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     

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.     

Characterization of the novel Listeria monocytogenes PCR serogrouping profile IVb-v1

The World Health Organization Collaborating Centre for Listeria (WHOCCL) has developed in 2004 a multiplex PCR assay that separates the 4 major Listeria monocytogenes serovars (1/2a, 1/2b, 1/2c, and 4b) into distinct PCR serogroups. A new PCR profile has been recently identified, constituted of amplified DNA fragments of prs, ORF2819, ORF2110 and lmo0737. Here we characterize 22 L. monocytogenes isolates of the WHOCCL collection with this PCR IVb variant 1 (IVb-v1) profile. The 22 isolates belong to the clinically predominant serovar 4b, exhibit 6 distinct pulsed-field gel electrophoresis ApaI/AscI combined profiles, and belong to 2 unrelated multilocus sequence types, indicating that the novel profile does not correspond to a recent clonal emergence. We have updated the WHOCCL serogroup-related PCR typing scheme to include this new profile.     

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.     

Control of Listeria monocytogenes growth in a ready-to-eat poultry product using a bacteriophage

A bacteriophage (phage) that infected strains of the species Listeria monocytogenes as well as Listeria ivanovii and Listeria welshimeri, but not Listeria grayi or Listeria innocua, was isolated from sheep faeces. The phage had a contractile tail and an icosohedral head indicating that it was a myovirus, and was morphologically similar to phage A511. At 30 °C, phages added at 5.2 × 10⁷ PFU ml⁻¹ prevented the growth in broth of L. monocytogenes present at approximately twice this concentration for 7 h, but re-growth occurred such that the concentration after 24 h incubation was similar in both control and phage-treated cultures. At the same temperature, but on the surface of vacuum-packed ready-to-eat chicken breast roll, there was an immediate 2.5 log₁₀ CFU cm⁻² reduction in pathogen concentration following addition of phages and then re-growth. However, at a temperature reflecting that at which a chilled food might be held (5°C), this re-growth was prevented over 21 days incubation. The data suggest a dose-dependent rapid reduction in pathogen concentration followed by no continued phage-mediated effect. These results, alongside other published data, indicate that a high concentration of phages per unit area is required to ensure significant inactivation of target pathogens on food surfaces.     

Improved Sample Preparation for Real-Time PCR Detection of Listeria monocytogenes in Hot-Smoked Salmon using Filtering and Immunomagnetic Separation Techniques

This work evaluated the application of filtration and immunomagnetic separation (IMS) as sample pretreatments for use in combination with real-time polymerase chain reaction (PCR) to detect and quantify Listeria monocytogenes in hot-smoked salmon. Salmon was artificially inoculated with L. monocytogenes at levels ranging from 8 × 10⁰ to 8 × 10⁵ cfu/g of sample, and homogenates obtained from these samples were filtered to recover bacterial cells without a pre-enrichment step. High recovery of bacterial cells was achieved using standard coffee filters. IMS significantly reduced the co-extraction of PCR inhibitors present in the samples to increase the assay sensitivity with regression line parameters applicable for quantification. The limit of detection and quantification were equal to 2 × 10¹–4 × 10¹ and 2 × 10² cfu/g of sample, respectively. The entire detection procedure could be completed within 3.5 h. This study demonstrated that coupling filtration and IMS with real-time PCR has contributed to improve the sensitivity of L. monocytogenes detection from hot-smoked salmon.     

Class I/Class IIa bacteriocin cross-resistance phenomenon in Listeria monocytogenes

Variants resistant to nisin A (vA), nisin Z (vZ), pediocin PA-1 (vP), divergicin M35 (vD) and to bacteriocin-like compounds produced by Bifidobacterium thermophilum subsp. infantis RBL67 (vB) were developed from Listeria monocytogenes LSD530. Lactic acid production, specific growth rate, potassium ion efflux, susceptibility to 13 antibiotics, cell-envelope fatty acid composition and bacteriocin cross-resistance were evaluated. Lactic acid production decreased to 75% or less of that by strain LSD530 for vP, vD and vB and to 20% or less for vA and vZ. Specific growth rates also decreased for all five variants. Acquired resistance to nisin A or Z increased resistance to pediocin and divergicin while vD showed increased resistance to nisin Z but decreased resistance to nisin A and vP exhibited increased resistance to nisin Z, pediocin and divergicin but decreased resistance to nisin A. Acquired bacteriocin resistance generally decreased antibiotic sensitivity, particularly to ampicillin, chloramphenicol, erythromycin and tetracycline. Palmitic acid (C(16:0)) in the cell wall fraction of all variants was significantly higher than in strain LSD530, accounting for 18%, 43%, 32%, 26%, 53% and 44% of the total fatty acids for LSD530, vP, vD, vB, vA, and vZ, respectively. The relationship between the acquisition of bacteriocin resistance, cross-resistance and pathogenicity of Listeria monocytogenes should be studied.     

