Evaluation Methods
A method for the rapid
quantification of anti-plaque
activity in vitro
Ritchie JA, Jones CL and
Prottey C
J Dent Res 1987, 66 (special issue),
859
Distal mesial plaque index: A
technique for assessing dental
plaque about the gingiva
Fischman S, Cancro LP,
Pretara-Spanedda P and
Jacobs D
Dental Hygiene 1987, 61,
404-409
The choice of an index system for
clinical testing is usually made in
consideration of the objectives of
the trial, size of the population,
period of the study and the type
and extent of change anticipated.
The objective of this study was to
evaluate a newly developed index
called the Distal Mesial Plaque
Index (DMPI) for sensitivity to
changing plaque state compared to
the established Modified Navy
Plaque Index (MN). The DMPI was
developed to reflect changes about
the proximal surfaces of the
gingival margin and this study
demonstrated a high correlation to
the MN. Correlations of the
total plaque scores to the total
weight of plaque removed was
appreciably higher for the DMPI
than the MN.
Periodontal awareness project
in the United Kingdom: CPITN
and self-assessment
Chesters RK, Dexter CH, Life JS,
Smales FC and van der Ouderaa F
Int Dent J 1987, 37, 218-221
CPITN score for each sextant was
used to determine treatment needs
for comparison with those
diagnosed by the specialist
periodontologists. The preliminary
results of this study are summarised
in Table 111. The four columns on
the left of Table 111 indicate the
percentage agreement between
the five experts for all these
activities, involving sponsored
research at a dental school and a
study with UK dentists, have
shown the potential of CPITN as a
screening method for patients in
general practice for periodontal
diseases. There is evidence to show
that patient-orientated
programmes have increased
periodontal awareness in the UK.
Development of a Rapid Human
Calculus Screening Model
Frienberg CA, Fernandez PM,
Lesetz M and Kanapka J
J Dent Res 1991, 70, 326
An In Vitro Plaque
Mineralisation Model for
Assessing Anti-calculus Agents
Bajor JS, Viccaro JP and Kanapka J
J Dent Res 1991, 70, 326
Comparison of Invasive and
Non-invasive Indices for
Gingivitis Measurement
Saxton CA, Putt MS* and
Soparker PM**
(*Department of Chemistry,
Indiana-Purdue University, Fort
Wayne, Indiana, USA)
(**Forsyth Dental Centre, Boston,
Massachusetts, USA)
J Dent Res 1991, 71, 327
A modified chemostat system to
study the ecology of oral
biofilms
Bradshaw DJ*, Marsh PD*, Watson
GK and Cummins D
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1992, 71, 674
A Biofilm model system which
simulates dental plaque
Bosko CA, Bajor J, Carson Rand
Schilling K
J Dent Res 1992, 71, 734
iIMAGEM 1
IMAGEM2
The use of CLSM for the study
of mixed plaque
Moss MC, Jones CL, Cummins D
and Howard CV*
(*Department of Human Anatomy
and Cell Biology, University of
Liverpool, Liverpool, UK)
J Dent Res 1992, 71, 601
Applications of confocal laser
scanning microscopy in in situ
mapping
Moss MC, Veiro JA, Singleton S,
Gregory DP, Birmingham JJ, Jones
CL, Cummins PG, Cummins D,
Miller RM, Sheppard RC*, Howard
VC** and Bhasker N*** (*Medical
Research Council, Laboratory of
Molecular Biology, Cambridge, UK)
(**Department of Human Anatomy
and Cell Biology, University of
Liverpool, Liverpool, UK)
(***Phase Separations Limited,
Deeside, Clwyd, UK)
Analyst 1993, 118, 1-9
A confocal laser scanning
microscope has been applied to the
detailed study of non-invasive
mapping on targeted systems. This
paper aims to show, by particular
reference to specific problems, the
wide ranging potential of the
technique for the analytical
community. The specific problems
successfully tackled by the
technique include characterization
of two types of inert support
bodies and the precursor/enzyme
distributions therein, the effect of
processing on dye penetration in
fibres, mapping the
interdependence and growth
patterns in bacterial and noninvasive
examination of epidermal
tissue layers.
Validation of a 21-day partial
mouth gingivitis model for
evaluating chemotherapeutic
dentifrices
Putt M*, van der Weijden**,
Kleber CJ* and Saxton CA
(*Health Science Research, Indiana
University-Purdue University, Fort
Wayne, USA
**Department of Periodontology,
Academic Centre for Dentistry,
Amsterdam, The Netherlands)
J Periodontal Res 1993, 28,
301-307
The experimental gingivitis model
has long been used to evaluate
chemotherapeutic agents in
mouthrinses. Only recently
however, has the model been
modified to test undiluted
dentifrices by using a toothshield
both to apply dentifrice and to
protect selected teeth from
toothbrushing. This also enables
participants to brush the rest of the
mouth and avoid the
unpleasantness of three weeks
without oral hygiene. Because of
its well-documented efficacy,
chlorhexidine was used in a
toothpaste formulation to
investigate the validity of the
partial-mouth, experimental
gingivitis model for evaluating
therapeutic dentifrices. Optimal
gingival health was established in
88 adults, who then were
randomly assigned to two equal
groups. A toothshield was
constructed for each subject to fit
the teeth of one mandibular
quadrant. During the trial
chlorhexidine or placebo dentifrice
was applied undiluted to the test
teeth via the toothshield, which
also prevented plaque removal
during brushing of the remaining
dentition. After 21 days, plaque
and gingivitis had developed in
both groups. However, the
chlorhexidine group had
significantly less plaque and
gingivitis than the placebo group.
No adverse soft tissue effects were
observed. This study demonstrated
that the partial-mouth,
experimental gingivitis model
allowed unhindered development
of plaque and gingivitis that was
comparable to whole-mouth
studies in which oral hygiene was
suspended for three weeks. By
corroborating with chlorhexidine, it
is concluded that this short-term
clinical model is valid for evaluating
the chemotherapeutic effect of
dentifrices.
A microassay for bacterial
adherence to hydroxyapatite
Schilling KM, Carson RG, Bosko
CA, Golikeri GD, Bruinooge A,
Hoyberg K, Waller AM and
Hughes NP
Colloids and Surfaces B:
Biointerfaces 1994, 3, 31-38
The aim of the present study was
to develop a microassay for
bacterial adherence to
hydroxyapatite (HA) which is more
convenient, reduces the required
amounts of materials, and removes
artifactual tube “wall effects”. To
form HA in situ on 96-well plates,
the wells were filled with 2.5mmol
1-1 calcium chloride, 7.5mmol 1-1
potassium phosphate, 250mmol 1-
1 triethanolamine (pH 7.4) and the
plates were incubated for 1.5h at
75°C. The solution was decanted
and the entire procedure was
repeated three more times. Analysis
of the mineral composition showed
the Ca/P ratio to be 1.63. X-ray
and electron diffraction analyses
indicated that all the mineral
formed was HA. Scanning electron
microscopy showed a continuous
layer of HA crystallites with
occasional aggregates and no
exposed polystyrene. Salivary
pellicles were formed on the HA
surfaces either with or without the
in situ synthesis of glucans. The
pellicle-coated HA plates were
incubated with radiolabelled
bacteria for 1.0h at 37°C.
Adherent bacteria were digested
with 1mmol 1-1 EDTA/1 N NaOH
for 1.0h at 37°C and were
quantitated by scintillation
counting. The results showed that
the Streptococcus gordinii,
Actinomyces naeslundii
(Actinomyces viscosus), and
Streptococcus mutans bound to HA
plates coated with saliva or saliva
plus glucans in a stereospecific
manner. The relative levels of
bacteria which attached to HA
plates paralleled the results found
with spheroidal whitlockite beads.
