Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Listeria monocytogenes in Cooked Ham.

Changing eating habits and rising demand of food have increased the incidence of foodborne diseases, particularly in industrialized countries. In this context, contaminated ready-to-eat food (RTE) may be a vehicle for the transmission of Listeria monocytogenes (L. monocytogenes), a foodborne pathogen responsible of listeriosis, a severe infectious disease involving humans and animals. It would be useful to have rapid detection methods to screen the presence of L. monocytogenes in food. In this study, a colorimetric Loop-mediated isothermal amplification (LAMP) assay was applied to the detection of L. monocytogenes in 37 experimentally contaminated RTE meat samples. The LAMP primers consisted of a set of six primers targeting eight regions on the hlyA gene; the assay was carried out in 30 min at 65 °C in a water bath. Amplification products were visualized by color change assessment. The results of colorimetric LAMP assays based on the hly gene obtained in this study were compared to microbiological cultural methods, real-time PCR and real-time LAMP PCR, which show 100% specificity and sensitivity. These data suggest that colorimetric LAMP assays can be used as a screen to detect L. monocytogenes in ready-to-eat meat food.

Phylogenetic and Evolutionary Genomic Analysis of Listeria monocytogenes Clinical Strains in the Framework of Foodborne Listeriosis Risk Assessment.

Listeria monocytogenes is one of the most important foodborne pathogens responsible for listeriosis, a severe disease with symptoms ranging from septicemia, meningoencephalitis, and abortion. Given the strong impact of listeriosis on human health and the difficulty of controlling L. monocytogenes along the food production chain, listeriosis has become a priority subjected to molecular surveillance in European Union/European Economic Area since 2007. From 2018, surveillance is based on whole-genome sequence using the core genome multilocus sequence type. The complete sequences of 132 clinical strains were used to define the evolutionary relatedness among subtypes of L. monocytogenes isolated in Italy from 2010 to 2016, allowing the identification of clades and/or clusters associated with outbreaks or sporadic cases of listeriosis. All the strains analyzed are clustered in lineages I and II, and the majority of the strains were classified as lineage II. A probable epidemic entrance in different years for every clade and cluster from each different region was defined. The persistence of the same specific clonal complexes of L. monocytogenes has been found over long periods; this may be related to the fact that some strains are able to survive better than others in a food production environment. Phylogenic studies, using whole-genome sequence data, are able to identify the emergence of highly persistent pathogenic variants, contributing to improving the hazard characterization of L. monocytogenes.

Hyper-Virulent Listeria monocytogenes Strains Associated With Respiratory Infections in Central Italy.

Listeria monocytogenes (Lm) is a foodborne pathogen causing listeriosis. Invasive forms of the disease mainly manifest as septicaemia, meningitis and maternal-neonatal infections. Lm-associated respiratory infections are very rare and little known. We reported two Lm respiratory infection cases occurred in Central Italy during the summer of 2020, in the midst of the SARS-CoV2 pandemic. In addition to collect the epidemiological and clinical characteristics of the patients, we used Whole Genome Sequencing to study the genomes of the Lm isolates investigating their virulence and antimicrobial profiles and the presence of genetic mobile elements. Both the strains belonged to hypervirulent MLST clonal complexes (CC). In addition to the Listeria Pathogenicity Island 1 (LIPI-1), the CC1 strain also carried LIPI-3 and the CC4 both LIPI-3 and LIPI-4. Genetic determinants for antimicrobial and disinfectants resistance were found. The CC1 genome presented prophage sequences but they did not interrupt the comK gene, involved in the phagosomal escape of Lm. None of the strains carried plasmids. Lm is an important, although rare, opportunistic pathogen for respiratory tract and lung infections. To avoid dangerous diagnostic delays of these severe clinical forms, it is important to sensitize hospital laboratories to this rare manifestation of listeriosis considering Lm in the differential diagnosis of respiratory infections.

Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products.

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment.

