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.     

Outbreak of clinical listeriosis in sheep: evaluation from possible contamination routes from feed to raw produce and humans

We report the results of clinical and microbiological investigations on Listeria monocytogenes infections in a flock of 55 sheep and describe the implications for the safety of the raw milk and raw-milk cheeses produced in the on-farm dairy. The outbreak was caused by feeding grass silage, which was contaminated with 5 log10 CFU L. monocytogenes/g. Clinically, although having been fed from the same batch of silage, abortive (nine ewes), encephalitic (one ewe) and septicaemic (four ewes) forms of listeriosis were observed during the outbreak phase. As the starting point of feeding the contaminated silage was known we could calculate an incubation period of 18+/-2 and 26 days for the abortive and the encephalitic form of listeriosis, respectively. Pathologically, the septicaemic cases suffered from Listeria accumulation at comparable numbers in visceral organs but not in the brain. Only a single ewe developed central nervous symptoms and a rhomb-encephalitis was immunohistologically confirmed. In this case the infection proceeded from the nasal mucosa into the brain, with no infections of the liver, spleen and other visceral organs. Sampling of the cheese production chain, the farm environment and the persons living at the farm revealed the exposure of a farm-worker to an isolate genetically indistinguishable from the outbreak clone, obviously through the consumption of faecally contaminated bovine raw milk. The cheese under processing was free of Listeria because, as a result of intensive consultations, the farmer ensured a proper acidification of the cheese. The epidemiological findings suggest that food safety matters should be assessed in any case where infection of food-producing animals with potential human pathogens is observed.     

李斯特氏菌因子血清的制备及食品检测的免疫磁性分离-PCR(MIPA)方法的建立

首先进行了李斯特氏菌因子血清的研制,制备出了可对所有李斯特氏菌分型的 １５ 个 Ｏ 抗原因子和４ 个 Ｈ 抗原因子的因子血清。利用复合因子血清的多克隆抗体包被磁性球,对食品中的单核细胞增多性李斯特氏菌进行免疫磁性分离,并与 Ｐ Ｃ Ｒ 方法相结合,建立了检测食品中单核细胞增多性李斯特氏菌的 Ｍ Ｉ Ｐ Ａ方法（免疫磁性分离—聚合酶链反应方法,ｍ ａｇｎ ｅｔｉｃ ｉｍ ｍ ｕｎｏｐｏｌｙｍ ｅｒａｓｅ ｃｈａｉｎ ｒｅａｃｔｉｏｎ ａｓｓａｙ）。对菌液、模拟样品的检测表明,本方法能够有效地克服食品基质、培养基成分和杂菌对 Ｐ Ｃ Ｒ 检验的干扰作用。食品样品在 Ｅ Ｂ增菌液中增菌 １２ ｈ 后,检测的敏感度达 ５ Ｃ Ｆ Ｕ／ｍ Ｌ,可以在 ２０ ｈ 内完成检测。本方法对实际食品样品的检测结果,与国家标准检验方法检测的结果一致。     

动物性食品中单核白细胞增多症李氏菌的检测

比较了4种增菌液和3种分离琼脂对动物性食品中单核白细胞增多症李氏菌的检测效果,以蛋白陈增菌液和胰蛋白陈琼脂为最佳.用这种方法检测了市售猪肉、分割鸡肉、鲜牛奶、奶皮、奶酪和奶豆腐中的单核白细胞增多症李氏菌,其检出率分别为6.7%,5.45%,5.0%,7.89%,27.03%和58.97%.     

Survival of Listeria monocytogenes in cheese made of unpasteurized goat milk

Food-born infections with Listeria monocytogenes have been reported during recent years, cheese being mentioned as one of the foods responsible. A classical opinion is that cheese represents a very inhospitable environment for pathogens due to antagonism by the starter culture of lactic-acid-producing organisms. In order to study the survival of L. monocytogenes in goat cheese, cheeses were made with the addition of L. monocytogenes cultures. The maximum survival time for L. monocytogenes was 18 weeks in two of the cheeses. It is concluded that L. monocytogenes has the ability to survive in semi-soft cheese made of unpasteurized goat milk during normal curing (2–3 months).     

