FEMS Microbiology Letters 120 (1994) 99-104
© 1994 Federation of European Microbiological Societies 0378-1097/94/$07.00
Published by Elsevier
99
FEMSLE 06036
Species-specificity of monoclonal antibodies
recognising Prevotella intermedia
and Prevotella nigrescens
D e i r d r e A. Devine ,,a Michelle A. P e a r c e
R o n a l d A. Dixon a and R u d o l f Gmiir d
Department
b Department
c Department
d Department
a
a,
Saheer E. G h a r b i a b,c H a r o u n N. Shah b
of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire, BD7 1DP, UK
of Microbiology, Eastman Dental Institute, University of London, UK
of Medical Microbiology, St Bartholemew's Hospital Medical College, University of London, UK
of Oral Microbiology and General Immunology, Dental Institute, University of Ziirich, Switzerland
(Received 9 March 1994; revision received and accepted 27 April 1994)
Abstract: Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in
disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P.
intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate
dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P.
nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens
reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but
not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified
biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens. These isolates
were not recognised by mAbs 39B11.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.
Key words: Prevotella intermedia; Prevotella nigrescens; Species-specific antibody; Monoclonal antibody; Species identification;
Multilocus enzyme electrophoresis
Introduction
Prevotella intermedia a n d Prevotella nigrescens
[1] a r e o b l i g a t e l y a n a e r o b i c G r a m - n e g a t i v e b a c t e r i a w h i c h c a n b e i s o l a t e d f r o m h e a l t h y a n d dis-
* Corresponding author. Tel: (0274) 383565; Fax: (0274)
309742.
SSDI 0 3 7 8 - 1 0 9 7 ( 9 4 ) 0 0 1 8 2 - Q
eased human oral sites, from the female genital
tract and from abscesses at a variety of body sites.
They share only 45% DNA homology but because they are phenotypically very similar they
have been identified for many years as two genotypes of the same species: Prevotella intermedia
and prior to that Bacteroides intermedius [2,3].
This heterogeneous species was proposed as one
of a group of bacteria causing human periodontal
100
disease [4-9] while some studies claimed it is
unimportant in the disease process [10,11]. This
controversy has arisen because few studies of P.
intermedia/B, intermedius have distinguished between the two genotypes, now classified as P.
intermedia and P. nigrescens [1]. Thus, while it is
possible that P. intermedia and P. nigrescens differ in their pathogenic potential, their ecology
and roles in periodontal disease are poorly understood and will remain so until more studies
properly distinguish between them.
Until recently, the only way to distinguish between P. intermedia and P. nigrescens was by
DNA homology studies. Shah and Gharbia [1]
separated them by multilocus enzyme electrophoresis (MLEE), as malate and glutamate
dehydrogenases (MDH and GDH, respectively)
from P. intermedia migrate faster than those from
P. nigrescens. However, these techniques are specialised and are not well suited to all laboratories
or to many clinical studies.
Gmiir and Wyss [12] developed a serotyping
scheme based on patterns of reactivity with four
monoclonal antibodies (mAbs) raised against
whole cells of P. intermedia / B . intermedius
[12,13]. DNA homology data available at the time
the mAbs were developed indicated that strains
belonging to genotype I (P. intermedia) reacted
with mAb 40BI3.2.2 whereas those from genotype
II (P. nigrescens) did not. However, this was based
on DNA homology data for only four strains of
genotype I and nine of genotype II. In this study
we aimed to investigate fully the species-specificity of the mAbs by determining their reactivity
patterns with 40 organisms which had been accurately identified using MLEE analysis of their
MDH and GDH enzymes.
Materials and Methods
Bacterial strains
All forty isolates had been biochemically identified as P. intermedia. Twenty-five have been
described with their sources in previous studies
[1,12-15] and were all originally isolated from
human oral sites. The reference strains, ATCC
25611, ATCC 25261 and ATCC 33563 (NCTC
9336) represent serotypes I, II and III, respectively, as defined by Gmiir and Guggenheim [13].
Nine isolates (prefix MH) were provided by M.
Haapasaalo (Department of Cariology, University
of Helsinki, Finland) and were from endodontic
infections and periodontal pockets. HST 1156-3,
HST 2160-1 and HST 2166-2 were provided by P.