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Distribution, virulence, genotypic characteristics and antibiotic resistance of Listeria monocytogenes isolated over one-year monitoring from two pig slaughterhouses and processing plants and their fresh hams.

Listeria monocytogenes contamination in raw pork and ready to eat foods is an important food safety concern, also for the increasing detection of antimicrobial-resistant isolates. Data on L. monocytogenes occurrence, persistence, distribution and genetic characterization in two different plants, namely in continuum from slaughtered pigs, environment and unfinished products (fresh hams) were observed by one-year monitoring and were integrated with their antimicrobial resistance patterns. A total of 98 samples out of the overall 1131 (8.7%) were positive for L. monocytogenes, respectively 2.6% and 13.2% in plants A and B: only three serotypes were identified, 1/2c (50%), 1/2b (36.7%) and 1/2a (13.27%), and strains were classified in 35 pulsotypes and 16 clusters by PFGE; a unique P-type was highlighted according to the detection of virulence genes. The contamination flow of L. monocytogenes has a low occurrence in slaughterhouse (Plant A = 1.1%, Plant B: 3.1%; p > 0.05) and increased throughout the processing chain with trimming area as the most contaminated (Plant A: 25%, Plant B: 57%; (p < 0.05)), both in the environment and in unfinished products (80% in hams before trimming in plant B). The dominant role of environmental contamination in post-slaughter processing is confirmed to be a significant cause of meat contamination by L. monocytogenes. Very high levels of resistance were observed for clindamycin (57%) and high resistance levels (>20-50%) to ciprofloxacin, oxacillin, levofloxacin and daptomycin, confirming the L. monocytogenes resistance trend to a wide range of antimicrobial agents. A total of 11 L. monocytogenes isolates were multidrug resistant and 7 out of them were isolated from slaughtered pigs. An interesting significant (p < 0.05) statistical correlation has been found between resistance to some antimicrobial agents and lineage/serotypes. Microbiological sampling of food and environments after sanitization are commonly used as verification procedure for the absence of L. monocytogenes in food plants and to give assurance of food safety, but strains characterization is necessary for industries to target specific control measures, like the enforcement of the hygiene program and of the control of operator activities, at least for permanent strains. The only presence of L. monocytogenes could not be considered as the conclusive assessment of a potential risk for public health, also in terms of emerging and emerged antimicrobial resistances.

Whole-Genome Sequence of Listeria monocytogenes Serovar 1/2a Strain IZSAM_Lm_15_17439_A144, Representative of a Human Outbreak in 2008.

Here, we report the genome sequence of Listeria monocytogenes serovar 1/2a strain IZSAM_Lm_15_17439_A144, isolated in Italy from a patient during a Listeria monocytogenes outbreak in 2008. This strain showed 98.9% sequence identity to a strain isolated in Canada in the same year.

A severe outbreak of listeriosis in central Italy with a rare pulsotype associated with processed pork products.

PURPOSE: From May 2015 to March 2016, an outbreak due to Listeria monocytogenes serotype 1/2a and clinical pulsotype never previously isolated in Europe occurred in central Italy, involving 24 confirmed clinical cases. The article provides a description of the outbreak and the investigation carried out by a multidisciplinary network. METHODOLOGY: Epidemiological and microbiological surveillance was conducted to confirm the outbreak and to detect the food vehicle of infection. The origin and destination of the implicated food and its ingredients were investigated by tracing-back and -forward investigation. RESULTS: Next-generation sequencing confirmed the unique outbreak strain. On 4 January 2016, a L. monocytogenes strain with pulsotype indistinguishable from that isolated from clinical cases in the outbreak was detected in a sample of hog head cheese purchased from a retail supermarket by one of the patients. The hog head cheese was produced by a small meat processing plant in the Marche region, where microbiological investigation confirmed environmental and food contamination by the outbreak strain. Plant production was suspended and all contaminated batches of the hog head cheese were withdrawn from the market by 19 February by local health authority. We subsequently observed a sharp decline in clinical cases, the last being reported on 11 March 2016. CONCLUSION: The key factor in the timely conclusion of this investigation was intersectoral collaboration among epidemiologists, microbiologists, veterinarians, statisticians and health and food safety authorities at national, regional and local levels.

