Schnellnachweis von Listeria monocytogenes mittels real-time PCR

Molecular biological methods like the real-time PCR are well suited for a safe and rapid detection of Listeria and especially of Listeria monocytogenes. The use of commercially available kits in combination with adapted sample preparation is advantageous for the routine testing in the food industry. In addition to its high specificity and sensitivity, its superior reproducibility and short time to result, the described method is easy to use. Depending on the food matrix, the result is available after 24 h to 48 h. The procedure consists of a short enrichment, a sample preparation to extract the DNA from the cells and the PCR. The data interpretation is carried out automatically and can be used directly for documentation purposes. Eingegangen: 31. Januar 2007; Angenommen: 8. Februar 2007     

PFGE characterisation and adhesion ability of Listeria monocytogenes isolates obtained from bovine carcasses and beef processing facilities

Listeria monocytogenes is a pathogen capable of adhering to many surfaces and forming biofilms, which may explain its persistence in food processing environments. This study aimed to genetically characterise L. monocytogenes isolates obtained from bovine carcasses and beef processing facilities and to evaluate their adhesion abilities. DNA from 29 L. monocytogenes isolates was subjected to enzymatic restriction digestion (AscI and ApaI), and two clusters were identified for serotypes 4b and 1/2a, with similarities of 48% and 68%, respectively. The adhesion ability of the isolates was tested considering: inoculum concentration, culture media, carbohydrate source, NaCl concentration, incubation temperature, and pH. Each isolate was tested at 10(8)CFUmL(-1) and classified according to its adhesion ability as weak (8 isolates), moderate (17) or strong (4). The isolates showed higher adhesion capability in non-diluted culture media, media at pH 7.0, incubation at 25°C and 37°C, and media with NaCl at 5% and 7%. No relevant differences were observed for adhesion ability with respect to the carbohydrate source. The results indicated a wide diversity of PFGE profiles of persistent L. monocytogenes isolates, without relation to their adhesion characteristics. Also, it was observed that stressing conditions did not enhance the adhesion profile of the isolates.     

Comparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy

Listeria monocytogenes ranks among the most frequent causes of death due to foodborne illness (20-30% case fatality rate).Discriminative subtyping methods are important to detect the relatedness of isolates and verify epidemiologic associations. AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. In this study, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance.A total of 103 unrelated L. monocytogenes strains isolated from different environmental and food sources were analyzed. Strains were isolated from samples obtained from food-production plants, supermarkets and small food markets in Piedmont, Italy.All methods clustered L. monocytogenes strains into two genetic lineages, Lineage I and II. The three methods were compared using the 82 isolates which were typeable with all techniques. The calculated pair-wise Pearson's correlation coefficients (r) showed close agreement between all three methods.Our findings suggest that the AFLP II method can be successfully used to subtype L. monocytogenes strains isolated from foods and food processing facilities.     

Assessing biofilm formation by Listeria monocytogenes strains

When a microtitre plate assay was used to quantify biofilm production by Listeria monocytogenes strains following growth in Tryptone Soy Broth (TSB) for 48 h at 20 degrees C, 127 of 138 strains (92.0%) were classified as weak, 9 of 138 strains (6.5%) as moderate and only 2 of 138 strains (1.5%) as strong biofilm formers. The strains included environmental, animal, food (persistent and sporadic strains) and clinical isolates previously typed using esterase electrophoresis (ESE) and multi-locus enzyme electrophoresis (MEE). Strains from different sources produced similar quantities of biofilm, whereas biofilm production by ESE type II strains, irrespective of source, was greater than that observed for other ESE types. No correlation between MEE type and biofilm production was observed. A Petri dish assay which allowed parallel quantification and microscopic examination of biofilms was used to examine biofilm formation by selected L. monocytogenes strains during growth in TSB for 14 days at 20 degrees C. Results from these assays showed that following prolonged incubation, some L. monocytogenes strains categorized as weak biofilm formers by the 48 h microtitre assay, were able to form biofilms similar in terms of quantity and structure to those produced by strains classified as strong or medium biofilm formers. Results from 14-day Petri dish assays confirmed 48 h microtitre assays regarding greater biofilm production by ESE type II strains compared to other ESE types of L. monocytogenes. Biofilm production was similar for ESE type II persistent and sporadic food isolates but reduced for ESE type II clinical strains.     

