Detection and prevalence of Listeria monocytogenes in the agricultural ecosystem

B. VAN RENTERGHEM, F. HUYSMAN, R. RYGOLE AND W. VERSTRAETE. 1991. The sensitivity of four different enrichment procedures to detect Listeria monocytogenes in the presence of high levels of Streptococcus faecalis was investigated. Defined mixed cultures of Strep. faecalis and L. monocytogenes gave better results with one-stage enrichment techniques. For manure samples, however, two-stage enrichment techniques gave the best performance. The so-called cold enrichment techniques were found to be unsatisfactory for samples from natural environments. The following materials were examined for the presence of L. monocytogenes: fresh pig faeces (16% positive), fresh cattle faeces (20% positive), stored liquid manure (0% positive), manured soil samples (0% positive) and ground water samples (5% positive). After 3 weeks of storage L. monocytogenes could be detected in only one of the initially nine positive fresh facces samples. Two months after inoculation of stored liquid pig manure, stored liquid cattle manure and soil with L. monocytogenes , this bacterium could not be traced in any of these materials. Radishes ( Raphanus sativus ) and carrots ( Daucus carota ), sown in soil inoculated with L. monocytogenes , were gathered after 3 months and examined for the presence of L. monocytogenes. Three of six radish samples were found to be positive. Remarkably, however, all carrot samples (six) were free of L. monocytogenes.     

Immobilization and Detection of Listeria monocytogenes

A procedure was developed for immobilization of Listeria monocytogenes cells on metal hydroxides coupled with detection and enumeration using an automated optical system. The results of the immobilization procedure (<1 h) and detection during overnight incubation agreed with calculated plate counts, and this technique is simple and rapid and provides samples that are ready for confirmation of the presence of the pathogen by rapid methods.     

Detection of Listeria species and Listeria monocytogenes using polymerase chain reaction

Five oligonucleotide sequences are described that were used as primers in the polymerase chain reaction (PCR) to amplify specific sequences from Listeria DNA. When all five primers were used in combination, three PCR products were possible; a Listeria specific product that occurs with DNA from any Listeria sp., a Listeria monocytogenes specific product that occurs only in the presence of DNA from this organism and a universal product that is found using DNA from any bacterial source. The occurrence of these PCR products was used as a diagnostic test on bacteria isolated from various food samples to detect Listeria sp. and L. monocytogenes.     

Detection of Listeria monocytogenes in Italian-style soft cheeses

AIMS: A rapid detection system specific for Listeria monocytogenes and based on the polymerase chain reaction (PCR) was developed. METHODS AND RESULTS: Primers annealing to the coding region of the actA gene, critically involved in virulence and capable of discrimination between two different alleles naturally occurring in L. monocytogenes, have been utilized. The procedure was applied to recover L. monocytogenes cells in artificially contaminated fresh Italian soft cheeses (mozzarella, crescenza and ricotta). Low levels of L. monocytogenes were detected in mozzarella and crescenza homogenates (0.04-0.4 and 4 CFU g(-1), respectively) whereas in ricotta the detection limit was higher (40 CFU g(-1)). CONCLUSIONS: This PCR-based assay is highly specific as primers used recognize the DNA from different L. monocytogenes strains of clinical and food origin, while no amplification products result with any other Listeria spp. strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted a low-cost and rapid procedure that can be appropriated for the detection in real time of low L. monocytogenes levels in soft cheese.     

Detection of Listeria monocytogenes using polyclonal antibody

Polyclonal antibody sensitive to Listeria was assayed for the detection of Listeria using two different methods, direct and sandwich enzyme linked immunosorbent assays (ELISAs). The direct ELISA uses anti-goat IgG antibody conjugated with horse-radish peroxidase, while the sandwich ELISA uses two antibodies both specific to Listeria antigens, one coated onto the microtitre plate and the other conjugated to horse-radish peroxidase. The results obtained show that the direct ELISA is superior to the sandwich ELISA in two distinct ways: (i) with direct ELISA the non- Listeria gave readings <0.2, whereas with sandwich ELISA it gave readings of 0.3–0.4; (ii) the direct ELISA is more cost-effective than the sandwich ELISA.     

Detection of Listeria monocytogenes and the toxin listeriolysin O in food

Listeria monocytogenes is an emerging bacterial foodborne pathogen responsible for listeriosis, an illness characterized by meningitis, encephalitis, and septicaemia. Less commonly, infection can result in cutaneous lesions and flu-like symptoms. In pregnant women, the pathogen can cause bacteraemia, and stillbirth or premature birth of the fetus. The mortality rate for those contracting listeriosis is approximately 20%. Currently, the United States has a zero tolerance policy regarding the presence of L. monocytogenes in food, while Canada allows only 100 cfu/g of food. As such, it is essential to be able to detect the pathogen in low numbers in food samples. One of the best ways to detect and confirm the pathogen is through the detection of one of the virulence factors, listeriolysin O (LLO) produced by the microorganism. The LLO-encoding gene (hlyA) is present only in virulent strains of the species and is required for virulence. LLO is a secreted protein toxin that can be detected easily with the use of blood agar or haemolysis assays and it is well characterized and understood. This paper focuses on some of the common methods used to detect the pathogen and the LLO toxin in food products and comments on some of the potential uses and drawbacks for the food industry.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