Braham (Research Centre in Oral Biology, University of Washington, USA); these were isolated
from vaginal sites and were identified biochemically and by gas liquid chromatography as P.
intermedia, but they were a-fucosidase negative
and did not hybridize with nucleic acid probes
raised against P. intermedia genotypes I and II
[16].
Growth conditions
All isolates were grown in modified FUM
medium [13], which was prepared and sterilised
as described by Loesche et al. [17] with the addition of 5% (v/v) sterile horse serum. Columbia
agar base (Oxoid CM311) or Blood agar base
(Oxoid CM55) supplemented with 5% (v/v) horse
blood were used as solid media. For ELISAs, 1
ml of an overnight broth culture was transferred
to 9 ml fresh FUM containing serum, which was
incubated for no more than 48 h. Cultures were
grown in prereduced media at 37°C in a Don
Whitley Compact or Mark III anaerobic workstation under an atmosphere of 80% nitrogen, 10%
hydrogen and 10% carbon dioxide. All isolates
were stored at -70°C in sterile glycerol (30%
v/v).
Multilocus enzyme electrophoresis (MLEE)
Differentiation of P. intermedia and P. nigrescens was acheived by determining the electrophoretic mobility of MDH and GDH enzymes
in cell-free extracts using the method of Shah and
Gharbia [1].
ELISA
Hybridoma culture supernatants of rat mAbs
37BI6.1, 39BI1.1.2 and 40BI3.2.2 were stored at
- 2 0 ° C . Broth cultures of bacteria were washed
twice in 0.9% (w/v) sodium chloride and were
resuspended in phosphate buffered saline (pH
101
7.2) to an optical density at 550 nm of 0.5 (2-3 ×
108 organisms ml-1). The whole cell ELISA technique described by Gmiir and Guggenheim [13]
was used to determine the antigenic reactivity
group of the 40 isolates.
Results
Twenty-four of the strains examined by MLEE
or DNA homology group determination were P.
nigrescens and 13 were P. intermedia. Determination of species by MLEE agreed with allocation
to genotypes by DNA homology for the 14 strains
which had been genotyped in earlier studies (Table 1; [1,12]. The GDH enzymes of the three
vaginal isolates, HST 1156-3, HST 2160-1 and
HST 2166-2, migrated slower than GDH from
both P. interrnedia and P. nigrescens, while their
MDH enzymes migrated more slowly than MDH
of P. intermedia but faster than that of P. nigrescens (Fig. 1).
The patterns of reactivity of all isolates with
mAbs 37BI6.1, 39BI1.1.2 and 40BI3.2.2 are shown
in Table 1. All 13 of the isolates identified as P.
intermedia reacted to high titres with the three
antibodies with strong colour intensity. The titre
for mAb 40BI3.2.2 was consistently in excess of
Table 1
Characterization of 40 strains identified biochemically as 'Prevotella intermedia' by DNA-DNA hybridization, MLEE analysis and
binding with monoclonal antibodies
ATCC25611T
B9
OMZ 248
OMZ 268
3b
FDC 581
BH 20/30
MUI 1, 65
MH 12, 16,
18, 21
ATCC25261
T588
OMZ 251
MUI 21, 24
ATCC33563 r
M107-74
FDC 377
G11 a-d
OMZ 265
BH 18/23
MUI 4, 15,
32, 36, 41,
44, 57, 77
MH 1, 4, 5,
19, 20
HST 1156-3,
2160-1,
2166-2
DNA homology
Species by
Binding of mAb:
group
MLEE
37BI.6.1
39BI1.1.2
40BI3.2.2
P.
P.
P.
P.
P.
P.
P. intermedia
+ + a
+ +
+ +
nd
++
++
++
P. intermedia
+ +
+ +
+ +
nd
++
++
++
+
+
+
+
+
+
+
+
intermedia
intermedia
intermedia
intermedia
intermedia
intermedia
P.
P.
P.
P.
intermedia
intermedia
intermedia
intermedia
+
+
+
+
intermedia
mgrescens
mgrescens
mgrescens
mgrescens
mgrescens
mgrescens
nlgrescens
mgrescens
ntgrescens
mgrescens
++
++
++
++
++
++
--
++
++
--
++
++
nd
P.
P.
P.
P.
P.
P.
P.
e.
P.
P.
