Concentration Of Listeria Monocytogenes Research Articles

Nanozyme-catalyzed and zwitterion-modified swabs based for the detection of Listeria monocytogenes in complex matrices

In recent years, nanozymes have been widely used in the field of biosensing and food safety testing due to their advantages of low cost, high stability, easy modification and adjustable catalytic activity. However, how to reduce the signal interference generated by reducing substances, macromolecules and colored substances in the food matrix in nanozymes-based colorimetric sensing is still a major challenge. In this paper, using Listeria monocytogenes as a model analyte, sodium sulfonyl methacrylate (SBMA) polymers were modified onto cotton swabs by photothermal polymerization and combined with Listeria monocytogenes-specific aptamer (Apt1) to prepare swabs that can specifically capture and isolate Listeria monocytogenes from complex matrices (SBMA/Apt1 cotton swab). In addition, in combination with the inhibitory effect of the aptamer (Apt2) on the oxidase activity of Mn3O4 NPs, a colorimetric biosensor based on nanozymes that can quantitatively, sensitively, and specifically identify Listeria monocytogenes in food products was constructed. The results showed that the colorimetric signal of the method was linear with the concentration of Listeria monocytogenes in the range of 2.83–2.83 × 105 CFU/mL, and the limit of detection was 2.64 CFU/mL, which can be used for the detection of Listeria monocytogenes in complex environments and food samples.

Phenotypic characterisation of Listeria monocytogenes in cow milk from three catchment areas of Goliati Area in Thyolo District, Malawi

Background: Listeria monocytogenes is one of the food borne pathogens that cause an illness known as Listeriosis upon ingestion of contaminated food. Outbreaks of Listeriosis have been reported in Canada, USA, Europe, South Africa, Ghana and other countries but there is limited data on outbreaks due to Listeria monocytogenes and its isolation from contaminated foods in Malawi. This prompted our interest to determine the presence of Listeria monocytogenes in cow milk from Goliati area, Thyolo district in Malawi. Objectives: To determine the presence and concentration of Listeria monocytogenes in cow milk from daily farmers around Goliati area in Thyolo district in Malawi. Methodology: Nine raw milk samples from three bulk centers and two processed milk samples were cultured on both Nutrient Agar and Brain Heart Infusion agar as primary cultures. A pure culture of Listeria monocytogenes was obtained by sub culturing the primary culture on Brain Heart Infusion agar media. Colonies from the pure culture were further identified using Gram staining, Catalase test, Motility test and Beta hemolysis test on blood agar to phenotypically identify Listeria monocytogenes. Results: There was a 100% growth rate of colonies suggestive of Listeria monocytogenes on all the nine samples. These colonies were observed to contain Gram positive, purple, short, rod shaped bacteria which was motile and catalase positive and it caused complete beta hemolysis on blood agar. Conclusion: In this study, isolates of Listeria monocytogenes were phenotypically identified in cow milk that is produce from Goliath Area in Thyolo district. This is a major public health concern for milk farmers and consumers in the area and beyond. The findings from this work calls for improved hygienic practices in the handling of milk and milk products to reduce contamination.

An Assessment of Listeriosis Risk Associated with a Contaminated Production Lot of Frozen Vegetables Consumed Under Alternative Consumer Handling Scenarios

Frozen foods do not support the growth of Listeria monocytogenes (LM) and should be handled appropriately for safety. However, consumer trends regarding preparation of some frozen foods may contribute to the risk of foodborne listeriosis, specifically when cooking instructions are not followed and frozen products are instead added directly to smoothies or salads. A quantitative microbial risk assessment model FFLLoRA (Frozen Food Listeria Lot Risk Assessment) was developed to assess the lot-level listeriosis risk due to LM contamination in frozen vegetables consumed as a ready-to-eat food. The model was designed to estimate listeriosis risk per serving and the number of illnesses per production lot of frozen vegetables contaminated with LM, considering individual facility factors such as lot size, prevalence of LM contamination, and consumer handling prior to consumption. A production lot of 1 million packages with 10 servings each was assumed. When at least half of the servings were cooked prior to consumption, the median risk of invasive listeriosis per serving in both the general and susceptible population was <1.0 × 10-16 with the median (5th, 95th percentiles) predicted number of illnesses per lot as 0 (0, 0) and 0 (0, 1) under the exponential and Weibull-gamma dose-response functions, respectively. In scenarios in which all servings are consumed as ready-to-eat, the median predicted risk per serving was 1.8 × 10-13 and 7.8 × 10-12 in the general and susceptible populations, respectively. The median (5th, 95th percentile) number of illnesses was 0 (0, 0) and 0 (0, 6) for the exponential and Weibull-Gamma models, respectively. Classification tree analysis highlighted initial concentration of LM in the lot, temperature at which the product is thawed, and whether a serving is cooked as main predictors for illness from a lot. Overall, the FFLLoRA provides frozen food manufacturers with a tool to assess LM contamination and consumer behavior when managing rare and/or minimal contamination events in frozen foods.

