APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 2006, p. 2439–2448
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 72, No. 4
Discovery of Natural Atypical Nonhemolytic Listeria seeligeri Isolates†
Dmitriy Volokhov,1* Joseph George,1Christine Anderson,1Robert E. Duvall,2and Anthony D. Hitchins2
Center for Biologics Evaluation and Research, Food and Drug Administration, Kensington, Maryland 20895,1and Center for
Food Safety and Applied Nutrition, Food and Drug Administration, College Park, Maryland 20740-38352
Received 4 October 2005/Accepted 20 January 2006
We found seven Listeria isolates, initially identified as isolates with the Xyl?Rha?biotype of Listeria
welshimeri by phenotypic tests, which exhibited discrepant genotypic properties in a well-validated Listeria
species identification oligonucleotide microarray. The microarray gives results of these seven isolates being
atypical hly-negative L. seeligeri isolates, not L. welshimeri isolates. The aberrant L. seeligeri isolates were
D-xylose fermentation positive, L-rhamnose fermentation negative (Xyl?Rha?), and nonhemolytic on blood
agar and in the CAMP test with both Staphylococcus aureus (S?reaction) and Rhodococcus equi (R?reaction).
All genes of the prfA cluster of L. seeligeri, located in the prs-ldh region, including the orfA2, orfD, prfA, orfE,
plcA, hly, orfK, mpl, actA, dplcB, plcB, orfH, orfX, orfI, orfP, orfB, and orfA genes, were checked by PCR and direct
sequencing for evidence of their presence in the atypical isolates. The prs-prfA cluster–ldh region of the L.
seeligeri isolates was approximately threefold shorter due to the loss of orfD, prfA, orfE, plcA, hly, orfK, mpl, actA,
dplcB, plcB, orfH, orfX, and orfI. The genetic map order of the cluster genes of all the atypical L. seeligeri isolates
was prs-orfA2-orfP-orfB-orfA-ldh, which was comparable to the similar region in L. welshimeri, with the excep-
tion of the presence of orfA2. DNA sequencing and phylogenetic analysis of 17 housekeeping genes indicated
an L. seeligeri genomic background in all seven of the atypical hly-negative L. seeligeri isolates. Thus, the novel
biotype of Xyl?Rha?Hly?L. seeligeri strains can only be distinguished from Xyl?Rha?L. welshimeri strains
genotypically, not phenotypically. In contrast, the Rha?Xyl?biotype of L. welshimeri would not present an
The bacterial genus Listeria is currently taxonomically sub-
divided into the following six species: Listeria monocytogenes,
L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, and L. grayi
(33). All Listeria species are ubiquitously distributed in the
natural environment and frequently isolated from different
biocenoses. Despite the ability of unambiguously distinguish-
ing the pathogenic Listeria species from the other unharmful
saprophytic Listeria spp., the identification of all Listeria iso-
lates to the species level is an important taxonomic issue.
Listeria spp. identified based on selective cultural enrichments
and isolation on selective agar growth media and subsequent
isolates can be easily distinguished from each other to the
species level by using the following markers: hemolysis (in
Christie-Atkins-Munch-Peterson [CAMP] test with Staphylo-
coccus aureus and Rhodococcus equi) and acid production from
D-xylose, L-rhamnose, mannitol, and alpha-methyl-D-manno-
side. As a result, extra phenotypic or genotypic methods are
rarely required (3, 14). The hemolysis and CAMP tests are
crucial steps for identification of the hemolytic L. monocyto-
genes, L. ivanovii, and L. seeligeri species as well as for their
differentiation from the nonhemolytic species, L. innocua, L.
welshimeri, and L. grayi. The gene hly that encodes hemolysin
is present within the prfA virulence gene cluster that is found
between prs and ldh in L. monocytogenes, L. ivanovii, and L.
seeligeri but is absent from the genomes of the nonhemolytic L.
innocua, L. welshimeri, and L. grayi species (7, 8, 33).
The occurrence of atypical or aberrant isolates in the indis-
pensable diagnostic tests (hemolysis and CAMP) can lead to
species misidentifications and taxonomic problems (15, 22).
The natural and artificially modified Listeria isolates that ex-
press weak hemolytic or nonhemolytic phenotypes may be at-
tributable to a constitutive or inducible type of hemolysin ex-
pression, inappropriate culturing conditions, or an alteration
or deletion of the hly or prfA gene inside the cluster, all of
which have been found in L. monocytogenes (19, 20, 29), but no
analogous information is available for other hemolytic species
such as L. ivanovii and L. seeligeri. Isolates with the natural
weak hemolytic or nonhemolytic phenotype of L. monocyto-
genes may be misclassified as L. innocua strains, but the avail-
ability of genomic information for these two species allows
accurate identification between them by using multiple-locus
sequence typing (16).
