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Journal o f Food Protection, Vol. 77, No. 8, 2014, Pages 1402-1406 doi: 10.4315/0362-028X. JFP-14-026 Copyright © , International Association for Food Protection
Research Note
Comparison of Listeria monocytogenes Isolates across the Island of Ireland DANIEL HURLEY, 1 LAURA LUQUE-SASTRE, 1 NIALL DeLAPPE,2 JOHN E. MOORE,3 MARTIN CORMICAN,2 KIERAN N. JORDAN,4 SEAMUS FANNING, 1 AND EDWARD M. FOX' 5* [UCD Centre fo r Food Safety, School o f Public Health, Physiotherapy and Population Science, University College Dublin, Republic o f Ireland;2Department o f Bacteriology, National University o f Ireland, Galway, Republic o f Ireland; 3Northern Ireland Public Health Laboratory, Department o f Bacteriology, Belfast City Hospital, Belfast BT9 AD, Northern Ireland, UK; 4Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Republic o f Ireland; and 5CSIRO Animal, Food and Health Sciences, Werribee, Victoria 3030, Australia MS 14-026: Received 16 January 2014/Accepted 1 April 2014
ABSTRACT Building a comprehensive knowledge base of the association of Listeria monocytogenes isolates across national food chains, clinical cases, and environments can play a key role in helping control the incidence of listeriosis. Today, many food chains cross national borders and are often shared by neighboring countries. This study characterized L. monocytogenes isolated from food samples in Northern Ireland and investigated whether similarities in the population and associations of L. monocytogenes strains exist in the neighboring countries of Northern Ireland and the Republic of Ireland, which together constitute the island of Ireland. Listeria monocytogenes isolates were characterized using serotyping and pulsed-field gel electrophoresis subtyping. This data was then interrogated against existing data for the Republic of Ireland, to identify any shared trends in the ecology and contamination patterns of L. monocytogenes strains. The results of this study indicated that contaminated food products often shared L. monocytogenes strains with other products. A total of six different strain subtypes were identified among 18 contaminated products. Overall strain diversity in positive samples was low, with no sample yielding more than one L. monocytogenes strain, as determined by pulsed-field gel electrophoresis subtyping. When comparisons against an Irish strain database were performed, many related strain subtypes were also shared by a variety of sources in the Republic of Ireland. This study highlights the potential benefits that a whole-island surveillance approach may present to food safety and public health in both Northern Ireland and the Republic of Ireland.
Listeria m onocytogenes is an im portant foodbom e pathogen that can cause severe listeriosis in hum ans, a disease with a high associated mortality o f up to 20% (3 ,1 8 , 22). Infections involving L. m onocytogenes are m ost com m on in the young, the old, and the im m unocom pro m ised (4 ,1 0 ). It is also o f significant im portance to pregnant w om en because it can infect the child in utero and m ay lead to m iscarriage (21). To reduce the associated burden to public health, m any countries have taken such m easures as raising aw areness am ong these groups regarding higher risk foods, introduction o f surveillance netw orks, and the establishm ent o f reference laboratories to characterize L. m onocytogenes isolates that occur in both clinical and food settings. For exam ple, PulseN et International is a surveil lance netw ork com prising m any centralized networks located globally (e.g., PulseN et USA, PulseN et AsiaPacific), w hich collect pulsed-field gel electrophoresis (PFGE) data that m ay be com pared throughout by these different m em ber netw orks (20). D ata on both clinical and
* Author for correspondence. Tel: + 61 3 9731 3407; Fax: + 61 3 9731 3201; E-mail: [email protected].
food isolates are fundam ental to effective outbreak response and epidem iological investigations. Listeriosis has been a notifiable disease in the Republic o f Ireland since 2004. To assist with clinical surveillance o f the causative agent, a reference laboratory was established at U niversity C ollege H ospital G alw ay, to characterize and curate clinical isolates. In the Republic o f Ireland, there are roughly 10 cases o f listeriosis annually (2007, w ith a spike o f 21 cases, being an exception to this) (9 ,1 2 ). C oordinated surveillance o f food isolates, how ever, w as not in place. To bridge the gap in inform ation betw een clinical and food isolates, an Irish L. m onocytogenes national database (ILm D B) was developed (5), incorporating standardized surveillance m ethods used in the PulseN et networks. This database w as populated w ith subtyping data relating to Irish L. m onocytogenes isolates, including serotype, PFG E subtype, and antibiotic sensitivity profile. Isolates were collected from a variety o f food sources, including meat, dairy, vegetable, and fish, in addition to farm and food production environm ents. This provided valuable epidem i ological data on L. m onocytogenes in a R epublic o f Ireland context and provided a foundation for im proved surveil lance. This database is prim arily used for com parative
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CHARACTERIZATION OF L. MONOCYTOGENES ISOLATES FROM IRELAND
TABLE 1. Number o f isolates analyzed from each food category included in this study Ready-to-eat category
No. of isolates (n = 90)
No. of positive samples (n = 18)
Meat Mixed (meat and vegetable) Vegetable Fish
60 15 10 5
12 3 2 1