Potentiometric Detection of Listeria monocytogenes via a Short Antimicrobial Peptide Pair-Based Sandwich Assay

Peptide-based sandwich assays are promising tools in molecular detection, but may be restricted by the availability of "pairs" of affinity peptides. Herein, a new potentiometric sandwich assay for bacteria based on peptide pairs derived from an antimicrobial peptide (AMP) ligand is demonstrated. As a model, the original AMP with a well-defined structure for Listeria monocytogenes (LM) can be split into two fragments to serve as the peptide pairs for the sandwich assay. The recognition and binding of the short peptide pairs to the target can be verified by circular dichroism, flow cytometry, fluorometry, and optical microscopy. The potentiometric magnetic bead-based sandwich assay is designed by using horseradish peroxidase as a label. The enzyme can catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine with H2O2 to induce a potential change on a polymeric membrane ion-selective electrode. Under optimal conditions, the concentration of LM can be determined potentiometrically in a linear range of 1.0 × 102 to 1.0 × 106 CFU mL-1 with a detection limit of 10 CFU mL-1 (3σ). The proposed sensing strategy expands the applications of peptides in the field of bioassays.

Concentration of Listeria monocytogenes in skim milk and soft cheese through microplate immunocapture

Microplate immunocapture is an inexpensive method for the concentration of foodborne pathogens using an antibody-coated microplate. The objective of this study was to determine the efficacy of microplate immunocapture as an alternative to traditional enrichment for concentrating Listeria monocytogenes to levels detectable with selective plating or real-time PCR. L. monocytogenes isolates serologically characterized as Type 1 (1/2a) and Type 4 (untypeable) were grown overnight and diluted to 100 to 106 colony-forming units (CFU)/mL. The isolates were used to optimize microplate immunocapture in tryptic soy broth with 0.6% yeast extract (TSBYE), skim milk, and queso fresco samples. Following microplate immunocapture, the bacteria were streaked onto polymyxin-acriflavine-LiCl-ceftazidime-aesculin-mannitol (PALCAM) agar, followed by incubation at 37 °C for 24 ± 2 h. The bacteria also underwent real-time polymerase chain reaction (PCR). The optimized microplate immunocapture method was tested in triplicate for its ability to capture L. monocytogenes in broth and food samples. Overall recovery rates for L. monocytogenes in food samples at cell populations of 100, 102, and 104 CFU/25 g using microplate immunocapture with real-time PCR were 88.9%, 94.4%, and 100%, respectively. Recovery in these matrices using microplate immunocapture with selective plating was comparatively lower, at 0%, 44.4%, and 100%, respectively. Conventional culture method showed 100% detection at each inoculation level. Microplate immunocapture combined with real-time PCR shows high potential to reduce the time required for detection, with concentration of L. monocytogenes to detectable levels within 1–4 h. The incorporation of a short enrichment step may improve recovery rates at low cell levels.

Challenge Test Improvement: Analytical Costs and Time Optimization

&lt;p&gt;&lt;em&gt;The study’s aim was to develop a quantitative risk assessment model of Listeria monocytogenes in liver chicken paté. The model was performed using the Integrated Challenge Test (Italian Journal of Food Safety, Vol. 1 N. 6 2012) with the objective to reduce the analytical cost and time. The challenge test was carried out on 3 different batches stored at 12°C and inoculated with a mix of Listeria monocytogenes strains. Lactobacillus spp. and Listeria monocytogenes plate counts were performed daily on each sample until the stationary phase was reached by both populations. The challenge test results at 12°C were input in the Combase DMfit software to determine the growth parameters of Listeria monocytogenes and lactic flora which showed mutual interaction. Then, using the Combase Predictor for Listeria monocytogenes and the FSSP (Food Spoilage and Safety Predictor) software for lactic flora, the growth parameters were extrapolated at 4°C and 8°C. The growth parameters of both populations at 4°C, 8°C and 12°C were then used to apply the model in order to predict the maximum daily concentration of Listeria monocytogenes. Model results were assessed against the results of an additional challenge test conducted with the same strain mix inoculum in 3 different batches stored for 4 days at 4°C, 4 days at 8°C and then 4 days at 12°C. The proposed model represents a reliable quantitative risk evaluation which provides realistic results with limited cost.&lt;/em&gt;&lt;/p&gt;

Impact of the prevalence of different pathogens on the performance of sampling plans in lettuce products

