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Comparison of Listeria monocytogenes strain types in Irish smoked salmon and other foods
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Int. J. Hyg. Environ.-Health 209 (2006) 527–534 www.elsevier.de/ijheh
Comparison of Listeria monocytogenes strain types in Irish smoked salmon and other foods Deborah Corcorana,b, David Clancyc, Michea´l O’Mahonya, Kathie Grantd, Evelyn Hylandc, Noel Shanaghyc, Paul Whytea, James McLauchlind, Anne Moloneyc, Se´amus Fanninga, a
Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College, Belfield, Dublin, Ireland Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork, Ireland c Regional Food Microbiology Laboratory, Department of Medical Microbiology, Waterford Regional Hospital, Waterford, Ireland d Health Protection Agency Food Safety Microbiology Laboratory, Centre for Infections, London, UK b
Received 21 December 2005; received in revised form 31 May 2006; accepted 4 June 2006
Abstract An investigation of Listeria monocytogenes in Irish retail smoked salmon products and other unrelated food products was undertaken. Serotyping and genotyping methods were applied. Twenty-six L. monocytogenes isolates cultured from ready-to-eat smoked salmon and an additional 20 L. monocytogenes isolates from various commercially available food products (other than smoked salmon) were compared. Four serotypes, 12 ribotypes, 12 amplified fragment length polymorphism (AFLP) types and 17 pulsed-field gel electrophoresis (PFGE) types were identified among the 46 isolates studied. Genotyping identified a single dominant strain that accounted for 65% of those cultured from smoked salmon and this strain was present in product obtained from three out of five of the manufacturers surveyed. When compared to the food products obtained from a variety of sources, those from smoked salmon appeared to cluster as a single group. In Irish smoked salmon this strain may have adapted, and be capable of persisting in this food product. All isolates were grouped into genetic lineages based on their EcoR1 ribotypes. The attendant risk to public health following consumption of these foods is discussed. r 2006 Elsevier GmbH. All rights reserved. Keywords: Listeria monocytogenes; Smoked salmon; Ribotyping; AFLP; PFGE
Introduction Listeria monocytogenes is a Gram-positive, motile, non-spore forming bacterium which grows over a wide range of conditions, including temperatures of 1–45 1C (Farber, 2000; Farber and Peterkin, 1991; Graves and Corresponding author. Tel.: +353 1 716 6082; fax: +353 1 716 6091. E-mail address: [email protected] (S. Fanning).
1438-4639/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2006.06.001
Swaminathan, 2001). The natural habitats of L. monocytogenes include water, soil and plant material, particularly plant material undergoing decay (Fenlon, 1999; Suihko et al., 2002). This organism has been recognized as an opportunistic pathogen of humans and animals for almost 90 years (Wesley, 1999). Food-borne L. monocytogenes was initially described in 1981 when contaminated coleslaw was implicated in an outbreak of listeriosis (Schlech et al., 1983). Since then L. monocytogenes has been implicated as an important
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food-borne pathogen associated with large outbreaks (Rocourt et al., 2000; Schlech, 2000). L. monocytogenes may be associated with a febrile gastroenteric syndrome and may cause serious invasive diseases, including septicaemia, meningitis and meningoencephalitis. Most invasive cases of human listeriosis occur in pregnant women, the unborn, neonates, the elderly and in immunocompromised individuals (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). Although not as prevalent as other food-borne illnesses listeriosis can lead to severe illness with a typical mortality rate of approximately 30% (VazquezBoland et al., 2001). This disease has been estimated to be the second and fourth most common cause of death from food-borne illness in the England and Wales, and in the USA, respectively (Adak et al., 2002; Mead et al., 1999). Several modes of transmission have been identified, but the primary route for humans is through consumption of contaminated food (Farber and Peterkin, 1991). A variety of food types have been implicated in disease transmission including unpasteurized cheese, meat and meat products and vegetables (Jemmi et al., 2002; Lyytikainen et al., 2000; Schlech, 2000). Seafoods have been identified as products posing a particular risk for food-borne listeriosis (Rocourt et al., 2000). Contaminated fish products (Farber et al., 2000; Jemmi et al., 2002) including cold smoked rainbow trout (Ericsson et al., 1997), smoked mussels (Miscrachi et al., 1991; Mitchell, 1991) and cooked shrimp (Riedo et al., 1994) have been reported. Previous studies from other countries have reported a variable but occasionally high prevalence of this pathogen in the fish smoking industry. For example an American study reported a prevalence of 7.8% in raw fish, 18.1% in samples from the processing environment, and 7.3% of finished product (Norton et al., 2001). Cold-smoking of fish typically involves temperatures in the region of 20–50 1C. Whilst phenolic and other volatile compounds in smoke may inhibit the growth of L. monocytogenes (Eklund et al., 2004; Jemmi and Keusch, 1992), smoked salmon is an example of a food product of animal origin consumed without any bactericidal thermal treatment. A Danish study reported contamination of 34–60% of cold smoked fish with L. monocytogenes, and large variability amongst processing establishments (Jorgensen and Huss, 1998). No similar data have been reported in Ireland. Characterization of isolates implicated in human disease outbreaks has established the similarity with strains present in implicated food products (Ericsson et al., 1997; Schlech et al., 1983). The present study reports the characterization of a collection of Irish L. monocytogenes isolates from vacuum-packed smoked salmon product(s). Additional isolates from a variety of unrelated food sources were included for comparison and analysed in parallel. These data identified the main L. monocytogenes strain types
associated with smoked salmon and various unrelated food products in Ireland at this time. These data may facilitate a better understanding of potential risk to public health following consumption of these food products.
Materials and methods Bacterial isolates Twenty-six L. monocytogenes isolates were obtained from sliced vacuum packed smoked salmon products taken from retail premises throughout Ireland during October, November and December, 2001. This study formed part of the Food Safety Authority of Ireland (FSAI) 4th Quarter National Survey conducted during 2001. Twenty L. monocytogenes isolates from various other food sources were included for comparison. All isolates are described in Table 1. Isolates were recovered using standard laboratory methods for the isolation of L. monocytogenes from food (ISO 11290-1:1996). All isolates were checked for purity on Colombia Blood Agar and stored on beads in cryopreservation fluid at 80 1C.
Serotyping All isolates were serotyped using standard methods as described previously by Seeliger and Hone (1979).