The HA plates eliminate wall
effects, simplify sample processing
and scintillation counting, require
smaller amounts of reagents, and
reduce radioactive and other
waste. Accordingly, HA-coated
microtitre plates represent an
improved format for studying oral
bacterial adhesion.
IMAGEM 3
Intra/inter examiner
reproducibility study of gingival
bleeding
van der Weijden GA*, Timmerman
MF*, Saxton CA, Russell JI,
Huntington E and
van der Velden U*
(*Department of Periodontology,
ACTA, Academic Centre for
Dentistry, Amsterdam, The
Netherlands)
J Periodontal 1994, 29, 236-241
Sulcular bleeding after stimulation
with a periodontal probe has been
associated with the presence of an
inflammatory lesion in the gingiva.
The objective of this study was to
evaluate the effect of the
placement of the probe in relation
to the tooth surface, and the effect
of standardising the probing
pressure on the intra- and interexaminer
reproducibility of the
gingival bleeding when using a
well-defined bleeding index. Four
different variations were defined:
parallel bleeding index (ParBI):
when the probe is run along the
marginal gingiva it is held parallel
to the tooth surface. Angulated
bleeding index (AngBI): the probe is
held at an angle of approximately
60° to the longitudinal axis of the
tooth and in contact with the
sulcular epithelium. Controlled
force parallel bleeding index (CFParBI)/
controlled force angulated
bleeding index (CF-AngBI): the
same procedure as described for
the Par BI and AngBI is followed,
but instead of a conventional
(WHO-ASH/Dentsply®) probe an
adjustable force probe (Brodontic®-
ASH/Dentsply), adjusted to 0.25N is
used. The results show that the
agreement both within and
between clinicians could hardly be
rated as ‘fair’ on a site basis.
Approximately one in every three sites
was given a different assessment on
the second examination. Agreement
on a quadrant basis was slightly
better. Furthermore, the direction
of the probing influences the
number of provoked bleeding sites.
Angulation of the probe produces
consistently less bleeding compared
with parallel placement of the
probe.
IMAGEM 4
Comparison of a conventional
and modified tooth stain index
Macpherson LMD*, Stephen KW*,
Schæfer F, Huntington E and
Joiner A
(*University of Glasgow Dental
Hospital and School, Glasgow UK)
J Clin Perio 27,(11), p854, 2000
A modified tooth stain index was
evaluated and the effect of dietary
factors on stain formation
determined. At baseline, 182 adult
volunteers were given a full oral
prophylaxis followed by stain
assessment using a modification of
the Lobene Index. This involved
assigning separate scores to the
mesial (M) and distal (D) sites of
each tooth, in addition to the
standard gingival area (G) and
tooth body (B). For each site, stain
intensity and areas were scored.
The sum of (intensity x area) scores
was calculated for all sites (GBMDvalue)
and for the gingival and
body sites only (GB-value). The
subjects used a standard abrasive
dentifrice and a soft toothbrush for
6 weeks. Stain was reassessed and
the average increase in GBMDvalue
was 20.9 (sd=9.9) (buccal
aspect) and 29.9 (sd=18.0) (lingual
aspect). The corresponding GBvalues
were 4.4 (sd=4.8) and 12.5
(sd=10.2). The coefficients of
variation of the GBMD index values
were consistently lower than those
of the GB values. Smoking and
tea-drinking were found to be the
only significant (p<0.001) factors
investigated for stain formation. It
is concluded that the modified
index may be well advantageous to
its conventional counterpart
because sites with most visible stain
are assessed separately and
because it may also offer higher
discriminatory power due to a lower coefficient of variation.
Laboratory Studies
Comparison of methods for
determining the prosthetic
metal ion of bacterial
superoxide dismutases in crude
extracts
McEuen AR* Hill HAO* Dring GJ
and Ingram GS
(*University of Oxford)
Dev Biochem 1980, 11a, 272-283
Three different types of superoxide
dismutase (SOD) have been
characterised on the basis of the
prosthetic metal ion. The copper
and zinc containing enzyme, with
two exceptions has been found
exclusively in eukaryotes, the
manganese-containing enzyme has
been found in both prokaryotes
and eukaryotes, and the ioncontaining
enzyme has been found
predominantly in prokaryotes,
though it has recently been
reported in eukaryotic algae and
higher plants. The manganese and
iron super-oxide dismutases are
closely related on the basis of
amino and sequence homology,
while the cuprozinc enzymes form
a phylogenetic class of their own.
Presented here are three methods
for the identification of the
prosthetic metal ion and superoxide
dismutases; the merits and
limitations of each method are
discussed.
Studies of a periodontal tissue
lesion in the rat treated or
untreated with chlorhexidine
digluconate
Kenworthy R and Baverel M
J
Clin Periodontol 1981, 8,
349-358
The studies currently reported
formed part of an enquiry whose
overall objective was to identify a
suitable animal model upon which
initial screening of compounds and
formulations with prophylactic
and/or therapeutic potential in
periodontitis could be carried out.
An earlier study (Rovin et al., 1966)
reported that periodontal
inflammation could be produced in
the conventional laboratory rat by
application of a ligature to the
molar teeth. The present paper
describes the pathological and
histo-pathological changes in the
periodontium following application
of ligatures to the molar teeth of
young rats, and the tissue
responses observed after topical
application of chlorhexidine
digluconate. Plaque formed rapidly
and an acute periodontitis was
induced; application of increasing
concentrations of chlorhexidine
digluconate gave a progressive
diminution in the severity of the
lesions. The major bacterial
components of the plaque were
actinomycetes and streptococci.
Zinc and its role as an antimicrobial
agent
Cummins D
Proceedings XXIV ICCC, Chemica
Chronika 1986 (special issue), 902
Effect of speciation of zinc on
metabolism of streptococcus
mutans
Watson GK, Jones S, Prottey C and
Cummins D
J Dent Res 1986, 65, 96
Metal ions and their
interactions with biological
fluids: speciation of trace metals
in saliva
Hurford, SR*, Christie GL*,
Williams DR*, Cordery CS,
Cummins D and Riley Pl
(*Department of Applied
Chemistry, UWIST, Cardiff, UK)
Proceedings XXIV ICCC, Chemica
Chronika 1986 (special issue), 738
The importance of the
speciation of zinc to its activity
as an anti-microbial agent
Cummins D, Jones S and
Watson GK
Proceedings ICBIC 3, Recueil
1987,106, 435
A comparison of calculus
inhibitory systems
Ingram GS and Carter P
J Dent Res 1987, 66, 191
The inhibition of facultative and
obligate anaerobic bacteria by
Triclosan
Ritchie JA and Jones CL
Anaerobes Today 1988, 240
Edited by JM Hardie and SP
Borriello
Importance of speciation of zinc
to its antimicrobial activity
Cummins D and Watson GK
J Dent Res 1988 (special issue), 67,
402
The effect of zinc and citrate
salts on hydroxyapatite
formation
Harrap GJ
J Dent Res 1988, 67, 320
Effects of anticalculus agents on
hydroxyapatite crystal growth
Ingram GS and Carter P
Caries Res 1988, 22,101
Use of a zinc-containing
dentrifice: Uptake of zinc by
hydroxyapatite and by dental
calculus
Ingram GS, Tan-Walker R, Gilbert
RJ and van der Ouderaa FJG
J Dent Res 1988, 67 (special issue),
402
The effect of zinc citrate on oral
bacteria in continuous culture
Jones CL, Purdell-Lewis D and
van der Ouderaa FJG
J Dent Res 1988 (special issue), 67,
327
Adsorption of zinc by plaque
and hydroxyapatite
Ingram GS, Tan-Walker R, Watson
GK and Gilbert RJ
Caries Res 1989, 23, 106
Delivery Systems for Agents in
Supra- and Sub-gingival Plaque
Control
van der Ouderaa F J G and
Cummins D
J Dent Res 1989, 68, 1617-1624
Delivery vehicles for anti-plaque
agents function to provide a
microbiologically, chemically, and
physically stable environment for
the drug prior to use, to optimize
bio-availability at the site of action
in an appropriate physical and
chemical form, to promote uptake
at oral receptor sites, and to
provide a dosage vehicle
compatible with the oral hygiene
methods and the expected degree
of compliance of the patient.