Prevalence and biofilm-forming ability of Listeria monocytogenes in New Zealand mussel (Perna canaliculus) processing plants

Greenshell™ mussels are New Zealand’s largest seafood export species. Some export markets require compliance with ‘zero’ tolerance legislation for Listeria monocytogenes in 25 g of product. Even though individually quick frozen (IQF) mussel products are labeled ‘to be cooked’, and are not classified as ready-to-eat, some markets still require them to comply with the strict policy. Three mussel processing plants were assessed for the pattern of L. monocytogenes contamination on raw material, environment, food contact surfaces, and in the final product. Cultures (n = 101) obtained from an industrial Listeria monitoring program from August 2007 to June 2009 were characterized by serotyping and pulsed field gel electrophoresis. Using the crystal violet method, isolates were assessed for their ability to form biofilms. This work confirmed the presence of L. monocytogenes in raw and processed product, and the importance of cross-contamination from external and internal environments. Processing plants had L. monocytogenes pulsotypes that were detected more than once over 6 months. No correlation was found between biofilm-forming ability and persistent isolates. Two pulsotypes (including a persistent one), were previously isolated in human cases of listeriosis in New Zealand, but none of the pulsotypes matched those involved in international outbreaks.     

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

A real-time PCR assay was designed to detect a 162-bp fragment of the ssrA gene in Listeria monocytogenes. The specificity of the assay for L. monocytogenes was confirmed against a panel of 6 Listeria species and 26 other bacterial species. A detection limit of 1-10 genome equivalents was determined for the assay. Application of the assay in natural and artificially contaminated culture enriched foods, including soft cheese, meat, milk, vegetables and fish, enabled detection of 1-5 CFU L. monocytogenes per 25g/ml of food sample in 30h. The performance of the assay was compared with the Roche Diagnostics 'LightCycler foodproof Listeria monocytogenes Detection Kit'. Both methods detected L. monocytogenes in all artificially contaminated retail samples (n=27) and L. monocytogenes was not detected by either system in 27 natural retail food samples. The method developed in this study has the potential to enable the specific detection of L. monocytogenes in a variety of food types in a time-frame considerably faster than current standard methods. The potential of the ssrA gene as a nucleic acid diagnostic (NAD) target has been demonstrated in L. monocytogenes. We are currently developing NAD tests based on the ssrA gene for a range of common foodborne and clinically relevant bacterial pathogens.     

Reliability of Listeria monocytogenes Identification by Specific PCR Assessed by Phenotypic and Genotypic Techniques

This study evaluates the possibility of using polymerase chain reaction (PCR) for rapid identification of food-borne Listeria monocytogenes as an alternative to API Listeria system and estimates the incidence of API Listeria misidentifications in food-borne Listeria species. A total of 198 strains, 11 L. monocytogenes, 28 other Listeria species, and 159 food isolates were phenotypically and genotypically characterized by API Listeria profiles and randomly amplified polymorphic DNA (RAPD) profiles, respectively. They were also tested for PCR amplification using genus- and species-specific primers. Clustering analysis of phenotypic and genotypic data showed discrepancies in species identification of some isolates by API Listeria profiles. Their identities were confirmed by 16S rDNA sequencing, and thus, it was revealed that 33% of Listeria innocua and 19% of Listeria welshimeri were misidentified as L. monocytogenes by API Listeria profiles. Reliable identification of L. monocytogenes was obtained by LM1–LM2 specific primers which allowed PCR amplification only in reference strains and isolates previously identified as L. monocytogenes by RAPD and 16S rDNA sequence analysis. These results corroborate the suitability of specific PCR as a rapid and accurate test for the identification of L. monocytogenes, avoiding misidentification with other Listeria species commonly found in food products.     