Various DNA typing methods have been used to distinguish
Listeria isolates at the species or subspecies level (23, 25, 28, 31,
37). Predominantly, DNA typing methods provide better spe-
cies and even strain definition than conventional phenotypic
tests and serotyping, and as a result, comprehensive character-
ization of prokaryotic species by analysis of diverse chromo-
somal loci is recommended and can provide bacteriologists
with uniform information for species definition as well as phy-
logenetic and ecological studies (34). This study uses different
genetic loci as phylogenetic and identification markers for the
characterization of nonhemolytic L. seeligeri isolates and to
distinguish them from L. welshimeri isolates.
In the present study, we describe seven unusual L. seeligeri
isolates which were initially identified as being the Xyl?Rha?
* Corresponding author. Mailing address: U.S. Food and Drug
Administration, Center for Biologics Evaluation and Research, Of-
fice of Vaccine Research and Review, Division of Viral Products,
Laboratory of Methods Development, HFM-470, 1401 Rockville Pike,
Rockville, MD 20852. Phone: (301) 827-8757. Fax: (301) 827-9531.
† Supplemental material for this article may be found at http://aem
biotype of L. welshimeri by bacteriological phenotypic tests.
Detailed analysis of the aberrant L. seeligeri isolates has shown
a unique organization of the prs-ldh cluster which is different
from that described for the species. Multilocus sequence typing
and phylogenetic analysis of various housekeeping genes
showed an L. seeligeri-specific genome background for all of
the isolates. Thus, we show here that these L. seeligeri isolates
represent a novel biotype within the taxon. This knowledge
provides critical information to allow the definition of L. seelig-
eri strains that may have evolved into a nonhemolytic biotype
and those that may be equally present in the environment
together with the typical hemolytic biotype of L. seeligeri but
misclassified as Xyl?Rha?L. welshimeri.
MATERIALS AND METHODS
Bacterial strains. The Listeria strains used in this study were obtained from the
Center for Food Safety and Applied Nutrition, U.S. Food and Drug Adminis-
tration, College Park, MD. Several L. welshimeri strains were provided by the
Institut Pasteur, Paris, France. Working cultures of the strains were maintained
on Trypticase soy agar supplemented with yeast extract at 5°C. Bacteria were
grown overnight on brain heart infusion plates (Difco, Detroit, Mich.) at 37°C.
Conventional Listeria identification tests. Identification of strains was done
according to the procedures in the FDA Bacteriological Analytical Manual (14).
All of these procedures were done at the Listeria Methods Research Laboratory,
Center for Food Safety and Applied Nutrition, FDA.
Total DNA preparation. Freshly grown bacteria were boiled in 1? Tris-EDTA
buffer (approximately 108cells/ml) for 10 min, followed by centrifugation at
14,000 ? g for 10 min to remove denatured proteins and bacterial membranes.
The presence of genomic DNA in all prepared samples was confirmed by 1%
agarose gel electrophoresis followed by staining with ethidium bromide.
PCR primers and microarray analysis. The primers used for PCR amplifica-
tion are listed in Table S1 in the supplemental material. Microarray analysis was
performed as described previously (36).
PCR amplification. The standard PCR mixture (50 ?l) contained 1.5 U of
HotStarTaq DNA polymerase, 1? reaction buffer supplemented with 2.5 mM
MgCl2(QIAGEN, Chatsworth, Calif.), 600 nM (each) forward and reverse
primers, a 200 ?M concentration of each deoxynucleoside triphosphate (dATP,
dGTP, dCTP, and dTTP), and 1 to 2 ?l of DNA template (approximately 0.2 ?g
of bacterial DNA). PCR was performed with a Gene Amp PCR system 9700
thermocycler (Applied Biosystems, Foster City, Calif.) under the following con-
ditions: initial activation at 95°C for 15 min, 40 cycles at 94°C for 40 s, 50°C for
40 s, and 72°C for 1 min per 1 kb, and a final extension at 72°C for 10 min. The
PCR products were separated by electrophoresis in 1% agarose gels containing
1? Tris-acetate-EDTA buffer and visualized by staining with ethidium bromide.
DNA sequencing. DNA sequencing was conducted by using an ABI PRISM
BigDye Terminator v3.1 cycle sequencing kit. Cycle sequencing reactions were
conducted according to the kit’s protocol. Reaction samples were then purified
with Centrisep spin columns (Princeton Separations, Adelphia, NJ) and dried
under a vacuum. Samples were sequenced using an ABI Prism 3100 genetic
Descriptive analysis of sequence data. Nucleotide diversity (?; average pair-
wise nucleotide difference/site), numbers of mutations, numbers of synonymous
mutations (ds), numbers of nonsynonymous mutations (dn), and dn/ds ratios
(ratio of the number of nonsynonymous substitutions/nonsynonymous site [dn] to
the number of synonymous substitutions/synonymous site [ds], with a Jukes-
Cantor correction of the Nei-Gojobori method ) were calculated using
MEGA, version 2.1. The selection pressure on a protein-encoding gene can be
measured by comparing the nonsynonymous substitution rate (dn) (amino acid
altering) to the synonymous substitution rate (ds) (silent, with no amino acid
change) to obtain the dn/ds ratio (?). An ? value of 1 indicates neutral evolution
(relaxed selective constraint; nonsynonymous changes have no associated fitness
advantage and are fixed at the same rate as synonymous changes), ? values of ?1
indicate purifying selection (strong functional constraint; nonsynonymous
changes are deleterious for protein function and are fixed at a lower rate than
synonymous changes), and ? values of ?1 indicate positive selection (adaptive
evolution; nonsynonymous changes are favored because they confer a fitness
advantage and are fixed at a higher rate than synonymous changes) (4).
