INTERNATIONAL
JOURNAL OF SYSTEMATIC
BACTERIOLOGY,
Jan. 1992, p. 69-73
0020-7713/92/010069-05$02.00/0
Copyright 0 1992, International Union of Microbiological Societies
Vol. 42, No. I
Listeria ivanovii subsp. londoniensis subsp nova
PATRICK BOERLIN,l JOCELYNE ROCOURT,* FRANCINE GRIMONT,3 PATRICK A. D. GRIMONT,3
CHRISTINE JACQUET,* A N D JEAN-CLAUDE PIFFARETTIl"
Istituto Cantonale Batteriologico, Via Ospedale 6 , 6904 Lugano, Switzerland, and Unit&d'Ecologie Bacte'rienne, Centre
National de Re'firence pour la Lysotypie et le Typage Mole'culaire de Listeria and WHO Collaborating Center for
Foodborne Listeriosis2 and Unite' des Ente'robacte'ries, Institut National de la Sante' et de la Recherche Me'dicale,
Unit&INSERM 199,3 Institut Pasteur, 75724 Paris Cedex 15, France
An analysis of 23 Listeria ivunovii strains in which we used multilocus enzyme electrophoresis at 18 enzyme
loci showed that this bacterial species could be divided into two main genomic groups. The results of DNA-DNA
hybridizations and rRNA gene restriction patterns confirmed this finding. The DNA homology data suggested
that the two genomic groups represent two subspecies, L. ivunovii subsp. ivanovii and L . ivanovii subsp.
londoniensis subsp. nov. The two subspecies can be distinguished biochemically on the basis of the ability to
ferment ribose and N-acetyl-P-D-mannosamine.The type strain of L. ivanovii subsp. londoniensis is strain CLIP
12229 (=CIP 103466).
Of the seven recognized Listeria species, only Listeria
monocytogenes and Listeria ivanovii are pathogenic (18).
Both of these organisms have been isolated from patients
with clinical symptoms, healthy carriers, and the environment, but L . ivanovii (formerly called L . monocytogenes
serovar 5 ) has been isolated less frequently than L. monocytogenes (22, 26, 29). L . ivanovii causes mainly abortion in
sheep (4, 9, 10, 11, 15); more rarely, it causes diseases in
bovines or in humans (21, 26). All L . ivanovii strains belong
to serovar 5, and reciprocally, all serovar 5 strains are
members of L . ivanovii. These organisms are members of the
only species in the genus Listeria which gives a positive
CAMP reaction with Rhodococcus equi and a negative
CAMP reaction with Staphylococcus aureus (24). They
produce a particularly wide zone of hemolysis on sheep
blood agar and produce acid from xylose but not from
D-mannitol, L-rhamnose, and a-methyl-D-mannoside (25).
Because of these characteristics, L . ivanovii strains can be
easily distinguished from strains of the other Listeria species.
From a taxonomic point of view, Ivanov suggested that L .
monocytogenes serovar 5 should be separated as a distinct
species from L . rnonocytogenes (11).In 1982, Seeliger et al.
also recommended that serovar 5 should be considered a
taxon that is distinct from L. monocytogenes (26). On the
basis of its phenotypic characteristics and the results of a
DNA homology study (20), the species L . ivanovii was
officially recognized in 1984 (25).
In the last few years, multilocus enzyme electrophoresis
(MEE) has been used successfully with bacteria to analyze
various epidemiologicand taxonomic problems (17,27). This
method allows not only differentiation of strains, but also
estimation of the genomic relatedness of strains, and their
affiliation with species or subspecies. We recently found that
a strain identified as L. ivanovii by using conventional
biochemical markers clearly belonged to a genomic group
that has not been described previously when it was examined by MEE. In this study, we analyzed more L . ivanovii
strains by using MEE and identified other members of this
group, which we characterized further by using their rRNA
gene restriction patterns and the results of DNA-DNA
hybridization experiments.
MATERIALS AND METHODS
In this study we used 3 L . monocytogenes strains, 2
Listeria innocua strains, 2 Listeria seeligeri strains, 2 Listeria welshimeri strains, 2 Listeria grayi strains, 2 Listeria
murrayi strains, and 23 L . ivanovii strains (Table 1). All of
these strains were registered in the Listeria Collection of the
Pasteur Institute (CLIP), Paris, France, or in the Special
Listeria Culture Collection (SLCC), Wiirzburg, Germany.
The methods which we used for species identification have
been described elsewhere (21). When needed, serotyping of
the strains was kindly performed by workers at the Swiss
National Listeria Reference Center, Lausanne, Switzerland,
who used the reference method (23).