Ribotyping Ribotyping was performed according to the manufacturer’s recommendations, using standard protocols designed for the Riboprinter (Du Pont Qualicons, Wilmington, DE). Briefly, pure cultures were streaked onto Columbia Blood Agar and incubated aerobically in a moist chamber overnight at 37 1C. Bacterial cells were suspended in lysis buffer and heat inactivated at 90 1C for 10 min, then lysed and placed in the Riboprinter. Following transfer to the Riboprinter genomic DNA was digested with the enzyme EcoR1, separated on a 0.8% (w/v) agarose gel, and transferred onto a nylon membrane. This solid support was then hybridized with a chemiluminescent rRNA probe. Probe patterns were then acquired as a gel image, normalized and assigned an identity number by the system software.
Pulsed-field gel electrophoresis (PFGE) Purification of template DNA for PFGE was performed as described previously (Graves and Swaminathan, 2001) with the following modifications. DNA was isolated and then restricted with Apa1 (Roche,
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Table 1. A summary of the phenotypic and genotypic characteristics identified among 46 L. monocytogenes isolates cultured from smoked salmon and other Irish food products Strain no.
Food type
Serotype
dup
Ribogroup
PFGE
AFLP
3699/01 F1577 F1599 F1710 F1722 F1725 F1753 F1843 F1930 1675/01 1036/01 10319/01 126/01 1367 1331 PF8092 PF8102 PF5854 1408/01 PF8119 PF5195 PF3400 PF5650 PF5684 PF5685 PF4753 PF4781 PF4783 PF4810 PF4811 PF3712 PF5143
S.S.-2 S.S.-1 S.S.-1 S.S.-1 S.S.-1 S.S.-2 S.S.-1 S.S.-1 S.S.-1 S.S.-anon S.S.-1 S.S.-4 S.S.-anon S.S.-2 S.S.-1 S.S.-1 S.S.-2 Coleslaw S.S.-2 S.S.-2 Ham S Ham C Ham B Ham B Ham B Turkey Turkey S Ham S Turkey S Ham C Tuna Salad Mussels/ prawns C Egg Mayo Coleslaw S.S.-anon S.S.-anon S.S.-anon S..S.-3 S.S.-anon S.S.-5 Smoked mackerel R Pasta Salad Ham B Ham S Ham C S.S.-1
1/2a 1/2a 1/2a 1/2a 1/2a 1/2a NT 1/2c 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a NT 1/2a 1/2a NT 1/2a 1/2b 1/2c 1/2c 1/2a 1/2a 1/2a 1/2a 1/2a 4b
1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1039 1039 1039 1039 1039 1039 1039 1045 1045 1045 1045 1045 1042
251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-14-2 251-14-2 251-14-2 251-14-2 251-14-2 251-11-1
A A A A A A A A A A A A A A A A A P D D G G G O O K K K K K I
A A A A A A NT A A A A A A A A A A I C B NT C B B B C C C B B G
1/2b 1/2b 4b 1/2a 1/2a 1/2a 4b 4b 1/2a
1042 1042 1042 1053 1053 1053 1038 1038 1045
251-11-1 251-11-1 251-11-1 251-50-4 251-50-4 251-50-4 251-16-3 251-16-3 251-11-4
L L Q B B B F F E
I M G B B B E E D
1/2a 4b NT 1/2a 4b 1/2c
1045 1038 1047 1062 1042 1053
251-11-4 251-15-7 251-12-8 251-12-3 251-12-1 251-15-3
J H M M N C
H F NT J K B
PF5396 PF5856 134/01 145/01 146/01 1563/01 1673/01 PF8136 rdrlPF4337 PF3706 PF5578 PF5167 PF5237 PF8072
Indianapolis, IN, USA). The bacteria were grown on nutrient agar plates at 37 1C for 24 h, and the agarose plug slices were incubated for 20–24 h with the restriction enzyme Apa1 at 30 1C according to the manufacturer’s instructions. The DNA restriction fragments in
the plug slices were resolved by electrophoresis through a 1% SeaKem Gold agarose gel in 0.5 TB buffer. The electrophoretic parameters used were the standard ones for L. monocytogenes (Graves et al., 1994), initial switch time 4.0 s; final switch time 40 s; run time 22 h; angle
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1201 gradient 6.0 V/cm; temperature 14 1C; ramping factor linear. After electrophoresis gels were stained for 30 min in 250 ml of deionized water containing 12 ml of ethidium bromide (10 mg/ml), then destained in distilled water and viewed under UV light. Lamda ladder PFGE marker (New England Biolabs (Hertfordshire, England, UK) was used for molecular weight size determination.
Amplified fragment length polymorphism (AFLP) analysis L. monocytogenes cultures were analysed by AFLP using a modification of the method described previously (Guerra et al., 2002). Isolates were cultured on blood agar and nucleic acid extracted using a MagNA Pure automatic nucleic acid extraction instrument (Total Nucleic Acid Isolation Kit or the Bacterial and Fungal DNA III isolation Kit, Roche Molecular Biochemicals, Lewes, UK) according to the manufacturer’s instructions. AFLP analysis comprised digestion of DNA with EcoR1, ligation with adapters ECO-AD1 and -AD2 followed by amplification by polymerase chain reaction (PCR) with the primer EcoR1-G. A no template control was included in each batch of tests. Banding patterns were resolved by running 13 ml of the amplified product in a 1.5% (w/v) agarose gel containing ethidium bromide, observed under UV transillumination and fluorescent bands were recorded with type 667 film (Polaroid, St. Albans, UK). Banding patterns of images of the ethidium bromide-stained gels were analysed by visual inspection.
Virulence lineage Isolates were categorized into three lineages I, II and III, correlating with potential human pathogenicity according to ribotype and serotype characteristics, as previously described (Nadon et al., 2001; Wiedmann et al., 1997). Lineage I representing the most potentially virulent group is comprised of serotypes 1/2b, 3b, 3c and 4b. Lineage II representing intermediate virulence was regarded as comprising serotypes 1/2a, 1/2c and 3a. Lineage III comprised of serotypes 4a and 4c.
Results Forty-six L. monocytogenes isolates were investigated using phenotype-based and genotyping methods. Twenty-six of these isolates had been cultured from product obtained from a number of smoked fish food processsors operating in Ireland. In addition, a further 20 L. monocytogenes from various food products other than smoked salmon were analysed in parallel to provide a comparative data set. Types of food product
included coleslaw, bacon (baked, cooked and smoked), egg mayonnaise, pasta salad, mussels/prawns, smoked mackerel and smoked turkey.
Serotyping All L. monocytogenes isolates were serotyped (Table 1). Four serotypes were identified among the 46 isolates. Most of the isolates were identified as serotype 1/2a representing 63% (n ¼ 29) of the collection. The next most common serotype among the collection was identified as 4b (13%, n ¼ 6), followed by 1/2c (9%, n ¼ 4), and 1/2b (6.5%, n ¼ 3). Flagellar antigen could not be determined for the isolates PF1753, PF5578 or PF 5195 and PF5854 was non-typeable by these methods (Table 1).
