The challenge of challenge testing to monitor Listeria monocytogenes growth on ready-to-eat foods in Europe by following the European Commission (2014) Technical Guidance document

European Regulation (EC) No. 2073/2005 lays down the microbiological criteria for certain microorganisms in foods and the implementing rules to be complied with by food business operators (FBOs) in Europe when implementing general and specific hygiene measures. In relation to Listeria monocytogenes, this regulation covers primarily ready-to-eat (RTE) food products, and requires different microbiological criteria depending on the ability of the food product to support growth of L. monocytogenes. In addition, this regulation establishes that food safety is the responsibility of the FBO. The FBO can conduct studies to evaluate the growth of L. monocytogenes that may be present in the product during the shelf-life under reasonably foreseeable storage conditions of distribution, storage and use in order to investigate compliance with the criteria throughout the shelf-life of the product. The European Union Community Reference Laboratory for L. monocytogenes published a revised technical guidance document in June 2014 for conducting shelf-life studies on L. monocytogenes in RTE foods. This review article describes the recently published European guidance document, with special focus on the design of challenge studies to determine the growth potential of L. monocytogenes on foods. Information is given particularly on what a challenge test is and when one is advisable. The factors to be considered and the laboratory methodology to be applied when performing a challenge test to determine the growth potential of L. monocytogenes in a defined food matrix are also described. Results of recent research articles applying challenge tests to determine the growth of L. monocytogenes in a range of foodstuffs are summarized and discussed. Finally, recommendations for obtaining data that can contribute to any further revision of the guidance document and for addressing the main challenges of challenge testing for FBOs are presented.     

Investigation of Listeria monocytogenes contamination pattern in local Chinese food market and the tracing of two clinical isolates by RAPD analysis

This study was undertaken to investigate the contamination pattern of Listeria monocytogenes in local Chinese food markets and to trace two clinical isolates. Random amplification polymorphic DNA (RAPD) was developed to track the source of L. monocytogenes in ready-to-eat (RTE) food products and from clinical origin. Three random primers, PB1, PB4 and HLWL74, were used to subtype all the L. monocytogenes strains isolated from RTE food products, fresh food products, environmental sewages in the markets and two clinical meningitis patients. It was shown that all the 49 isolates could be classified into 5, 4 and 4 types using these three random primers, PB1, PB4, and HLWL74, respectively. Twenty-seven composite profiles were identified by a combination of the three primers. The same composite profiles of L. monocytogenes could be found both in the fresh food products, environmental sewages and RTE food products, suggesting that the L. monocytogenes in the RTE food products may come from those in the fresh food products or sewage in the same market. The composite profiles of the two clinical isolates were the same as those of strains isolated from RTE food products, indicating that the disease might have resulted from the consumption of the RTE food products contaminated with L. monocytogenes. The results show that RAPD could be a powerful tool for the investigation of contamination pattern of L. monocytogenes in Chinese food markets and also for tracking the source of L. monocytogenes in clinical patients.     

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.     

Listeria monocytogenes in retail establishments: Contamination routes and control strategies

In the past two decades, serious outbreaks of foodborne disease were caused by Listeria monocytogenes, a pathogen frequently found in delicatessens at retail. Although the prevalence of listeriosis is not high, the severity of disease is significant, with high hospitalization and mortality rates. Potential sources of L. monocytogenes and food contamination routes in retail and food service operations include incoming raw materials, food products, food handlers, customers, vendors, and environmental sources. Risk mitigation strategies for L. monocytogenes should be based on integrated control along the food chain continuum, from farm to retail establishment.     

