ORAL MICRO FLORA

Presented by – Dr. Shubham Gupta

Preceptor – Dr. Anju Aggarwal

CONTENTS

• Introduction
• Definition
• History
• Classification
• Various forms of Oral flora, their location & infections caused by them
• Advantages & disadvantages of oral flora

• Risk factors associated with oral flora
• Acquisition of oral flora
• Introduction of micro flora due to various prosthodontic procedures
• Disinfection
• Candidiasis
• Denture stomatitis
• Angular chelitis
• Peri implantitis
• Conclusion

Introduction

• The first simple forms of life appeared on earth for more than three billion years ago. Their descendants have changed and developed.
• Microscopic forms of life are present in vast numbers in nearly every environment known, i.e soil, water, air, food etc.

Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition

• Since the conditions in normal human body also favours their growth, they are found in various body cavities of human beings.

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• Fortunately, the majority of these micro organisms are not harmful.

DEFINITION

Oral microbiology is the study of micro organisms of oral cavity and the interactions between the oral micro organisms with each other and with the host.

Oral micro flora :

Refers to the population of microorganisms that inhabit mucous membranes of normal healthy oral cavity. These microbial types are referred collectively as the Normal/ indigenous or resident floras.

Opportunistic infections : An infection by a microorganism that normally does not cause disease but becomes pathogenic when the body's immune system is impaired.

Shafer’s Oral Pathology,, 6th edition

Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition

• The mouth is inhabited by an indigenous "normal” micro flora that is composed of over 500 species— “the majority still uncultivable!!!”.

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• Certain microbial types are constantly found in the specific oral areas.

HISTORY

• Antonie van Leeuwenhoek (1632‑1723) - Father of bacteriology and protozoology. With the help of simplest microscope which he constructed himself, he described different types of microbes as animalcules.

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• F. Muller- classified the bacteria in 1773 and 1788, and coined new terms ‑ vibro and monas.

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• Ehrenberg- established the new genus Bacterium. The origin of the word Bacterium was from a Greek word ‘bactos’ meaning staff.

Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition

• Edward Jenner (1749-1825) – Developed technique of vaccination. He studied two disease small pox & cow pox
• Louis Pasteur (1822-1895) –Known as father of Microbiology and Fermentation processes. Defined the theory of spontaneous generation and established that living microorganism are responsible for chemical changes that occur during fermentation .He confirmed that some certain microbes were responsible for formation of different molecules such as acetic acid and lactic acid.

• Robert Koch (1822 -1910)-Father of Bacteriology. He was a German doctor who made several discoveries in the field of microbiology
• main are –Bacteria can be isolated and causes Disease. He developed technique of pure culture discovered bacterium which caused anthrax in cattles and demonstrated its progression .
• He developed koch postulates Koch postulates are

1)Particular organism can always be found in association with a particular disease, but not in healthy individual.

2) The organism can grow in the laboratory itself.

3)the pure growing culture will produce the same disease when placed back into a new susceptible animal.

4)it is possible to recover the organism from the sick animal and grow them in pure culture.

• Paul ehrlich (1854-1915) father of chemotherapy and pioneer in the field of immunology. He was a german chemist and student of koch who came with the chemical explanation of immunity. He invented different types of stains like analine. His major field of research was disease immunization.

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CLASSIFICATION

1.Depending upon staining� Gram positive – Resist decolourisation and retain the primary stain

• appearing violet.
• They have thicker cell wall that prevents the decolorisation by organic acid.

Gram Negative – Decolorized by organic solvents and take up the counter stain appearing red.

2.Depending upon morphology:

1. Coccus�2. Bacillus�3. Coccobacilli�4. Fusiformbacilli

5. Vibrio

6. Spirillum

7. Spirochete

3.Depending upon influence of oxygen on growth and viability.

1.Aerobes: Required O2 for growth.

2.Anaerobes:�  Obligate: Grows in the absence of O2.�  Facultative: Grow in the presence or absence of O2.

3.Microaerophilic

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Acquisition of Microflora in oral cavity (baveja/ samarnayake)

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• The infant mouth is sterile at birth.
• Within few hours of birth, streptococci sp (salivarius) establish themselves through mothers mouth or environment.

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Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition

• With the eruption of teeth, there is hard surfaces and gingival crevices are available for colonization.
• Simultaneously, there is also increase in number of anerobic microbes like prevotella, porphyromonas.