Review--Persistence of Listeria monocytogenes in food industry equipment and premises

To understand why Listeria monocytogenes may persist in food industry equipment and premises, notably at low temperature, scientific studies have so far focused on adhesion potential, biofilm forming ability, resistance to desiccation, acid and heat, tolerance to increased sublethal concentration of disinfectants or resistance to lethal concentrations. Evidence from studies in processing plants shows that the factors associated with the presence of L. monocytogenes are those that favor growth. Interestingly, most conditions promoting bacterial growth were shown, in laboratory assays, to decrease adhesion of L. monocytogenes cells. Good growth conditions can be found in so-called harborage sites, i.e. shelters due to unhygienic design of equipment and premises or unhygienic or damaged materials. These sites are hard to eliminate. A conceptual model of persistence/no persistence based on the relative weight of growth vs. outcome of cleaning and disinfection is suggested. It shows that a minimum initial bacterial load is necessary for bacteria to persist in a harborage site and that when a low initial bacterial charge is applied, early cleaning and disinfection is the only way to avoid persistence. We conclude by proposing that there are no strains of L. monocytogenes with unique properties that lead to persistence, but harborage sites in food industry premises and equipment where L. monocytogenes can persist.     

Multiple-locus variable number of tandem repeat analysis (MLVA) of Listeria monocytogenes directly in food samples

Listeria monocytogenes is the etiologic agent of listeriosis responsible for severe and fatal infections in humans. Listeria contamination occurs quite often in a wide range of foods due to its ubiquitous nature. Isolates need to be characterized to a strain level for accurate diagnosis of Listeria infection, epidemiological studies, investigation of outbreaks and effective prevention and control of food-borne listeriosis. The purpose of this research was to evaluate the multiple-locus variable number of tandem repeat analysis (MLVA) for sub-typing L. monocytogenes isolates in pure cultures and in food matrices. Two multiplex PCR assays were formulated to amplify six specific loci using fluorescently-labeled primers; and the amplicons were analyzed by capillary electrophoresis. The MLVA method resulted in 34 unique DNA fingerprint patterns from 46 L. monocytogenes isolates of 10 serotypes which had 29 or 30 PFGE patterns with a single restriction enzyme and 34 AFLP patterns. The MLVA patterns of the 46 isolates remained unchanged in the presence of pre-enriched food matrices including sausage, ham, chicken, milk and lettuce. The MLVA method successfully typed L. monocytogenes strains spiked in cheese, roast beef, egg salad and vegetable samples after 48 h enrichment at the initial inoculation levels of 1-5 CFU per 25 g of food or higher. The limits of detection (typing) of the MLVA method were 10³-10⁴ CFU/mL of pre-enriched food broth when evaluated using post-spiked sausage, ham, chicken, milk and lettuce samples. The MLVA method was simple, highly discriminatory, and easy to perform with portable (numerical) results. To our knowledge, this is the first report that describes the application of the MLVA method directly to food samples and demonstrates the possibility to obtain rapid and accurate subtyping results before an isolate is obtained.     

Edible chitosan films on ready-to-eat roast beef for the control of Listeria monocytogenes

The use of chitosan as an edible film was evaluated for its antimicrobial activity against Listeria monocytogenes (LM) on the surface of ready-to-eat (RTE) roast beef. L. monocytogenes, decimally diluted to give an initial inoculation of >6.50logCFU/g, was inoculated onto the surface of RTE roast beef cubes, and air-dried. The samples were dipped into chitosan (high or low molecular weights) solutions dissolved with acetic or lactic acid at 0.5% (w/v) or 1% (w/v) then bagged and refrigerated at 4 degrees C. The bacterial counts were determined on days 0, 7, 14, 21, and 28. The samples were spread plated onto modified Oxford agar plates and incubated at 37 degrees C for 48h. An initial 6.50logCFU/g of L. monocytogenes inoculated onto the surface of the non-coated RTE roast beef increased too >10logCFU/g by day 28. On day 14, L. monocytogenes counts were significantly different for all the chitosan-coated samples from the control counts by 2-3logCFU/g and remained significantly different on day 28. Our results have shown that the acetic acid chitosan coating were more effective in reducing L. monocytogenes counts than the lactic acid chitosan coating. Our study indicated that chitosan coatings could be used to control L. monocytogenes on the surface of RTE roast beef.     