Ribotyping Twelve distinct ribotypes were defined among the 46 L. monocytogens isolates analysed. All ribotypes were assigned on the basis of the corresponding EcoR1 restriction profile after probing with the standard riboprobe. Banding patterns obtained consisted of DNA fragments ranging in size from 1.5- through 6kbp. A composite illustration of 12 profiles is given in Fig. 1. The most common profile identified with this method was defined as DUP-ID group 1062 and identified as ribogroup 251-14-7. This profile accounted for 18 of the 46 isolates (39%) and included the serotypes 1/2a and 1/2c (Table 1). Within this grouping, 15 of the isolates were serotype 1/2a. These isolates were cultured from product obtained from three known smoked salmon processors (designated S.S.-1, -2 and -4) along with two other anonymous manufacturers (S.S.-anon). Two of the four serotypes, 1/2a and 1/2c were associated with the former processor (S.S.-1), while serotype 1/2a alone was linked with S.S.-2. The isolate designated PF5854, which was cultured from a coleslaw sample, represented the only non-smoked salmon isolate within this ribogrouping. The DUP-ID group 1062 was also assigned to a single isolate, PF5167, obtained from a smoked bacon source and which did not form part of this grouping. In this case PF5167 was also serotyped as 1/2a (Table 1). Ribogroup 251-12-2 represented the next most prevalent group, accounting for seven isolates (15%). This ribogroup contained three serogroups 1/2a (n ¼ 3), 1/2b ðn ¼ 1Þ and 1/2c ðn ¼ 2Þ. Two of the three 1/2a serotypes were associated with smoked fish product. Isolates from foods other than smoked salmon were the predominant isolates in this ribotype. The remaining 21 isolates were distributed into 10 ribogroupings. One-third of these were cultured from smoked salmon. DUP-ID 1045 accounted for ribogroup
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Fig. 1. Screen captured image showing the 12 different ribogroups identified in the course of this study. The image displays the ribogroup number along with the corresponding normalized fragment pattern data for each ribogroup.
251-14-2 ðn ¼ 5Þ all of which were serotype 1/2a. Ribogroup 251-11-1 ðn ¼ 4Þ contained two serotypes 1/2b and 4b. Three isolates of serotype 1/2a, were designated as ribogroup 251-50-4. A further four isolates, two of which were serotype 4b and two 1/2a, were assigned to ribogroups 251-16-3 and 251-11-4, respectively. The final 5 isolates were assigned different DUP-ID numbers and were assigned to different ribogroupings.
Pulsed-field gel electrophoresis When applied to purified genomic DNA the restriction endonuclease Apa1 provided macrorestriction profiles which could be compared to discriminate among the 46 L. monocytogenes isolates. Banding patterns produced 17 distinct DNA fragment profiles, which were nominally designated according to the Tenover Rules as A through Q. Each profile was comprised of between 13 and 17 DNA fragments, with sizes ranging from 48.5 to 776 kbp (Fig. 2 shows composite profiles of some of these DNA fingerprints). PFGE profile A was the most common (n ¼ 17; 37%) in this collection, comprising entirely of smoked salmon-derived isolates of similar serotype and ribogroup (Table 1 and Fig. 2). Another five isolates were grouped as profile K the next most common banding pattern and in this case these isolates corresponded to serotype 1/2a and ribogroup 251-14-2 and were only found in isolates from foods other then smoked salmon. The remaining 24 isolates were represented by PFGE types B through J and L through Q. In each case the profile was assigned to between one and three isolates. In the current data set
the PFGE profiles of isolates from smoked salmon product were not shared with those from other food products (Table 1).
Amplified fragment length polymorphism analysis Purified genomic DNA prepared from all isolates was used for AFLP analysis. Following EcoR1 digestion, 12 distinct AFLP profiles designated A through M, were obtained (data not shown). In analogy with the PFGE data, AFLP profile A ðn ¼ 16Þ was the most common profile identified in this data set, again found only in the smoked salmon-derived isolates, and representing serotypes 1/2a or 1/2c). AFLP profile B accounted for 9 of the isolates (of serotypes 1/2a, 1/2b and 1/2c), C ðn ¼ 6Þ, E, I and G (n ¼ 2 each), D, J, H, M, K and F ðn ¼ 1Þ. Among the smoked salmon isolates, five AFLP profiles were assigned. In contrast to PFGE, some AFLP profiles were found in both the smoked salmon and the unrelated food product isolates, while three isolates F1753, PF5578 and PF5195 were non-typeable by AFLP.
Virulence lineage All samples for which a serotype was available were within either Lineage I or II. Two of the 26 smoked salmon isolates (8%) were serotype 4b and thus Lineage I. Seven of the 20 isolates from the unrelated food products (35%) were Lineage I. Thirty-seven of the 46 isolates in the current data set (80%) were categorized as Lineage II.
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Fig. 2. Pulsed-Field Gel Electrophoresis representing 16 out of the 26 smoked fish isolates investigated. The predominant PFGE profile is indicated by the grey bars at the foot of the gel. Lane 1 ¼ 1036/01; (A), lane 2 ¼ 10319/01; (A), lane 3 ¼ 126/01; (A), lane 4 ¼ 134/01; (B), lane 5 ¼ 145/01; (B), lane 6 ¼ 146/01; (B), lane 7 ¼ 1367; (A), lane 8 ¼ 1331; (A), lane 9 ¼ PF8072; (C), lane 10 ¼ PF8102; (A), lane 11 ¼ F8119; (D), lane 12 ¼ F8136; (E), lane 13 ¼ PF8092, (A), lane 14 ¼ 3699/01; (A), lane 15 ¼ 1577; (A) and lane 16 ¼ 1599 (A). M represents a set of molecular weight markers. Letters in parenthesis denote the PFGE profile grouping assigned to each isolate.
