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Environmental Microbiology (2014) 16(2), 405–416

doi:10.1111/1462-2920.12342

The ubiquitous nature of Listeria monocytogenes clones: a large-scale Multilocus Sequence Typing study

Jana K. Haase,1† Xavier Didelot,2 Marc Lecuit,3,4 Hannu Korkeala,5 L. monocytogenes MLST Study Group‡ and Mark Achtman1,6* 1 Environmental Research Institute, University College Cork, Cork, Ireland. 2 Department of Infectious Disease Epidemiology, Imperial College London, London, UK. 3 Institut Pasteur, Biology of Infection Unit, National Reference Centre and WHO collaborating centre for Listeria, Inserm Unit 1117, Paris, France. 4 Division of Infectious Diseases and Tropical Medicine, Necker-Enfants Malades University Hospital, APHP, Paris, France. 5 University of Helsinki, Helsinki, Finland. 6 Warwick Medical School, University of Warwick, Coventry CV4 7AL, UK. Summary Listeria monocytogenes is ubiquitously prevalent in natural environments and is transmitted via the food chain to animals and humans, in whom it can cause life-threatening diseases. We used Multilocus Sequence Typing (MLST) of ∼2000 isolates of L. monocytogenes to investigate whether specific associations existed between clonal complexes (CCs) and the environment versus diseased hosts. Most CCs (72%) were not specific for any single source, and many have been isolated from the environment, food products, animals as well as from humans. Our results confirm that the population structure of L. monocytogenes is largely clonal and consists of four lineages (I–IV), three of which contain multiple CCs. Most CCs have remained stable for decades,

Received 30 August, 2013; accepted 16 November, 2013. *For correspondence. E-mail [email protected]; Tel. (+44) 247657 5592; Fax (+44) 247657 4637. †Present address: UIC Department of Microbiology and Immunology, 835 S. Wolcott Ave., Chicago, IL 60612, USA. ‡Listeria monocytogenes MLST study group: Alexandre Leclercq (Institut Pasteur, Paris, France), Kathy Grant (Health Protection Agency, London, UK), Martin Wiedmann (Cornell University Ithaca, Ithaca, New York, U.S.A.) and Petra Apfalter (Austrian Agency for Health and Food Safety, Vienna, Austria).

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd

but one epidemic clone (CC101) was common in the mid-1950s and very rare until recently when it may have begun to re-emerge. The historical perspective used here indicates that the central sequence types of CCs were not ancestral founders but have rather simply increased in frequency over decades. Introduction Listeria monocytogenes are Gram-positive bacteria that are ubiquitous in soil, vegetation and other environmental sources (Weis and Seeliger, 1975; Moshtaghi et al., 2003; Sauders et al., 2006; 2012), and also cause invasive disease with high mortality rates in animals and humans. Infection arises when the food chain includes vegetables (Heisick et al., 1989) and fruit (Cosgrove et al., 2012) that are contaminated by environmental sources and/or wild animals. Intermittent outbreaks of human disease associated with the consumption of contaminated dairy (Linnan et al., 1988; Miettinen et al., 1999a; Lyytikainen et al., 2000), fishery (Ericsson et al., 1997; Miettinen et al., 1999b) and meat products (de Valk et al., 2001; Olsen et al., 2005) can result when food-processing plants are contaminated for extended time periods and when such food products were stored for long periods of time prior to consumption. However, it remains uncertain whether all environmental isolates of L. monocytogenes are potentially capable of causing disease in humans and animals, or whether individual clades are particularly virulent. Bacterial isolates from infected humans and contaminated food have been compared by pulsed-field gel electrophoresis (PFGE) in attempts to identify identical or closely related genotypes that are present in both sources (Miettinen et al., 1999a,b; de Valk et al., 2001; Cosgrove et al., 2012; Cartwright et al., 2013). The results indicated that some PFGE genotypes have only been rarely isolated or may be particularly common only in specific environments, whereas others are widely distributed (Fugett et al., 2007). These widely distributed PFGE types tend to be uniform by PFGE, ribotyping and multilocus sequence analysis (Cantinelli et al., 2013), and are associated with distinctive genomic sequences (Nelson et al., 2004; Chen et al., 2011). PFGE types that have caused outbreaks in multiple locations since the 1980s have been referred to