analysis of L. monocytogenes isolated from the Republic of Ireland, although it has also been used for comparative analysis with subtyping information from other European countries and from the United States (5, 19). The island of Ireland consists of two countries: the Republic of Ireland and Northern Ireland. As such, many food products are distributed and sold in both countries, and the ingredients for these may be sourced from either. In Northern Ireland, there are roughly three to five cases of human listeriosis annually (16). There is, however, no centralized laboratory to characterize L. monocytogenes isolates, as is in place in the Republic of Ireland. Isolates from Northern Ireland that require typing for epidemiolog ical purposes are sent to the Listeria Reference Laboratory at Public Health England, which is situated at Colindale, London, UK. There is little information available to compare the epidemiology of listeriosis across the entire island of Ireland, apart from incidence rates. The purpose of this study was to characterize L. monocytogenes isolates from Northern Ireland using serotyping and PFGE analysis and to compare this to Republic of Ireland isolates to gain insight into epidemiol ogy and food-chain associations across the entire island of Ireland. This work aimed to provide greater insight into shared public health issues of listeriosis across both countries and to examine the value of a coordinated crossborder surveillance network. M A TER IA LS A N D M E TH O D S Isolates used in this study. In this study, 90 L. monocyto genes isolates obtained from 18 different food samples in Northern Ireland were characterized (Table 1). The isolates were collected using the standard ISO 11290-1 method for the detection of L. monocytogenes from food sources (13). Briefly, 25 g of sample was added to 225 ml of half Fraser broth and was incubated at 30°C for 24 h; then 10 pi of this enrichment was plated on Brilliance Listeria agar and Oxford agar, and 100 pi of the enrichment was added to 10 ml of Fraser broth (all media from Oxoid, Ltd., Basingstoke, UK) and was incubated at 37°C for 48 h. This was plated on BLA and Oxford agar plates as previously described. Presumptive-positive Listeria colonies were confirmed according to the ISO 11290 method. Five isolates were taken from each of the 18 positive food samples, to determine the diversity of L. monocytogenes strains in each sample. All isolates were taken from foods tested in the years 2009 or 2012. The isolates were collected from retail food products by the Health and Social Care Microbiology Strain Repository, MicroARK culture collection (Belfast, Northern Ireland). Serotyping of isolates. Serotyping of all isolates was performed by a combination of O-antisera testing (Denka Seiken,
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Tokyo, Japan) and the PCR method of Doumith et al. (2004), as previously described (2, 6). To visualize PCR products, 10 pi of the completed reaction mix was run on a 1% (wt/vol) agarose gel and was stained with SYBR safe DNA stain (Invitrogen, Paisley, UK). PFGE of Listeria isolates. Subtyping of L. monocytogenes isolates was performed using PFGE as per the standard PulseNet protocol (17), with the following exceptions: sodium dodecyl sulfate was not included in the 1% SeaKem Gold plug agarose and electrophoresis was performed on a CHEF Mapper (Bio-Rad, UK) for 21 h. These modifications did not affect comparability of the generated profiles with those generated by the original protocol, and the modified procedure has been validated in-house (data not shown). The restriction enzymes A id and Apal were used to generate the PFGE profiles. Five L. monocytogenes isolates from each individual sample were analyzed to determine strain diversity in the samples. The generated PFGE profiles were analyzed using BioNumerics v6.5 (Applied Maths, Sint-Martens-Latem, Belgium), using the Dice coefficient and unweighted pair group method with arithmetic mean analysis, with “ optimization” and “ tolerance” settings of 1%. Comparative analysis of L. monocytogenes isolates. PFGE fingerprints generated in this study were interrogated against the BioNumerics-based ILmDB database, previously generated to archive Irish L. monocytogenes isolates (5). This analysis used the Dice coefficient and unweighted pair group method with arithmetic mean analysis, with “ optimization” and “ tolerance” settings of 1%. A 92% similarity cut-off threshold was used to select isolates for inclusion in the cross-country comparison, to identify indistin guishable or closely related clones, typically with one to two band differences (21). The ILmDB contains subtyping information on L. monocytogenes isolates from many sources, including clinical, food, and food production environments (meat, dairy, vegetable, fish), as well as environmental isolates (e.g., farms). This includes data such as PFGE subtyping, serotyping, and antibiotic sensitivities. RESULTS AND D ISC U SSIO N A ctive surveillance o f the L. monocytogenes biom e is an im portant com ponent in preventing disease and controlling the public health burden caused by the organism in com m unities. Surveillance o f L. monocytogenes can include m any aspects. A t its m ost fundam ental, this m ay com prise food and environm ental sam pling for the presence or absence o f the organism , w hich can identify food batches that are a risk to the public or identify areas in a local environm ent, such as a farm or food processing facility, that m ay be colonized by the organism and represent a risk factor for cross-contam ination to the food chain. Serotyping can be em ployed in epidem iological investigations as a tool to rapidly screen potential outbreak strains, by identifying potential isolates m atching (or differing from) the serotype o f the outbreak strain (1). By utilizing m ore advanced scientific tools, such as strain subtyping, a far m ore advanced understanding o f the epidem iology o f the organism can be gleaned (8). In relation to a localized environm ent, it can identify putative transfer routes that are responsible for cross contam ination o f the organism , reveal contam ination events, and identify persistent isolates that can increase the risk of contam ination o f food products or food chains. T he m ost advanced surveillance systems, how ever, represent national, international, or global approaches, w hich com bine all these