Discussion This study employed a variety of typing methods to compare L. monocytogenes isolates from smoked salmon, with those isolated from other food products. One subtype (serogroup 1/2a, Ribotype 251-14-7 DUP 1062, PFGE A and AFLP A) was dominant in the smoked salmon isolates, representing 65% ðn ¼ 17Þ of the total number of smoked salmon isolates investigated (Table 1). Neither PFGE nor AFLP profile A were detected in isolates from other food products, while ribotype 251-14-7 occurred only once. These apparently clustered organisms were isolated from three of the five smoked salmon manufacturers involved in the current study. The finding of a single predominant subtype of L. monocytogenes in smoked fish is consistent with previous reports (Dauphin et al., 2001; Norton et al., 2001; Vitas and Garcia-Jalon, 2004). The current study provides further evidence of adaptation of a single clonal type of L. monocytogenes to persist in the fish smoking industry, either on the product during processing, or within the processing environment with subsequent product contamination. The serotype of the dominant subtype in smoked salmon in the current study, 1/2a, was consistent with previous studies which found serogroup to account for 93% of L. monocytogenes isolates from smoked salmon (Dauphin et al., 2001; Vitas and Garcia-Jalon, 2004). The next most common banding profile identified in the current study (Ribotype 251-14-2 DUP 1045, PFGE K) was found primarily in isolates from foods other than smoked salmon including both turkey and ham pro-
ducts. This finding contrasts with a previous study which found DUP 1045 to persist in the smoked-fishprocessing environment (Norton et al., 2001). All five isolates in this group were also serotype 1/2a. AFLP divided the group into two profiles B and C that may indicate that AFLP is not as discriminatory a method for L. monocytogenes as PFGE and ribotyping, as these AFLP profiles were also found in the smoked salmon isolates. The diversity of subtypes found amongst the 20 isolates from various food products (4 serotypes, 11 PFGE profiles, 9 ribotypes 11 AFLP profiles) highlights the need for efficient reliable subtyping methods to effectively identify the various genetic strains that can be found in food products. Wiedmann et al. (1997) previously investigated the relationship between ribotyping and virulence potential of a collection of L. monocytogenes. Three lineages designated I through III were defined based on ribo- and serotype characteristics (Nadon et al., 2001). These definitions were applied to this collection in an attempt to infer pathogenic potential. The low percentage (8%) of lineage I strains identified in the smoked salmon isolates correlates with the finding that smoked fish and other seafood’s are infrequently associated with human listeriosis (Jinneman et al., 1999). A higher percentage of isolates from the unrelated food sources were of lineage I, including isolates from food sources such as coleslaw, tuna salad and egg salad. While the present data set may not be entirely representative of the real situation in Ireland, our data indicates that the smoked fish products may pose a lower risk to public health compared to other types of food.
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The serotypes 1/2a, 1/2b and 4b have been implicated in the majority of human clinical listeriosis cases (McLauchlin, 1990; Pinner et al., 1992). Conventional methods such as serotyping for species identification are often used as an initial screening method, but are of limited value in detailed epidemiological investigations. Molecular subtyping methods such as the genotyping methods employed in this study have the potential utility of further resolving isolates into clonal groupings. These techniques have applicability in characterizing virulence of isolates, tracing pathogens along the foodchain, and tracking alterations in pathogen genome over time. A study conducted by the WHO validated PFGE as an excellent and reproducible method for subtyping of L. monocytogenes (Brosch et al., 1994). Internationally standardized protocols for this PGFE should facilitate inter-laboratory and international comparison of isolated pathogens (Graves and Swaminathan, 2001). However, PFGE requires specialized equipment and human resources, and is relatively time consuming, therefore is not used routinely for the initial screening of isolates. Ribotyping is a rapid, technically straightforward, method of subtyping which has been sufficiently automated to minimize operator involvement. However, ribotyping is an expensive procedure, both in terms of initial capital outlay for specialized equipment, along with substantial running costs. Furthermore, previous studies have shown that the ability of ribotying to discriminate within the serogroup 4b strains may not be adequate for epidemiological investigations (Graves et al., 1994). AFLP fingerprinting has been used successfully for typing and classifying a number of bacteria strains (Savelkoul et al., 1999). Ribotyping combined with PFGE are shown to be complimentary typing methods when compared to ribotyping and AFLP or PFGE and AFLP. Previous studies have indicated the value of combining phenotypic and genotypic approaches to investigation of listeriosis outbreaks. In the current data set, serotyping combined with both ribotyping and PFGE produced a high level of discrimination. Combined in parallel, these subtyping tools appear to provide a powerful epidemiological approach to characterize L. monocytogenes strains isolated from animals, foods, the food production environment and human clinical disease.
Acknowledgements Colleagues at the Food Safety Authority of Ireland (FSAI) are acknowledged for providing financial support to the WRH Laboratory.
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Anttila, V.J., Johansson, T., Rantala, L., Aalto, T., Korkeala, H., Siitonen, A., 2000. An outbreak of Listeria monocytogenes serotype 3a infections from butter in Finland. J. Infect. Dis. 181, 1838–1841. McLauchlin, J., 1990. Distribution of serovars of Listeria monocytogenes isolated from different categories of patients with listeriosis. Eur. J. Clin. Microbiol. Infect. Dis. 9, 210–213. Mead, P.S., Slutsker, L., Dietz, V., McCaig, L.F., Bresee, J.S., Shapiro, C., Griffin, P.M., Tauxe, R.V., 1999. Food-related illness and death in the United States. Emerg. Infect. Dis. 5, 607–625. Miscrachi, A., Watson, A.J., Coleman, D., 1991. Listeria in smoked mussels. Commun. Dis. Intell. 15, 427. Mitchell, D.L., 1991. A case cluster of listeriosis in Tasmania. Commun. Dis. Intell. 15, 427. Nadon, C.A., Woodward, D.L., Young, C., Rodgers, F.G., Wiedmann, M., 2001. Correlations between molecular subtyping and serotyping of Listeria monocytogenes. J. Clin. Microbiol. 39, 2704–2707. Norton, D.M., McCamey, M.A., Gall, K.L., Scarlett, J.M., Boor, K.J., Wiedmann, M., 2001. Molecular studies on the ecology of Listeria monocytogenes in the smoked fish processing industry. Appl. Environ. Microbiol. 67, 198–205. Pinner, R.W., Schuchat, A., Swaminathan, B., Hayes, P.S., Deaver, K.A., Weaver, R.E., Plikaytis, B.D., Reeves, M., Broome, C.V., Wenger, J.D., 1992. Role of foods in sporadic listeriosis, II: Microbiologic and epidemiologic investigation. The Listeria Study Group. J. Am. Med. Assoc. 267, 2046–2050. Riedo, F.X., Pinner, R.W., Tosca, M.L., Cartter, M.L., Graves, L.M., Reeves, M.W., Weaver, R.E., Plikaytis, B.D., Broome, C.V., 1994. A point-source foodborne listeriosis outbreak: documented incubation period and possible mild illness. J. Infect. Dis. 170, 693–696. Rocourt, J., Jacquet, C., Reilly, A., 2000. Epidemiology of human listeriosis and seafoods. Int. J. Food Microbiol. 62, 197–209.
