IJSEM Papers in Press. Published March 5, 2014 as doi:10.1099/ijs.0.052720-0
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Five new species of Listeria (L. floridensis sp. nov, L. aquatica sp. nov., L. cornellensis sp.
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nov. L. riparia sp. nov., and L. grandensis sp. nov.) from agricultural and natural
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environments in the United States
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Henk C. den Bakker,1* Steven Warchocki,1 Emily M. Wright,1 Adam F. Allred,2 Christina
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Ahlstrom,3† Clyde S. Manuel,3‡ Matthew J. Stasiewicz,1 Angela Burrell,2 Sherry Roof,1 Laura K.
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Strawn,1 Esther Fortes,1§ Kendra K. Nightingale,4 Daniel Kephart,2 and Martin Wiedmann1
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1
Department of Food Science, Cornell University, Ithaca, NY 14853, USA
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2
Life Technologies, 2130 Woodward Street, Austin, TX 78744, USA
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3
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, USA
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4
Department of Animal and Food sciences, Texas Tech University, Lubbock, TX 79409, USA
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† Present address: Production Animal Health, University of Calgary, Alberta, Canada
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‡ Present address: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina
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State University, Raleigh, NC 27695-7624, USA
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§ Present address: Hinton State Laboratory Institute, Massachusetts Department of Public Health,
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Jamaica Plain, Massachusetts, USA
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Category: New Taxa (Firmicutes and Related Organisms)
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Running Title: Novel taxa of Listeria from environmental samples
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Correspondence: Henk C. den Bakker, [email protected], Phone: (+1) 607 255-1266, Fax: (+1)
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607 254-4868
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains TTU
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A1-0210T, TTU A1-0212T, FSL S10-1187T, FSL S10-1188T, and FSL S10-1204T are JX961634,
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JX961635, JX961636, JX961637, and JX961638, respectively. The GenBank/EMBL/DDBJ
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accession number for the genome sequences of strains TTU A1-0210T, TTU A1-0212T, FSL S10-
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1187T, FSL S10-1188T, and FSL S10-1204T are AODE00000000, AODD00000000,
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AODF00000000, AOCG00000000, and AODL00000000, respectively. The versions described in
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this paper are the first versions, AODE01000000, AODD01000000, AODF01000000,
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AOCG01000000, and AODL01000000.
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1
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Abstract:
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Sampling of agricultural and natural environments in two US states (Colorado and
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Florida) yielded 18 Listeria-like isolates that could not be identified as previously
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described species using traditional methods. Using whole genome sequencing and
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traditional phenotypic methods we identified five new species, each with a genome wide
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average blast nucleotide identity (ANIb) of less than 85% to currently described species.
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Phylogenetic analysis based on 16S rDNA sequences and amino acid sequences of 31
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conserved loci showed the existence of four well-supported monophyletic clades within
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the genus Listeria; (i) a clade representing L. monocytogenes, L. marthii, L. innocua, L.
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welshimeri, L. seeligeri and L. ivanovii, which we refer to as Listeria sensu stricto, (ii), a
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clade consisting of L. fleischmannii and two newly described species, L. aquatica sp. nov.
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(type strain FSL S10-1188T =DSM 26686T =LMG 28120T =BEI NR-42633T) and L.
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floridensis sp. nov. (type strain FSL S10-1187T =DSM 26687T =LMG 28121T =BEI NR-
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42632T), (iii) a clade consisting of L. rocourtiae, L. weihenstephanensis, and three new
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species, L. cornellensis sp. nov. (type strain TTU A1-0210T = FSL F6-0969T =DSM
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26689T =LMG 28123T =BEI NR-42630T), L. grandensis sp. nov. (type strain TTU A1-
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0212T =FSL F6-0971T =DSM 26688T =LMG 28122T =BEI NR-42631T) and L. riparia
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sp. nov. (type strains FSL S10-1204T =DSM 26685T =LMG 28119T = BEI NR- 42634T),
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and a clade containing L. grayi. Genomic and phenotypic data suggest the novel species
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are nonpathogenic.
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The genus Listeria was first described by Pirie (1940), and at present comprises 10
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species, Listeria monocytogenes (Pirie, 1940), Listeria grayi (Errebo Larsen & Seeliger,
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1966), Listeria innocua (Seeliger, 1981), Listeria welshimeri (Rocourt & Grimont, 1983),
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Listeria seeligeri (Rocourt & Grimont, 1983), Listeria ivanovii (Seeliger et al., 1984),
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Listeria marthii (Graves et al., 2010), Listeria rocourtiae (Leclercq et al., 2010), Listeria
2
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fleischmannii (Bertsch et al., 2013), and Listeria weihenstephanensis (Lang Halter et al.,
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2013). Additionally, two subspecies have been recognized within L. ivanovii (subsp.
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ivanovii and subsp. londoniensis (Boerlin et al., 1992)), L. grayi (subsp. grayi and subsp.
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murrayi (Stuart & Welshimer, 1973)) and L. fleischmannii (subsp. fleischmannii and
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subsp. coloradonensis (Den Bakker et al., 2013)).
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During sampling projects of agricultural and natural environments in the US states
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Florida and Colorado, we isolated 70 isolates, predominantly from water, with a colony
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morphology on Listeria monocytogenes Plating Medium (LMPM) reminiscent of
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Listeria. These isolates could be placed in the Listeriaceae by phylogenetic analysis of
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partial sequences of 16S rDNA and 52 isolates could be assigned to L. fleischmannii (32
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isolates from Florida and 20 isolates from Colorado), however, 18 isolates did not
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phylogenetically cluster within previously described species and therefore could not be
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classified as any of the previously described species. Based on further genotypic
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characterization of these isolates, including by sigB sequencing (Sauders et al., 2012), one
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or two isolates per putative new taxon were selected for further phenotypic and genomic
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characterization (see Fig. S1 for more information).
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Draft genome sequences were assembled as detailed in Den Bakker et al. (2013) from Ion
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PGM™ instrument reads obtained for (i) Brochothrix thermosphacta ATCC 11509T and
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B. campestris ATCC 43754T type strains, (ii) the type strains of L. monocytogenes ATCC
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15313T, L.weihenstephanensis DSM 24698T, L. rocourtiae CIP 109804T, L. grayi subsp.
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murrayi ATCC 25401T, (iii) one additional strain of L. fleischmannii (FSL S10-1203),
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and (iv) five isolates that showed Listeria-like characteristics, but could not be classified
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to species by traditional approaches. Briefly, de novo assembly was performed using the
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MIRA assembler (Chevreux 1999), and homopolymers were corrected using a
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homopolymer correction script (available at http://tinyurl.com/oke6xaa); this script uses
3
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output from VarScan (Koboldt et al., 2012) and BWA (Li & Durbin, 2009) to obtain a
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consensus about the homopolymer length, which is then used to correct the
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homopolymers. De novo assemblies (draft genomes) of Ion Torrent reads ranged in total
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size from 2.37 (Brochothrix campestris) to 3.37 (L. weihenstephanensis) Mbp (see Table
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S1 for total draft genome lengths and additional genome assembly statistics). Comparison
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of the draft genome obtained here for L. monocytogenes ATCC 15313T and a recently
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finished genome of L. monocytogenes SLCC5850 (Kuenne et al., 2013), a strain closely
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related to the type strain (McLauchlin & Rees, 2009), showed the draft genome covers
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97.3% of the length of the finished genome. We thus conclude that the draft genomes
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obtained in this study are of high quality and suitable for comparative analysis. These
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draft genomes also will provide a starting point for future in-depth genomic and
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evolutionary analyses of the genus Listeria.
100 101
BLAST Average nucleotide identities (ANIb) were calculated based on draft genome
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sequences and publicly available genome sequences using JSpecies (Richter & Rosselló-
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Móra, 2009). Additionally, BLAST average amino acid identities (AAI) (Konstantinidis
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& Tiedje, 2005) were calculated using the AAI.rb script from the Enve-omics package
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(http://enve-omics.ce.gatech.edu/). Pairwise comparisons between currently recognized
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species in Listeria sensu stricto yielded ANIb and AAI values < 95% for comparisons
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between different species (Fig. 1; Table S2); an ANIb value of 9596% generally
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corresponds to 70% DNA-DNA hybridization and is typically used as a cut-off value for
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species delineation in bacterial taxonomy (Richter & Rosselló-Móra, 2009). Our data also
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support the conclusions from previous studies that ANIb values are robust even with
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fragmented unfinished draft assemblies. This robustness is demonstrated by an ANIb
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value of 99.94% for the comparison between the draft genome for the L. monocytogenes
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type strain ATCC 15313T (a draft genome obtained in this study) and the finished
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genome for L. monocytogenes SLCC5850. For all genomes representing previously
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reported species, ANIb values supported prior species classification, despite the mixed
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use of finished and draft genomes (Fig. 1). Most importantly, our analyses showed that
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all isolates representing new species proposed in this study show ANIb values < 84% and
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AAI values < 87.9% (Table S2) when their genomes are compared to each other and to
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phylogenetically related previously described species (L. fleischmannii, L. rocourtiae and
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L. weihenstephanensis). While the high 16S sequence similarity (> 99%) between L.
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rocourtiae CIP 109804T, L. weihenstephanensis DSM 24998T, L. cornellensis TTU A1-
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0210T, L. grandensis TTU A1-0212T and L. riparia FSL S10-1204T could be interpreted
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as suggesting that these taxa represent ecotypes of a single species (Cohan, 2001;
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Konstantinidis et al., 2006), ANIb and AAI values, and shared gene content do not
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support this hypothesis. AAI and ANIb values between these species range from 83.2 to
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88.6% and from 77.7 to 83.2%, respectively, and the proportion of shared gene content
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between each species pair ranges from 63 to 75%, justifying recognition as individual
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species (Fig. S2).
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Phylogenetic analysis of (i) 16S rDNA sequences (Fig. S2), and (ii) the concatenated
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sequences of 31 aa sequences (Wu & Eisen, 2008)(Fig. 2) were performed as previously
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described (Den Bakker et al., 2013), with the exception that Gblocks version 0.91b
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(Castresana, 2000) was used to remove ambiguous sites from the aa sequence alignment.
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Maximum parsimony (MP) analyses were performed in PAUP* version 4.0b10
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(Wilgenbusch & Swofford, 2003). Phylogenies obtained from the MP (data not shown)
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did not differ from those found by maximum likelihood (ML) based analyses, and hence
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the results of the ML analyses are discussed here. Both 16S rDNA and aa sequence
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phylogenies confirmed placement of the novel taxa in the genus Listeria, and revealed
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four well-supported clades (100 bootstrap support [BS]) within the genus Listeria; (i) a
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clade containing L. monocytogenes and related species (Listeria sensu stricto: L. marthii,
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L. innocua, L. welshimeri, L. seeligeri and L. ivanovii), (ii) a clade consisting of L.