• A second childhood starts (in terms of oral bacterial colonization) when all teeth are lost due to senility.
• Introduction of prosthetic appliance at this stage changes the microbial composition once again.

Samaranayake LP, Hunjan M, Jennings KJ. Carriage of oral flora on irreversible hydrocolloid and elastomeric impression materials. J Prosthet Dent. 1991;65(2):244-249.

• Growth of candida sp is particularly increased after introduction of acrylic dentures.

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• Also prevelance of staphylococcus aureus and lactobacilli is high in those aged above 70 years

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Patil S, Rao RS, Sanketh DS, Amrutha N. Microbial flora in oral diseases. J Contemp Dent Pract. 2013;14(6):1202-1208. Published 2013 Nov 1.

Various groups of flora, their sites and the infections caused respectively

Gram positive streptococcus

• Mutans group- seen on tooth surfaces, known for causing dental caries.
• Salivarius group – seen on dorsum of tongue and in saliva. They help in lowering pH.

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Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition

Mitis group : eg streptococcus mitis, sanguinis.

• Seen mainly in dental plaque, tongue, cheek.
• They are known for causing dental caries.

Anaerobic streptococci : eg Peptostreptococcus anaerobius

• Seen on teeth, especially on carious dentine.
• Known for causing periodontal and dentoalveolar abscess in mixed culture.

Gram positive rods and filaments

• Actinomyces : eg actinomyces odontolyticus,naeslundii.
• actinomyces georgie/gerencseria are minor components of healthy gingival flora.
• Actinimyces israelli is an opportunistic pathogen.

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• Actinomyces naeslundii is implicated in causing root surface caries.

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Lactobacillus : eg lactobacillus acidophillus/ fermentum

• Most common oral inhabitants
• Seen in dental plaque biofilms

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Gram negative cocci : eg neisseria, veillonella

• Isolated in low numbers.
• They are rarely associated with disease.

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Gram negative rods

• Eg Haemophilus parainfluenze/ haemolyticus
• Known for causing dentoalveoloar infections, sialedenitis and infective endocarditis.

Aggregatibacter : eg actinomycetemcomitans.

• Isolated from periodontal pockets.
• Known for causing localised/ aggressive periodontitis.

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Capnocytophaga

• Seen in plaque, mucosal surfaces, saliva
• They are known to cause periodontal destruction and opportunistic infections in immunocompromised patients

Gram negative anaerobes

• Porphyromonas : eg P gingivalis/endodontalis
• Isolated from gingival crevices, subgingival plaque
• Known to cause chronic periodontitis and dentoalveoloar abscess.

Prevotella : eg prevotella intermedia /corporis/oris

• Isolated from dental plaque, periodontal pockets
• Cause chronic periodontitis and dentoalveolar abscess.

Fusobacterium : Eg F.nucleatum

• Most commonly found in normal gingival crevices
• Known to cause to periodontal infections and also has a major role in causing ANUG and dentoalveolar abscess.

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Treponema : Eg T.denticola/ vincentii

• Found in gingival crevices
• Causative agent in ANUG and periodontal destruction.

“Red complex” bacteria : It consists of

• Porphyromonas Gingivalis
• Treponema Denticola and
• Tannerella Forsythia (formerly known as bacteroids)

Fungi and protozoa present in oral cavity (Baveja)

Fungi :

• Candida albicans
• C. Tropicalis
• C. Pseudotropicalis
• Cryptococcus sp.

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Protozoa :

• Entamoeba gingivalis
• Trichomonas tenax.

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Advantages & Disadvantages of micro flora

Slot , Contemporary Oral Microbiology and Immunology

ADVANTAGES

• They constitute a protective host defense mechanism by occupying ecological niches.
• The oral flora contribute to immunity.
• The normal flora may antagonize other bacteria.
• Mains indigenous flora contributes in synthesis of vitamins (B and K) through digestion of food.

DISADVANTAGES

They can cause disease:

• When individuals become immunocompromised or debilitated.
• When they change their usual anatomic location.
• The oral flora of humans may harm their host since some of these bacteria are pathogens or opportunistic pathogens

Risk factors associated with normal oral flora is

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• Dental plaque

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• Dental caries

• Periodontal diseases

THE ORAL ECOSYSTEM �

TO DEAL WITH THE TAXONOMIC DIVERSITY BACTERIAL SPECIES CAN BE DIVIDED ON THE BASIS OF PREVALENCE INTO: �

• Indigenous flora
• Supplemental flora
• Transient flora

Indigenous flora:

• It comprises those species that are almost always present in high numbers in a particular site.
• They are compatible with the host and have entered into a stable relationship with the host.