Savelkoul, P.H., Aarts, H.J., de Haas, J., Dijkshoorn, L., Duim, B., Otsen, M., Rademaker, J.L., Schouls, L., Lenstra, J.A., 1999. Amplified-fragment length polymorphism analysis: the state of an art. J. Clin. Microbiol. 37, 3083–3091. Schlech, W.F., 2000. Foodborne listeriosis. Clin. Infect. Dis. 31, 770–775. Schlech 3rd, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., Johnson, S.E., King, S.H., Nicholls, E.S., Broome, C.V., 1983. Epidemic listeriosis – evidence for transmission by food. N. Engl. J. Med. 308, 203–206. Seeliger, H.P.R., Hone, K., 1979. Seotyping of Listeria monocytogenes and related species. In: Bergan, T., Norris, J.R. (Eds.), Methods in Microbiology. Academic Press, London. Suihko, M.L., Salo, S., Niclasen, O., Gudbjornsdottir, B., Torkelsson, G., Bredholt, S., Sjoberg, A.M., Gustavsson, P., 2002. Characterization of Listeria monocytogenes isolates from the meat, poultry and seafood industries by automated ribotyping. Int. J. Food Microbiol. 72, 137–146. Vazquez-Boland, J.A., Kuhn, M., Berche, P., Chakraborty, T., Dominguez-Bernal, G., Goebel, W., Gonzalez-Zorn, B., Wehland, J., Kreft, J., 2001. Listeria pathogenesis and molecular virulence determinants. Clin. Microbiol. Rev. 14, 584–640. Vitas, A.I., Garcia-Jalon, V.A., 2004. Occurrence of Listeria monocytogenes in fresh and processed foods in Navarra (Spain). Int. J. Food Microbiol. 90, 349–356. Wesley, I.V., 1999. Listeriosis in animals. In: Ryser, E.T., Marth, E.H. (Eds.), Listeria, Listeriosis and Food Safety. Marcel Dekker, New York, pp. 39–73. Wiedmann, M., Bruce, J.L., Keating, C., Johnson, A.E., McDonough, P.L., Batt, C.A., 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. Immun. 65, 2707–2716.
Int. J. Hyg. Environ.-Health 209 (2006) 527–534 www.elsevier.de/ijheh
Comparison of Listeria monocytogenes strain types in Irish smoked salmon and other foods Deborah Corcorana,b, David Clancyc, Michea´l O’Mahonya, Kathie Grantd, Evelyn Hylandc, Noel Shanaghyc, Paul Whytea, James McLauchlind, Anne Moloneyc, Se´amus Fanninga, a
Centre for Food Safety, School of Agriculture, Food Science and Veterinary Medicine, University College, Belfield, Dublin, Ireland Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork, Ireland c Regional Food Microbiology Laboratory, Department of Medical Microbiology, Waterford Regional Hospital, Waterford, Ireland d Health Protection Agency Food Safety Microbiology Laboratory, Centre for Infections, London, UK b
Received 21 December 2005; received in revised form 31 May 2006; accepted 4 June 2006
Abstract An investigation of Listeria monocytogenes in Irish retail smoked salmon products and other unrelated food products was undertaken. Serotyping and genotyping methods were applied. Twenty-six L. monocytogenes isolates cultured from ready-to-eat smoked salmon and an additional 20 L. monocytogenes isolates from various commercially available food products (other than smoked salmon) were compared. Four serotypes, 12 ribotypes, 12 amplified fragment length polymorphism (AFLP) types and 17 pulsed-field gel electrophoresis (PFGE) types were identified among the 46 isolates studied. Genotyping identified a single dominant strain that accounted for 65% of those cultured from smoked salmon and this strain was present in product obtained from three out of five of the manufacturers surveyed. When compared to the food products obtained from a variety of sources, those from smoked salmon appeared to cluster as a single group. In Irish smoked salmon this strain may have adapted, and be capable of persisting in this food product. All isolates were grouped into genetic lineages based on their EcoR1 ribotypes. The attendant risk to public health following consumption of these foods is discussed. r 2006 Elsevier GmbH. All rights reserved. Keywords: Listeria monocytogenes; Smoked salmon; Ribotyping; AFLP; PFGE
Introduction Listeria monocytogenes is a Gram-positive, motile, non-spore forming bacterium which grows over a wide range of conditions, including temperatures of 1–45 1C (Farber, 2000; Farber and Peterkin, 1991; Graves and Corresponding author. Tel.: +353 1 716 6082; fax: +353 1 716 6091. E-mail address: [email protected] (S. Fanning).
1438-4639/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2006.06.001
Swaminathan, 2001). The natural habitats of L. monocytogenes include water, soil and plant material, particularly plant material undergoing decay (Fenlon, 1999; Suihko et al., 2002). This organism has been recognized as an opportunistic pathogen of humans and animals for almost 90 years (Wesley, 1999). Food-borne L. monocytogenes was initially described in 1981 when contaminated coleslaw was implicated in an outbreak of listeriosis (Schlech et al., 1983). Since then L. monocytogenes has been implicated as an important
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food-borne pathogen associated with large outbreaks (Rocourt et al., 2000; Schlech, 2000). L. monocytogenes may be associated with a febrile gastroenteric syndrome and may cause serious invasive diseases, including septicaemia, meningitis and meningoencephalitis. Most invasive cases of human listeriosis occur in pregnant women, the unborn, neonates, the elderly and in immunocompromised individuals (Farber and Peterkin, 1991; Vazquez-Boland et al., 2001). Although not as prevalent as other food-borne illnesses listeriosis can lead to severe illness with a typical mortality rate of approximately 30% (VazquezBoland et al., 2001). This disease has been estimated to be the second and fourth most common cause of death from food-borne illness in the England and Wales, and in the USA, respectively (Adak et al., 2002; Mead et al., 1999). Several modes of transmission have been identified, but the primary route for humans is through consumption of contaminated food (Farber and Peterkin, 1991). A variety of food types have been implicated in disease transmission including unpasteurized cheese, meat and meat products and vegetables (Jemmi et al., 2002; Lyytikainen et al., 2000; Schlech, 2000). Seafoods have been identified as products posing a particular risk for food-borne listeriosis (Rocourt et al., 2000). Contaminated fish products (Farber et al., 2000; Jemmi et al., 2002) including cold smoked rainbow trout (Ericsson et al., 1997), smoked mussels (Miscrachi et al., 1991; Mitchell, 1991) and cooked shrimp (Riedo et al., 1994) have been reported. Previous studies from other countries have reported a variable but occasionally high prevalence of this pathogen in the fish smoking industry. For example an American study reported a prevalence of 7.8% in raw fish, 18.1% in samples from the processing environment, and 7.3% of finished product (Norton et al., 2001). Cold-smoking of fish typically involves temperatures in the region of 20–50 1C. Whilst phenolic and other volatile compounds in smoke may inhibit the growth of L. monocytogenes (Eklund et al., 2004; Jemmi and Keusch, 1992), smoked salmon is an example of a food product of animal origin consumed without any bactericidal thermal treatment. A Danish study reported contamination of 34–60% of cold smoked fish with L. monocytogenes, and large variability amongst processing establishments (Jorgensen and Huss, 1998). No similar data have been reported in Ireland. Characterization of isolates implicated in human disease outbreaks has established the similarity with strains present in implicated food products (Ericsson et al., 1997; Schlech et al., 1983). The present study reports the characterization of a collection of Irish L. monocytogenes isolates from vacuum-packed smoked salmon product(s). Additional isolates from a variety of unrelated food sources were included for comparison and analysed in parallel. These data identified the main L. monocytogenes strain types
associated with smoked salmon and various unrelated food products in Ireland at this time. These data may facilitate a better understanding of potential risk to public health following consumption of these food products.
