A novel real-time PCR-based method for the detection of Listeria monocytogenes in food

AIMS: A new real-time PCR-based method was developed for the detection of Listeria monocytogenes in food. METHODS AND RESULTS: A two-step enrichment involving a 24-h incubation in half-Fraser broth followed by a 6-h subculture in Fraser broth was used, followed by cell lysis and real-time PCR with primers and a TaqMan probe previously developed in our laboratory. When the method was evaluated with 144 naturally contaminated food samples, 44 were detected as positive by the PCR-based method and 42 by the standard method EN ISO 11290-1. With 61 food samples artificially contaminated at a level of 10(0) CFU per 25 g, 61 and 58 positive samples were detected by the respective methods. CONCLUSIONS: The developed real-time PCR-based method facilitated the detection of L. monocytogenes in food on the next day after the sample reception, with a reduction of false-positive results because of dead bacterial cells and false-negative results because of PCR inhibitors. SIGNIFICANCE AND IMPACT OF THE STUDY: The method can be used for L. monocytogenes detection in food as a faster alternative to current methods.     

PCR detection of Listeria monocytogenes: a study of multiple factors affecting sensitivity

AIMS: To test, under comparable conditions, several parameters affecting sensitivity of PCR detection in order to establish a PCR procedure suitable for the routine detection of Listeria monocytogenes in food. METHODS AND RESULTS: Beef samples artificially inoculated were used to determine sensitivity of PCR detection under different parameters. As few as 1 CFU g(-1) were detected by DNA extraction using a DNeasy Tissue Kit (Qiagen GmhH, Hilden, Germany) of 1 ml aliquot and PCR amplification with primers directed to the hlyA gene. This PCR protocol was applied in 60 naturally contaminated foods, comparing two enrichment procedures with the traditional culture method. The highest number of positives was recorded by PCR following a 24-h pre-enrichment step at 30 degrees C and a 24-h enrichment step at 37 degrees C. Afterwards, it was applied in 217 naturally contaminated foods and 56 of them tested positive for L. monocytogenes in which only 17 tested positive using the culture method. CONCLUSIONS: The PCR procedure described has proved to be a rapid and sensitive method suitable for the routine analysis of different types of food. SIGNIFICANCE AND IMPACT OF THE STUDY: The method proposed for the detection of L. monocytogenes, has been validated in naturally contaminated food and is suitable to implement in the food industry.     

Prevalence and lineages of Listeria monocytogenes in Chinese food products

AIMS: This study was undertaken to investigate the prevalence and lineages of Listeria monocytogenes in different kinds of food products in local Chinese markets. METHODS AND RESULTS: A total of 2686 food samples and 645 water samples were collected and L. monocytogenes was isolated from 2.28% (76 of 3331) of all samples. The prevalence of L. monocytogenes (14 of 290, 4.83%) in raw meat products was significantly higher than that in other raw food products (P < 0.05). Among 844 ready-to-eat (RTE) food samples, 21 samples were positive for L. monocytogenes. RTE packaged food products from two supermarkets had a prevalence ranging from 0.00% to 25.00%. The prevalence of L. monocytogenes in meat products of freshly slaughtered hogs was 0.95% (four of 420), significantly lower than that in raw meat products in the retail markets (P < 0.05). Ten isolates were recovered from 645 water samples, which were collected after hands washing by shopkeepers or waiters. A total of 38 isolates were randomly selected for lineage classification based on the nucleotide variation of actA gene. Eighty percentage of isolates from RTE food products belonged to Lineage II while only 20% belonged to Lineage I. CONCLUSIONS: Food products in Chinese markets are contaminated with L. monocytogenes. Raw meat products have the highest contamination rates among all the raw food samples. RTE food products are more likely to be contaminated with Lineage II strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented here show the main contamination sources of L. monocytogenes in Chinese food products.     