P.
nd
nd
nd
nd
P. nigrescens
P. nigrescens
P. nigrescens
nd
+
+
+
+
+
+
+
+
+
+
+
+
++
_b
--
++
±c
--
++
++
++
++
++
±
±
±
±
±
-
P. nigrescens
+ +
+
nd
P. nigrescens
+ +
+
nd
Unknown
+ +
P.
P.
P.
P.
P.
nigrescens
nigrescens
nigrescens
nigrescens
nigrescens
m
nd. not determined; a + +, maximum dilution of antibody solution giving significant antibody binding was between 1:1280 and
> 1 : 20480; b --, no binding of mAb; c +, maximum dilution of antibody solution giving significant antibody binding was between
1 : 10 and 1 : 640, low colour intensity.
102
Fig. 1. Multilocus enzyme electrophoresis of isolates identified biochemically as 'P. intermedia' (a) afer staining for malate
dehydrogenase, (b) after staining for glutamate dehydrogenase. Lane 1: P. intermedia ATCC 25611; Lane 2: P. nigrescens ATCC
33563; Lane 3: HST 2166-2; Lane 4: HST 1156-3; Lane 5: HST 2160-1.
10,000 for most isolates. None of the 24 strains
identified as P. nigrescens reacted with mAb
40BI3.2.2. Only five P. nigrescens had strong reactions with m A b 39BI1.1.2, mostly giving titres
of between 2560 and 5120, which were generally
lower than the titres with P. intermedia isolates.
The remaining 19 P. nigrescens had a weak reaction with m A b 39BI1.1.2 (titres from 10 to 640).
The three vaginal isolates, H S T 1156-3, H S T
2160-1 and H S T 2166-2, reacted to titres of 2560,
5120 and 2560 respectively with mAb 37BI6.1 but
none reacted with m A b 39BI1.1.2 or 40BI3.2.2.
Discussion
Very few studies have distinguished between
P. intermedia and P. nigrescens because of the
lack of an easily applied and interpreted method.
This study has shown that m A b 40BI3.2.2, which
recognises a 150 kDa protein [12], provides such
an easy method because it is specific for strains of
P. intermedia and does not react with P. nigrescens. The positive reaction seen between P.
intermedia isolates and this antibody is strong and
easy to detect even at quite low concentrations of
antibody. As m A b 40BI3.2.2 is species-specific it
has the potential to be a very useful tool in
studies of the ecology and epidemiology of P.
intermedia and P. nigrescens, allowing differentiation between the two organisms without any specialised D N A or protein extraction procedures.
Conventional methods of detection usually involve culture of the organisms of interest but
techniques of enumeration of P. intermedia/P.
nigrescens in oral samples are problematical and
a high number of false negatives can be recorded
[18]. Thus, the direct examination of clinical sam-
103
pies without the need for culturing would give the
most accurate results. The usefulness of mAb
40BI3.2.2 in direct examination of plaque samples using immunofluorescence has been examined [19,20]. While the other mAbs were useful
for this, mAb 40BI3.2.2 gave only weak immunofluorescence despite having proven high
affinity for its target, making its usefulness in
quantitative immunofluorescence studies questionable. The binding of mAb 40BI3.2.2 to P.
intermedia is still most reliably detected using
ELISA of pure cultures [20] but immunofluorescence may be a useful adjunct. This antibody-antigen reaction can behave unpredictably in other
tests: the antigen could not be localised by immunogold labelling and electron microscopy due
to a high level of background labelling and almost
no reaction with the bacterial cell surface [14].
P. nigrescens can be divided into two serogroups based on the weak interaction between
some strains and mAb 39BI1.1.2 [12,13]. Devine
et al. [14] reported that this weak interaction was
not seen in ELISAs with control strains of known
serotype, and also that interpretation of results
with mAbs 39BI1.1.2 and 40BI3.2.2 was difficult
due to high levels of background labelling of
wells: as a result a minority of organisms were
assigned to the wrong serotype. This problem of
interpretation was not seen in more recent ELISA
assays where the weak reactions with mAb
39BI1.1.2 were usually easily detected, and positive reactions with mAb 40BI3.2.2 were very clear.
The reasons for the problems in earlier experiments are unknown.