应用环介导等温扩增技术快速检测单核细胞增生李斯特菌的研究

目的建立环介导等温扩增技术快速检测单核细胞增生李斯特菌。方法根据单核细胞增生李斯特菌(LM)hlyA基因序列中的保守区域,采用在线引物设计软件Primer Explorer4.0进行设计,获得一套特异性的环介导等温扩增(LAMP)引物,对单核细胞增生李斯特菌hlyA基因进行LAMP扩增,并与常规PCR方法进行比较。结果建立的LAMP方法能成功扩增出梯形条带,LAMP检测单核细胞增生李斯特菌纯培养物和人工染菌的灵敏度为5.44×102cfu/mL,而对照PCR检测的灵敏度为5.44×104cfu/mL。对10株细菌进行LAMP扩增,仅单核细胞增生李斯特菌得到阳性结果。从DNA提取到报告结果,耗时仅1h。结论 LAMP检测单核细胞增生李斯特菌灵敏度高,特异性强,耗时短,方法简便,有望发展成为快速检测食品中单核细胞增生李斯特菌的有效手段。     

Optimization of a culture technique for the isolation of Listeria monocytogenes from faecal samples

A culture technique employing cold enrichment at 4 degrees C followed by selective enrichment and plating at higher temperatures (30 degrees C) was used to isolate Listeria monocytogenes from faecal samples. The samples were held at 4 degrees C for 15 weeks and cultured weekly to assess the sensitivity of the culture after cold storage for different lengths of time. No media, Listeria selective enrichment broth (LSEB), nutrient broth (NB) and saline were used as cold storage medium. Cold storage increased the frequency of Listeria positive samples. The sensitivity of the culture for Listeria spp. and L. monocytogenes was 72 and 94%, and 56 and 61% after third and seventh week of cold storage, respectively. When the results of third and seventh week of cold storage were combined, the sensitivity was 100% for Listeria spp. and 94% for L. monocytogenes. LSEB and NB as storage medium increased Listeria positive samples after the first week of cold storage but did not maintain the increase thereafter while saline had an adverse effect on the growth of the bacteria. However, samples held in no media in a pilot study involving monthly sampling of a herd revealed better results. Detection limit of the culture media was also investigated. The lowest concentration detected by culture media was 3.17 organisms/ml. This was seven organisms/g for known Listeria positive sample. The faecal samples spiked with 10-fold dilutions of L. monocytogenes and held at 4 degrees C revealed that the sample spiked with 3.17 x 10-1 cfu/ml organisms resulted in growth after the second week of cold storage. The results suggest that the culture technique employing cold enrichment followed by selective enrichment and plating is more sensitive, the storage of faecal samples in no media when compared with the samples in storage medium, LSEB, NB and saline, during cold enrichment is a better application and culture of faeces, immediately after collection, at third and seventh week of cold enrichment produce more satisfactory results.     

A direct plating method for monitoring the contamination of Listeria monocytogenes in silage.

Twenty-two silage samples were analyzed for the presence of L. monocytogenes using five Listeria selective plating media, with and without previous selective enrichment step. L. monocytogenes was recovered from 3 samples by both procedures, but direct plating allowed the quantification of Listeria population. Two of these positive samples were implicated in outbreaks of listeriosis in sheep; the L. monocytogenes population in these samples was about 10(6) cells/g. The L. monocytogenes population in the other positive sample was 10(3) cells/g. Direct isolation of L. monocytogenes was only possible from LPM, PALCAM and LSAMm media. MOX and LSM media were not selective enough to allow direct Listeria isolation. In our hands, LSAMm was the most suitable plating medium for the direct isolation and specific quantification of L. monocytogenes from silage employing a red blood cells overlay technique.     