406 J. K. Haase et al. as epidemic clones (ECs) (Chen et al., 2007; Cheng et al., 2008; Knabel et al., 2012; Lomonaco et al., 2013). It is difficult to evaluate the uniqueness of ECs in the absence of additional data on the broad global population structure of L. monocytogenes. Combined with epidemiological investigations, PFGE typing can be a valuable tool for recognizing common source outbreaks (Cartwright et al., 2013). However, it is unsuitable for inferring genetic relationships within and between lineages because changes in PFGE pattern seem to reflect changes in the accessory genome, including transient bacteriophages (Cheng et al., 2008; Zhou et al., 2013), which occur stochastically and are not predictable. Broader genetic relationships are more appropriately studied by the use of multiple sequences from the core genome, such as are used for Multilocus Sequence Typing (MLST) (Maiden et al., 1998), and which are thought to represent neutral markers of descent whose temporal microevolution proceeds in a clock-like fashion. MLST has been used to investigate the broad population structure of L. monocytogenes (Salcedo et al., 2003; Nightingale et al., 2005; Ragon et al., 2008). The results confirmed the existence of four discrete genetic lineages, Lineages I–IV, each of which can cause human disease (Bundrant et al., 2011; den Bakker et al., 2012). Initial results indicated that human isolates were often members of Lineage I, whereas environmental, farm and food isolates were preferentially in Lineage II (Sauders et al., 2006). However, Lineage II-specific serotypes (1/2a, 1/2c) (Ragon et al., 2008) were isolated at least as frequently as serotypes in Lineage I (1/2b, 3b, 4b) from invasive human disease in Finland (Lukinmaa et al., 2003) and other European countries, and include the ECs ECIII and ECV (Cantinelli et al., 2013) as well as ECVII (see later discussion). Lineages III and IV are generally isolated only rarely (Piffaretti et al., 1989; Rasmussen et al., 1995; Ward et al., 2008) and have not been investigated yet in great detail. MLST based on seven housekeeping gene fragments was used by Ragon and colleagues (2008) to assign 360 isolates to arbitrarily numbered sequence types (STs) that differ from each other at one or more genes, and to assign groups of single locus variants (SLVs) to so-called clonal complexes (CCs). Subsequent analyses of other isolates from multiple continents showed that many of the CCs are globally distributed and that the ECs described earlier each belong to a discrete CC or ST (ChenalFrancisque et al., 2011; Cantinelli et al., 2013). These studies are important because they introduced a consistent language for population groupings, and a public website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/ Lmono.html) to store the body of information that is being accumulated by the global community. However, these studies and other smaller studies focused predominantly

on Lineages I and II, and on recent isolates from infected humans and their foodstuffs. As a result, they did not address whether all lineages and CCs of L. monocytogenes are capable of infecting humans, or whether CCs are common in the environment that are not associated with disease. We therefore used a robotically facilitated pipeline for MLST (O’Farrell et al., 2012) to investigate the diversity within a large collection of isolates from various sources that spanned multiple decades and continents (Haase et al., 2011) as well as additional recent isolates from humans, wild and domesticated animals and the environment. Results Strain collection We performed MLST on 1154 Listeria strains that span considerable temporal and geographical diversity, and which included numerous environmental isolates in addition to food and disease isolates. These were chosen from the historical ‘Special Listeria Culture Collection’ (SLCC) (Haase et al., 2011) and the extensive strain collection at the Health Protection Agency (HPA) Colindale, London, and were supplemented with environmental isolates from Finland, the USA (Sauders et al., 2006), as well as Austria and Ireland. We also included ten isolates from Lineage IV (Ward et al., 2010) because we anticipated that only few such isolates would be included in the other collections. We analysed a total of 1997 isolates by also including all published data from 843 isolates that were on the MLST website at the end of 2012 (Ragon et al., 2008; Chenal-Francisque et al., 2011; Leclercq et al., 2011; Adgamov et al., 2012; Knabel et al., 2012; Nilsson et al., 2012; Wang et al., 2012) (Table 1). Our collection of 1154 isolates represented 249 MLST STs and 50 CCs (Table 1), including data from 37 L. innocua within the Seeliger collection, which had previously been improperly classified as L. monocytogenes (Table 1). Of the 249 STs, 156 (62%) were novel, representing 41% of the combined total of 385 STs among all 1997 isolates. Novel STs were found in all four Lineages of L. monocytogenes as well as L. innocua but were particularly dominant in Lineage IV and L. innocua, from which only few isolates had been previously studied by MLST. Rarefaction curves of the progressive identification of STs for random samples from the entire set of 1997 isolates showed that the number of STs is still increasing as additional strains are tested (Supporting Information Fig. S1A). Numerous additional STs are therefore to be expected as even more isolates are tested. In contrast, only 12 new CCs were identified, and the number of CCs in Lineages I and II reached saturation after sampling only relatively few strains from these lineages (Supporting Information Fig. S1B). It is therefore unlikely that many