Optimization of a Real Time PCR based method for the detection of Listeria monocytogenes in pork meat.

The aim of this study was to optimize a Real-Time PCR protocol for a rapid detection of Listeria monocytogenes in pork meat, using reduced volumes of primary selective enrichment broth and times of incubation to decrease the cost and time for analysis. Forty-five samples of pork meat were artificially contaminated with two different levels of L. monocytogenes (1-10 CFU per sample and 10-100 CFU per sample), homogenized in three different volumes of Half Fraser Broth (1:3; 1:5 and 1:10) and incubated at 30°C ± 1°C for 5h, 8h and 24h. The detection was conducted in parallel by Real-Time PCR and the ISO standard 11290-1 methods. L. monocytogenes was detected in all the samples after 24h by Real-Time PCR method, also using reduced volumes of Half Fraser Broth. This represents a clear advantage as the time to final detection and the inherent costs were significantly reduced compared to the ISO reference method. All samples artificially contaminated were correctly detected also after 8 of incubation at 30°C ± 1°C in Half Fraser Broth and 24h in Fraser Broth at 37°C ± 1°C using cultural method.

Reducing time in the analysis of Listeria monocytogenes in meat, dairy and vegetable products.

The microbiological standard for detection of Listeria monocytogenes relies on several cultural steps and requires 7 days for final confirmation, and due to food distribution and market demands, there is a prevailing need for an alternative methodology for its detection. The aim of this study was to compare different detection strategies based on real-time PCR (RTi-PCR) for a rapid and sensitive detection in an ample range of food products: raw pork and poultry meat, raw sheep milk cured cheese, and ready to eat lettuce salad. Four parameters were evaluated to reduce the time and cost for final results: the initial sample size (25 and 50 g), the dilution of the sample (1:3; 1:5 and 1:10 dilutions in Half Fraser broth), the incubation times (6, 10 and 24h) and the bacterial DNA extraction (simple boiling of the culture after washing the bacterial pellet, the use of the Chelex resin, and a commercial silica column. The results obtained demonstrate that a combination of an incubation in Half-Fraser for 24h of a 1:10 diluted-25 g-sample coupled to a DNA extraction using a commercial silica column and a real-time PCR assay detected down to 2-4 L. monocytogenes CFU per sample in less than 27 h in different types of food products. This RTi-PCR-based method is fully compatible with the ISO standard, providing results more rapidly and cost-effectively. The results were confirmed in a large number of naturally contaminated food samples with at least the same analytical performance as the reference method.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

European validation of a real-time PCR-based method for detection of Listeria monocytogenes in soft cheese.

The classical microbiological method for detection of Listeria monocytogenes requires around 7 days for final confirmation, and due to perishable nature of RTE food products, there is a clear need for an alternative methodology for detection of this pathogen. This study presents an international (at European level) ISO 16140-based validation trial of a non-proprietary real-time PCR-based methodology that can generate final results in the following day of the analysis. This methodology is based on an ISO compatible enrichment coupled to a bacterial DNA extraction and a consolidated real-time PCR assay. Twelve laboratories from six European countries participated in this trial, and soft cheese was selected as food model since it can represent a difficult matrix for the bacterial DNA extraction and real-time PCR amplification. The limit of detection observed was down to 10 CFU per 25 of sample, showing excellent concordance and accordance values between samples and laboratories (>75%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (82.75%, 96.70% and 97.62%, respectively) when the results obtained for the real-time PCR-based methods were compared to those of the ISO 11290-1 standard method. An interesting observation was that the L. monocytogenes detection by the real-time PCR method was less affected in the presence of Listeria innocua in the contaminated samples, proving therefore to be more reliable than the reference method. The results of this international trial demonstrate that the evaluated real-time PCR-based method represents an excellent alterative to the ISO standard since it shows a higher performance as well as reduce the extent of the analytical process, and can be easily implemented routinely by the competent authorities and food industry laboratories.

