TAXONOMIC DESCRIPTION

Núñez-Montero et al., Int J Syst Evol Microbiol

DOI 10.1099/ijsem.0.002596

Listeria costaricensis sp. nov.

Kattia Núñez-Montero,1† Alexandre Leclercq,2,3,4† Alexandra Moura,2,3,4† Guillaume Vales,2,3,4 Johnny Peraza,1
a5,6,7,*‡ and Marc Lecuit2,3,4,8,*
Javier Pizarro-Cerd!

Abstract
A bacterial strain isolated from a food processing drainage system in Costa Rica fulfilled the criteria as belonging to the
genus Listeria, but could not be assigned to any of the known species. Phylogenetic analysis based on the 16S rRNA gene
revealed highest sequence similarity with the type strain of Listeria floridensis (98.7 %). Phylogenetic analysis based on
Listeria core genomes placed the novel taxon within the Listeria fleishmannii, L. floridensis and Listeria aquatica clade (Listeria
sensu lato). Whole-genome sequence analyses based on the average nucleotide BLAST identity (ANI<80 %) indicated that this
isolate belonged to a novel species. Results of pairwise amino acid identity (AAI>70 %) and percentage of conserved proteins
(POCP>68 %) with currently known Listeria species, as well as of biochemical characterization, confirmed that the strain
constituted a novel species within the genus Listeria. The name Listeria costaricensis sp. nov. is proposed for the novel
species, and is represented by the type strain CLIP 2016/00682T (=CIP 111400T=DSM 105474T).

With the rapid development of whole genome sequencing collected from a food processing drainage system in the
technologies, the genus Listeria has expanded since 2009 to province of Alajuela, Costa Rica, in August 2015, and char-
reach 17 species with validly published names [1]. Recently, acterized using the method described by Hitchins and
the genus has been subdivided into two major groups [1– others [20]. Briefly, selective enrichment was performed
5]: (i) Listeria sensu stricto, constituted by the species Liste- using buffered Listeria enrichment broth (BLEB; Oxoid)
ria monocytogenes [6], L. innocua [7], L. welshimeri [8], with subsequent selective plating in modified Oxford Liste-
L. seeligeri [8], L. ivanovii [9], and L. marthii [10] and (ii) ria selective agar (MOX; Oxoid). Typical Listeria species
Listeria sensu lato, presumably non-pathogenic and consti- colonies growing on MOX agar were characterized by PCR
tuted by the species L. grayi [11, 12], L. rocourtiae [13], using the primers List-univ 1 and List-univ 2, as described
L. fleischmannii [3, 14], L. weihenstephanensis [15], L. flori- [21]. Positive polymerase chain reaction (PCR) amplifica-
densis, L. aquatica, L. cornellensis, L. riparia, L. grandensis tion indicated that this isolate belonged to the Listeria
[4], L. booriae and L. newyorkensis [16]. The two species genus, and the new isolate was sent to WHOCCL for further
L. monocytogenes and L. ivanovii are pathogenic to humans identification.
and animals [17]. Due to the rarity of cases of L. ivanovii
Species identification of the isolate was attempted using
infection, only L. monocytogenes represents a worldwide
matrix-assisted laser desorption ionization time-of-flight
public health concern [18, 19].
mass spectometry (MALDI-TOF MS), based on the mass
In 2016, the World Health Organization Collaborating Cen- spectra obtained from full protein extraction on a MicroFlex
tre for Listeria (WHOCCL), Paris, France, received isolate LT system (Bruker Daltonics), according to the manufac-
CLIP 2016/00682T (originally designated ES106) from the turer’s instructions [22]. Spectral analyses against the MBT
Institute of Technology of Costa Rica. The isolate was library (DB-5989 MS; Bruker Daltonics), which is limited to