Criteria for delivery in most
reported studies relate primarily to
gross microbiological and clinical
parameters rather than to
concentrations of the drug
delivered. Few reports identify the
nature of the oral reservoirs of any
of the individual anti-plaque agents
studied so far. Comparison of the
effectiveness of delivery of single
agents via different routes is a
prerequisite to the optimization of
effective delivery vehicles for
specific applications, and yet
surprisingly few data of this type
exist. Key factors which affect the
delivery and, hence, efficacy of
anti-plaque agents in vivo include
the pH and state of ionization of
the drug, the solubility and rate of
solubilization of the drug (both in
the vehicle and at the site of
action), physical and chemical
interactions between the drug and
its excipients, and last, but not
least, drug dosage and dosing
protocol.
Many vehicles have been
successfully used for administration
of doses of topical agents of widely
differing chemical natures for
supragingival plaque control.
Examples are alcoholic rinses,
dentifrices, aqueous and nonaqueous
gels, impregnated dental
floss, chewing gum, irrigators, and,
recently, sustained-release devices.
Sub-gingival plaque control has
traditionally been achieved by
systemic administration of drugs
such as tetracycline and
metronidazole. Delivery to the oral
cavity is thus obtained via gingival
sulcus fluid and saliva. Experimental
systems for direct delivery into
periodontal pockets via dialysis
tubing or acrylic strips have also
been described, with some groups
recently reporting biodegradable
controlled-release devices. In
addition, irrigators have been used.
These local delivery systems have
the potential to deliver the antiplaque
effect at much lower total
body load of the drug.
Differences between the
distribution of epidermal
growth factor receptor in
human skin and oral mucosa,
detected by immunohistology
and EGF binding studies
Partridge M*, Smith CG and
Green MR
(*Charing Cross Sunley Research
Centre, Lurgan Avenue,
Hammersmith, London)
Epithelia 1989, 1, 179-190
The localisation of the EGF-receptor
in normal human skin and oral
mucosa from different sites has
been determined using two
different light microscopic methods
and an immuno-ultrastructural
technique. Immunoreactive
receptors were localised using a
monoclonal antibody against the
human receptor. Accessible,
unoccupied receptors were
visualised by 125I-EGF binding
studies. Basal cells were always
found to express EGF-receptors,
while suprabasal cells showed
equivalent, variable or no receptor
expression. As cell proliferative
capacity is predominantly
associated with basal cells, it is
concluded that the expression of
EGF-receptors is a necessary but
insufficient indicator of cell
proliferative potential. Marked
tissue differences in the patterns of
EGF-receptor expression on
epidermis, orthokeratinised,
parakeratinised and nonkeratinised
oral mucosa were consistently
observed. Explanations for these
variations are discussed, and it is
proposed that the pattern of EGFreceptor
expression may be related
to the type of epithelial
keratinisation. Nuclear EGFreceptors,
predominantly associated
with chromatin, were seen using
immunoelectron microscopy in all
tissues examined suggesting a role
for the EGF-receptor in gene
regulation.
Computer model relating
chemistry to biological activity
of metal anti-plaque agents
Cummins D and Watson GK
J Dent Res 1989, 68, 1702-1705
It is clear from literature reviews
that the clinical and microbiological
activity observed for zinc, copper,
tin and silver compounds are
variable and not well understood.
We propose that much of this
variation in biological response can
be rationalized if due account is
taken of the chemistry of the metal
ion.
The use of computer simulation
offers a new approach to
understanding of the delivery,
distribution and bio-availability of
metal ions. Application to studies
on the distribution of stannous in
solution in the presence of fluoride,
and the importance of zinc
speciation for antimicrobial action
of zinc have demonstrated the
value and the power of computer
simulation to aid our
understanding of the action of
metal-based anti-plaque agents.
Anti-bodies for the detection of
proteoglycan fragments from
alveolar bone
Al Bayathy H*, Embery G*,
Waddington RJ* and Harrap G
(*Department of Basic Dental
Science, University of Wales,
Cardiff)
J Dent Res 1990, 69, 980
Antibacterial testing of metal
ions using a chemically defined
medium
Ritchie JA and Jones CL
Letters in Applied Microbiology
1990, 11, 152-154
Data from in-vitro tests on
potential germicides can be greatly
influenced by the culture medium.
The bioavailability and biochemical
reactivity of the biocides can be
influenced by chemical interference
with media components (Spooner
& Sykes, 1972). Bird et al (1985)
showed that metal ions are
particularly prone to chemical
interferences. A chemically defined
solid medium has been developed
to monitor the antibacterial activity
of metal ions. The minimum
inhibitory concentrations of zinc
and silver have been determined
against a range of bacteria using
this medium.
Crevicular Fluid, Interleukin-1B
and 1gG Subclasses in
Periodontitis
Wilton J*, Bampton J*, Hurst T**,
Powell J and Caves J
(*Medical Research Council Dental
Unit, London
**London Hospital Dental School,
London)
J Dent Res 1991, 70, 353
Crevicular Fluid IgG Subclasses
in Health and Periodontitis
Powell J, Alexander A* and
Smales F**
(*Institute of Naval Medicine,
Gosport, Hampshire, UK)
(**London Hospital Medical
College, London, UK)
J Dent Res 1991, 70,353
Comparison Between CLSM and
SE for the Study of Plaque
Jones CL, Cummins D, Cummins PG
and Howard CV*
(*University of Liverpool, UK)
J Dent Res 1991, 70, 368
Anti-plaque Agents. Rationale
and Prospects for Prevention of
Gingivitis and Periodontal
Disease
van der Ouderaa FJG
J Clin Periodontol 1991, 18,
447-454
Oral health surveys have shown
that even in countries with
established patterns of oral hygiene
habits, most individuals have
relatively poor gingival health. This
is due to a low interest in
complying with oral health
procedures. A number of factors
are apparent when investigating
compliance to oral hygiene habits,
viz only approximately 50% of the
population brushes twice a day or
more, brushing time is probably
much too short and use of dental
floss is not very prevalent. Studies
of the effect of motivation on oral
hygiene suggest that improvements
can be achieved, but these are not
maintained unless motivation is
continuously reinforced. This
suggests that topically applied antiplaque
agents should be used to
augment mechanical plaque
control. A number of product
forms are available to delivery antiplaque
agents i.e., mouthrinses,
dentifrices, aqueous gels, and
additionally floss, chewing gum
and lozenges. Any product form
should provide a physically,
chemically and microbiologically
stable environment for the agent
concerned. It should facilitate
optimal bioavailability of the agents
at the site of action and encourage
patient compliance. Anti-plaque
agents for topical administration
should have the following
properties: high intrinsic efficacy
against a broad spectrum of oral
organisms, toxicological and
ecological safety, oral substantivity,
no adverse reactions and good
chemical stability. A number of
classes of anti-plaque agents have
been identified such as positively
charged organic molecules, metal
salts, phenols, enzymes, peroxides,
sugar substitues, fluorides and
surface modifying agents. In order
to achieve optimal bioavailability,
the agent to be dosed should be
compatible with the product form
used. The two major product forms
are rinses and dentifrices. With
respect to rinses as the product
form, the formulation excipients
are generally compatible with
positively charged organic agents,
phenolic agents and some metal
salts. In the case of dentifrice
formulations, interactions of the
agent with the product excipients
limit compatibility to some metal
salts and non-charged phenolic
agents, Triclosan being a well
researched example. The
combination of metal salts, in
particular zinc, and the phenolic
agent Triclosan has been shown to
represent a further powerful route
to enhance systems for daily
plaque control.
