Amplified fragment length polymorphism (AFLP) analysis of Clostridium perfringens for epidemiological typing

International Journal of Food Microbiology 56 (2000) 21–28 www.elsevier.nl / locate / ijfoodmicro

Amplified fragment length polymorphism (AFLP) analysis of Clostridium perfringens for epidemiological typing 1

J. McLauchlin*, G. Ripabelli , M.M. Brett, E.J. Threlfall Division of Gastrointestinal Infections, Public Health Laboratory Service Central Public Health Laboratory, 61 Colindale Ave, London NW9 5 HT, UK Received 13 November 1999; accepted 6 January 2000

Abstract Thirty-five Clostridium perfringens isolates from patients and foods implicated in seven outbreaks of suspected Cl. perfringens food poisoning together with five unrelated incidents were analysed by serotyping and amplified fragment length polymorphism (AFLP). Despite minor band differences, AFLP was found to be highly reproducible and 16 different profiles (each unique to the 12 incidents) were recognised. The results from both serotyping and AFLP analysis identified exactly the same groups of related cultures. It is concluded that AFLP can provide a rapid, sensitive and reproducible method for the typing of Cl. perfringens for outbreak investigation.  2000 Elsevier Science B.V. All rights reserved. Keywords: AFLP; Clostridium perfringens; Epidemiological typing

1. Introduction Clostridium perfringens is probably the most widely occurring bacterial pathogen and is widespread in soil and the intestinal contents of animals and humans (Hathaway and Johnson, 1998). It is consequently common in a variety of foods, particularly meat and poultry, and the endospores produced by this bacterium will survive some cooking processes. Although previously suspected as a *Corresponding author. Tel.: 1 44-020-8200-4400; fax: 1 44020-8200-8264. E-mail address: [email protected] (J. McLauchlin) 1 Current address: University of Molise, Department of Animal, Plant and Environmental Science, Chair of Hygiene, Via de Sanctis, 86100 Campobasso, Italy.

cause of food poisoning, the importance of Cl. perfringens as a cause of diarrhoea was first conclusively shown in 1946 by Cravitz and Gillmore who reproduced the illness in both man and animals by oral administration of live cultures (Berry and Gilbert, 1991). It is now established that the diarrhoeal disease is caused by the production of enterotoxin during sporulation in the intestine (Berry and Gilbert, 1991). Cl. perfringens causes a range of diarrhoeal diseases which can be foodborne, infectious or antibiotic associated. Foodborne infection occurs 8– 24 h after consumption of food containing large numbers of vegetative cells, and results in severe diarrhoea and abdominal pain with a duration of 10–24 h. Fatalities occasionally occur among debilitated persons, particularly geriatric patients. Out-

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breaks are generally caused by meat and poultry dishes which were cooked too far in advance and / or improperly stored. Between 1980 and 1995 more than 17 500 cases of Cl. perfringens food poisoning were reported to the Public Health Laboratory Service in England and Wales, the annual totals varied between 342 and 1716 cases per year (PHLS, unpublished data). However a recent study of infectious intestinal disease in England suggests that these totals seriously underestimates the incidence of this disease (Tompkins et al., 1999). There are several laboratory criteria which confirm a diagnosis of Cl. perfringens food poisoning and these include the isolation of the same strain of the organism from the majority of patient’s faeces, and if available, the incriminated food, together with the detection of Cl. perfringens enterotoxin in the faeces. Hence, for both diagnosis of Cl. perfringens food poisoning, and for methods to facilitate epidemiological investigations, there is a need for suitable discriminatory typing systems for this bacterium. For epidemiological typing of this organism both phenotypic and genotypic methods have been investigated, including the use of bacteriocins, bacteriophages, plamid analysis (Schalch et al., 1998), pyrolysis mass spectrometry (Sissons et al., 1992), ribotyping (Forsblom et al., 1995; Schalch et al., 1999) and pulsed-field gel electrophoresis (Schalch et al., 1999). However, because these methods have inherent problems such as an inability to type some organisms, expense and complex methodologies and analysis, they have not been widely adopted for outbreak investigation. A serological typing scheme based on the use of ‘in-house’ produced antisera