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

MLST of L. monocytogenes

407

Table 1. Numbers of isolates, STs and CCs. Previously publisheda

This study

Total

Groupings

Isolates

STs (novel)

CCs (novel)

Isolates

STs

CCs

Isolates

STs (% novel)

CCs (% novel)

Lineage I Lineage II Lineage III Lineage IV L. innocua Total

510 552 41 14 37 1,154

84 (45) 110 (64) 22 (15) 11 (11) 22 (21) 249 (156)

14 (1) 28 (6) 5 (2) 0 4 (3) 50 (12)

472 342 24 0 5 843

102 94 13 0 4 213

13 22 3 0 2 40

982 894 65 14 42 1,997

154 (29) 164 (39) 30 (50) 11 (100) 26 (81) 385 (41)

14 (7) 28 (21) 5 (40) 0 5 (60) 52 (23)

a. Published by Ragon and colleagues (2008), Chenal-Francisque and colleagues (2011), Leclercq and colleagues (2011), Adgamov and colleagues (2012), Knabel and colleagues (2012), Nilsson and colleagues (2012), and Wang and colleagues (2012).

new CCs remain to be discovered from these two Lineages. However, multiple new CCs probably exist in Lineages III and IV, and in L. innocua, because their rarefaction curves have not yet reached a plateau.

Genetic diversity Our assignments of STs to Lineages and species were based on a phylogenetic tree of the concatenated sequences of the seven MLST gene fragments (Supporting Information Fig. S2).This tree was constructed with CLONALFRAME (Didelot and Falush, 2007), which calculates genealogies after excluding the significantly clustered single nucleotide polymorphisms (SNPs) that are markers of recombination events with other bacteria. Comparable results were obtained with a neighbourjoining tree (Saitou and Nei, 1987) of the same data. A striking feature of the phylogeny is the clear delineation between the four Lineages, which differ from each other by 56–168 fixed nucleotide differences, and between 184 and 221 from L. innocua (Fig. 1). We did not identify a single intermediate strain that straddled these delineations. Although some SNPs are shared between Lineages or between the two species, all allelic sequences are Lineage-specific except for strains CLIP85 (Ragon et al., 2008), CLIP98 (Ragon et al., 2008) and VIMVH333 (Adgamov et al., 2012) that contain one or two alleles from one Lineage and the others from a second Lineage. Those strains were excluded from the subsequent analyses presented here because they might represent experimental errors and were not obtainable for testing. The phylogenetic tree (Supporting Information Fig. S2) showed that concatenated sequences in Lineage II diversified earlier than those in Lineage I. Lineages III and IV diversified even earlier, and the oldest split was between L. monocytogenes and L. innocua. These varying branch lengths are accompanied by large differences in genetic diversity (π) (Supporting Information Table S1), which differs by twofold between Lineages I and II despite comparable numbers of alleles (and STs). The pairwise distances between concatenated sequences from Lineage

I are quite homogeneous (Supporting Information Fig. S3A), but the distances within Lineage II form a bimodal distribution because of a subset of sequences with greater branch lengths (Supporting Information Fig. S3B). More CCs were identified in Lineage II than in Lineage I (Table 1), and these two Lineages accounted for 94% of all isolates. Finally, population genetic analyses confirmed that the Lineages are discrete (Supporting Information Fig. S4) and showed that genetic diversity is largely due to mutation rather than recombination (Supporting Information Appendix S1).