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Isolate
Food Source
Date Isolated
Serotype Clade
L/09/1
Garlic and chive croquettes
19-Jan-09
l/2 c
L/09/32
Diced cooked chicken
27-Jan-09
l/2 c
20-Jan-09
1/2 a
Cl
L/09/13
Chicken tikka
L/09/52
Sweet chilli chicken sandwich 02-Feb-09
l/2 a
L/09/57
Chicken tikka
02-Feb-09
l/2 a
L/09/64
Diced chicken
02-Feb-09
l/2 a
L/09/7
Sweet chilli chicken
20-Jan-09
l/2 a
L/09/23
Cooked ham
23-Jan-09
l/2 a
L/09/30
Cooked chicken
23-Jan-09
l/2 a
L/09/38
BBQ chicken
29-Jan-09
l/2 a
L/09/66
Egg and onion sandwich
03-Feb-09
l/2 a
L/09/75
Cooked chicken
03-Feb-09
l/2 a
L/09/76
Cooked chicken breast
04-Feb-09
l/2 a
L/12/1
Cooked chicken
2012
4b
L/12/6
Tuna
2012
4b 4b
L/09/16
Chicken sandwich
20-Jan-09
L/09/43
Cooked chicken
29-Jan-09
4b
L/09/50
Chicken sandwich
29-Jan-09
4b
C2
C3
C4
C5
FIGURE 1. Similarity dendrogram of each individual PFGE subtype identified in positive food samples. Combined Asc/ and Apa/ restriction subtyping clustered the 18 strains into six distinct clades, labelled Cl to C5. Genetic lineage groupings are marked. aspects to allow rapid responses to listeriosis outbreaks to minimize the impact on public health on a large scale (4,11). The data contained in these systems can also provide valuable insight into, for example, the high frequency of some strains in certain food chains or their widespread distribution through many environments (5, 8). Currently, in the Republic of Ireland, there are two reference laboratories: the National Salmonella, Shigella and Listeria Reference Laboratory, which receives and curates clinical isolates; and the Dairy Science Laboratory of the Department of Agriculture, which curates food and environmental isolates. In Northern Ireland, however, the Health and Social Care MicroARK culture collection exists to preserve and archive all clinical and food isolates. Previous outbreaks of listeriosis have sometimes involved more than one strain; the fact that foods may be contaminated with more than one strain, a multitude of which may be capable of eliciting severe disease in humans, can complicate the response by public health authorities (7, 14). For the purposes of this study, each unique combined PFGE subtype (i.e., A id and ApaT) has been treated as relating to an individual strain. Isolates sharing the same combined PFGE subtypes are identified as the same L. monocytogenes strain. Comparative analysis of all five isolates taken from each sample indicated that no individual sample contained more than one strain, based on serotyping and PFGE subtyping results. It is possible, however, that another strain may have been present but was outcompeted by the dominant strain in the sample and, so, was not detected. Nonetheless, the possibility of strain diversity in samples should be taken into consideration. Strains isolated from foods in this study were identified as one of three different serotypes (overall distribution in parentheses): l/2a (61%), 4b (28%), and l/2c (11%). This trend is shared by many countries, with these serotypes, along with serotype l/2b, among the most common found in
both food and disease incidence (3, 5). Distinct genetic lineages have been previously described for L. monocyto genes strains (15). The majority of serotype 4b strains identified to date are representative of lineage I, whereas serotype l/2a and l/2c strains are classified among lineage II strains. Clustering of the strains in this study by PFGE analysis (Fig. 1) clearly separated the lineage I and II strains into two distinct groupings; the lineage I group contained the clades C4 and C5, and a second distinct lineage II group contained the C l, C2, and C3 clades. Although serotype 4b strains are present in lineages I, III, and IV, the close similarity of serotype 4b strains in this study with lineage I isolates from the Republic of Ireland suggests that these Northern Ireland strains are lineage I. Overall strain diversity, as determined by PFGE analysis, was relatively low, with strains from all 18 samples falling into one of five PFGE clades (each clade containing a single, indistinguish able PFGE subtype, with the exception of C l, discussed below). PFGE profiling of strains by AscI restriction separated the 18 strains into five different PFGE subtypes (a strain was defined as having a unique PFGE profile, as determined by A id and Apal PFGE analysis). Apal restriction divided these strains into six different PFGE subtypes. Combined comparative analysis using unbiased A id and Apal subtyping results yielded five different clades, C l to C5 (Fig. 1). It is unclear whether this is reflective of dominant L. monocytogenes strain types among Northern Ireland food chains, or whether this low diversity may possibly be due to a smaller subset of businesses involved in production of the foods tested. Such knowledge will require additional testing of a wide variety of food products. Interestingly, 14 of the 18 samples that tested positive contained chicken; strains isolated from products containing chicken were identified in all five PFGE clades. Only one of these clades, C 1, had a highly similar (98%, Fig. 2) Republic of Ireland strain originating from a sample containing
J. Food Prot., Vol. 77, No. 8
CHARACTERIZATION OF L. MONOCYTOGENES ISOLATES FROM IRELAND
Database 8 PFGE-Apal
PFGE-Ascl
[No. o f isolates] A
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Isolated fro m * C
D
DE
F
M M E RM V
Year o f isolation
Clade
FIGURE 2. Comparative analysis o f isolates from both Northern Ireland and the Republic o f Ireland. A similarity cut-off threshold o f 92% was used, following dendrogram clustering ofPFGE subtyping data. Among Republic o f Ireland strains, PFGE subtypes were found that were indistinguishable from, or highly related to, four o f the six Northern Ireland strain subtypes .Each sample type that contained the particular strain is marked with a closed black box (■ ). A, animal; C, clinical; D, dairy food; DE, dairy environment; F ,fish; M, meat; ME, meat environment; RM, ready-to-eat food (mix); V, vegetable.