recognising capsular antigens is currently used in the PHLS Food Safety Microbiology Laboratory (FSML), and this identifies more than 200 serotypes (Stringer et al., 1980, 1982, 1985). However, because of the difficulties in producing antisera, the inability of some strains to produce recognised antigens, and for occasional cultures which autoagglutinate (Berry and Gilbert, 1991) there is a need for additional methods to type this organism. Amplified fragment length polymorphism (AFLP) (Zabeau and Vos, 1993; Vos et al., 1995; Janssen et al., 1996) has been successfully applied to the epidemiological typing of bacteria from several different gram-negative and gram-positive genera (Keim et al., 1997; Picardeau et al., 1997; Gibson et al., 1998; Boumedine and Rodolakis, 1998; Struelens et al., 1998; Geornaras et al., 1999; Duim et al., 1999; Van Eldere et al., 1999). This method involves the digestion of total purified bacterial DNA using a restriction enzyme, followed by the ligation of the resulting fragments to a double stranded oligonucleotide adapter which is complementary to the base sequence of the restriction site. The adapters are designed such that the original restriction sites are not restored after ligation, thus preventing further restriction digestion. Since the adapters are not phosphorylated, adapter-to-adapter ligation is prevented. 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 original target DNA (Fig. 1). The resulting DNA fragments amplified by PCR are then analysed by gel electrophoresis.

Fig. 1. HindIII restriction site, adapters and primer sequences for AFLP typing of Cl. perfringens. (1) Fragment produced from HindIII digestion (recognition site A↓AGCTT). (2) Sequences of the two complementary oligonucleotides forming the adapter which is ligated to each end of each restriction fragments. The underlined nucleotides indicate the bases inserted into the adapter sequence to eliminate the restriction of this site after ligation. (3) Sequence of the primer used in subsequent amplification step. X indicates the selective base inserted into the primer.

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The purpose of this study was to develop a simple AFLP procedure for typing Cl. perfringens, and compare this system with the results obtained using serotyping coupled with epidemiological data.

2. Materials and methods

2.1. Cultures of Cl. perfringens Thirty five cultures of Cl. perfringens were selected from the FSML culture collection and were preserved at 2 408C on glass beads in glycerol medium (Microbank, Pro-Lab Diagnostics, Neston, UK). The growth of cultures was achieved using gas jars and the Anaerobic Gas Generation System (Oxoid Ltd, Basingstoke, UK). The identity of all cultures was confirmed by characteristic colonial morphology on Colombia blood agar, a positive Nagler reaction, and these were analysed by serotyping as described previously (Berry and Gilbert, 1991; Stringer et al., 1980, 1982, 1985). The isolates were recovered from 12 separate incidents: brief epidemiological information was available for each incident (Table 1).

2.2. DNA extraction For AFLP, cultures were grown on 5% horse blood columbia agar (Oxoid, Basingstoke, UK) and incubated at 378C overnight. Total cellular DNA was extracted using a modification of the method of Boom et al. (1990). Approximately 100–150 mg wet weight of bacterial cells (equivalent to the growth harvested from one 9-cm diameter petri-dishes) was washed twice by centrifugation in 1 ml of ice cold sterile physiological saline, and suspended in 900 ml of L6 buffer (10 M guanidinium thiocyanate in 0.1 M Tris–HCl, pH 6.4 plus 35 mM EDTA, pH 8, 2% (w / v) Triton X-100) plus 60 ml of isoamyl alcohol and 0.3 g of 0.1-mm diameter zirconium beads (Stratech Scientific Ltd, Luton, UK). The tubes were vortex mixed for 1 min at maximum speed and left at room temperature for 5 min before centrifugation at 13 400 3 g for 15–30 s. The particulate material was discarded and 100 ml of size-fractionated silica (Severn Biotech Ltd, Kidderminster, UK) was then added to the supernatant which was gently mixed for 10 min at room temperature. The suspension was centrifuged as above and the supernatant discarded.

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The silica was washed by centrifugation, twice with 200 ml of L2 buffer (10 M guanidinium thiocyanate in 0.1 M Tris–HCl, pH 6.4), twice with 200 ml of 80% cold ethanol and once with 200 ml of cold acetone, after which the pellet was dried for 10 min at 568C. This was then resuspended in 100 ml of sterile distilled water, vortexed briefly and incubated for 5 min in a 568C. The supernatant was collected by centrifugation as above and the DNA concentration and purity estimated by absorbance at 230, 260 and 280 nm as described previously (Gibson et al., 1998).