Ecological distributions of genotypes and temporal dynamics We compared the relative frequencies of isolation of the isolates from humans, animals, food products or the environment among the 131 STs with two or more isolates (Supporting Information Fig. S5 and Table S2). Only 24% of these STs were from a single source category. Fortyfive per cent were isolated from two source categories, 17% from three and 14% from all four. These frequencies were expected to be biased by the numbers of isolates, and indeed, almost all STs containing isolates from only one or two sources each included less than 10 strains (Supporting Information Fig. S5). However, even among STs with less than 10 isolates, most had originated in two or more sources. And almost all STs with 20 or more isolates were from three or four sources. These observations tend to contradict a common belief that virulence is a special attribute of rare ECs of L. monocytogenes. Instead, some clades (STs) are indeed more common than others, but they do not seem to be specific for any single source in comparisons of the environment with infections of wild and food animals or humans. The details of associations between CC and source are shown within the context of a minimal spanning tree for allelic distances between STs in Lineages I (Fig. 2A) and II (Fig. 2C). The same minimal trees are also represented in Figs 2B and D, except that they are coloured by date of isolation. The CCs that correspond to the seven ECs

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

408 J. K. Haase et al. Fig. 1. Minimal spanning tree of 1997 isolates based on the concatenated sequences. Each circle represents one ST, and the diameters of the circles reflect the numbers of isolates from that ST. The designations of prominent CCs containing multiple isolates are indicated within the central circle. The numbers of fixed mutations (shared mutations) between lineages are shown next to the dashed lines.

Lineage IV 56 (20) 168 (22) 9

Lineage III

101

141 (5)

14

121 20

155

37

69

8

129 (54)

21

7

221 (31) 18

Lineage II

106 (52) 3

80 (47) 5

59 6

2

1

4

184 (44)

Lineage I

195 (47)

195 (35)

L. innocua

L. monocytogenes and L. innocua Fixed mutation: 97 Shared mutations: 109

polymonomutations morphic morphic (No.) sites (No.) sites (No.) 142 Lineage I 3127 154 192 Lineage II 3091 201 170 Lineage III 3118 174 104 Lineage IV 3184 104 704 L. monocytogenes 2559 843 230 L. innocua 3058 236 Grouping

(CC1:ECI; CC2:ECIV; CC5:ECVI; CC6:ECII; CC7:ECVII; CC8:ECV; CC11:ECIII) (Ragon et al., 2008; ChenalFrancisque et al., 2011; Knabel et al., 2012; Cantinelli et al., 2013; Lomonaco et al., 2013) were each isolated from multiple sources, as is typical for the majority of L. monocytogenes. Indeed, the only CC with a clear predominance of human isolates was CC101, which has not previously been recognized as an EC possibly because 62% of CC101 strains were isolated between 1953 and 1956, and it has only rarely been isolated since the 1950s. However, in a very recent report, ST38 of CC101 was responsible for 31/132 cases of listeriosis in humans that occurred between 2006 and 2010 in Lombardy, Italy (Mammina et al., 2013). We note that the old isolates were from the Seeliger collection, which was strongly biased to human isolates, and an association of CC101 with humans may reflect that bias. None of the other CCs contained a large number of isolates from the 1950s, but all that are common today have persisted over decades and were frequently isolated from all source categories. Despite our observation that almost all STs were isolated from multiple sources, the quantitative distributions

according to source do differ between Lineages I and II. Both lineages contain similar numbers of isolates from the environment and from food, but animal isolates were significantly more frequent in Lineage II (Fisher’s exact test P < 10−14) (Supporting Information Table S3), whereas Lineage I contains a higher frequency of human isolates (Fisher’s exact test P < 10−16). We also identified several human isolates from Lineages III and IV, and very surprisingly, even from L. innocua (Fig. 3). We considered the possibility that a putative human source for isolates in Lineages III and IV, and L. innocua represented strain mix-ups. To test this possibility, we performed MLST on a second stab or lyophil stock culture from 12 human isolates in Lineages III and IV, which resulted in identical MLST assignments. This approach was not possible for the human isolates within L. innocua because duplicate stock cultures did not exist. However, the consistency of their epidemiological attributions argue against the possibility of strain mixups because four of the six human isolates of L. innocua were in three distinct STs of CC526 and were annotated as having been isolated in Pecs, Hungary in 1969. This