Anti-plaque Dentifrices: Current
Status and Prospects
van der Ouderaa FJG and
Cummins D
Int Dent J 1991, 41, 117-123
A dentifrice can, in principle, be an
effective delivery vehicle for antiplaque
agents, provided
bioavailability of the agent
concerned can be achieved,
because the agent is applied and
delivered at the site of action and
favourable patient compliance can
be obtained. It is widely assumed
that anti-plaque agents need to be
retained in the oral cavity after
application (‘substantivity’). To use
a dentifrice as a dosing vehicle
successfully the anti-plaque agents
have to be compatible with the dentifrice formulation to achieve
release from the formulation and
subsequent retention of the antiplaque
agent after application.
Recently, progress has been
achieved with a selection of
compatible anti plaque agents for
inclusion in a dentifrice such as
metal ions and non-charged
phenolic agents. Studies on longterm
unsupervised brushing have
shown favourable anti-plaque and
anti-gingivitis activity from antiplaque
agents dosed from a
dentifrice.
Routes to Chemical Plaque
Control
Cummins D
Biofouling 1991, 4, 199-207
A logical approach to the
prevention of periodontal disease is
through excellent supragingival
plaque control. Such control is not
generally achieved by mechanical
oral hygiene procedures alone.
Thus, there is a clear rationale for
the use of anti-plaque agents to
augment mechanical means. The
principal routes to chemical plaque
control are to prevent colonization
of the tooth surface, to inhibit the
growth of microorganisms, to
prevent plaque maturation, to
modify plaque biochemistry and to
modify plaque ecology to a less
pathogenic flora. The most
successful approach to date has
been to use broad spectrum
antimicrobial agents to reduce
plaque growth and, in
consequence, plaque metabolism.
Currently, only the chlorhexidine
and Listerine mouthwashes and a
toothpaste containing zinc citrate
and Triclosan have proved efficacy
in controlled clinical trials. Their
effectiveness is a result of good
oral substantivity as well as broad
spectrum antimicrobial activity.
All other routes to chemical plaque
control are in the early stages of
development. Several approaches
have shown sufficient
potential to warrant further
research. As understanding of the
role of specific plaque
microorganisms in the aetiology of
disease increases, more rational,
sophisticated approaches to
chemical plaque control become
feasible. In particular, routes to
control specific pathogenic
organisms may become a reality.
Anti-plaque agents for Supragingival
Plaque Control –
Rationale and Outlook
van der Ouderaa FJG
Recent advances in
Periodontology II
Eds: Gold S I, Midda M and
Mutlu S
Excerpta Medica Amsterdam 1991,
129-136
Agents for daily use to control
plaque and gingivitis have been
successfully formulated in product
systems such as dentifrice and
mouthrinses. A number of rinse
and gel systems containing
positively charged agents are
available. These agents are
however incompatible with
dentifrices. In contrast metal salts
and non-charged agents, such as
Triclosan, are bio-available from this
vehicle. Good results have also
been obtained using a combination
of two agents i.e., Triclosan and
zinc citrate.
Thermodynamic Formation
constants and Solid State
Properties for the Interaction of
Zn(II) Ions with Citrate,
Phenolsulphate,
Monofluorophosphate and
Saccharinate
Christie GL**, Cummins D, Duffield
JR, Hurford SR***, Morris CR*,
Riley PI, Vesay JA* and
Williams DR*
(*School of Chemistry, University of
Wales, Cardiff, UK)
(**Kingston Polytechnic, Surrey,
UK) (***Department of Chemistry,
Manchester Polytechnic,
Manchester, UK)
J Inorg Biochem 1991, 42,
273-287
The role of zinc as a therapeutic
ingredient of dentifrices is reviewed
and chemical speciation modelling
is suggested as a means of
optimizing the beneficial effects.
The solid state chemistry of zinc
citrate, monofluorophosphate, and
saccharinate is reported as FTIR
spectra. Concentration formation
constants were measured by
solution potentiometry.
Inhibition of Acid Production by
Streptococcus Mutans NCTC
10449 by Zinc and the Effect of
Metal Speciation
Watson GK, Cummins D and
van der Ouderaa F J G
Caries Res 1991, 25, 431-437
Ionic zinc as zinc sulphate was
strongly inhibitory to acid
production from glucose by
washed cell suspensions of
Streptococcus mutans NCTC 10449
in a pH-stat assay (50% inhibition
at 0.1mM zinc). Zinc was adsorbed
to the cells (up to 8µg/mg cells).
Several other zinc salts that
dissociated to free zinc ions were
also strongly inhibitory. Zinc in a
partially complexed form as zinc
citrate was less strongly inhibitory
(50% inhibition at 0.2mM zinc),
and adsorption of zinc was lower
(up to 3µg/mg cells). Stoichiometric
addition of ethylenediaminetetraacetic
acid (EDTA) to assays
containing zinc sulphate completely
removed inhibition and adsorption
of zinc. A good correlation
between inhibition of acid
production and adsorption of zinc
was found, and zinc adsorption
was correlated with the levels of
free zinc ion in the assay mixtures.
The strongly anionic complexes of
zinc, Zn(CIT)-, Zn(CIT)42_ and
Zn(EDTA)2–, were not inhibitory
and did not adsorb to the bacterial
cells.
Anti-calculus effect of a
dentifrice containing 0.5% zinc
citrate trihydrate
Segreto VA*, Collins EM*,
D’Agostino R**, Cancro LP**,
Pfeifer HJ*** and Gilbert RJ
(*University of Texas Health Science
Centre, San Antonio, TX)
(**Department of Mathematics,
University of Boston, Boston, MA)
(***4230 Secor Road, Toledo, OH,
USA).
Community Dent Oral Epidemiol,
1991, 19, 29-31
A double blind three-month
anticalculus clinical study was
conducted to determine the
magnitude of the anti-tartar effect
to be derived from use of a 0.5%
zinc citrate dentifrice. From a
population of 1600 subjects
exhibiting calculus, 1210 subjects
(aged 18 and over) were selected
and received a dental prophylaxis.
The subjects were initially stratified
on the basis of calculus score, age,
and sex, then allocated randomly
to a 3-month usage period of one
of two treatment groups. These
were a dentifrice containing 0.5%
zinc citrate or a control dentifrice
without zinc citrate. Calculus was
assessed using the Volpe-Manhold
Index. Oral soft tissue status was
assessed throughout the course of
the study. At the conclusion of the
study 964 subjects had completed
the trial: 486 using the zinc citrate
dentifrice and 478 using the
control. The results showed that
the mean calculus scores in the
group using the dentifrice
containing zinc citrate were 13.7%
lower than those of the group
using the control dentifrice. This
finding was statistically significant
(p<0.05). No adverse effects related
to the use of either dentifrice were
observed.