chicken. This suggests that integration of the epidemiological data of both countries will allow a greater understanding of the spread of certain strains through different food chains. Clade C l contained two different strains, which shared a high similarity. These strains were indistinguishable by AycI analysis, and only Apal separated them into distinct, but closely related, strains. This highlights how genetic diversifi cation (e.g., through insertion events, such as phage integration into the chromosome, or through genetic mutations) may cause individual species of L. monocytogenes to diversify into different subspecies. The physiological or clinical significance of this diversification could vary; although L. monocytogenes may be separated into different species, the potential clinical significance of the new strain subtype may not be altered. Thus, understanding characteristics of highly related strains, such as its virulence, may provide insights into the clinical significance of other highly related strains. Clades C2 and C3 contained the two most common strain subtypes identified (from five and six different food sources, respectively). All food sources containing the C2 strain subtype contained chicken, and four of the six foods in the C3 clade contained chicken, indicating that this may be an important vector for human transmission in Northern Ireland. Among Republic of Ireland isolates, 37 indistinguishable strains were identified, found in meat production environ mental samples and in fish and vegetable sources, when PFGE data was compared with C3 strains. This suggests a wide distribution of this strain through different food chains. Clade C4 contained the two isolates from 2012, with one originating in a chicken product and the other isolate found in tuna. This PFGE subtype was closely related to clinical isolates from cases in the Republic of Ireland in the years 2007 and 2009 (indistinguishable A id PFGE profiles); the subtype was also identified in cheese, as well as in bovine fecal samples. Taken together, this dataset of sources from both countries indicates that these clinically relevant strains may be widespread in food chains across the island of Ireland and, as such, may be regarded as a high-risk strain. The PFGE subtype of clade C5 strains was found in three different food sources, all of which contained chicken. This PFGE subtype showed high similarity to isolates from clinical cases in the Republic of Ireland in the years 2005, 2008, and
2009 (indistinguishable by AscI analysis). Interestingly, this PFGE subtype has not been previously linked with any food type in the Republic of Ireland and has only been associated with isolates from human patients with listeriosis. This highlights the potential of combined surveillance across Northern Ireland and the Republic of Ireland to identify possible food sources that may be important in epidemiological investigation of cases of listeriosis, which may not be apparent when data from one country is studied in isolation. When examined temporally, individual strain subtypes identified through PFGE analysis were not seen across multiple years: all isolates from 2009 contained one of five strain subtypes, and the strain type characteristic of clade C4 was exclusively found in 2012. Since the sample size of this study was small, however, this likely does not reflect the overall distribution of these strain types through different food chains or environments, over time. Combining this data with information from the Republic of Ireland, however, may contribute to an improved understanding of ecological distribution of L. monocytogenes strains. Although Northern Ireland and the Republic of Ireland are separate countries with their own indigenous food businesses, many food production businesses may also supply across the border to the neighboring counfry; and, similarly, ingredients suppliers may supply to businesses on both sides of the border. To examine whether L. monocyto genes populations were distinct when comparing that of both countries, the PFGE subtyping data of isolates from foods in Northern Ireland were analyzed by scrutinizing against the ILmDB, which contains an extensive record of L. monocy togenes isolates from the Republic of Ireland, obtained from a wide variety of foods and environments. A similarity dendrogram was generated by applying a 92% similarity cut-off threshold to comparative analysis of PFGE data of all isolates from Northern Ireland and the Republic of Ireland. Interestingly, strains that were highly related to or indistin guishable from four of the six strain types identified among the Northern Ireland isolates had previously been identified in the Republic of Ireland (Fig. 2). This indicates that both countries likely share some very similar epidemiological trends in terms of strain diversity and suggests that a coordinated cross-border surveillance of L. monocytogenes
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would provide much value to public health. This is particularly prudent as three of these four strain types were implicated in cases of clinical disease in the Republic of Ireland, indicating that these contaminated food products may be at higher risk for causing illness. Note that A.vcl analysis, when taken alone, did not differentiate certain strains (e.g., C4 and C5 strain PFGE subtypes could only be distinguished from otherwise indistinguishable Republic of Ireland strains through Apal analysis). This highlights the importance of including a second restriction enzyme when using PFGE analysis for outbreak investigations, for example. Previous work has identified highly similar or indistin guishable clones (as determined by PFGE analysis) across different countries ( J , 19), suggesting that more data is needed to investigate whether the similarities between L. monocyto genes strains across Northern Ireland and the Republic of Ireland are due to shared aspects along the food chain or are simply due to the existence of stable global clones. Another interesting observation when comparing strains from both countries was the wide variety of sources from which they were isolated, with a combination of two to five different foods and environments associated with most strains. This widespread distribution may be responsible for a higher frequency of these strains contaminating food products. It is worth noting that these strains were also identified over long periods of time (up to 9 years), indicating that these strains may have colonized niches along the food production chain, resulting in recontamination over time. In conclusion, although the isolate set for this study was limited, it has identified many similarities in the ecology of L. monocytogenes strains shared by both Northern Ireland and the Republic of Ireland and emphasizes the value of a whole-island surveillance strategy. Such a strategy will help reinforce listeriosis public health safety across all communities of Ireland.
7.
8.
9.
10. 11.
12.
13.
14.
15.
16.
ACKNOWLEDGMENTS This project was supported by .ra/efood, through the Listeria Knowledge Networks. The authors acknowledge the help of Marion Dalmasso and Avelino Alvarez-Ordonez for their contribution to the aspects on strain comparison.