Author affiliations: 1Centro de Investigación en Biotecnología, Escuela de Biología, Instituto Tecnológico de Costa Rica, Cartago, Costa Rica; 2Institut
Pasteur, National Reference Center and WHO Collaborating Center for Listeria, Paris, France; 3Institut Pasteur, Biology of Infection Unit, Paris,
France; 4Inserm U1117, Paris, France; 5Institut Pasteur, Bacteria-Cell Interactions Unit, Paris, France; 6Inserm U604, Paris, France; 7INRA USC2020,
Paris, France; 8Paris Descartes University, Sorbonne Paris Cit! e, Division of Infectious Diseases and Tropical Medicine, Institut Imagine, Necker-
Enfants Malades University Hospital, APHP, Paris, France.
*Correspondence: Javier Pizarro-Cerd! a, [email protected]; Marc Lecuit, [email protected]
Keywords: Listeria; Costa Rica; new taxa; whole genome sequencing; ANI; AAI; POCP.
Abbreviations: AAI, average amino acid BLAST identity; ANI, average nucleotide BLAST identity; BAM, Bacteriological Analytical Manual; BHI, brain
heart infusion; BLAST, Basic Local Alignment Search Tool; BLEB, buffered Listeria enrichment broth; MOX, modified Oxford agar; N50, minimum con-
tig length covering 50% of the genome; PCR, polymerase chain reaction; PI-PLC, phosphatidylinositol-specific phospholipase C; POCP, percentage of
conserved proteins; WHOCCL, World Health Organization Collaborating Centre for Listeria.
†These authors contributed equally to this work.
‡Present address: Institut Pasteur, Yersinia Research Unit, Paris, France.
The GenBank/EMBL/DDBJ accession numbers for the draft genome sequence of strain CLIP 2016/00682T are FXUT01000001–FXUT01000044.
One supplementary figure is available with the online version of this article.

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eight Listeria species [22], were inconclusive. The genome similarity (98.7–99.0 %; [29, 30]). Maximum-likelihood
of isolate CLIP 2016/00682T was then sequenced after DNA phylogeny based on the amino acid sequences of Listeria
extraction (DNeasy Blood and Tissue kit; Qiagen), and core genes (Fig. 2) placed the novel taxon within the
library preparation (Nextera XT DNA Sample kit; Illumina), L. fleishmannii, L. floridensis and L. aquatica clade (Listeria
using a NextSeq 500 (2!150 bp) platform (Illumina), sensu lato).
according to the manufacturer’s protocol, as described [23].
Species identification was determined by whole-genome
The data obtained followed the quality standards for use of
pairwise average nucleotide and amino acid BLAST iden-
this platform for taxonomic purposes [24]. The draft
tity comparisons (ANI and AAI, respectively) against Lis-
genome assembly was obtained from high-quality reads
teria species reference genomes deposited at the NCBI
(final coverage 108!) using CLC Assembly Cell 4.3.0 (Qia-
database, using the enveomics package [31]. The percent-
gen) as described [23], and annotated with Prokka v.1.9
age of conserved proteins (POCP) was calculated as
[25]. The draft assembly contained 44 contigs, with a total
described by Qin et al. [32]. ANI analysis revealed that
length of 3.18 Mb and N50 length of 224 949 bp.
strain CLIP 2016/00682T shared less than 80 % genome
Phylogenetic analyses were performed based on 16S rRNA sequence similarity with all known Listeria species
gene sequence comparisons and on the concatenated (Fig. 3), lower than the proposed genomic species cut-off
deduced amino acid sequences of 243 core genes present in of 95 % [33]. Further analysis based on the deduced pro-
all Listeria species, defined in this study using Roary v.3.11 teomes revealed highest AAI with L. fleischmannii subsp.
[26] and a BLASTP identity cut-off of 80 %. Sequences were fleischmannii (two-way AAI of 70.40 %, based on 2041
aligned using MUSCLE v.3.8 [27] and maximum-likelihood orthologous proteins) and L. fleischmannii subsp. cornel-
phylogenetic trees were inferred by using IQ-Tree v.1.5 lensis (two-way AAI of 70.09 %, based on 2056 ortholo-
[28]. 16S rRNA gene sequence analysis revealed highest gous proteins), above the 60 % cut-off proposed for
similarity with the type strain of L. floridensis (98.71 %; genus delineation [34]. The pairwise POCP, which also
Fig. 1), i.e. at the lower cut-off value previously proposed for takes into account the size of deduced proteomes [32],
species delineation based on 16S rRNA gene sequence between strain CLIP 2016/00682T and L. fleischmannii