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

MLST of L. monocytogenes

A

B

Lineage I

409

Lineage I

315 195 345 288

6

1 ECII

491

54

2

4

ECI

3 ECIV

5 59

ECVI

1921-1923 1933-1935

animal environment

Number of isolates

88

1948-1950

161

food

Lineage II

unknown

369

C

1963-1965

56 25 13 1

human

Lineage II

1978-1980

D 366

1993-1995

9

2008-2011

204

199

29 20

ECV

21 177

18 362

14

14 391

8

155

11

399

19

90

ECIII

16

ECVII

7 121

26 37

398

475 361 101

persistent over decades

recent expansion

historical expansion

Fig. 2. Minimal spanning tree of STs in Lineages I (A, B) and II (C, D). Each ST is indicated by a circle whose size reflects the number of isolates, and the CC designations are indicated by numbers in A and C. Line connecting STs indicate single (thick lines) and double (thin lines) locus variants that differ by one or two alleles respectively. Parts A and C are coloured by source, whereas parts B and D reflect the same data but coloured by year of isolation. Coloured arrows highlight previous and recent expansions of CCs or persistence of clones over several decades, and are labelled with designations for ECs I–VII according to Ragon and colleagues (2008), Chenal-Francisque and colleagues (2011), Cantinelli and colleagues (2013), Lomonaco and colleagues (2013).

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

410 J. K. Haase et al. Fig. 3. Minimal spanning tree of STs in Lineages III and IV and L. innocua coloured by source. Details are as in Fig. 2.

observation is reminiscent of the diversity seen in other CCs, including some of the ECs. The fifth human isolate of L. innocua was also from Pecs in 1969 but belonged to a different CC (CC532), and the sixth human isolate was isolated in Peru in 1977 and belonged to ST530. These observations suggest that multiple CCs of L. innocua are capable of causing rare disease in humans, contradicting a common belief that this species is not pathogenic. Founder genotypes Many CCs in multiple bacterial species consist of a central ST with multiple isolates that is linked to a surrounding cloud of single or double locus variants, each containing only few isolates. It is a common perception that the central ST represents the ancestral genotype of the variants (Feil et al., 2004). However, our results did not support this interpretation because SLVs tended to be old (e.g. CC9 or CC7; Fig. 2C). We examined the frequency of isolates over time that were within the central ST among CCs with at least three STs and a predominant central ST. Of 44 strains that were isolated prior to 1951, only three belonged to the central ST, whereas higher frequencies were observed thereafter (Fig. 4C). It has been suggested by Cantinelli and colleagues (2013) that older SLVs are predominantly due to mutations in the ldh gene. Our data confirm this observation (Fig. 4A), but they also show that only 7/32 strains isolated before 1941 were in the central ST when the six other MLST genes were examined (Fig. 4B). These observations suggest that

today’s central STs were not the ancestral genotypes and that L. monocytogenes has become more homogeneous since the 1950s because of the expansion of nonancestral STs. An alternative explanation for our observations would be that they reflect mutations that accumulated progressively during prolonged laboratory storage, such as in stab cultures maintained at room temperature for laboratory derivatives of Salmonella enterica serovar Typhimurium strain LT-2 (Eisenstark, 2010) and as inferred specifically for ldh mutations in L. monocytogenes (Cantinelli et al., 2013). However, our data also do not support this hypothesis. We performed MLST on 12 paired isolates of Lineages III and IV (described earlier) plus 10 other strains from independent stabs that were in the Seeliger collection at University College Cork (UCC) or within a copy of that collection that had been maintained at the Institut Pasteur since the 1970s. Sixteen of 19 isolates yielded identical MLST STs, and three other pairs of isolates each differed by one SNP in one gene fragment (ldh or dat) (Supporting Information Tables S4 and S5). This frequency of SNPs in pairs of matched stabs (15.8%) is so different from the frequency of isolates prior to 1951 in non-central STs (41/44 = 93%) that laboratory mutations acquired during long-term storage in stab cultures at room temperature are unlikely to account for our observations. Further support is provided because some of the old strains share the same ST as more modern strains, whereas laboratory mutations are most likely to occur only once.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

MLST of L. monocytogenes

A. ldh

37

90

54

229

315

222

21

29

83

55

164

87

237

322

221

45

33

48

107

70

139

75

218

298

206

45

30

139

78

20

411

78

B. 6 genes

C. 7 genes

Proportion of isolates in Founder STs

7

22

11 88

2

1

1

2 7

18 10

2

47

119

5

86 8

22

11 80

1

1 1

1921−1930 1931−1940 1941−1950 1951−1960 1961−1970 1971−1980 1981−1990 1991−2000 2001−2010

Dates of Isolation Fig. 4. Histogram of relative frequencies of isolates in central STs. Analyses were performed on all isolates with known dates within 22 CCs with a predominant central ST and at least two other STs. The figure indicates the proportion of isolates that were in the central ST (black) versus isolates in non-central STs (white) segregated into bins reflecting their dates of isolation. The absolute numbers of isolates are indicated within the bars. A. Data based exclusively on ldh. B. Data based on six MLST genes other than ldh. C. Data from all seven genes in the MLST scheme.