MEE. Lysate preparation, electrophoresis, and enzyme
selective staining were done as described by Selander et al.
(27). Electrophoresis preparations for aconitase, alanine
dehydrogenase, glutamic-oxalacetic transaminase, nucleoside phosphorylase, L-phenylalanyl-L-leucine peptidase, and
6-phosphogluconate dehydrogenase were run in buffer system A (Tris citrate, pH 8.0). Electrophoresis preparations
for NADP-dependent glutamate dehydrogenase, glucose-6phosphate dehydrogenase, lactate dehydrogenase, phosphoglucose isomerase, and mannose phosphate isomerase were
run in buffer system B (Tris citrate, pH 6.7). Electrophoresis
preparations for acid phosphatase, adenylate kinase, catalase, fumarase, NAD-dependent glyceraldehyde-3-phosphate dehydrogenase, indophenol oxidase, and phosphoglucomutase were run in buffer system F (Tris maleate, pH 8.2).
Specific staining for catalase was performed as described by
Harris and Hopkinson (8). The statistical analysis of the data
was done with a computer program designed by T. S.
Whittam and R. K. Selander as described elsewhere (27).
DNA-DNA hybridization. DNA-DNA hybridization was
performed at 60°C by using the S1 nuclease-trichloroacetic
acid method described by Grimont et al. (7) and Rocourt et
al. (21). Bacteria were lysed as described below. Cells were
grown for 48 h at 37°C in six Roux flasks containing 150 ml
of Columbia agar, harvested, and washed in 20 ml of 0.1X
SSC (Ix
SSC is 0.15 M NaCl plus 0.015 M sodium citrate).
They were then incubated for 1 h at 37°C in 6 ml of a
lysozyme solution (10 mM sodium phosphate-20% sucrose
[pH 7.01 containing 0.2% lysozyme [Appligene, Illkirch,
France]), lysed by adding 48 ml of a proteinase K solution
(10 mM Tris-HC1 [pH 8.01, 1 mM EDTA, 1.25% sodium
* Corresponding author.
69
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INT.J. SYST.BACTERIOL.
BOERLIN ET AL.
TABLE 1. Analysis of L. ivanovii strains: with MEE, DNA-DNA hybridization, and rRNA gene restriction patterns
SDecies
SerovaP
ET~
Strain
96 DNA
with labeled DNA
from' :
Strain
SLCC
3769
L. ivanovii
L . ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L . ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L. ivanovii
L . ivanovii
L. ivanovii
L . ivanovii
L . ivanovii
L. ivanovii
L . ivanovii
L. ivanovii
L. ivanovii
L . ivanovii
L . monocytogenes
L. monocytogenes
L. monocytogenes
L. innocua
L. innocua
L. seeligeri
L. seeligeri
L. welshimeri
L . welshimeri
L. grayi
L. grayi
L. murrayi
L. murrayi
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
1/2
1l2a
4b
6a
6b
1/2b
us
6b
6a
NA
NA
NA
NA
CLIP 8457
CLIP 12547
CLIP 8459
CLIP 9441
CLIP 2300
SLCC 2098
CLIP 12590
SLCC 3769
CLIP 8181
SLCC 4723
SLCC 4728
SLCC 4729
CLIP 257
CLIP 12510T
SLCC 3887
SLCC 4054
SLCC 4306
SLCC 3765
CLIP 2737
CLIP 12065
CLIP 1347
CLIP 12229
CLIP 6645
CLIP 14531T
CLIP 12498
CLIP 12505
CLIP 12511T
CLIP 12512
CLIP 12513T
SLCC 3990
CLIP 12514T
SLCC 5332
CLIP 125MT
CLIP 640
CLIP 125MT
CLIP 12516
1 (I)
1 (I)
1 (I)
1 (I)
2 (I)
3 (1) 100 (0.3)
4 (I)
4 (I) 100 (0.0)
5 (I)
6 (I)
6 (I)
7 (I) 103 (1.1)
8 (I)
9 (I) 98 (0.6)
10 (I) 106 (1.5)
11 (I) 99 (0.6)
12 (I)
13 (11) 60 (4.6)
14 (11) 58 (6.4)
15 (11) 58 (5.3)
16 (11) 64 (4.9)
17 (11) 62 (3.1)
18 (11) 64 (4.7)
16
Restriction
patterns
Acid production
from:
Origin'
S t r ~ ~ ~EcoRI
~ l HindIII
P
Ribose
EIVl
61 (3.6)
HIVl
EIVl HIVl
EIV2 HIVl
70 (5.0)
EIVl
EIVl
EIVl
HIVl
HIVl
HIV1
65 (4.8)
EIVl
HIV2
66 (5.6)
68 (5.9)
81 (6.2)
EIVl HIVl
EIV 1 HIVl
EIVl HIVl
107 (0.0)
91 (0.6)
94 (0.2)
106 (0.0)
100 (0.0)
107 (0.6)
E N 3 HIVl
HIVl
EIV3 HIVl
EIV3 HIVl
+
+
t
+
+
+
+
+
+
+
+
+
+
+
+
+
+_
EIV4 HIV3
-
-
16
16
16
18
42 (7.3)
37
17
19
4
3
8
15
N-acety l-P-D-
mannosamined
France, sheep (abortion)
France, sheep (abortion)
France, sheep (abortion)
Germany
Belgium, human
Australia, sheep (liver)
France, cheese
Germany, environment
Italy
Germany, bovine (nose)
Germany, bovine (nose)
Germany
France, human (feces)
SLCC 2739T, ATCC 19119T
Germany, environment
Germany, cow (feces)
Bulgaria
Germany, corn leaves
Czechoslovakia
Belgium, goat
France, dormouse
France, food
Switzerland
ATCC 15313T
ATCC 35152
ATCC 19115
ATCC 33O9OT
ATCC 33091
ATCC 35967T
ATCC 35897T
ATCC 19120T
ATCC 25401T
ATCC 25402
US, undesignated serovar; NA, not applicable.