Limitation in the detection of Listeria monocytogenes in food in the presence of competing Listeria innocua

Aims: Detectability of Listeria monocytogenes at 10⁰ CFU per food sample in the presence of Listeria innocua using standard microbiological detection was evaluated and compared with the real‐time PCR‐based method. Methods and Results: Enrichment in half‐Fraser broth followed by subculture in Fraser broth according to EN ISO 11290‐1 was used. False‐negative detection of 10⁰ CFU L. monocytogenes was obtained in the presence of 10¹ CFU L. innocua per sample using the standard detection method in contrast to more than 10⁵ CFU L. innocua per sample using real‐time PCR. Identification of L. monocytogenes on the chromogenic medium by the standard procedure was impossible if L. innocua was able to overgrow L. monocytogenes by more than three orders of magnitude after the enrichment in model samples. These results were confirmed using naturally contaminated food samples. Conclusions: Standard microbiological method was insufficient for the reliable detection of 10⁰ CFU L. monocytogenes in the presence of more than 10⁰ CFU of L. innocua per sample. On the other hand, if the growth of L. monocytogenes was sufficient to reach the concentration equal to the detection limit of PCR, the amount of the other microflora present in the food sample including L. innocua was not relevant for success of the PCR detection of L. monocytogenes. Significance and Impact of the Study: After the enrichment, the PCR detection is more convenient than the standard one as PCR detection is not compromised by other present microflora.     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。     

Simultaneous quantitative detection of Listeria spp. and Listeria monocytogenes using a duplex real-time PCR-based assay

We report a duplex real-time PCR-based assay for the simultaneous quantitative detection of Listeria spp. and the food-borne pathogen Listeria monocytogenes. The targets of this single tube reaction were the 23S rDNA and hly genes of Listeria spp. and L. monocytogenes, respectively. Our assay was efficient, 100% selective (i.e., it allowed accurate simultaneous identification of 52 L. monocytogenes and 120 Listeria spp. strains through the FAM-labelled hly and the VIC-labelled 23S rDNA probes, respectively); and had a detection limit of one target molecule in 100% (23S rDNA) and 56% (hly) of the reactions. Simultaneous quantification was possible along a 5-log dynamic range, with an upper limit of 30 target molecules and R2 values > 0.995 in both cases. Our results indicate that this assay based on the amplification of the 23S rDNA gene can accurately quantify any mixture of Listeria species and simultaneously unambiguously quantify L. monocytogenes.     

Proteomic identification of Listeria monocytogenes surface‐associated proteins

This study aimed to identify proteins exposed on the surface of Listeria monocytogenes cells for diagnostic reagent development. Brief trypsin treatment of L. monocytogenes cells followed by peptide separation and identification by nano‐LC and online‐MS/MS was performed. In parallel, as a negative control, proteins secreted into the digest buffer as well as proteins from cell lysis were identified. One hundred and seventy‐four proteins were identified in at least two of three trials in either the negative control or during cell digest. Nineteen surface, 21 extracellularly secreted, 132 cytoplasmic, and two phage proteins were identified. Immunofluorescence microscopy of L. monocytogenes cells revealed the surface localization of two potential candidates for L. monocytogenes isolation and detection: lipoprotein LMOf2365_0546 and PBPD1 (LMOf2365_2742). In this report, we present the first data set of surface‐exposed L. monocytogenes proteins currently available. The data have been deposited to the ProteomeXchange Consortium with identifier PXD000035.     

Human heat-shock protein 60 receptor-coated paramagnetic beads show improved capture of Listeria monocytogenes in the presence of other Listeria in food