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rocourtiae, L. weihenstephanensis, L. cornellensis sp. nov, L. grandensis sp. nov. and L.
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riparia sp. nov. (iii) a clade consisting of L. fleischmannii, L. floridensis sp. nov. and L.
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aquatica sp. nov., and (iv) a clade consisting of both subspecies of L. grayi. The 16S
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analysis (Fig. S3) further indicates that a previously unidentified Listeria-like bacterium
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(referred to as ‘unidentified bacterium isolate MB405’ in Fig. S3) from Belgium (Rijpens
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et al., 1998) is a putative sister species of L. aquatica sp. nov. Phylogenetic analysis of
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individual genes, such as iap (Fig. S4), also reveal the subdivision of Listeria into four
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distinct clades. The phylogenetic relationships between the four clades are unresolved in
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the 31 aa loci tree (Fig. 1, BS values < 60), however the 16S rDNA sequence analysis
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suggests that the L. rocourtiae clade and the L. grayi clade are sistergroups (Fig. S3, 85
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BS). AAI values between members of each phylogenetically distinct clade range from 67
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to 70% (Fig. S2). While Konstantinidis and Tiedje (2005; 2007) show AAI between 65
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and 72% coincide with traditional genus delimitations in bacterial taxonomy, we do not
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propose classification of these clades into separate genera at this point, including because
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reclassification of L. grayi into a new genus has previously been unsuccessful and
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controversial (McLauchlin & Rees, 2009).
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For the methyl red (MR) and Voges-Proskauer (VP) tests, all Listeria isolates were
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cultured on BHI (Becton, Dickinson & Co.) agar aerobically for 18 h at 30 °C. A single
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colony was suspended in 5 ml of MR-VP broth (pH: 6.9(±0.2); Becton, Dickinson &
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Co.); inoculated tubes were incubated aerobically for 5 days at 35 °C. MR and VP test
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procedures were carried out according to the manufacturer’s guidelines (Becton,
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Dickinson & Co procedure # L007474). Results are listed in the species descriptions and
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Table 1.
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Testing for catalase activity was performed according to (Bacteriological Analytical
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Manual (BAM) protocol R12 (U.S. Food and Drug Administration, 2013). An isolated
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colony from BHI agar was emulsified in 1 drop of 3% hydrogen peroxide on a glass
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slide. Bubbling within 15 s was interpreted as a positive result for the catalase test.
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Results were independently scored by two individuals and are listed in the species
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descriptions and Table 1.
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To test nitrate reduction, isolates were grown in nitrate broth (BAM Media M108; [U.S.
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Food and Drug Administration, 2013]) with Durham tubes for 48 h at 30 °C non-shaking.
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Durham tubes allowed detection of gas production at 24 and 48 h, which is indicative of
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N2 production. Reduction of nitrate to nitrite was tested by adding 50 µl of bacterial
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culture grown in nitrate broth to 0.9 ml phosphate buffer (50 mM, pH 7.4), followed by
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the addition of 1 ml sulfanilic acid/HCL (5.0 g sulfanilic acid, 100 ml concentrated HCL,
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400 ml H20; final pH -0.38) and 1 ml 0.58% N-(1-Naphthyl) ethylenediamine. An
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immediate color change to deep pink through purple indicated the production of nitrite.
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For all isolates, nitrite reduction was determined using a protocol comparable to the
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nitrate reduction protocol described above, however, KNO2 was substituted for KNO3 in
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BAM Media M108. Nitrite reduction was negative if the reaction solution produced
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magenta to purple coloration. If an isolate reduces nitrate to an oxidation state lower than
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nitrite, the nitrate reduction test will produce a false negative. Therefore, separately
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testing nitrite reduction and the generation of N2 (Durham tubes) measures the reduction
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of nitrate to nitrite. All non-motile species were found to be able to reduce nitrate, with
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the exception of L. floridensis. None of the new species reported here was able to reduce
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nitrite.
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Hemolysis for each isolate was determined using the CAMP test as described in BAM
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(U.S Food and Drug Administration, 2013) and performed in duplicate. To test for PI-
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PLC activity, single colonies from BHI agar plates were sub-cultured to Listeria
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monocytogenes Plating Medium (LMPM; R&F Laboratories). PI-PLC activity positive
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strains color blue on LMPM medium. None of the new species described here showed
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hemolytic activity or PI-PLC activity, suggesting these species lack the genes (i.e., hly
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and plcA) associated with virulence in L. monocytogenes and L. ivanovii. Virulence
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genes (i.e., genes found in the prfA-cluster (Schmid et al., 2005)) or LIPI-2 (Domínguez-
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Bernal et al., 2006) were absent from the draft genomes, further supporting that none of
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the new species described here are pathogenic.
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API Listeria and API 50 CH (with API 50 CHB/E medium) kits (bioMerieux) were
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utilized for further phenotypic characterization, using bacterial colonies taken from BHI
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agar plates. API Listeria tests were incubated for 24 h at 34 °C for all isolates (within the
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manufacturer recommended temperature range of 36±2 °C). API 50 CH tests were
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incubated for 48 h at 37 °C for all Listeria isolates. A mineral oil cover was not used,
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since all species studied here are facultative anaerobes. Results were recorded at 48 h,
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unless there were positive or variable results at 24 h with a corresponding negative result
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at 48 h, in which case 24 h results were reported (as instructed in the API 50 CHB/E
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manual). Results are summarized in the species descriptions and Table 1. Results for the
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individual isolates are reported in Table S3.
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To test growth characteristics, isolates were grown in BHI broth at six different
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temperatures (4, 7, 22.5, 30, 37, and 41°C). The Synergy™ H1 hybrid multi-mode
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microplate reader (BioTek Instruments Inc., Winooski, Vermont) was employed to test
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growth at 22.5±0.5 °C, 30±0.1 °C, 37±0.1 °C, and 41±0.1 °C (temperature tolerances as
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provided by manufacturer). Colony inoculated cultures were grown for 24 h in BHI
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broth; an aliquot of these cultures (20 µl) was used to inoculate Costar 96 well flat bottom
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plates (clear polystyrene) prefilled with 180 µl of BHI. Inoculated plates were incubated
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at 22.5 °C for 48 h, 30 °C for 41 h, 37 °C for 26 h, and 41 °C for 41 h in the Synergy™
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H1 reader. Optical density at 600 nm (OD600) was measured after 10 s orbital shaking as
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follows: every 2 min for the first 20 min, every 5 min for the next 40 min, every 10 min
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for the remainder of the experiment. Growth was quantified by finding the first time the
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cells had increased by 0.6 log (c.f.u./ml), i.e. a 4-fold increase in cell number. Measured
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increases in log(OD600) were converted to increases in log (c.f.u./ml) using the slope of
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the strain specific linear function relating log(OD600) to log (c.f.u./ml). The linear
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function was created with a series of eight 2-fold serial dilutions of a 24 h culture of each
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strain, performed in a microtiter plate, and the resulting OD600 values for each dilution.
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The slope of the resulting best fit lines averaged 0.92 log (OD600)/log (c.f.u./ml), with
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average R2 of 0.99. For the measurement of growth characteristics at temperatures below
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ambient temperature, BHI culture tubes were inoculated at 103 c.f.u./ml (± 0.3 log) and
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incubated at 4 °C (low: 3°, high: 9 °C) and 7 °C (6.8±0.2 °C) in a refrigerated incubator.
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Cultures were spiral plated on BHI agar (to determine c.f.u./ml) every 24 hours for 12
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days. Growth was quantified by calculating the time for cells to increase by 1.0 log
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(c.f.u./ml), i.e. a 10-fold increase in cell number, by linear interpolation of the measured
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points immediately before and after the observed 1.0 log increase. Growth characteristics
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are reported in the species descriptions and additional data on growth characteristics of
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the individual isolates can be found in Table S4.
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Motility was determined in Motility Test Medium (prepared according to BAM Media
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M103 [U.S. Food and Drug Administration, 2013]). For each isolate, 13 mm tubes
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containing Motility Test Medium (MTM) were inoculated 1 cm under the surface, with a
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stab, from a colony on BHI agar. Isolates were incubated aerobically for 7 days at 22 °C,
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30 °C, or 37 °C or for 10 days at 4 °C. An isolate was considered motile if cloudy growth
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beyond the stab was observed in addition to the characteristic mushroom or umbrella-
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shaped growth. Results are reported in Table 1 and the species descriptions. L.
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monocytogenes 10403S was included as a positive control, while L. fleischmannii DSM
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24998T was included as a negative control. In addition to the motility test, draft genomes
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of the type strains sequenced were queried using BLAST (Altschul et al., 1990) for genes
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associated with motility in L. monocytogenes (Table S5). Consistent with observations of
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the motility test, no flagellar motility associated genes were found in the new species
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described here.
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Light microscopic observations were performed on Listeria isolates that were cultured on
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BHI agar aerobically for 18 h at 30 °C. Stationary-phase bacteria were examined on agar-
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coated slides and viewed by phase-contrast microscopy using an Olympus BX61
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microscope (Olympus). Digital photographs were captured using the SlideBook™
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v4.0.2.2 software (Intelligent Imaging Innovations) and length-width ratios were based
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on the average length and width of five bacteria. Results are reported in Table 1 and the
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species descriptions. Photographs of the individual isolates can be found in Figure S5.
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Description of Listeria floridensis sp. nov.
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Listeria floridensis (flo.rid.en'sis. N.L. fem. adj. floridensis of or belonging to Florida, the
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state in the United States from which the first strain was isolated).
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Cells are 0.6 by 1.3 – 1.9 µm in size, length/width ratio 2.7, Gram-positive, straight rods
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with rounded ends. No growth occurs at temperatures of 7 °C and below. Optimum
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growth temperature 37 – 41 °C. No motility at 4, 22, 30, or 37 °C. Voges-Proskauer
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negative, and catalase-positive. CAMP-negative. Nitrate reduction negative, nitrite-
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reduction negative. Other characteristics are reported in Table 1. L. floridensis is
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currently the only member of the genus Listeria that both lacks motility and is unable to
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reduce nitrate. A unique API-Listeria numerical profile (2 710) was observed for the type
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strain.
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10
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Type strain (FSL S10-1187T), isolated from running water in Florida, USA, is deposited
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in DMSZ (DSM 26687T), BCCM/LMG (LMG 28121T), and BEI (BEI NR-42632T). The
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DNA G+C content of the type strain is 41.8 mol% (determined by genome sequencing).