The ETs are listed in the same order as in Fig. 1; cluster designations are indicated in parentheses.
' Percentage of relative binding at 60°C. The values in parentheses are AT,,, values (in degrees Celsius).
After 18 to 24 h.
' ATCC, American Type Culture Collection, Rockville, Md.
dodecyl sulfate, 20 mg of proteinase K [Appligene]), and
incubated overnight at 37°C. The DNA was finally purified
by sequential phenol-chloroform extractions. DNAs from
strains SLCC 3769 and CLIP 12229T (T = type strain) were
radioactively labeled by nick translation.
rRNA gene restriction patterns. rRNA gene restriction
patterns were determined as described by Jacquet et al. (12)
by using cloned rDNA (genes coding for rRNA) from Bacillus subtilis as the probe (3). Restriction enzymes EcoRI and
HindIII were used to digest L . ivanovii DNA.
For biochemical tests, we used API 50CH galleries (API
System, Montalieu-Vercieu, France). Additional tests for
fermentation of trehalose, ribose, and N-acetyl-P-D-mannosamine were performed by using a 1% solution of substrate in broth containing (per liter) 10 g of bacteriological
peptone (Oxoid), 5 g of Lab-Lemco (Oxoid), 3 g of NaC1,
and 10 ml of a 0.2% bromthymol blue or 0.2% phenol red
solution. The pH was adjusted at 7.0 with NaOH, and the
solution was filtered to sterility. The methyl red test was
performed in MR-VP medium (Difco).
RESULTS
The 23 L . ivanovii strains which we studied by using MEE
were assigned to 18 electrophoretic types (ETs) (Table 1and
Fig. 1). A cluster analysis of these ETs showed that they
were clearly divided into two main genomic groups (clusters
I and 11) (Fig. 1).Clusters I and I1 were separated at a genetic
distance of 0.92, whereas the greatest genetic distances
within clusters I and I1 were 0.34 and 0.17, respectively.
Eight alleles were monomorphic and specific for cluster I
(acid phosphatase, adenylate kinase, alanine dehydrogenase,
glutamic-oxalacetic transaminase , glucose-6-phosphate dehydrogenase, mannose phosphate isomerase, nucleoside phosphorylase, phosphoglucose isomerase) and 12 alleles were
monomorphic and specific for cluster I1 (acid phosphatase,
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VOL. 42, 1992
LISTERIA IVANOVll SUBSP. LONDONIENSIS SUBSP. NOV.
71
ET
1
II
-
11
12
_c: 13
14
r- 15
16
I
I?
I
I
a
1.0
a9
0.8
1
a7
0.6
0.5
c2.4
I
I
I
0.3
0.4
0.2
0.1
I
o
GENETIC DISTANCE
FIG. 1. Genetic relationships among 18 ETs of L . ivanovii. The
dendrogram was generated by using the average-linkage method of
clustering from a matrix of pairwise coefficients of genetic distances,
based on electrophoretically demonstrable allelic variations at 18
enzyme loci. ET 3,4, 7,9,10, and 11(cluster I) strains belong to one
hybridization group, and ET 13 to 18 (cluster 11) strains belong to
another hybridization group (see Table 1).
adenylate kinase, alanine dehydrogenase, catalase, NADPdependent glutamate dehydrogenase, glutamic-oxalacetic
transaminase, NAD-dependent glyceraldehyde-3-phosphate
dehydrogenase, glucose-6-phosphatedehydrogenase, nucleoside phosphorylase, 6-phosphogluconate dehydrogenase,
phosphoglucose isomerase, phosphoglucomutase).