The occurrence of Listeria in the production of processed meat, salami and mettwurst

In six Swiss meat-processing plants 206 samples of cured and air-dried beef (Bündnerfleisch), salami and Mettwurst were analyzed for the presence of Listeria spp. Samples were taken during the fabrication, fermentation and drying of the products. Out of 44.7% of all samples Listeria spp. could be detected. 6.8% turned out to be L. monocytogenes, 37.4% L. innocua and 0.5% L. seeligeri. Listeria spp. were found in all production stages of the tested foods. The concentration of L. monocytogenes was always less than or equal to 20 MPN/g. 86% of the isolated strains formed part of the serogroup 1/2 and 14% of the serogroup 4. Listeria spp. could only be found on the surface of Bündnerfleisch. Both, L. monocytogenes and L. innocua were able to survive the maturation process of salami, even when the initial concentration was very low. The ripening was more often survived by L. innocua than by L. monocytogenes. It appeared that Mettwurst had the highest contamination rate of Listeria spp. (94.4%), followed by salami (46.7%) and Bündnerfleisch (23.1%). The corresponding proportions for L. monocytogenes were 8.0% (salami), 5.8% (Bündnerfleisch) and 0% (Mettwurst). Listeria spp. positive samples were found in every examined plant, L. monocytogenes in five of therm. The Listeria spp. contamination rates moved from 10.0% to 86.2%, those of L. monocytogenes from 0% to 12.1%.     

Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil: occurrence and interference of indigenous microbiota in their isolation and development

This study aimed to verify the occurrence of Listeria monocytogenes and Salmonella spp. in raw milk produced in Brazil. On account of the poor microbiological quality of this product, possible interference from the indigenous microbiota in these pathogens was also evaluated. Two-hundred and ten raw milk samples were collected in four important milk-producing areas in Brazil, tested for L. monocytogenes and Salmonella spp. presence, and for enumeration of indicator microorganisms: mesophilic aerobes, total coliforms and Escherichia coli. The interference of the indigenous microbiota in the isolation procedures was also tested, as well the frequency of naturally occurring raw milk strains with antagonistic activity against both pathogens. The pathogens were not isolated in any raw milk sample, but poor microbiological quality was confirmed by the high levels of indicator microorganisms. When present at high levels, the indigenous microbiota generated an evident interference in the methodologies of L. monocytogenes and Salmonella spp. isolation, mainly when the pathogens appeared at low levels. Three-hundred and sixty raw milk strains were tested for antagonistic activity against both pathogens, and 91 (25.3%) showed inhibitory activity against L. monocytogenes and 33 (9.2%) against Salmonella spp. The majority of the antagonistic strains were identified as Lactic Acid Bacteria species, mainly Lactococcus lactis subsp. lactis and Enterococcus faecium, known by antimicrobial substance production.     

Determination of the minimum enrichment time for the qualitative detection of Listeria monocytogenes in minced pork meat using multiplex-PCR, microarray and ELISA

A qualitative detection of Listeria monocytogenes was performed in spiked minced pork meat using ELISA, Multiplex-PCR, Microarray and cultural reference method. Minced pork meat in batches of 10 or 25 g was spiked with 25 cfu Listeria monocytogenes per gram and incubated in selective enrichment solutions. During enrichment samples were collected continiously and a Listeria monocytogenes-ELISA, a Multiplex-PCR with electrophoretical detection (Listeria duplex) and a PCR with detection by Microarray (NUTRI Chip) were performed.The enrichment time after which all sub-samples were positive was defined as minimum enrichment time. With the cultural reference method Listeria monocytogenes was detected in 100 % of the samples after a total analysis time of 5 days. With the ELISA kit used in this study positive results were achieved after enrichment for 24 h. Multiplex-PCR with electrophoretical detection was positive after only 16 h of enrichment.The most sensitive detection method was the microarray. Using this technique, an enrichment time of 10 h was sufficient to get positive results in all samples.     

