Direct Identification of Listeria spp by molecular methods.
Identificazione diretta della Listeria spp, attraverso metodi molecolari
Barocci S.*, Malvestiti A.**, Valli M. B.*
[* Istituto Zooprofilattico Sperimentale del'Umbria e delle Marche; ** Università degli Studi di Camerino]
Summary: in the last decades, the widespread use of ready-to-eat industrial foods, has greatly increased the incidence of foodborne Listeriosis in subjects whose immunity is impaired. The aim of the present study is to optimize the molecular multiplex-PCR-based method described by Buber et al (1999) in order to detect and identify all the species of the genus Listeria simultaneously within 2-3 days, from food samples, or maximum within 3-4 hours, from colony on agar plates.
Riassunto: nell'ultimo decennio, la diffusione dell'industria dei cibi pronti ha incrementato l'incidenza delle tossinfezioni alimentari sostenute da Listeria, specialmente in soggetti immunodepressi.
Lo scopo del presente studio è di indicare dei metodi per ottimizzare il metodo multiplex-PCR descritto da Buber et al (1999), allo scopo di rilevare simultaneamente tutte le specie appartenenti al genere Listeria in 2-3 giorni, da campioni di cibo, (det. entro 3-4 ore da colonie coltivate su agar gel).
INTRODUCTION
The genus Listeria comprises six species: L. monocytogenes, L. innocua, L. seeligeri, L. welshimeri, L. ivanovii and L. grayi. L. monocytogenes is the only species of the genus that has been involved in food-borne outbreaks of Listeriosis in humans and it is the only one considered to be of public health significance.
However, infections with L. ivanovii and L. seeligeri may occur.
Human foodborne listeriosis represents a relatively rare but clinically serious disease, it has the second highest fatality rate (21%) and the highest hospitalisation rate (90%) of all food-borne pathogens (Centres for Disease Controls and Prevention, 2001).
In humans, clinical symptoms range from influenza-like vomiting, nausea and diarrhoea to septicaemia, meningitis (or meningoencephalitis), and in pregnant women the infection may result in abortion or premature birth. In animals, pathogenesis of Listeriosis is almost similar to human being.
Current microbiological standard methods to detect this bacteria's family have all in common two necessary phases of investigation: isolation and identification.
The isolation method adopted by IZSUM (ISO 11290 certification) is based on a pre-enrichment in HFB (Half Fraser Broth) followed by subsequent selective enrichment in FB (Fraser Broth). After incubation, cultures are streaked onto selective agar plates, just to isolate the presumed colonies of L. monocytogenes. In this study ALOA (Agar Listeria acc. to Ottaviani & Agosti) and OXFORD (Listeria Oxford Agar Base) were used.
Colonies, grown on agar plate, are selected for a further identification step, using the API Listeria kit (BioMérieux®, France).
Traditional culture-based assays are laborious, time-consuming and require up to 1 week for species identification. Moreover, because of the high phenotypic similarity in Listeria genus, differentiation between species is not always reached. For these reasons, little is known about the occurrence and distribution of Listeria species other than L. monocytogenes.
To overcome these problems, since the late 1980s several molecular approaches to detect Listeria spp have been developed. However, most of them enable a more rapid detection of the genus Listeria and/or only Listeria monocytogenes, thus lacking the ability to easily characterize all the species of Listeria.
In this paper we described a novel molecular method to directly identify all the members of the genus Listeria. A multplex-PCR strategy coupled whit RFLP allowed a simultaneous identification of Listeria spp in food samples, based on specific gel electrophoresis migration patterns.
The method described could represent an interesting tool to monitor the spread of the genus Listeria in food and in environmental samples and for epidemiological purposes as well.
MATERIALS AND METHODS
The Listeria species used for the molecular investigations are: L. monocytogenes, L. innocua, L. welshimeri, L. ivanovii and L. seeligeri. L. grayi is excluded from the study because it has a limited diffusion and it has not clinical relevance.
Stored at -20°C in Microbanks® beads (Pro-Lab Diagnostics, Canada), each species has been re-suspended in a non-selective enrichment broth; after incubation (37°C for 12 hours), valued an estimable growth, the broth is streaked on Oxford and ALOA plates to check the strains' pureness.
DNA extraction
Two distinct DNA extraction methods were used: the first one employed the QIAamp® DNA Mini kit, produced by QIAGEN, while the second one was a simple direct water boiling of colonies grown on agar plates.
The aim of comparing the two methods is to verify if the simplest, rapid and economic boiling method gives results similar to those obtained by the commercial kit, considered as reference (not only for quantity, but also for quality of DNA extracted).
