MICROBIOLOGY

1. MICROBIOLOGY Prepared by: Dr. D. Boyd, DDS Oral & Maxillofacial Pathologist Associate Professor Reference: Kaplan Review Notes

2. INTRODUCTION • NORMAL MICROBIAL FLORA • Properties: • Population of microbes that usually reside in the body. • Resident flora: fixed population that will repopulate if disturbed, e.g S mutans (caries) • Transient flora: from environs & reside in body without invasion, & prevent infection by more pathogenic organisms (bacterial interference)

3. Distribution of Normal Microbial Flora • LocationMajor Organism • Skin Propionibacteria acnes, Staph epidermis & aureus, diptheroids • Oral cavity viridians Streptococcus, Prevotella melaninogenicus, Actinomyces, Peptostreptococcus, other anaerobes • Nasopharynx:oral microbes, Staph pneumonia, Hemophilus, Nisseria meningitis • Vagina Childbearing age: Lactobacillus, yeast, Streptococcus

4. Microbial Virulence Factors • Gene products required for establishment in the host. • Gene products located on chromosomes, or plasmid or transposons (mobile genetic material) • Primary pathogens virulence factors disease. • Opportunistic pathogens (environ or normal flora)  disease ONLY if host is immunocompromized

5. Categories of Virulence Factors • Enzyme production Hyaluronidasebreaks down Hyaluronic acid digestion of tissue Protease digests protein  spread of infection Coagulase allows coagulation of fibrinogen  clot Collagenase breaks down collagen (connective tissue)

6. Categories of Virulence Factors • 2. Toxins • Exotoxins • heat-labile proteins with specific enzymatic activities • Produced by both Gram positive & negative microbes • Released extracellularly • Often sole cause of disease

7. Categories of Virulence Factors • Exotoxins: • Domains with discrete biologic functions  maximal toxicity • A - B toxins • B subunit bind to host tissue Glycoproteins • A subunit enzymatically attack susceptable

8. Categories of Virulence Factors • Endotoxins • Heat-stable lipopolysaccharide molecule • Located on outer membrane of Gram negative microbes • When released by cell lysis  Lipid A portion  septic shock (fever, acidosis, hypotension, complement consumption, and disseminated intravascular coagulation DIC)

9. Categories of Virulence Factors • 3.Surface components • Protect organism from immune response • Polysaccharide capsule of H influenzae • Acidic polysaccharide capsule of Streptococcus pneumoniae • Adhesins  attach microb to cell of host • Filamentous appendages (fimbriae, pili)  attach microb to cell of host • Flagella  motility

10. Classification & Identification of Bacteria • General properties • Prokaryotic = single –cell organism • 70 S ribrosomes • Naked, single, circular chromosome of double stranded DNA the replicates bi-directionally) • No true nucleus (no nuclear membrane) • No membrane bound organelles • Cell wall is rigid, peptidoglycan layer  Gram positive or negative, Acid fast (resist acid de-coloration e.g Mycobacteria)

11. Classification & Identification of Bacteria • Mycoplasma + Ureaplasm • Only bacteria that do NOT have cell walls • Chlamydia’s cell wall lack muramic acid • Both resistant to beta-lactam antibiotic (penicillin + cephalosporins) • Prokaryotic flagella do NOT have: • 9 + 2 microtubles arangement • microtubules

12. Classification of Bacteria • Biomedical characteristics • Bordetella pertussis grows only onBordet- Gengou agar) • E. coli ferment only Lactose sugar • M. tuberculosis produces Naicin • Serologic reactivity with specific Antibodies in diagnostic immunoassays • Bacteriophage typing used in tracing source of epidemics • Animal pathogenicity & Antibiotic sensitivity

13. Bacterial Structure • 1. CELL ENVELOPE • Gram positivesmooth surface with 3 layers • Cytoplasmic membrane = smooth surface • Thick layer of: • Peptidogylcan • Lipoteichoic acids • Polysaccharides • Teichoic acid (sometimes • Outer capsule (sometimes)

