8: Microbiology and Immunology

CHAPTER 8 Microbiology and Immunology

MICROBIOLOGY

Microorganisms are life forms that normally CANNOT be seen with the unaided human eye. Possess characteristics common to ALL cellular life in terms of physiology, morphology, reproduction. Inhabit MOST niches of environment. Normal inhabitants of human body (normal flora). A medically IMPORTANT microorganism is one that inhabits human host and is considered a pathogen (causes disease).

image • See CD-ROM for Chapter Terms and WebLinks.

Microbiology Classification

Microbiology spectrum includes prokaryotes, eukaryotes, viruses. Classification of prokaryotes and eukaryotes employs Latin binomial system of genus and species. Ultrastructures have important MAIN differences.

A. Prokaryotes (bacteria) possess a relatively simple cellular structure and are MORE primitive cells.

1. Propagate by binary fission (split into two).
2. Operationally classified into divisions and classes based primarily on morphological and physiological characteristics; divided into two major taxa: eubacteria and archaeobacteria.

a. Eubacteria (true bacteria): comprise bacteria of MOST importance to medicine; smallest bacteria.

(1) Mycoplasmas: smallest free-living bacteria.
(2) Rickettsias and chlamydials, obligate intracellular parasites: smallest members of bacteria.

b. Archaeobacteria: primitive nonpathogenic bacteria normally associated with extreme or unusual environments (e.g., thermophilic, halophilic, methanogenic bacteria).

B. Eukaryotes: possess relatively complex cellular structure, MORE highly evolved than prokaryotes.

1. Fungi: NOT photosynthetic; include number of pathogenic molds and yeasts.
2. Protozoa and helminths: NOT photosynthetic; include some pathogens.
3. Algae: photosynthetic; do NOT include pathogens.

C. Viruses: acellular life forms that are principally composed of proteins and nucleic acid; NOT a cell, so cannot be classified as either prokaryote or eukaryote.

1. ALL are parasites and require viable (living) host cell for replication; replicate once inside the cell and NOT by binary fission.
2. Classification by shape (symmetry), type of nucleic acid, type of host cell, type of vector (agent of transmission), and/or associated disease produced.

Microbial Metabolism

Microbial metabolism includes sum total of all biochemical reactions that occur in the cell.

• See Chapter 3, Anatomy, Biochemistry, and Physiology: body metabolism.
A. Exoenzymes:

1. Enzymes that are excreted into external cellular environment.
2. Break down polymers and other molecules into subunits that are small enough to be transported into the cell.
3. May be potent exotoxins (e.g., Clostridium botulinum and C. tetani produce lipases that affect central nervous system [CNS]).
4. Examples:

a. Carbohydrases degrade polysaccharides to individual sugar residues.
b. Proteases degrade proteins to amino acids.
c. Lipases degrade lipid molecules.
d. Hemolysins lyse red blood cells.

B. Transport of molecules for metabolism:

1. Includes transport of individual molecules to the cell and concentration of these molecules within the cell.
2. Includes four MAIN transport mechanisms:

a. Passive diffusion: random movement of molecules into and out of the cell, progressing from higher to lower concentration gradient (e.g., diffusion of water); cellular energy and carrier proteins are NOT involved.
b. Facilitated diffusion: transport of molecules, such as sugars, mediated by carrier protein that follows a concentration gradient; cellular energy is NOT expended.
c. Active transport: mediated by a carrier protein, requires expenditure of cellular energy to move molecules into the cell against a concentration gradient.
d. Group translocation (phosphotransferase system): moves molecules into the cell against concentration gradient:

(1) Carrier protein and energy (form of phosphoenol pyruvate) are involved, and substrate (e.g., glucose molecule) becomes phosphorylated in the process.
(2) Molecule becomes negatively charged (e.g., glucose-phosphate), then retained inside the cell.
(3) MOST efficient and MOST common form of transport in bacteria.