Aims: To investigate the suitability of human Hsp60, a receptor for Listeria adhesion protein (LAP), on paramagnetic beads (PMB) to capture Listeria monocytogenes from food in the presence of other Listeria to facilitate rapid and specific detection of this pathogen. Methods and Results: Commercially available streptavidin‐coated PMBs were linked with biotinylated Hsp60 (PMB‐Hsp60), and the bacterial capture efficiency from pure culture and meat samples was determined. Capture rate was also compared with the monoclonal antibody (MAb)‐C11E9‐coated beads (PMB‐C11E9) and the commercial Dynabeads anti‐Listeria. Captured cells were detected and quantified by plating on selective medium, quantitative real‐time PCR (qPCR) and a light‐scattering sensor. Overall, all ligand‐coated beads had similar capture efficiency (varied from 1·8 to 9·2%) for L. monocytogenes under the conditions employed, and the minimum cell number required to achieve such capture was 10³ CFU ml^?1. PMB‐Hsp60 had significantly greater capture efficiency for pathogenic Listeria (P < 0·0001) than the nonpathogenic Listeria. In contrast, PMB‐C11E9 and Dynabeads anti‐Listeria had similar capture efficiency for both. The efficacy of all PMBs to capture L. monocytogenes in the presence of Listeria innocua from food matrices was compared. Although Dynabeads anti‐Listeria had the overall best capture efficiency, PMB‐Hsp60 was able to selectively capture L. monocytogenes even in the presence of 10–100‐fold more L. innocua cells from enriched meat samples. Conclusions: Data show that the human cell receptor, Hsp60, is suitable for the capture of pathogenic Listeria on PMB in the presence of other Listeria in food. Significance and Impact of the Study: As pathogen interaction with host cells is highly specific, host cell receptors could be used as alternate capture molecules on PMB to aid in specific detection of pathogens.     

Modification of a virulence-associated phenotype after growth of Listeria monocytogenes on food

AIM: To assess the effect of different foods, which have been implicated or not in cases of listeriosis, on the in vitro virulence-associated phenotype level of different Listeria monocytogenes strains. METHODS AND RESULTS: The virulence-associated phenotype level of L. monocytogenes was studied with the in vitro cell test based on a plaque-forming assay with a human adenocarcinoma cell line (HT-29) monolayer. Three strains of L. monocytogenes were grown in preparations (homogenate, 1-mum filtrate or 0.2-mum filtrate) of different food extracts ['rillettes' (potted minced pork), milk, raw salmon and cold-smoked salmon] or in a control medium, brain heart infusion (BHI). The bacterial suspensions grown in food extracts or in BHI at 37 degrees C were diluted with their growth medium (food extract or BHI) or with minimum essential medium before seeding on confluent HT-29 cell monolayers. Filtration of food extracts had no significant effect on the plaque numbers formed by the bacteria. A significant decrease in the plaque numbers was noted for the three strains when they grew in the rillettes extracts, compared with the other food extracts and BHI. The levels of in vitro virulence-associated phenotype of the strains after growth in the rillettes extract were similar to or lower than that of the hypovirulent internal reference strain L. monocytogenes 442. After growth in milk and cold-smoked salmon, the impact on virulence-associated phenotype depended on the strain. In contrast, plaque-forming assay indicated increased virulence-associated phenotype when the strains were switched from a nutrient-rich medium (food extract or BHI) to a minimum essential medium. CONCLUSIONS: In vitro virulence-associated phenotype level of the studied strains grown in BHI or cold-smoked salmon was the same as the control virulent strain EGD. In contrast, the nutrients present in rillettes may therefore substantially reduce the number of plaques but not the growth of L. monocytogenes. The utilization of minimum essential medium as diluent attenuates changes the effect of the food extract on virulence-associated phenotype in vitro. SIGNIFICANCE AND IMPACT OF THE STUDY: In the experimental design of this study, we showed that the nature of the food could affect the in vitro virulence-associated phenotype level of L. monocytogenes.     

Conductivity and pH dual detection of growth profile of healthy and stressed Listeria monocytogenes