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Journal o f Food Protection, Vol. 77, No. 8, 2014, Pages 1402-1406 doi: 10.4315/0362-028X. JFP-14-026 Copyright © , International Association for Food Protection
Research Note
Comparison of Listeria monocytogenes Isolates across the Island of Ireland DANIEL HURLEY, 1 LAURA LUQUE-SASTRE, 1 NIALL DeLAPPE,2 JOHN E. MOORE,3 MARTIN CORMICAN,2 KIERAN N. JORDAN,4 SEAMUS FANNING, 1 AND EDWARD M. FOX' 5* [UCD Centre fo r Food Safety, School o f Public Health, Physiotherapy and Population Science, University College Dublin, Republic o f Ireland;2Department o f Bacteriology, National University o f Ireland, Galway, Republic o f Ireland; 3Northern Ireland Public Health Laboratory, Department o f Bacteriology, Belfast City Hospital, Belfast BT9 AD, Northern Ireland, UK; 4Teagasc Food Research Centre, Moorepark, Fermoy, County Cork, Republic o f Ireland; and 5CSIRO Animal, Food and Health Sciences, Werribee, Victoria 3030, Australia MS 14-026: Received 16 January 2014/Accepted 1 April 2014
ABSTRACT Building a comprehensive knowledge base of the association of Listeria monocytogenes isolates across national food chains, clinical cases, and environments can play a key role in helping control the incidence of listeriosis. Today, many food chains cross national borders and are often shared by neighboring countries. This study characterized L. monocytogenes isolated from food samples in Northern Ireland and investigated whether similarities in the population and associations of L. monocytogenes strains exist in the neighboring countries of Northern Ireland and the Republic of Ireland, which together constitute the island of Ireland. Listeria monocytogenes isolates were characterized using serotyping and pulsed-field gel electrophoresis subtyping. This data was then interrogated against existing data for the Republic of Ireland, to identify any shared trends in the ecology and contamination patterns of L. monocytogenes strains. The results of this study indicated that contaminated food products often shared L. monocytogenes strains with other products. A total of six different strain subtypes were identified among 18 contaminated products. Overall strain diversity in positive samples was low, with no sample yielding more than one L. monocytogenes strain, as determined by pulsed-field gel electrophoresis subtyping. When comparisons against an Irish strain database were performed, many related strain subtypes were also shared by a variety of sources in the Republic of Ireland. This study highlights the potential benefits that a whole-island surveillance approach may present to food safety and public health in both Northern Ireland and the Republic of Ireland.
Listeria m onocytogenes is an im portant foodbom e pathogen that can cause severe listeriosis in hum ans, a disease with a high associated mortality o f up to 20% (3 ,1 8 , 22). Infections involving L. m onocytogenes are m ost com m on in the young, the old, and the im m unocom pro m ised (4 ,1 0 ). It is also o f significant im portance to pregnant w om en because it can infect the child in utero and m ay lead to m iscarriage (21). To reduce the associated burden to public health, m any countries have taken such m easures as raising aw areness am ong these groups regarding higher risk foods, introduction o f surveillance netw orks, and the establishm ent o f reference laboratories to characterize L. m onocytogenes isolates that occur in both clinical and food settings. For exam ple, PulseN et International is a surveil lance netw ork com prising m any centralized networks located globally (e.g., PulseN et USA, PulseN et AsiaPacific), w hich collect pulsed-field gel electrophoresis (PFGE) data that m ay be com pared throughout by these different m em ber netw orks (20). D ata on both clinical and
* Author for correspondence. Tel: + 61 3 9731 3407; Fax: + 61 3 9731 3201; E-mail: [email protected].
food isolates are fundam ental to effective outbreak response and epidem iological investigations. Listeriosis has been a notifiable disease in the Republic o f Ireland since 2004. To assist with clinical surveillance o f the causative agent, a reference laboratory was established at U niversity C ollege H ospital G alw ay, to characterize and curate clinical isolates. In the Republic o f Ireland, there are roughly 10 cases o f listeriosis annually (2007, w ith a spike o f 21 cases, being an exception to this) (9 ,1 2 ). C oordinated surveillance o f food isolates, how ever, w as not in place. To bridge the gap in inform ation betw een clinical and food isolates, an Irish L. m onocytogenes national database (ILm D B) was developed (5), incorporating standardized surveillance m ethods used in the PulseN et networks. This database w as populated w ith subtyping data relating to Irish L. m onocytogenes isolates, including serotype, PFG E subtype, and antibiotic sensitivity profile. Isolates were collected from a variety o f food sources, including meat, dairy, vegetable, and fish, in addition to farm and food production environm ents. This provided valuable epidem i ological data on L. m onocytogenes in a R epublic o f Ireland context and provided a foundation for im proved surveil lance. This database is prim arily used for com parative
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TABLE 1. Number o f isolates analyzed from each food category included in this study Ready-to-eat category
No. of isolates (n = 90)
No. of positive samples (n = 18)
Meat Mixed (meat and vegetable) Vegetable Fish
60 15 10 5
12 3 2 1