C. Metabolism:

1. Involves metabolic pathways; once inside the cell, a molecule (e.g., glucose) enters metabolic pathways.
2. Includes two major classes of metabolic reactions:

a. Catabolic reactions: degradative reactions that:

(1) Produce energy (adenosine triphosphate [ATP]).
(2) Produce carbon skeletons.
(3) Produce reducing power.

b. Anabolic reactions: biosynthetic reactions that:

(1) Use up energy (ATP).
(2) Use up carbon skeletons.
(3) Use up reducing power.

D. Oxidation-reduction reactions: employed in cellular metabolism:

1. Compound is oxidized when gives up or loses electrons; reduced when receives or gains electrons.
2. Reactions are simultaneous; when molecule is oxidized, another is simultaneously reduced.

E. Energy production (catabolism):

1. Bacteria produce cellular energy, MAINLY in form of ATP, by passing pair of electrons through the cell.
2. Molecule that gives up the pair of electrons and becomes oxidized is called the electron donor.
3. Molecule that ultimately receives these electrons after they pass through the cell is called the final electron acceptor.
4. Molecules that accept and transfer these electrons (within the cell) from the donor to the final electron acceptor are called electron carriers; typical electron carrier involved in energy metabolism is nicotine adenine dinucleotide (NAD).
5. When the electron donor (e.g., glucose) becomes oxidized, NAD receives the pair of electrons (and protons) and becomes reduced to NADH2; however, the cell is limited in number of oxidized NAD molecules; soon all would become reduced to NADH2 and energy metabolism would cease.
6. The reduced NADH2 molecule MUST be able to donate its electrons to an acceptor to regenerate oxidized NAD; thus defines fermentation and respiration:

a. Fermentation: organic molecule serves as final electron acceptor.
b. Respiration: inorganic molecule serves as final electron acceptor.

F. Fermentation: energy-inefficient process that results in the production of two ATPs from the partial oxidation of one glucose molecule.

1. Strictly anaerobic process:

a. Glucose (electron donor) enters glycolytic pathway.
b. Glycolysis occurs, resulting in oxidation of 1 glucose molecule (C6) into 2 pyruvate molecules (C3) and net production of 2 reduced NADH2 molecules and 2 molecules of ATP, which is referred to as substrate level phosphorylation.
c. Pyruvate, organic compound, is central “hub” of fermentation reactions and serves as final electron acceptor in simplest fermentation; reduced NADH2 donates its electrons directly to pyruvate, reducing it to lactic acid (C3), which is released into external environment.

2. Accomplished by microorganisms capable of carrying out fermentations that result in a wide variety of metabolic end products, including CO2, H2, organic acids, and alcohols.
3. Ability or inability of organism to ferment different substrates is one of KEY features that enable distinction among bacterial species.

G. Respiration: energy-efficient process that results in production of 38 ATPs from complete oxidation of 1 glucose molecule.

1. Can be either aerobic or anaerobic process:

a. Glucose (C6) enters the glycolytic pathway, with same result as in fermentation: 2 ATP, 2 NADH2, and 2 pyruvate (C3) molecules are formed.
b. Pyruvate becomes decarboxylated and bound to coenzyme A, resulting in acetyl-CoA, CO2, and production of 1 reduced NADH2 molecule.
c. Acetyl-CoA feeds into tricarboxylic acid (TCA) cycle; one turn of the TCA cycle results in production of 2 CO2 molecules and 4 reduced NADH2 molecules.
d. Second pyruvate molecule generated by glycolysis undergoes same series of reactions; at this point glucose is completely oxidized to 6 molecules of CO2; 2 ATPs have been produced and a total of 12 reduced NADH2s have been formed; cells then regenerate oxidized NAD and generate MORE energy by means of the electron transport system.
e. Electron transport chain is made up of a series of electron transfer molecules (cytochromes) that are located in cytoplasmic membrane:

(1) Reduced NADH2 donates its electrons and protons to electron transport chain, becoming reoxidized to NAD.
(2) Passage of the electrons through electron transport chain results in production of 3 ATP molecules, producing oxidative phosphorylation.
(3) At the end of electron transport chain the electrons and protons combine with O2, reducing it to H2O.
(4) Overall, there are a total of 12 reduced NADH2 molecules, which results in the production of 36 ATPs (3 × 12) in addition to the 2 ATPs produced from glycolysis, for a total production of 38 ATPs from 1 glucose molecule.