99 L. monocytogenes NCTC10357T (X56153)

67 L. innocua ATCC 33090T (X98527)
L. welshimeri ATCC 35897T (DQ065846)
99 73 L. marthii NR-9579T (EU545982)
0.01
L. ivanovii ivanovii CLIP 12510T (X98528)
L. seeligeri ATCC 35967T (DQ065845)
86 L. rocourtiae CIP 109804T (FJ557241)
97
L. newyorkensis FSL M6-0635T (KM066002)
82
L. cornellensis FSL F6-969T (JX961634)
89 Listeria
L. grandensis FSL F6-971T (JX961635)
92
L. weihenstephanensis DSM 24698T (FR850019)
99
L. riparia FSL S10-1204T (JX961638)
99 L. booriae FSL A5-0281T (KM066001)
L. grayi grayi DSM 20601T (X98526)
97 L. aquatica FSL S10-1188T (NR_125646)
85 L. fleischmannii LU2006-1T (JN093101)
L. floridensis FSL S10-1187T (NR_125645)
89
L. costaricensis CLIP 2016/00682T (FXUT01000000)
Brochothrix thermosphacta NCDO1676T (X56155)
100 Brochothrix campestris ATCC 43754T (NR_044824)

Fig. 1. Phylogenetic analysis of the 16S rRNA gene based on the maximum-likelihood method. Distance estimation was based on the
Kimura two-parameter model [45]. Selected members of the genus Brochothrix were used as an outgroup. Positions containing gaps
and missing data were eliminated, resulting in a total of 1071 positions. Branch lengths represent the number of nucleotide substitu-
tions per site and bootstrap percentages of 1000 replicates are shown. GenBank/EMBL/DDBJ accession numbers are provided in
parentheses. Bar, 0.01 nucleotide substitutions per site.

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63 L. monocytogenes F2365 (NC_002973)

100 L. monocytogenes FSL J1-208 (NZ_CM001469)
100 L. monocytogenes HCC23 (NC_011660)
100 L. monocytogenes FSL F6-684T (KN050647)
0.020
100 L. monocytogenes EGD-e (NC_003210)
100 Listeria
L. marthii FSL S4-120T (CM001047) sensu
100 stricto
L. innocua CLIP 11262 (NC_003212)
100 L. welshimeri SLCC 5334T (AM263198)
100 L. ivanovii ivanovii PAM55 (NC_016011)
98 L. ivanovii londoniensis WSLC30151 (CP009575)
100
L. seeligeri SLCC 3954T (FN557490)
L. grayi grayi DSM 20601T (NZ_GL538352)
100 L. grayi murrayi FSL F6-1183 (AODG01000001)
100 L. booriae FSL A5-0281T (JNFA01000001)
L. riparia FSL S10-1204 (AODL01000001)
100 L. newyorkensis FSL M6-0635T (JNFB01000001)
100 L. cornellensis FSL F6-0969 (AODE01000001)
100
81 Listeria
L. rocourtiae FSL F6-920 (AODK01000001)
sensu
L. grandensis FSL F6-971 (AODD01000001)
100 lato
L. weihenstephanensis FSL R9-0317 (AODJ01000001)
100 L. fleischmannii LU2006-1T (ALWW01000001)
100 L. fleischmannii coloradonensis TTUM1-001T (AGUG01000001)
L. floridensis FSL S10-1187T (AODF01000001)
100 100 L. aquatica FSL S10-1188T (AOCG01000001)
L. costaricensis CLIP 2016/00682T (FXUT01000000)

Fig. 2. Maximum-likelihood phylogenetic analysis based on the concatenated amino acid sequences of 243 core genes present in all
Listeria species. Distance estimation was obtained by the Whelan and Goldman model [46]. Branch lengths represent the number of
amino acid substitutions per site and bootstrap percentages of 1000 replicates are shown. GenBank/EMBL/DDBJ accession numbers
are provided in parentheses. Bar, 0.02 amino acid substitutions per site.