In this study, growth of Listeria monocytogenes in a low conductivity growth medium (LCGM) was simultaneously monitored by conductivity and pH measurements. Detection times obtained from the conductivity and pH growth curves were inversely related to the initial concentration of L. monocytogenes in the medium. Linear responses were found by plotting detection times obtained from both conductivity and pH growth curves as a function of initial cell concentration in the range of 10(2) to 10(7) cfu/mL. The detection time was approximately 12 and 2 h for 10(2) and 10(7) cfu/mL of viable L. monocytogenes, respectively, using the conductivity growth curves, whereas it was approximately 1 h less using the pH growth curves. This dual detection system was used for evaluating the growth of acid-, temperature-, and salt-treated L. monocytogenes in the medium. Acid stress at pH 2 and 3 for 3 h caused approximately 12 and 4 h delay in the detection time on pH growth curves, while stress at pH 5 for 3 h did not cause a significant delay in detection time. Delay in detection times was also observed for L. monocytogenes cells exposed to 45 degrees C for more than 1 h (2 and 6 h). Exposure to 10% NaCl for 3 h did not cause visible delay in the detection time. These observations on detection times for stressed L. monocytogenes had a consistent trend with the cell number decrease determined by surface plating method.     

Evaluation of a new chromogenic agar for the detection of Listeria in food

AIM: Rapid identification of Listeria in food is important in protecting consumers from infection. The development of chromogenic media such as agar Listeria according to Ottaviani and Agosti (ALOA) has allowed more rapid detection of Listeria monocytogenes, with presumptive identification of this pathogenic species after only 24 h of incubation. The aim of this study was to evaluate Oxoid chromogenic Listeria agar (OCLA) in comparison with ALOA and a traditional, nonchromogenic medium, Oxford agar. METHODS AND RESULTS: Media were compared using pure cultures, spiked food samples and naturally contaminated samples. Whilst development of typical colony morphology took 48 h on Oxford agar, Listeria spp. were frequently detected after 24 h of incubation on OCLA and ALOA. There was no significant difference in recovery between the two chromogenic media. CONCLUSIONS: Results indicate that OCLA gives equivalent recovery of Listeria spp. compared with ALOA. Whilst L. monocytogenes was frequently detected after 24 h of incubation, a 48-h incubation time was necessary to ensure detection of both L. monocytogenes and other Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has shown that a commercially available chromogenic medium other than ALOA is appropriate for use in the international standard method. The commercial availability of more than one medium will facilitate the more widespread use of the method, thus increasing confidence in the ability to detect L. monocytogenes in food in the presence of other Listeria spp.     

Performance testing of six chromogenic ALOA-type media for the detection of Listeria monocytogenes

Aims:  The aim of the study was to test the performance of commercially available chromogenic plating media for detection and enumeration of the food-borne pathogen Listeria monocytogenes . A wide range of chromogenic media similar to Agar Listeria according to Ottaviani and Agosti (ALOA) were compared using PALCAM agar, according to van Netten et al.
Methods and Results:  Six chromogenic media similar to ALOA were challenged for inclusivity and exclusivity. Additionally, the ability of chromogenic agars to facilitate growth of stressed L. monocytogenes strains and mixed cultures with competitive non-Listeria strains was estimated. Finally, we tested the detection and enumeration of L. monocytogenes in artificially inoculated and naturally contaminated food samples. The results of this study indicated that chromogenic media are a good supplementation to PALCAM agar. A single application is not advisable, as the specificity of chromogenic agars is frequently insufficient (50·0–88·9%), particularly in food samples with a complex microflora.
Conclusions:  The competitive flora of food samples is able to overgrow low numbers of L. monocytogenes , especially in half-Fraser enrichment. This might lead to the underestimation of L.   monocytogenes positive samples.
Significance and Impact of the Study:  Although many evaluation studies of chromogenic agar have been published recently, harmonized validation strategies are lacking. This survey provides a new concept for stepwise testing of plating media.     

Isolation and 2‐D‐DIGE proteomic analysis of intracellular and extracellular forms of Listeria monocytogenes