Listeria monocytogenes in Aquatic Food Products—A Review

With the increased demand for lightly preserved and/or ready‐to‐eat (RTE) food products, the prevalence of the foodborne pathogen Listeria monocytogenes has increased, which is a public health concern. The goal for this review is to discuss the incidence, epidemiological importance, and contamination routes of L. monocytogenes in various aquatic ecosystems, seafood products, and processing environments and to summarize the data obtained since the 1990s. L. monocytogenes primarily enters the food‐production chain by cross‐contamination in production plants, making this pathogen a major threat to the seafood industry. This pathogen generally contaminates food products at low or moderate levels, but the levels involved in listeriosis outbreaks are significantly higher. The majority of isolates from aquatic products belong to serotype 1/2a, and outbreaks have been linked to highly similar or even indistinguishable strains. Several seafood‐processing plants are colonized by specific “in‐house” flora containing special DNA subtypes of L. monocytogenes. In such cases, L. monocytogenes populations can persist and/or multiply despite the inherent obstacles to their growth in food preservation and manufacturing operations. Therefore, food‐processing facilities must be designed carefully with an emphasis on effective cleaning and disinfecting operations in the production line.     

Efficacy of pulsed light for shelf-life extension and inactivation of Listeria monocytogenes on ready-to-eat cooked meat products

Pulsed light (PL) was tested for its utility to improve the microbial quality and safety of ready-to-eat cooked meat products. Vacuum-packaged ham and bologna slices were superficially inoculated with Listeria monocytogenes and treated with 0.7, 2.1, 4.2 and 8.4 J/cm². PL treatment at 8.4 J/cm² reduced L. monocytogenes by 1.78 cfu/cm² in cooked ham and by 1.11 cfu/cm² in bologna. The effect of PL on lipid oxidation and sensory properties was also investigated. The 2-thiobarbituric acid values were very low and chromaticity parameters were within the normal values reported for cooked meat products. PL at 8.4 J/cm² did not affect the sensory quality of cooked ham, while treatments above 2.1 J/cm² negatively influenced the sensory properties of bologna. The combination of PL and vacuum packaging provided ham with an additional shelf-life extension of 30 days compared with only vacuum packaging. The shelf-life of bologna was not extended by PL.

Industrial relevance

The efficacy of pulsed light for the decontamination of surfaces offers excellent possibilities to ensure food safety and to extend shelf-life of ready-to-eat (RTE) products. The results of this study indicate that Listeria monocytogenes can be reduced by approximately 2 log cfu/cm² in RTE cooked ham and 1 log cfu/cm² in bologna using a fluence of 8.4 J/cm². This dose does not affect the sensory properties of ham and triples its shelf-life when compared with conventional RTE products. On the contrary, fluences above 2.1 J/cm² are not suitable for the treatment of bologna since sensory quality is modified.     

Antibacterial activity of egg yolk antibody (IgY) against Listeria monocytogenes and preliminary evaluation of its potential for food preservation

BACKGROUND: Egg yolk antibody (IgY) is a unique type of immunoglobulin found in egg yolks, and many reports have described its ability to inhibit corresponding antigen bacteria. The objective of this study was to evaluate the antibacterial activity of IgY specific to Listeria monocytogenes, an important food pathogen to both humans and animals, as well as its potential use for food preservation. RESULTS: Specific IgY was generated by immunising Leghorn chickens with whole cells of L. monocytogenes, and its inhibitory effect on bacterial growth was tested in liquid medium and food samples. After 8 h of incubation with specific IgY, there was a significant decrease in the growth (absorbance at 600 nm) of L. monocytogenes in comparison with controls. IgY also inhibited the growth of L. monocytogenes inoculated onto fresh or smoked salmon samples. Compared with those of blanks, numbers of L. monocytogenes were reduced by more than 2 log units after 15 days of storage at 6 ± 1 °C in the presence of specific IgY. CONCLUSION: The results suggest the potential application of specific IgY as a natural antimicrobial agent for food preservation. Copyright © 2011 Society of Chemical Industry     

Probabilistic models for the prediction of target growth interfaces of Listeria monocytogenes on ham and turkey breast products