279 280
Description of Listeria aquatica sp. nov.
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Listeria aquatica (a.qua’ti.ca L. fem. adj. aquatica, found in water, aquatic).
282 283
Cells are 0.6 – 0.7 by 1.5 – 2.4 µm in size, average length-width ratio 3.2, Gram-positive,
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straight rods with rounded ends. No growth occurs at temperatures of 7 °C and below.
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Optimum growth temperature range 37 – 41 °C. No motility at 4, 22, 30, or 37 °C.
286
Voges-Proskauer negative for type strain, positive for strain FSL S10-1181. Catalase-
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positive. CAMP-negative. Nitrate reduction positive, nitrite-reduction negative. Other
288
characteristics are reported in table 1. L. aquatica is currently the only member of the
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genus Listeria that is unable to ferment D-Maltose. Among non-motile Listeria species it
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is unique in its ability to ferment D-tagatose. A unique API-Listeria numerical profile (6
291
731) was observed for the type strain and FSL S10-1181.
292 293
Type strain (FSL S10-1188T) was isolated from running water in Florida (USA) and is
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deposited in DMSZ (DSM 26686T), BCCM/LMG (LMG 28120T) and BEI (BEI NR-
295
42633T). The DNA G+C content of the type strain is 40.9 mol% (determined by genome
296
sequencing).
297 298
Description of Listeria cornellensis sp. nov.
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Listeria cornellensis (cor.nell.en'sis. N.L. fem. adj. cornellensis, named after Cornell, the
300
university where most of the research was performed that led to the discovery of the
301
Listeria species described in this study).
302
11
303
Cells are 0.4 – 0.7 by 2.4 – 3.8 µm in size, average length-width ratio 5.3, Gram-positive,
304
straight rods with rounded ends. Optimal growth at 30 – 37 °C. No motility at 4, 22, 30,
305
or 37 °C. Voges-Proskauer negative, catalase-positive. CAMP-negative. Nitrate-
306
reduction positive, nitrite-reduction negative. Other characteristics are indicated in Table
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1. While phylogenetically and genomically distinct, this species resembles L. grandensis
308
for the phenotypic characteristics recorded here. Among the non-motile Listeria species
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L. cornellensis and L. grandensis are the only non-motile Listeria species that are L-
310
rhamnose negative in the API CH50 test (performed at 37° C), however, the API Listeria
311
test strip (incubated at 34° C) showed acidification of L-rhamnose for L. grandensis, and
312
for one of the two L. cornellensis strains tested. An operon involved in rhamnose
313
utilization could be found in the draft genome of L. grandensis, but was absent from the
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draft genome of the type strain of L. cornellensis, suggesting an absence of rhamnose
315
utilization for the type strain of L. cornellensis and temperature dependent ability of L.
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grandensis to acidify L-rhamnose. L. cornellensis can be further distinguished from L.
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grandensis by weak acidification of D-lactose. API-Listeria numerical profile 2 330 was
318
observed for the two isolates (TTU A1-0210T and FSL F6-0970) of this species; this
319
numerical profile is associated with L. ivanovii according to the API-Listeria manual
320
(bioMerieux; version 04/2007).
321 322
Type strain (TTU A1-0210T), isolated from water in Colorado, USA, is deposited in
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DMSZ (DSM 26689T), BCCM/LMG (LMG 28123T) and BEI (BEI NR-42630T). The
324
DNA G+C content of the type strain is 42.5 mol% (determined by genome sequencing).
325 326
Description of Listeria grandensis sp. nov.
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Listeria grandensis (grand.en’sis, N.L. fem. adj. of or belonging to Grand, the county
328
where the type strain was isolated).
329
12
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Cells are 0.6 – 0.7 by 2.0 – 3.1 µm in size, average length-width ratio 4.0, Gram-positive,
331
straight rods with rounded ends. Optimal growth temperature 30 – 37 °C. No motility at
332
4, 22, 30, or 37 °C. Voges-Proskauer negative, catalase-positive. CAMP-negative.
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Nitrite-reduction negative and nitrate-reduction positive. Other characteristics are
334
reported in Table 1. See L. cornellensis for differentiation of L. grandensis from this
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species. A unique API-Listeria numerical profile (2 730) was observed for the type strain.
336 337
Type strain (TTU A1-0212T), isolated from water in Colorado, USA, is deposited in
338
DMSZ (DSM 26688T), BCCM/LMG (LMG 28122T) and BEI-resources (BEI NR-
339
42631T). The DNA G+C content of the type strain is 43.0 mol% (determined by genome
340
sequencing).
341 342
Description of Listeria riparia sp. nov.
343
Listeria riparia (ri.pa’ri.a L. fem. adj. riparia, of the bank of a river or stream).
344 345
Cells are 0.5 – 0.7 by 2.3 – 3.7 µm in size, average length-width ratio 4.8, Gram-positive,
346
straight rods with rounded ends. Optimum growth temperature 37 – 41 °C. No motility at
347
4, 22, 30, or 37 °C. Voges-Proskauer negative, and catalase-positive. CAMP-negative.
348
Nitrite-reduction negative and nitrate-reduction positive. Other characteristics are
349
reported in Table 1. Can be differentiated from other non-motile Listeria species by a
350
combination of α-Mannosidase activity, and the ability to acidify L-rhamnose, D-
351
galactose and L-arabinose. A unique API-Listeria numerical profile (6 710) was observed
352
for the isolates tested (FSL S10-1204T and FSL S10-1219).
353 354
Type strain (FSL S10-1204T), isolated from running water in Florida, USA, is deposited
355
in DSMZ (DSM 26685T), BCCM/LMG (LMG 28119T) and BEI-resources (BEI NR-
13
356
42634T). The DNA G+C content of the type strain is 41.9 mol% (determined by genome
357
sequencing).
358 359
Acknowledgements. This project was supported by USDA NIFA Special Research
360
Grant 2010-34459-20756 as well as USDA NIFA National Integrated Food safety
361
initiative Grants 2008-51110-04333 and 2008-51110-04688. We thank Dr. Esther Angert
362
(Cornell University, Department of Microbiology) for the help with the microscopy. We
363
would like to thank Dr. Catherine Donnelly (University of Vermont) and Drs. Arnout de
364
Bruin and Wilma Jacobs (RIVM, the Netherlands) for their help with the VP test. We
365
would further like to thank Barbara Bowen for her help with some experiments.
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403–410. Bertsch, D., Rau, J., Eugster, M. R., Haug, M. C., Lawson, P. A., Lacroix, C. & Meile, L. (2013). Listeria fleischmannii sp. nov., isolated from cheese. Int J Syst Evol Microbiol 63, 526–532. Bille, J., Catimel, B., Bannerman, E., Jacquet, C., Yersin, M. N., Caniaux, I., Monget, D. & Rocourt, J. (1992). API Listeria, a new and promising one-day system to identify Listeria isolates. Appl Environ Microbiol 58, 1857–1860. Boerlin, P., Rocourt, J., GRIMONT, F., Grimont, P. A. D., Jacquet, C. & Piffaretti, J. C. (1992). Listeria ivanovii subsp. londoniensis subsp. nov. Int J Syst Bacteriol 42, 69–73. Castresana, J. (2000). Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol 17, 540–552. Cohan, F. M. (2001). Bacterial species and speciation. Systematic Biology 50, 513–524. Den Bakker, H. C., Manuel, C. S., Fortes, E. D., Wiedmann, M. & Nightingale, K. K. (2013). Genome sequencing identifies Listeria fleischmannii subsp. coloradonensis subsp. nov., isolated from a ranch. Int J Syst Evol Microbiol 63, 3257–3268. Domínguez-Bernal, G., Müller-Altrock, S., González-Zorn, B., Scortti, M., Herrmann, P., Monzó, H. J., Lacharme, L., Kreft, J. & Vazquez-Boland, J. A. (2006). A spontaneous genomic deletion in Listeria ivanovii identifies LIPI-2, a species-specific pathogenicity island encoding sphingomyelinase and numerous internalins. Mol Microbiol 59, 415–432. Graves, L. M., Helsel, L. O., Steigerwalt, A. G., Morey, R. E., Daneshvar, M. I., Roof, S. E., Orsi, R. H., Fortes, E. D., Milillo, S. R. & other authors. (2010). Listeria marthii sp. nov., isolated from the natural environment, Finger Lakes National Forest. Int J Syst Evol Microbiol 60, 1280–1288. Koboldt, D. C., Zhang, Q., Larson, D. E., Shen, D., McLellan, M. D., Lin, L., Miller,
14
394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439
C. A., Mardis, E. R., Ding, L. & Wilson, R. K. (2012). VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22, 568–576. Konstantinidis, K. T., Ramette, A. & Tiedje, J. M. (2006). The bacterial species definition in the genomic era. Philos Trans R Soc Lond, B, Biol Sci 361, 1929–1940. Konstantinidis, K. T. & Tiedje, J. M. (2005). Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187, 6258–6264. Konstantinidis, K. T. & Tiedje, J. M. (2007). Prokaryotic taxonomy and phylogeny in the genomic era: advancements and challenges ahead. Curr Opin Microbiol 10, 504– 509. Kuenne, C., Billion, A., Mraheil, M. A., Strittmatter, A., Daniel, R., Goesmann, A., Barbuddhe, S., Hain, T. & Chakraborty, T. (2013). Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome. BMC Genomics 14, 47. Lang Halter, E., Neuhaus, K. & Scherer, S. (2013). Listeria weihenstephanensis sp. nov., isolated from the water plant Lemna trisulca taken from a freshwater pond. Int J Syst Evol Microbiol 63, 641–647. Leclercq, A., Clermont, D., Bizet, C., Grimont, P. A. D., Le Flèche-Matéos, A., Roche, S. M., Buchrieser, C., Cadet-Daniel, V., Le Monnier, A. & other authors. (2010). Listeria rocourtiae sp. nov. Int J Syst Evol Microbiol 60, 2210–2214. Li, H. & Durbin, R. (2009). Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754–1760. McLauchlin, J. & Rees, C. (2009). Genus I. Listeria Pirie 1940a 383AL. In, 2nd edn. pp. 244–257. Springer, New York: Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3 (The Firmicutes). Pirie, J. H. (1940). The Genus Listerella Pirie. Science 91, 383. Richter, M. & Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106, 19126–19131. Rijpens, N., Vlaemynck, G., Rossau, R., Herman, L. & Jannes, G. (1998). Unidentified Listeria-like bacteria isolated from cheese. Lett Appl Microbiol 27, 198– 202. Rocourt, J. & Grimont, P. A. D. (1983). Listeria welshimeri sp. nov. and Listeria seeligeri sp. nov. Int J Syst Bacteriol 33, 866–869. Sauders, B. D., Overdevest, J., Fortes, E., Windham, K., Schukken, Y., Lembo, A. & Wiedmann, M. (2012). Diversity of Listeria species in urban and natural environments. Appl Environ Microbiol 78, 4420–4433. Schmid, M. W., Ng, E. Y. W., Lampidis, R., Emmerth, M., Walcher, M., Kreft, J., Goebel, W., Wagner, M. & Schleifer, K.-H. (2005). Evolutionary history of the genus Listeria and its virulence genes. Syst Appl Microbiol 28, 1–18. Seeliger, H. P. R. (1981). Apathogene Listerien: L. innocua sp.n. (Seeliger et Schoofs, 1977). Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Orig A 249, 487-493. Seeliger, H. P. R., Rocourt, J., Schrettenbrunner, A., Grimont, P. A. D. & Jones, D. (1984). Listeria ivanovii sp. nov. Int J Syst Bacteriol 34, 336–337. Stuart, S. E. & Welshimer, H. J. (1973). Intrageneric relatedness of Listeria Pirie. Int J Syst Bacteriol 23, 8–14. U.S. Food and Drug Administration (2013). Bacteriological Analytical Manual.
