal sample of the household members suggesting that consumption of even low contaminated food items (<100 CFU/g) results in Listeria shedding after the passage through the gut.

Genome Sequencing of Listeria monocytogenes “Quargel” Listeriosis Outbreak Strains Reveals Two Different Strains with Distinct In Vitro Virulence Potential

A large listeriosis outbreak occurred in Austria, Germany and the Czech Republic in 2009 and 2010. The outbreak was traced back to a traditional Austrian curd cheese called “Quargel” which was contaminated with two distinct serovar 1/2a Listeria monocytogenes strains (QOC1 and QOC2). In this study we sequenced and analysed the genomes of both outbreak strains in order to investigate the extent of genetic diversity between the two strains belonging to MLST sequence types 398 (QOC2) and 403 (QOC1). Both genomes are highly similar, but also display distinct properties: The QOC1 genome is approximately 74 kbp larger than the QOC2 genome. In addition, the strains harbour 93 (QOC1) and 45 (QOC2) genes encoding strain-specific proteins. A 21 kbp region showing highest similarity to plasmid pLMIV encoding three putative internalins is integrated in the QOC1 genome. In contrast to QOC1, strain QOC2 harbours a vip homologue, which encodes a LPXTG surface protein involved in cell invasion. In accordance, in vitro virulence assays revealed distinct differences in invasion efficiency and intracellular proliferation within different cell types. The higher virulence potential of QOC1 in non-phagocytic cells may be explained by the presence of additional internalins in the pLMIV-like region, whereas the higher invasion capability of QOC2 into phagocytic cells may be due to the presence of a vip homologue. In addition, both strains show differences in stress-related gene content. Strain QOC1 encodes a so-called stress survival islet 1, whereas strain QOC2 harbours a homologue of the uncharacterized LMOf2365_0481 gene. Consistently, QOC1 shows higher resistance to acidic, alkaline and gastric stress. In conclusion, our results show that strain QOC1 and QOC2 are distinct and did not recently evolve from a common ancestor.

Physical Characteristics of Six New Thermocyclers

Since the publication of the first article describing PCR, thermocyclers have become a staple in academic and industrial laboratories (1). The thermocycler is a programmable cycling incubator that performs repeated PCR steps of DNA denaturation, primer annealing, and primer elongation at defined intervals. Rapid heat transfer from the heating block to the in-tube sample liquid ensures a high efficiency of amplicon multiplication; therefore, a thermal processor should guarantee temperature uniformity for all samples within an individual run as well as run-to-run repeatability. PCR-based protocols can give unsatisfying results (2)(3). Several collaborative studies have shown weak reproducibility with random amplified polymorphic DNA (RAPD) protocols (4)(5). One reason might be the influence of the thermocycler on amplification efficiency. Despite its striking importance for PCR, the literature on thermocyclers is scarce. Some studies were published on the first generation of cyclers (6)(7)(8). Others determined the amplification efficiency but did not evaluate the physical characteristics (9)(10). The impact on PCR of the variation within thermocyclers, with regard to their thermocycling settings, has not been fully determined. The goal of this study was to define the physical characteristics of performance of the latest generation of thermocyclers and to discuss the influence of the physical properties on amplification efficiency. Six new thermocyclers were selected for this performance study: ( A ) Gene Amp 9700 (Applied Biosystems), ( B ) Multicycler PTC 200 (MJ Research, Inc.), …

A case of sporadic ovine mastitis caused by Listeria monocytogenes and its effect on contamination of raw milk and raw-milk cheeses produced in the on-farm dairy

We describe a case of listerial mastitis in a flock of 130 sheep. The animals were housed at a farm where the bulk raw ewe milk was processed to produce raw milk soft cheese. List. monocytogenes was shed from the right mammary complex. Shedding was observed over a period of 99 d. A mean level of 4-56 x 10(4) cfu (colony forming units) Listeria monocytogenes/ml was recovered from the raw milk originating from the infected udder. The numbers ranged from 9 x10(1) to 2.95 x 10(5). The bulk milk was contaminated by approx. 5.7 x 10(3) cfu/ml. In the cheese product, 2.0 x 10(2) cfu List. monocytogenes/g were constantly detectable for a period of 7 d post manufacture. The starter culture used for coagulation had a pivotal influence on the behaviour of List. monocytogenes during cheesemaking. Using the same mesophilic buttermilk culture as used by the farmer allowed numbers of Listeria to increase 60-fold within 12 h owing to a delayed acidification of the bulk milk. Addition of a thermophilic yogurt culture reduced the numbers of Listeria within 8 h of incubation.

Important Vectors for Listeria monocytogenes Transmission at Farm Dairies Manufacturing Fresh Sheep and Goat Cheese from Raw Milk

The aim of this study was to determine the transmission routs of Listeria spp. in dairy farms manufacturing fresh cheese made from ovine and caprine raw milk and to evaluate the impact of Listeria monocytogenes mastitis on raw milk contamination. Overall, 5,799 samples, including 835 environmental samples, 230 milk and milk product samples, and 4,734 aseptic half-udder foremilk samples were collected from 53 dairy farms in the dairy intensive area of Lower Austria. Farms were selected for the study because raw milk was processed to cheese that was sold directly to consumers. A total of 153 samples were positive for Listeria spp., yielding an overall prevalence of 2.6%; L. monocytogenes was found in 0.9% of the samples. Bulk tank milk, cheese, and half-udder samples were negative for Listeria spp. Because none of the sheep and goats tested positive from udder samples, L. monocytogenes mastitis was excluded as a significant source of raw milk contamination. L. monocytogenes was detected at 30.2% of all inspected farms. Swab samples from working boots and fecal samples had a significantly higher overall prevalence (P < 0.001) of L. monocytogenes (15.7 and 13.0%, respectively) than did swab samples from the milk processing environment (7.9%). A significant correlation was found between the prevalence of L. monocytogenes in the animal and in the milk processing environment and the silage feeding practices. Isolation of L. monocytogenes was three to seven times more likely from farms where silage was fed to animals throughout the year than from farms where silage was not fed to the animals.