The vaginal isolates used in this study were
shown previously to be atypical in that they failed
to react with RNA probes prepared against nucleic acid of strains of genotypes I or II of P.
interrnedia (i.e.P. intermedia and P. nigrescens),
and they were a-fucosidase negative [16]. MLEE
provides a definitive distinction between P. intermedia and P. nigrescens [1] but MDH and GDH
enzymes from the vaginal isolates did not have
migration rates which corresponded to either
species. The reactivity of the three strains with
the mAbs used here also indicates that they are
not P. intermedia or P. nigrescens, and together
these results strengthen the proposal made by
Moncla et al. [16] that they represent a previously
undescribed species which is nonetheless biochemically similar to P. intermedia and P. nigrescens. Further studies of these and other organisms identified as P. intermedia, and isolated
from non-oral sites, are in progress.
Previous studies of organisms identified as
Bacteroides intermedius from healthy and diseased oral sites have used mAbs 37BI6.1,
39BI1.1.2 and 40BI3.2.2 [20,21]. At this time the
groups distinguished by the mAbs were described
as serotypes I (recognised by all mAbs), II (no
reaction with mAb 40BI3.2.2) and III (weak reaction with mAb 39BI1.1.2 and none with mAb
40BI3.2.2). Isolates belonging to serotype I comprised a significantly higher proportion of organisms from deep periodontal pockets compared
with healthy shallow sites [21]. Conversely
serotypes II and III increased in prevalence in
root abscesses while serotype I did not [20]. As
strains of serotype I have now been shown to be
P. intermedia whereas those of serotypes II and
III are P. nigrescens, this is a strong indication
that P. intermedia and P. nigrescens differ in their
oral habitats and possibly their pathogenicity.
A few other studies have attempted to distinguish between genotypes of P. intermedia, now P.
intermedia and P. nigrescens, and they also indicate that these species may differ in prevalence in
specific periodontal diseases. Strains belonging to
genotype I were more commonly isolated from
periodontal disease of adults than genotype II
[22], and other studies indicated that genotype I
increases in prevalence in periodontal pockets
compared with healthy sites [23,24]. Genotype II
may be associated with other distinct disease conditions, such as juvenile periodontitis [22,25],
acute necrotizing ulcerative gingivitis [26] and endodontic infections [24]. In some of these studies
the methods used only tentatively distinguished
between the two species/genotypes and strong
claims were not made by the authors. Thus, while
there is reason to believe that P. intermedia and
P. nigrescens differ in their pathogenic potential,
their ecology and roles in periodontal disease are
poorly understood and will remain so until more
studies properly distinguish between the two
species. Monoclonal antibody 40BI3.2.2 distin-
104
guishes well between P. intermedia and P. nigrescens and will be a powerful tool in such
studies.
Acknowledgements
We would like to thank Mrs Madge Hollowood and Mrs Lynne Keeble for their excellent
technical assistance. We are also grateful to Drs.
M. Haapasalo, P. Handley and Ms P. Braham for
their generous provision of strains. Michelle
Pearce is supported by a grant from the SERC.
This work was presented in part at the 8th Society for Anaerobic Microbiology Symposium, July
1993.
References
1 Shah, H.N. and Gharbia, S.E. (1992) Biochemical and
chemical studies on strains designated Prevotella interrnedia and proposal of a new pigmented species, Prevotella
nigrescens sp. nov. Int. J. Syst. Bacteriol. 42, 542-546.
2 Johnson, J.L. and Holdeman, L.V. (1983) Bacteroides intermedius comb. nov. and descriptions of Bacteroides corporis sp. nov. and Bacteroides levii sp. nov. Int. J. Syst.
Bacteriol. 33, 15-25.
3 Shah, H.N. and Collins, M.D. (1990) Prevotella, a new
genus to include Bacteroides melaninogenicus and related
species formerly classified in the genus Bacteroides. Int. J.
Syst. Bacteriol. 40, 205-208.
4 Slots, J. (1979) Subgingival microflora and periodontal
disease. J. Clin. Periodontol. 6, 351-382.
5 Slots, J. (1986) Bacterial specificity in adult periodontitis-a
summary of recent work. J. Clin. Periodontol. 13, 912-917.
6 Zambon, J.J., Reynolds, H.S. and Slots, J. (1981) Blackpigmented Bacteroides spp. in the human oral cavity.
Infect. Immun. 32, 198-203.
7 Moore, W.E.C., Holdeman, L.V., Cato, E.P., Smibert,
R.M., Burmeister, J.A., Palcanis, K.G. and Ranney, R.R.
(1985) Comparative bacteriology of juvenile periodontitis.
Infect. Immun. 48, 507-519.