Three other pairs of strains differed at all seven MLST alleles as well as in serovar (Supporting Information Table S4). The serotypes of the three strains at the Institut Pasteur differed from those in the original Seeliger records, whereas STs of the strains tested at UCC were consistent with those records. We interpret these results as strain mix-ups, which may have occurred during transfer of the copy of the Seeliger collection to the Institut Pasteur. Discussion The results described here are based on a survey by MLST of ∼2000 isolates of L. monocytogenes and L. innocua, the largest MLST analysis yet performed with these species. We identified numerous new STs and novel alleles but only few new CCs. Similarly, the novel alleles raised the number of polymorphic sites per allele by 5–12% versus previous estimates (Ragon et al., 2008), but the overall nucleotide diversity (π) remained constant. The general population structure, including the existence of four Lineages, was also con-

firmed by the additional data. Because of the breadth of sampling presented here, which encompasses differing geography, source and date of isolation, this population structure of L. monocytogenes is unlikely to change again except for additional STs and a limited number of novel CCs. Importantly, intermediate strains that cannot be assigned to one of the four Lineages should be very rare. It is therefore possibly appropriate to now develop a rapid and cheap alternative to MLST for the classification of L. monocytogenes based on SNP-based typing assays that target informative mutations within the MLST gene fragments. A SNP typing scheme based on MLST genes could be designed to target all known CCs or to focus on historical or newly emerging ECs, and then expanded with time as novel CCs are identified. SNP typing assays that classify lineages and ECs have been previously described (Ward et al., 2008; 2010). However, those targeted SNPs were predominantly located in virulence associated genes, such as inlA or hly, which might be subject to selection for homoplastic mutations that can yield unreliable assignments.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

412 J. K. Haase et al. Seventy-two per cent of the STs in L. monocytogenes that contained at least two isolates were isolated from two to four sources (Supporting Information Fig. S5). This result is as expected if human infections resulted from the random transmission of a saprophyte from the environment through farms and infected livestock to food products that then infect the consumer. In contrast, although Pseudomonas aeruginosa is also ubiquitous, only 19% of its STs have been isolated from infected humans, indicating variable virulence potentials among isolates in the environment (Kidd et al., 2012). However, even though most STs of L. monocytogenes were isolated from multiple sources, human isolates were concentrated in Lineage I, and animal isolates were more often associated with Lineage II. Environmental and food isolates are equally distributed between the two lineages (Supporting Information Table S3), suggesting a differential specificity for human and non-human hosts in Lineages I and II respectively. Large-scale genomic and transcriptomic studies of matched isolates sharing the same ST from different hosts might potentially yield insights into mechanisms for host specificity. Listeria innocua is generally considered to be nonpathogenic for humans. However, at least one human patient died after bacteraemia caused by a nonhaemolytic L. innocua (Perrin et al., 2003), and multiple L. innocua are haemolytic because they carry the prfA pathogenicity cluster (Johnson et al., 2004; Volokhov et al., 2007; Milillo et al., 2012; Moreno et al., 2012). We identified six strains from human infections that were L. innocua according to MLST. These isolates had been scored as L. monocytogenes in the Seeliger records during a period where the test for haemolysis was the primary distinction between the two species, suggesting that they are haemolytic. Our observations suggest that not all L. innocua are harmless for humans and that contamination of food products by this species might be an occasional source of human disease. However, these conclusions are based on old laboratory records that could not be independently confirmed and additional isolates of L. innocua from humans should be compared by phylogenetic and population genetic analyses with larger numbers of L. innocua from the environment in order to determine whether particular lineages with L. innocua are more virulent than others. CC101 isolates were isolated frequently in the mid 1950s, both in Europe and in North America, predominantly from human disease. Only few environmental isolates were included in the Seeliger collection, and it is unclear whether CC101 was also common in the environment at that time. In recent years, CC101 has been isolated from Peru and Morocco as well as from a wild bird in Finland, confirming its broad geographical distribution. CC101 has not yet been designated as an EC because it

has been isolated only rarely in recent times, but a dramatic upsurge in the frequency of invasive listeriosis caused by CC101 recently in Lombardy (Mammina et al., 2013) may mark its resurgence again after years of dormancy. In summary, this study confirmed the clonal population structure of L. monocytogenes and its subdivision into four lineages, all of which can cause human disease. It has expanded the number of known STs and CCs, and shown that these are widely distributed among multiple sources. ECs that cause human disease correspond to STs and CCs that are very common in the environment. We have also identified a historical EC that caused extensive human disease in the 1950s and now seems to be re-emerging.