14. Bacterial Structure • Gram negative (complex cell envelope) • Cytoplasmic membrane (inner membrane) • Periplasmic space (containing peptidoglycan • Outer membrane • Tri-layered anchored to cell membrane by lipoprotein • Endotoxin (LPS, somatic O antigen, core polysaccharide) • Protein porin channels • Capsule (sometimes)

15. Bacterial Structure 2. PLASMA (cell) MEMBRANE Function as osmotic barrier 60 – 70% protein 30 – 40% lipid (fat) Carbohydrate (small amounts) Bacterial electron transport chain (cytoplasmic membrane) Membrane polyribosome-DNA Mesosomes = convoluted structures of cell membrane important in cell division)

16. Bacterial Structure • 3. CYTOPLASMIC STRUCTURES • 1. Nucleoid region = circular chromosome of double-stranded DNA • Lack introns, histones, nuclear membrane • 2. Ribosomes • 70% RNA (ribonucleic acid), 30% protein • 70S ribosome attached to messenger RNA  proteins • 70 S complex  subunits 50S + 20S

17. Bacterial Structure • 3. Polyamines located in Ribosomes • Prevent dissociation of the 70S ribosome • 4. Cytoplasmic granules store: • Glycogen • Lipid (poly-bete-hydroxybutyrate) • Phosphate (volutin granules)

18. Bacterial Structure • Spores (endospores): • Bacillus & Clostridium species • Promote survival • Resit heat + drying • Highly dehydrated + refractile • Convert to vegetative state via germination when environ favorable • Bacillus species used to check Autoclave used in Sterilizing instruments

19. Bacterial Growth • Depends on • Available nutrients • External environs e.g Temperature • Growth rate of specific species • Lag phase = period of no growth, adapting • Exponential phase • Steady state of growth, • Continues until nutrients are depleted or toxic wastes products accumulate • Antibiotics maximally effective

20. Bacterial Growth • Stationary phase occurs when nutrients are exhausted or toxins accumulate (cell loss = cell formation) • Phase of decline occur when death rate increases due to cell starvation or sensitivity to toxins

21. Survival in Oxygen • Used to classify bacteria • All bacteria produce Superoxideion (O2-) in the presence of Oxygen. • Superoxide dismutase + O2- Hydrogen peroxide (H2O2) • Catalase or Peroxidase + H2O2 H2O + O2 • Obligate Anaerobeslack these enzymes therefore Oxygen is toxic to them. (Clostridium & Bacteroides) • Facultative organisms grow with or without oxygen.

22. Energy Production • Requires a source of Carbon • Fastidious bacteria lost their metabolic machinery and need many additional requirements. • Siderophores = Iron (Fe3+) chelating compound essential for bacterial growth.

23. Mechanisms of Energy Production • 1. Fermentation • Anaerobic degradation of glucose to obtain ATP • Less efficient than respiration • Used by most Obligate Anaerobes & all Streptococcus species

24. Mechanisms of Energy Production • 2. Respiration • Completely oxidizes Organic fuels • Requires an Electron Transport Chain to drive the synthesis of ATP • Produces 20 times as much ATP • Requires terminal electron acceptor (TEA) • Oxygen, nitrate, fumarate • Obligate Aerobes • Uses respiration only • Must use O2 as TEA (M. tuberculosis)

25. Sporulation • Spore • Dormant structure capable of surviving long period of unfavorable environs. • Capable of re-establishing the vegetative state. • Resistant to radiation, drying, disinfectants. • Thermal resistance due to high content of Calcium & Dipicolinic acid in the core.

26. Sporulation • Bacillus & Clostridiumspecies. • Inhibition of sporulation related to Guanosine Tri-phosphate (GTP) pool. • Carbon & Nitrogen important • Germination & Outgrowth occur when environs & nutrition (glucose, nucleic acid, amino acids) allow.