Anti-microbial activity of
stannous and stannic
Watson GK, Waterfield PC and
Cummins D
J Dent Res 1992, 71, 708
Identification of salivary zinc
binding proteins
Davey H*, Embery G*, Cummins D
and Weir AJ
(*Department of Basic Dental
Science, University of Wales Coll
Med Dent School, Cardiff, UK)
J Dent Res 1992, 71, 323
Effect of pre-treatment with
saliva or glucosyltransferases on
biofilm development
Bradshaw DJ*, Marsh PD*, Watson
GK and Cummins D
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1992, 71, 731
Pedocin PA-1 from pediococcus
acidilactici PAC1.0: mode of
action and genetics
Chikindas ML, Garcia MJ*, Driessen
AJM*, Marugg JD, Ledeboer AM,
Abee T*, Konings WN*, Venema
G, Kok J*
(*Departments of Genetics and
Microbiology, University of
Groningen, The Netherlands)
FEMS Microbiology Reviews, 1993,
12, 123
Bacterial adhesion to apatite
materials: Electrokinetic aspects
Yelloji Rao MK*, Somasundaran
P*, Schilling KM, Carson R and
Ananthapadmanabhan KP
(*Henry Krumb School of Mines,
Columbia University, NY, USA)
Colloid Surfaces A: Physico Chem
1993, 79, 293-300
The role of the electrokinetic
properties of Streptococcus sanguis
and Actinomyces naeslundii in
determining their adhesion to
apatite minerals is examined. It is
shown that bacterial adhesion onto
a mineral surface takes place even
when both the surfaces are
negatively charged, and that the
adhered layer is resistant to
washing. Also, the mineral fines
after exposure to bacteria exhibit
zeta potential values which are in
between those of the mineral and
the bacteria, but lie closer to that
of the mineral. In the absence of salivary proteins, the inorganic
species present in saliva alter the
magnitude of the surface charge
but do not affect the bacterial
adhesion process. Dissolved apatite
mineral species (in supernatant
solutions) are also shown to
decrease the magnitude of the zeta
potential of the bacteria. The
observed adsorption of negatively
charged bacteria onto negatively
charged minerals suggests that
electrostatic interactions are not
the primary factors determining
adhesion.
The effects of Triclosan and zinc
citrate, alone and in
combination, on a community
of oral bacteria grown in vitro
Bradshaw DJ*, Marsh PD*, Watson
GK and Cummins D
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1993, 72, 25-30
A mixed-culture chemostat system
has been used as a more stringent
laboratory system for evaluation of
the antimicrobial effects of
Triclosan and zinc citrate. The
inhibitors were added alone, and in
combination, as a pulse (a high
initial inhibitor concentration which
decreased with time) or as a dose
(concentration of inhibitor
increased with time) to give
maximum concentrations of
34.5µmol/L Triclosan and
39.8µmol/L zinc citrate. When
dosed, Triclosan inhibited A.
viscosus and all five Gram-negative
species, whereas zinc citrate had
less effect, probably due to
complexation by media
components. Similar effects were
seen when Triclosan was pulsed,
except that S. mutans was the
most sensitive Gram-positive
species and V. dispar was
unaffected. However, when the
inhibitors were dosed or pulsed in
combination, marked
complementary and additive
inhibitory effects were observed,
particularly against Gram- and
negative species, although S.
gordonii S. oralis were relatively
unaffected. The data confirm that
increased effects can be obtained
with suitable combinations of
antimicrobial agents and suggest
that, under certain conditions, apparently broad-spectrum
antimicrobial agents may be acting
more selectively than hitherto
suspected.
Effect of oxygen and innoculum
composition on development of
mixed culture biofilms
Bradshaw DJ*, Marsh PD*, Watson
GK and Schilling K
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1994, 73, 849
Effect of pre treatment with
parotid saliva in the
development of mixed culture
systems
Bradshaw D*, Marsh PD*, Watson
GK and Schilling K
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
Caries Res 1994, 28, 182.
The targetting of phospholid
liposomes to bacteria
Jones M*, Kaszuba M*, Reboiras
MD**, Lyle IG, Hill KJ*, Song YH*,
Wilmot SW* and Creeth JE
(*School of Biological Sciences,
University of Manchester
**Depatmento de Quimica,
Faculdad de Ciencias, Universidad
Autonoma de Madrid)
Biochimica et Biophysica 1994,
1196 (1), 57-64
Phospholipid liposomes have been
prepared from phospholipid
mixtures including
dipalmitoylphosphatidylcholine/pho
sphatidylinositol (DPPC/PI) and
DPPC/ dipalmitoylphosphatidylglycerol
(DPPC/DPPG) mixtures and
targeted to adsorbed biofilms of
the skin-associated bacteria
Staphylococcus epidermidis and
Proteus vulgaris and the oral
bacterium Streptococcus sanguis.
The effects of time, liposome
concentration and density of
bacteria in the biofilm have been
studied in detail for Staphylococcus
epidermidis. The targeting (as
assessed by the apparent
monolayer coverage of the biofilms
by liposomes) to the biofilms was
found to be sensitive to the mol%
of PI and DPPG in the liposomes
and optimum levels of PI were
found for targeting to each
bacterium. The use of PI and DPPGcontaining
liposomes for the
delivery of the bactericide,
Triclosan, to biofilms of
Staphylococcus epidermidis was
studied as a function of the
amount of Triclosan carried by the
liposomes. All the liposome systems
tested inhibited the growth of
bacteria from the biofilms after
brief (two min) exposure to
Triclosan-carrying liposomes. At low
Triclosan levels bacterial growth
inhibition by Triclosan-carrying
liposomes exceeded that by an
equivalent level of free Triclosan.
After short periods (min) of
exposure of biofilms to Triclosancarrying
liposomes the bactericide
was shown to preferentially
concentrate in the biofilms relative
to its liposomal lipid carrier. The
results suggest that phospholipid
liposomes with appropriately
chosen lipid composition have
potential for the targeting and
delivery of bactericide to bacteria.
Immunological characteristics of
a synthetic peptide associated
with a catalytic domain of
mutans streptococcal
glucosynltransferase
Smith DJ*, Taubman MA*, King
WF*, Eida S, Powell JR and
Eastcott J
(*Department of Immunology,
Forsythe Dental Centre,
Boston, USA)
Infection and Immunity 1994, 62,
5470-5476
The immunogenicity of a multiple
antigenic peptide construct
consisting of four copies of the
synthetic 21-mer peptide
DANFDSIRVDAVDNVDADLLQ was
measured. The composition of this
peptide was derived from a
sequence in the N-terminal region
of mutans streptococcal
glucosyltransferases (GTFs)
containing an aspartic acid
implicated in catalysis. The peptide
(CAT) construct was synthesized as
a tetramer on a lysine backbone
and subcutaneously injected into
Sprague-Dawley rats for polyclonal
antibody formation or
intraperitoneally injected into
BALB/c mice, and then spleen cell
fused with Sp2/0Ag14 murine
myeloma cells for monoclonal
antibody formation. The resulting
rat antisera and mouse monoclonal
antibodies reacted with CAT and
with native GTF isozymes from
Streptococcus sobrinus and
Streptococcus mutans (in enzymelinked
immunosorbent assay and
Western blot [immunoblot]
analyses). Functional inhibition of
the water-insoluble glucan
synthetic activity of S. sobrinus
GTF-1 was demonstrated with an
immunoglobulin M anti-CAT
monoclonal antibody (>80%
inhibited) and with rat sera
(approximately 17% inhibited). The
monoclonal antibody preparation
also modestly inhibited the watersoluble
glucan synthetic activity of
an S. mutans GTF mixture. These
results suggest that the CAT
peptide contains B-cell epitopes
that are similar to those of intact
mutans streptococcal GTFs and has
the potential to elicit antibody that
can inhibit GTF function. Thus,
sequences within this peptide
construct may have value for
inclusion in a synthetic dental caries
vaccine.