2.3. Restriction endonuclease digestion, ligation of adapters and PCR Restriction endonuclease digestion and ligation was performed using a modification of the method of Gibson et al. (1998). Briefly, 4 mg of DNA was added to 24 U of HindIII (Gibco Life Technologies, Paisley, UK), 5 mM spermidine trihydrochloride (Sigma, Poole, UK), resuspended with water to a final volume of 20 ml and incubated overnight at 378C. An aliquot of 5 ml of the digested DNA was added to 0.2 mg of the adapter oligonucleotides ADH1 and ADH2 (Gibco Life Technologies), 1 U of T4 DNA ligase and ligase buffer (Gibco Life Technologies) in a final volume of 20 ml and incubated at room temperature for 3 h. Ligated DNA was heated to 808C for 10 min, diluted 1 / 5 in sterile distilled water, and 5 ml were used for each PCR reaction. PCR reactions were performed in 50 ml final volumes and contained: 5 ml of ligated DNA; 2.5 mM MgCl 2 ; 300 ng of primer (either HI-A, HI-C, HI-G or HI-T; Gibco Life Technologies) and 1.25 U of Taq DNA polymerase in 1 X PCR buffer (Gibco Life Technologies). The mixture was subjected to an initial denaturing step of 948C for 4 min, followed by 35 cycles of 1 min at 948C, 1 min 608C and with 2.5 min at 728C. The base sequences of the ligation and amplification primers together with their relative position with respect to the HindIII restriction site are shown in Fig. 1. The amplified products were analysed on a 1.5% agarose gel containing ethidium bromide, observed by UV transillumination and photographed using Type 667 film (Polaroid Ltd, St Albans, UK). A 100-bp ladder (Gibco Life Technologies) was included at least twice on each electrophoresis gel. Banding patterns were assessed visually consider-

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Table 1 Results of AFLP analysis of 35 Cl. perfringens from seven food-poisoning and five other unrelated incidents a Incident number

Description

Source

Serotype

AFLP pattern

1

Routine food examination

Cooked chicken

27

A

2

Outbreak of diarrhoea in a mental health unit

Faeces, patient 1 Faeces, patient 2 Faeces, patient 3 Faeces, patient 4

52 NT 61 NT

B B C D

3

Outbreak of diarrhoea in a residential home

Faeces, patient 1 Faeces, patient 2 Faeces, patient 2 Faeces, patient 3 Faeces, patient 3 Faeces, patient 4 Faeces, patient 5 Faeces, patient 5 Faeces, patient 5 Faeces, patient 6 Faeces, patient 7 Faeces, patient 7

28 27 NT* 27 28 28 28 28 NT* 27 27 NT*

E I 11 I 11 I E E E E E I I I

4

Diarrhoea and abdominal pain in 16 people 13.5 h after consumption of a restaurant meal

Cooked chicken Rice mix

NT NT

L L

5

Diarrhoea and abdominal pain in $ 29 people, 12–14 h after consumption of a restaurant meal

Faeces Cooked chicken Cooked chicken Cooked chicken

63,66,72 1,44 1,44 1,44

M O O O

6

Diarrhoea in 16 people in residential home associated with cooked pork products

Cooked pork

NT

P

7

Diarrhoea in 39 people associated with consumption of beef bourgignon at wedding reception

Faeces Beef bourgignon

11 11

Q 11 Q

8

Diarrhoea in 12 people 10 to 13 h after consumption of a meal in an Indian restaurant Cl. perfringens was isolated at 10 4 –10 5 cfu / g of food

Beef curry Beef curry Faeces Faeces Faeces

55 55 55 55 55

R21 R R R R

9

Asymptomatic

Faeces

NT

S

10

Asymptomatic

Faeces

11

T

11

Asymptomatic

Faeces

TW9

U

No data

Faeces

71

V

12 a

NT 5 non-typable; * 5 rough cultures. AFLP types are designated by A-V, and each pattern varied by two or more bands from all other patterns. Similar profiles which varied by # 2 bands are designated by the same letter plus a superscript suffix denoting the number (either 1 or 2 ) of differences.

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ing only strong and moderately stained fragments. Analyses of the AFLP banding patterns were performed blindly and under code with respect to the results of serotyping and epidemiological data.