Materials and methods Bacterial isolates Twenty-six L. monocytogenes isolates were obtained from sliced vacuum packed smoked salmon products taken from retail premises throughout Ireland during October, November and December, 2001. This study formed part of the Food Safety Authority of Ireland (FSAI) 4th Quarter National Survey conducted during 2001. Twenty L. monocytogenes isolates from various other food sources were included for comparison. All isolates are described in Table 1. Isolates were recovered using standard laboratory methods for the isolation of L. monocytogenes from food (ISO 11290-1:1996). All isolates were checked for purity on Colombia Blood Agar and stored on beads in cryopreservation fluid at 80 1C.
Serotyping All isolates were serotyped using standard methods as described previously by Seeliger and Hone (1979).
Ribotyping Ribotyping was performed according to the manufacturer’s recommendations, using standard protocols designed for the Riboprinter (Du Pont Qualicons, Wilmington, DE). Briefly, pure cultures were streaked onto Columbia Blood Agar and incubated aerobically in a moist chamber overnight at 37 1C. Bacterial cells were suspended in lysis buffer and heat inactivated at 90 1C for 10 min, then lysed and placed in the Riboprinter. Following transfer to the Riboprinter genomic DNA was digested with the enzyme EcoR1, separated on a 0.8% (w/v) agarose gel, and transferred onto a nylon membrane. This solid support was then hybridized with a chemiluminescent rRNA probe. Probe patterns were then acquired as a gel image, normalized and assigned an identity number by the system software.
Pulsed-field gel electrophoresis (PFGE) Purification of template DNA for PFGE was performed as described previously (Graves and Swaminathan, 2001) with the following modifications. DNA was isolated and then restricted with Apa1 (Roche,
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Table 1. A summary of the phenotypic and genotypic characteristics identified among 46 L. monocytogenes isolates cultured from smoked salmon and other Irish food products Strain no.
Food type
Serotype
dup
Ribogroup
PFGE
AFLP
3699/01 F1577 F1599 F1710 F1722 F1725 F1753 F1843 F1930 1675/01 1036/01 10319/01 126/01 1367 1331 PF8092 PF8102 PF5854 1408/01 PF8119 PF5195 PF3400 PF5650 PF5684 PF5685 PF4753 PF4781 PF4783 PF4810 PF4811 PF3712 PF5143
S.S.-2 S.S.-1 S.S.-1 S.S.-1 S.S.-1 S.S.-2 S.S.-1 S.S.-1 S.S.-1 S.S.-anon S.S.-1 S.S.-4 S.S.-anon S.S.-2 S.S.-1 S.S.-1 S.S.-2 Coleslaw S.S.-2 S.S.-2 Ham S Ham C Ham B Ham B Ham B Turkey Turkey S Ham S Turkey S Ham C Tuna Salad Mussels/ prawns C Egg Mayo Coleslaw S.S.-anon S.S.-anon S.S.-anon S..S.-3 S.S.-anon S.S.-5 Smoked mackerel R Pasta Salad Ham B Ham S Ham C S.S.-1
1/2a 1/2a 1/2a 1/2a 1/2a 1/2a NT 1/2c 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a 1/2a NT 1/2a 1/2a NT 1/2a 1/2b 1/2c 1/2c 1/2a 1/2a 1/2a 1/2a 1/2a 4b
1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1062 1039 1039 1039 1039 1039 1039 1039 1045 1045 1045 1045 1045 1042
251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-14-7 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-12-2 251-14-2 251-14-2 251-14-2 251-14-2 251-14-2 251-11-1
A A A A A A A A A A A A A A A A A P D D G G G O O K K K K K I
A A A A A A NT A A A A A A A A A A I C B NT C B B B C C C B B G
1/2b 1/2b 4b 1/2a 1/2a 1/2a 4b 4b 1/2a
1042 1042 1042 1053 1053 1053 1038 1038 1045
251-11-1 251-11-1 251-11-1 251-50-4 251-50-4 251-50-4 251-16-3 251-16-3 251-11-4
L L Q B B B F F E
I M G B B B E E D
1/2a 4b NT 1/2a 4b 1/2c
1045 1038 1047 1062 1042 1053
251-11-4 251-15-7 251-12-8 251-12-3 251-12-1 251-15-3
J H M M N C
H F NT J K B
PF5396 PF5856 134/01 145/01 146/01 1563/01 1673/01 PF8136 rdrlPF4337 PF3706 PF5578 PF5167 PF5237 PF8072
Indianapolis, IN, USA). The bacteria were grown on nutrient agar plates at 37 1C for 24 h, and the agarose plug slices were incubated for 20–24 h with the restriction enzyme Apa1 at 30 1C according to the manufacturer’s instructions. The DNA restriction fragments in
the plug slices were resolved by electrophoresis through a 1% SeaKem Gold agarose gel in 0.5 TB buffer. The electrophoretic parameters used were the standard ones for L. monocytogenes (Graves et al., 1994), initial switch time 4.0 s; final switch time 40 s; run time 22 h; angle
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1201 gradient 6.0 V/cm; temperature 14 1C; ramping factor linear. After electrophoresis gels were stained for 30 min in 250 ml of deionized water containing 12 ml of ethidium bromide (10 mg/ml), then destained in distilled water and viewed under UV light. Lamda ladder PFGE marker (New England Biolabs (Hertfordshire, England, UK) was used for molecular weight size determination.
