Oral microbiology Veronika Holá Institute for Microbiology Faculty of Medicine, Masaryk University  and St. Anne´s Faculty Hospital in Brno TZKM, spring 2018 Oral microbilogy • Oral cavity • Dental plaque • Dental caries • Parodontitis • Systemic infections • Infections manifested in oral cavity • Biofilms in GP office 2 • Aterosclerosis of coronary vessels • Stroke • Diabetes mellitus • Pre‐term delivery • Low birth weight • Aesophagal carcinoma Oral health consequences 3 Oral microbiology started with Leeuwenhoek 4 (a) Leeuwenhoek´s drawings & charts; (b) Campylobacter rectus; (c) Selenomonas sputigena ; (d) oral cocci;  (e) Treponema denticola; (f) Leptotrichia buccalis Animacules • Anthony van Leeuwenhoek – 1632‐1723 Description of dental plaque 5 Microflora of the oral cavity 6 • More than 700 genera • Resident & transient • Ecological system • Biofilm formation • Important for health • Relation to etiology of dental caries,  parodontitis, halitosis… Oral microflora 7 • Specific environment • Mucosal surfaces  • Sulcar liquid • Communicates with outer environment • Influenced by ingested food Ecosystem of oral cavity 8 • Lips • Buccal mucosa & soft palate • Tongue • Supragingival teeth surfaces • Sulci gingivales Oral cavity as microbial biotop 9 • Lips – Well aerated, dry environment • Buccal mucosa & soft palate – Detachment – Rich in nutrients, well aerated – Biofilm layer weaker • Surface of the tongue  – Rich colonisation – Transient microorganisms Particular surfaces in oral cavity I. 10 – Papillar region ‐ anaerobes • Teeth  – Ideal surface for bacteria, that form dental plaque • Mucous membrane of sulcus gingivalis – Differs according to conditions of sulci      • Artificial teeth and dental implantates – Specific microflora Particular surfaces in oral cavity II. 11 • Sulcar fluid • Rich in nutrients Sulcus gingivalis 12 • Mix of aerobic and anaerobic species • Α‐haemolytic streptococci • S. mutans group – S. mutans – Less frequent S. sobrinus, S. cricetus and S. rattus • S. salivarius group – S. salivarius, S. vestibularis – Mucous colonies – Can cause endocarditis Streptococci in the oral cavity I. 13 • S. mitis group – S. mitis , S. oralis, S. peroris • Can cause sepsis lenta – S. sanguinis and S. gordonii – S. sanguinis • Can cause IE • S. anginosus group – S. anginosus (S. milleri), S. constellatus – S. c.  constellatus and S. c. pharyngis, and S.  intermedius Streptococci in the oral cavity II. 14 • Proteolytic • Coaggregation with fusobacteria,  porphyromonades • Close relationship to P. gingivalis • Motility   • Chemotaxis • Adhesins, invasins • Haemolysins Treponema denticola 15 wikipedia.org/wiki/Treponema • Inflammatory starters • Highly proteolytic • Short & long fimbriae • Quickly resides sulcus gingivalis Porphyromonas gingivalis 16 • Crossfeeding with T. denticola • Releases outer membrane vesicles for the invasion of Tannerella forsythia • Fimbriae – coculture with S. gordonii • Interaction between T. forythia and P. gingivalis • Member of the red complex of periodontal  pathogens Invasion of T. forsythia into cells, Inagaki 2006, confocal laser microscopy Tannerella forsythia 17 • N. subflava, N. sicca, N. mucosa • Staphylococci, micrococci • Eikenella • Veillonella • Other genera – Propionibacter, Enterococcus – less than 1% Other G+ and G‐ bacteria 18 • Actinomyces sp.  • Lactobacillus sp.– less than 1% • Mycoplasma pneumoniae, M. hominis, M.  salivarium Mycoplasmas, actinomyces,  lactobacilli 19 • Products of metabolism  • Fusobacterium nucleatum protects P.  gingivalis • Bacteriocins – natural antibiotics Interactions of microorganisms 20 • Susceptibility depends on microorganism • Some of them more resistant to phagocytosis • Metastatic infections Protection against immunity system 21 • Entamoeba gingivalis, Trichomonas tenax • Candida sp.   – Immunocompromised patients – ECC relation??? Parasites and fungi in oral cavity 22 Dental plaque 23 • Dental plaque ‐ adherent microbial layer • Can not be washed • Composition variable • According to the location – Supragingival – Subgingival • Sometimes also classified as coronary, fissural,  supragingival and subgingival Plaque 24 • Sessile microbial community • Cells irreversibly attached to a substratum or  interface or to each other • Highly resistant to outer influences Definition of biofilm 25 • It is composed of numerous bacteria • In disease ‐ shift • Open architecture similar to other biofilms Dental plaque 26 Biofilm architecture 27 (a) channel (b) fungoid structure Oral microflora 28 • Bacteria in plaque communicate mutually – Physically – Biochemically – Quorum sensing Mutual relations between biofilm  bacteria 29 Relations of bacteria in the plaque 30 http://dentalplaque.wikispaces.com/Formation • E.g. anaerobic Fusobacterium nucleatum – Early colonizers of the tooth surface – Anaerobic late colonizers Coaggregation in plaque 31 • Pellicula • Surface of enamel, source of some ions  (calcium, phosphorus) • Glycoproteins ‐ receptors for adhesion of G+  cocci and rods • Exopolysaccharid production • Bacterial metabolism in plaque Dental plaque development mechanisms I. 32 • In bottom layers – mineralisation • Finally ca. 80% minerals • Bacterial metabolism • pH decreases to < 5.5 – enamel  demineralisation • Subgingival calculus ‐ mostly G– microorganisms • Calculus is porous Dental plaque development mechanisms II. 33 http://www.intelligentdental.com/2011/12/03/what‐are‐the‐causes‐of‐tartar‐on‐human‐teeth/ Dental tartar 34 • Less than 24 hours: Streptococci prevalent • Days: G+ rods and filamentous microorganisms • Week: Columns/microcolonies of coccoid microbes – rods and filamentous microbes attached • Three weeks: filamentous microbes prevalent,  „corn‐cob“ formation Development of dental plaque 35 • Microcolonies development Oral microflora 36 Confocal laser microscopy – two-species biofilm, Kolenbrander et al., 2002 Bacterial complexes in the oral cavity 37 source: Socransky et al. 1998 • Actinomyces sp.  • Supragingival plaque • Subgingival plaque • Supragingival plaque ‐ reservoir for infections of subgingival space Distribution of microorganisms 38 • Adherent plaque – adherent to  the dental root • Non‐adherent plaque – between adherent plaque and gingival surface Subgingival plaque 39 Subgingival plaque 40 Adherent part Non-adherent part Rather Gram- positive Rather Gram- negative Rather non-motile Rather motile Rather facultative anaerobes Rather strict anaerobes Supragingival plaque 41 • Actinomyces sp.  • Supragingival plaque • Periodontal pathogens can be occasionally found in supragingival plaque Supragingival plaque 42 43 • Different and fluctuating composition • Streptococci • Candida sp.  • Anaerobes G+ rods, but also G‐ cocci ‐ Veillonella sp. • Commonly also staphylococci, mostly STAU Dental plaque on dental plates 44 Microbiology of dental caries 45 • Bounded destruction of tooth tissue • Multifactorial illness • Affection Dental caries 46 • Tooth profile • Structure of enamel • Saliva Endogenous factors 47 • Direct relation • Saccharose • Consumption • Also glucose, galactose, lactose, soluble amyls Nutrition 48 • Specific plaque hypothesis • Unspecific plaque hypothesis • Most of microbes present have biochemically cariogenic potential – But S. mutans, lactobacilli and actinomycetes have stronger potential Microbial factors 49 • Most commonly S. mutans c, e & f and S.  sobrinus d & g • Often isolated from enamel just before caries formation • Correlation of their numbers and progression of caries lesion • Glucans formation • Survive and multiply in low pH • Create low pH environment very rapidly ‐ demineralization S. mutans group 50 • High numbers in caries lesions • Correlation of caries activity and their numbers in plaque and saliva • Multiply in low pH • Form lactic acid BUT • In the initial caries lesions commonly not  present Lactobacilli 51 • Actinomycetes relation to dental root caries Actinomycetes 52 • Higher numbers