Gram positive cell wall characteristics

1.  Linear polymers of NAG and NAM linked with beta 1,4 bond; teichoic acid and lipoteichoic acid (major role as anchoring sites to cytoplasmic membrane)

2.  Teichoic acid- only found in G + cell walls- major antigens

Gram negative cell wall components

Envelope- external layer outer membrane- bilayer structure composed of phospholipid, proteins, and LPS

-Most abundant protein of outer membrane: porin- subunits form channel that allows passage of small molecules. 

LPS= endotoxin; only in gram neg bacteria; Composed of Lipid A, core polysaccharide, and O-specific polysaccharide antigen

Cause shock and fever

-Periplasmic gel= between inner surface of outer membrane and outer surface of inner plasma membrane- contains hydrolytic enzymes and binding proteins involved in transport and chemotactic receptor activities.

Gram Staining

-Both G+ and G- take up CV and Iodine

- Thin peptidoglycan and outer membrane layers of G-s does not stick to dye.

-Counterstain: safranin

G+=> purple

G-=> pink/red

What is a Mesosome?

• Intracellular component in cytosol

• invagination of cytoplasmic membrane
• attached to nucleoid

• Associated with division septa

How is serologic reactivity an approach to taxonomy classification?

Presence of:

• Specific cell wall LPS ( O antigen)

• Flagella (H antigen)

• capsular (K antigen)

When would you use PCR?

ID of particular pathogen which occurs in low concentration

• Determine taxonomy of bacteria residing in oral cavity or Rapid ID of bacteria in STDs

Describe microarray technique in genetic classification.

• Portion of Single stranded DNA from specific gene attacted to each square chip
• Each square represents different gene on bacterial chromosome
• Used to determine if gene is being expressed and define quorum sensing
• If gene is expressed, RNA copies will be present in cell; mRNA is extracted, labeled and added to chip
• Homologous nucleic acids will hybridize

What is generation time?

Average time needed to double bacterial population. 

Determined during log phase growth

• Grows only under conditions of high reducing intensity
• Lacks metabolic enzyme for using oxygen
• Lacks enzyme for processing toxic oxygen products (catalase)
• Metabolism involves anaerobic respiration

Aerotolerant anaerobe

• common for oral cavity
• will grow in presence of O2 but does not use it as an electron acceptor

Facultative Anaerobe

Capable of growing under both aerobic and anaerobic conditions

• Will employ aerobic respiration when O2 is present
• Metabolism includes fermentation, aerobic, and anaerobic respiration
• Can process oxygen products

Obligate aerobes

Require oxygen for growth

• metabolism is aerobic respiration
• Can process toxic oxygen products

Microaerophiles

• grows best at low O2 levels

Oxidase test

biochemical test

• oxidase enzyme- catalyzes oxidation or reduced cytochrome by molecular oxygen
• oxidase test reagents= artifical electron acceptors --> Kovac's reagent

Anaerobic respiration

• NO3, SO4, or CO2 can serve as terminal electron acceptors. 
• Large group of facultative anaerobes are nitrate reducers: predominantly hetertrophys

• E. Coli and Staphylococcus Aureus

• Nitrate reduction test: determines ability of organism to reduce nitrate to nitrites or free nitrogen gas

Fermentation

-NO respiratory chain or cytochromes

• requires organic compound as terminal electron acceptor
• Obligate, aerotolerant, and facultative anaerobes
• incomplete oxidation of carbs is common
• fermentative end products: lactic acid, ethanol, acetic acid, butyric acid, butanol, propionic acid or acetone. 

Transformation

• naked DNA derived from one cell or virus is taken up by another cell and recombines with genome
• Competency: ability to incorporate DNA
• short period during log phase growth
• Membranes aren't fully fused in log phase

Transduction

• DNA of one cell is introduced into another cell by viral infection
• In process of replication, cellular DNA may "accidentally" be incorporated into new infectious viruses.
• Generalized transduction: any region of host genome can be transduced; only chromosomal DNA
• Specialized transduction:only specific regions of chromosome are transduced, particles carry both chromosomal DNA and phage DNA.

   

Conjugation

• transfer DNA by direct contact between cells
• Sex pilus: sex factor F= extrachromosomal, autonomously replicating DNA molecule (plasmid)
• F promotes conjugation and gene exchange
• Male: F+; Female: F-
• F+ conjugation: F contains fertility information plus limited number of genes- promotes formation of sex pilus; F- receives single stranded copy of F which becomes double-stranded and circular, converting F- to F+

Transposons

• segments of double-stranded DNA that can move from place to place within a genome or between genomes or plasmids or phages genomes
• Can cause mutations
• can carry genes; do not carry genes controlling own replication, but possess insertion sequence elements responsible for transposability

Insertion Sequences

Small (1000 BP) segments of double-stranded DNA that code for site specific recombination

• When these sequences integrate into a gene, function or activity is altered. 