Phylogenetic analysis of sequences and construction of phylogenetic trees.
The genes in the prs-ldh region and other housekeeping gene sequences were
compared to the GenBank nucleotide and protein databases using the BLASTN
and BLASTP algorithms at GenBank (6). Nucleotide and deduced amino acid
sequences for each gene were initially aligned with CLUSTALX (2). Inter- and
intraspecies similarity score matrixes for each gene were generated using
MEGA, version 2.1, and BioEdit software (http://www.megasoftware.net and
http://www.mbio.ncsu.edu/BioEdit/bioedit.html, respectively). Phylogenetic and
molecular evolutionary analyses were conducted using MEGA, version 2.1. Ge-
netic distances were calculated by the Kimura two-parameter and Tamura-Nei
models. The method of Nei and Gojobori was applied to the various sequences
to obtain synonymous and nonsynonymous distances (multiple substitutions ad-
justed by the Jukes-Cantor formula). Phylogenetic trees were constructed and
compared using neighbor-joining, maximum parsimony, and minimum evolution
algorithms (18). The same gene sequences of other bacteria were used as out-
groups for phylogenetic comparisons of some housekeeping genes, and bootstrap
analyses were performed with 1,000 replicates.
Nucleotide sequence accession numbers. The GenBank accession numbers of
the sequences determined for this study are DQ153172 to DQ153182,
DQ154289, DQ154290, DQ151663 to DQ151667, DQ093572 to DQ093578,
DQ091845 to DQ091849, DQ091851, DQ092637 to DQ092642, DQ091852,
DQ091853, DQ091833 to DQ091844, DQ086415 to DQ086421, DQ083394 to
DQ083400, DQ065839 to DQ065846, DQ060335 to DQ060361, DQ016504 to
DQ016510, AY994168 to AY994175, AY994176 to AY994187, AY878348,
AY822470 to AY822475, AY785381, AY785382, AY748441 to AY748446,
AY753217 to AY753221, AY729917 to AY729926, AY521653, AY521654,
AY553865 to AY553869, and AY352074 to AY352076.
Generic testing. Seven isolates (LS159, LS165, LS160,
SE107, SE116, LS166, and 2436KA) of L. seeligeri were origi-
nally obtained from food (raw milk and crabmeat) and envi-
ronmental (salt marsh/estuarine water or sediment) samples by
Listeria selective enrichment and selective isolation. Each iso-
late had a typical Listeria colonial appearance on esculin-con-
taining PALCAM and Oxford agars. Each isolate was a gram-
positive bacillus. Each isolate was motile, exhibiting tumbling
motility in wet mounts and producing an umbrella pattern in
motility-stab agar. Each isolate was positive in catalase, methyl
red, and Voges-Proskauer tests as well as in esculin, maltose,
and glucose fermentation tests. The isolates were negative in
oxidase and indole tests. Triple sugar iron agar reactions were
an acid slant and butt with no gas or hydrogen sulfide. There-
fore, it was concluded that each isolate is a Listeria species. All
seven isolates were nonhemolytic by sheep blood agar stab
testing and by CAMP hemolysis enhancement testing (S?and
R?reactions with Staphylococcus aureus and Rhodococcus
equi, respectively). The negative hemolysis reactions suggested
that the isolates were not L. ivanovii, L. monocytogenes, or L.
seeligeri. The isolates did not produce acid from L-rhamnose
and mannitol but did from D-xylose. Together, the hemolysis
and sugar reactions suggested that the strains were L. welshi-
meri Rha?Xyl?strains. Serotyping of the original isolate of
2436KA with polyclonal antibodies had shown that it was se-
rotype 4, but no serotyping had been performed on the rest of
the original isolates.
Identification of Listeria strains by oligonucleotide microar-
ray. Two primer sets, LisF-LisR and IsoF-IsoR, were used for
duplex PCR amplification of the Listeria species-specific alleles
of the iap and hly genes, respectively (36). No synthesis of
detectable amounts of amplicons was observed for the hly gene
with any of these isolates, whereas these isolates were positive
in iap-specific PCR amplifications. All seven isolates, previ-
ously identified as L. welshimeri, were reidentified as L. seeligeri
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