Abstract: This study developed growth/no growth models for predicting growth boundaries of Listeria monocytogenes on ready‐to‐eat cured ham and uncured turkey breast slices as a function of lactic acid concentration (0% to 4%), dipping time (0 to 4 min), and storage temperature (4 to 10 °C). A 10‐strain composite of L. monocytogenes was inoculated (2 to 3 log CFU/cm²) on slices, followed by dipping into lactic acid and storage in vacuum packages for up to 30 d. Total bacterial (tryptic soy agar plus 0.6% yeast extract) and L. monocytogenes (PALCAM agar) populations were determined on day 0 and at the endpoint of storage. The combinations of parameters that allowed increases in cell counts of L. monocytogenes of at least l log CFU/cm² were assigned the value of 1, while those limiting growth to <1 log CFU/cm² were given the value of 0. The binary data were used in logistic regression analysis for development of models to predict boundaries between growth and no growth of the pathogen at desired probabilities. Indices of model performance and validation with limited available data indicated that the models developed had acceptable goodness of fit. Thus, the described procedures using bacterial growth data from studies with food products may be appropriate in developing growth/no growth models to predict growth and to select lactic acid concentrations and dipping times for control of L. monocytogenes. Practical Application: The models developed in this study may be useful in selecting lactic acid concentrations and dipping times to control growth of Listeria monocytogenes on cured ham and uncured turkey breast during product storage, and in determining probabilities of growth under selected conditions. The modeling procedures followed may also be used for application in model development for other products, conditions, or pathogens.     

Quantifying Listeria monocytogenes prevalence and concentration in minced pork meat and estimating performance of three culture media from presence/absence microbiological testing using a deterministic and stochastic approach

Listeria monocytogenes poses a serious threat to public health, and the majority of cases of human listeriosis are associated with contaminated food. Reliable microbiological testing is needed for effective pathogen control by food industry and competent authorities. The aims of this work were to estimate the prevalence and concentration of L. monocytogenes in minced pork meat by the application of a Bayesian modeling approach, and also to determine the performance of three culture media commonly used for detecting L. monocytogenes in foods from a deterministic and stochastic perspective. Samples (n = 100) collected from local markets were tested for L. monocytogenes using in parallel the PALCAM, ALOA and RAPID'L.mono selective media according to ISO 11290-1:1996 and 11290-2:1998 methods. Presence of the pathogen was confirmed by conducting biochemical and molecular tests. Independent experiments (n = 10) for model validation purposes were performed. Performance attributes were calculated from the presence–absence microbiological test results by combining the results obtained from the culture media and confirmative tests. Dirichlet distribution, the multivariate expression of a Beta distribution, was used to analyze the performance data from a stochastic perspective. No L. monocytogenes was enumerated by direct-plating (<10 CFU/g), though the pathogen was detected in 22% of the samples. L. monocytogenes concentration was estimated at 14–17 CFU/kg. Validation showed good agreement between observed and predicted prevalence (error = −2.17%). The results showed that all media were best at ruling in L. monocytogenes presence than ruling it out. Sensitivity and specificity varied depending on the culture-dependent method. None of the culture media was perfect in detecting L. monocytogenes in minced pork meat alone. 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. Furthermore, the problem of observing zero counts may be overcome by applying Bayesian analysis, making the determination of a test performance feasible.     

Emergence of Antibiotic Resistance in Listeria monocytogenes Isolated from Food Products: A Comprehensive Review

Listeria monocytogenes is an opportunistic pathogen that has been involved in several deadly illness outbreaks. Future outbreaks may be more difficult to manage because of the emergence of antibiotic resistance among L. monocytogenes strains isolated from food products. The present review summarizes the available evidence on the emergence of antibiotic resistance among L. monocytogenes strains isolated from food products and the possible ways this resistance has developed. Furthermore, the resistance of food L. monocytogenes isolates to antibiotics currently used in the treatment of human listeriosis such as penicillin, ampicillin, tetracycline, and gentamicin, has been documented. Acquisition of movable genetic elements is considered the major mechanism of antibiotic resistance development in L. monocytogenes. Efflux pumps have also been linked with resistance of L. monocytogenes to some antibiotics including fluoroquinolones. Some L. monocytogenes strains isolated from food products are intrinsically resistant to several antibiotics. However, factors in food processing chains and environments (from farm to table) including extensive or sub‐inhibitory antibiotics use, horizontal gene transfer, exposure to environmental stresses, biofilm formation, and presence of persister cells play crucial roles in the development of antibiotic resistance by L. monocytogenes.     