Listeria monocytogenes in Ready-to-Eat Seafood and Potential Hazards for the Consumers.

The risk of exposure to Listeria monocytogenes (L. monocytogenes) when consuming Ready-to-Eat (RTE) seafood was assessed in the Veneto Region (Italy). Thirty-eight samples were analyzed, each sample consisted of three subunits belonging to the same batches. The first of the three units was examined immediately, the second was stored at +4°C (for all of its shelf-life) and the third at +10°C (for the latter third of its shelf-life) before the analysis. Chemical-physical and microbiological parameters were tested simultaneously. Culture results showed the presence of viable L. monocytogenes in 9 (23,68%) of the 38 samples analysed, 3 (33,33%) of which with a concentration >100 cfu/g. PCR tests yielded 12 L. monocytogenes positive samples. Semipreserves with aw (water activity) and pH values that favour L. monocytogenes growth were the only ones to result positive to microbiological and PCR tests. Temperature proved to be an important factor as it limits the growth of L. monocytogenes, including products with potentially high competitive microbial charges. Four different serotypes were recovered and ribotyping has helped to highlight the genomic variability of L. monocytogenes strains in food. This supports the hypothesis that L. monocytogenes continues to evolve genetically to the detriment of phenotypic conservation.

Listeria monocytogenes serotypes in human infections (Italy, 2000-2010).

BACKGROUND: In developed countries invasive listeriosis is an infection of great concern to public health to due its clinical severity and high fatality rate, despite its low incidence. In Europe, statistically significant increasing trends in listeriosis notification rates from 2005 to 2009 were noted in Austria, Denmark, Hungary, Italy, Spain and Sweden. MATERIALS AND METHODS: The standardized techniques based on phenotype to typing Listeria monocytogenes is the serotyping. In Europe, as elsewhere in the world, about 95% of L. monocytogenes strains isolated from clinical and food samples belongs to serovars 1/2a, 1/2b, 1/2c and 4b. RESULTS: The target of this work is to draw attention to this important and atypical foodborne disease, reporting epidemiological data and serotypes distribution of 251 human L. monocytogenes isolates reported during 2000-2010 to Veterinary Public Health and Food Safety Department of Istituto Superiore di Sanità, focusing on epidemiological trend of invasive listeriosis in Lombardia, a North Italian Region. The serotypes most frequently identified are 1/2a, 4b, 1/2b (in total 92%), but the detection of uncommon serotypes is not missing (1/2c, 3a, 3b, 4d). CONCLUSIONS: In Italy the surveillance laboratory network, as well as the foodborne disease network (ENTER-NET), has revealed in the last 11 years an increase trend of listeriosis cases reported likewise with results of Notificable National Infectious Disease surveillance System. This is probably due to a real increase of listeriosis, even if there is a greater sensitivity of the network in some regions.

PCR experion automated electrophoresis system to detect Listeria monocytogenes in foods.

Listeria monocytogenes is frequently found as a contaminant in raw and ready-to-eat foods. The ability of L. monocytogenes to multiply at refrigeration temperatures and to grow in a wide range of pH values is of particular concern for food safety. According to the European Union regulation on microbiological criteria for foodstuffs, L. monocytogenes must be absent in some categories of ready-to-eat foods. The standard microbiological method for L. monocytogenes detection in foods (ISO 11290-1: 1996 (ISO, International Organization for Standardization)) is cost and time consuming. Developments of rapid, cost-effective and automated diagnostic methods to detect food-borne pathogens in foods continue to be a major concern for the industry and public health. The aim of this study was the development of a rapid, sensitive and specific molecular detection method for L. monocytogenes. To this purpose, we have applied a capillary electrophoresis method to a PCR protocol (PCR-EES (EES, experion automated electrophoresis system)) for detecting L. monocytogenes in food. In particular, a microfluidic chip-based automated electrophoresis system (experion automated electrophoresis system, Bio-Rad Laboratories, USA) was used for the rapid and automatic analysis of the amplicons. Fifty naturally contaminated samples were analysed with this method and the results were compared with those obtained with ISO method. Moreover, the microfluidic chip-based automated electrophoresis system was compared with classical gel electrophoresis (PCR-CGE). The results showed that after 24 h of culture enrichment, the PCR-EES showed a relative accuracy of 100% with ISO, while using PCR-CGE decreased it down to 96%. After 48 h of enrichment, both PCR-EES and PCR-CGE showed an accuracy of 100% with ISO.