Prevalence and level of Listeria monocytogenes and other Listeria species in retail pre-packaged mixed vegetable salads in the UK

As part of the European Commission (EC) co-ordinated programme for 2005, a study of pre-packaged ready-to-eat (RTE) mixed salads containing meat or seafood ingredients from retail premises was undertaken in the UK to determine the frequency and level of Listeria monocytogenes in these products. Almost all (99.8%; 2682/2686) samples were of satisfactory/acceptable microbiological quality. Two (0.1%) samples exceeded EC legal food safety criteria due to the presence of L. monocytogenes in excess of 100 cfu g(-1) (1.7 x 10(2), 9.9 x 10(2)cfu g(-1)) while another two (0.1%) were unsatisfactory due to L. welshimeri levels over 100 cfu g(-1) (1.2 x 10(3), 6.0 x 10(3) cfu g(-1)). Overall contamination of Listeria spp. and L. monocytogenes found in samples of mixed salads in the UK was 10.8% and 4.8%, respectively. Almost twice as many salad samples with meat ingredients were contaminated with Listeria spp. and L. monocytogenes (14.7% and 6.0%, respectively) compared to samples with seafood ingredients (7.4% and 3.8%, respectively). Pre-packaged mixed salads were contaminated with Listeria spp. and L. monocytogenes more frequently when: collected from sandwich shops; not packaged on the premises; stored or displayed above 8 degrees C. This study demonstrates that the control of L. monocytogenes in food manufacturing and at retail sale is essential in order to minimize the potential for this bacterium to be present in mixed salads at the point of consumption at levels hazardous to health.     

免疫捕捉PCR和直接PCR技术检测单核细胞增生性李斯特菌比较研究

将免疫学技术与分子生物学技术相结合,建立了免疫捕捉PCR(IC-PCR),并与传统的直接PCR(Direct-PCR)进行比较。结果显示:纯菌液中IC-PCR检测灵敏度(104CFU/mL)是Direct-PCR灵敏度(105CFU/mL)的10倍;模拟带菌实验结果表明:IC-PCR最低可检测到5×101个细菌/PCR反应体系(即104CFU/mL),检测限比Direct-PCR(5×103个细菌/PCR反应体系即106CFU/mL)高100倍;特异性实验、干扰实验结果表明IC-PCR可特异检测单核细胞增生性李斯特菌(Listeria monaytogenes,LM),而和18株其他常见食源性致病菌无交叉反应。IC-PCR具有快速、经济、灵敏、特异等优点,是一种适合食品安全监管部门、食品企业的LM快速检测方法。     

Genetic variation of Listeria monocytogenes isolates from domestic and imported foods in Japan

Phylogenetic analyses were carried out on a total of 118 Listeria monocytogenes isolates from foods or food processing environments, and 7 isolates from listeriosis patients in Japan to evaluate the genetic variation in the pathogen in this country. Isolates of serotypes 1/2a, 1/2b and 4b were mainly examined to assess the risk of exposure of humans to L. monocytogenes from foods in Japan. The nucleotide sequences of the part of the iap gene that contains the region encoding the threonine-asparagine repeat units were determined in order to construct phylogenetic trees of the isolates investigated. A phylogram showed high genetic diversity among lineage 2 isolates, while the lineage 1 isolates showed clonal characteristics. The results of the genetic analyses suggested the presence of rare putative lineage 3 isolates and epidemic clone I (ECI) isolates in foods in Japan. The results showed that ECI was also isolated from listeriosis patients. The genetic variation in L. monocytogenes in Japan reported here suggests the necessity of monitoring the pathogen in foods and environments in addition to surveillance of listeriosis patients.     