subspecies (POCP=68 %) was also higher than the genus b-glucosidase activity, and not surrounded by a white halo,
boundary cut-off of 50 %. These results confirmed that denoting the absence of phosphatidylinositol-specific phos-
the new isolate represents a novel Listeria taxon. pholipase C (PI-PLC) activity (Table 1).
In silico PCR serogrouping [35] was positive for the prs gene Strain CLIP 2016/00682T did not show the presence of a
(serogroup L, typical of Listeria species with the exception capsule after performing the India ink test as described by
of L. monocytogenes), although negative by multiplex PCR Hughes and Smith [36]. Gram staining, catalase and oxidase
due to primer mismatches. activities, respiratory characteristics and endospore forma-
tion were tested as described by Guillet and others [18]. The
Isolate CLIP 2016/00682T was also characterized at the pheno-
isolate was a Gram-stain-positive, rod-shaped bacterium
typic and biochemical levels (Table 1; methods adapted from
that was oxidase-negative, non-spore-forming and faculta-
[16]).
tively anaerobic. Strain CLIP 2016/00682T was catalase-neg-
Strain CLIP 2016/00682T was grown on BHI agar plates, ative, in contrast to all other 17 Listeria species. Expression
on agar Listeria according to Ottaviani and Agosti (ALOA; of catalase is actually a major phenotypic characteristic of
bioM!erieux) and on MOX (Oxoid) agar plates after incuba- the genus Listeria, although there are reports of catalase-
tion for 24 h at 30 or 37 " C. Colonies on BHI were opaque, negative L. monocytogenes strains [37, 38]. The lack of cata-
flat and yellow-pigmented, with a diameter of 0.5–1.0 mm. lase activity in strain CLIP 2016/00682T was consistent with
The production of a yellow pigment in strain CLIP 2016/ the absence of a catalase gene in the draft genome.
00682T (Fig. S1, available in the online version of this To determine growth characteristics, isolates were grown on
article) is atypical of Listeria species. On MOX agar, colo- BHI agar and in BHI broth at 4 " C for 10 days and at 22, 30,
nies were dimpled, round and pewter-coloured with black 37 and 42 " C for 7 days. Growth was considered positive if
haloes. On ALOA agar, colonies were blue, due to there was an increase in cell number of at least 1.0 log (c.f.u.
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L. monocytogenes F2365
L. monocytogenes EGD-e
L. monocytogenes FSL F6-684T
L. monocytogenes HCC23
L. monocytogenes FSL J1-208 Listeria
L. marthii FSL S4-120T sensu
stricto
L. innocua CLIP 11262T
L. welshimeri SLCC 5334T
L. ivanovii ivanovii PAM55
L. ivanovii londoniensis WSLC30151
L. seeligeri SLCC 3954T
L. grayi grayi DSM 20601T
L. grayi murrayi FSL F6-1183
L. aquatica FSL S10-1188T
L. costaricensis CLIP 2016/00682T
L. floridensis FSL S10-1187T
L. fleischmannii coloradonensis TTUM1-001T
L. fleischmannii fleischmannii LU2006-1T Listeria
sensu
L. cornellensis FSL F6-0969
lato
L. newyorkensis FSL M6-0635T
L. grandensis FSL F6-971
L. weihenstephanensis FSL R9-0317
L. rocourtiae FSL F6-920
L. booriae FSL A5-0281T
L. riparia FSL S10-1204
100
80

85

90

95

Fig. 3. UPGMA clustering based on the genomic average nucleotide difference (ANI). The vertical dashed bar represents the proposed
95 % ANI species cut-off that correlates with the 70 % DNA–DNA hybridization threshold [33]. The scale bar represents the percentage
similarity.