Effect of oxygen and flow-rate
on bacterial survival and biofilm
development
Bradshaw DJ*, Marsh PD*, Watson
GK, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1995, 74, 853
An in vitro biofilm system for
study of plaque ecology and
physiology
Watson GK, Halliday D, Albiston L,
Singleton S, Allison C
J Dent Res 1995,74, 853
Identification of dissimilatory
sulfate reduction in the human
oral cavity
Holt J, Allison C, Willis CL*,
Gibson GR*
(*Institute of Food Research,
University of Reading,
Reading, UK)
J Dent Res 1995, 74, 846
Optical Imaging and
characterisation of oral biofilms
structures using vital stains and
specific antibody probes
Singleton S, Albiston L, Treloar R,
Mahers E, Hodgson R, Watson G K,
Schilling K and Allison C
The Life and Death of Biofilm Eds:
Wimpenny J, Handley P, Gilbert P
and Lappin- Scott H Bioline, Cardiff
1995, 33-36
Oral biofilms exhibit a diversity of
physiological and biochemical
characteristics super-imposed on a
complex structure. A prerequisite
for studies of the role of biofilm
microstructure in biofilm-specific
activities is a facility for
non-destructive analysis. In this
work confocal laser scanning
microscopy (CLSM) has been used
in conjunction with vital stains and
species-specific monoclonal
antibody (Mab) probes to
investigate oral biofilm structures
formed in vitro. Methods have
been developed for the application
of optical microscopy
in combination with fluorescent
probes and image analysis
programs to investigate early
colonisation of model surfaces by
oral bacteria. In particular,
sampling of relatively large areas
may provide information on
changing patterns of colonisation
by specific organisms against the
total biofilm biomass and a
parameterised description of the
process as a function of time and
spatial distribution. Studies
undertaken using 3D image sets
generated by CLSM of smaller
representative samples have
demonstrated (i) confocal imaging
and enumeration of putatively
viable organisms in 3D structures,
(ii) confocal imaging and
enumeration of Mab-labelled
bacteria in biofilms and (iii)
parameterisation of 3D structural
distribution characteristics of
representative biofilm segments.
These methods form the basis of
fundamental investigations into
biofilm structure and the
development of an ability to characterise biofilm structures using
statistically relevant data.
IMAGEM 5
The effect of conditioning films
on adhesion
Bradshaw DJ*, Marsh PD*, Watson
GK and Schilling K
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
The Life and Death of Biofilm
Eds: Wimpenny J, Handley P,
Gilbert P and Lappin-Scott H
Bioline, Cardiff 1995, 47-52
A well-characterised chemostat
system, containing 10 oral bacteria,
has been modified to allow the
study of biofilms growing on the
surface of hydroxyapatite (HA)
discs. This paper describes
experiments examining the effect
of pre-treatment of HA discs with
bacterial supernatants containing
glucosyltransferase (GTF activity,
human parotid or whole saliva, on
the development of these oral
biofilms. Treatment with crude GTF
preparations from Streptococcus
mutans or from S. sanguis
produced increases in the total CFU
recoverable from discs, in
particular numbers of P. gingivalis
and S. mutans. In addition, S.
sanguis GTF enhanced numbers of
F. nucleatum, V. dispar and S.
sanguis cells in biofilms,
while treatment with S. mutans
GTF produced little effect on these
species. Parotid saliva reduced
streptococci, but enhanced
numbers of F. nucleatum in 1 hour
biofilms; no effects on total
numbers or on individual species
were apparent after 4 days. Whole
saliva had little effect on total
numbers in biofilms, but did have
selective effects. Numbers of
streptococci were increased, and
Gram
Growth, incidence and activities
of dissimilatory sulfate-reducing
bacteria in the human oral
cavity
Willis CL*, Gibson GR*, Allison C,
Macfarlane S* and Holt JS
(*Dunn Clinical Nutrition Centre,
University of Cambridge, UK)
FEMS Microbiology Letters 1995,
129, 267-272
Viable counts and activities of
sulfate-reducing bacteria were
determined in the oral cavities of 12
healthy volunteers. Of these, 10
harboured viable sulfate-reducing
bacteria populations. Six separate
sites were sampled: the posterior
tongue, anterior tongue, mid buccal
mucosa, vestibular mucosa,
supragingival plaque and subgingival
plaque. Sulfate reducing bacteria
occurred in all areas, with the highest
incidence in supragingival plaque.
Viable counts and sulfate-reducing
activities in each of the regions varied
from 0 to 108cfu (g wet weight)-1
and from 0 to 50nmol (g wet
weight)-1 h-1, respectively. As
sulfate-reducing bacteria can be
detected in the oral cavity, they may
potentially be involved in terminal
oxidative processes carried out by the
microflora of the mouth.
Requirement for FlhA in flagella
assembly and swarm-cell
differentiation by Proteus
Mirabilis
Gygi D*, Bailey MJ*, Allison CA
and Hughes C*
(*Department of Pathology,
University of Cambridge,
Cambridge, UK)
Molecular Microbiol 1995, 15,
761-9
Swarming by Proteus mirabilis is
characterized by cycles of rapid
population migration across
surfaces, following differentiation
of typical rods into long, aseptate
swarm-cells that over express
flagella and virulence factors,
particularly haemolysin. A
non-swarming Tn5phoA mutant
was unable to synthesize flagella,
to fully elongate or to induce high
levels of the toxin. The mutation
lay within a 2091bp gene encoding
a homologue of the Escherichia coli
FlhA belonging to a family of
proteins that are required for
assembly of flagella or virulence
proteins and that are suggested to
act either directly in membrane
translocation and/or in regulating
synthesis of the export apparatus.
In trans expression of multicopy
FlhA restored cell elongation and
migration and generated
differentiation-specific
hyperexpression of flagellin and
toxin genes to levels above those
seen in the wild-type strain.
Transcription of FlhA was strongly
induced during differentiation,
from its own putative sigma 28
promoter. The results suggest a
mechanistic coupling of flagella
assembly and swarm-cell
differentiation.
Coaggregation between
Prevotella nigrescens and
Prevotella intermedia with
Actinomyces naeslundii strains
Cookson AL*, Handley PS*, Jacob
AE*, Watson GK and Allison C
(*Dept. Microbiology, University of
Manchester, Manchester, UK)
FEMS Microbiology Letters 1995
132, 291-296
Using a visual coaggregation assay,
43% (6 of 14) of Prevotella
nigrescens and 50% (4 of 8) of
Prevotella intermedia strains
coaggregated with Actinomyces
naeslundii strains which
represented the six Actinomyces
coaggregation groups (A to F). For
both species, coaggregation
occurred most frequently with A.
naeslundii strains from
coaggregation groups C, D and E.
No coaggregation was observed
with Actinomyces israelii,
Actinomyces odontolyticus or the
six oral Streptococcus species.
Coaggregation was not
inhibited by lactose, saliva or
serum. Pretreatment of Prevotella
strains with heat, SDS and
proteinase K abolished
coaggregation when the treated
cells were added to untreated
Actinomyces strains. The same
pretreatment of the Actinomyces
strains had no effect on their ability
to coaggregate with untreated
Prevotella strains. Pretreatment of
all coaggregating P. nigrescens
strains with trypsin abolished
coaggregation, whereas the
coaggregation ability of the P.
intermedia and Actinomyces strains
was resistant to trypsin
pretreatment. Pretreatment of the
strains of both Prevotella species
and the Actinomyces with
periodate abolished coaggregation
in all cases. These results suggest
that the Prevotella strains each
possess a protein coaggregation
adhesin, which for the P.
intermedia strains is resistant to
trypsin, that interacts with a
non-protein receptor on the A.
naeslundii strains.
A modified chemostat system to
study the ecology of oral
biofilms
Bradshaw DJ*, Marsh PD*,
Schilling KM and Cummins D
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Appl Bact 1996 80, 124-130
Previously, we developed a
chemostat system to study the
behaviour and properties of a
community of up to 10 species of
oral bacteria. The present study
describes modification of this
system to incorporate removable
and replaceable hydroxyapatite (the
major mineral in human dental
enamel) disks on which biofilms
could develop. Hydroxyapatite disks
were immersed in the chemostat
for known time periods, and the
bacterial content of biofilms
determined by viable counting.
Initial deposition rates were rapid,
with all 10 species detected after
1h, and the numbers of bacteria in
biofilms continued to increase for
21d. The species composition of
biofilms reflected that of the
surrounding fluid phase, and
showed only limited signs of the
type of ‘species succession’ which
is observed in developing dental
plaque in vivo, although anaerobic
species increased in proportion in
older biofilms. Four-day biofilms
showed the least variability and
were chosen as the ‘standard
biofilm’ for more detailed study.
