INTERNATIONAL JOURNALOF SYSTEMATIC BACTERIOLOGY,
OCt. 1988, p. 335-339
0020-7713/88/040335-05$02.00/0
Copyright 0 1988, International Union of Microbiological Societies
Vol. 38, No. 4
Streptococcus vestibularis sp. nov. from the Human Oral Cavity
R. A. WHILEY" AND J. M. HARDIE
Department of Oral Microbiology, The London Hospital Medical College, London El 2AD, United Kingdom
Thirteen strains of gram-positive, catalase-negative, chain-forming cocci isolated from the vestibular mucosa
of human oral cavities were compared with other oral streptococcal species. The new strains were unusual in
that they formed acid from lactose, from salicin, and usually from cellobiose, but not from mannitol, sorbitol,
inulin, or raffinose, and infrequently formed acid from trehalose. They hydrolyzed esculin, urea, and starch
but not arginine, formed hydrogen peroxide, and were usually Voges-Proskauer positive. They did not produce
extracellular glucan or fructan from sucrose. The strains were compared by analyzing long-chain fatty acids,
by determining whole-cell polypeptide patterns by sodium dodecyl sulfate-polyacrylamide gel electrophoresis,
and by performing deoxyribonucleic acid (DNA)-DNA hybridizations. They possessed fatty acid profiles with
major amounts of hexadecanoic (C16:O) and octadecenoic (C18:lw7; cis-vaccenic) acids together with
tetradecanoic (C14:O; myristic), hexadecenoic (C16:l; palmitoleic), octadecanoic (C18:O; stearic), octadecenoic
(C18:lw9; oleic), and eicosenoic (C20:1) acids, as shown by capillary gas-liquid chromatography. The sodium
dodecyl sulfate-polyacrylamide gel electrophoresis polypeptide patterns of the isolates showed some similarity
to the patterns obtained for strains of Streptococcus salivarius subsp. salivarius, although they were,
nevertheless, clearly distinguishable from the latter. DNA-DNA hybridization studies demonstrated that the
new strains are more closely related to S. salivan'us than to the other species of oral streptococci, but are
sufficientlydissimilar to warrant separate species status. The name Streptococcus vestibularis is proposed. The
DNA base composition is 38 to 41 mol% guanine plus cytosine. The type strain is strain MM1(= NCTC 12166).
The viridans streptococci are a heterogeneous group of
bacteria that comprise the majority of the streptococci found
in mouths and upper respiratory tracts (23,24). The presence
of atypical and unidentifiable strains is characteristic of
studies on the classification of this group of bacteria undertaken over the past 20 years (6,8, 10,11, 18,22,26,32). One
such example is the seven alpha-hemolytic strains which
comprised group IV of the Carlsson numerical taxonomic
study of a collection of oral streptococci (8). These strains
were isolated mainly from the vestibular mucosa of human
oral cavities (7) and were described as producing acid from
lactose, salicin, and arbutin, but not from mannitol, sorbitol,
melibiose, raffinose, or inulin. ThLy produced hydrogen
peroxide but did not produce ammonia from arginine. The
relationship of this group of strains to previously recognized
species of the genus Streptococcus was uncertain.
Colman (9) cited the Carlsson group IV strains as a likely
example of the as-yet-unrecognized species of viridans
streptococci that are undoubtedly present in humans. In a
more recent numerical taxonomic study (6), one of the
Carlsson group IV strains, strain LV71, clustered with
strains of Streptococcus salivarius, while in the same study
another group IV strain, strain LV81, was included in a
cluster called oral group 11, which was later named Streptococcus oralis ( 5 ) .
During the course of a taxonomic study of oral streptococci undertaken in our laboratory (Whiley, Ph.D. thesis,
University of London, London, 1987), two representative
strains of group IV, strains OP81 and LV71, were found to
be very similar in biochemical and physiological characteristics, as well as in whole-cell polypeptide patterns as
determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). These two strains
remained unassigned to any of the previously recognized
species of oral viridans streptococci (Streptococcus mutans,
Streptococcus sobrinus, Streptococcus rattus, Streptococ-
* Corresponding author.
cus cricetus, Streptococcus sanguis, Streptococcus mitis,
Streptococcus oralis, and Streptococcus anginosus). Preliminary deoxyribonucleic acid (DNA)-DNA hybridization experiments indicated that these strains shared a high degree of
sequence homology (>SO%) and, with the exception of S .
salivarius, shared little homology with the other species of
oral streptococci.
In the present study six of the original strains from group
IV of the Carlsson study (8) were compared with seven
similar strains isolated in our laboratory and with other
representative strains of human oral streptococci by using
biochemical and physiological tests, SDS-PAGE polypeptide patterns, long-chain fatty acid analysis by capillary
gas-liquid chromatography, and DNA-DNA hybridization.