Inhibition of phagolysosome fusion

Mycobacterium tuberculosis

Resistance to lysosomal enzymes

Salmonella Typhimurium

Hyaluronidase extracellular enzyme

• spreading factor

• affects ability of pathogen to penetrate tissues of host by hydrolyzing hyaluronic acid

Collagenase

• extracellular enzyme

• spreading factor that breaks down connective tissue of muscle

Coagulase

• Causes coagulation of fibrinogen

• Fibrin coats cell walls of bacteria, protecting them against action of phagocytes (immunologically invisible)

• involved in walling off process

Kinases

• Breaks down fibrin; dissolves clots from by body to isolate infection

• Allows dissemination of bacteria

Urease

• Splits urea to form ammonium hydroxide, raises pH of urine and facilitates formation of renal calculi (kidney stones)

• Promotes colonization of Helicobacter in stomach

Endotoxins

Example: LPS in gram -

• Lipid A= active portion
• induce fever, initiate complement cascade
• activate B lymphocytes
• stimulate production of TNF, IL-1, and prostaglandins
• Effects of exposure to LPS include fever, hypotension, shock, and death

Exotoxins- 3 types

• Site of action is more localized than endotoxin

1. A-B exotoxins
2. Membrane-active exotoxins
3. super-antigens

A-B exotoxin

• B subunit= binding specificity to host cell
• A subunit= determines mechanism of action

1. neurotoxins= tetanus and botulinum toxins
2. cytotoxins= diphtheria toxin inhibits protein synthesis
3. enterotoxins= cholera toxin and E. coli cause diarrhea

Membrane-active exotoxins

• act directly on cell's surface to lyse or kill

1. leukocidins= damage membranes of neutrophils and macrophages
2. lecithinase= enzyme destroys plasma membranes- especially RBCs
3. hemolysins- destroy red blood cells and other tissue cells by producing pores in membranes

Super-antigens

• type of exotoxin
• bind to MHC class II on antigen-presenting cells and T cells
• association causes release of high levels of IL-2 which leads to release of other cytokines

• Example: Staphylococcus aureus-mediated toxic shock syndrom toxin-1 (TSST-1)

Bacteria of concern in dentistry and routes of transmission

Neisseria gonorroheae

Treponema pallidum

Mycobacterium tuberculosis

Streptococcus pyogenes

Alcohol disinfectants

For skin: use 70% ethyl alcohol, alcohols plus aldehyes for surface use

Aldehyde disinfectants

• Glutaraldehyde alone or in combination with others
• Commonly used in dentistry

• "Cold Sterilization"
  

Bisguanide disinfectants

• Chlorhexidine- commonly used in dentistry for surgical scrub, as an anti-plaque agent and as a denture disinfectant.
• Effectiveness: absorption on to hydroxyapatite and salivary mucus

Halogen disinfectant

• Hypochlorites and poviodine= oxidizing agents
• cheap, effective
• corrode metal and quickly inactivated by organic matter

Phenolic disinfectants

• do not irritate skin- used for gross decontamination

• not easily inactivated by organic material

• poorly virucidal and sporicidal

Dental unit water lines

• used for handpieces, ultrasonic scalers, and air/water syringes

Latex Agglutination

• immunologic tests- either antigen or IgG antibody is nonspecifically bound to surface of latex bead
• Addition of specific antigen or antibody visibly agglutinates milk-white suspension

Enzyme Immunoassay (EIA)

ELISA

• antibody bound to solid support
• Antigen containing sample layered over sensitized solid phase
• Enzyme-labeled Ab is added to form sandwich
• Bound enzyme plus substrate results in color change indicating presence of Ag
• Direct vs. indirect test

Determining minimum inhibitory concentration (MIC)

Highest dilution of antimicrobial agent that is able to inhibit growth of test organism; quantitative estimate of antibiotic sensitivity.

Disk agar diffusion (DAD) (Kirby-Bauer)

• bacterial inoculum- applied to agar plate and then paper disks to which antimicrobial agents have been added are placed on agar surface

• zones of growth inhibition around discs indicate sensitivity to the antibiotic, whereas growth of the organism up to the disc indicates resistance.

Etest

-common susceptibility test

• paper strips containing continuous gradient of antimicrobial-agent concentrations- placed on inoculated agar surface
• After incubation: elliptical zone of inhibition forms as antimicrobial agent inhibits growth
• MIC= where growth intersects Etest strip

Penicillins

• Affect cell wall synthesis
  
• b-lactam ring: 5-member thiazolidine ring joined to b-lactam ring to which is attached a side chain; side chain determines antibacterial properties.
• Mechanism of action: inhibit last step of cell wall synthesis, in which transpeptidation reaction results in cross-linking of adjacent peptidoglycan strands (polymerization)
• Penicillin-binding proteins= occur in cytoplasmic membrane; responsible for transpeptidation reaction
• B-lactam ring portion binds to protein=prevent polymerization
• when binding occurs, lysins are released which degrae formed peptidoglycan
• penicilloic acid- inactive product formed when B-lactamases cleave ring

Types of Penicillins

1. Antistaphylococcal penicillins: resistant to beta-lactamase
2. Aminopenicillins: beta-lactamase sensitive
3. antipseudomonas penicillins: beta-lactamase sensitive