ml#1) over 14 days. Strain CLIP 2016/00682T showed draft genome of strain CLIP 2016/00682T, suggesting that it
growth between 22 and 42 " C but not at 4 " C. is not pathogenic.
Motility was tested by stab-inoculation in mannitol-mobil- Biochemical tests with API Listeria strips (bioM!erieux) and
ity semi-solid agar (Bio-Rad) and incubation at different the API50CH system (bioMe!rieux) were performed as rec-
growth temperatures (4, 22 and 37 " C) for 10 days under ommended by the manufacturer [41]. API Listeria tests were
aerobic conditions. L. monocytogenes ATCC 35152T and recorded after incubation at 37 " C for 24 h. API50CH tests
L. booriae CIP 111022T were used as positive and negative were recorded after 2, 5, 10 and 15 days of incubation at
controls, respectively. In contrast to other Listeria sensu lato 30 " C. Results are summarized in Table 1. Strain CLIP 2016/
species [4, 16], strain CLIP 2016/00682T was motile (only at 00682T matched the API Listeria numerical profile 2730 of
37 " C), giving a typical umbrella-like growth pattern, due to Listeria ivanovii (identification at 99.7 %; bioM!erieux API
the presence of the flagella biosynthesis regulon typical of web database, version 04/2017) with divergent tests for D-ary-
Listeria sensu stricto. Nitrate reduction and Voges–Proska- lamidase and glucose-1-phosphate.
uer tests were performed as described by Guillet and others
[18]. Nitrate reductase tests were positive (Table 1), whereas Interestingly, according to its phenotypic profile (Table 1),
nitrite reduction was negative. The Voges–Proskauer reac- strain CLIP 2016/00682T clustered together with Listeria
tion was positive (Table 1). grayi and the subgroup of Listeria sensu stricto (Fig. 4).
No haemolysis was detected using either Columbia agar Susceptibility to a wide range of antibacterial agents was
plates containing 5 % defibrinated horse blood (bioM!erieux) determined with the disc diffusion method on Mueller–
or the Christie, Atkins, Munch-Petersen (CAMP) test on Hinton agar plates (Bio-Rad), using the interpretative criteria
Columbia agar containing 5 % defibrinated sheep blood and recommendations from the French Microbiology Society
(bioM!erieux) as described by Guillet and others [18]. The and the European Committee on Antibiotic Susceptibility
lack of haemolysis was consistent with the absence of Liste- Testing [42, 43]. Strain CLIP 2016/00682T was sensitive to
ria pathogenicity islands (LIPI-1 to -4) [39, 40] within the penicillin G, ampicillin, amoxicillin, imipenem, kanamycin,
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Table 1. Biochemical characteristics of species of the genus Listeria based on observations made in this study and on previously published studies [16]
+, Positive; (+), weakly positive; #, negative; V, variable (between replicates and/or between strains); v!, variable between studies (possibly due to differ-
ences in incubation times and temperatures between studies); ND, not determined or not recorded. Taxa: Lco, L. costaricensis CLIP 2016/00682T (this
study); Lmo, L. monocytogenes 10403S (data from [5] and [14]); Lin, L. innocua FSL S4-378 (data from [5] and [14]); Lse, L. seeligeri (data from [5] and
[14]); Liv, L. ivanovii ATCC BAA-678 (data from [5, 14]); Lws, L. welshimeri (data from [5, 14, 41]); Lma, L. marthii FSL S4-120T (data from [4]); Lgy, L. grayi
ATCC 19120T and ATCC 25401T (data from [4]); Lro, L. rocourtiae CIP 109804T (data from [4]); Lwp, L. weihenstephanensis DSM 24698T (data from [4]);
Lcn, L. cornellensis TTU A1-0210T and FSL F6-0970 (data from [4]); Lri, L. riparia FSL S10-1204T and FSL S10-1219 (data from [4]); Lgd, L. grandensis
TTU A1-0212T (data from [4]); Lfc, L. fleischmannii strains DSM 24998T, ATCC BAA-2414T, FSL F6-1019, FSL S10-1186, FSL S10-1203 and FSL S10-
1220 (data from [4]); Laq, L. aquatica strains FSL S10-1188 T and FSL S10-1181 (data from [4]); Lfo, L. floridensis strain FSL S10-1187 T (data from [4]);
Lny, L. newyorkensis strains FSL M6-0635T and A5-0209 (data from [16]); Lbo, L. booriae strains FSL A5-0279T and FSL A5-0281 (data from [16]). All
species/strains are positive for aesculin and acid production from N-acetylglucosamine, amygdalin, arbutin, cellobiose, D-fructose, D-mannose and sali-
cin. All species/strains are negative for nitrite reduction and acid production from D-adonitol, D-arabinose, glycogen, methyl b-D-xylopyranoside, potas-
sium 2-ketogluconate and raffinose.