In addition, because of the relatively close relationship
initially observed between the vestibular strains and S .
salivarius and the recent demonstration of high levels of
DNA-DNA homology between S . salivarius and strains of
Streptococcus thermophilus (19, 25), three strains of S .
thermophilus were included in this study.
MATERIALS AND METHODS
Bacterial strains. The strains used are listed in Table 1. All
streptococci from the mucosa of vestibules were isolated by
using the procedure described by Carlsson (7). Strains OP81,
LV81, LV71, HV81, OV71, and PV91 were from the study
of Carlsson (8), and strains JW3, MMIT (T = type strain),
AS2, SS2, BN3, RW3, and JM8 were isolated in our laboratory.
Biochemical and physiological characteristics. The 13
strains from vestibular mucosa were tested for acid production from the following carbohydrates at concentrations of
0.5% (wt/vol): glucose, sucrose, fructose, maltose, galactose, arabinose , raffinose, lactose, salicin, glycerol, mannitol, sorbitol, cellobiose, melezitose, xylose, melibiose, adonitol, trehalose, amygdalin, and inulin. Previously described
methods were used for these tests (31). Production of acetoin
(Voges-Proskauer test) was determined as described by
335
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336
INT. J. SYST.BACTERIOL.
WHILEY AND HARDIE
TABLE 1. DNA-DNA relatedness between strains from vestibular mucosa and other oral streptococci“
G+C content
(mol%)
Unlabeled DNA from:
Vestibular mucosa isolates
OP81
LV81
LV71
HV81
OV71
PV91
JW3
M M ~ ~
AS2
ss2
BN3
RW3
S . salivarius subsp. salivarius strains
NCTC 86MT
NCTC 8606
M36
B242
S . salivarius subsp. thermophilus strains
NCDO 573T (= ATCC 19258T)
NCDO 2075
NCDO 1409
S . mutans NCTC 10449T(= ATCC 25175T)
S. anginosus NCTC 10713T(= ATCC 33397T)
S. sanguis NCTC 7863T (= ATCC 10556T)
S. mitis NCTC 3165T (= ATCC 33399T)
S. oralis LVGIT (= NCTC 11427T)“
% Homology with 3H-labeled DNA from:
Strain
OP81
Strain
NCTC 8618’r
Strain
NCDO 573T
39
39
38
40
40
39
39
39
40
41
40
40
100
108
72
93
79
92
81
90
86
68
74
79
57
47
40
41
41
48
64
60
43
44
56
46
51
44
44
51
41
54
48
43
48
42
42
57
41
41
39
40
49
53
36
40
100
82
90
67
44
N T ~
NT
37
40
39
39
37
37
42
38
40
57
62
45
11
7
13
6
16
59
45
59
3
2
2
13
14
100
87
75
2
14
13
1
2
Hybridizations were performed under optimum conditions at 60°C.
NT, Not tested.
L‘ Labeled DNA from strain LV71 also showed only 10% homology with representative strain FW69 of “Streptococcus mitior” genetic group 2 of Coykendall
and Munzenmaier (14).
a
Cowan (12). Tests for esculin hydrolysis and the production
of ammonia from arginine were performed as previously
described (3); the production of ammonia from arginine was
detected by adding Nessler reagent.
Urease production was detected by the method of Christensen as described by Cowan (12). The formation of hydrogen peroxide was tested on heated 2.5% (by volume) horse
blood agar supplemented with 1% (wthol) glucose and
0.01% (wthol) o-dianisidine by using the method of Whittenbury (35). Positive colonies were detected when the
surrounding medium became dark brown or black. Starch
hydrolysis was tested on brain heart infusion medium (Difco
Laboratories, Detroit, Mich.) supplemented with 0.2% (wt/
vol) soluble starch and 1.2% (wthol) Oxoid no. 1 agar
(Oxoid Ltd., Hampshire, United Kingdom). After 5 days
anaerobic incubation plates were flooded with Lugols iodine.
The hydrolysis of starch was indicated by the appearance of
clear zones around the colonies. For growth in the presence
of 10 and 40% (wthol) bile, oxgall (Difco) was added at the
appropriate concentration to blood agar base plates (agar
blood base no. 2; Oxoid Ltd.). Growth in the presence of 4
and 6.5% (wthol) NaCl was tested on blood agar base plates
(agar blood base no. 2; Oxoid Ltd.) to which the appropriate
amount of NaCl had been added. Formation of intracellular
polysaccharide was tested by methods described previously
(1). Extracellular polysaccharide production was tested by
growing strains on 5% sucrose medium (TYC medium; Lab
M, Lancaster, United Kingdom) and on mitis salivarius agar
(Difco) for 4 days both aerobically and anaerobically.