Growth potential of Listeria monocytogenes strains in mixed ready-to-eat salads

In this study, a microbiological challenge test in three artificially contaminated retail mixed mayonnaise-based ready-to-eat salads stored at refrigerator temperatures (3 °C and 7 °C) for 48 h was carried out. Shrimp-tomato salad, smoked ham salad and garlic cheese salad were separately contaminated by a suspension of particular Listeria monocytogenes strains. The number of L. monocytogenes, Enterobacteriaceae, staphylococci and total plate count (CFU/g) was determined. Listeria monocytogenes growth potential in the salads was calculated and evaluated.A significant increase in total plate count and L. monocytogenes count throughout storage of all three investigated salads was found. Enterobacteriaceae levels were high at the beginning in all salads but significantly (p < 0.05) decreased throughout the experiment depending on the temperature.All investigated L. monocytogenes strains demonstrated growth at both temperatures but expressed different growth potential. Especially garlic cheese salad and smoked ham salad were able to support the growth of Listeria. Shrimp-tomato salad supported growth the least. The growth potential increased with the increasing temperature and exceeded 0.5 log₁₀ CFU/g in many cases. If the potential for growth is > 0.5 log₁₀ CFU/g, food products can potentially endanger human health. Reference strain (ATCC 7644) showed the least growth potential almost in all cases in comparison with strains isolated from frozen pollock loins and from thermally treated specialty sausage containing preservatives. To eliminate the occurrence of microbiological risks, the shelf-life of the studied salads was estimated.     

Molecular Subtyping and Tracking of Listeria monocytogenes in Latin-Style Fresh-Cheese Processing Plants

Latin-style fresh cheeses, which have been linked to at least 2 human listeriosis outbreaks in the United States, are considered to be high-risk foods for Listeria monocytogenes contamination. We evaluated L. monocytogenes contamination patterns in 3 Latin-style fresh-cheese processing plants to gain a better understanding of L. monocytogenes contamination sources in the manufacture of these cheeses. Over a 6-mo period, 246 environmental samples were collected and analyzed for L. monocytogenes using both the Food and Drug Administration (FDA) method and the Biosynth L. monocytogenes detection system (LMDS). Finished cheese samples from the same plants (n = 111) were also analyzed by the FDA method, which was modified to include L. monocytogenes plating medium (LMPM) and the L. monocytogenes confirmatory plating medium (LMCM) used in the LMDS method. Listeria monocytogenes was detected in 6.3% of cheese and 11.0% of environmental samples. Crates, drains, and floor samples showed the highest contamination rates, with 55.6, 30.0, and 20.6% L. monocytogenes positive samples, respectively. Finished products and food contact surfaces were positive in only one plant. The FDA method showed a higher sensitivity than the LMDS method for detection of L. monocytogenes from environmental samples. The addition of LMPM and LMCM media did not further enhance the performance of the FDA method for L. monocytogenes detection from finished products. Molecular subtyping (PCR-based allelic analysis of the virulence genes actA and hly and automated ribotyping) was used to track contamination patterns. Ribotype DUP-1044A, which had previously been linked to a 1998 multistate human listeriosis outbreak in the United States, was the most commonly identified subtype (20/36 isolates) and was isolated from 2 plants. This ribotype was persistent and widespread in one factory, where it was also responsible for the contamination of finished products. We hypothesize that this ribotype may represent a clonal group with a specific ability to persist in food processing environments. While previous listeriosis outbreaks were linked to Latin-style fresh cheeses made from unpasteurized milk, the presence of this organism in pasteurized cheese products illustrates that persistent environmental contamination also represents an important source of finished product contamination.     