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     

Inactivation of Listeria monocytogenes on agar and processed meat surfaces by atmospheric pressure plasma jets

An apparatus for generating atmospheric pressure plasma (APP) jet was used to investigate the inactivation of Listeria monocytogenes on the surface of agar plates and slices of cooked chicken breast and ham. He, N₂ (both 7 L/min), and mixtures of each with O₂ (0.07 L/min) were used to produce the plasma jets. After treatment for 2 min with APP jets of He, He + O₂, N₂, or N₂ + O₂, the numbers of L. monocytogenes on agar plates were reduced by 0.87, 4.19, 4.26, and 7.59 log units, respectively. Similar treatments reduced the L. monocytogenes inoculated onto sliced chicken breast and ham by 1.37 to 4.73 and 1.94 to 6.52 log units, respectively, according to the input gas used with the N₂ + O₂ mixture being the most effective. Most APP jets reduced the numbers of aerobic bacteria on the meat surfaces to <10² CFU/g, and the numbers remained below that level of detection after storage at 10 °C for 7 days. The results indicate that APP jets are effective for the inactivation of L. monocytogenes on sliced meats and for prolonging the shelf-life of such foods.     

Control of meatborne Listeria monocytogenes and Brochothrix thermosphacta by a bacteriocinogenic Brochothrix campestris ATCC 43754

The effect of a bacteriocinogenic Brochothrix campestris ATCC 43754 upon the growth of Brochothrix thermosphacta and a 4 strain mixture of Listeria monocytogenes was determined in All Purpose Tween (APT) broth and on pork adipose tissue discs at 4 degrees C. Inocula were prepared to give initial numbers of B. campestris of 6-7 log cfu/ml or cm(2) and 3-4 log cfu/ml or cm(2) of B. thermosphacta and L. monocytogenes. Adipose tissue discs were evaluated by a sensory panel to determine the intensity and acceptability of any off-odours produced during the growth of B. campestris. During co-culture in APT broth with B. campestris the growth of B. thermosphacta or L. monocytogenes was 4 log cycles less than growth in its absence. B. campestris showed limited growth on inoculated pork adipose tissue, increasing from initial numbers of about 6 log cfu/cm(2) to a maximum of 7 log cfu/cm(2) within 7d. B. campestris at numbers of 7 log cfu/cm(2) produced slight off-odours but these were not perceived by the panel as unacceptable. When co-inoculated on adipose tissue discs with B. campestris the numbers of B. thermosphacta or L. monocytogenes was limited to about 2-3 log units less than the numbers attained in its absence. B. campestris ATCC 43754 may be useful for meat preservation because it can inhibit B. thermosphacta and L. monocytogenes in situ while producing little change in the sensory properties of the product.     

EMA结合实时荧光PCR方法检测单核细胞增生李斯特氏菌

建立叠氮溴化乙锭（EMA）结合实时荧光PCR（qPCR）方法检测单核细胞增生李斯特氏菌（Listeria monocytogenes）活菌。以李斯特溶血素O（LLO）基因hly设计引物、TaqMan探针,用李斯特属典型菌、沙门氏菌等56株致病菌株验证特异性,不同质量浓度EMA处理进行qPCR检测。尝试脱氧胆酸钠溶液（SD）强化抑制效果,73份不同的人工污染食品、环境样本（卤鸡肉、牛奶、肉馅、垃圾渗滤液）测试实用性。结果表明,引物探针准确检测L. monocytogenes,对其他菌株无特异性扩增。EMA最适质量浓度2.5 μg/mL,经过15 min光激活与死菌DNA共价结合明显抑制了扩增,方法检出限为150 CFU/mL。SD处理L. monocytogenes活菌Ct值增加。与传统培养法比较,EMA-qPCR方法检测100 %准确,操作简单、省时高效,在食品、环境方面应用前景广阔。     

Bioprotective Leuconostoc strains against Listeria monocytogenes in fresh fruits and vegetables