• They do not compromise the host’s survival.
• Eg Streptococcus,Actinomyces, Neisseria

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Supplemental flora:

• It comprises species that are nearly always present, but in low numbers (<1%).
• These organisms may become indigenous if the environment changes.

Transient flora:

• It comprises organisms “just passing through” a host.
• Bacteria present in food or drink may be temporarily established in the mouth.
• They do not persist long and quickly disappear.
• However overt pathogens appear to quickly pass from the transient stage to a predominant stage.

HOST-BACTERIA INTERRELATIONSHIPS

• Symbiosis: When both the host and the bacteria benefit from their inter-relationship, it is termed “symbiotic”.
• Antibiosis: It is the opposite of symbiosis relationship.
• When bacteria cause an infection that is combatted by the defence system of the host, the relationship is said to be antibiotic.

• This relationship is very unstable for both the host and the pathogenic bacteria.

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Amphibiosis:

• Rosebury introduced the term “amphibiotic” to describe an intermediate state in which the host and its flora exist in a form of stable balance with each other.
• Most of the oral flora are thought to exist in an amphibiotic relationship with their host.

MICROBIOLOGY OF DENTAL CARIES

S. mutans, Lactobacilli and Actinomyces are more important than others.

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S. mutans:

• The feature that supports its role as cariogenic organisms are its : Rapid generation time, Acidogenic nature, Production of extra cellular polysaccharides from sucrose.
• Isolated in high numbers in caries active mouth in incipient lesions.
• S.mutans is able to utilize dietary sucrose to enhance colonization.

Lactobacilli

• for many years was believed to be the causative agent of dental caries as: High numbers were obtained in most enamel caries.
• Able to synthesis extra cellular and intra cellular polysaccharides form glucose.
• They produces lactic acid at pH < 5 .
• They were however absent from incipient lesions while present is significant numbers in developed caries.
• Thus, they were categorized as secondary invaders which caused progression of caries due to their acidogenic and aciduric properties.

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Introduction to micro organisms due to various clinical procedures

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Impression procedures

According to a study conducted by

• Lakshman et al, the carriage and persistence of oral flora on irreversible hydrocolloids and elastomeric impressions materials , it was documented that :
• Retention of bacteria on irreversible hydrocolloids was two to five fold high than the other elastomeric impression materials.

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Samaranayake LP, Hunjan M, Jennings KJ. Carriage of oral flora on irreversible hydrocolloid and other elastomeric impression materials. J Prosthet Dent. 1991;65(2):244-249.

• In case of disinfected irreversible hydrocolloid the organisms were totally destroyed in less than 3 mins.
• It was concluded that total bacterial load of impression surface is relatively low and decreases rapidly after impression making.
• But disinfection of the impression surface is mandatory to prevent any cross contamination.

The organisms isolated were mainly

• bacillus species 70%
• Staphylococci 30%
• Streptococci 10%

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Subgingival flora during fixed prosthodontic procedures

Flemmig, T. F., Sorensen, J. A., Newman, M. G., & Nachnani, S.. Gingival enhancement in fixed prosthodontics. Part II: Microbiologic findings. The Journal of Prosthetic Dentistry, 65(3), 365–372.

In the study to assess subgingival/ marginal micro flora during fixed prosthodontic procedure and effect of adjunctive rinsing with chlorhexidine 0.12%

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• Subgingival and marginal plaque were analysed at baseline, before crown preparation(2weeks) and after crown cementation (2 weeks)- showed no changes

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• Rinsing with chlorhexidine significantly reduced putative periodontal pathogens as compared to control cases.

• Chlorhexidine was a useful adjunct during fixed prosthodontic procedures in maintaining and establishing microflora compatible with periodontal health.

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• In contrast, a study conducted by Dhanraj et al to Evaluate Subgingival Microflora in All Ceramic Restorations with Subgingival Heavy Chamfer Finish Lines showed that

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• there was a significant increase in sulcular microbial flora at varying intervals of time!!!

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• The possible reason for increase in microbial population includes distension of gingiva by the subgingivally placed all ceramic restoration with mild possible over contouring in the cervical area.