Comparison of direct colony count methods and the MPN-method for quantitative detection of Listeria in model and field conditions]

In order to compare the plate count method for quantitating Listeria, as published in the "Official Collection of Testing Methods" in section 35 LMBG (L. 00.00-22), to an MPN-method for Listeria based on the same mediums, these two detection methods for Listeria were tested in three sets of experiments and a routine sample status evaluation. A pure broth culture of L. monocytogenes, artificially with L. monocytogenes contaminated ground meat, artificially contaminated and cold stored ground meat as well as 77 ground beef samples from Berlin retail food stores were used in the four trials. The detection limit of the MPN-method is about 66% lower than the plate count method allowing detection of a clearly greater number of Listeria-positive samples from naturally contaminated ground meat. The MPN-method yielded more Listeria spp.-positive samples (rel. 43%) and more L. monocytogenes-positive samples (rel. 21%) versus the colony count method based on the results from the field trial using ground beef samples from retail food stores in Berlin. Nevertheless the standardized colony count method is preferred over the MPN-method for routine use because of its slightly higher productivity and much smaller variation in the results. However, the MPN-method is preferable for epidemiological studies because of the significance of the lower detection level. The random sampling evaluation of ground beef from retail stores indicated that 39% of the samples were Listeria spp.-positive and 31% were L. monocytogenes-positive when using the colony count method. A total of 56% of the meat samples were found to be Listeria spp.-positive and 38% L. monocytogenes-positive when the MPN-method was used. Population levels ranged from 10 to 580 cfu/g (Listeria spp.-positive samples) and from 10 to 270 cfu/g (L. monocytogenes-positive samples) for the colony count method. The MPN-method yielded population levels of 3.6 to 930 MPN/g for Listeria spp.-positive samples and 3.6 to 150 MPN/g for L. monocytogenes-positive samples. L. monocytogenes strains isolated using the colony count method belonged to the following serovars: 1/2a (46%), 1/2b (13%), 1/2c (33%), 3b (4%) and 4c (4%). A similar serovar isolation pattern was found for L. monocytogenes-positive MPN-tubes. The most common serotype was 1/2a (43%), followed by 1/2c (32%) and 1/2b (14%). The serotypes 3c, 4b and 4c were all isolated 4% of the time.     

Epidemiological studies on the occurrence of Listeria monocytogenes in the feces of dairy cattle

Seasonal variation in the fecal shedding of Listeria spp. in dairy cattle was examined by collecting a total of 3,878 fecal samples during a period of two years. The prevalences of Listeria spp. and L. monocytogenes were higher during the indoor season (12.7% and 9.2%, respectively) than in samples collected from the animals on pasture (5.3% and 3.1%, respectively). The highest frequencies of Listeria spp. (19.4%) and L. monocytogenes (16.1%) were detected in December. Listeriae were isolated from at least one of the dairy cows from 45.8% of the 249 herds examined. 2.9% of the 314 milk samples collected from the farm bulk tanks on 80 dairy farms on four different occasions yielded L. monocytogenes. The seasonal occurrence of these bacteria in milk reflected the frequencies of Listeria in the fecal material but not those in the main roughage used; grass silage and pasture grass. Fecal material is considered to be a potential source of contamination of raw milk by L. monocytogenes. Investigation of the numbers of viable Listeria organisms in different animal fodders is considered essential in further epidemiological studies of these bacteria.     