in most of supragingival plaques Veillonela • Actinomycetes – A. naeslundii, A. odontolyti‐ cus, A. gerensceriae • Low numbers of S. mutans group & lactobacilli • In the microflora higher ratio of G‐ species Dental root caries 53 Microbiology of parodontitis 54 • Anaerobic environment • Rich in nutrients Sulcus gingivalis I. 55 • Mix of aerobic and anaerobic species  • RED COMPLEX bacteria… Red complex bacteria 56 Tannerella forsythia Porphyromonas gingivalis Treponema denticola feed, coaggregation support of adhesion and invasion RED COMPLEX adherence + FUSOBACTERIA Responsibility for parodontitis Sulcus gingivalis II. 57 ANAEROBES Aggregatibacter (Actinobacillus) actinomycetemcomitans Actinomyces – A. gerencseriae, A. georgiae Fusobacterium – F. nucleatum, F. alocis, F. sulci Prevotella nigrescens Porphyromonas gingivalis, P. endodontalis Treponema denticola, T. vincentii, pectinovarum, socranskii Tannerella forsythia Wolinella succinogenes Selenomonas sputigena AEROBES Streptococcus anginosus, Streptococcus constellatus subsp. constellatus, Streptococcus constellatus subsp. pharyngis, Streptococcus intermedius Relationship of bacterial communities  in parodontitis 58source: Socransky et al. 1998 • The red complex  – Porphyromonas gingivalis – Tannerella forsythia – Treponema denticola • Categorized together based on their  association with severe forms of periodontal  disease  • Exhibit very strong relationship with pocket  depth Subgingival microbial complexes I. 59 • The orange complex – Fusobacterium nucleatum ‐ Eubacterium nodatum – Prevotella intermedia ‐ Campylobacter showae – Prevotella nigrescens ‐ Campylobacter gracilis – Peptostreptococcus micros ‐ Campylobacter rectus – Streptococcus constellatus • P. intermedia is always detected in the presence of F.  nucleatum in subgingival plaque samples from deep pockets  • All species in the orange complex showed significant  association with increasing pocket depth Subgingival microbial complexes II. 60 • The yellow complex – Streptococcus sanguis – Streptococcus oralis – Streptococcus mitis – Streptococcus gordonii – Streptococcus intermedius • Bacteria not directly associated Subgingival microbial complexes III. 61 • The purple and green complexes  • Present in the periodontal pocket but not  significantly associated with signs of  periodontal disease progression • Seems to prepare the way for the other  bacterial complexes • The blue complex • Not associated with progression of  periodontal disease Subgingival microbial complexes IV. 62 Systemic infections related to the  oral microflora 63 • Systemic illnesses – Metastatic infections • Bacteria – Metastatic injury • Toxins – Metastatic inflammation • Antigens Systemic infections 64 Metastatic infection 65 • Via transient bacteremia – Subacute infective endocarditis – Acute bacterial myocarditis – Brain abscess – Cavernous sinus thrombosis – Sinusitis – Lung abscess/infection – Ludwig's angina – Orbital cellulitis – Skin ulcer – Osteomyelitis – Prosthetic joint infection Metastatic injury 66 • Circulation of oral microbial toxins  – Cerebral infarction – Acute myocardial infarction – Abnormal pregnancy outcome – Persistent pyrexia – Idiopathic trigeminal neuralgia – Toxic shock syndrome – Systemic granulocytic cell defects – Chronic meningitis Metastatic inflammation 67 • Immunological injury from oral organisms  – Behçet's syndrome – Chronic urticaria – Uveitis – Inflammatory bowel disease – Crohn's disease • Weak data – Propionibacterium acnes – Peptostreptococcus prevotii – Fusobacterium nucleatum – Prevotella intermedia – Saccharomyces cerevisiae – Actinomyces israelii – Streptococcus intermedius – Streptococcus sanguis Bacteraemia 68 • P. gingivalis ‐ linked to rheumatoid arthritis • T. forsythia ‐ identified in atherosclerotic  lesions Other consequences 69 Subacute infectious endocarditis 70 • Endocarditis  • Commonly ‐ S. sanguis and S. gordonii, S.  oralis, S. peroris • Rarely ‐ S. salivarius, S. vestibularis • Difficult and long‐term treatment Subacute infectious endocarditis 71 Risk of IE I. • Low – Defect of heart atrium septum – Implantation of pacemaker – After bypass surgery • Medium – Congenital heart defects – Provisional surgery of heart defects – Rheumatoid valve defects – Prolaps MI valve – Hypertrophic obstruction cardiomyopathy – After surgical correction of cardiovascular defect Risk of IE II. • High – Heart valve replacement – After IE – After surgical correction of cardiovascular deffect Therapy recommendation • ATB prophylaxis • Medium risk – Adults: AMC 2g in 1 dose 1 hr prior to intervention – Children: AMC 50mg/kg, max. 3g in 1 dose 1 hr prior to intervention • High risk – Adults: AMC 2g in 1 dose 1 hr prior to intervention, followed by 750 mg/6 hrs (7 doses) – Children: AMC 50mg/kg, max. 3g in 1 dose 1 hr prior to intervention, followed by 15 mg/kg/6 hrs (7 doses) Therapy recommentation • In PNC allergy alternatively CLI (600mg, children 15 mg/kg 1 hr prior to intervention) • In high risk continue with 300 mg/6hrs (7 doses), children 7,5 mg/kg/6hrs (7 doses) Biofilms in GP office 76 • Growth in the planctonic form – In the fluid environment the isolated microbial cells freely float  • Growth in the biofilm form – Natural tendency to stick to each other and to  solid surfaces – Form a community Two forms of microbial growth 77 • Planctonic form – Common in the laboratory (e.g. in broth) • Biofilm form – Primary in the natural environment because it is  more advantageous for the microbes Which form is more frequent? 78 • Sessile microbial community • Exhibit an altered phenotype  • Highly resistant to outer influences Definition of biofilm 79 • Waterlines = ideal environment for microbial colonization → biofilm • Source of microbes • Consequence of biofilm in dental unit  waterlines Biofilm in GP/dental office I.  80 • Microbes found in waterlines in significant concentrations – Pseudomonads – Legionellae • Evidence suggests exposure of patients and  dental staff to potential bacterial pathogens via dental unit waterlines Biofilm in GP/dental office II.  81 • No efficient way of controlling biofilms • Main interim recommendation – Run water for several minutes at the beginning of  each clinic day – Run high‐speed handpieces for 30 seconds after  use on each patient – 60°C waterlines treatment Biofilm in GP office III.  82 Infections manifested in oral  cavity 83 Overview 84 • HSV • Primary infection on small children – Inaparent – Older children – gingivostomatitis • Acyclovir • Dentists ‐ paronychium Viroses in oral cavity I. 85 • VZV – chickenpox – Rapidly bursting vesicules, erythrema on the palate & uvula – Herpes zoster – pain similar to teeth pain • EBV  – Small petechia on the borders of soft and bony  palate, pharyngitis, pseudomembranous tonsilitis,  pronounced neck lymphadenitis – Edema of Waldeyer lymphatic bow – In HIV specific – hairy leukoplakia Viroses in oral cavity II. 86 • HHV 6  – Exanthema subitum – Erythrematous papuli • Coxackieviruses – Herpangina – ulcerative lesions on tonsili, soft  palate and uvula, similar to herpetic, 1‐2 mm,  grayish surface, surrounded by erythrema Viroses in oral cavity III. 87 • Morbilli – Prodromal period – Koplik´s marks Koplik´s marks Hand‐foot‐mouth disease Hairy leukoplakia Viroses in oral cavity IV. 88 • First indicator – mycotic infections • Viral infections • Bacterial infections • Cervical lymphadenopathy and lymphomas HIV 89 • Oral candidosis – opportunistic pathogen • Treatment – local ATM, autovaccines Mycotic infections 90 • Pseudomembranous candidosis – soor • Erythrematous (atrophic) candidosis – Acute – Chronic • Hyperplastic candidosis • Angular candidosis Mycotic infections 91 • Gonorrhoea • Scarlatina Bacterial infections I.  92 • Syphillis • Late congenital – Hutchinson's teeth – Hard palate defect – Fournier teeth Bacterial infections III.  93 • Diphteria – Pseudomembranous tonsilitis or laryngitis Bacterial infections II. 94 95