Variability in the bacterial
composition of 4-d biofilms was
comparable both within a single
chemostat run and between
independent chemostat runs.
Glucose pulsing in the absence of
pH control resulted in the selection
of cariogenic species; the
disruption of the biofilm
community was less marked than
that of the equivalent planktonic
culture. The model system has
considerable potential in studying
the effects of a variety of factors
on biofilm development, as well as
in comparing the efficacy of
antimicrobial systems against
biofilms.
Effect of oxygen, inoculum
composition and flow rate on
development of mixed
culture oral biofilms
Bradshaw DJ*, Marsh PD*,
Allison C, Schilling K M
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
Microbiology 1996,142, 623-9
The effect of aeration on the
development of a defined biofilm
consortium of oral bacteria was
investigated in a two-stage
chemostat system. An inoculum
comprising 10 species, including
both facultatively anaerobic and
obligately anaerobic bacteria, and
species associated with oral health
and disease, was inoculated into an
anaerobic first-stage chemostat
vessel. The effluent from this
chemostat was linked to an
aerated [200ml C02/air (5:95v/v)
min-1] second-stage vessel, in
which removable hydroxyapatite
discs were inserted to allow biofilm
formation. Comparisons were
made of planktonic and biofilm
communities in the aerated
second-stage vessel by means of
viable counts. Both planktonic and
early biofilm communities were
dominated by Neisseria subflava,
comprising >40% of total c.f.u. in
the fluid phase, and >80% of c.f.u.
in 2h biofilms. Obligate anaerobes
persisted in this mixed culture, and
succession in biofilms led them to
predominate only after 7d. Despite
the continuous addition of air, the
dissolved oxygen tension (dO2)
within the culture remained low
(<5% of air saturation), and the
redox potential (Eh) was -275mv. In
order to assess the significance of
the presence of N. subflava in
community development, a
subsequent experiment omitted
this aerobe from the inoculum, to
produce a nine-species culture. The
planktonic phase was
predominated by three
streptococcal species, Prevotella
nigrescens and Fusobacterium
nucleatum. Biofilms again
underwent successional changes,
with anaerobes increasing in
proportion with time. In contrast to
the culture including
N. subflava, dO2 was 50-60% of
air saturation, and the Eh was
+50mV. In the final experiment, the
rate of addition of first-stage
culture was reduced to 1/10 of that
in the previous experiment, in order
to determine whether anaerobes
were growing, rather than merely
persisting in the aerated culture.
IMAGEM 6
The data for the planktonic phase
indicated that the anaerobes were
growing in aerated (dO2 40-50t, Eh
+100mV) conditions. Once again,
anaerobes increased in proportion
in older biofilms. The study
indicates that mixed cultures can
protect obligate anaerobes from
the toxic effects of oxygen, both in
the biofilm and planktonic modes
of growth.
Potential role for coaggregation
in microbial
community-development
in vitro
Bradshaw DJ*, Marsh P D*,
Watson G K, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1996, 75,1507
Potential role for coaggregation
in microbial community
development in vitro
Bradshaw DJ*, Marsh PD*, Watson
GK, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1996, 75, 206
Role of
fusobacterium-nucleatum in
anaerobe survival in microbial
communities
Marsh PD*, Bradshaw DJ*,
Watson GK, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1997, 76,1700
Evidence for multi-species
interactions in oral biofilm
development
Bradshaw DJ*, Marsh PD*, Watson
GK, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
J Dent Res 1997, 76,1058
Interactions between facultative
and obligate anaerobes in oral
biofilm development
Bradshaw DJ*, Marsh PD*, Watson
GK, Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
Advances in Dent Res 1997,
11,187
Modulation of physiological
responses and population
structure of mixed culture
oral biofilms grown in vitro
Allison C, Watson G K,
Singleton S, Schilling K,
Cummins D
Advances in Dent Res 1997,
11,191
positive and Gram-negative rods
were reduced in early biofilms.
Little effect on either total or
specific counts was detected after
4 days.
The results show the value of the
model system in examining
molecules which are important in
initial biofilm attachment and its
subsequent development. As
expected, younger biofilms were
highly dependent on attachment
events, whereas older biofilms were
influenced more by biofilm growth.
Further experiments will
utilise better defined molecules and
treatments, and examine other
factors which may be important in
determining the development of
dental plaque.
Structural analysis of oral
biofilms using optical
microscopy methods
Labarbe R, Treloar R, Watson GK
and Allison C Biofilms: Community Interactions
and Control. Eds: Wimpenny J,
Handley P, Gilbert P, Lappin-Scott H
and Jones M
Bioline, Cardiff 1997, 11-14
This study was undertaken to
develop optical microscopy
methods for the characterisation of
biofilm structures grown under
different physiological conditions.
Confocal microscopy was used to
non invasively image a series of
fluorescently labelled mixed species
oral biofilms grown under a range
of nutrient availabilities. After
restoring the images using a
nearest neighbour deconvolution
algorithm to remove some
microscopy artifacts, image
processing techniques were used to
extract binary microstuctures from
the 3D datasets. A set of
measurements was produced for
each object in these
microstructures and a database of
all measurements generated. This
database was interrogated using
exploratory visual data mining
techniques in an attempt to track
structural changes between the
different samples. Clear differences
in biofilm structural parameters
were noted under different
physiological conditions.
Inter-species interactions in
microbial communities Bradshaw DJ*, Marsh PD*, Watson
GK and Allison C
(*Centre for Applied Microbiology
and Research, Salisbury, UK)
Biofilms: Community Interactions
and Control. Eds: Wimpenny J,
Handley P, Gilbert P, Lappin-Scott H
and Jones M
Bioline, Cardiff 1997, 63-71
Mixed cultures were grown in
chemostats on a habitat-simulating
growth medium including hog
gastric mucin (HGM), in order to
study the community behaviour of
oral consortia. The chemostat
design was varied to allow
development of biofilms in an
aerated second-stage vessel.
Obligate anaerobes were able to
survive and grow in relatively
oxidised environments, but only in
the presence of facultatively
anaerobic species, in both the
biofilm and planktonic phase.
Further experiments suggested that
Fusobacterium nucleatum could
allow coaggregation of obligate
anaerobes with facultative species,
thereby facilitating close metabolic
interactions. Anaerobe survival was
later shown to be greatly reduced
in aerated conditions in the
absence of F. nucleatum. Studies
on an increasingly complex series
of mixed cultures correlated the
greater enzymatic capabilities of
the communities in degrading
HGM with increasing bacterial
numbers. Added species which
increased the HGM metabolism of
the community allowed increases in
numbers of extant species, which
were already present. Many
community interactions which are
often ascribed to the biofilm mode
of growth may also occur readily in
the planktonic phase as a result of
community interactions.
Colonisation and interactions of
Pseudomonas species in binary
culture
Brading MG, Boyle J* and
Lappin-Scott HM*
(*School of Biological Sciences,
University of Exeter, Exeter, UK)
Biofilms: Community Interactions
and Control. Eds: Wimpenny J,
Handley P, Gilbert P, Lappin-Scott H
and Jones M
Bioline, Cardiff 1997, 73-79
Two environmental isolates,
Pseudomonas fluorescens and
P. putida have been used to study
colonisation and interactions at the
surface of two closely related
organisms. A recirulating batch
culture system was attached to a
Modified Robbins Device. Silastic
rubber was used as the
colonisation surface and surfaces
were removed at regular time
intervals for viable count
assessment. In simultaneous
colonisation of the two organisms,
P. fluorescens maintained an
advantage over
P. putida for intial attachment. In
challenge of a monoculture biofilm
with a second organism,
P. fluorescens became incorporated
into a P. putida biofilm faster than
P. putida was detected in P.
fluorescens biofilms. However, one
species did not preclude the other
from being incorporated into the
biofilms. The possible advantages
P. fluorescens has for initial
colonisation will be discussed,
together with other environmental
factors which may affect the
interactions of the two organisms
at the surface.