8 van Steenbergen, T.J.M., van Winkelhoff, A.J., van der
Velden, U. and de Graaff, J. (1989) Taxonomy, virulence
and epidemiology of black-pigmented Bacteroides species
in relation to oral infections. Infection 17, 194-196.
9 Haapasalo, M., Ranta, H., Ranta, K. and Shah, H.N.
(1986) Black-pigmented Bacteroides spp. in human apical
periodontitis. Infect. Immun. 53, 149-153.
10 White, D. and Mayrand, D. (1981) Association of oral
Bacteroides with gingivitis and adult periodontitis. J. Periodont. Res. 16, 259-265.
11 van Winkelhoff, A.J., Carl6e, A.W. and de Graaff, J.
(1985) Bacteroides endodontalis and other black-pigmented
Bacteroides species in odontigenic abscesses. Infect. Immun. 49, 494-497.
12 Gmiir, R. and Wyss, C. (1985) Monoclonal antibodies to
characterize the antigenic heterogeneity of Bacteroides
intermedius. In: Monoclonal antibodies against bacteria
(Macario, A.J.L. and Conway de Macario, E., Eds.), pp.
91-119. Academic Press, London.
13 Gmiir, R. and Guggenheim, B. (1983) Antigenic heterogeneity of Bacteroides intermedius as recognised by monoclonal antibodies. Infect. Immun. 42, 459-470.
14 Devine, D.A., Gmiir, R. and Handley, P.S. (1989) Ultrastructure, serogrouping and localization of surface antigens of Bacteroides intermedius. J, Gen. Microbiol. 135,
967-979.
15 Devine, D.A. and Handley, P.S. (1989) The relationship
between coaggregation properties and surface structures
of Bacteroides intermedius. Microb. Ecol. Health Dis. 2,
267-278.
16 Moncla, B.J., Braham, P., Rabe, L.K. and Hillier, S.L.
(1991) Rapid presumptive identification of black-pigmented Gram-negative anaerobic bacteria by using 4methylumbelliferone derivatives. J. Clin. Microbiol. 29,
1955-1958.
17 Loesche, W.J., Hockett, R.N. and Syed, S.A. (1972) The
predominant cultivable flora of tooth surface plaque removed from institutionalized subjects. Arch. Oral Biol. 17,
1311-1325.
18 Dahl~n, G., Renvert, S., Wikstr6m, M. and Egelberg, J.
(1990) Reproducibility of microbiological samples from
periodontal pockets. J. Clin. Periodontol. 17, 73-77.
19 Gmiir, R. (1988) Applicability of monoclonal antibodies to
quantitatively monitor subgingival plaque for specific bacteria. Oral Microbiol. Immunol. 3, 187-191.
20 Gmiir, R. (1992) Habilitation thesis. University of Ziirich,
Switzerland.
21 Dahl~n, G., Wikstr6m, M., Renvert, S., Gmiir, R. and
Guggenheim, B. (1990) Biochemical and serological characterization of Bacteroides interrnedius strains isolated
from the deep periodontal pocket. J. Clin. Microbiol. 28,
2269-2274.
22 Moore, L.V.H., Moore, W.E.C., Cato, E.P. Smibert, R.M.,
Burmeister, J.A., Best, A.M. and Ranney, R.R. (1987)
Bacteriology of human gingivitis. J. Dent. Res. 66, 989-995.
23 Hanazawa, S., Takana, S., Kin, M., Amano, S., Nakada,
K., Masuda, T. and Kitano, S. (1990) Application of monclonal antibodies to the detection of black-pigmented Bacteroides spp. in subgingival plaques by immunoslot blot
assay. J. Clin. Microbiol. 28, 2248-2252.
24 Gharbia, S.E., I-Iaapasalo, M., Shah, H.N., Kotiranta, A.,
Lounatmaa, K., Pearce, M.A. and Devine, D,A.J. Periodontol. 65, in press.
25 Nakazawa, F., Zambon, J.J., Reynolds, H.S. and Genco,
R.J. (1988) Serological studies of oral Bacteroides intermedius. Infect. Immun. 56, 1647-1651.
26 Chung, C.P., Nisengard, R.J., Slots, J. and Genco, R.J.
(1983) Bacterial IgG and IgM antibody titres in acute
necrotizing ulcerative gingivitis. J. Periodontol. 54, 557562.