High hydrostatic pressure processing of sliced fermented sausages: A quantitative exposure assessment for Listeria monocytogenes

Fermented sausages have traditionally been considered to be safe products from a microbiological point of view, mainly due to nitrite addition, their low a_w and reduced pH. However, post-process contamination during slicing and packaging operations may increase microbial concentration and prevalence on final products. A stochastic simulation modelling approach was conducted to determine the extent of Listeria monocytogenes survival on sliced chorizo submitted or not to high hydrostatic pressure (HHP) treatments after post-process contamination (i.e., cross-contamination during slicing). A probabilistic model comprising nine steps from mixing of raw materials to consumption was constructed. The effects of various initial levels of L. monocytogenes in the meat batter (−1.43–3 log cfu/g), HHP treatments (400–600 MPa/18 °C for 0–12 min) and nitrite concentrations (0–150 ppm) on L. monocytogenes distribution were assessed by means of the application of predictive models, literature information and data obtained experimentally. Once implemented, the probabilistic model was simulated by using Monte Carlo analysis. The probability distribution of L. monocytogenes contamination levels was determined for various scenarios. Model outputs showed that cross-contamination during slicing was an important source contributing to increase pathogen prevalence and concentration on final products, with transferred levels equal to 0.59 ± 0.48 log cfu/g. Under all simulated scenarios, formulation and storage conditions, the level of L. monocytogenes on sliced vacuum-packed chorizo at the consumption phase was estimated to be lower than 100 cfu/g and pressure treatments at 600 MPa for 10–12 min would result in non-contaminated packs. Overall, the probabilistic model developed in this study from raw material reception up to the end of the shelf-life (i.e., 90 days) of sliced fermented sausages is proposed as a suitable tool to determine combinations of HHP treatments and nitrite concentrations ensuring the compliance with microbiological criteria.     

Investigation of some factors affecting the antibacterial activity of rosemary extracts in food models by a food microdilution method

The activity of rosemary phenolic extracts against Listeria monocytogenes and Escherichia coli was evaluated in 50% food models of meat, vegetable and dairy products in relation to some factors that can affect MIC (minimal inhibitory concentration) values (inoculum level, proportion of food, temperature) using a new food microdilution method. It was shown that the interactions of meat and milk components with plant extracts reduced the antibacterial effectiveness of rosemary extracts. MIC values for L. monocytogenes were lower than for E. coli in all tested conditions. A lower inoculation level caused a decrease in MIC values for E. coli but an increase in MIC values for L. monocytogenes in control media. In food models, MIC values were higher or equal to MIC values in control media regardless of bacterial type. We showed that the food microdilution method represents a simple, rapid, reproducible and inexpensive method for testing the antimicrobial efficiency of plant extracts in food systems.     

Susceptibility of Listeria monocytogenes to high pressure processing: A review

Listeria monocytogenes has been regarded as an emerging food pathogen responsible for listeriosis, a serious disease given its high mortality rate. The need for better food processing methods has led to an increased interest in high pressure processing (HPP), a novel nonthermal method presented as “producer” of safer food products. This review provides an overview of the effects of HPP on Listeria monocytogenes and on L. innocua, with the latter often used as an amenable surrogate for the pathogenic species. The factors that affect the susceptibility of listeriae to HPP, as well as the long-term implications of postprocessing recovery, are discussed in the perspective of the use of HPP to improve the safety of potential food vehicles.     