Dhanraj, M. & Selvaraj, Anand & Ariga, Padma. (2013). Evaluation of Subgingival Microflora in All Ceramic Restorations with Subgingival Heavy Chamfer Finish Lines. The Journal of Indian Prosthodontic Society.

• The variation in gingival crevicular fluid flow to newly placed retainers could have facilitated increased microbial colonization.
• The subsequent increase in microbial population can be attributed to gingival remodeling.

Tooth preparation

• The quality of dental unit water is of considerable importance since the patients, dentists and the dental personal are constantly exposed to aerosols.

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• They may be ingested either in form of aerosols or through contaminated surgical wounds.

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• Dental aerosols, irrigants and coolant water entering the patients mouth contain large number of microbes including gram negative bacteria, fungi, protozoas and saprophytes.

• Also abnormal nasal flora among dental personnel has been attributed to water system contamination, as a result of continuous inhalation of aerosols on daily and long term basis.

Change in microbial flora in subgingival restoration due to various restorative materials

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• The relationship between subgingival dental restoration and periodontal health was assessed for 3 different materials - amalgam, GIC and composites

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• No significant changes were found among amalgam and GIC.

Paolantonio, Michele & D'ercole, Simonetta & Perinetti, Giuseppe & Tripodi, Domenico & Catamo, Giovanni & Serra, Emanuela & Bruè, Claudia & Piccolomini, Raffaele. (2004). Clinical and microbiological effects of different restorative materials on the periodontal tissues adjacent to subgingival class V restorations. 1-Year results. Journal of clinical periodontology.

• Where as in composite resin group there was significant increase in total bacterial counts.

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• This indicated that composite resins have some negative effect on quantity and quality of subgingival plaque.

• Sampio et al observed in vitro that retained denture adhesives also showed microbial contamination
• Some sp like strep. oralis, prevotella oralis fusobacterium nucleatum, strep. Mutans, candida albicans play a role in halitosis, a common problem in denture wearers.

Sampaio-Maia, B., Figueiral, M. H., Sousa-Rodrigues, P., Fernandes, M. H., & Scully, C. (2011). The effect of denture adhesives on Candida albicans growth in vitro.

DISINFECTION

• the disinfection of dental impression should now be considered as a routine procedure in dental surgeries and dental laboratories.
• There are several ways in which an impression material can be disinfected.

The popular methods :

• Spray disinfection is a simple and convenient method, however there are some concerns that this procedure may not ensure thorough disinfection of all the impression material.

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• Immersion disinfection is perhaps considered to be a more reliable method which should ensure a more even contact between the disinfectant and the impression material.
• Ex household bleach (1:10 dilution), iodophors, synthetic phenols, 2 % gluteraldehyde, sodium hypochlorite etc

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Laser disinfection : A range of lasers is now available for use in dentistry.

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• The diode laser can directly contact the implant surfaces without inducing melting, cracking, or crater formation.

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• The 810-nm diode laser, does not damage titanium surfaces, which is useful when uncovering submerged implants, and can be used to treat bacterial induced peri-implantitis

Photoactivated dye techniques have been developed which use low power lasers to elicit a photochemical reaction.

OPPORTUNISTIC INFECTIONS WITH PROSTHODONTIC CONSIDERATION

Candidiasis (shafers)

Synonms: monoliasis, thrush

• Candidiasis is caused by candida albicans.
• It is the most common inhabitant of oral cavity, gastro intestinal cavity and female genital tract of clinically healthy persons.
• This disease is said to be the one of the most opportunistic infection in the world.

Shafer’s Oral Pathology,, 6th edition

• It’s occurrence has remarkably increased due to prevelent use of antibiotics and other drugs.
• Hence its most common occurrence in patients suffering from leukemia ,lymphomas and other tumours.
• It is reported that more than 90% of the HIV infected individuals develop oral candidiasis.

• C. albicans is an oral commensal in as many as 40% to 65% of healthy adult mouths.

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• The papillated dorsal surface of the tongue and palatal mucosa beneath a maxillary denture are favored reservoir sites.

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• Oral candidal infection almost always involves a compromised host. It may be local or systemic.

Zegarelli DJ. Fungal infections of the oral cavity. Otolaryngol Clin North Am.26(6):1069-1089.

• Some have even implicated advanced age and the female gender as being mild predisposing factors.

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• Furthermore, the C. albicans infection itself can depress a host's immune system.

