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.     

Effects of sewage treatment on the removal of Listeria monocytogenes

Two sewage treatment plants in Baghdad, Iraq, were investigated to assess the effects of the different treatment stages on the removal of Listeria monocytogenes . The bacteria were severely affected after the activation and digestion stages at both plants. A dramatic decrease in numbers of listerias after each of these two stages was noticed during the cold months (September-January). The organisms were able to survive these treatments and were present in the final effluent and even in low numbers in the sewage sludge cake. Sufficient dewatering of sewage sludge is recommended to obtain sewage free of listerias. Improvements in the isolation procedure of L. monocytogenes from such heavily contaminated material is also discussed.     

Effects of sewage treatment on the removal of Listeria monocytogenes

Two sewage treatment plants in Baghdad, Iraq, were investigated to assess the effects of the different treatment stages on the removal of Listeria monocytogenes. The bacteria were severely affected after the activation and digestion stages at both plants. A dramatic decrease in numbers of listerias after each of these two stages was noticed during the cold months (September-January). The organisms were able to survive these treatments and were present in the final effluent and even in low numbers in the sewage sludge cake. Sufficient dewatering of sewage sludge is recommended to obtain sewage free of listerias. Improvements in the isolation procedure of L. monocytogenes from such heavily contaminated material is also discussed.     

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.     

Identification and enumeration of Listeria monocytogenes by nonradioactive DNA probe colony hybridization.

A plasmid containing the cloned listeriolysin gene of Listeria monocytogenes was used as a probe to identify Listeria strains by DNA colony hybridization. The probe DNA was labeled with horseradish peroxidase in the presence of glutaraldehyde. After the hybridization and wash procedures, the hybrid molecules were detected by luminescence, which resulted from the oxidation of luminol by a horseradish peroxidase-hydrogen peroxide-coupled reaction. Of the 150 Listeria strains and 16 non-Listeria strains examined, the probe hybridized only with L. monocytogenes. The technique was also used to enumerate L. monocytogenes in artificially contaminated foods.     

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 and prevalence of Listeria monocytogenes in the agricultural ecosystem

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 faeces 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.     

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.     

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.     

Storage effects of sewage sludge cake on the survival of Listeria monocytogenes

Sewage sludge cake is widely used as an agricultural fertilizer in Iraq. Listeria monocytogenes was shown to be present in small numbers in this material despite sewage treatments. In an attempt to reduce the numbers of this pathogen in this sewage end product, the survival of L. monocytogenes was monitored in a heap of sewage sludge cake stored for over 23 weeks on farm land. The organisms were reduced in numbers and eliminated to undetectable limits during 8 weeks of storage under subtropical weathering and did not recover even 2 months after disappearance. Dewatering processes seem to have some affect on the survival of the bacteria. Therefore, solar dewatering by heaping the sewage sludge cake and exposing it to sun for no less than 8 weeks is recommended to obtain a listeria-free product.     

Storage effects of sewage sludge cake on the survival of Listeria monocytogenes

Sewage sludge cake is widely used as an agricultural fertilizer in Iraq. Listeria monocytogenes was shown to be present in small numbers in this material despite sewage treatments. In an attempt to reduce the numbers of this pathogen in this sewage end product, the survival of L. monocytogenes was monitored in a heap of sewage sludge cake stored for over 23 weeks on farm land. The organisms were reduced in numbers and eliminated to undetectable limits during 8 weeks of storage under subtropical weathering and did not recover even 2 months after disappearence. Dewatering processes seem to have some affect on the survival of the bacteria. Therefore, solar dewatering by heaping the sewage sludge cake and exposing it to sun for no less than 8 weeks is recommended to obtain a listeria-free product.     

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.     

Modeling the lag time of Listeria monocytogenes from viable count enumeration and optical density data

The following two factors significantly influence estimates of the maximum specific growth rate ( micro (max)) and the lag-phase duration (lambda): (i) the technique used to monitor bacterial growth and (ii) the model fitted to estimate parameters. In this study, nine strains of Listeria monocytogenes were monitored simultaneously by optical density (OD) analysis and by viable count enumeration (VCE) analysis. Four usual growth models were fitted to our data, and estimates of growth parameters were compared from one model to another and from one monitoring technique to another. Our results show that growth parameter estimates depended on the model used to fit data, whereas there were no systematic variations in the estimates of micro (max) and lambda when the estimates were based on OD data instead of VCE data. By studying the evolution of OD and VCE simultaneously, we found that while log OD/VCE remained constant for some of our experiments, a visible linear increase occurred during the lag phase for other experiments. We developed a global model that fits both OD and VCE data. This model enabled us to detect for some of our strains an increase in OD during the lag phase. If not taken into account, this phenomenon may lead to an underestimate of lambda.     

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。     

Survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer

The survival of Listeria monocytogenes in raw milk treated in a pilot plant size pasteurizer was investigated. Raw milk was inoculated with different initial concentrations of L. monocytogenes and heated at temperatures ranging from 69° to 73°C. Listerias were not isolated from any of the milk samples immediately after thermal treatment. They were isolated, however, from 46.6% of heated samples (none from samples heated at 73°C) after variable periods at refrigeration temperature. The results suggest that a low number of listerias survive some thermal treatments, but a cold enrichment is necessary to repair the thermally injured cells and detect these organisms in milk. The importance of the isolation technique in the recovery of listerias from pasteurized milk samples is discussed.     

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.