Download Division of Oral Microbiology

Annual Report March 2006 – March 2007
Division of Oral Microbiology
Head: Dr M Patel
Oral Microbiology is a specialized division that deals with Microbiology related to Dentistry.
The staff members are self trained general microbiologist. It is a rare specialty and there are
only 5-6 oral microbiologists in this country. This division provides service to the Dental
hospital as well as private dentists in the form of diagnostic services, to the University in the
form of undergraduate and postgraduate student teaching and research. University of the
Witwatersrand is the only university in this country that runs a fully developed teaching
program containing oral microbiology as a subject for Dental students.
Diagnostics Services:
This laboratory performs caries activity tests, Microbiology of periodontal pocket specimen,
pus swabs, culture for Candida, Microbiology of denture wearer and root canals. This
laboratory also provides infection control monitoring service to the dental hospital.
Teaching: The division run three undergraduate courses to BDS I (45 students), BDS III (40
students) and OH I (12 students) each year. Course work teaching for the M Dent, a
postgraduate degree is also done each year. Four research reports for M Dent degree were
completed in 2006-2007.
Departmental Research Interests
The main research interest of this division is HIV and oral Candidiasis. The research projects
in 2006-2007 and their progress reports are as under.
Research Projects
Antifungal effect of mouth rinses on oral Candida counts and salivary flow in HIVinfected patients
Researchers: Dr Mrudula Patel, Mrs Jo-Anne Shackleton, Prof Maeve M Coogan.
Complete, Submitted to a journal for a possible publication. Funding: Colgate
Palmolive Pty Ltd, 3M South Africa
Oral candidiasis causes problems in HIV infection because patients are prone to xerostomia
and are predisposed to Candida infection. Alternatives to antifungals are being sought as
resistance to these agents develop. This study investigated the ability of commercial mouth
rinses and sodium bicarbonate to reduce salivary Candida and to improve the saliva flow of
HIV positive patients. 150 HIV patients without oral candidiasis were examined for oral lesions
initially and after two, four and twelve weeks. Unstimulated saliva was collected; the volume
was measured and cultured for yeasts. Subjects were provided with mouth rinses containing
either Benzydamine hydrochloride, Benzydamine hydrochloride with chlorhexidine gluconate,
Triclosan with Sodium fluoride, 5% sodium bicarbonate or placebo and asked to rinse twice
daily for 12 weeks. The effect of the mouth rinses and placebo on Candida counts and saliva
flow was analysed using ANOVA. A total of 108 patients completed the trial, 35 missed
appointments, 4 died, 2 developed oral candidiasis and one herpatic lesion. Triclosan/fluoride
decreased the Candida count more than the placebo (p=0.005) while
chlorhexidine/benzydamine hydrochloride (p=0.001) and triclosan/fluoride mouthrinses
(p=0.002) increased the salivary flow during the initial 4 weeks. The most effective mouth
rinse triclosan/fluoride decreased oral Candida counts and increased saliva flow.
The antifungal activity of the plant Dodonaea viscosa on Candida albicans
isolated from HIV positive patients
Researchers: Dr Mrudula Patel, Prof MM Coogan. Completed, Will be submitted for a
possible publication. Funding: Surplus previous research funds
The antifungal treatment of oral candidiasis associated with HIV infection has resulted in
resistance. An alternative is antimicrobial mouth rinses. Dodonaea viscosa var. angustifolia
(PLE) a South African plant is used traditionally to treat oral candidiasis. This study
determined the minimum inhibitory concentration (MIC) and the time taken by PLE to kill C.
albicans and compared it with chlorhexidine gluconate (CHX) and triclosan (TRN).
Forty one strains of C. albicans were investigated, 20 from HIV positive patients, 20 from HIV
negative subjects and Candida albicans ATCC 90028. The MICs of an acetone extract of
PLE, CHX and TRN were measured using a microtitre double dilution technique, and the time
taken to kill 99.5% of the strains was determined. The MICs of PLE, CHX and TRN were
6.25-25 mg/ml, 0.008-0.16mg/ml and 0.0022-0.009 mg/ml respectively. PLE killed all the test
strains within 30 seconds and CHX 40% of the isolates from HIV positive patients and 20% of
strains from HIV negative subjects in one minute. During the same time TRN killed 55% and
35% of isolates from HIV positive and HIV negative patients. D. viscosa var. angustifolia has
antifungal properties and is more effective than commercially available mouth rinses.