15
440 441 442 443 444 445 446 447 448 449
http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnaly ticalManualBAM/default.htm. Wilgenbusch, J. C. & Swofford, D. (2003). Inferring evolutionary trees with PAUP*. Curr Protoc Bioinformatics Chapter 6, Unit 6.4. Wu, M. & Eisen, J. A. (2008). A simple, fast, and accurate method of phylogenomic inference. Genome Biol 9, R151.
450
Fig. 1 UPGMA cluster diagram based percent average nucleotide difference (=
451
100%ANIb: see Chan et al., 2012). The scale bar indicates the % ANIb. The grey
452
vertical bar marks the 5% average nucleotide difference, or 95% ANIb, a value which has
453
shown to correlate to the traditional DNA-DNA hybridization value of 70%. New species
454
are printed bold.
Figure legends
455 456
Fig. 2 Maximum likelihood phylogeny based on concatenated amino acid sequences of
457
31 core genes. Values on the branches represent bootstrap values based on a 1000
458
replicates. Bootstrap values < 70 are not indicated. Novel species are printed in bold font.
459
16
460 461
Table 1. Physiological characteristics of Listeria species based on observations made in this study and the current literature.
462
Species: 1, L. monocytogenes (strain 10403S, additional data from McLauchlin & Rees, 2009 and Bertsch et al., 2012); 2, L. innocua
463
(strain FSL S4-378, additional data from McLauchlin & Rees, 2009 and Bertsch et al., 2012); 3, L. seeligeri (data from McLauchlin &
464
Rees, 2009 and Bertsch et al., 2012); 4, L. ivanovii (strain ATCC BAA-678, additional data from McLauchlin & Rees, 2009 and
465
Bertsch et al., 2012); 5, L. welshimeri (data from Bille et al. (1992), McLauchlin & Rees, 2009 and Bertsch et al., 2012); 6, L. marthii
466
(strain FSL S4-120T); 7, L. grayi (strains ATCC 19120T, ATCC 25401T); 8, L. rocourtiae (strain CIP 109804T); 9, L.
467
weihenstephanensis (strain DSM 24698T); 10, L. cornellensis (strains TTU A1-0210T, FSL F6-0970); 11, L. riparia (strains FSL S10-
468
1204T, FSL S10-1219); 12, L. grandensis (strain TTU A1-0212T); 13. L. fleischmannii (strains DSM 24998T, ATCC BAA-2414T, FSL
469
F6-1019, FSL S10-1186, FSL S10-1203, FSL S10-1220); 14. L. aquatica (strains FSL S10-1188T, FSL S10-1181); 15. L. floridensis
470
(strain FSL S10-1187T). All species/strains are catalase and methyl red positive. All strains are positive for acid production from
471
esculin ferric citrate, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-fructose, D-mannose. All Listeria species
472
were negative for nitrite reduction and acid production from D-arabinose, D-adonitol, methyl-ßD-xylopyranoside, D-RAFfinose,
473
glycogen, L-fucose, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. +, Positive; (+) weakly positive;
474
-, negative; v, variable (between replicates and/or between strains); V! variable between studies, possibly due to differences in
475
incubation times and temperatures between studies; ND, not done or not reported.
Characteristic
1
2
3
4
5
6
7
8
Voges-Proskauer
+
+
+
+
+
+
+
-
Nitrate Reduction
-
-
-
-
-
-
v
Motility
+
+
+
+
+
+
Methyl Red
+
+
+
+
+
Hemolysis
+
-
+
+
Arylamidase
-
+
+
α-Mannosidase
+
+
PI-PLC
+
-
10
11
12
13
14
15
-
-
-
-
-
v
-
+
+
+
+
+
+
+
-
+
-*
-*
-
-
-
-
-
-
+
+
v
v
v
+
v
+
+
+
-
-
-
-
-
-
-
-
-
-
-
v
v
-
+
-
-
-
-
-
-
-
-
-
-
+
+
v
+
-
-
+
-
-
+
-
-
+
-
-
-
-
-
-
-
-
-
-
-
Acidification of:
17
9
D-Arabitol
+
+
+
+
+
+
+
-
+
-
-
v
+
-
-
D-Xylose
-
-
+
+
+
-
-
+
+
+
+
+
+
+
+
L-Rhamnose
+
v
-
-
v
-
-
+
+
-
+
-
+
+
+
α-Methyl-D-glucoside
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
D-Ribose
-
-
-
+
-
-
+
+
-
+
v
+
+
+
-
Glucose-1-phosphate
-
-
-
v
-
-
-
-
-
-
-
-
-
-
-
D-Tagatose
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
D-Mannitol
-
-
-
-
-
-
+
+
+
-
v
-
v
-
-
Sucrose
+
+
+
+
+
-
-
-
-
-
-
-
v
-
-
D-Turanose
-
v
-
-
-
+
-
-
-
-
-
-
v
-
-
Glycerol
v
+
+
+
+
-
v
+
+
v
v
-
+
v
-
D-Galactose
v
-
-
v
-
-
+
+
-
-
+
-
-
-
+
L-arabinose
-
-
-
-
-
-
-
-
-
v
+
-
-
+
+
V!
V!
-
V!
-
V!
V!
-
-
-
-
-
v
-
-
Inositol
-
-
-
-
-
-
-
-
-
-
v
-
v
v
-
Methyl α-D-mannopyranoside
-
-
ND
-
ND
-
+
-
-
-
-
-
v
-
-
D-Maltose
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
D-Lactose
+
+
+
+
+
+
+
+
V!
(+)
+
-
+
-
+
D-Melibiose
V!
v
-
-
-
v
-
+
-
-
v
-
v
-
-
Inulin
V!
V!
-
-
-
-
-
-
-
-
-
-
-
-
-
D-Melozitose
v
v
v
v
v
-
-
-
-
-
-
-
v
-
-
D-Lyxose
v
v
-
-
v
-
v
-
-
-
-
-
-
v
+
D-glucose
V!
V!
+
V!
+
V!
+
+
+
+
+
+
+
+
+
L-sorbose
476
* Motility was reported for these species in the original publications (Lang Halter et al., 2013;
477
Leclercq et al., 2010) of these species. We did not observe motility in the type strains of these
478
species, nor did we find genes encoding a flagellar apparatus in the genomes of the type strains
18
479
(see Table S4). In agreement with our observations, Bertsch et al. (Bertsch et al., 2013) did not
480
observe motility in the type strain of L. rocourtiae.
481
19
Figure 1 L. aquatica FSL S10-1188T L. floridensis FSL S10-1187T L. fleischmannii subsp. coloradensis FSL S10 -1203 L. fleischmannii subsp. coloradensis TTU M1-001T L. fleischmannii subsp. fleischmannii DSM 24998T L. monocytogenes ATCC 15313T L. monocytogenes EGD-e L. monocytogenes F2365 L. monocytogenes HCC23 L. monocytogenes FSL J1-208 L. marthii FSL S4-120T L. innocua Clip11262 L. innocua FSL S4-378 L. innocua FSL J1-023 L. welshimeri SLCC5334 T L. ivanovii subsp. londoniensis ATCC 49954T L. ivanovii subsp. ivanovii PAM 55 L. seeligeri FSL N1-067 L. seeligeri FSL S4-171 L. seeligeri SLCC3954T L. cornellensis TTU A1-0210T L. rocourtiae CIP 109804T L. grandensis TTU A1-0212T L. weihenstephanensis DSM 24699T L. riparia FSL S10-1204T L. grayi subsp. grayi DSM20601T L. grayi subsp. murrayi ATCC 25401T B. campestris ATCC 43754T B. thermosphacta ATCC 11509T 65
75
85
Listeria
Brochothrix
95
Fig. 1 UPGMA cluster diagram based percent average nucleotide difference (= 100% -ANIb: see Chan et al., 2012). The scale bar indicates the % ANIb. The grey vertical bar marks the 5% average nucleotide difference, or 95% ANIb, a value which has shown to correlate to the traditional DNA-DNA hybridization value of 70%. New species are printed bold.
L. monocytogenes EGD-e
Figure 2
84
L. monocytogenes F2365 L. marthii FSL S4-120 T L. innocua Clip11262 L. welshimeri serovar 6b str. SLCC5334 T
100
L. seeligeri serovar 1/2b str. SLCC3954 T L. ivanovii subsp. ivanovii str. PAM55 L. weihenstephanensis DSM 24699T 85 100 100
L. cornellensis TTU A1-0210T
Listeria
L. rocourtiae CIP 109804T L. grandensis TTU A1-0212T L. riparia FSL S10-1204T
100 100 100
L. fleischmannii FSL S10 -1203 L. fleischmannii subsp. coloradensis TTU M1-001T L. fleischmannii subsp. fleischmannii DSM 24998T
100 100
L. aquatica FSL S10-1188 T L. floridensis FSL S10-1187 T
96 100
L. grayi subsp. murrayi ATCC 25401T L. grayi subsp. grayi DSM 20601T B. campestris ATCC 43754T
100
B. thermosphacta ATCC 11509T 100
Brochothrix Staphylococcus epidermidis ATCC 12228 Staphylococcus haemolyticus JCSC1435
100
Staphylococcus aureus subsp. aureus USA300 TCH1516
96
Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305 Enterococcus faecalis V583 0.05
Fig. 2 Maximum likelihood phylogeny based on concatenated amino acid sequences of 31 core genes. Values on the branches represent bootstrap values based on a 1000 replicates. Bootstrap values < 70 are not indicated. Novel species are printed in bold font.