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CLASSIFICATION OF ORAL CANDIDIASIS (AS PROPOSED BY SAMARANAYAKE 1991 & MODIFIED BY AXELL ET ALL 1997)

Primary oral candiadiasis

• Acute forms
• Erythematous
• Chronic forms
• Hyperplastic
• Nodular plaque like
• Pseudomembranous
• Candida associated lesions
• Denture stomatitis
• Angular chelitis
• Median rhomboid glossitis

Shafer’s Oral Pathology,, 6th edition

SECONDARY ORAL CANDIDIASIS

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• ORAL MANIFESTATIONS OF SYSTEMIC MUCOCUTANEOUS CANDIDIASIS AS A RESULT OF DISEASES SUCH AS THYMIC APLASIA AND CANDIDIASIS ENDOCRINOPATHY SYNDROME.

Denture stomatitis : It is characterized as inflammation and erythema of the oral mucosal areas covered by the denture.

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• It is a form of chronic atrophic candidiasis.
• Usually asymptomatic except for the soreness.
• Palatal mucosa is most commonly effected.

GENDREAU, L., & LOEWY, Z. G. (2011). EPIDEMIOLOGY AND ETIOLOGY OF DENTURE STOMATITIS. JOURNAL OF PROSTHODONTICS, 20(4), 251–260. 

Etiological factors include

• poor denture hygiene
• continual and night time wearing of dentures,
• accumulation of denture plaque, and bacterial and yeast colonization of denture surface.
• In addition, poor-fitting dentures can increase mucosal trauma leading to inflammation.

TYPES :

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• Newton´s type I: pin-point hyperaemic lesions (localized simple inflammation)
• Newton´s type II: diffuse erythema confined to the mucosa contacting the denture (generalized simple inflammation)
• Newton´s type III: granular surface (inflamatory papillary hyperplasia)

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Newtons stage 1

Newtons stage 2

Newtons stage 3

Angular Cheilitis : Angular cheilitis is the term used for an infection involving the lip commissures.

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• the majority of cases are Candida associated
• Other possible etiologic cofactors include
• reduced vertical dimension
• nutritional deficiency

• Co-infection with Staphylococcus and beta hemolytic Streptococcus.

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SALERNO, CARMEN & PASCALE, MICHELANGELO & CONTALDO, MARIA & ESPOSITO, VINCENZO & BUSCIOLANO, MAURIZIO & MILILLO, LUCIO & GUIDA, AGOSTINO & PETRUZZI, MASSIMO & SERPICO, ROSARIO. (2011). CANDIDA-ASSOCIATED DENTURE STOMATITIS. MEDICINA ORAL, PATOLOGÍA ORAL Y CIRUGÍA BUCAL. 16. E139-43.

Treatment

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1) General Instructions:

• Maintain good hygiene of dentures.
• No wearing Dentures over night.

2) Relining and rebasing of ill fitting dentures

3) Withdrawal or change of antibiotics if patient is on long term use

4) Surgical excision of hyperplastic issues & reconstruction of new dentures

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• The use of sodium hypochlorite as an overnight denture soak has been shown to eliminate denture plaque and recent investigations have demonstrated that microwave irradiation of denture is bactericidal and candidacidal.

Local and systemic antifungal therapy

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Topical :

• clotrimazole 10mg tablet used 3-4 times daily
• Nystatin suspension 1,00,000 units/cc 1 teaspoon dissolved in ¼ cup water and used as oral rinse.
• Amphotericin B: 5-10ml as oral rinse and expectorated ,3-4 times daily
• 0.2% Chlorhexidine solution as oral rinse

Topical creams/ointments:

• nystatin, ketaconozole or cotrimazole useful In angular chelitis

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• 1% Chlorhexidine gels can be useful in denture stomatitis

Systemic

• Nystatin 250 mg TDS 2 weeks
• Ketaconazole 200mg tab once daily for 2 weeks
• Fluconazole/ itraconazole 100 mg taken once daily for 2 weeks

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Note Azoles interact with oral hypoglycemics, anti epileptics, digoxin, anti coagulants, immunosuppresive drugs

Note

Antifungal treatment can eradicate C. albicans contamination and relieve stomatitis symptoms, but unless dentures are decontaminated and their cleanliness maintained, stomatitis will recur when antifungal therapy is discontinued.

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Peri Implantitis : Defined as inflammation of soft tissues surrounding the implant and progressive bone loss.