27. Genetic Transfer • Movement of genetic material into Host • 1. Transformation • Uptake & Integration of naked DNA • Once inside the cell homologous re-combination with chromosome of the recipient must occur • Induced in the Lab with Salt & Heat shock, which force cells to take up plasmids carrying genes of interest. • Streptococcus, Haemophilius, Neisseria gonorrhea, Helicobacter pylori (stomach ulcers)

28. Genetic Transfer • 2. Transduction • Phage-mediated transfer of bacterial DNA • Generalized: • bacterial DNA mistakenly packaged into empty phage head • Recombination occur

29. Genetic Transfer • Transduction (cont) • Specialized: • Lysogenic phage integrated into bacterial chromosome excises itself • Accidentially takes up chromosomal DNA • Phage replicates  bacterial gene picked up replicates • Genes carried into cells that the progeny virus infected • Occurs most often

30. Genetic Transfer • 3. Conjugation (bacterial sex) • Direct transfer of bacterial DNA • Requires cell to cell contact • Most important mechanism for widespread transfer of genetic information between bacteria. • Plasmid mediated. (Extrachromosomal piece of circular DNA that can replicate itself) • Carries genes that encode resistance to antibiotics + virulence factors • Narrow-host-range, broad-host-range plasmids

31. Genetic Transfer • 3. Conjugation (continued) • Narrow-host-range exist in single species • Broad-host-range transfer between different genera • Conjugated plasmid code for genes involved in transfer between cells • Non-conjugated plasmids require help of conjugated plasmid

32. Genetic Transfer • 4. Insertion Sequences are small pieces of DNA that code for the enzyme Transposase which allow pieces to jump into & out of DNA • Transposons consist of: • Two insertion sequences flanking an antibiotic resistance genes • Frequently associated with mutiple drug resistance plasmids

33. Dental Clinic Microbiology • Sterilization • Most commonly used • Bacteria + Fungi + Viruses + Spores killed • Disinfection • Killing of pathogenic organisms + most microbes in general. • Will NOT kill spores • Disinfected instruments are NOT sterile but safe to use

34. Classification of Instruments • Critical Instruments • Pierce mucous membrane or enter sterile tissues • Scalpel blades, Periodontal scalers, Endodontic files, Handpieces • Must be Sterilized or Disposable • Semi-critical Instruments • Touch mucous membrane • Mouth mirrors, Explorers, Xray instruments

35. Sterilization Methods • Autoclave (Steam)dull or corrode sharp edges • 121 degrees F for 20 – 30 minutes, 15psi • Dry Heat (Driclave) – maintain sharp edges • 160 degrees F for 1 – 2 hours • Ethylene oxide (Chemiclave) 8 – 12 hours • Used for heat-sensitive instruments • “Cold Sterilization (misnomer) • Uses long-term disinfectants • Spores are NOT killed unless placed in Glutaraldehyde 12 – 15 hours

36. Sterilization Methods • All instruments must be clean & free of debris • Failure to Strelize due to: • Autoclave over packed • Time insufficient (wrapped instrument need more time) • Autoclave cycle interrupted (power cut) • Tissue (protein) left on instruments

37. Material Method & Action • Material/Method Action • Steam Heat & Protein denaturation Disinfectants Alcohol & Phenols Protein denaturation • Glutaraldehyde Alkylation protein / DNA & Ethylene oxide • Detergent Membrane disruption • Chlorohexidine Membrane disruption • Peroxides & Hg & I Oxidize Sulfhydryl bond • Soap Emulsifcation of Fat

38. Disinfectant Guidelines • Must be Environmental Protection Agency (EPA) approve • Must kill “benchmark” organism Mycobacterium tuberculosis • Must have Dental Association seal of approval for use on dental instruments • Disinfectants used on materials & surfaces • Antiseptics used on live tissue • Hepatitis A & Mycobacterium hard to kill on surfaces

39. Sterilization Monitors • Sterilizers should be checked weekly • Process Indicator(Does NOT show sterilization) • Shows that sufficient Temperature was reached for a specific period of time. • Color change strip or section on autoclave bag. • Biological Monitors (Legal requirement) • Spore strips of Bacillus sp loaded with instruments & cultured after autoclaving. • “Control strip” used to show viability of spores.