哈尔滨市鲜肉中单核细胞增生性李斯特菌的分离鉴定及耐药性分析

为了解哈尔滨市单核细胞增生性李斯特茵(Lm)的污染状况及耐药状况.在哈尔滨市市场随机采集158份鲜肉样品,采用显色培养基分离,API试剂条和PCR鉴定等方法对样品中的Lm进行分离鉴定,并通过Kirby-Barer法测定分离菌株对24种抗生素的耐药性.结果从鲜肉中共分离到Lm 23株,检出率为14.56%,其中鲜猪肉检出率最高,达20.00%(14/70);23株分离菌株中耐药菌株为22株,耐药率高达95.65%.这表明哈尔滨市鲜肉中存在一定程度的Lm污染,并且分离菌株存在较严重的耐药现象.应加强控制动物饲料亚治疗抗生素的使用并严格遵守休药期,防止耐药菌株产生进而控制食源性疾病的发生.     

The diagnosis of Listeria encephalitis in ruminants using cultural and immunohistological techniques. I. Comparison of different selective media and culture techniques for the detection of Listeria from ruminant brains]

The selective L-PALCAMY differential enrichment broth, the Listeria enrichment broth of the International Dairy Federation, Oxford Listeria selective agar, and PALCAM Listeria selective agar were comparatively examined in the cultural isolation of Listeria spp. from ten ruminant brains. The L-PALCAMY medium proved to be superior to the IDF broth in both selectivity and productivity for Listeria spp. in the brain samples, which were also contaminated with other bacteria. The Oxford and PALCAM agars corresponded in their productivity for Listeria spp. The latter, however, was more selective than the Oxford agar. Bacterial counts of up to 1.2 x 10(9) CFU/g of brain stem sample were made from Listeria monocytogenes (L.m.), and up to 6.2 x 10(4) CFU/g from Listeria innocua. A total of 164 brains from ruminants showing CNS disturbances and/or pathoanatomical CNS alterations were examined using L-PALCAMY medium, and Oxford and PALCAM agar. L.m. could be isolated from 29 of the brains, and Listeria innocua from five. Cultural isolation of both Listeria spp. occurred in one brain. Of 27 brains containing L.m., which were also examined using cold enrichment, L.m. was isolated in 59.3% of the cases with direct culture, in 81.5% of the cases using selective warm enrichment, and in 77.8% of the cases by means of selective cold enrichment. Five cases each were identified solely by cold or warm enrichment, respectively. In investigations of further 69 ruminant brains the number of brains shown to contain L.m. could be increased from seven to 13 by means of selective cold enrichment for three months.     

石河子地区动物性食品中单核细胞增生李斯特菌的检测

目的了解新疆石河子地区动物性食品中单核细胞增生李斯特氏菌(LM)污染状况。方法在石河子地区选取5个具有代表性动物性食品零售点,对最常食用的生鲜猪肉、牛肉、羊肉、鸡肉、冻鸡肉、冻虾和冻带鱼8类动物性食品进行随机采样,采用病原分离培养和PCR法对样品中的单核细胞增生李斯特氏菌进行检测。结果检测8类249份食品样品,细菌分离鉴定阳性样品12份,平均阳性率4.82%;PCR法检测阳性样品36份,平均阳性率为14.46%。结论石河子动物性食品中LM的污染比较普遍,尤以冻鸡肉和冻虾LM污染较重,新鲜猪肉、牛肉和羊肉LM污染程度较轻。     

产单核细胞李斯特菌的PFGE分子分型分析

对50株不同来源的产单核细胞李斯特菌（Lm）进行PFGE分型,比较食源、环境、人源菌株之间的相似性。结果显示,同一市场内,环境污水分离株和新鲜食品分离株表现出较高同源性,新鲜食品分离株与即食性（RTE）食品分离株相似性很高,临床脑膜炎病人的1株Lm与RTE食品分离株在进化上显示出了很近的亲缘关系。50株Lm的分子分型分析提示了菌株从市场环境污水-新鲜食品-RTE食品-人的传播模式,为食源性疾病溯源追踪提供了新的思路。     

Listeria monocytogenes in food]