1
Five new species of Listeria (L. floridensis sp. nov, L. aquatica sp. nov., L. cornellensis sp.
2
nov. L. riparia sp. nov., and L. grandensis sp. nov.) from agricultural and natural
3
environments in the United States
4 5
Henk C. den Bakker,1* Steven Warchocki,1 Emily M. Wright,1 Adam F. Allred,2 Christina
6
Ahlstrom,3† Clyde S. Manuel,3‡ Matthew J. Stasiewicz,1 Angela Burrell,2 Sherry Roof,1 Laura K.
7
Strawn,1 Esther Fortes,1§ Kendra K. Nightingale,4 Daniel Kephart,2 and Martin Wiedmann1
8 9
1
Department of Food Science, Cornell University, Ithaca, NY 14853, USA
10
2
Life Technologies, 2130 Woodward Street, Austin, TX 78744, USA
11
3
Department of Animal Sciences, Colorado State University, Fort Collins, CO 80523, USA
12
4
Department of Animal and Food sciences, Texas Tech University, Lubbock, TX 79409, USA
13
† Present address: Production Animal Health, University of Calgary, Alberta, Canada
14
‡ Present address: Department of Food, Bioprocessing and Nutrition Sciences, North Carolina
15
State University, Raleigh, NC 27695-7624, USA
16
§ Present address: Hinton State Laboratory Institute, Massachusetts Department of Public Health,
17
Jamaica Plain, Massachusetts, USA
18 19
Category: New Taxa (Firmicutes and Related Organisms)
20
Running Title: Novel taxa of Listeria from environmental samples
21 22
Correspondence: Henk C. den Bakker, [email protected], Phone: (+1) 607 255-1266, Fax: (+1)
23
607 254-4868
24 25
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains TTU
26
A1-0210T, TTU A1-0212T, FSL S10-1187T, FSL S10-1188T, and FSL S10-1204T are JX961634,
27
JX961635, JX961636, JX961637, and JX961638, respectively. The GenBank/EMBL/DDBJ
28
accession number for the genome sequences of strains TTU A1-0210T, TTU A1-0212T, FSL S10-
29
1187T, FSL S10-1188T, and FSL S10-1204T are AODE00000000, AODD00000000,
30
AODF00000000, AOCG00000000, and AODL00000000, respectively. The versions described in
31
this paper are the first versions, AODE01000000, AODD01000000, AODF01000000,
32
AOCG01000000, and AODL01000000.
33
1
34
Abstract:
35 36
Sampling of agricultural and natural environments in two US states (Colorado and
37
Florida) yielded 18 Listeria-like isolates that could not be identified as previously
38
described species using traditional methods. Using whole genome sequencing and
39
traditional phenotypic methods we identified five new species, each with a genome wide
40
average blast nucleotide identity (ANIb) of less than 85% to currently described species.
41
Phylogenetic analysis based on 16S rDNA sequences and amino acid sequences of 31
42
conserved loci showed the existence of four well-supported monophyletic clades within
43
the genus Listeria; (i) a clade representing L. monocytogenes, L. marthii, L. innocua, L.
44
welshimeri, L. seeligeri and L. ivanovii, which we refer to as Listeria sensu stricto, (ii), a
45
clade consisting of L. fleischmannii and two newly described species, L. aquatica sp. nov.
46
(type strain FSL S10-1188T =DSM 26686T =LMG 28120T =BEI NR-42633T) and L.
47
floridensis sp. nov. (type strain FSL S10-1187T =DSM 26687T =LMG 28121T =BEI NR-
48
42632T), (iii) a clade consisting of L. rocourtiae, L. weihenstephanensis, and three new
49
species, L. cornellensis sp. nov. (type strain TTU A1-0210T = FSL F6-0969T =DSM
50
26689T =LMG 28123T =BEI NR-42630T), L. grandensis sp. nov. (type strain TTU A1-
51
0212T =FSL F6-0971T =DSM 26688T =LMG 28122T =BEI NR-42631T) and L. riparia
52
sp. nov. (type strains FSL S10-1204T =DSM 26685T =LMG 28119T = BEI NR- 42634T),
53
and a clade containing L. grayi. Genomic and phenotypic data suggest the novel species
54
are nonpathogenic.
55 56
The genus Listeria was first described by Pirie (1940), and at present comprises 10
57
species, Listeria monocytogenes (Pirie, 1940), Listeria grayi (Errebo Larsen & Seeliger,
58
1966), Listeria innocua (Seeliger, 1981), Listeria welshimeri (Rocourt & Grimont, 1983),
59
Listeria seeligeri (Rocourt & Grimont, 1983), Listeria ivanovii (Seeliger et al., 1984),
60
Listeria marthii (Graves et al., 2010), Listeria rocourtiae (Leclercq et al., 2010), Listeria
2
61
fleischmannii (Bertsch et al., 2013), and Listeria weihenstephanensis (Lang Halter et al.,
62
2013). Additionally, two subspecies have been recognized within L. ivanovii (subsp.
63
ivanovii and subsp. londoniensis (Boerlin et al., 1992)), L. grayi (subsp. grayi and subsp.
64
murrayi (Stuart & Welshimer, 1973)) and L. fleischmannii (subsp. fleischmannii and
65
subsp. coloradonensis (Den Bakker et al., 2013)).
66 67
During sampling projects of agricultural and natural environments in the US states
68
Florida and Colorado, we isolated 70 isolates, predominantly from water, with a colony
69
morphology on Listeria monocytogenes Plating Medium (LMPM) reminiscent of
70
Listeria. These isolates could be placed in the Listeriaceae by phylogenetic analysis of
71
partial sequences of 16S rDNA and 52 isolates could be assigned to L. fleischmannii (32
72
isolates from Florida and 20 isolates from Colorado), however, 18 isolates did not
73
phylogenetically cluster within previously described species and therefore could not be
74
classified as any of the previously described species. Based on further genotypic
75
characterization of these isolates, including by sigB sequencing (Sauders et al., 2012), one
76
or two isolates per putative new taxon were selected for further phenotypic and genomic
77
characterization (see Fig. S1 for more information).
78 79
Draft genome sequences were assembled as detailed in Den Bakker et al. (2013) from Ion
80
PGM™ instrument reads obtained for (i) Brochothrix thermosphacta ATCC 11509T and
81
B. campestris ATCC 43754T type strains, (ii) the type strains of L. monocytogenes ATCC
82
15313T, L.weihenstephanensis DSM 24698T, L. rocourtiae CIP 109804T, L. grayi subsp.
83
murrayi ATCC 25401T, (iii) one additional strain of L. fleischmannii (FSL S10-1203),
84
and (iv) five isolates that showed Listeria-like characteristics, but could not be classified
85
to species by traditional approaches. Briefly, de novo assembly was performed using the
86
MIRA assembler (Chevreux 1999), and homopolymers were corrected using a
87
homopolymer correction script (available at http://tinyurl.com/oke6xaa); this script uses
3
88
output from VarScan (Koboldt et al., 2012) and BWA (Li & Durbin, 2009) to obtain a
89
consensus about the homopolymer length, which is then used to correct the
90
homopolymers. De novo assemblies (draft genomes) of Ion Torrent reads ranged in total
91
size from 2.37 (Brochothrix campestris) to 3.37 (L. weihenstephanensis) Mbp (see Table
92
S1 for total draft genome lengths and additional genome assembly statistics). Comparison
93
of the draft genome obtained here for L. monocytogenes ATCC 15313T and a recently
94
finished genome of L. monocytogenes SLCC5850 (Kuenne et al., 2013), a strain closely
95
related to the type strain (McLauchlin & Rees, 2009), showed the draft genome covers
96
97.3% of the length of the finished genome. We thus conclude that the draft genomes
97
obtained in this study are of high quality and suitable for comparative analysis. These
98
draft genomes also will provide a starting point for future in-depth genomic and
99
evolutionary analyses of the genus Listeria.
100 101
BLAST Average nucleotide identities (ANIb) were calculated based on draft genome
102
sequences and publicly available genome sequences using JSpecies (Richter & Rosselló-
103
Móra, 2009). Additionally, BLAST average amino acid identities (AAI) (Konstantinidis
104
& Tiedje, 2005) were calculated using the AAI.rb script from the Enve-omics package
105
(http://enve-omics.ce.gatech.edu/). Pairwise comparisons between currently recognized
106
species in Listeria sensu stricto yielded ANIb and AAI values < 95% for comparisons
107
between different species (Fig. 1; Table S2); an ANIb value of 9596% generally
108
corresponds to 70% DNA-DNA hybridization and is typically used as a cut-off value for
109
species delineation in bacterial taxonomy (Richter & Rosselló-Móra, 2009). Our data also
110
support the conclusions from previous studies that ANIb values are robust even with
111
fragmented unfinished draft assemblies. This robustness is demonstrated by an ANIb
112
value of 99.94% for the comparison between the draft genome for the L. monocytogenes
113
type strain ATCC 15313T (a draft genome obtained in this study) and the finished
114
genome for L. monocytogenes SLCC5850. For all genomes representing previously
4
115
reported species, ANIb values supported prior species classification, despite the mixed
116
use of finished and draft genomes (Fig. 1). Most importantly, our analyses showed that
117
all isolates representing new species proposed in this study show ANIb values < 84% and
118
AAI values < 87.9% (Table S2) when their genomes are compared to each other and to
119
phylogenetically related previously described species (L. fleischmannii, L. rocourtiae and
120
L. weihenstephanensis). While the high 16S sequence similarity (> 99%) between L.
121
rocourtiae CIP 109804T, L. weihenstephanensis DSM 24998T, L. cornellensis TTU A1-
122
0210T, L. grandensis TTU A1-0212T and L. riparia FSL S10-1204T could be interpreted
123
as suggesting that these taxa represent ecotypes of a single species (Cohan, 2001;
124
Konstantinidis et al., 2006), ANIb and AAI values, and shared gene content do not
125
support this hypothesis. AAI and ANIb values between these species range from 83.2 to
126
88.6% and from 77.7 to 83.2%, respectively, and the proportion of shared gene content
127
between each species pair ranges from 63 to 75%, justifying recognition as individual
128
species (Fig. S2).