40. Universal Precautions • All patients are assumed to be infectious. • Equal Disinfectant/Sterilization/Cleaning procedures for all patients • Preparation of Rooms Instruments & Materials depend on Procedure NOT Patient. • Hand Washing: • Single most effective measure for infection control • Arrival & leaving work, before & between patients, before & after going to restroom

41. Procedures Included in Universal Precautions • Sterilization of MOST instruments. • Disinfection of semi-critical instruments + “Touch and Splash” surfaces. • Barrier methods (Gloves, Masks, Face Shields, Plastic Chair Covers, Light Handle Covers) • Disposable instruments (Saliva Ejectors, Prophy angles, etc)

42. HISTORY OF UNIVERSAL PRECAUTIONS • 1970s: Hepatitis B “clusters”traced to dental offices • 1980s: HID disease refocused dental profession • Much easier to contract HBV than HIV • Approximate conversion rate after needle stick: • HBV 30% • HCV 3% • HIV 0.3%

43. VACCINATION • Hepatitis B vaccination MUST be offered Free to all Dental Health Care Workers • Three injections (0 month, 1 month, 6 months) • Cannot contract disease from vaccination, not made from human blood products. • Recombivax: • Made HBsAg • Produced by Yeast

44. Gram Positive CocciStaphlococcus & Streptococcus • STAPHLOCOCCUS • Genus Characteristics & Classification • Gram positive • Divide in perpendicular plane  Clusters • Relatively resistant to Heat & Drying • Metabolically • Facultative organism • Possess Superoxide dismutaese & Calalase

45. Staphylococcus (cont) • Clinically: • S. aureus only is pathogenic • +ve Coagulase Test identifies S. aureus • S. epidermidis most numerous on the Skin • Coagulase test negative • Also S. Saprophyticus • Urinary tract infection in sexually active women • Treatment: Penicillin

46. Staphylococcus aureus • Common infectious agent in humans • NOT part of normal flora • Transient in Nasopharynx, Skin, Vagina (30%) • Host defense = PMNs • NO protective immunity  repeated infection • Virulence factors include: • Protein A Binding protein Coagulase • DNAse Staphylokinase Lipase • Hyaluronidase Exotoxins (hemolysis)

47. Staphylococcus aureus (cont) • Conditions Commonly Caused by S. aureus • Direct Infection of Skin: • Folliculitis Furuncle Carbuncle Abscess • Cellulitis Wound infection • Systemic Infection: • Osteomyelitis Endocarditis • Lung abscess pneumonia • Toxic-mediateddisease: • Food poisoning Scalded skin syndrome • Bullous impetigo Toxic shock syndrome

48. Staphylococcus aureus (cont) Treatment: • Penicillinase-resistant penicillin: • Amoxicillin, Methicillin, Nafcillin • Cephalosporin (first generation) • Vancomycin (methicillin-resistant S. aureus) • VRSA have been discovered

49. Staphylococcus epidermidis Common nosocomial pathogen (Hospital based) Associated with: IVs Catheters Prosthetic devices Major virulence factor: Exopolysacharidebiofilm (slime) Difficult for Immune system to detect Treatment = Vancomycin

50. Streptococcus Know Very Well • Genus Characteristics • Gram positive cocci, grow in Chains • Metabolically: • Aerotolerant Anaerobes (facutative anaerobes) • Energy from fermentation only (lackcytochromes)  Lactic acid • Lack Catalase (cytochrome-containing enzyme degrades Hydrogen peroxide  oxygen + HOH • Medically most important Streptococcus are auxotrophs (require vitamin, amino acids for growth, not free living in the environs)