RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE, KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 / NAME OF THE CANDIDATE AND ADDRESS(IN BLOCK LETTERS) / DR. RESHMA JOY A .
POST GRADUATE STUDENT, DEPARTMENT OF ORAL PATHOLOGY AND MICROBIOLOGY,
COORG INSTITUTE OF DENTAL SCIENCES,KK CAMPUS, MAGGULA,
VIRAJPET-571218.
2 / NAME OF THE INSTITUTION / COORG INSTITUTE OF DENTAL SCIENCES, VIRAJPET-571218.
3 / COURSE OF STUDY AND SUBJECT / MASTER OF DENTAL SURGERY,
ORAL PATHOLOGY AND MICROBIOLOGY.
4 / DATE OF ADMISSION OF COURSE / 30\05\2013
5
6 / TITLE OF THE TOPIC:
INHIBITORY ACTIVITY OF LACTOBACILLI AND BIFIDOBACTERIUM ON CANDIDA STRAINS. AN IN-VITRO STUDY.
BRIEF RESUME OF THE INTENDED WORK:
6.1 Need for the study:
Candida species are a normal commensal in the oral cavity being seen in 30-60% of healthy individuals. Overgrowth of Candida species or a frank infection which is termed as oral candidiasis usually is a result of disequilibrium in oral microbiota or due to impaired immunity. The frequency of invasive fungal infections has increased over the past few years concomitant with the rise in at-risk populations of patients.1
Candida infection includes a spectrum of clinical presentation starting from the prototypical pseudomembranous candidiasis to chronic mucocutaneous candidiasis. A wide range of therapeutic interventions have been proposed to treat oral Candida infections with variable results. Medical interventions like systemic antifungal agents are fraught with potential side-effects.2
Probiotics are living micro-organisms added to food which beneficially affect the host by improving the microbial balance. Probiotics have been successfully used in treating several conditions like diarrhoea and bacterial vaginosis.3 Use of probiotic strains to treat oral candidiasis is an interesting prospect, which negates the side effects of conventional antifungals and is well tolerated by the patients.
The present study employs Lactobacillus strains: L. reuteri, L. rhamnosus and Bifidobacterium strains: B. bifidum, B. longum; with the aim to investigate their ability to inhibit growth of Candida in- vitro.
6.2 Review of literature:
1. In an in-vitro study done to determine the antifungal activity of four strains of lactobacilli and bifidobacteria of human origin, it was concluded that there is high titratable acidity during joint cultivation of the strains of lactobacilli or bifidobacteria and C. albicans, the growth of pathogens was influenced slightly.2
2. Study done by Natalia F Gil in the year 2010, vaginal lactobacilli isolates were studied to screen microorganisms with probiotic properties against Candida species and were tested for their ability of auto-aggregation, coaggregation with C. albicans, C. glabrata, C. krusei, and C. tropicalis, adhesion to Caco-2 epithelial cells and production of lactic acid and hydrogen peroxide. It was concluded that four strains of lactobacilli inhibited candida exhibiting auto-aggregation, co-aggregation, ability to adhere to epithelial cells and produce lactic acid and hydrogen peroxide.3
3. An in-vitro study was done to investigate the ability of selected lactobacilli strains used in eight commercially available probiotic products to inhibit growth of oral S. mutans and Candida albicans and it was concluded that the selected probiotic strains showed a significant but somewhat varying ability to inhibit growth of S. mutans and C. albicans.4
4. The hypothesis that cheese containing probiotics bacteria (L.rhamnosus, propionibacterium freudenreichii ssp) can reduce the prevalence against oral Candida was tested in vivo on elderly people and it was found that cheese containing probiotics were successful in reducing the risk of high yeast counts by 75%.5
5. The study by R Doug Wagner et al in the year 2012 used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1W and Lactobacillus reuteri RC-14W and challenged with C. albicans. The results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes.6
6.3 Objectives of the study:
1. To investigate the ability of Lactobacillus strains: L. reuteri, L. rhamnosus to inhibit the growth of Candida spp. in-vitro.
2. To investigate the ability of Bifidobacterium strains: B. bifidum, B. longum to inhibit the growth of Candida spp. in-vitro.
7 / MATERIALS AND METHODS:
7.1 SOURCE OF DATA
The in-vitro study will be conducted in the Department of Oral Pathology and Microbiology, Coorg Institute of Dental Sciences, Virajpet.