analysis of L. monocytogenes isolated from the Republic of Ireland, although it has also been used for comparative analysis with subtyping information from other European countries and from the United States (5, 19). The island of Ireland consists of two countries: the Republic of Ireland and Northern Ireland. As such, many food products are distributed and sold in both countries, and the ingredients for these may be sourced from either. In Northern Ireland, there are roughly three to five cases of human listeriosis annually (16). There is, however, no centralized laboratory to characterize L. monocytogenes isolates, as is in place in the Republic of Ireland. Isolates from Northern Ireland that require typing for epidemiolog ical purposes are sent to the Listeria Reference Laboratory at Public Health England, which is situated at Colindale, London, UK. There is little information available to compare the epidemiology of listeriosis across the entire island of Ireland, apart from incidence rates. The purpose of this study was to characterize L. monocytogenes isolates from Northern Ireland using serotyping and PFGE analysis and to compare this to Republic of Ireland isolates to gain insight into epidemiol ogy and food-chain associations across the entire island of Ireland. This work aimed to provide greater insight into shared public health issues of listeriosis across both countries and to examine the value of a coordinated crossborder surveillance network. M A TER IA LS A N D M E TH O D S Isolates used in this study. In this study, 90 L. monocyto genes isolates obtained from 18 different food samples in Northern Ireland were characterized (Table 1). The isolates were collected using the standard ISO 11290-1 method for the detection of L. monocytogenes from food sources (13). Briefly, 25 g of sample was added to 225 ml of half Fraser broth and was incubated at 30°C for 24 h; then 10 pi of this enrichment was plated on Brilliance Listeria agar and Oxford agar, and 100 pi of the enrichment was added to 10 ml of Fraser broth (all media from Oxoid, Ltd., Basingstoke, UK) and was incubated at 37°C for 48 h. This was plated on BLA and Oxford agar plates as previously described. Presumptive-positive Listeria colonies were confirmed according to the ISO 11290 method. Five isolates were taken from each of the 18 positive food samples, to determine the diversity of L. monocytogenes strains in each sample. All isolates were taken from foods tested in the years 2009 or 2012. The isolates were collected from retail food products by the Health and Social Care Microbiology Strain Repository, MicroARK culture collection (Belfast, Northern Ireland). Serotyping of isolates. Serotyping of all isolates was performed by a combination of O-antisera testing (Denka Seiken,
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Tokyo, Japan) and the PCR method of Doumith et al. (2004), as previously described (2, 6). To visualize PCR products, 10 pi of the completed reaction mix was run on a 1% (wt/vol) agarose gel and was stained with SYBR safe DNA stain (Invitrogen, Paisley, UK). PFGE of Listeria isolates. Subtyping of L. monocytogenes isolates was performed using PFGE as per the standard PulseNet protocol (17), with the following exceptions: sodium dodecyl sulfate was not included in the 1% SeaKem Gold plug agarose and electrophoresis was performed on a CHEF Mapper (Bio-Rad, UK) for 21 h. These modifications did not affect comparability of the generated profiles with those generated by the original protocol, and the modified procedure has been validated in-house (data not shown). The restriction enzymes A id and Apal were used to generate the PFGE profiles. Five L. monocytogenes isolates from each individual sample were analyzed to determine strain diversity in the samples. The generated PFGE profiles were analyzed using BioNumerics v6.5 (Applied Maths, Sint-Martens-Latem, Belgium), using the Dice coefficient and unweighted pair group method with arithmetic mean analysis, with “ optimization” and “ tolerance” settings of 1%. Comparative analysis of L. monocytogenes isolates. PFGE fingerprints generated in this study were interrogated against the BioNumerics-based ILmDB database, previously generated to archive Irish L. monocytogenes isolates (5). This analysis used the Dice coefficient and unweighted pair group method with arithmetic mean analysis, with “ optimization” and “ tolerance” settings of 1%. A 92% similarity cut-off threshold was used to select isolates for inclusion in the cross-country comparison, to identify indistin guishable or closely related clones, typically with one to two band differences (21). The ILmDB contains subtyping information on L. monocytogenes isolates from many sources, including clinical, food, and food production environments (meat, dairy, vegetable, fish), as well as environmental isolates (e.g., farms). This includes data such as PFGE subtyping, serotyping, and antibiotic sensitivities. RESULTS AND D ISC U SSIO N A ctive surveillance o f the L. monocytogenes biom e is an im portant com ponent in preventing disease and controlling the public health burden caused by the organism in com m unities. Surveillance o f L. monocytogenes can include m any aspects. A t its m ost fundam ental, this m ay com prise food and environm ental sam pling for the presence or absence o f the organism , w hich can identify food batches that are a risk to the public or identify areas in a local environm ent, such as a farm or food processing facility, that m ay be colonized by the organism and represent a risk factor for cross-contam ination to the food chain. Serotyping can be em ployed in epidem iological investigations as a tool to rapidly screen potential outbreak strains, by identifying potential isolates m atching (or differing from) the serotype o f the outbreak strain (1). By utilizing m ore advanced scientific tools, such as strain subtyping, a far m ore advanced understanding o f the epidem iology o f the organism can be gleaned (8). In relation to a localized environm ent, it can identify putative transfer routes that are responsible for cross contam ination o f the organism , reveal contam ination events, and identify persistent isolates that can increase the risk of contam ination o f food products or food chains. T he m ost advanced surveillance systems, how ever, represent national, international, or global approaches, w hich com bine all these