The effect of Dodonea angustifolia on the adherence of yeast isolated from HIV
positive and HIV negative patients to oral epithelial cells
Researchers: Dr Mrudula Patel. Progress: 50% of the laboratory work is completed.
Preliminary results were presented at a local conference. Funding: Surplus previous
research funds
Adhesion of C. albicans to epithelial surface is considered as a critical step in the
pathogenesis of oral candidiasis. Candida albicans isolated from HIV positive patients are
known to be more virulent than from HIV negative patients. The leaves of an indigenous
South African plant Dodonaea angustifolia are used as a traditional remedy for treating oral
thrush. Our laboratory has determined the MIC and the time taken to kill Candida albicans.
This study investigated the effect of crude extract of this plant on the adherence of yeast
isolated from HIV positive and HIV negative patients to oral epithelial cells.
Acetone extract was prepared using Dodonaea angustifolia leaves. Yeast cells were treated
with sub inhibitory concentration of plant extract and mixed with freshly isolated oral epithelial
cells. Water instead of plant extract was used for the control. After 3 hours of incubation, the
epithelial cells were harvested, washed, slides were prepared and gram stain was performed.
Ten isolates from each group were studied. Adherent yeast cells per 100 epithelial cells were
measured for each test including the controls. Results of exposed and unexposed cells and
the results of the isolates from both the groups were compared using student t test.
The results showed that the plant extract reduce the adherence of yeast cells to the oral
epithelial cells significantly (p=<0.001). There was no difference in the results of the strains
isolated from the two study groups. Dodonaea angustifolia has ability to reduce the virulence
of C. albicans isolated from HIV positive and HIV negative patients.
The effect of this plant on the proteinase production, one of the virulence by C. albicans will
be studied in order to complete this project.
Methods: 20 isolates from HIV positive and 20 from HIV negative patients with virulence
properties of proteinase production will be selected from our culture collection. These cultures
will be exposed to Dodonaea angustifolia crude extract and sterile distilled water for three
hours. These cultures will be planted on to Yeast Carbon Base media, incubated at 30o C for
3, 5 and 7 days. The plates will be examined for proteinase production. Results from plant
extract and water will be compared.
Antibacterial effects of a controlled and sustained release formulation of chlorine
dioxide on artificially contaminated irreversible hydrocolloid impression material
Researchers: Dr Ismail Hamisi Rweyendela, Dr Mrudula Patel. Collaborator:
Department of Prosthodontics, Progress: Completed, in the preparation for a
publication. Funding: Dental Hospital
Dental impressions are potential contamination pathways, and need to be disinfected to
protect staff as well as patients. Irreversible hydrocolloid impressions are difficult to disinfect
because of their dimensional instability and unfavourable physical characteristics. The aim of
this study was to evaluate the antimicrobial efficacy of a chlorine dioxide brand - Aseptrol® on irreversible hydrocolloid impression material.
Irreversible hydrocolloid blocks contaminated with Candida albicans, Staphylococcus aureus,
Pseudomonas aeruginosa, Streptococcus mutans and Bacillus subtilis spores were placed
either in sterile distilled water as control, or disinfected by immersion in 48-ppm Aseptrol®
solution to which had been added organic matter. Immersion times were 30 seconds, one
minute, three minutes and five minutes. After exposure, test blocks were placed in sterile
0.5% sodium thiosulphate neutraliser solution to harvest surviving organisms.
Bacterial counts of the control and test blocks were determined using the serial dilution
technique followed by culturing on appropriate media, and were used to determine the
percentage reduction for each organism and exposure time. The results showed that 30seconds immersion in 48ppm Aseptrol® solution consistently achieved significant reduction
(99.99%) in all tests on vegetative organisms but not on spores, which required 3 minutes.
Further tests at 30sec intervals were then carried out for the B. subtilis spores, and it was
found that the same concentration of Aseptrol® achieved a 99.99% reduction in spores after
1½ minutes’ exposure time.