2019年北京市食源性单增李斯特菌的分子特征和耐药性研究

研究2019年北京市食源性单增李斯特菌进行分子特征和耐药性。 方法 使用多重PCR进行血清群分型、对分离的56株菌进行脉冲场凝胶电泳（pulsed field gel electrophoresis,PFGE）、多位点序列分析（multilocus sequence typing, MLST）,使用微量肉汤法检测菌株的耐药性。 结果 56株单增李斯特菌中有28株分离自冷锅串串和中式凉拌菜,1/2a, 3a是主要的血清群。56株菌共分为29个PFGE带型、14个ST型,ST9、ST5、ST8、ST121是其主要的ST型别。2株菌对环丙沙星耐药,1株菌对四环素耐药。结论 冷锅串串和中式凉拌菜是单增李斯特菌污染的高危食品。北京市食品来源单增李斯特菌的ST型别与国内其他地方食品来源菌株一致,与北京市临床分离株一致。     

Longitudinal monitoring of Listeria monocytogenes contamination patterns in a farmstead dairy processing facility

Contamination of dairy products with Listeria monocytogenes is a concern because multiple human listeriosis outbreaks have been linked to contaminated cheese and dairy products. Dairy production on farmstead operations may be a particular concern because L. monocytogenes is also an animal pathogen that can be shed by ruminants with and without clinical symptoms; physical proximity between production animal and dairy processing facilities may thus provide a higher risk for introduction of L. monocytogenes into the dairy production process. To better understand the risks of L. monocytogenes contamination associated with farmstead dairy production, samples from a farmstead dairy processing operation and the milking barn of the directly adjacent dairy sheep operation were tested for L. monocytogenes over a 3-yr period. Prevalence of L. monocytogenes for samples collected on the farm (n = 85) and the dairy production facility (n = 674) was 9.4 and 2.7%, respectively. Molecular subtyping using automated EcoRI ribotyping of L. monocytogenes isolates revealed that distinct subtypes were associated with the dairy production facility and the farm's milking parlor. Although a total of 5 and 4 different ribotypes were identified among isolates obtained from the dairy production facility and the milking parlor, respectively, only 1 ribotype (DUP-1030A) was isolated from both. Different ribotypes were predominant among isolates from the dairy production facility (ribotype DUP-1052A, representing 15 of 18 isolates) and the farm's milking parlor (ribotype DUP-1039A, representing 4 of 8 isolates); each of these ribotypes appeared to persist over time in the respective area. Our data support that i) in farmstead dairy processing facilities, L. monocytogenes present on the farm can largely be prevented from being introduced into the processing facility; and ii) L. monocytogenes can persist on farm and in processing areas, providing a potential high-risk source for contamination. Preventing cross contamination between dairy production and processing facilities and control of persistent L. monocytogenes are thus critical to assuring the microbial safety of farmstead dairy products.     

Initial adhesion of Listeria monocytogenes to solid surfaces under liquid flow

Some strains of the food borne pathogen Listeria monocytogenes persist in food processing environments. The exact reason behind this phenomenon is not known, but strain differences in the ability to adhere to solid surfaces could offer an explanation. In the present work, initial adhesion of nine strains of L. monocytogenes was investigated under liquid flow at two levels of shear stress on six different surfaces using a flow chamber set-up with microscopy measurements. The surfaces tested were glass and PVC, and glass coated with beef extract, casein, and homogenised and unhomogenised milk. In addition, the effect of prior environmental stress (5% NaCl, low nutrient availability) on initial adhesion was investigated. The hydrophobicity of the investigated surfaces was determined by contact angle measurements and the surface properties of the investigated L. monocytogenes strains were determined using Microbial Adhesion To Solvents (MATS). All surfaces with the exception of PVC were found to be hydrophilic. Strain differences were found to significantly influence the initial adhesion rate (IAR) of all nine strains to all the surfaces (p < 0.05) at both low and high shear stress. Furthermore, there was a significant effect of the surfaces tested (p < 0.05) in the adhesion ability of almost all strains. The IAR was affected by flow rate (shear stress) as seen by a decrease in adhesion at high shear stress for most strains. A significant effect of interactions between strain-surface and strain-shear stress (p < 0.001) was observed but not of interactions between surface-shear stress. No correlation between surface hydrophobicity and IAR was observed. Addition of 5% NaCl during propagation resulted in a decrease in IAR whilst propagation in low nutrient media caused an increase indicating a general change in surface characteristics under these conditions. Known persisting strains did not display general better adherence.     