Novel Approach for Assessing Performance of PCR Cyclers Used for Diagnostic Testing

ABSTRACT As part of a large international project for validation and standardization of PCR, the influence of thermocyclers on PCR was tested. Six brand-new, Peltier technology-driven 96-well thermocyclers were subjected to a novel and stringent in-tube (not block) physical testing. The temperature was directly monitored in PCR tubes containing 50 μl of distilled water at 13 different block positions. The certified temperature accuracy of the measurement system was ±0.3°C. Finally, the results of the physical testing were compared to those of an amplification efficiency study running an in-house PCR assay. The cyclers did not perform within the manufacturer′s specification. Premature timing, under- and overshooting, and spatial variation of heat transfer were found to be the critical factors. The physical testing allowed us to distinguish accurate from less-accurate (2/6) cyclers. The lack of thermal homogeneities became most evident at the denaturation level during the first 15 s. At the time point zero, the accurate cyclers showed temperature deviations of 0.5 to 1.5°C, whereas less-accurate cyclers failed to reach the set temperature by 13 to 20°C. Consequently, the two less-accurate cyclers could not gain positive PCR results by running an in-house PCR assay. However, by modifying the original temperature protocol by increasing the denaturation temperature and time, the amplification efficiency of these two cyclers could be improved significantly. The results have implication for laboratories using diagnostic PCR testing.

Fluctuation in contamination dynamics of L. monocytogenes in quargel (acid curd cheese) lots recalled during the multinational listeriosis outbreak 2009/2010.

For the first time it has been possible to determine the contamination level of Listeria monocytogenes in the very cheese lots of acid curd cheese that caused a multinational outbreak between 2009/2010. The listeriosis outbreak accounted for 34 clinical cases and eight deaths. The cheese, which was distributed in Austria, Germany, the Czech Republic, Poland and Slovakia, was recalled on the 23rd January 2010. All recalled lots were immediately investigated after call back from the retail market. The company manufactured two different cheese types, (i) red smear ripened--and (ii) mold coated/white veined--acid curd cheese. Depending on the lot production dates, cheese samples (n=1045) were analyzed at three different time points: (i) beginning to mid shelf-life (lot nos. 15-18; production period 5.1.2010-13.1.2010); (ii) end of shelf-life (lot nos. 9-18; production period 21.12.2009-13.1.2010) and, (iii) ≤46days after the expiry date (lot nos. 1-18; production period 1.12.2009-13.1.2010). Qualitative and quantitative examinations of cheese samples were performed according to ISO 11290-1&2. Examination of the samples, according to ISO 11290-1, resulted in 16 L. monocytogenes positive (red smear type) and two negative lots (mold coated type). These results were confirmed by a combined enrichment/real-time PCR method. The contamination values obtained by quantitative ISO 11290-2 varied from ≤log 2 cell forming units (CFU)/g to log 8.1CFU/g. Three out of sixteen L. monocytogenes positive lots revealed a contamination level of ≤log 2CFU/g at the beginning of their shelf-life when stored at 4°C. Nevertheless, by increasing the storage life and/or the storage temperature (15, 22°C) the contamination level could be raised to between log 5 and log 6CFU/g. Our data indicate that 81.3% (13/16) of the recalled red smear quargel cheese lots were highly contaminated with L. monocytogenes. All this implies that the main contamination of the quargel cheese took place during the red smear process and that quargel cheese can easily support growth of L. monocytogenes.

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.

Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes.

Aims:  The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food‐borne pathogen Listeria monocytogenes. A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al.

Melamine Milk Powder and Infant Formula Sold in East Africa

This is the first study proving the existence of melamine in milk powder and infant formula exported to the African market. A total of 49 milk powder batches were collected in Dar-es-Salaam (Tanzania, East Africa), the center of international trade in East Africa, which serves as a commercial bottleneck and shipment hub for sub-Saharan, Central, and East Africa. Two categories of samples were collected between October and December 2008, immediately after the melamine contamination of Chinese products became public: (i) market brands of all international companies supplying the East African market and (ii) illegally sold products from informal channels. Melamine concentration was determined with the AgraQuant Melamine Sensitive Assay. Despite the national import prohibition of Chinese milk products and unlabeled milk powder in Tanzania, 11% (22 of 200) of inspected microretailers sold milk powder on the local black market. Manufacturers could be identified for only 55% (27) of the 49 investigated batches. Six percent (3 of 49) of all samples and 11% (3 of 27) of all international brand name products tested revealed melamine concentrations up to 5.5 mg/kg of milk powder. This amount represents about twice the tolerable daily intake as suggested by the U.S Food and Drug Administration. Based on our study, we can assume that the number of affected children in Africa is substantial.