3. Results Initial experiments were performed to assess the suitability of the four selective primers HI-A, HI-C, HI-G and HI-T using five cultures randomly selected from different incidents. Results with one culture (pattern D from incident 2) are shown in Fig. 2. Using all four primers, the numbers of bands varied from 7 to . 25, and the intensity of the staining decreased with increasing numbers of fragments generated. The quality of fragments generated by HI-T and HI-C primers was low and were less distinct than those from HI-A and HI-G. The primer

Fig. 2. AFLP patterns produced by Cl. perfringens cultures with HI-G primer. Lanes 1, 6, 11 and 16 contain a 100-bp DNA ladder. Lanes 2–5 represent patterns A, B, C and L; lanes 7–10 represent patterns M, O, I and I; lanes 12–15 represent the initial experiment with the culture from incident 2 with primers HI-A, HI-T, HI-C and HI-G (pattern D).

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HI-A gave a more defined banding pattern, but showed limited variation between the five cultures tested. HI-G was therefore selected for further evaluation. All cultures were then tested using HI-G. Each culture generated between 9 and 14 DNA fragments of between approximately 200–1500 bp. No bands were common to all of the cultures tested. To initially assess the reproducibility of the system, PCR products stored at 48C for . 6 weeks and which were representative of each banding pattern were re-electrophoresed and identical banding patterns were recognised. DNA from three cultures was re-extracted, ligated and amplified after 20 serial subcultures: identical banding patterns were obtained with two of the cultures and a single band was lost with the third. Amongst all of the 35 cultures tested, 16 AFLP profiles were observed (designated A-V) which varied by two or more bands from all other patterns (Figs. 2 and 3). Similar profiles which

Fig. 3. AFLP patterns produced by Cl. perfringens cultures with HI-G primer. Lanes 1, 6, 11 and 16 contain a 100-bp DNA ladder. Lanes 2–5 represent patterns P, S, T and Q 11 ; lanes 7–10 represent patterns Q, U, V and R21 ; lanes 12–15 all represent pattern R.

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varied by # 2 bands are designated by the same letter plus a superscript suffix denoting the number (either 1 or 2 ) of differences. For example, the AFLP patterns obtained with isolates from patients 2 and 7 associated with incident 3 were identical except for a single extra band; the AFLP patterns were therefore designated I and I 11 in the isolates from patients 7 and 2, respectively. The distribution of the AFLP patterns between each of the 12 incidents is shown in Table 1. Each incident yielded a unique AFLP profile: more than one profile was detected in three of the incidents. The results of serotyping and AFLP identified exactly the same groups of related cultures. For example, in incident 5, both serotyping and AFLP showed that all three food isolates were indistinguishable, but these differed from the isolate from faeces. All five cultures associated with incident 8 are shown in Fig. 3: with the exception of a single band different in one culture, identical banding patterns were obtained with all isolates. Isolates from outbreak 2 (Table 1) yielded three different serotypes and AFLP types. There was incomplete evidence that this outbreak was due to Cl. perfringens, and these isolates may be reflecting the normal faecal flora. Three cultures were analysed from incident 3 which showed rough colonial forms, all of which could not therefore be serotyped: one of the cultures reacted weakly with the type 27 antisera. All three cultures yielded either AFLP profile E or I which were also identified in other cultures associated with this incident: rough and smooth cultures recovered from patient 7 showing an identical AFLP banding pattern.

4. Discussion Molecular methods based on the recognition of enzyme-generated DNA fragment patterns and the characterisation of DNA sequences are becoming increasingly used for the epidemiological typing of a wide range of bacteria. Amongst the techniques using PCR or restriction endonucleases, AFLP has a number of advantages and is easier to perform and requires simpler and less expensive equipment than PFGE. AFLP is potentially more reproducible than random amplification of polymorphic DNA (RAPD)