Experimental procedures Bacterial isolates A strain collection of 1154 Listeria isolates was assembled. This included 622 isolates from the SLCC (Haase et al., 2011). It also included isolates from the collections at the University of Helsinki (n = 178), Cornell University Ithaca (n = 149), HPA, Colindale, London (n = 155), Austrian Agency for Health and Food Safety (n = 32), and University Hospital Galway (n = 8) (Supporting Information Table S5). The criteria for strain selection from the SLCC were: (i) isolation before 1950, (ii) isolation from the environment (soil, vegetation, sewage and water) or wild animals (bat, bird, buffalo, cat, deer, dog, fly, fox, horse, llama, monkey, rodent, tick and wild boar), and/or (iii) isolation in Africa, Asia, Oceania or South America. The criteria for selecting isolates from the HPA were that they were isolated before 1950 from the environment or a wild bird or mammal, and/or from outside Europe and North America. The strain collection in Helsinki encompasses numerous isolates obtained from wild birds and other environmental sources. Because environmental isolates from the USA were under-represented, we included 149 such samples from the collection at Cornell University Ithaca (Sauders et al., 2006). Forty additional environmental samples were obtained from the Austrian and Irish reference laboratories. Finally, 10 strains from the Agricultural Research Service Culture Collection (NRRL) in Illinois were included in this analysis because they have been shown to belong to Lineage IV by multiple criteria (Roberts et al., 2006; Ward et al., 2008; 2010; den Bakker et al., 2010). Our final strain collection that was tested by MLST comprises 369 strains from the environment, 193 strains from human disease, 147 strains from food products and factories, and 363 strains from various animals including wild animals such as foxes, birds and elks (n = 284). The continents represented were: Africa (n = 14), Asia (n = 38), Europe (n = 578), North America (n = 219), Oceania (n = 88) and South America (n = 75). The strains span a time period of isolation of nine decades (1921–1949: 24; 1950–1959: 124, 1960–1969: 189; 1970–1979: 147; 1980–1989: 185; 1990– 1999: 157; 2000–2011: 274). Finally, published MLST data from 843 additional strains (Ragon et al., 2008;

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

MLST of L. monocytogenes Chenal-Francisque et al., 2011; Leclercq et al., 2011; Adgamov et al., 2012; Knabel et al., 2012; Nilsson et al., 2012; Wang et al., 2012), which were present on the MLST website (http://www.pasteur.fr/recherche/genopole/PF8/mlst/ Lmono.html), were also included for phylogenetic and population genetic analyses, resulting in MLST data from a total of 1997 strains (Supporting Information Table S6). All bacterial strains that were typed by MLST at UCC were single colony isolated and transferred into BHI broth with supplements for frozen storage at −80°C as previously described (Haase et al., 2011). Selected strains were reserotyped at the World Health Organization laboratory for Listeria at the Institute Pasteur, Paris and the Austrian Agency for Health and Food Safety, Vienna (Supporting Information Tables S7 and S8).

MLST DNA extraction, PCR and sequencing were performed as described (O’Farrell et al., 2012). Gene fragments of the MLST scheme described by Ragon and colleagues (2008) were amplified and sequenced with newly designed primers (Supporting Information Table S9). Primer redesign was necessary because previously published primers were located too close to the trimmed allelic sequences, resulting in sequence reads that were too short and did not cover the full allele in both sequencing directions. For MLST of some L. innocua strains, it was necessary to reduce the annealing temperatures from 50 to 47°C to amplify the gene fragments dat and lhkA with the new primers. When amplification was unsuccessful under those conditions, the primers described by Ragon and colleagues (2008) were used, often also requiring lowering the annealing temperature to 47°C.