7.2 MATERIALS
1. Candida strains: C. albicans,
C. krusei,
C. tropicalis,
C. parapsilosis.
2. Bifidobacterium strains: B. bifidum,
B. longum.
3. Lactobacillus strains: L. reuteri,
L. rhamnosus.
4. Man Rogosa Sharpe medium.
5. Sabouraud dextrose agar.
6. Filter paper disks.
7. Anaerobic jar.
8. Agar plates.
9. Incubator.
10. Vortex mixer.
11. pH meter.
12. Test tubes.
13. Spectrophotometer.
7.3 METHOD:
1. Bacterial strains and culture conditions:
Two probiotic Lactobacilli strains: L. reuteri, L. rhamnosus, will be cultured on MRS agar medium for 24 hours at 35° C anaerobically.
Two probiotic Bifidobacterium: B. longum, B. bifidum, will be cultured on MRS medium for 24-48 hours at 37° C anaerobically.
Four strains of Candida: C. albicans, C. krusei, C. tropicalis, C. parapsilosis will be cultured on Sabourad’s dextrose agar aerobically overnight and will be transferred to 2 ml of Sabourad’s dextrose broth and incubated for 16-20 hrs the following day.
The inhibitory efficacy will be assessed through agar overlay interference tests.
Method of collection of data:
Sample size: The in-vitro study consists of 4 test groups of probiotic strains and will tested for interference with 4 groups of Candida strains. All the groups will be assessed as triplicates.
Sample selection: Not Applicable.
Inclusion criteria: Not Applicable.
Exclusion criteria: Not Applicable.
Data collection procedure: The probiotic strains and the Candida strains will be procured and cultured on the respective media. The inhibitory efficacy will be assessed through agar overlay interference tests.
Study variables and parameters:
1. Inhibition score as assessed through agar overlay interference test.
2. Surface pH changes in culture plates
7.4 Does the study require any investigations or interventions to be conducted on the patients or other humans or animals? ( If so please describe briefly)
Not applicable
7.5 Has Ethical clearance been obtained from your institution in case of 7.3?
Not applicable
8. / LIST OF REFERENCES:
1) Scully C, El-Kabir M, Samaranayake LP. Candida and oral candidosis: a review. CROBM 1994; 5(2): 125-57.
2) Gil NF, Martinez RCR, Gomes BC, Nomizo A, De Martinis ECP. Vaginal lactobacilli as potential probiotics against Candida spp. Braz J Microbiol 2010; 41: 6 -14.
3) Hasslof P, Hedberg M, Twetman S, Stecksen-Blicks C. Growth inhibition of oral mutans streptococci and candida by commercial probiotic lactobacilli-an in vitro study. BMC Oral Health 2010; 10:18.
4) Denkova R, Yanakieva V, Denkova Z, Nikolova V, Radeva V. In vitro inhibitory activity of bifidobacterium and lactobacillus strains against Candida albicans. BJVM 2013; 16 (3): 186-97.
5) Hatakka K, Ahola AJ, Yli-knuuttila H, Richardson M, Poussa T, Meurman JH, Korpela R. Probiotics reduce the prevalence of oral candida in elderly- A randomized controlled trial. J Dent Res 2007; 86(2): 125-30.
6) Wagner RD, Johnson SJ. Probiotic lactobacillus and estrogen effects on vaginal epithelial gene expression responses to candida albicans. J Biomed Sci 2012; 19: 58.
9. / SIGNATURE OF THE CANDIDATE /
(Dr. RESHMA JOY A.)
10. / REMARKS OF THE GUIDE
11. / NAME & DESIGNATION OF (IN BLOCK LETTERS)
11.1 GUIDE
11.2 SIGNATURE / DR. SUDHEENDRA U.S.
(PROFESSOR)
11.3 CO- GUIDE (IF ANY)
11.4 SIGNATURE
11.5 HEAD OF THE DEPARTMENT
11.6 SIGNATURE / DR. SHASHIDARA R.
(PROFESSOR& HOD)
12. / 12.1 REMARKS OF THE CHAIRMAN & PRINCIPAL
12.2 SIGNATURE / DR. SEQUEIRA PETER SIMON (PRINCIPAL)