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Isolate
Food Source
Date Isolated
Serotype Clade
L/09/1
Garlic and chive croquettes
19-Jan-09
l/2 c
L/09/32
Diced cooked chicken
27-Jan-09
l/2 c
20-Jan-09
1/2 a
Cl
L/09/13
Chicken tikka
L/09/52
Sweet chilli chicken sandwich 02-Feb-09
l/2 a
L/09/57
Chicken tikka
02-Feb-09
l/2 a
L/09/64
Diced chicken
02-Feb-09
l/2 a
L/09/7
Sweet chilli chicken
20-Jan-09
l/2 a
L/09/23
Cooked ham
23-Jan-09
l/2 a
L/09/30
Cooked chicken
23-Jan-09
l/2 a
L/09/38
BBQ chicken
29-Jan-09
l/2 a
L/09/66
Egg and onion sandwich
03-Feb-09
l/2 a
L/09/75
Cooked chicken
03-Feb-09
l/2 a
L/09/76
Cooked chicken breast
04-Feb-09
l/2 a
L/12/1
Cooked chicken
2012
4b
L/12/6
Tuna
2012
4b 4b
L/09/16
Chicken sandwich
20-Jan-09
L/09/43
Cooked chicken
29-Jan-09
4b
L/09/50
Chicken sandwich
29-Jan-09
4b
C2
C3
C4
C5
FIGURE 1. Similarity dendrogram of each individual PFGE subtype identified in positive food samples. Combined Asc/ and Apa/ restriction subtyping clustered the 18 strains into six distinct clades, labelled Cl to C5. Genetic lineage groupings are marked. aspects to allow rapid responses to listeriosis outbreaks to minimize the impact on public health on a large scale (4,11). The data contained in these systems can also provide valuable insight into, for example, the high frequency of some strains in certain food chains or their widespread distribution through many environments (5, 8). Currently, in the Republic of Ireland, there are two reference laboratories: the National Salmonella, Shigella and Listeria Reference Laboratory, which receives and curates clinical isolates; and the Dairy Science Laboratory of the Department of Agriculture, which curates food and environmental isolates. In Northern Ireland, however, the Health and Social Care MicroARK culture collection exists to preserve and archive all clinical and food isolates. Previous outbreaks of listeriosis have sometimes involved more than one strain; the fact that foods may be contaminated with more than one strain, a multitude of which may be capable of eliciting severe disease in humans, can complicate the response by public health authorities (7, 14). For the purposes of this study, each unique combined PFGE subtype (i.e., A id and ApaT) has been treated as relating to an individual strain. Isolates sharing the same combined PFGE subtypes are identified as the same L. monocytogenes strain. Comparative analysis of all five isolates taken from each sample indicated that no individual sample contained more than one strain, based on serotyping and PFGE subtyping results. It is possible, however, that another strain may have been present but was outcompeted by the dominant strain in the sample and, so, was not detected. Nonetheless, the possibility of strain diversity in samples should be taken into consideration. Strains isolated from foods in this study were identified as one of three different serotypes (overall distribution in parentheses): l/2a (61%), 4b (28%), and l/2c (11%). This trend is shared by many countries, with these serotypes, along with serotype l/2b, among the most common found in
both food and disease incidence (3, 5). Distinct genetic lineages have been previously described for L. monocyto genes strains (15). The majority of serotype 4b strains identified to date are representative of lineage I, whereas serotype l/2a and l/2c strains are classified among lineage II strains. Clustering of the strains in this study by PFGE analysis (Fig. 1) clearly separated the lineage I and II strains into two distinct groupings; the lineage I group contained the clades C4 and C5, and a second distinct lineage II group contained the C l, C2, and C3 clades. Although serotype 4b strains are present in lineages I, III, and IV, the close similarity of serotype 4b strains in this study with lineage I isolates from the Republic of Ireland suggests that these Northern Ireland strains are lineage I. Overall strain diversity, as determined by PFGE analysis, was relatively low, with strains from all 18 samples falling into one of five PFGE clades (each clade containing a single, indistinguish able PFGE subtype, with the exception of C l, discussed below). PFGE profiling of strains by AscI restriction separated the 18 strains into five different PFGE subtypes (a strain was defined as having a unique PFGE profile, as determined by A id and Apal PFGE analysis). Apal restriction divided these strains into six different PFGE subtypes. Combined comparative analysis using unbiased A id and Apal subtyping results yielded five different clades, C l to C5 (Fig. 1). It is unclear whether this is reflective of dominant L. monocytogenes strain types among Northern Ireland food chains, or whether this low diversity may possibly be due to a smaller subset of businesses involved in production of the foods tested. Such knowledge will require additional testing of a wide variety of food products. Interestingly, 14 of the 18 samples that tested positive contained chicken; strains isolated from products containing chicken were identified in all five PFGE clades. Only one of these clades, C 1, had a highly similar (98%, Fig. 2) Republic of Ireland strain originating from a sample containing
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Database 8 PFGE-Apal
PFGE-Ascl
[No. o f isolates] A
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Isolated fro m * C
D
DE
F
M M E RM V
Year o f isolation
Clade
FIGURE 2. Comparative analysis o f isolates from both Northern Ireland and the Republic o f Ireland. A similarity cut-off threshold o f 92% was used, following dendrogram clustering ofPFGE subtyping data. Among Republic o f Ireland strains, PFGE subtypes were found that were indistinguishable from, or highly related to, four o f the six Northern Ireland strain subtypes .Each sample type that contained the particular strain is marked with a closed black box (■ ). A, animal; C, clinical; D, dairy food; DE, dairy environment; F ,fish; M, meat; ME, meat environment; RM, ready-to-eat food (mix); V, vegetable.