analysis, and does not require Southern-blotting followed by probing with labelled nucleotides such as is required by ribotyping (Janssen et al., 1996) or insertion sequence typing (IST; Stanley and Saunders, 1996) We have reported here an initial evaluation of a simple AFLP method for the epidemiological typing of Cl. perfringens. The use of an extraction using zirconium beads, guanidinium thiocyanate and activated silica (Boom et al., 1990) produces pure DNA suitable for AFLP, and is more rapid than previously described methods (Janssen et al., 1996; Gibson et al., 1998). In addition, DNA extracted by this method is also suitable for the AFLP typing of bacteria in other gram-positive genera (G. Ripabelli, unpublished data). Although minor variations of one fragment difference were detected, the AFLP method described here for Cl. perfringens was found to be reproducible and applicable to the analysis of all strains. The results presented here suggest that the method is highly discriminatory since a unique AFLP profile was recognised in each different incident and 16 different patterns were recognised amongst the 35 cultures tested. The observation that different AFLP patterns were detected in isolates of the same serotype but recovered from different incidents also suggests that this system is highly discriminatory. Because of the widespread distribution of this bacterium, several strains of Cl. perfringens may be simultaneously present in both food and faeces, and it was very encouraging that the same related strains were identified by AFLP typing as by serotyping. In Cl. perfringens, roughness of cultures may be associated with the loss of the capsular antigen. Cultures can be induced to revert to smooth forms by passage through experimentally infected animals (Stringer et al., 1982). It is particularly encouraging that both non-typable and rough strains can be analysed by AFLP and that within the non-typable cultures, seven different AFLP profiles were recognised. Since AFLP is less labour intensive than other molecular techniques, the technique is particularly applicable to the analysis of large numbers of isolates. Hence AFLP typing, together with serotyping, is likely to prove a valuable additional tool to investigate the epidemiology of Cl. perfringens food poisoning. For comparisons of limited numbers of isolates

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within an individual outbreak, the visual method for analysis of banding patterns as used here is probably suitable for future use. However, because of both the diversity and the complexity of patterns generated, comparisons over longer time periods (including between and within incidents) will require the application of computer assisted gel analysis systems. We are currently commencing evaluation of computer-assisted analysis of banding patterns. Future studies will be targeted at evaluation of the stability and reproducibility of this AFLP method together with inter-laboratory trials to establish the validity of the method for the rapid subtyping of Cl. perfringens for outbreak investigation.

Acknowledgements One of us (G.R.) was partially funded by a shortterm mobility grant from the Consiglio Nazionale delle Ricerche, Rome, Italy. The technical expertise and helpful discussion from Dr J.R. Gibson, M.D. Hampton and S. Sweeney of the PHLS Division of Gastrointestinal Infections is gratefully acknowledged. The helpful discussion and critical comments from Professor G.M. Grasso (University of Molise, Campobasso, Italy) is also acknowledged.

References Berry, P.R., Gilbert, R.J., 1991. Clostridium perfringens type A food poisoning. In: Duerden, B.I., Draser, B.S. (Eds.), Anaerobes in Human Disease, London, Arnold, pp. 362–371. Boom, R., Sol, C.J.A., Salimans, M.M.M., Jansen, C.L., Wertheim van Dillen, P.M.E., van der Noordaa, J., 1990. Rapid and simple method for purification of nucleic acids. J. Clin. Microbiol. 28, 495–503. Boumedine, K.S., Rodolakis, A., 1998. AFLP allows the identification of genetic markers of ruminant Chlamydia psittaci strains useful for typing and epidemiological studies. Res. Microbiol. 149, 735–744. Duim, B., Wassenaar, T.M., Rigter, A., Wagenaar, J., 1999. Highresolution genotyping of Campylobacter strains isolated from poultry and humans with amplified fragment length polymorphism fingerprinting. Appl. Environ. Microbiol. 65, 2369– 2375. Forsblom, B., Palmu, A., Hirvonen, P., Jousimies-Somer, H., 1995. Ribotyping of Clostridium perfringens isolates. Clin. Infect. Dis. 20, S323–S324. Geornaras, I., Kunene, N.F., von Holy, A., Hastings, J.W., 1999. Amplified fragment length polymorphism fingerprinting of