Amplified fragment length polymorphism (AFLP) analysis L. monocytogenes cultures were analysed by AFLP using a modification of the method described previously (Guerra et al., 2002). Isolates were cultured on blood agar and nucleic acid extracted using a MagNA Pure automatic nucleic acid extraction instrument (Total Nucleic Acid Isolation Kit or the Bacterial and Fungal DNA III isolation Kit, Roche Molecular Biochemicals, Lewes, UK) according to the manufacturer’s instructions. AFLP analysis comprised digestion of DNA with EcoR1, ligation with adapters ECO-AD1 and -AD2 followed by amplification by polymerase chain reaction (PCR) with the primer EcoR1-G. A no template control was included in each batch of tests. Banding patterns were resolved by running 13 ml of the amplified product in a 1.5% (w/v) agarose gel containing ethidium bromide, observed under UV transillumination and fluorescent bands were recorded with type 667 film (Polaroid, St. Albans, UK). Banding patterns of images of the ethidium bromide-stained gels were analysed by visual inspection.
Virulence lineage Isolates were categorized into three lineages I, II and III, correlating with potential human pathogenicity according to ribotype and serotype characteristics, as previously described (Nadon et al., 2001; Wiedmann et al., 1997). Lineage I representing the most potentially virulent group is comprised of serotypes 1/2b, 3b, 3c and 4b. Lineage II representing intermediate virulence was regarded as comprising serotypes 1/2a, 1/2c and 3a. Lineage III comprised of serotypes 4a and 4c.
Results Forty-six L. monocytogenes isolates were investigated using phenotype-based and genotyping methods. Twenty-six of these isolates had been cultured from product obtained from a number of smoked fish food processsors operating in Ireland. In addition, a further 20 L. monocytogenes from various food products other than smoked salmon were analysed in parallel to provide a comparative data set. Types of food product
included coleslaw, bacon (baked, cooked and smoked), egg mayonnaise, pasta salad, mussels/prawns, smoked mackerel and smoked turkey.
Serotyping All L. monocytogenes isolates were serotyped (Table 1). Four serotypes were identified among the 46 isolates. Most of the isolates were identified as serotype 1/2a representing 63% (n ¼ 29) of the collection. The next most common serotype among the collection was identified as 4b (13%, n ¼ 6), followed by 1/2c (9%, n ¼ 4), and 1/2b (6.5%, n ¼ 3). Flagellar antigen could not be determined for the isolates PF1753, PF5578 or PF 5195 and PF5854 was non-typeable by these methods (Table 1).
Ribotyping Twelve distinct ribotypes were defined among the 46 L. monocytogens isolates analysed. All ribotypes were assigned on the basis of the corresponding EcoR1 restriction profile after probing with the standard riboprobe. Banding patterns obtained consisted of DNA fragments ranging in size from 1.5- through 6kbp. A composite illustration of 12 profiles is given in Fig. 1. The most common profile identified with this method was defined as DUP-ID group 1062 and identified as ribogroup 251-14-7. This profile accounted for 18 of the 46 isolates (39%) and included the serotypes 1/2a and 1/2c (Table 1). Within this grouping, 15 of the isolates were serotype 1/2a. These isolates were cultured from product obtained from three known smoked salmon processors (designated S.S.-1, -2 and -4) along with two other anonymous manufacturers (S.S.-anon). Two of the four serotypes, 1/2a and 1/2c were associated with the former processor (S.S.-1), while serotype 1/2a alone was linked with S.S.-2. The isolate designated PF5854, which was cultured from a coleslaw sample, represented the only non-smoked salmon isolate within this ribogrouping. The DUP-ID group 1062 was also assigned to a single isolate, PF5167, obtained from a smoked bacon source and which did not form part of this grouping. In this case PF5167 was also serotyped as 1/2a (Table 1). Ribogroup 251-12-2 represented the next most prevalent group, accounting for seven isolates (15%). This ribogroup contained three serogroups 1/2a (n ¼ 3), 1/2b ðn ¼ 1Þ and 1/2c ðn ¼ 2Þ. Two of the three 1/2a serotypes were associated with smoked fish product. Isolates from foods other than smoked salmon were the predominant isolates in this ribotype. The remaining 21 isolates were distributed into 10 ribogroupings. One-third of these were cultured from smoked salmon. DUP-ID 1045 accounted for ribogroup
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Fig. 1. Screen captured image showing the 12 different ribogroups identified in the course of this study. The image displays the ribogroup number along with the corresponding normalized fragment pattern data for each ribogroup.
251-14-2 ðn ¼ 5Þ all of which were serotype 1/2a. Ribogroup 251-11-1 ðn ¼ 4Þ contained two serotypes 1/2b and 4b. Three isolates of serotype 1/2a, were designated as ribogroup 251-50-4. A further four isolates, two of which were serotype 4b and two 1/2a, were assigned to ribogroups 251-16-3 and 251-11-4, respectively. The final 5 isolates were assigned different DUP-ID numbers and were assigned to different ribogroupings.
Pulsed-field gel electrophoresis When applied to purified genomic DNA the restriction endonuclease Apa1 provided macrorestriction profiles which could be compared to discriminate among the 46 L. monocytogenes isolates. Banding patterns produced 17 distinct DNA fragment profiles, which were nominally designated according to the Tenover Rules as A through Q. Each profile was comprised of between 13 and 17 DNA fragments, with sizes ranging from 48.5 to 776 kbp (Fig. 2 shows composite profiles of some of these DNA fingerprints). PFGE profile A was the most common (n ¼ 17; 37%) in this collection, comprising entirely of smoked salmon-derived isolates of similar serotype and ribogroup (Table 1 and Fig. 2). Another five isolates were grouped as profile K the next most common banding pattern and in this case these isolates corresponded to serotype 1/2a and ribogroup 251-14-2 and were only found in isolates from foods other then smoked salmon. The remaining 24 isolates were represented by PFGE types B through J and L through Q. In each case the profile was assigned to between one and three isolates. In the current data set
the PFGE profiles of isolates from smoked salmon product were not shared with those from other food products (Table 1).