单核细胞增生李斯特氏菌的PCR检测方法

研究比较了裂解法、热煮沸法、试验盒法提取单增李斯特氏菌DNA的效果 ,认为Promega试剂盒法提取的DNA ,得率高、纯度好。同时根据单增李斯特氏菌的毒力相关基因的 5对引物 ,进行了引物的特异性研究 ,结果发现inlA引物、inlB引物、plcA引物特异性好 ,可以用于食品中单增李斯特氏菌的检测     

Staphylococcus aureus and Listeria monocytogenes in Norwegian raw milk cheese production

The aim of this study was to survey the presence of Staphylococcus aureus and Listeria monocytogenes during the cheese making process in small-scale raw milk cheese production in Norway.The prevalence of S. aureus in bovine and caprine raw milk samples was 47.3% and 98.8%, respectively. An increase in contamination during the first 2-3 h resulted in a 73.6% prevalence of contamination in the bovine curd, and 23 out of 38 S. aureus-negative bovine milk samples gave rise to S. aureus-positive curds. The highest contamination levels of S. aureus were reached in both caprine and bovine cheese after 5-6 h (after the first pressing). There was no contamination of L. monocytogenes in caprine cheeses and only one (1.4%) contaminated bovine cheese.This work has increased our knowledge about S. aureus and L. monocytogenes contamination during the process of raw milk cheese production and gives an account of the hygiene status during the manufacture of Norwegian raw milk cheeses.     

Reduction of counts of Listeria monocytogenes in cheese by means of high hydrostatic pressure

Inactivation of Listeria monocytogenes (strains NCTC 11994 and Scott A) was evaluated in model cheeses submitted to 10 min HHP treatments of 300, 400 or 500 MPa at 5 or 20 degrees C. Counts were measured immediately after high hydrostatic pressure (HHP) treatment (day 1) and after 2, 15 and 30 days of storage at 8 degrees C. Both strains behaved significantly different after 400 and 500 MPa, being NCTC 11994 more sensitive. Scarce differences were found among final values at both HHP treatment temperatures. Initial reductions (log cfu/g) for 400 MPa at 20 degrees C were 2.9 +/- 0.2 for strain NCTC 11994 and 1.5 +/- 0.2 for Scott A. They reached after 30-day storage 5.3 +/- 0.2 and 4.6 +/- 0.4 log cfu/g for NCTC 11994 and Scott A, respectively. For 500 MPa treatments, day-1 reductions of both strains were around 5-log cfu/g, and counts fell below quantification limit after 30 days. Injured cells (around 0.8-log cfu/g) were mostly observed in 400 MPa treated samples on days 1 and 2. Starter cells suffered higher inactivation and injury. For 20 degrees C treatments, its final counts (log cfu/g) at 300, 400 and 500 MPa were: 8.5 +/- 0.2, 5.4 +/- 0.3 and 2.5 +/- 0.1, respectively. These figures evidence the HHP potential to improve safety of cheese products.     

Development of an Internal Amplification Control Using Multiplex PCR for the Detection of Listeria monocytogenes in Food Products

The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection, it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/μL. Following an enrichment procedure, the minimum number of organisms detected in a spiked food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased the reliability of the PCR for food diagnostic purposes.