The pathogenicity of Listeria monocytogenes is related to its ability of invading and multiplying in eukaryotic cells. Its main virulence factors are now well characterized, but limited proteomic data is available concerning its adaptation to the intracellular environment. In this study, L. monocytogenes EGD (serotype 1/2a) grown in human THP‐1 monocytes (24 h) were successfully separated from host organelles and cytosolic proteins by differential and isopycnic centrifugation. For control, we used cell homogenates spiked with bacteria grown in broth. Proteomes from both forms of bacteria were compared using a 2‐D‐DIGE approach followed by MALDI‐TOF analysis to identify proteins. From 1684 distinct spots, 448 were identified corresponding to 245 distinct proteins with no apparent contamination of host proteins. Amongst them, 61 show underexpression (stress defense; transport systems, carbon metabolism, pyrimidines synthesis, D ‐Ala‐D ‐Ala ligase) and 22 an overexpression (enzymes involved in the synthesis of cell envelope lipids, glyceraldehyde‐3‐phosphate, pyruvate and fatty acids). Our proteomic analysis of intracellular L. monocytogenes (i) suggests that bacteria thrive in a more favorable environment than extracellularly, (ii) supports the concept of metabolic adaptation of bacteria to intracellular environment and (iii) may be at the basis of improved anti‐Listeria therapy.     

Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples

AIMS: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. METHODS AND RESULTS: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml(-1) contamination rate. CONCLUSIONS: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. SIGNIFICANCE AND IMPACT OF THE STUDY: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.     

The characterization of Listeria spp. isolated from food products and the food‐processing environment

Aim: To enhance the information pertaining to the epidemiology of a collection of 378 Listeria spp. isolates obtained from several food‐processing plants in Ireland over a 3‐ year period (2004–2007). Methods and results: The collection was characterized by pulsed‐field gel electrophoresis (PFGE). The most prevalent pulse‐type was PFGE profile I (n = 14·5%) that consisted mainly of environmental Listeria spp. samples. Serotyping of 145 Listeria monocytogenes isolates was performed. The most common serovar was 1/2a and comprised 57·4% (n = 77) of the L. monocytogenes collection. The other serovars were as follows: 4b (14·1%, n = 19), 1/2b (9·7%, n = 13), 4c (4·4%, n = 6) and 1/2c (6·7%, n = 9), respectively. Eleven isolates were identified as non‐Listeria spp., the remaining ten L. monocytogenes isolates were nontypeable. The antimicrobial susceptibility testing revealed the antibiotic that isolates displayed the most resistance to was gentamicin (5%) followed by sulfamethoxazole‐trimethoprim (2%), tetracycline and ciprofloxacin (1·5%). Conclusions: The subtyping has indicated the diversity of the Listeria spp. The presence of serotype 1/2a, 1/2b and 4b in both raw and cooked ready‐to‐eat food products is a public health concern, as these serotypes are frequently associated with foodborne outbreaks and sporadic cases of human listeriosis. In addition, the emergence of antimicrobial‐resistant L. monocytogenes isolates could have serious therapeutic consequences. Significance and Impact of Study: The molecular subtyping and the further characterization of these isolates may be valuable particularly in the context of a suspected common source outbreak in the future.     

Mechanistic study of membrane concentration and recovery of Listeria monocytogenes

Detection of the foodborne pathogen Listeria monocytogenes requires that food samples be processed to remove proteins and lipids, concentrate microorganisms to a detectable concentration, and recover the concentrated cells in a small volume compatible with micron-scale biochips. Mechanistic considerations addressed in this research include the roles of membrane structure, pore size, and detergents in maximizing recovery of cells from a complex biological fluid. The fluid in this case was a food sample (hotdog extract) inoculated with L. monocytogenes. This study showed how membrane filtration using a syringe filter is able to concentrate L. monocytogenes by 95x with up to 95% recovery of living microorganisms by concentrating 50 mL of food sample into a volume of 500 microL. Tween 20 was added to the sample to prevent irreversible adsorption of the microorganism to the membrane and thereby help to ensure high recovery. Comparison of polycarbonate, mixed cellulose, nylon, and PVDF membranes with 0.2 to 0.45 microm pores showed the 0.2 microm polycarbonate membrane with straight through, mono-radial pores gives the highest recovery of living microorganisms. The mixed cellulose, nylon, and PVDF membranes have a fibrous structure whose characteristic openings are much larger than their effective pore size cut-offs of 0.22 or 0.45 microm. We define conditions for rapid membrane-based cell concentration and recovery that has the potential to supplant enrichment steps that require a day or more. This approach has the added benefit of facilitating examination of a large amount of fluid volume by reducing its volume to a range that is compatible with the microliter scales of biochip or other biosensor detection systems.     