Prevalence and concentration of Listeria monocytogenes, Salmonella spp. and enteric pathogenic viruses (namely Hepatitis A—HAV, and noroviruses genogroup I—NoVGI and genogroup II—NoVGII) were determined in raw and RTE lettuce from a Spanish processing premise. Fifteen samplings were made from September 2010 to February 2012 (n=600 samples). Sampling strategies for pathogen detection were suggested by the characterization of the uncertainty in prevalence associated with the performance of two-class attributes sampling plans (c=0). A probabilistic model was run (1000 iterations) using a Bayesian approach with a conjugate beta distribution considering the impact of taking different number of samples on the proportion of positive samples and lots (within- and between-lot prevalence). No enumeration results were obtained for the pathogens tested. Presence of L. monocytogenes and NoVGII in RTE lettuce (10%) and NoVGI and NoVGII in unprocessed lettuce (10%) was obtained in the tested lots during cold season. Results evidenced that, as the number of samples increased, the probability of rejecting a contaminated lot became higher, yielding right-skewed distributions with values close to 1. According to our results, 25 samples would result in 80% of rejected lots, while 95% confidence level would be reached with n>100. However, although those levels would imply a unrealistic high number of samples making the application of the sampling plan unfeasible, these results might be useful for food operators and risk managers to know the underlying distributions of microbial contamination together with potential control measures to be applied to assure a safer production of minimally processed vegetables.

Rapid detection of Listeria monocytogenes in milk by self-assembled electrochemical immunosensor

Listeria monocytogenes (LM) is a gram-positive food-borne pathogen, which can cause serious disease. Rapid screening plays an important role in the prevention and identification of LM to cause food poisoning. In this study, we describe a novel electrochemical immunosensor by self-assembled monolayers (SAM)-modified gold (Au) electrodes for the detection of LM. Mouse monoclonal antibody of LM was immobilized on the SAM through a stable acyl amino ester intermediate generated by the co-addition of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then, a direct sandwich format was developed, in which a polyclonal antibody for LM was conjugated to horseradish peroxidase (HRP) as the enzyme label. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) were employed to characterize the stepwise assembly of the immunosensor. In the presence of H2O2, HRP catalyzes the oxidation of thionine, whose back electrochemical reduction was detected on an Au electrode at −0.3V for 200s with chronoamperometry measurements versus an Ag/AgCl reference electrode. The reaction conditions were optimized, such as antibody concentration, immune reaction time, activation time, and blocking time. Under optimized conditions, when the concentration of LM is ranging from 102 to 106CFU/ml, the response current which is obtained from the labeled HRP to thionine–H2O2 system and the amount of LM are linearly related (R2=0.9883). There was no need for sample pretreatment when applied to detect LM in milk. Compared with plate count assay, it was found that the relative error of two methods was lower than 8%. The sensor is a straightforward and reliable method for analysis of LM with a simple operation, sensitivity at a low cost.

RETRACTED: Bayesian inference for quantifying Listeria monocytogenes prevalence and concentration in minced pork meat from presence/absence microbiological testing

The purpose of this work was to estimate the prevalence and concentration of Listeria monocytogenes in minced pork meat by the application of a Bayesian modeling approach. Samples (n=100) collected from local markets were tested for L.monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media. Presence of the pathogen was confirmed through biochemical and molecular tests. Independent experiments (n=10) for validation purposes were performed. No L.monocytogenes was enumerated by direct-plating (<10CFU/g), though the pathogen was detected in 22% of the samples. Sensitivity and specificity varied depending on the culture method. L.monocytogenes concentration was estimated at 14-17CFU/kg. Validation showed good agreement between observed and predicted prevalence (error=-2.17%). The use of at least two culture media in parallel enhanced the efficiency of L.monocytogenes detection. Bayesian modeling may reduce the time needed to draw conclusions regarding L.monocytogenes presence and the uncertainty of the results obtained.

Prevalence and Concentration of Listeria monocytogenes in Sliced Ready-to-Eat Meat Products in the Hellenic Retail Market

The aim of this work was to estimate the prevalence and concentration of Listeria monocytogenes in packaged precut (slices or cubes) ready-to-eat (RTE) meat products available in the Hellenic retail market. Samples of these RTE meat products (n = 209) were taken from local supermarkets during a 3-month period and analyzed for the presence of L. monocytogenes with an automated enzymatic qualitative immunoassay followed by biochemical confirmation of positive results. The concentration of the pathogen in the positive samples was also determined. Seventeen samples (8.1%) were positive for L. monocytogenes. Eight (47.1%) of these 17 samples were from the same manufacturer; 36.4% of the products tested from this manufacturer were positive for L. monocytogenes. When bacon samples were not considered, the estimated prevalence of L. monocytogenes in sliced RTE meat products was much lower (3.1%). The L. monocytogenes populations in all positive samples were low, ≤10 CFU/g. In 64.7% of the L. monocytogenes–positive samples, other Listeria species, including L. innocua and L. welshimeri, were also present at <10 to 690 CFU/g. These results indicate that L. monocytogenes is present in low numbers but is in a considerable proportion of the packaged precut RTE meat products that are sold in the Hellenic retail market. Cooked ham and bacon cut in cubes were the sample types most often contaminated with L. monocytogenes. The higher level of handling (e.g., cutting) associated with these products may further increase the risk of contamination with L. monocytogenes.