• When the inflammation is restricted to soft tissue with no signs of loss of supporting bone it is termed as peri implant mucositis
• And when there is involvement of both soft tissues and progressive loss of supporting bone it is termed as peri implantitis.

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• Microbial plaque is the major factor which causes inflammation in soft tissues around implants.

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• Gram negative anaerobic rods, spirochetes and fusiform bacteria are found in high proportions at peri implant sites.

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• Atleast 10% of Implant failures have been suggested due to peri implantitis.

• The peri-implant area is colonized by a large variety of oral microbial complexes.

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• The microflora of the oral cavity prior to implant placement determines the composition of the microflora in the peri-implant area.

• Implants involved in peri-implantitis are colonized with large amounts of Gram-negative anaerobic bacteria.
• Also, Actinobacillus actinomycetemcomitans can be isolated from these lesions. Thus, the microflora of peri-implantitis lesions resembles that of adult or refractory periodontitis.

Also the incomplete removal of residual cement in sub gingival space around implants is thought to be a cause of inflammation of soft tissues due to two factors

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• Roughness of cement used can cause soft tissue inflammation
• Surface topography may provide a positive environment for bacterial attachment.

Treatment :

Non surgical therapy involves

• Local removal of plaque deposits and polishing of all accessible surface.
• Subgingival irrigation with 0.12% chlorhexidine
• Broad spectrum antimicrobial therapy
• Irradiation with soft lasers has shown promising results in destruction of bacterial cells.

Surgical techniques

• Once the inflammatory process is under control, then an attempt can be made to improve or re establish osseo integration.
• Full thickness mucoperiosteal flap is raised to allow thorough cleaning of contaminated implant surfaces.

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• The stabilization of intra osseous peri implant defect with bone substitute/ bone graft/ bio active substance.

• The micro flora in oral cavity are present with balance with other eco systems.
• Introduction of prosthetic appliance changes the microbial composition.
• Mucosal surfaces beneath Acrylic dentures show increased candida growth because of anaerobic micro environment created.
• Risk factors associated with normal oral flora is dental plaque, dental caries, periodontal diseases.

Conclusion

• Disinfection of impression is mandatory as it can prevent cross contamination.
• Chlorhexidine was a useful adjunct during fixed prosthodontic procedures in maintaining and establishing microflora compatible with periodontal health.
• The use of sodium hypochlorite as an overnight denture soak has been shown to eliminate denture plaque and recent investigations have demonstrated that microwave irradiation of denture is bactericidal and candidacidal.
• Laser disinfection can be of useful in treating bacterial induced peri implantitis.

REFERENCES

• Shafer’s Oral Pathology,, 6th edition
• Ananthnarayan and Paniker’s, Texbook Of Microbiology, 7th edition
• Slot , Contemporary Oral Microbiology and Immunology
• Grossman 12th edition
• Gendreau, L., & Loewy, Z. G. (2011). Epidemiology and Etiology of Denture Stomatitis. Journal of Prosthodontics, 20(4), 251–260. 

• Salerno, Carmen & Pascale, Michelangelo & Contaldo, Maria & Esposito, Vincenzo & Busciolano, Maurizio & Milillo, Lucio & Guida, Agostino & Petruzzi, Massimo & Serpico, Rosario. (2011). Candida-associated denture stomatitis. Medicina oral, patología oral y cirugía bucal. 16. e139-43.
• Samaranayake LP, Hunjan M, Jennings KJ. Carriage of oral flora on irreversible hydrocolloid and elastomeric impression materials. J Prosthet Dent. 1991;65(2):244-249.
• Dhanraj, M. & Selvaraj, Anand & Ariga, Padma. (2013). Evaluation of Subgingival Microflora in All Ceramic Restorations with Subgingival Heavy Chamfer Finish Lines. The Journal of Indian Prosthodontic Society.

• Paolantonio, Michele & D'ercole, Simonetta & Perinetti, Giuseppe & Tripodi, Domenico & Catamo, Giovanni & Serra, Emanuela & Bruè, Claudia & Piccolomini, Raffaele. (2004). Clinical and microbiological effects of different restorative materials on the periodontal tissues adjacent to subgingival class V restorations. 1-Year results. Journal of clinical periodontology.
• Sampaio-Maia, B., Figueiral, M. H., Sousa-Rodrigues, P., Fernandes, M. H., & Scully, C. (2011). The effect of denture adhesives on Candida albicans growth in vitro.

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