Listeria monocytogenes: Strain Heterogeneity,Methods, and Challenges of Subtyping

Listeria monocytogenes is a food‐borne bacterial pathogen that is associated with 20% to 30% case fatality rate. L. monocytogenes is a genetically heterogeneous species, with a small fraction of strains (serotypes 1/2a, 1/2b, 4b) implicated in human listeriosis. Monitoring and source tracking of L. monocytogenes involve the use of subtyping methods, with the performance of genetic‐based methods found to be superior to phenotypic‐based ones. Various methods have been used to subtype L. monocytogenes isolates, with the pulsed‐field gel electrophoresis (PFGE) being the gold standard. Although PFGE has had a massive impact on food safety through the establishment of the PulseNet, there is no doubt that whole genome sequence (WGS) typing is accurate, has a discriminatory power superior to any known method, and allows genome‐wide differences between strains to be quantified through the comparison of nucleotide sequences. This review focuses on the different techniques that have been used to type L. monocytogenes strains, their performance challenges, and the tremendous impact WGS typing could have on the food safety landscape.     

Construction of food and water borne pathogens' dose-response curves using the expanded Fermi Solution

Abstract: Theoretically, the relationship between the number of pathogens that cause acute infection if settling in the gut, N, and that initially ingested, M, can be constructed from the survival probabilities at the different “stations” along the digestive tract. These probabilities are rarely known exactly, but their ranges can be estimated. If for a given N one generates estimates of M using random probabilities within these ranges, the estimates’ distribution will be approximately lognormal and its cumulative (CDF) form will represent the pathogen's dose–response curve. The distribution's logarithmic mean and standard deviation can be calculated from the ranges with a formula and used to plot the curve. The method was used to generate dose–response curves of hypothetical food and waterborne pathogens and calculate their infective dose (ID) at 5%, 50%, and 95% probability. The curves were compatible with the Beta Poisson model and robust against minor perturbations in the underlying probabilities’ ranges. The calculation and plotting procedure was automated and posted on the Internet as a freely downloadable interactive Wolfram Demonstration. It allows the user to generate, modify, examine, and compare dose–response curves, and to calculate their characteristics, by moving sliders on the screen.     

The fate of Listeria monocytogenes during the manufacture of Camembert-type cheese: A comparison between raw milk and milk treated with high hydrostatic pressure

Camembert-type cheese was produced from: raw bovine milk; raw milk inoculated with 2 or 4 log CFU/ml Listeria monocytogenes; raw milk inoculated with L. monocytogenes and subsequently pressure-treated at 500 MPa for 10 min at 20 °C; or uninoculated raw milk pressure-treated under these conditions. Cheeses produced from both pressure-treated milk and untreated milk had the typical composition, appearance and aroma of Camembert. Curd and cheese made from inoculated, untreated milk contained large numbers of L. monocytogenes throughout production. An initial inoculum of 1.95 log CFU/ml in milk increased to 4.52 log CFU/g in the curd and remained at a high level during ripening, with 3.85 log CFU/g in the final cheese. Pressure treatment inactivated L. monocytogenes in the raw milk at both inoculum levels and the pathogen was not detected in any of the final cheeses produced from pressure-treated milk. Therefore high pressure may be useful to inactivate L. monocytogenes in raw milk that is to be used for the production of soft, mould-ripened cheese.

Industrial relevance

This paper demonstrates the potential of high pressure (HP) for treatment of raw milk to be used in the manufacture of soft cheeses. HP treatment significantly reduced the level of Listeria monocytogenes in the raw milk and so allowed the production of safer non-thermally processed camembert-like soft cheese.     

Influence of organic acid concentration on survival of Listeria monocytogenes and Escherichia coli 0157:H7 in beef carcass wash water and on model equipment surfaces