SD-PMA-ddPCR检测食品中单核细胞增生李斯特氏菌

【目的】检测食品中单核细胞增生李斯特氏菌活菌。【方法】利用脱氧胆酸钠(SD)对受损细胞预处理,然后使叠氮溴化丙锭(PMA)进入受损细胞与DNA发生共价交联,提取细菌基因组DNA进行微滴式数字PCR(dd PCR)检测。【结果】0.1%SD和5.0 mg/L PMA协同作用,可以有效抑制108 CFU/m L的单核细胞增生李斯特氏菌死菌DNA的PCR扩增。经过SD和PMA对样品预处理,dd PCR可以在死菌存在条件下,定量检测鸡肉中单核细胞增生李斯特氏菌活菌,消除了"假阳性"结果的出现。活菌灵敏度检测结果显示:SD-PMA-dd PCR的灵敏度为2.0 copies/20μL。SD-PMA-dd PCR方法精密度和稳定性良好。【结论】SD-PMA-dd PCR在检测食源性致病菌方面有巨大的发展空间。     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

微流控振荡流PCR快速检测单增李斯特氏菌

一种高通量振荡流PCR微流控技术已被成功开发,借此来快速检测食品致病菌-单增李斯特氏菌(L.monocytogenes).该PCR微流控装置主要由基于LabView的温度控制与采集系统、三个铜加热块以及聚四氟乙烯(PTFE)毛细管等构成.在该微流控装置上,三个铜块维持PCR反应所需要的三个温度,而包埋在铜块沟槽中的PT...     

Detection of Listeria monocytogenes by using the polymerase chain reaction.

A method was developed for detection of Listeria monocytogenes by polymerase chain reaction amplification followed by agarose gel electrophoresis or dot blot analysis with a 32P-labeled internal probe. The technique identified 95 of 95 L. monocytogenes strains, 0 of 12 Listeria strains of other species, and 0 of 12 non-Listeria strains.     

Biodiversity of the species Listeria monocytogenes and the genus Listeria

This review describes the Listeria monocytogenes genome sequences available today and their comparison with that of Listeria innocua and Listeria welshimeri by highlighting their characteristic features and common traits. The diversity present among them is analysed with emphasis on putative virulence and host-pathogen interaction related functions. Then large-scale studies comparing gene content of Listeria and how these studies contributed to typing applications will be discussed. Finally, evolutionary conclusions and future perspectives in Listeria genomics are presented.     

Listeria monocytogenes in Nature

Samples from 12 farms were examined during two successive spring and early autumn seasons. L. monocytogenes was isolated from vegetation or soil taken from 11 of the 12 farms and from 6 of the 7 nonagricultural sites. A total of 27 strains were isolated from the 19 sites. The organism was not isolated from any of the autumn collections.     

Detection of hemolytic Listeria monocytogenes by using DNA colony hybridization

A fragment of about 500 base pairs of the beta-hemolysin gene from Listeria monocytogenes was used to screen different bacterial strains by DNA colony hybridization. The cells in the colonies were lysed by microwaves in the presence of sodium hydroxide. Of 52 different strains of Listeria species screened, only the DNA from beta-hemolytic (CAMP-positive) strains of L. monocytogenes hybridized with this probe.     

Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq

Listeria monocytogenes was isolated from a sewage treatment plant in Baghdad, Iraq, at all stages of treatment. The treatment processes did not yield a sewage sludge cake or a final discharge free of listerias. The agricultural practice of using such sewage products as fertilizers could become a route of spreading the organism in Iraq, particularly by infecting animals that consume vegetation in fields spread with such sewage. Dewatering of sewage reduced the number of L. monocytogenes but long periods of exposure to sun would be needed to obtain a 'safe' sewage sludge cake.     

Detection and enumeration of Listeria monocytogenes in a sewage treatment plant in Iraq

Listeria monocytogenes was isolated from a sewage treatment plant in Baghdad, Iraq, at all stages of treatment. The treatment processes did not yield a sewage sludge cake or a final discharge free of listerias. The agricultural practice of using such sewage products as fertilizers could become a route of spreading the organism in Iraq, particularly by infecting animals that consume vegetation in fields spread with such sewage. Dewatering of sewage reduced the number of L. monocytogenes but long periods of exposure to sun would be needed to obtain a 'safe' sewage sludge cake.     

DNA Extraction and PCR Methods for the Detection of Listeria monocytogenes in Cold-Smoked Salmon

Protocols for the specific detection of Listeria monocytogenes in cold-smoked salmon were developed. PCR was used as the method of detection. Inhibitors of PCR present in the food samples were removed by ether extraction or column purification, or their effect was overcome by the use of Tween 20 as an enhancer. These protocols are many times more rapid than conventional detection methodologies and also have the potential for automation.