Direct Identification in Food Samples of Listeria spp. and Listeria monocytogenes by Molecular Methods

Abstract

A new molecular approach for the detection and identification of Listeria spp. and Listeria monocytogenes in food is presented here. The method is based on the PCR amplification of a fragment of the iap gene from the five species belonging to the genus and on the analysis of the PCR products obtained by denaturing gradient gel electrophoresis (DGGE). The protocol was first optimized by using strains from international collections. Based on the differences present in the sequences amplified, it was possible to obtain species-specific DGGE migration that allowed fast and easy identification of L. monocytogenes, L. innocua, L. welshimeri, L. seeligeri, and L. ivanovii. Moreover, for L. monocytogenes serotypes, partial differentiation was possible. The optimized protocol was used for identification of Listeria strains traditionally isolated from food and for direct detection and identification of Listeria members in food after an overnight enrichment. Identification of 48 food isolates and direct detection of Listeria spp. in 73 food samples show the potential of the method that can be used as a fast screening test to investigate the presence of Listeria spp. and L. monocytogenes in food.The genus Listeria comprises a group of ubiquitous, grampositive, nonsporulating bacteria. Previous results of DNA-DNA hybridization experiments unambiguously demonstrated that the species formerly called "Listeria monocytogenes" in fact contained five genomic groups deserving the species rank: and L. seeligeri (48, 49). This subdivision has proven its usefulness for practical and epidemiological purposes, especially when problems caused by food contamination resulted in human listeriosis (29). Only the hemolytic species of Listeria, L. monocytogenes, L. ivanovii, and L. seeligeri, are associated with human pathogenicity. L. monocytogenes is the only species of the genus Listeria that has been involved in known food-borne outbreaks of listeriosis. L. ivanovii has been described to be involved in human pathology only rarely, and L. seeligeri has been reported only once to be the cause of meningitis in a nonimmunocompromised adult (35).Several large outbreaks of listeriosis have been associated with contaminated commercial foodstuffs, such as vegetables, milk, and meat products, on which these bacteria can multiply even at low temperatures (52). Usually, the presence of any Listeria species in food is an indicator of poor hygiene (40).Significant efforts have been dedicated to the development of enrichment media and protocols for L. monocytogenes and Listeria sp. isolation (1,10,11,15,24,47). Ideal enrichment media would facilitate recovery of injured Listeria cells and enrichment of Listeria spp. and L. monocytogenes over competing microflora. In traditional culture-based assays, it becomes very difficult to detect L. monocytogenes at any level when it is greatly outnumbered by other Listeria spp., such as L. innocua, which appears to be present together with L. monocytogenes (10, 51). Consequently, detection of Listeria spp. is oft...

Prevalence of Shiga toxin-producing Escherichia coli in food products of animal origin as determined by molecular methods

“…Pathogenic and spoilage microorganisms (with particular emphasis on enterobacteria) as well as technologically important microorganisms (mainly lactic acid bacteria and coagulase negative staphylococci), were considered. All strains belonged to the culture collection of the University of Turin and comprised international culture collection strains and strains isolated from foods and previously identified by sequencing of partial 16S rRNA gene (Cocolin et al, 2000(Cocolin et al, , 2002Rantsiou et al, 2006Rantsiou et al, , 2008 . The amplification, for all four genes, was carried out in 25 µl final volume, containing 12.5 µl of the 2X FluoCycle mix for probe (Euroclone, Milan, Italy), 1 µl of 10 µM of each of the primers, 0.625 µl of 10 µM of the probe and 1 µl of DNA.…”

Rapid Discrimination of Listeria monocytogenes Strains by Microtemperature Gradient Gel Electrophoresis

“…Standard TGGE/DGGE analysis requires, on average, 4 h for electrophoresis (6,20), whereas in the current study, the miniaturized -TGGE apparatus required only 7 min to resolve the amplicons. Even taking into account the time required to extract the genomic DNA and perform the PCR amplification, it was possible to complete the entire process within 2 h, which is a substantial improvement over the times required for other typing methods.…”

“…In previous studies, TGGE/DGGE analysis of 16S rRNA or inl gene fragments was used to detect the presence of L. monocytogenes in certain foods (6,20). These studies sought to distinguish L. monocytogenes from other Listeria spp., such as L. innocua.…”

“…Many studies have used these methods to analyze microbiological populations based on differences in amplified regions of the 16S rRNA gene (24,25). They have also been used to discriminate L. monocytogenes from other Listeria species, such as Listeria innocua (6,20). Although TGGE/DGGE has adequate discrimination power and is reproducible, it is time-consuming, generally requiring ϳ4 h per run.…”

High-Throughput Sequencing Technologies and new Approaches in Food Microbiology

“…The introduction of culture independent methods has allowed to understand and to overcome the limitation of microbial cultivation [3]. It was applied in food microbiology as tools to analyze foodstuff microbiota, to study fermentation and food spoilage processes and also to investigate the ecology of food-borne pathogens [4,5].…”