Cephalosporins

• Affect cell wall
• produced by Fungi
• 6-member dihydrothiazine ring joined to a beta-lactam ring; similar mode of action as penicillins
• broader spectrum than penicillins
• Basic structure allows moderate resistance to beta-lactamases but not cephalosporinases

Carbapenems

• affect cell wall synthesis
• Beta-lactam with carbon substituted for sulfur
• Imipenem

Monobactam

• affect cell wall synthesis
• mode of action: similiar to beta-lactams
• Example: aztreonam

B-lactamase inhibitors

• "suicide" inhibitors of beta-lactamases
• beta-lactams that bind to and inactivate beta-lactamase enzymes
• Examples: clavulanic acid and fulvactam
• Clavulanate + amoxicillin= augmentin
• clavulanate + ticarcillin= tigmentin

Glycopeptide antibiotics

• affect cell wall synthesis

• inhibit use of lipid-linked cell wall intermediates in assembly of linear peptidoglycan molecule
• Used for multi-resistant Gram + bacteria
• Not effective for Gram - because it will not pass outer membrane
• Example= Vancomycin

Polypeptides

• Affect cell wall synthesis

1. bacitracin= topically applied; gram +;  targets a lipid carrier responsible for moving peptidoglycan precursors through cytoplasmic membrane to cell wall
2. polymixins= fatty acid portion penetrates into hydrophobic portion of outer membrane of gram - bacteria; ring portion interacts with LPS and phospholipids; can cause serious nephrotoxicity; used for external treatment of localized infections (eye and skin)

Cell wall agents for mycobacteria

Quinolones

• Interfere with nucleic acid synthesis
• mechanism of action: inhibit DNA replication by inhibiting the supercoiling of DNA; binds to DNA gyrases
• Nalidixic acid, ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin

Rifamycins

• interfere with nucleic acid synthesis
• bind to DNA-dependent RNA polymerase and prevent RNA synthesis (transcription level)
• Rifampin= mycobacterium tuberculosis
• Rifabutin= mycobacterium avium

Metronidazole

• interfere with nucleic acid synthesis
• reduced form acts as free radical that damages bacterial DNA
• Anaerobic bacteria produce nitroreductase that reduces compound when taken up by organisms
• no significant activity against aerobic or facultatively anaerobic bacteria

Tetracyclines

• inhibit protein synthesis
• Doxycycline= commonly used in dentistry- periochips and localized aggressive periodontitis
• Bacteriostatic
• Can cause tooth staining in developing teeth

Clindamycin

• inhibit protein synthesis
• commonly used for anaerobic, Gram negs
• common prophylactic in dentistry (broad spectrum)

Metabolic antagonists

1. Sulfonamides= structural analog of PABA- interferes with microbial growth by competitively inhibiting incorporation of PABA into folic acid.
2. Trimethroprim= structural analog of dihydrofolic acid- inhibits synthesis of folic acid

Diptheria Vaccine

Tetanus Vaccine

Corynebacterium diphtheria and Clostridium tetani

Both are toxoid components for children and adults

Pertussis vaccine

Bordetella pertussis

Acellular or killed whole cell components for children

Vaccines for:

Hib Meningitis/epiglottitis

Pneumococcal otitis media/meningitis

Meningococcal disease

All are conjugate components (capsular polysaccaride + protein)

Haemophilus influenza type B-> kids

Streptococcus pneumonia-> kids

Neisseria meningitides A and C-> high risk groups military, college students

Pneumococcal pneumonia/meningitis vaccine

Streptococcus pneumonia

Capsular polysaccharides components for high risk groups- Elderly

Staphylococcus strains (3 types)

1. S. aureus (coagulase +)
2. S. epidermidis (coagulase -)
3. S. saprophyticus (coagulase -)

typically cause skin infections;

serious occur in compromised hosts

Staphylococcus

Gram positive

Clusters

Catalase positive

Susceptible to lysostaphin (cleaves glycine-glycine linkages)

Staphylococcus

Extracellular enzymes for S. aureus

1. coagulase
2. hyaluronidase

Toxins for S. aureus

1. Alpha toxins- membrane active toxin- lyse RBCs
2. enterotoxins- resistant to gastric enzymes, heating; superantigens which act on neural receptors in GI tract; food poisoning
3. exfoliative toxins- "scalded-skin syndrome"- young children; lysis of intracellular attachment at granular layer of epithelium
4. toxic shock syndrom toxin-1 (TSST-1)- pyyrogenic, lethal hypotension, rash, multiple organsystems; superantigen; direct toxic effects endothelial cells resulting in capillary leakage and hypotension

Clinical Manifestations of S. aureus

1.  skin infections- most common bacterial infections in humans- folliculitis, boils, carbuncles, and impetigo; staph scalded-skin syndrom

2. Food poisoning- severe cramping, abdominal pain, nausea, vomitting, and diarrhea

3.  Toxic shock syndrome- acute febrile illness- hypotension, vascular coagulation, renal failure, acidosis, and rash; vaginal association in females and focal staph petechial infection in males.