SDS-PAGE of whole-cell extracts. The polypeptide patterns
of strains were obtained by PAGE of whole-cell extracts in
the presence of SDS, using the discontinuous buffer system
of Laemmli (27, 33).
Analysis of long-chain fatty acids. Fatty acids were extracted from lyophilized cells by the whole-cell acid methanolysis and thin-layer procedure of Minnikin et al. (28) and
were analyzed by capillary gas-liquid chromatography, using
methods described previously (34).
DNA analyses. DNA was extracted and purified from
approximately 2 to 3 g (wet weight) of bacterial cells by the
method of Garvie (20). For base composition determinations
the DNA was finally dialyzed in standard I X SSC (0.15 M
NaCl plus 0.015 M trisodium citrate, pH 7.0) and the
guanine-plus-cytosine (G+ C) content was determined from
the thermal melting point of the sample (2).
DNA-DNA hybridizations were performed by the S1
nuclease method of Shah et al. (30), which was based on the
method of Crosa et al. (17).
RESULTS AND DISCUSSION
SDS-PAGEof whole-cell extracts. The polypeptide patterns
of six vestibular strains together with those of S . salivarius
subsp. salivarius, S . salivarius subsp. thermophilus, and
other oral streptococci are shown in Fig. 1. All 13 new
strains possessed identical patterns. The patterns obtained
for strains isolated from the vestibular mucosa and those
obtained for S . salivarius subsp. salivarius are similar with
respect to polypeptides having molecular weights of 35,000
and below. However, the regions of the patterns in the
molecular weight range above 35,000 share few common
bands. It is interesting that the patterns obtained for the
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VOL. 38, 1988
STREPTOCOCCUS VESTIBULARIS SP. NOV.
337
FIG. 1. SDS-PAGE polypeptide patterns of strains isolated from oral vestibular mucosa and other oral streptococcal species. Lanes a to
d, S . salivarius subsp. salivarius NCTC 86MT, NCTC 8606, M36, and B242, respectively; lanes e to j,vestibular mucosa strains OP81, PV91,
HV81, MMIT, SS2, and RW3, respectively; lanes k to m, S. salivarius subsp. thermophilus NCDO 573=, NCDO 1409, and NCDO 2075,
respectively; lane n, S. sanguis, NCTC 7863T; lane 0,S. mitis, NCTC 3165T; lane p, S. oralis, LVGIT; lane q, S. anginosus NCTC 10713T.
Molecular weights (x103) are indicated on the left.
three strains of S . salivarius subsp. thermophilus showed
little resemblance to those obtained for either the new strains
or the strains of S . salivarius subsp. salivarius.
Long-chain fatty acid analysis. The long-chain fatty acid
prQfiles of the vestibular strains were composed of major
amounts of hexadecanoic (C16:O; palmitic) and octadecenoic
(C18:107; cis-vaccenic) acids together with tetradecanoic
(C14:O; myristic), hexadecenoic (C16:1; palmitoleic), octadecanoic, (C18:O; stearic), octadecenoic (C18:109; oleic),
and eicosenoic (C20:l) acids. The presence of eicosenoic
acids differentiates these strains from several other species
of oral streptococci ( S . sanguis, S . mitis, S . oralis) and
provides further evidence of the close relationship between
the vestibular strains and S. salivarius. The presence of
eicosenoic acids in S . salivarius and closely related strains is
in accord with the findings of Farrow and Collins (19).
DNA analyses. The G + C contents of the vestibular strains
ranged from 38 to 41 mol% (Table 1).
Relatively high DNA relatedness values were observed
among the vestibular strains, S. salivarius subsp. salivarius,
and S . salivarius subsp. thermophilus. However, the degrees
of DNA homology observed among these taxa were sufficiently low for the new strains to be considered a separate
species (Table 1).The reduced level of homology observed
TABLE 2. Differential phenotypic characteristics of S. vestibularis and other viridans streptococcia
G +C
Acid produced from:
Hydrolysis of
content
of DNA
(mol%) Inulin Raffinose Lactose Trehalose Arginine Esculin Starch Urea
Species or
subspecies
S . vestibularis
S . salivarius subsp.
salivarius
S . salivarius subsp.
thermophilus
S . sanguis
S . mitis
S . oralis
S.anginosus
~~~
3841
3942
3740
4046
38-39
3740
3640
-b
+/+d
+
-
-
+
-/+
-/+
+/-
+
-
+
+
+
+
-
+
+
+
-
-
-
+
+
+
+/-
+e
+e
+
+g
-/+c
-/+
+
+/-/+
+
+
+/-
+
Production of
Acetoin
+
-
-
-
+
-
-
+
-
-/+
-/+
+
+e
+
+/-
-
+/-c
-/+
+/-
+/-
-
-/+d
-
-/+
NT~ -/+
-
+g
-
-
H 2 0 2 Glucan Fructan
+d
+
-
-e
+/-
-
-
Growth
at 45°C
-/+d
+
-/+d
-/+
NT
-
~~
Data taken from references 6, 8, 10, 11, 13, 16, 19, 21, 26, 28, and 29.