129 130
Phylogenetic analysis of (i) 16S rDNA sequences (Fig. S2), and (ii) the concatenated
131
sequences of 31 aa sequences (Wu & Eisen, 2008)(Fig. 2) were performed as previously
132
described (Den Bakker et al., 2013), with the exception that Gblocks version 0.91b
133
(Castresana, 2000) was used to remove ambiguous sites from the aa sequence alignment.
134
Maximum parsimony (MP) analyses were performed in PAUP* version 4.0b10
135
(Wilgenbusch & Swofford, 2003). Phylogenies obtained from the MP (data not shown)
136
did not differ from those found by maximum likelihood (ML) based analyses, and hence
137
the results of the ML analyses are discussed here. Both 16S rDNA and aa sequence
138
phylogenies confirmed placement of the novel taxa in the genus Listeria, and revealed
139
four well-supported clades (100 bootstrap support [BS]) within the genus Listeria; (i) a
140
clade containing L. monocytogenes and related species (Listeria sensu stricto: L. marthii,
141
L. innocua, L. welshimeri, L. seeligeri and L. ivanovii), (ii) a clade consisting of L.
5
142
rocourtiae, L. weihenstephanensis, L. cornellensis sp. nov, L. grandensis sp. nov. and L.
143
riparia sp. nov. (iii) a clade consisting of L. fleischmannii, L. floridensis sp. nov. and L.
144
aquatica sp. nov., and (iv) a clade consisting of both subspecies of L. grayi. The 16S
145
analysis (Fig. S3) further indicates that a previously unidentified Listeria-like bacterium
146
(referred to as ‘unidentified bacterium isolate MB405’ in Fig. S3) from Belgium (Rijpens
147
et al., 1998) is a putative sister species of L. aquatica sp. nov. Phylogenetic analysis of
148
individual genes, such as iap (Fig. S4), also reveal the subdivision of Listeria into four
149
distinct clades. The phylogenetic relationships between the four clades are unresolved in
150
the 31 aa loci tree (Fig. 1, BS values < 60), however the 16S rDNA sequence analysis
151
suggests that the L. rocourtiae clade and the L. grayi clade are sistergroups (Fig. S3, 85
152
BS). AAI values between members of each phylogenetically distinct clade range from 67
153
to 70% (Fig. S2). While Konstantinidis and Tiedje (2005; 2007) show AAI between 65
154
and 72% coincide with traditional genus delimitations in bacterial taxonomy, we do not
155
propose classification of these clades into separate genera at this point, including because
156
reclassification of L. grayi into a new genus has previously been unsuccessful and
157
controversial (McLauchlin & Rees, 2009).
158 159
For the methyl red (MR) and Voges-Proskauer (VP) tests, all Listeria isolates were
160
cultured on BHI (Becton, Dickinson & Co.) agar aerobically for 18 h at 30 °C. A single
161
colony was suspended in 5 ml of MR-VP broth (pH: 6.9(±0.2); Becton, Dickinson &
162
Co.); inoculated tubes were incubated aerobically for 5 days at 35 °C. MR and VP test
163
procedures were carried out according to the manufacturer’s guidelines (Becton,
164
Dickinson & Co procedure # L007474). Results are listed in the species descriptions and
165
Table 1.
166 167
Testing for catalase activity was performed according to (Bacteriological Analytical
168
Manual (BAM) protocol R12 (U.S. Food and Drug Administration, 2013). An isolated
6
169
colony from BHI agar was emulsified in 1 drop of 3% hydrogen peroxide on a glass
170
slide. Bubbling within 15 s was interpreted as a positive result for the catalase test.
171
Results were independently scored by two individuals and are listed in the species
172
descriptions and Table 1.
173 174
To test nitrate reduction, isolates were grown in nitrate broth (BAM Media M108; [U.S.
175
Food and Drug Administration, 2013]) with Durham tubes for 48 h at 30 °C non-shaking.
176
Durham tubes allowed detection of gas production at 24 and 48 h, which is indicative of
177
N2 production. Reduction of nitrate to nitrite was tested by adding 50 µl of bacterial
178
culture grown in nitrate broth to 0.9 ml phosphate buffer (50 mM, pH 7.4), followed by
179
the addition of 1 ml sulfanilic acid/HCL (5.0 g sulfanilic acid, 100 ml concentrated HCL,
180
400 ml H20; final pH -0.38) and 1 ml 0.58% N-(1-Naphthyl) ethylenediamine. An
181
immediate color change to deep pink through purple indicated the production of nitrite.
182
For all isolates, nitrite reduction was determined using a protocol comparable to the
183
nitrate reduction protocol described above, however, KNO2 was substituted for KNO3 in
184
BAM Media M108. Nitrite reduction was negative if the reaction solution produced
185
magenta to purple coloration. If an isolate reduces nitrate to an oxidation state lower than
186
nitrite, the nitrate reduction test will produce a false negative. Therefore, separately
187
testing nitrite reduction and the generation of N2 (Durham tubes) measures the reduction
188
of nitrate to nitrite. All non-motile species were found to be able to reduce nitrate, with
189
the exception of L. floridensis. None of the new species reported here was able to reduce
190
nitrite.
191 192
Hemolysis for each isolate was determined using the CAMP test as described in BAM
193
(U.S Food and Drug Administration, 2013) and performed in duplicate. To test for PI-
194
PLC activity, single colonies from BHI agar plates were sub-cultured to Listeria
195
monocytogenes Plating Medium (LMPM; R&F Laboratories). PI-PLC activity positive
7
196
strains color blue on LMPM medium. None of the new species described here showed
197
hemolytic activity or PI-PLC activity, suggesting these species lack the genes (i.e., hly
198
and plcA) associated with virulence in L. monocytogenes and L. ivanovii. Virulence
199
genes (i.e., genes found in the prfA-cluster (Schmid et al., 2005)) or LIPI-2 (Domínguez-
200
Bernal et al., 2006) were absent from the draft genomes, further supporting that none of
201
the new species described here are pathogenic.
202 203
API Listeria and API 50 CH (with API 50 CHB/E medium) kits (bioMerieux) were
204
utilized for further phenotypic characterization, using bacterial colonies taken from BHI
205
agar plates. API Listeria tests were incubated for 24 h at 34 °C for all isolates (within the
206
manufacturer recommended temperature range of 36±2 °C). API 50 CH tests were
207
incubated for 48 h at 37 °C for all Listeria isolates. A mineral oil cover was not used,
208
since all species studied here are facultative anaerobes. Results were recorded at 48 h,
209
unless there were positive or variable results at 24 h with a corresponding negative result
210
at 48 h, in which case 24 h results were reported (as instructed in the API 50 CHB/E
211
manual). Results are summarized in the species descriptions and Table 1. Results for the
212
individual isolates are reported in Table S3.
213 214
To test growth characteristics, isolates were grown in BHI broth at six different
215
temperatures (4, 7, 22.5, 30, 37, and 41°C). The Synergy™ H1 hybrid multi-mode
216
microplate reader (BioTek Instruments Inc., Winooski, Vermont) was employed to test
217
growth at 22.5±0.5 °C, 30±0.1 °C, 37±0.1 °C, and 41±0.1 °C (temperature tolerances as
218
provided by manufacturer). Colony inoculated cultures were grown for 24 h in BHI
219
broth; an aliquot of these cultures (20 µl) was used to inoculate Costar 96 well flat bottom
220
plates (clear polystyrene) prefilled with 180 µl of BHI. Inoculated plates were incubated
221
at 22.5 °C for 48 h, 30 °C for 41 h, 37 °C for 26 h, and 41 °C for 41 h in the Synergy™
222
H1 reader. Optical density at 600 nm (OD600) was measured after 10 s orbital shaking as
8
223
follows: every 2 min for the first 20 min, every 5 min for the next 40 min, every 10 min
224
for the remainder of the experiment. Growth was quantified by finding the first time the
225
cells had increased by 0.6 log (c.f.u./ml), i.e. a 4-fold increase in cell number. Measured
226
increases in log(OD600) were converted to increases in log (c.f.u./ml) using the slope of
227
the strain specific linear function relating log(OD600) to log (c.f.u./ml). The linear
228
function was created with a series of eight 2-fold serial dilutions of a 24 h culture of each
229
strain, performed in a microtiter plate, and the resulting OD600 values for each dilution.
230
The slope of the resulting best fit lines averaged 0.92 log (OD600)/log (c.f.u./ml), with
231
average R2 of 0.99. For the measurement of growth characteristics at temperatures below
232
ambient temperature, BHI culture tubes were inoculated at 103 c.f.u./ml (± 0.3 log) and
233
incubated at 4 °C (low: 3°, high: 9 °C) and 7 °C (6.8±0.2 °C) in a refrigerated incubator.
234
Cultures were spiral plated on BHI agar (to determine c.f.u./ml) every 24 hours for 12
235
days. Growth was quantified by calculating the time for cells to increase by 1.0 log
236
(c.f.u./ml), i.e. a 10-fold increase in cell number, by linear interpolation of the measured
237
points immediately before and after the observed 1.0 log increase. Growth characteristics
238
are reported in the species descriptions and additional data on growth characteristics of
239
the individual isolates can be found in Table S4.
240 241
Motility was determined in Motility Test Medium (prepared according to BAM Media
242
M103 [U.S. Food and Drug Administration, 2013]). For each isolate, 13 mm tubes
243
containing Motility Test Medium (MTM) were inoculated 1 cm under the surface, with a
244
stab, from a colony on BHI agar. Isolates were incubated aerobically for 7 days at 22 °C,
245
30 °C, or 37 °C or for 10 days at 4 °C. An isolate was considered motile if cloudy growth
246
beyond the stab was observed in addition to the characteristic mushroom or umbrella-
247
shaped growth. Results are reported in Table 1 and the species descriptions. L.
248
monocytogenes 10403S was included as a positive control, while L. fleischmannii DSM
249
24998T was included as a negative control. In addition to the motility test, draft genomes
9
250
of the type strains sequenced were queried using BLAST (Altschul et al., 1990) for genes
251
associated with motility in L. monocytogenes (Table S5). Consistent with observations of
252
the motility test, no flagellar motility associated genes were found in the new species
253
described here.
254 255
Light microscopic observations were performed on Listeria isolates that were cultured on
256
BHI agar aerobically for 18 h at 30 °C. Stationary-phase bacteria were examined on agar-
257
coated slides and viewed by phase-contrast microscopy using an Olympus BX61
258
microscope (Olympus). Digital photographs were captured using the SlideBook™
259
v4.0.2.2 software (Intelligent Imaging Innovations) and length-width ratios were based
260
on the average length and width of five bacteria. Results are reported in Table 1 and the
261
species descriptions. Photographs of the individual isolates can be found in Figure S5.