Phylogenetic analyses Bionumerics V.6.5 (Applied Maths, Sint-Martens-Latem, Belgium) was used for sequence assembly, analysis and the construction of minimal spanning trees. Individual strains were assigned to lineages of L. monocytogenes or L. innocua on the basis of concatenated sequences of the seven MLST gene fragments using a neighbour-joining phylogeny (Saitou and Nei, 1987) in BIONUMERICS and using CLONALFRAME (Didelot and Falush, 2007). The assignments were consistent between the two methods. Ratios of recombination to mutation were calculated with CLONALFRAME using the standard settings, except that differing numbers of iterations were used: Lineage I, 20 000; Lineage II, 40 000; all L. monocytogenes plus L. innocua isolates, 100 000. These numbers of iterations were chosen due to difference in convergence properties based on comparisons of two independent runs. MEGA V5 (Tamura et al., 2011) was used to calculate dN/dS ratios (ω) with the Nei–Gojobori method and Jukes–Cantor correction. The homoplasy index was calculated with PAUP, and nucleotide diversity distance (π) was calculated with DnaSP V5.10.01 (Librado and Rozas, 2009). Rarefaction curves were constructed with R version 2.10.1 (R Development Core Team, 2004). The Bayesian analysis software STRUCTURE (Pritchard et al., 2000) with the linkage model was applied to a data set made of one representative of each of 385 unique STs. This

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approach assumes that the observed data arose through admixture between K ancestral populations. The linkage model was chosen because it accounts for linkage between closely located sites (Falush et al., 2003). Three independent runs were performed for each value of K from 3 to 7. Each run consisted of 200 000 iterations, of which 100 000 iterations were discarded to allow the algorithm to converge. Comparisons between runs using the same values of K showed good consistency, indicating good convergence and adequate mixing. We used two distinct techniques to determine the optimal value of K. The first approach was to search for a plateau in the marginal likelihood achieved by the different values of K (Supporting Information Fig. S6), and the second one was the method described by Evanno and colleagues (2005) that is based on the second derivative of the likelihood (Supporting Information Fig. S7). Both approaches indicated that K = 4, which was used in the results presented in Supporting Information Fig. S4.

Acknowledgements We thank Todd Ward for supplying reference strains of Lineage IV and Martin Cormican for additional strains from Ireland, Zhemin Zhou for assistance in figures and discussions, and Angela McCann for support with CLONALFRAME and PAUP. The project was funded by the Science Foundation Ireland (05/FE1/B882).

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Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Fig. S1. Rarefaction curves of the identification of 385 STs (A) and 52 CCs (B) in a sample of 1997 isolates. Fig. S2. CLONALFRAME radial phylogenetic tree of the concatenated sequences from 385 STs indicating the assignments to four Lineages within L. monocytogenes and to L. innocua. Fig. S3. Pairwise nucleotide mismatch distributions of concatenated sequences for each ST within Lineages I (154 STs) and II (164 STs). Fig. S4. Sources of ancestry of each unique ST from four ancestral populations according to the linkage model of

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416

416 J. K. Haase et al. STRUCTURE. Each ST is represented by a single line with the ST designation at the top consisting of coloured stacked bars that indicate the proportion of ancestry from each of four populations (dark blue, dark red, yellow and cyan). The ST designations are red, green, blue and mauve for Lineages I–IV, and black for L. innocua. The tree at the top is a similarity tree based on the STRUCTURE ancestry profiles. Fig. S5. Frequencies of STs according to sources and numbers of isolates among 131 STs with ≥ 2 isolates. The X axis represents the number of isolates within each ST. The Y axis represents the number of sources (human, animal, environment and food products) from which that ST was isolated. The sizes of the dots represent the number of STs with identical X and Y coordinates. Fig. S6. Estimating K for STRUCTURE (plateau method). The estimated log probability of the data according to the linkage model of STRUCTURE is plotted as a function of K, the number of ancestral populations. According to the manual for STRUCTURE, the optimal value of K corresponds to the point where the graph starts to plateau, which is K = 4 in this case. Fig. S7. Estimating K for STRUCTURE (second derivative method). The optimal value of K was estimated by the

procedure of Evanno and colleagues (2005), which corresponds to the modal value in the bottom right plot, which is K = 4 in this case. Table S1. Genetic diversity of seven MLST alleles among 1997 isolates. Table S2. Number of isolates per source category for each ST with more than one isolate. Table S3. Numbers of isolates per Lineage according to source. Table S4. Two independent stock cultures tested by MLST with discrepant results. Table S5. Two independent stock cultures tested by MLST with congruent results. Table S6. Strain information and MLST results for 1997 strains. Table S7. Strains with confirmed serotype. Table S8. Strains with corrected serotype. Table S9. List of primers for amplification and sequencing of MLST gene fragments. Appendix S1. Population structure and recombination.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 405–416