Distribution of serotypes and pulsotypes of Listeria monocytogenes from human, food and environmental isolates (Italy 2002-2005).

This work was undertaken to study the serotypes and pulsotypes of 674 Listeria monocytogenes isolates from human (57), food (558) and environmental (59) sources, collected from different Italian geographical areas during 2002-2005, to determine whether certain subtypes were associated with certain foods and more often involved in cases of listeriosis, and to determine possible geographical or temporal associations. Eleven different L. monocytogenes serotypes were found in the food, environmental and human isolates. Most isolates belonged to only four serotypes (1/2a, 1/2b, 1/2c, 4b). The isolates were divided into 133 distinct AscI pulsotypes grouped into 26 pulsogroups. Pulsogroups ranged from a minimum of 2 up to 212 isolates, and contained 1-19 different pulsotypes. When associations between subtypes and isolates from specific foods selected as being most frequently involved in cases of listeriosis were tested some of these associations were highly significant but not exclusive, indicating that there was no close correlation between specific subtypes and specific food products. Despite the limitations of this study (few human isolates versus many food isolates prevalently collected from one food category), we believe that a large-scale database of L. monocytogenes subtypes and a timely epidemiological investigation can facilitate risk assessment and outbreak detection and control.

Distribution of epidemic clonal genetic markers among Listeria monocytogenes 4b isolates.

Recent genome sequencing of isolates of Listeria monocytogenes serotype 4b implicated in some major outbreaks of foodborne listeriosis has revealed unique genetic markers in these isolates. The isolates were grouped into two distinct epidemic clones, ECI and ECII. In the present study, selected ECI- and ECII-specific genetic markers were detected in 16 and 15 of 89 L. monocytogenes 4b isolates, respectively. The ECI markers were found in 6 of 34 clinical isolates, 9 of 50 food isolates, and 1 of 5 environmental isolates, and the ECII markers were detected in 7 of 34 clinical isolates, 7 of 50 food isolates, and 1 of 5 environmental isolates. Hence, of the isolates with the epidemic clonal genetic markers, 38% (13 of 34) were of clinical origin, 32% (16 of 50) were of food origin, and 40% (2 of 5) were of environmental origin. The predominance of the epidemic clonal markers among the clinical and environmental isolates supports the hypothesis that these markers are correlated with the pathogenic potential of strains and with their environmental persistence. Several isolates had only one epidemic clonal marker, either the ECI-specific marker 133 or the ECII-specific marker 4bSF18. Pulsed-field gel electrophoresis analysis revealed higher genomic diversity among the strains with ECII-like characteristics than among those strains carrying the ECI-specific genetic markers.

Incidence of Listeria monocytogenes in food and environmental samples in Italy between 1990 and 1999: serotype distribution in food, environmental and clinical samples.

We report the findings of the study of 4185 food samples and 958 environmental samples collected in Italy in the period 1990-1999 and tested for the presence of Listeria monocytogenes. The strains isolated were biochemically and serologically characterised. We found a fairly high percentage of L. monocytogenes contamination in food (12.8%), whereas the level of contamination was lower in the environment (environment and work surfaces in food processing plants) (6.1%). Serotyping showed a prevalence of a few serotypes (i.e., 1/2a, 1/2b, 1/2c and 4b), which were the same as those found in clinical samples collected during outbreaks and from sporadic cases of listeriosis reported in Italy in the period considered. The geographical distribution of the strains of L. monocytogenes isolated from food samples is very similar to that of the clinical strains.