262 263
Description of Listeria floridensis sp. nov.
264
Listeria floridensis (flo.rid.en'sis. N.L. fem. adj. floridensis of or belonging to Florida, the
265
state in the United States from which the first strain was isolated).
266 267
Cells are 0.6 by 1.3 – 1.9 µm in size, length/width ratio 2.7, Gram-positive, straight rods
268
with rounded ends. No growth occurs at temperatures of 7 °C and below. Optimum
269
growth temperature 37 – 41 °C. No motility at 4, 22, 30, or 37 °C. Voges-Proskauer
270
negative, and catalase-positive. CAMP-negative. Nitrate reduction negative, nitrite-
271
reduction negative. Other characteristics are reported in Table 1. L. floridensis is
272
currently the only member of the genus Listeria that both lacks motility and is unable to
273
reduce nitrate. A unique API-Listeria numerical profile (2 710) was observed for the type
274
strain.
275
10
276
Type strain (FSL S10-1187T), isolated from running water in Florida, USA, is deposited
277
in DMSZ (DSM 26687T), BCCM/LMG (LMG 28121T), and BEI (BEI NR-42632T). The
278
DNA G+C content of the type strain is 41.8 mol% (determined by genome sequencing).
279 280
Description of Listeria aquatica sp. nov.
281
Listeria aquatica (a.qua’ti.ca L. fem. adj. aquatica, found in water, aquatic).
282 283
Cells are 0.6 – 0.7 by 1.5 – 2.4 µm in size, average length-width ratio 3.2, Gram-positive,
284
straight rods with rounded ends. No growth occurs at temperatures of 7 °C and below.
285
Optimum growth temperature range 37 – 41 °C. No motility at 4, 22, 30, or 37 °C.
286
Voges-Proskauer negative for type strain, positive for strain FSL S10-1181. Catalase-
287
positive. CAMP-negative. Nitrate reduction positive, nitrite-reduction negative. Other
288
characteristics are reported in table 1. L. aquatica is currently the only member of the
289
genus Listeria that is unable to ferment D-Maltose. Among non-motile Listeria species it
290
is unique in its ability to ferment D-tagatose. A unique API-Listeria numerical profile (6
291
731) was observed for the type strain and FSL S10-1181.
292 293
Type strain (FSL S10-1188T) was isolated from running water in Florida (USA) and is
294
deposited in DMSZ (DSM 26686T), BCCM/LMG (LMG 28120T) and BEI (BEI NR-
295
42633T). The DNA G+C content of the type strain is 40.9 mol% (determined by genome
296
sequencing).
297 298
Description of Listeria cornellensis sp. nov.
299
Listeria cornellensis (cor.nell.en'sis. N.L. fem. adj. cornellensis, named after Cornell, the
300
university where most of the research was performed that led to the discovery of the
301
Listeria species described in this study).
302
11
303
Cells are 0.4 – 0.7 by 2.4 – 3.8 µm in size, average length-width ratio 5.3, Gram-positive,
304
straight rods with rounded ends. Optimal growth at 30 – 37 °C. No motility at 4, 22, 30,
305
or 37 °C. Voges-Proskauer negative, catalase-positive. CAMP-negative. Nitrate-
306
reduction positive, nitrite-reduction negative. Other characteristics are indicated in Table
307
1. While phylogenetically and genomically distinct, this species resembles L. grandensis
308
for the phenotypic characteristics recorded here. Among the non-motile Listeria species
309
L. cornellensis and L. grandensis are the only non-motile Listeria species that are L-
310
rhamnose negative in the API CH50 test (performed at 37° C), however, the API Listeria
311
test strip (incubated at 34° C) showed acidification of L-rhamnose for L. grandensis, and
312
for one of the two L. cornellensis strains tested. An operon involved in rhamnose
313
utilization could be found in the draft genome of L. grandensis, but was absent from the
314
draft genome of the type strain of L. cornellensis, suggesting an absence of rhamnose
315
utilization for the type strain of L. cornellensis and temperature dependent ability of L.
316
grandensis to acidify L-rhamnose. L. cornellensis can be further distinguished from L.
317
grandensis by weak acidification of D-lactose. API-Listeria numerical profile 2 330 was
318
observed for the two isolates (TTU A1-0210T and FSL F6-0970) of this species; this
319
numerical profile is associated with L. ivanovii according to the API-Listeria manual
320
(bioMerieux; version 04/2007).
321 322
Type strain (TTU A1-0210T), isolated from water in Colorado, USA, is deposited in
323
DMSZ (DSM 26689T), BCCM/LMG (LMG 28123T) and BEI (BEI NR-42630T). The
324
DNA G+C content of the type strain is 42.5 mol% (determined by genome sequencing).
325 326
Description of Listeria grandensis sp. nov.
327
Listeria grandensis (grand.en’sis, N.L. fem. adj. of or belonging to Grand, the county
328
where the type strain was isolated).
329
12
330
Cells are 0.6 – 0.7 by 2.0 – 3.1 µm in size, average length-width ratio 4.0, Gram-positive,
331
straight rods with rounded ends. Optimal growth temperature 30 – 37 °C. No motility at
332
4, 22, 30, or 37 °C. Voges-Proskauer negative, catalase-positive. CAMP-negative.
333
Nitrite-reduction negative and nitrate-reduction positive. Other characteristics are
334
reported in Table 1. See L. cornellensis for differentiation of L. grandensis from this
335
species. A unique API-Listeria numerical profile (2 730) was observed for the type strain.
336 337
Type strain (TTU A1-0212T), isolated from water in Colorado, USA, is deposited in
338
DMSZ (DSM 26688T), BCCM/LMG (LMG 28122T) and BEI-resources (BEI NR-
339
42631T). The DNA G+C content of the type strain is 43.0 mol% (determined by genome
340
sequencing).
341 342
Description of Listeria riparia sp. nov.
343
Listeria riparia (ri.pa’ri.a L. fem. adj. riparia, of the bank of a river or stream).
344 345
Cells are 0.5 – 0.7 by 2.3 – 3.7 µm in size, average length-width ratio 4.8, Gram-positive,
346
straight rods with rounded ends. Optimum growth temperature 37 – 41 °C. No motility at
347
4, 22, 30, or 37 °C. Voges-Proskauer negative, and catalase-positive. CAMP-negative.
348
Nitrite-reduction negative and nitrate-reduction positive. Other characteristics are
349
reported in Table 1. Can be differentiated from other non-motile Listeria species by a
350
combination of α-Mannosidase activity, and the ability to acidify L-rhamnose, D-
351
galactose and L-arabinose. A unique API-Listeria numerical profile (6 710) was observed
352
for the isolates tested (FSL S10-1204T and FSL S10-1219).
353 354
Type strain (FSL S10-1204T), isolated from running water in Florida, USA, is deposited
355
in DSMZ (DSM 26685T), BCCM/LMG (LMG 28119T) and BEI-resources (BEI NR-
13
356
42634T). The DNA G+C content of the type strain is 41.9 mol% (determined by genome
357
sequencing).
358 359
Acknowledgements. This project was supported by USDA NIFA Special Research
360
Grant 2010-34459-20756 as well as USDA NIFA National Integrated Food safety
361
initiative Grants 2008-51110-04333 and 2008-51110-04688. We thank Dr. Esther Angert
362
(Cornell University, Department of Microbiology) for the help with the microscopy. We
363
would like to thank Dr. Catherine Donnelly (University of Vermont) and Drs. Arnout de
364
Bruin and Wilma Jacobs (RIVM, the Netherlands) for their help with the VP test. We
365
would further like to thank Barbara Bowen for her help with some experiments.
366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393
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C. A., Mardis, E. R., Ding, L. & Wilson, R. K. (2012). VarScan 2: somatic mutation and copy number alteration discovery in cancer by exome sequencing. Genome Res 22, 568–576. Konstantinidis, K. T., Ramette, A. & Tiedje, J. M. (2006). The bacterial species definition in the genomic era. Philos Trans R Soc Lond, B, Biol Sci 361, 1929–1940. Konstantinidis, K. T. & Tiedje, J. M. (2005). Towards a genome-based taxonomy for prokaryotes. J Bacteriol 187, 6258–6264. Konstantinidis, K. T. & Tiedje, J. M. (2007). Prokaryotic taxonomy and phylogeny in the genomic era: advancements and challenges ahead. Curr Opin Microbiol 10, 504– 509. Kuenne, C., Billion, A., Mraheil, M. A., Strittmatter, A., Daniel, R., Goesmann, A., Barbuddhe, S., Hain, T. & Chakraborty, T. (2013). Reassessment of the Listeria monocytogenes pan-genome reveals dynamic integration hotspots and mobile genetic elements as major components of the accessory genome. BMC Genomics 14, 47. Lang Halter, E., Neuhaus, K. & Scherer, S. (2013). Listeria weihenstephanensis sp. nov., isolated from the water plant Lemna trisulca taken from a freshwater pond. Int J Syst Evol Microbiol 63, 641–647. Leclercq, A., Clermont, D., Bizet, C., Grimont, P. A. D., Le Flèche-Matéos, A., Roche, S. M., Buchrieser, C., Cadet-Daniel, V., Le Monnier, A. & other authors. (2010). Listeria rocourtiae sp. nov. Int J Syst Evol Microbiol 60, 2210–2214. Li, H. & Durbin, R. (2009). Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754–1760. McLauchlin, J. & Rees, C. (2009). Genus I. Listeria Pirie 1940a 383AL. In, 2nd edn. pp. 244–257. Springer, New York: Bergey’s Manual of Systematic Bacteriology, 2nd edn, vol. 3 (The Firmicutes). Pirie, J. H. (1940). The Genus Listerella Pirie. Science 91, 383. Richter, M. & Rosselló-Móra, R. (2009). Shifting the genomic gold standard for the prokaryotic species definition. Proc Natl Acad Sci USA 106, 19126–19131. Rijpens, N., Vlaemynck, G., Rossau, R., Herman, L. & Jannes, G. (1998). Unidentified Listeria-like bacteria isolated from cheese. Lett Appl Microbiol 27, 198– 202. Rocourt, J. & Grimont, P. A. D. (1983). Listeria welshimeri sp. nov. and Listeria seeligeri sp. nov. Int J Syst Bacteriol 33, 866–869. Sauders, B. D., Overdevest, J., Fortes, E., Windham, K., Schukken, Y., Lembo, A. & Wiedmann, M. (2012). Diversity of Listeria species in urban and natural environments. Appl Environ Microbiol 78, 4420–4433. Schmid, M. W., Ng, E. Y. W., Lampidis, R., Emmerth, M., Walcher, M., Kreft, J., Goebel, W., Wagner, M. & Schleifer, K.-H. (2005). Evolutionary history of the genus Listeria and its virulence genes. Syst Appl Microbiol 28, 1–18. Seeliger, H. P. R. (1981). Apathogene Listerien: L. innocua sp.n. (Seeliger et Schoofs, 1977). Zentralbl Bakteriol Parasitenkd Infektionskr Hyg Abt 1 Orig A 249, 487-493. Seeliger, H. P. R., Rocourt, J., Schrettenbrunner, A., Grimont, P. A. D. & Jones, D. (1984). Listeria ivanovii sp. nov. Int J Syst Bacteriol 34, 336–337. Stuart, S. E. & Welshimer, H. J. (1973). Intrageneric relatedness of Listeria Pirie. Int J Syst Bacteriol 23, 8–14. U.S. Food and Drug Administration (2013). Bacteriological Analytical Manual.