In vitro antimicrobial properties of a mouthrinse containing salt, glycerine, potassium
nitrate and alum
Researchers: Dr Nozizwe Ndlovu, Dr Mrudula Patel. Progress: Completed, will be
submitted for a possible publication. Collaborator: Department of Prosthodontics,
Dental Hospital. Funding: Faculty Endowment research funds
Patients who have received radiation therapy due to oral cancers often present with
complications such as salivary dysfunction, mucositis, oft tissue necrosis, infections and
dental caries. The aim of this project was to investigate the antimicrobial properties of an
experimental mouth rinse which also contains analgesic and anticaries compounds and can
be used in the management of patients with oral cancers after radiation therapy.
The experimental mouth rinse contained a mixture of 30% glycerine (antimicrobial), 7%
potassium nitrate (analgesic) and 0.025% sodium fluoride (anticaries agent). The minimal
inhibitory concentration (MIC) of these ingredients were tested against Candida albicans,
Staphylococcus aureus and Streptococcus mutans. MICs of commercially available
mouthrinses corsodyl containing chlorhexidine gluconate and Plax containing triclosan and
NaF were also determined. Percentage kill over 1, 2 and 3 minute exposure to all three mouth
rinses was also studied.
The MICs of glycerine were 10%, 25% and 10% for C. albicans, Staph aureus and Strept
mutans respectively. The MIC of corsodyl was <0.02 mg/ml for all the test organisms.
Ptassium nitrat, sodium fluoride and alum did notshow any antimicrobial effects. The MIC of
plax was 0.02 mg/ml, <0.002 mg/ml and 0.005 mg/ml for C. albicans, Staph aureus and
Strept mutans respectively. Combining glycerine, potassium nitrate and sodium fluoride into a
mixture did not affect the antimicrobial properties of these constituents. The mixture killed
99.78, 99.88 and 99.98% of C albicans, 61.74, 70.64 and 85.09% Staph aureus and 91.72,
99.47 and 99.99% of Strept mutans after 1, 2 and 3 minutes respectively. Corsodyl killed
98.98, 99.97 and 99.99% of C albicans, 95.83%, 99.68 and 99.97% of Staph aureus and
99.98, 99.96 and 99.99% of Strept mutans fter 1, 2 and 3 minutes respectively. Plax showed
similar results.
A mouth rinse effective in relieving oral symptoms in patients receiving radiation therapy
needs to show some antimicrobial activity against common pathogens, whilst at the same
time having a palliative effect. This study has shown that the experimental mouth rinse will
fulfil these requirements. The experimental mouth rinse was found to be cheaper in
comparison to corsodyl and plax.
A prospective comparative study of potential risk factors between Ludwig’s angina and
localized odontogenic
Researchers: Dr Thoganthiren Perumal Chettiar, Dr Mrudula Patel. Progress:
Completed, in the preparation for a publication. Collaborator: Dept of Maxillo-facial and
Oral Surgery, Dental Hospital. Funding: Faculty Endowment research funds
Odontogenic abscesses and Ludwig’s angina are infections commonly seen by maxillofacial
surgeons. Both infections have periapical or periodontal origin and caused by oral bacteria.
Ludwig’s angina is an aggressive and fast spreading infection compared to odontogenic
abscess. The origin and the responsible bacteria of these infections are similar but the
development and response is different in patients. There is no comprehensive study that has
investigated the bacterial and host factors involved in the development of there infections.
The aim of this study was to compare the presence of bacteria and enzymes in to the pus
samples collected from patients with odontogenic abscess and Ludwig’s angina. Furthermore,
various haematological and immunological tests were also compared between the two study
groups. Forty two patients presenting with localized odontogenic abscesses and 15 with
Ludwig’s angina were selected. Patient was examined according to standard protocol and
history was recorded. Bloods were collected for haematology and immunology tests and pus
was collected for microbiology and enzymatic tests.
The results showed that highly virulent bacteria such as Staphylococcus aureus and black
pigmented bacteroides were prevalent, increase in C - reactive protein, white blood cell count,
IL6 and decrease in urea, circulating immune complexes and IgE in patients with Ludwig’s
angina.
Development of Ludwig’s angina could be due to the aggressive bacteria, their byproducts
and high immune response compared to the odontogenic abscesses.