Fibronectin‐binding protein,FbpA, is the adhesin responsible for pathogenesis of Listeria monocytogenes infection

The role of fibronectin binding protein A (FbpA) in Listeria monocytogenes infection and its pathogenesis were studied in vivo and in vitro by constructing a fbpA‐deficient mutant of L. monocytogenes (ΔfbpA). In vivo, ΔfbpA was less pathogenic in mutant mice than was wild‐type L. monocytogenes. FbpA did not affect the amounts of various virulence‐determining factors, including internalin B and listeriolysin O. However, adherence to, and invasion of, mouse hepatocytes by the ΔfbpA mutant were reduced. In contrast, adherence to, but not invasion of, the ΔfbpA mutant to macrophages was attenuated. Fibronectin contributed to the efficient adherence and invasion of wild‐type L. monocytogenes, but not to those of the ΔfbpA mutant. Attenuation of adhesion and uptake of the ΔfbpA mutant were reversed by overexpression of FbpA in it. FbpA was not involved in intracellular growth, autophagy induction or actin tail formation. Thus, the present findings clearly show that FbpA acts as an important adhesion molecule of L. monocytogenes, especially regarding hepatocytes, without modulating the expression of other virulence factors that have been implicated in the pathogenesis of L. monocytogenes infection.     

Behaviour of Listeria monocytogenes in packaged water buffalo mozzarella cheese

Aims: The aim of the study was to evaluate the behaviour of Listeria monocytogenes in the conditioning liquid of packaged water buffalo mozzarella cheese (WBMC). Methods and Results: The conditioning liquid was contaminated with L. monocytogenes, and the contaminated samples were stored at four different storage temperatures: 5 and 10°C for 22 days; 20°C for 9 days; 20°C for 3 days and then at 5°C for 6 days. The results showed that L. monocytogenes concentration decreased when contaminated samples were stored at 5°C. When WBMC was stored at 20°C and at 10°C, L. monocytogenes started to grow after a lag phase of 3 and 10 days, respectively. When samples were stored at variable temperature conditions, L. monocytogenes numbers showed a lag phase of 5 days. Conclusions: Use of a conditioning liquid characterized by acidity and a correct storage temperature is able to counteract pathogen replication during shelf life. A high concentration of lactic acid bacteria was associated with effective control of L. monocytogenes but the role of lactic acid bacteria in WBMC conditioning liquid requires further investigation. Significance and Impact of the Study: According to European regulations, food producers should be able to justify decision‐making on the shelf life assigned to their products, taking into account reasonable storage conditions and use by consumers. The results of the trial yielded information for producers of WBMC and similar cheeses for decision‐making on product shelf life.     

Characterization of isolates of Listeria monocytogenes from sludge using pulsed‐field gel electrophoresis and virulence assays

Aims: To study the diversity and virulence of Listeria monocytogenes isolated from sludge. Methods and Results: A total of 60 isolates of L. monocytogenes from sludge were characterized by serotyping, PFGE typing and using in vitro and in vivo virulence assays. The PFGE patterns were compared with those of food and human isolates to determine whether specific group clones are associated with environmental samples. The 60 isolates gave 44 different combined ApaI/AscI PFGE patterns. The PFGE patterns of most isolates were similar or very similar to those of epidemic isolates. The majority (93%) of isolates were found to be virulent by plaque‐forming assay and by mouse virulence assay. Conclusions: Our findings suggest that L. monocytogenes strains found in non‐sanitized sludge are virulent and represent a potential health hazard. Although no case of listeriosis related to sludge spread onto agricultural land has been reported, particular attention to this pathogen is needed. Significance and Impact of the study: This is the first study dealing with the characterization of L. monocytogenes isolates from non‐sanitized sludge samples by molecular typing methods and in vitro and in vivo virulence assays. Our findings provide relevant information for evaluating the health risks associated with spreading sludge onto agricultural land.