DNA-DNA hybridization of the DNAs from 12 L . ivanovii
strains with labeled DNAs from strains SLCC 3769 and
CLIP 12229T (representatives of clusters I and 11, respectively) confirmed the division of the species into two genomic groups (Table l). Each DNA hybridization group
corresponded to one of the clusters found by using MEE.
The levels of DNA homology between L . ivanovii SLCC
3769 and other strains belonging to cluster I ranged from 98
to 106% (mean, 101%; difference in melting temperature
[AT,] range, 0 to 1.5"C), and the levels of DNA homology
between strain SLCC 3769 and strains belonging to cluster TI
ranged from 58 to 64% (mean, 61%; AT, range, 3.1 to 6.4"C)
(Table 1). Conversely, strain CLIP 12229T exhibited 61 to
81% DNA homology with strains belonging to cluster I
(mean, 68%; AT, range, 3.6 to 6.2"C) and 91 to 107%
homology with strains belonging to cluster I1 (mean, 101%;
ATm range, 0.0 to 0.6"C). Strain CLIP 12229T exhibited 3 to
42% DNA homology with members of other Listeria species
(Table 1). The AT, for strain CLIP 12229T and the L .
seeligeri type strain (level of homology, 42%) was 7.3"C.
When the L . ivanovii DNAs were digested with EcoRI,
two restriction patterns for rRNA genes (patterns EIVl and
EIV2) were associated with cluster I and two other patterns
(patterns EIV3 and EIV4) were associated with cluster I1
(Table 1 and Fig. 2 and 3). There was no overlapping of
patterns between the two clusters when preparations were
assayed with this restriction enzyme. When Hind111 was
used, we observed three digestion patterns for the rRNA
genes (Table 1 and Fig. 3 and 4). One of these patterns
(pattern HIV1) was present in both clusters. None of these
patterns was found with other Species of the genus Listeria
(12).
tl.6
FIG. 2. Autoradiogram of Southern blot of EcoRI-digested
whole-cell DNAs obtained from L . ivanovii strains belonging to
genomic groups I and I1 after hybridization with a 16s rDNA probe.
Lane 1, pattern EIVl (strain CLIP 8457); lane 2, pattern EIV2
(strain SLCC 2098); lane 3, pattern EIV3 (strain CLIP 1347); lane 4,
pattern EIV4 (strain CLIP 6645).
When strains SLCC 3769 and CLIP 12229Twere tested as
representatives of clusters I and 11, respectively, by using
API 50CH galleries, these strains showed differences only in
the fermentation of trehalose and ribose. When all of the L .
ivanovii strains were tested for acid production from
trehalose, no correlation was observed between the results
of the test and the genomic groups (data not shown). After 24
h, all of the cluster I strains except strain SLCC 4306 were
positive for fermentation of ribose (Table 1); strain SLCC
4306 produced only a faint acidification of the medium after
48 to 72 h. All of the cluster I1 strains remained clearly
negative for this reaction. Conversely, all of the cluster I1
strains were positive for acid production from N-acetyl-P-Dmannosamine, and all of the cluster I strains were negative
for this test after 18 to 24 h (but not after 48 h). The
differences observed in the methyl red test results did not
correlate with the genetic groups (data not shown).
DISCUSSION
Listeria species are phenotypically similar ( 5 , 13), and
only a limited number of biochemical tests allow workers to
differentiate them (21). Most of these organisms can be
separated more distinctly by genetic methods (1, 20); the
exceptions are L . grayi and L . murrayi, which probably form
a unique species (19). Until now, no phenotypic subgroups
have been found within L . ivanovii ( 5 ) . Differences between
strains belonging to this taxon have been observed for the
following characteristics: acid production from galactose,
melezitose, sucrose, and trehalose (2,24, 26) and the methyl
red test (2). In a previous DNA-DNA hybridization study
(20), nine L . ivanovii strains were shown to be genetically
very similar (99 +- 5% DNA homology; AT,, less than
1.2"C).
With MEE, allelic variations at several enzyme loci are
revealed by differences in the electrophoretic mobilities of
the gene products and are used to distinguish genotypes (or
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72
INT. J . SYST.BACTERIOL.
BOERLIN ET AL.