Early Colonisation during development of oral biofilms
Watson GK, Singleton S, Treolar R and Allison C
Biofilms: Community Interactions
and Control. Eds: Wimpenny J,
Handley J, Gilbert P, Lappin-Scott H
and Jones M Bioline, Cardiff 1997,
81-86
Early events during colonisation of
the hydroxyapatite surface by an
oral consortium have been studied in vitro using image analysis and
quantitative characterisation of the
position, size and shape of
individual cells and groups of cells.
Fluorescent staining allowed
detection of both general biomass
and individual members of the
consortium using specific
antibodies. The results suggest that
initial colonisation by single or
small groups of cells is followed by
a non-linear increase of biomass
and formation of non-random
aggregates of cells. Distribution of Veillonella parvula was never
random during the colonisation
process and the results suggest a
role for coaggregation in the early
colonisation process.
Oral anaerobes cannot survive oxygen stress without interacting with facultative/aerobic species as a microbial community
Bradshaw DJ*, Marsh PD*, Watson GK and Allison C (*Centre for Applied Microbiology and Research, Salisbury, UK)
Letters in Applied Microbiology
1997, 25, 385-387
Anaerobic bacteria are found
commonly as components of mixed
culture biofilms in many aerated
habitats, including the mouth.
Previous studies showed that
anaerobes could survive in
planktonic and biofilm communities
in aerated conditions when part of
a community including facultative
and/or aerobic species, and the
numbers and proportions of
anaerobic species increased as
biofilms aged. When the obligate
anaerobes were grown in the
absence of aerobic/facultative
species, however, they were unable
to grow in either the planktonic or
biofilm culture. The mean survival
times of organisms in the aerated
culture containing four anaerobic
species varied from around 5min
for Fusobacterium nucleatum and Veillonella dispar, to less than 4min
for Porphyromonas gingivalis and Prevotella nigrescens. In addition,
in this culture, the biofilm mode of
growth did not provide a haven for
these bacteria in the absence of
oxygen-consuming species.
Effect of conditioning films on oral microbial biofilm development Bradshaw DJ*, Marsh PD*, Watson GK, Allison C (*Centre for Applied Microbiology and Research, Salisbury, UK) Biofouling 1997,11, 217-226
The aim of the study was to
evaluate the influence of either
host or microbially-derived
conditioning films on biofilm
formation. Hydroxyapatite (HA)
discs were pre-treated either with
parotid saliva (2h incubation), or
with crude glucosyltransferase
(GTF) preparations (3h incubation
in culture supernatant of Streptococcus sanguis or Streptococcus mutans, with
sucrose), and then immersed in a
mixed culture of oral bacteria
grown in continuous culture. Discs
were removed from the culture
after 4d, and biofilms were
detached with a sterile probe.
Numbers of bacteria in the biofilms
on pre-treated discs were
determined by differential viable
counts, and results were compared
with untreated discs. Pre-treatment
with GTF from S. sanguis or S. mutans increased
the total CFU on discs immersed in
the culture for 4d by 29% and
95% respectively, although these
differences were not statistically
significant. The two GTF
preparations had differential
effects, with the S. sanguis preparation increasing
the numbers of S. sanguis,
Streptococcus oralis and
S. mutans, whereas the S. mutans
preparation increased numbers of
S. sanguis, Veillonella dispar,
Fusobacterium nucleatum,
S. mutans and Porphyromonas
gingivalis. It was also notable that
each GTF preparation markedly
enhanced colonisation of the
species from which it was derived.
Parotid saliva treatment produced
no differences in biofilm counts
after 4d. In a further experiment,
parotid saliva-treated discs
immersed in the culture for only Ih
showed a highly significant
increase (p<0.002) of 2.6-fold in
CFU compared with untreated
controls, with the greatest effect
seen on counts of F. nucleatum.
Bacterial products can form a
conditioning film, and may have as
important a role in the degree and
pattern of oral biofilm development
as do host molecules.
IMAGEM 7
Methods for microscopic characterization of oral biofilms: Analysis of colonization, microstructure, and molecular transport phenomena
Singleton S, Treloar R, Warren P, Watson GK, Hodgson R, Allison C
Adv Dent Res. 1997, 11,133-149
Assessment of the role of biofilm
microstructure in biofilm-specific
activities requires non-destructive
measurement techniques for
parameterization of structural
characteristics in parallel with
relevant biochemical and
physiological data. This paper
briefly reviews some current
methods for biofilm structural
analysis, with emphasis on new
developments in optical imaging
and mathematical modeling
methods. Fluorescence imaging
studies of bacterial colonization
events occurring on exposed model
tooth surfaces indicated that
bacterial adhesion to sessile
organisms was of central
importance to the early
colonization process and that this
occurred in a non-random manner.
Structural studies of mature
biofilms by confocal microscopy
demonstrated the spatial
distribution of individual species
using fluorescent antibodies.
Biofilms grown under different
physiological conditions exhibited
differences in structure, and
methods were developed for
parameterizing the spatial
orientations of the bacteria.
Diffusive processes within biofilm
microstructures were studied using
a random walk model in both 2-D
and 3-D. Modeling of convective
flow within biofilm microstructures
was achieved by application of
lattice Boltzmann methodology.
The scratch hardness of in vitro formed pellicle
Joiner A, Linden JA* and Hutchings IM
(*University of Cambridge, UK)
The effects of histatin-derived basic antimicrobial peptides on oral biofilms
Helmerhorst EJ, Hodgson R, van’t Hof W*, Veerman ECI*, Allison C and Amerongen AVN*
(*Academic Centre for Dentistry
(ACTA) Vrije University, Department
of Oral Biochemistry, van der
Boechorststraat 7, 1081,
Amsterdam)
J Dent Res 78: (6) 1245-1250, Jun
1999
Susceptibility of bacteria to
antimicrobial agents is strongly
reduced by the formation of
complex biofilms. We investigated
whether synthetic histatin analogs
with broad-spectrum antibacterial
activity in vitro were also active
against these complex mixtures of
bacteria, as present in saliva and
plaque. In a simplified model
system for dental plaque,
hydroxyapatite discs were placed in
a continuous culture system
comprised of Streptococcus
mutans, S. sanguis, S. salivarius,
Actinomyces naeslundii, Veillonella
parvula, Fusobacterium nucleatum,
and Prevotella intermedia. Ex situ
treatment of the biofilms formed
on these discs with 100 mu g/mL
of peptide dhvar4 significantly
reduced facultative anaerobic, total
anaerobic, and obligate anaerobic
Gram-negative counts with 0.8,
0.5, and 0.5 log units, respectively.
El vivo treatment of salivary
bacteria gave reductions of 0.4,
0.7, and 1.5 log units, respectively.
For ex vivo treatment of plaque
bacteria, reductions of 0.4, 0.4,
and 1.4 log units, respectively, were
found. In both saliva and plaque
samples, obligate anaerobic Gramnegative
bacteria were significantly
more susceptible to dhvar4 than
facultatively anaerobic or anaerobic
bacteria as a whole (p = 0.013 and
p = 0.018, for salivary bacteria, and
p = 0.021 and p = 0.020 for
plaque bacteria, respectively).
Although the oral bacteria are
protected by biofilm formation, the
synthetic histatin analog caused a
significant reduction of viable
counts in a model for oral biofilm
as well as in isolated oral biofilms.
Effect of histatin-derived basic antimicrobial peptides on oral biofilms
Helmerhorst EJ*, Hodgson R, van’t Hof W*, Veerman ECI*, Allison C and Amerongen AVN*
(*Academic Centre for Dentistry
(ACTA) Vrije University, Department
of Oral Biochemistry, van der
Boechorststraat 7, 1081,
Amsterdam)
|