Characteristic Lco Lmo Lin Lse Liv Lws Lma Lgy Lro Lwp Lcn Lri Lgd Lfc Laq Lfo Lny Lbo

Motility + + + + + + + + – – – – – – – – – –
Nitrate reduction + – – – – – – V + + + + + + + – + +
Voges–Proskauer reaction + + + + + + + + – – – – – – V – – –
Catalase # + + + + + + + + + + + + + + + + +
Haemolysis # + – + + – – – – – – – – – – – – –
D-Arylamidase # – + + V V – + – – – – – – – – – –
a-Mannosidase # + + – – + + V + – – + – – + – – +
Phosphatidylinositol-specific # + – – + – – – – – – – – – – – – –
phospholipase C (PI-PLC)
Acid production from:
D-Arabitol + + + + + + + + – + – – V + – – – +
D-Galactose + V – – V – – + + – – + – – – + + +
D-Glucose + v! v! + v! + v! + + + + + + + + + + +
Glycerol + V + + + + – V + + V V – + V – + +
L-Fucose + # # # # # # # # # # # # # # # # #
Lactose + + + + + + + + + v! (+) + – + – + + +
Maltose + + + + + + + + + + + + + + – + + +
L-Rhamnose + + V – – V – – + + – + – + + + V +
D-Ribose + – – – + – – + + – + V + + + – + V

Sucrose + + + + + + – – – – – – – V – – – –
Methyl a-D-glucoside + + + + + + + + + + + + + + – + + +
Methyl a-D-mannose + – – ND – ND – + – – – – – V – – – –
Potassium 5-ketogluconate + – – – – – – – – – – – – – – – – –
D-Xylose + – – + + + – – + + + + + + + + + +
L-Arabinose # – – – – – – – – – V + – – + + + +
Glucose-1-phosphate # – – – V – – – – – – – – – – – – –
Inositol # – – – – – – – – – – V – V V – – –
Inulin # v! v! – – – – – – – – – – – – – – –
D-Lyxose # V V – – V – V – – – – – – V + – –
D-Mannitol # – – – – – – + + + – V – V – – + +
Melezitose # V V V V V – – – – – – – V – – – –
Melibiose # v! V – – – V – + – – V – V – – – +
L-Sorbose # v! v! – v! – v! v! – – – – – V – – – –
D-Tagatose # – – – – + – – – – – – – – + – – –
Turanose # – V – – – + – – – – – – V – – – –

streptomycin, gentamicin, fosfomycin, rifampicin, erythro- Thus, on the basis of the molecular findings described as
mycin, levofloxacin, moxifloxacin, clindamycin, tetracycline, well as the phenotypic distinctiveness of strain CLIP
chloramphenicol, sulphonamides, trimethoprim and vanco- 2016/00682T, we propose that this strain should be classi-
mycin. However, it showed resistance to nalidixic acid and fied as a member of a novel species of the genus Listeria
cefotaxime, and intermediate resistance to ciprofloxacin and for which the name Listeria costaricensis sp. nov. is
fusidic acid, as for other Listeria species [44]. proposed.

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L. monocytogenes genus Listeria by the absence of catalase reaction, production

L. innocua of acid from potassium 5-ketogluconate and production of a
L. welshimeri Listeria yellow pigment on BHI.
sensu
L. marthii stricto The type strain, CLIP 2016/00682T(=CIP 111400T=DSM
L. ivanovii 105474T) was isolated in August 2015 from the drainage
L. seeligeri system of a food-processing plant in the province of Ala-
L. grayi juela, northern Costa Rica. The genomic DNA G+C content
L. costaricensis of the type strain is 43.7 mol%, as determined by genome
L. fleischmannii
sequencing.
L. weihenstephanensis
L. cornellensis Funding information
Listeria This work was supported by Institut Pasteur, INSERM and the Vice-
L. grandensis
sensu rrectoría de Investigación y Extensión-Instituto Tecnológico de Costa
L. booriae lato Rica (Research Project VIE-1510068).
L. riparia
Acknowledgements
L. rocourtiae We thank the P2M platform (Institut Pasteur, Paris, France) for
L. newyorkensis genome sequencing and the Leibniz Institute DSMZ – German Collec-
tion for Microorganisms and Cell Cultures GmbH (Braunschweig, Ger-
L. floridensis many) and the Collection of Institut Pasteur (Paris, France) for the
L. aquatica deposit of the type strain in their collections.
70

80

90

100

Conflicts of interest
The authors declare that there are no conflicts of interest.

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