chicken. This suggests that integration of the epidemiological data of both countries will allow a greater understanding of the spread of certain strains through different food chains. Clade C l contained two different strains, which shared a high similarity. These strains were indistinguishable by AycI analysis, and only Apal separated them into distinct, but closely related, strains. This highlights how genetic diversifi cation (e.g., through insertion events, such as phage integration into the chromosome, or through genetic mutations) may cause individual species of L. monocytogenes to diversify into different subspecies. The physiological or clinical significance of this diversification could vary; although L. monocytogenes may be separated into different species, the potential clinical significance of the new strain subtype may not be altered. Thus, understanding characteristics of highly related strains, such as its virulence, may provide insights into the clinical significance of other highly related strains. Clades C2 and C3 contained the two most common strain subtypes identified (from five and six different food sources, respectively). All food sources containing the C2 strain subtype contained chicken, and four of the six foods in the C3 clade contained chicken, indicating that this may be an important vector for human transmission in Northern Ireland. Among Republic of Ireland isolates, 37 indistinguishable strains were identified, found in meat production environ mental samples and in fish and vegetable sources, when PFGE data was compared with C3 strains. This suggests a wide distribution of this strain through different food chains. Clade C4 contained the two isolates from 2012, with one originating in a chicken product and the other isolate found in tuna. This PFGE subtype was closely related to clinical isolates from cases in the Republic of Ireland in the years 2007 and 2009 (indistinguishable A id PFGE profiles); the subtype was also identified in cheese, as well as in bovine fecal samples. Taken together, this dataset of sources from both countries indicates that these clinically relevant strains may be widespread in food chains across the island of Ireland and, as such, may be regarded as a high-risk strain. The PFGE subtype of clade C5 strains was found in three different food sources, all of which contained chicken. This PFGE subtype showed high similarity to isolates from clinical cases in the Republic of Ireland in the years 2005, 2008, and
2009 (indistinguishable by AscI analysis). Interestingly, this PFGE subtype has not been previously linked with any food type in the Republic of Ireland and has only been associated with isolates from human patients with listeriosis. This highlights the potential of combined surveillance across Northern Ireland and the Republic of Ireland to identify possible food sources that may be important in epidemiological investigation of cases of listeriosis, which may not be apparent when data from one country is studied in isolation. When examined temporally, individual strain subtypes identified through PFGE analysis were not seen across multiple years: all isolates from 2009 contained one of five strain subtypes, and the strain type characteristic of clade C4 was exclusively found in 2012. Since the sample size of this study was small, however, this likely does not reflect the overall distribution of these strain types through different food chains or environments, over time. Combining this data with information from the Republic of Ireland, however, may contribute to an improved understanding of ecological distribution of L. monocytogenes strains. Although Northern Ireland and the Republic of Ireland are separate countries with their own indigenous food businesses, many food production businesses may also supply across the border to the neighboring counfry; and, similarly, ingredients suppliers may supply to businesses on both sides of the border. To examine whether L. monocyto genes populations were distinct when comparing that of both countries, the PFGE subtyping data of isolates from foods in Northern Ireland were analyzed by scrutinizing against the ILmDB, which contains an extensive record of L. monocy togenes isolates from the Republic of Ireland, obtained from a wide variety of foods and environments. A similarity dendrogram was generated by applying a 92% similarity cut-off threshold to comparative analysis of PFGE data of all isolates from Northern Ireland and the Republic of Ireland. Interestingly, strains that were highly related to or indistin guishable from four of the six strain types identified among the Northern Ireland isolates had previously been identified in the Republic of Ireland (Fig. 2). This indicates that both countries likely share some very similar epidemiological trends in terms of strain diversity and suggests that a coordinated cross-border surveillance of L. monocytogenes
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would provide much value to public health. This is particularly prudent as three of these four strain types were implicated in cases of clinical disease in the Republic of Ireland, indicating that these contaminated food products may be at higher risk for causing illness. Note that A.vcl analysis, when taken alone, did not differentiate certain strains (e.g., C4 and C5 strain PFGE subtypes could only be distinguished from otherwise indistinguishable Republic of Ireland strains through Apal analysis). This highlights the importance of including a second restriction enzyme when using PFGE analysis for outbreak investigations, for example. Previous work has identified highly similar or indistin guishable clones (as determined by PFGE analysis) across different countries ( J , 19), suggesting that more data is needed to investigate whether the similarities between L. monocyto genes strains across Northern Ireland and the Republic of Ireland are due to shared aspects along the food chain or are simply due to the existence of stable global clones. Another interesting observation when comparing strains from both countries was the wide variety of sources from which they were isolated, with a combination of two to five different foods and environments associated with most strains. This widespread distribution may be responsible for a higher frequency of these strains contaminating food products. It is worth noting that these strains were also identified over long periods of time (up to 9 years), indicating that these strains may have colonized niches along the food production chain, resulting in recontamination over time. In conclusion, although the isolate set for this study was limited, it has identified many similarities in the ecology of L. monocytogenes strains shared by both Northern Ireland and the Republic of Ireland and emphasizes the value of a whole-island surveillance strategy. Such a strategy will help reinforce listeriosis public health safety across all communities of Ireland.
7.
8.
9.
10. 11.
12.
13.
14.
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16.
ACKNOWLEDGMENTS This project was supported by .ra/efood, through the Listeria Knowledge Networks. The authors acknowledge the help of Marion Dalmasso and Avelino Alvarez-Ordonez for their contribution to the aspects on strain comparison.
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