27

pseudomonas strains from a poultry processing plant. Appl. Environ. Microbiol. 65, 3828–3833. Gibson, J.R., Slater, E., Xerry, J., Tompkins, D.S., Owen, R.J., 1998. Use of an amplified-fragment length polymorphism technique to fingerprint and differentiate isolates of Helicobacter pylori. J. Clin. Microbiol. 36, 2580–2585. Hathaway, C.L., Johnson, E.A., 1998. Clostridium: the sporebearing anaerobes. In: Balows, A., Duerden, B.I. (Eds.), Systematic Bacteriology, Topley and Wilson’s Microbiology and Microbial Infections, Vol. 2, London, Arnold, pp. 731–782. Janssen, P., Coopman, R., Huys, G., Swings, J., Bleeker, M., Vos, P., Zabeau, M., Kersters, K., 1996. Evaluation of the DNA fingerprinting method AFLP as a new tool for bacterial taxonomy. Microbiology 142, 1881–1893. Keim, P., Kalif, A., Schupp, J., Hill, K., Travis, S.E., Richmond, K., Adair, D.M., Hugh-Jones, M., Kuske, C.R., Jackson, P., 1997. Molecular evolution and diversity in Bacillus anthracis as detected by amplified fragment length polymorphism markers. J. Bacteriol. 179, 818–824. Picardeau, M., Prod’Hom, G., Raskine, L., LePennec, M.P., Vincent, V., 1997. Genotypic characterization of five subspecies of Mycobacterium kansasii. J. Clin. Microbiol. 35, 25–32. Schalch, B., Eisgruber, H., Schau, H.P., Wiedmann, M., Stolle, A., 1998. Strain differentiation of Clostridium perfringens by bacteriocin typing, plasmid profiling and ribotyping. Zentralbl Veterinarmed [B] 45, 595–602. Schalch, B., Sperner, B., Eisgruber, H., Stolle, A., 1999. Molecular methods for the analysis of Clostridium perfringens relevant to food hygiene. FEMS Immunol. Med. Microbiol. 24, 281–286. Sissons, P.R., Kramer, J.M., Brett, M.M., Freeman, R., Gilbert, R.J., Lightfoot, N.F., 1992. Application of pyrolysis mass spectrometry to the investigation of outbreaks of food poisoning and non-gastrointestinal infection associated with Bacillus species and Clostridium perfringens. Int. J. Food Microbiol. 17, 57–66. Stanley, J., Saunders, N., 1996. DNA insertion sequences and the molecular epidemiology of Salmonella and Mycobacterium. J. Med. Microbiol. 45, 236–251. Stringer, M.F., Turnbull, P.C.B., Gilbert, R.J., 1980. Application of serological typing to the investigation of outbreaks of Clostridium perfringens food poisoning 1970–1978. J. Hyg. 84, 443–456. Stringer, M.F., Watson, G.N., Gilbert, R.J., 1982. Clostridium perfringens type A: serological typing and methods for the detection of enterotoxin. In: Corry, J.E.L., Roberts, D., Skinner, F.A. (Eds.), Isolation and Identification Methods For Food Poisoning Organisms, Society For Applied Bacteriology Technical Series, Vol. No. 7, Academic Press, London, pp. 111–135. Stringer, M.F., Watson, G.N., Gilbert, R.J., Wallace, J.G., Hassall, J.E., Tanner, E.I., Webber, P.P., 1985. Faecal carriage of Clostridium perfringens. J. Hyg. 95, 277–288. Struelens, M.J., De Gheldre, Y., Deplano, A., 1998. Comparative and library epidemiological typing systems: outbreak investigations versus surveillance systems. Infect. Control Hosp. Epidemiol. 19, 565–569. Tompkins, D.S., Hudson, M.J., Smith, H.R., Elgin, R.P., Wheeler, J.G., Brett, M.M., Owen, R.J., Brazier, J.S., Cumberland, P., King, V., Cook, P.E., 1999. A study of infectious intestinal

28

J. McLauchlin et al. / International Journal of Food Microbiology 56 (2000) 21 – 28

disease in England: microbiological findings in cases and controls. Comm. Dis. Publ. Hlth. 2, 108–113. Van Eldere, J., Janssen, P., Hoefnagels-Schuermans, A., van Lierde, S., Peetermans, W.E., 1999. Amplified-fragment length polymorphism analysis versus macro-restriction fragment analysis for molecular typing of Streptococcus pneumoniae isolates. J. Clin. Microbiol. 37, 2053–2057.

Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., Kuiper, M., Zabeau, M., 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23, 4407–4414. Zabeau, M., Vos, P., 1993. Selective Restriction Fragment Amplification: A General Method For DNA Fingerprinting, European Patent Office, Munich, Germany, EP 0 534 858.