Amplified fragment length polymorphism analysis Purified genomic DNA prepared from all isolates was used for AFLP analysis. Following EcoR1 digestion, 12 distinct AFLP profiles designated A through M, were obtained (data not shown). In analogy with the PFGE data, AFLP profile A ðn ¼ 16Þ was the most common profile identified in this data set, again found only in the smoked salmon-derived isolates, and representing serotypes 1/2a or 1/2c). AFLP profile B accounted for 9 of the isolates (of serotypes 1/2a, 1/2b and 1/2c), C ðn ¼ 6Þ, E, I and G (n ¼ 2 each), D, J, H, M, K and F ðn ¼ 1Þ. Among the smoked salmon isolates, five AFLP profiles were assigned. In contrast to PFGE, some AFLP profiles were found in both the smoked salmon and the unrelated food product isolates, while three isolates F1753, PF5578 and PF5195 were non-typeable by AFLP.
Virulence lineage All samples for which a serotype was available were within either Lineage I or II. Two of the 26 smoked salmon isolates (8%) were serotype 4b and thus Lineage I. Seven of the 20 isolates from the unrelated food products (35%) were Lineage I. Thirty-seven of the 46 isolates in the current data set (80%) were categorized as Lineage II.
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Fig. 2. Pulsed-Field Gel Electrophoresis representing 16 out of the 26 smoked fish isolates investigated. The predominant PFGE profile is indicated by the grey bars at the foot of the gel. Lane 1 ¼ 1036/01; (A), lane 2 ¼ 10319/01; (A), lane 3 ¼ 126/01; (A), lane 4 ¼ 134/01; (B), lane 5 ¼ 145/01; (B), lane 6 ¼ 146/01; (B), lane 7 ¼ 1367; (A), lane 8 ¼ 1331; (A), lane 9 ¼ PF8072; (C), lane 10 ¼ PF8102; (A), lane 11 ¼ F8119; (D), lane 12 ¼ F8136; (E), lane 13 ¼ PF8092, (A), lane 14 ¼ 3699/01; (A), lane 15 ¼ 1577; (A) and lane 16 ¼ 1599 (A). M represents a set of molecular weight markers. Letters in parenthesis denote the PFGE profile grouping assigned to each isolate.
Discussion This study employed a variety of typing methods to compare L. monocytogenes isolates from smoked salmon, with those isolated from other food products. One subtype (serogroup 1/2a, Ribotype 251-14-7 DUP 1062, PFGE A and AFLP A) was dominant in the smoked salmon isolates, representing 65% ðn ¼ 17Þ of the total number of smoked salmon isolates investigated (Table 1). Neither PFGE nor AFLP profile A were detected in isolates from other food products, while ribotype 251-14-7 occurred only once. These apparently clustered organisms were isolated from three of the five smoked salmon manufacturers involved in the current study. The finding of a single predominant subtype of L. monocytogenes in smoked fish is consistent with previous reports (Dauphin et al., 2001; Norton et al., 2001; Vitas and Garcia-Jalon, 2004). The current study provides further evidence of adaptation of a single clonal type of L. monocytogenes to persist in the fish smoking industry, either on the product during processing, or within the processing environment with subsequent product contamination. The serotype of the dominant subtype in smoked salmon in the current study, 1/2a, was consistent with previous studies which found serogroup to account for 93% of L. monocytogenes isolates from smoked salmon (Dauphin et al., 2001; Vitas and Garcia-Jalon, 2004). The next most common banding profile identified in the current study (Ribotype 251-14-2 DUP 1045, PFGE K) was found primarily in isolates from foods other than smoked salmon including both turkey and ham pro-
ducts. This finding contrasts with a previous study which found DUP 1045 to persist in the smoked-fishprocessing environment (Norton et al., 2001). All five isolates in this group were also serotype 1/2a. AFLP divided the group into two profiles B and C that may indicate that AFLP is not as discriminatory a method for L. monocytogenes as PFGE and ribotyping, as these AFLP profiles were also found in the smoked salmon isolates. The diversity of subtypes found amongst the 20 isolates from various food products (4 serotypes, 11 PFGE profiles, 9 ribotypes 11 AFLP profiles) highlights the need for efficient reliable subtyping methods to effectively identify the various genetic strains that can be found in food products. Wiedmann et al. (1997) previously investigated the relationship between ribotyping and virulence potential of a collection of L. monocytogenes. Three lineages designated I through III were defined based on ribo- and serotype characteristics (Nadon et al., 2001). These definitions were applied to this collection in an attempt to infer pathogenic potential. The low percentage (8%) of lineage I strains identified in the smoked salmon isolates correlates with the finding that smoked fish and other seafood’s are infrequently associated with human listeriosis (Jinneman et al., 1999). A higher percentage of isolates from the unrelated food sources were of lineage I, including isolates from food sources such as coleslaw, tuna salad and egg salad. While the present data set may not be entirely representative of the real situation in Ireland, our data indicates that the smoked fish products may pose a lower risk to public health compared to other types of food.
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The serotypes 1/2a, 1/2b and 4b have been implicated in the majority of human clinical listeriosis cases (McLauchlin, 1990; Pinner et al., 1992). Conventional methods such as serotyping for species identification are often used as an initial screening method, but are of limited value in detailed epidemiological investigations. Molecular subtyping methods such as the genotyping methods employed in this study have the potential utility of further resolving isolates into clonal groupings. These techniques have applicability in characterizing virulence of isolates, tracing pathogens along the foodchain, and tracking alterations in pathogen genome over time. A study conducted by the WHO validated PFGE as an excellent and reproducible method for subtyping of L. monocytogenes (Brosch et al., 1994). Internationally standardized protocols for this PGFE should facilitate inter-laboratory and international comparison of isolated pathogens (Graves and Swaminathan, 2001). However, PFGE requires specialized equipment and human resources, and is relatively time consuming, therefore is not used routinely for the initial screening of isolates. Ribotyping is a rapid, technically straightforward, method of subtyping which has been sufficiently automated to minimize operator involvement. However, ribotyping is an expensive procedure, both in terms of initial capital outlay for specialized equipment, along with substantial running costs. Furthermore, previous studies have shown that the ability of ribotying to discriminate within the serogroup 4b strains may not be adequate for epidemiological investigations (Graves et al., 1994). AFLP fingerprinting has been used successfully for typing and classifying a number of bacteria strains (Savelkoul et al., 1999). Ribotyping combined with PFGE are shown to be complimentary typing methods when compared to ribotyping and AFLP or PFGE and AFLP. Previous studies have indicated the value of combining phenotypic and genotypic approaches to investigation of listeriosis outbreaks. In the current data set, serotyping combined with both ribotyping and PFGE produced a high level of discrimination. Combined in parallel, these subtyping tools appear to provide a powerful epidemiological approach to characterize L. monocytogenes strains isolated from animals, foods, the food production environment and human clinical disease.
Acknowledgements Colleagues at the Food Safety Authority of Ireland (FSAI) are acknowledged for providing financial support to the WRH Laboratory.
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