Acid-adapted cultures of Escherichia coli O157:H7 and Listeria monocytogenes were inoculated in meat decontamination spray-washing runoff fluids in order to evaluate their survival and potential to form biofilms on stainless steel coupons. The cultures (10⁷ cfu ml⁻¹) and stainless steel coupons were exposed to mixtures of water and organic acid washings (composites of each of 2% acetic acid or lactic acid washings with water washings from meat decontamination in proportions of 1/9, 1/49, 1/99 [vol/vol]) or to water washings for up to 14 days at 15°C. E. coli O157:H7 formed biofilms and remained detectable (1.3 log cfu cm⁻²) on stainless steel for up to 4 d in the 1/9 dilution (pH 3.17–3.77) of the organic acid washings, and persisted throughout storage (14 d) in the 1/49 (pH 3.96–4.33) and 1/99 (pH 4.34–6.86) dilution of the organic acid washings. L. monocytogenes populations were unable to form detectable (<1.3 log cfu cm⁻²) biofilms in the 1/9 and 1/49 dilutions of both organic acid washings for up to 14 d; however, by day-14 in the 1/99 dilution of the washings, the pathogen was able to attach at detectable levels (2.7 to 3.4 logs). The pH effects of lower concentrations (1/49 or 1/99) of acidic washings decreased over time due to the formation of amine compounds produced by the natural meat flora, allowing resuscitation of the acid-stressed pathogen survivors. The resuscitation of acid-stressed pathogens may potentially enhance their survival and prevalence in biofilms and thus more attention should be focused on avoiding or minimizing the collection of decontamination runoff fluids on food contact equipment surfaces.     

The probability of growth of Listeria monocytogenes in cooked salmon and tryptic soy broth as affected by salt,smoke compound,and storage temperature

The objectives of this study were to examine and model the probability of growth of Listeria monocytogenes in cooked salmon containing salt and smoke (phenol) compound and stored at various temperatures. A growth probability model was developed, and the model was compared to a model developed from tryptic soy broth (TSB) to assess the possibility of using TSB as a substitute for salmon. A 6-strain mixture of L. monocytogenes was inoculated into minced cooked salmon and TSB containing 0–10% NaCl and 0–34 ppm phenol to levels of 10^2–3 cfu/g, and the samples were vacuum-packed and stored at 0-–25 °C for up to 42 days. A total 32 treatments, each with 16 samples, selected by central composite designs were tested. A logistic regression was used to model the probability of growth of L. monocytogenes as a function of concentrations of salt and phenol, and storage temperature. Resulted models showed that the probabilities of growth of L. monocytogenes in both salmon and TSB decreased when the salt and/or phenol concentrations increased, and at lower storage temperatures. In general, the growth probabilities of L. monocytogenes were affected more profoundly by salt and storage temperature than by phenol. The growth probabilities of L. monocytogenes estimated by the TSB model were higher than those by the salmon model at the same salt/phenol concentrations and storage temperatures. The growth probabilities predicted by the salmon and TSB models were comparable at higher storage temperatures, indicating the potential use of TSB as a model system to substitute salmon in studying the growth behavior of L. monocytogenes may only be suitable when the temperatures of interest are in higher storage temperatures (e.g., >12 °C). The model for salmon demonstrated the effects of salt, phenol, and storage temperature and their interactions on the growth probabilities of L. monocytogenes, and may be used to determine the growth probability of L. monocytogenes in smoked seafood.     

Development of a nucleic acid lateral flow immunoassay for simultaneous detection of <Emphasis Type="Italic">Listeria</Emphasis> spp. and <Emphasis Type="Italic">Listeria</Emphasis><Emphasis Type="Italic">monocytogenes</Emphasis> in food

We present a new nucleic acid lateral flow immunoassay (NALFIA) for the assessment of listeria contamination. The detection procedure starts with enrichment of sample in Half Fraser broth (24 h). Following isolation of DNA, a duplex PCR is performed with two labelled primer sets, one generic and directed to a specific sequence of the gene encoding 16S rRNA from Listeria spp. and the other specific and directed to a part of the prfA gene encoding the central virulence gene regulator from the food pathogen Listeria monocytogenes (3.5 h). The PCR solution is directly added to the one-step assay device and the appearance of a grey/black line is indicative of the presence of specific amplicons (max 15 min). In all tests performed, the method correctly identified L. monocytogenes and strains of Listeria spp. PCR material of over 20 food samples was tested by NALFIA. The method proved to be useful for the detection of L. monocytogenes in different kinds of food samples.