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http://www.fda.gov/Food/ScienceResearch/LaboratoryMethods/BacteriologicalAnaly ticalManualBAM/default.htm. Wilgenbusch, J. C. & Swofford, D. (2003). Inferring evolutionary trees with PAUP*. Curr Protoc Bioinformatics Chapter 6, Unit 6.4. Wu, M. & Eisen, J. A. (2008). A simple, fast, and accurate method of phylogenomic inference. Genome Biol 9, R151.
450
Fig. 1 UPGMA cluster diagram based percent average nucleotide difference (=
451
100%ANIb: see Chan et al., 2012). The scale bar indicates the % ANIb. The grey
452
vertical bar marks the 5% average nucleotide difference, or 95% ANIb, a value which has
453
shown to correlate to the traditional DNA-DNA hybridization value of 70%. New species
454
are printed bold.
Figure legends
455 456
Fig. 2 Maximum likelihood phylogeny based on concatenated amino acid sequences of
457
31 core genes. Values on the branches represent bootstrap values based on a 1000
458
replicates. Bootstrap values < 70 are not indicated. Novel species are printed in bold font.
459
16
460 461
Table 1. Physiological characteristics of Listeria species based on observations made in this study and the current literature.
462
Species: 1, L. monocytogenes (strain 10403S, additional data from McLauchlin & Rees, 2009 and Bertsch et al., 2012); 2, L. innocua
463
(strain FSL S4-378, additional data from McLauchlin & Rees, 2009 and Bertsch et al., 2012); 3, L. seeligeri (data from McLauchlin &
464
Rees, 2009 and Bertsch et al., 2012); 4, L. ivanovii (strain ATCC BAA-678, additional data from McLauchlin & Rees, 2009 and
465
Bertsch et al., 2012); 5, L. welshimeri (data from Bille et al. (1992), McLauchlin & Rees, 2009 and Bertsch et al., 2012); 6, L. marthii
466
(strain FSL S4-120T); 7, L. grayi (strains ATCC 19120T, ATCC 25401T); 8, L. rocourtiae (strain CIP 109804T); 9, L.
467
weihenstephanensis (strain DSM 24698T); 10, L. cornellensis (strains TTU A1-0210T, FSL F6-0970); 11, L. riparia (strains FSL S10-
468
1204T, FSL S10-1219); 12, L. grandensis (strain TTU A1-0212T); 13. L. fleischmannii (strains DSM 24998T, ATCC BAA-2414T, FSL
469
F6-1019, FSL S10-1186, FSL S10-1203, FSL S10-1220); 14. L. aquatica (strains FSL S10-1188T, FSL S10-1181); 15. L. floridensis
470
(strain FSL S10-1187T). All species/strains are catalase and methyl red positive. All strains are positive for acid production from
471
esculin ferric citrate, N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose, D-fructose, D-mannose. All Listeria species
472
were negative for nitrite reduction and acid production from D-arabinose, D-adonitol, methyl-ßD-xylopyranoside, D-RAFfinose,
473
glycogen, L-fucose, potassium gluconate, potassium 2-ketogluconate and potassium 5-ketogluconate. +, Positive; (+) weakly positive;
474
-, negative; v, variable (between replicates and/or between strains); V! variable between studies, possibly due to differences in
475
incubation times and temperatures between studies; ND, not done or not reported.
Characteristic
1
2
3
4
5
6
7
8
Voges-Proskauer
+
+
+
+
+
+
+
-
Nitrate Reduction
-
-
-
-
-
-
v
Motility
+
+
+
+
+
+
Methyl Red
+
+
+
+
+
Hemolysis
+
-
+
+
Arylamidase
-
+
+
α-Mannosidase
+
+
PI-PLC
+
-
10
11
12
13
14
15
-
-
-
-
-
v
-
+
+
+
+
+
+
+
-
+
-*
-*
-
-
-
-
-
-
+
+
v
v
v
+
v
+
+
+
-
-
-
-
-
-
-
-
-
-
-
v
v
-
+
-
-
-
-
-
-
-
-
-
-
+
+
v
+
-
-
+
-
-
+
-
-
+
-
-
-
-
-
-
-
-
-
-
-
Acidification of:
17
9
D-Arabitol
+
+
+
+
+
+
+
-
+
-
-
v
+
-
-
D-Xylose
-
-
+
+
+
-
-
+
+
+
+
+
+
+
+
L-Rhamnose
+
v
-
-
v
-
-
+
+
-
+
-
+
+
+
α-Methyl-D-glucoside
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
D-Ribose
-
-
-
+
-
-
+
+
-
+
v
+
+
+
-
Glucose-1-phosphate
-
-
-
v
-
-
-
-
-
-
-
-
-
-
-
D-Tagatose
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
D-Mannitol
-
-
-
-
-
-
+
+
+
-
v
-
v
-
-
Sucrose
+
+
+
+
+
-
-
-
-
-
-
-
v
-
-
D-Turanose
-
v
-
-
-
+
-
-
-
-
-
-
v
-
-
Glycerol
v
+
+
+
+
-
v
+
+
v
v
-
+
v
-
D-Galactose
v
-
-
v
-
-
+
+
-
-
+
-
-
-
+
L-arabinose
-
-
-
-
-
-
-
-
-
v
+
-
-
+
+
V!
V!
-
V!
-
V!
V!
-
-
-
-
-
v
-
-
Inositol
-
-
-
-
-
-
-
-
-
-
v
-
v
v
-
Methyl α-D-mannopyranoside
-
-
ND
-
ND
-
+
-
-
-
-
-
v
-
-
D-Maltose
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
D-Lactose
+
+
+
+
+
+
+
+
V!
(+)
+
-
+
-
+
D-Melibiose
V!
v
-
-
-
v
-
+
-
-
v
-
v
-
-
Inulin
V!
V!
-
-
-
-
-
-
-
-
-
-
-
-
-
D-Melozitose
v
v
v
v
v
-
-
-
-
-
-
-
v
-
-
D-Lyxose
v
v
-
-
v
-
v
-
-
-
-
-
-
v
+
D-glucose
V!
V!
+
V!
+
V!
+
+
+
+
+
+
+
+
+
L-sorbose
476
* Motility was reported for these species in the original publications (Lang Halter et al., 2013;
477
Leclercq et al., 2010) of these species. We did not observe motility in the type strains of these
478
species, nor did we find genes encoding a flagellar apparatus in the genomes of the type strains
18
479
(see Table S4). In agreement with our observations, Bertsch et al. (Bertsch et al., 2013) did not
480
observe motility in the type strain of L. rocourtiae.
481
19
Figure 1 L. aquatica FSL S10-1188T L. floridensis FSL S10-1187T L. fleischmannii subsp. coloradensis FSL S10 -1203 L. fleischmannii subsp. coloradensis TTU M1-001T L. fleischmannii subsp. fleischmannii DSM 24998T L. monocytogenes ATCC 15313T L. monocytogenes EGD-e L. monocytogenes F2365 L. monocytogenes HCC23 L. monocytogenes FSL J1-208 L. marthii FSL S4-120T L. innocua Clip11262 L. innocua FSL S4-378 L. innocua FSL J1-023 L. welshimeri SLCC5334 T L. ivanovii subsp. londoniensis ATCC 49954T L. ivanovii subsp. ivanovii PAM 55 L. seeligeri FSL N1-067 L. seeligeri FSL S4-171 L. seeligeri SLCC3954T L. cornellensis TTU A1-0210T L. rocourtiae CIP 109804T L. grandensis TTU A1-0212T L. weihenstephanensis DSM 24699T L. riparia FSL S10-1204T L. grayi subsp. grayi DSM20601T L. grayi subsp. murrayi ATCC 25401T B. campestris ATCC 43754T B. thermosphacta ATCC 11509T 65
75
85
Listeria
Brochothrix
95
Fig. 1 UPGMA cluster diagram based percent average nucleotide difference (= 100% -ANIb: see Chan et al., 2012). The scale bar indicates the % ANIb. The grey vertical bar marks the 5% average nucleotide difference, or 95% ANIb, a value which has shown to correlate to the traditional DNA-DNA hybridization value of 70%. New species are printed bold.
L. monocytogenes EGD-e
Figure 2
84
L. monocytogenes F2365 L. marthii FSL S4-120 T L. innocua Clip11262 L. welshimeri serovar 6b str. SLCC5334 T
100
L. seeligeri serovar 1/2b str. SLCC3954 T L. ivanovii subsp. ivanovii str. PAM55 L. weihenstephanensis DSM 24699T 85 100 100
L. cornellensis TTU A1-0210T
Listeria
L. rocourtiae CIP 109804T L. grandensis TTU A1-0212T L. riparia FSL S10-1204T
100 100 100
L. fleischmannii FSL S10 -1203 L. fleischmannii subsp. coloradensis TTU M1-001T L. fleischmannii subsp. fleischmannii DSM 24998T
100 100
L. aquatica FSL S10-1188 T L. floridensis FSL S10-1187 T
96 100
L. grayi subsp. murrayi ATCC 25401T L. grayi subsp. grayi DSM 20601T B. campestris ATCC 43754T
100
B. thermosphacta ATCC 11509T 100
Brochothrix Staphylococcus epidermidis ATCC 12228 Staphylococcus haemolyticus JCSC1435
100
Staphylococcus aureus subsp. aureus USA300 TCH1516
96
Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305 Enterococcus faecalis V583 0.05
Fig. 2 Maximum likelihood phylogeny based on concatenated amino acid sequences of 31 core genes. Values on the branches represent bootstrap values based on a 1000 replicates. Bootstrap values < 70 are not indicated. Novel species are printed in bold font.
