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.

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.

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.

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.

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 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.