EM
EIV2
ElV4
IIII
i II
I lil
lil
HIVi
(b]
I1
Ill
II
I I
I
I
I
I
I
i
I Ill
Ill
I1111
HIV2
HIV3
FIG. 3. Schematic representation of EcoRI (a) and HindIII (b) rRNA gene restriction patterns for L . ivunovii strains belonging to genomic
groups 1 and 11. Dashed lines indicate faint bands.
ETs) and to estimate the levels of relatedness of strains (27).
Using this method, we divided the 23 L . ivanovii strains
which we analyzed into two major clusters (clusters I and
11). The same division was revealed by the results of the
DNA-DNA hybridization experiments, which also demonstrated that L . ivanovii cluster I1 strains do not belong to one
of the six other Listeria species (Table 1).Strains that exhibit
70% or more DNA homology and AT,,, values of less than
5°C are considered to belong to the same bacterial species
(28). Strains that exhibit less than 50% DNA homology and
AT,,, values of more than 7°C are generally considered
members of different species. However, pairing strains be-
FIG. 4. Autoradiogram of Southern blot of HindIII-digested
whole-cell DNAs obtained from L . ivanovii strains belonging to
genomic groups I and I1 after hybridization with a 16s rDNA probe.
Lane 1, pattern HIVl (strain CLIP 1347); lane 2, pattern HIV2
(strain SLCC 4729); lane 3, pattern HIV3 (strain CLIP 6645).
longing to one cluster with strains belonging to the other
cluster led to intermediate DNA homology values and AT,,,
values (Table 1).Thus, genomic groups I and I1 (corresponding to clusters I and I1 [Fig. 11) should be considered to be
two distinct subspecies of L . ivanovii. This result is not in
agreement with the genetic distance of 0.92 obtained by
MEE, which is significantlygreater than the distance of 0.7
that is empirically considered to be sufficient to distinguish
two species. DNA-DNA hybridization is based on the whole
bacterial genome, whereas only a portion of the genome is
considered when MEE is used. This may occasionally lead
to bias in estimates of genetic relatedness and could explain
the overestimated genetic distance between clusters I and I1
obtained by MEE.
Analysis of rRNA gene restriction patterns has been
proposed as a potential tool for bacterial taxonomy (6, 16).
When we digested L . ivanovii DNAs with EcoRI, we observed no overlap between the patterns obtained for the two
strain clusters; therefore, these results support the division
of this species into two genomic groups. However, the
relatively close genetic relatedness determined by DNADNA hybridization could explain the fact that the two
genomic groups shared a common rRNA gene restriction
pattern (pattern HIV1) when the DNAs were digested with
HindIII (Table 1).
The biochemical assays showed that ribose fermentation
is not a constant characteristic of L . ivanovii strains and,
consequently, cannot be used as a differential test for the
identification of this species as previously proposed (14).
However, the ability to ferment ribose and the ability to
produce acid from N-acetyl-P-D-mannosamine within 24 h
correlate with the placement of the strains in clusters I and I1
(Table 1). Therefore, these biochemical reactions can be
used to identify the subspecies of L . ivanovii. Only one
isolate (strain SLCC 4306) gave an unclear reaction for
ribose fermentation. This exception among the 17 strains
belonging to cluster I was probably due to a mutation that led
to poor expression of the phenotype.
In conclusion, our results clearly demonstrate that L .
ivanovii contains two genomic groups of strains at the
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LISTERIA ZVANOVZZ SUBSP. LONDUNZENSZS SUBSP. NOV.
VOL. 42, 1992
subspecies level, which can be distinguished by the ability to
degrade ribose and N-acetyl-P-D-mannosamine. Therefore,
we propose that genomic group I should be named Listeria
ivanovii subsp. ivanovii and genomic group I1 should be
named Listeria ivanovii subsp. londoniensis subsp. nov.
Description of Listeria ivanovii subsp. londoniensis subsp.
nov. Listeria ivanovii subsp. londoniensis (1on.don.i.en’sis.
N.L. fern. adj. londoniensis, from London, Ontario, Canada,
where E. G. D. Murray and R. G. E. Murray worked on
Listeria). Most of the characteristics are similar to those of
L . ivanovii (25), except that L . ivanovii subsp. londoniensis
does not produce acid from ribose but produces acid from
N-acetyl-P-D-mannosamine after 18 to 24 h of incubation at
37°C. The type strain is strain CIP 103466 (=CLIP 12229).
ACKNOWLEDGMENTS
We thank N. El Sohl for supplying the rDNA probe and E.
Ageron and B. Catimel for technical help in the DNA-DNA hybridization experiments and DNA extraction, respectively.
This research was supported by a grant from the Swiss Federal
Veterinary Office and by grant 31-9396.88 from the Swiss National
Science Foundation.
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