+, >80% of strains positive; -, <20% of strains positive; +I-, 50 to 79% of strains positive; -/+, 21 to 49% of strains positive.
Type strain MM1 (= NCTC 12166) does not produce acid from trehalose and is weakly positive for the production of acetoin (Voges-Proskauer test).
Results reported from different laboratories vary.
Reactions reported for type strain NCTC 3165 (= ATCC 33399) only (21).
f N T , Not tested.
g Hemolytic strains of S. anginosus usually do not produce acid from lactose or hydrolyze esculin.
a
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338
INT.J. SYST.BACTERIOL.
WHILEY AND HARDIE
between S. salivarius subsp. salivarius and S . salivarius
subsp. therrnophilus compared with higher values obtained
previously by other workers who used the membrane filter
method under optimum hybridization conditions (19, 25) is
characteristic of the S1 nuclease method (4, 14, 15). These
results suggest that the higher levels of DNA relatedness
between these two taxa reported previously may have been
at least partly due to the presence of partial hybrids, which
are eliminated when S1 nuclease is used (4, 14, 15) and that
these strains also should be regarded as belonging to separate species. Further work with larger numbers of isolates
will be required to verify this.
Description of Streptococcus vestibularis sp. nov. The description below is based on the results of the present work
together with data from the study of Carlsson (8). Streptococcus vestibularis (ves.tib.u.1ar’i.s. L. noun vestibulurn,
entrance hall or forecourt, referring to the vestibule of the
mouth) cells are gram-positive, catalase-negative cocci, approximately 1 km in diameter, that grow in chains. Colonies
grown anaerobically at 37°C for 3 days on mitis salivarius
agar are usually 2 to 3 mm in diameter, dark blue, matte, and
umbonate with undulate edges. Colonies grown aerobically
on mitis salivarius agar are 1to 2 mm in diameter, dark blue,
convex, and glossy with entire edges. Strain LV71 is an
exception, producing glossy, entire colonies even under
anaerobic conditions. Growth on TYC agar both anaerobically and aerobically produces colonies 1 to 2 mm in diameter that are white, convex, and glossy with entire edges.
Acid is produced from N-acetylglucosamine, arbutin, fructose, galactose, glucose, lactose, maltose, mannose, salicin,
and sucrose. Acid is not produced from adonitol, arabinose,
dextrin, dulcitol, fucose, glycerol, glycogen, inositol, inulin,
mannitol, melezitose, melibiose, raffinose, ribitol, ribose,
sorbitol, starch, or xylose. Most strains produce acid from
cellobiose and amygdalin. Few strains produce acid from
trehalose or D-glucosamine hydrochloride. Only strains
HV81, OP81, and LV8l ferment trehalose; these are the
same three isolates which are unable to ferment cellobiose
and amygdalin and constitute a separate biotype from the
majority of the strains examined.
Urease and hydrogen peroxide are produced. Most strains
produce acetoin and hydrolyze esculin and starch. The type
strain, strain MM1, is weakly positive in the Voges-Proskauer test and hydrolyzes both esculin and starch. Ammonia
is not produced from arginine. Growth does not occur at 10
or 45”C, in the presence of 4% (wthol) NaC1, or in the
presence of 0.0004% (wt/vol) crystal violet. Most strains
grow in the presence of 10% (wt/vol) bile but not in the
presence of 40% (wt/vol) bile. Strain MMIT does not grow in
10% bile. Polysaccharide is not produced intra- or extracelMarly. All strains produce alpha-hemolysis on horse blood
agar. The cellular long-chain fatty acids consist of major
amounts of hexadecanoic (C16:0; palmitoleic) and octadecenoic (C18:107; cis-vaccenic) acids together with tetradecanoic (C14:O; myristic), hexadecenoic (C16:1; palmitoleic),
octadecanoic (C18:O; stearic), octadecenoic ((218:109; oleic)
and eicosenoic (C20:l) acids. This organism has been isolated mainly from the vestibular mucosa of human oral
cavities. The G+C content is 38 to 40 mol%. The type strain
is strain MM1 (= NCTC 12166).
Phenotypic characteristics for differentiating S. vestibularis from other oral streptococci are shown in Table 2.
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