Polimos

System. Appl. Microbiol. 25, 456–461 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/sam

Amplified Fragment Length Polymorphism (AFLP) Analysis of Listeria monocytogenes
M. M. GUERRA1, F. BERNARDO1, and J. MCLAUCHLIN2,*
1 2

CIISA / Laboratório de Inspecção Sanitária, Faculdade de Medicina Veterinária Universidade Técnica de Lisboa, Portugal Food Safety MicrobiologyLaboratory, Division of Gastrointestinal Infections, Public Health Laboratory Service Central Public Health Laboratory, UK

Received: July 11, 2002

Summary
An agarose gel based single enzyme AFLP method using EcoR1 digestion of Listeria monocytogenes DNA was developed for epidemiological typing. The method was evaluated with 84 L. monocytogenes cultures, and results were compared with thoseobtained with serotyping, phage-typing and cadmium and arsenic resistance typing. The AFLP method was reproducible and 14 different banding patterns comprising between five and eight DNA fragments were produced. All except two of the AFLP patterns were serotype specific. Different AFLP patterns were recognised within serovar 4b (four patterns), 1/2a (two patterns), 1/2b (six patterns): singlepatterns were obtained from cultures of serovars 1/2c, 3a, 3b and 3c. There were associations with AFLP results and those from phage-typing and cadmium and arsenic resistance typing, although each method showed some independence. This preliminary evaluation suggests that this AFLP method will be useful for epidemiological typing of L. monocytogenes. Key words: Listeria monocytogenes – amplified fragmentlength polymorphism (AFLP) – epidemiological typing

Introduction
Listeriosis attracted considerable attention during the 1980s following a series of large foodborne outbreaks and a general increase in incidence in both Europe and North America (MCLAUCHLIN, 1996). The numbers of cases of listerioisis has now declined in most of Northern Europe and North America. The reported incidence inEngland and Wales was 1–2 cases per million of the population between 1995–1999 (SMERDON et al., 2001) and in France 5.4 cases per million in 1997 (GOULET et al., 2001). However, after the mid 1990s, large outbreaks of human foodborne listeriosis have continued to occur in the USA (ANON, 1999; ANON, 2000; ANON 2001), France (DE VALK et al., 2001), Italy (AURELI et al., 2000) and Finland (Maijala et al.,2001). It has been estimated that in the USA, listeriosis is the second largest identified cause of death from a foodborne disease (MEAD et al., 1999). Epidemiological typing is an essential tool for the surveillance of listeriosis, including the recognition of outbreaks, and therefore justifies continued analysis of the causative organism, Listeria monocytogenes. Amplified fragment lengthpolymorphism (AFLP) has successfully been applied to bacteria from a wide variety of different genera (ZABEAU and VOS, 1993; VOS et al., 1995; JANSSEN et al., 1996; GIBSON et al., 1998). This method involves the digestion of total purified bacterial DNA using one or more restriction enzymes, followed by the ligation of the resulting fragments to a double stranded oligonucleotide adapter complementary tothe base sequence of the restriction site. The adapters are designed such that the original restriction site is not restored after ligation, thus preventing further restriction digestion. Selective amplification by PCR of sets of these fragments is achieved using primers corresponding to the contiguous base sequences in the adapter, restriction site plus one or more nucleotides in the originaltarget DNA. The resulting patterns of PCR-amplified DNA fragments are then analysed by electrophoresis. AFLP analysis of L. monocytogenes has been described previously (AARTS et al., 1999; RIPABELLI et al., 2000). One of these systems (AARTS et al., 1999) utilised
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AFLP analysis of Listeria monocytogenes

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an automatic laser fluorescence sequencer which...