As in recent years laboratory diagnostics of listeria has become part of food microbiology, the frequency of occurrence of the bacteria Listeria monocytogenes has been followed in various kinds of foods for a year. A total of 51 strains of L. monocytogenes (7.2%) was isolated from 700 kinds of samples (raw milk, pasteurized milk, meat surface, poultry, cheeses, thermally not treated meat products, food--industry machinery). As can be seen in Tab. I, the highest number of strains was isolated from meat surfaces (13.5%), followed by meat--industry machinery (12.72%), poultry (10%) and cheeses (5%). The lower numbers of strains were found out in thermally not treated meat products (3.8%) and in raw milk (3.3%). Pasteurized milk did not contain any strains. Our findings in raw milk (3.3%) and in pasteurized milk (0) are in agreement with the data cited e. g. by authors from the USA (Lovett et al., 1987), who mention the value of 4.2% in raw milk and the zero value in pasteurized milk. The percentage of strains monitored in cheeses (5%) can be evaluated as low as the assortment of investigated cheeses was small (all strains were isolated from soft ripening cheeses). German authors (Tham et al., 1988) speak about the 2.5% percentage of L. monocytogenes strains; this is in keeping with our findings. The findings in thermally not treated meat products (3.8%) can be evaluated as low although the number of strains found in raw meat was high.(ABSTRACT TRUNCATED AT 250 WORDS)     

Presence and coincidence of Listeria spp., motile aeromonads and Yersinia enterocolitica in a cold-smoked salmon processing and packing plant

Environmental samples and samples of partially processed fish from a cold-smoked salmon processing and packing plant, and product samples purchased from retail outlets, were examined for the presence of Listeria monocytogenes and other Listeria spp., motile aeromonads and Yersinia enterocolitica. Listeria spp. were not isolated from raw fish but were a contaminant of processed and partially processed fish. Listeria spp. were also detected in 18.8% of surfaces in contact with fish. Unlike Listeria spp., motile aeromonads were isolated from the raw fish but not from the finished product. They were more frequently isolated from environmental sources than Listeria spp. Yersinia enterocolitica was not isolated from any of the samples tested. Little evidence was found to show a coincidence of motile aeromonads and Listeria spp., since only one subset of samples showed such a link. It is concluded that contamination by Listeria spp. was from environmental sources at the processing plant at, or beyond, the slicing stage. Reducing the number of wet areas and special cleaning and sanitation considerations for a contaminated site (the freezer seal) are suggested as ways of reducing contamination of the product.     

Occurrence,Antibiotic Resistance and Molecular Characterization of Listeria monocytogenes in the Beef Chain in the Republic of Ireland

This study investigated the occurrence, concentration and key characteristics of Listeria monocytogenes in beef chain samples (n = 1100) over a 2‐year period (July 2007–June 2009). Listeria monocytogenes was isolated from bovine hides (27%), pre‐chill carcasses (14%) and ground beef (29%), but not from ready‐to‐eat (RTE) beef. The concentration of the pathogen in the majority (95%) of contaminated samples was low and detected by enrichment only. The highest concentrations recovered (100–200 CFU/g) were in ground beef samples. The most commonly isolated serotype group was 1/2a (58%) followed by 4b (12%), 1/2b (10%) and 1/2c (6%). A small portion (<5%) isolates had demonstrated resistance to key anti‐microbials including ampicillin, vancomycin and gentamycin which are recommended treatment options for listeriosis. Pulsed‐field gel electrophoresis showed indistinguishable profiles for a number of isolates recovered from the hide and carcass (after slaughter and dressing) of the same animals, highlighting the role of hides as a source of contamination. Equally, indistinguishable pulsotypes for isolates recovered at different stages and time points (up to 6 months apart) in the beef chain demonstrated the persistence of specific clones in the factory, process and distribution environments. Overall, the study demonstrated a high prevalence of clinically significant L. monocytogenes entering and progressing along the beef chain and highlights the needs to control cross‐contamination during beef processing and distribution and the need for thorough cooking of raw beef products.