Multiplex PCR
Bubert A. et al. have selected a combination of five different primers that allow the differentiation of Listeria species with a single multiplex PCR reaction followed by gel analysis. For this purpose, the iap gene, common to all members of the genus Listeria, has been used as PCR target because it has conserved regions at the 5' and 3' ends, while the internal portions is species-specific. The iap gene of L. monocytogenes encodes the extracellular protein p60, a hydrolase required for septum separation in the latest step during cell division.
By a simple reaction of amplification, the method enables the simultaneous detection of Listeria species and differentiation between L. monocytogenes, L. innocua, and a group containing very rarely occurring species (Siwi group: L. seeligeri, L. welshimeri and L. ivanovii).
The primers used were:
MonoA (5'-CAAACTGCTAACACAGCTACT-3') specific for L. monocytogenes;
Ino-2 (5'-ACTAGCACTCCAGTTGTTAAAC-3') specific for L. innocua;
Siwi-2 (5'-TAACTGAGGTAGCGAGCGAA-3') specific for L. ivanovii, L. seeligeri and L. welshimeri group;
Lis1B (5'-TTATACGCGACCGAAGCCAAC-3') common to all species.
Anyway, this combination of primers can't differentiate between the three species L. ivanovii, L. seeligeri and L. welshimeri (Buber et al, 1999).
In our study we have improved such a molecular strategy introducing a restriction analysis that enables the simultaneous detection of Listeria species.
By GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) and BLAST analysis (http://www.ncbi.nlm.nih.gov/BLAST/), the complete iap gene sequences of each Listeria species selected were obtained and theoretical specificity of all the primers used were controlled.
Restriction analysis
The restriction analysis of the iap gene PCR products permit the identification of each Listeria genus species. Moreover, it enables not only to strengthen furthermore the discrimination obtained by multiplex PCR, but also to differentiate specifically the three species grouped in Siwi.
In order to find an appropriate restriction enzyme that could allow the discrimination of all species, we used Restrictionmapper available on line (www.restrictionmapper.org). Through the restriction tool of this website, it was possible to find a unique endonuclease (DdeI) ideal for this purpose and to simulate a virtual digest of our PCR products, as reported in Fig. 1.
The endonuclease used in this analysis, DdeI, recognizes and cleaves the following DNA-sequence:
The quantity of DNA is critical for restriction analysis: the experimental evidences suggested that the digested PCR products are estimable only, when after electrophoresis the bands of amplified DNA show an intensity equal to that of 500 bp band (corresponding approximately to 97 ng) of 1 ?L of 500 ?g/mL DNA ladder used (100 bp DNA ladder, New England BioLabs, USA).
When this occurs, it is possible to visualize clear bands of the restriction products.
RESULTS
In all Listeria species the iap gene which encodes the extracellular protein p60 contains a variable region whit a small tandem repeat domain. In fact, the PCR results showed products only approximately comparable to those indicated in the paper of Bubert et al.
That's why, in this study, we obtained PCR products with an average in bp as shown in the following table:
Species
Attended PCR
product (Bubert, 1999)
PCR product*
(BLAST average)
L. monocytogenes
660
715
L. innocua
870
1000
Siwi group
(seeligeri, ivanovii, welshimeri)
1200
1350
* The values here reported have been calculated as the mean of all the theoretical amplified sequences of all the species (using all the primers previously described) found in GenBank in December 2005. In Fig. 1 and Fig. 2 we demonstrated that these are the real amplicons found amplifying all the strains tested in our Lab.
Both DNA extraction methods guarantee comparable and repeatable results of PCR analysis (Fig. 2 and Fig. 3).
For what concerns the enzymatic restriction reaction, it was possible to verify the superposition of restriction patterns resulted from the analysis carried out in our Lab and the virtual digest products obtained using Restrictionmapper. Both of them showed the follow electrophoretic prospect:
Even if DdeI endonuclease hasn't cutting sites in the iap gene of L. monocytogenes, the enzymatic restriction is a good discriminating tool for all Listeria species (Fig. 4).
During the year 2005 214 DNA were extracted from colonies grown on selective agar plates, derived from official food products, and subjected to PCR and RFLP analysis. The molecular protocol developed in this study, enabled the identification of Listeria monocytogenes in 153, of Listeria innocua in 56 and of L. welshimeri in 5 of all the analysed colonies.
The technique here described allows to detect all the species of the genus Listeria as a support for traditional isolation methods and could be applied both in Food Safety and in Animal Health.
CONCLUSIONS
The research data described in this paper has allowed:
the optimization of an effective technique of preventive-screening of Listeria spp;
- the confirmation of biochemical tests and/or identification kit for the identification of all the species of the genus Listeria;
- the rapid detection of all Listeria species within 2-3 days, by matrix, or maximum within 3-4 hours, by colony grown on agar plates;
- the evaluation of the efficacy of directly boiling colonies from agar plates as DNA extraction method and the comparability, according to the results, with commercial methods (such as QIAamp® DNA Mini kit), minimizing the cost of the whole analysis.
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