Ten Leuconostoc mesenteroides and one Ln. citreum strains isolated from fresh fruit and vegetables were tested for their antagonistic capacity against Listeria monocytogenes. Genetic differences among strains were analyzed by Random Amplified Polymorphic DNA (RAPD). All the isolates clustered together and differed from the type strain Ln. mesenteroides ATCC 8293 as well as from Ln. fallax and Ln. citreum. Organic acids, hydrogen peroxide and bacteriocins were detected as main inhibition mechanisms. Characterization of culture supernatants from the bacteriocinogenic strains, CM135 and CM160 revealed a high resistance of antibacterial activity to temperature and pH, and a bactericidal mode of action against L. monocytogenes. Produced bacteriocins belonged to the Class IIa and sequencing of genes showed complete homology with mesentericin Y105. A study of the effect of the relative dose of pathogen and LAB on control of L. monocytogenes in wounds of Golden Delicious apples and Iceberg lettuce leaf cuts was performed. A comparison of the dose of bioprotective strain needed for a ten fold reduction of the viable pathogen concentration (ED(90)) revealed that strain CM160 was the most effective against L. monocytogenes. ED(90) values varied from 1.3.10(4) to 5.0.10(5) cfu.g(-1) or wound, at ranges of pathogen levels from 1.0.10(3) to 5.0.10(4) cfu.g(-1) of lettuce or wound of apple. The efficiency of the strains was also calculated as the ratio of the ED(90) value to the pathogen dose inoculated. The lowest ratio was found for strain CM160 at 5 to 50 cells of LAB per cell of pathogen. The strain offers potential application for prevention of the presence of L. monocytogenes in fresh fruit and vegetables.     

食品中单核细胞增生性李斯特氏菌PCR快速检测

通过扩增hly基因建立检测单核细胞增生性李斯特氏菌(Lm)的PCR方法.该方法具有较强的特异性,35株经传统方法鉴定的菌株PCR结果均为阳性,而其他三种同属异种菌,包括英诺克李斯特氏菌、绵羊李斯特氏菌和威尔斯李斯特氏菌及非李斯特氏菌均未扩增出特异性的片段.PCR方法对上Lm纯培养物的最低检测限为7.3CFU/μl,对模拟污染的生猪肉和蔬菜的检测低限为4CFU/g,牛奶为4CFU/ml.应用该方法对285份食品样品检测,17份样品Lm呈阳性,结果与常规的分离培养方法完全一致.该种方法具有敏感、特异、快速及准确的优点,可用于食品中Lm的快速检测.     

Combined effect of packaging atmosphere and storage temperature on growth of Listeria monocytogenes on ready-to-eat shrimp

Cooked, peeled, and deveined shrimp were inoculated with a 5 strain mixture of Listeria monocytogenes and packaged in air, vacuum, and a 100% carbon dioxide modified atmosphere. The packaged shrimp were then stored at 3, 7, and 12 degrees C for 15 days to monitor the growth of L. monocytogenes and psychrotrophic bacteria. Uninoculated shrimp were also subjected to sensory evaluation by a trained panel to measure odor and appearance over the storage period. Results demonstrated that shrimp packaged in CO(2) and stored at 3 degrees C did not permit growth of L. monocytogenes during the 15-day storage period, while all other packaging/temperature combinations allowed for multiplication of the bacterium. Carbon dioxide packaging also resulted in the slowest growth of psychrotrophic bacteria and resulted in shrimp having acceptable sensory odor and appearance scores at the end of storage. When strict temperature control is difficult, such as during processing, transportation, retail display, or home use, additional antimicrobial hurdles may be necessary to ensure safety.     

数字PCR定量检测食品中单核细胞增生李斯特氏菌方法的研究

目的建立微滴式数字PCR技术(droplet digital PCR,ddPCR)定量检测食品中单核细胞增生李斯特氏菌的分析方法。方法选择基因hly A为靶序列,设计PCR扩增引物和Taq Man探针,优化反应条件和反应体系,通过细菌分离和dd PCR方法对靶标基因的检测特异性、灵敏度和重复性进行实验,并对定量结果进行分析。结果 ddPCR反应中的最佳探针浓度为5 pmol/μL,特异性好,检出限为(3.6±0.1)copies/20μL,重复性实验良好,标准偏差为0.067%。ddPCR的拷贝数(copy number)与细菌密度(colony forming units,CFU/mL)形成了较好的线性关系。结论本研究建立dd PCR的拷贝数和菌液密度或菌落数的线性关系,可以为单核细胞增生李斯特氏菌的快速定量检测提供参考。     

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.