H. Differences between aerobes and anaerobes:

1. Aerobic respiration:

a. Carried out by bacterium that can use O2 as the final electron acceptor in respiration.
b. Involves a combination of O2, electrons, and protons at the terminus of the electron transport chain, resulting in the formation of toxic intermediates, superoxide anions (O2–), and hydrogen peroxide (H2O2).
c. Requires bacterium that can grow aerobically to possess two enzymes for removal (conversion) of these toxic products.

(1) Superoxide dismutase: converts O2– into H2O2.
(2) Catalase (peroxidase): subsequently converts H2O2 into H2O and O2.

2. Anaerobic respiration:

a. Carried out by bacteria that possess electron transport chain but lack either or both superoxide dismutase and catalase and are unable to remove toxic by-products of aerobic respiration.
b. Uses oxidized inorganic compounds other than O2 as final electron acceptor; some common inorganic compounds used in anaerobic respiration include sulfate (SO4), sulfur (S), nitrate (NO3), and carbon dioxide (CO2).

I. Anaerobic fermentation enables bacteria that lack components of, or do NOT have, electron transport chain to grow anaerobically by means of fermentation.
J. Biosynthesis (anabolism): fermentation and respiration produce energy for driving biosynthetic (anabolic) reactions of the cell for necessary synthesis of nucleic acids, proteins, carbohydrates, and lipids.

Microbial Observation

Observation of microorganisms may be either macroscopic or microscopic, depending on total number of organisms.

A. Macroscopic observation is necessary when organisms are in large numbers:

1. Turbidity (cloudiness) in liquid cultures.
2. Colonial morphology occurs when grown on solid surface such as agar agar Petri dish:

a. Defined as mass of cells that arise from single cell.
b. May provide distinguishing characteristics of organism in terms of color (pigmentation), texture, size, shape.

B. Microscopic observation of individual cells is required because of small size:

1. Bacteria may range in size from ∼10 μm (size of a red blood cell) to 0.1 μm (micrometer = 10-6 of a meter); viruses are even smaller; range in size from ∼0.25 μm to as small as 15 nm (nanometer = 10-9 of a meter).
2. Compound microscopes (two or more lenses) provide enlargement (magnification) and allow distinguishing of fine details (resolving power).
3. Resolving power is dependent on wavelength of light and employed with decreasing wavelength, resulting in higher resolving power.

C. Bright field microscopy (via compound microscope): uses visible light and oil immersion:

1. MOST commonly used microscopy type (magnification = ∼1000× and resolution = ∼0.25 μm).
2. Specimens MUST be stained to be observed.

D. Dark field microscopy: observes living, unstained specimens; oblique light increases contrast so specimens appear bright against dark background.
E. Phase-contrast microscopy: observes unstained specimens; contrast is amplified by detection of small differences in refractive indices between specimen and surrounding medium; some dental offices offer this to show dental biofilm during oral hygiene discussion.
F. Fluorescence microscopy: visualizes objects that fluoresce, i.e., emit light, when exposed to light of different wavelength:

1. Detects fluorescent objects illuminated by ultraviolet or near-ultraviolet light; specimens absorb light and emit visible light.
2. Fluorescent dyes conjugated with specific antibodies are basis of immunofluorescence; employed in identification of microorganisms and BEST for tracking of antigen-antibody complexes.

G. Electron microscopy (EM): takes advantage of short wavelength of electrons:

1. Greatly increases magnification and resolving power.
2. BEST for observation of cellular ultrastructure and viruses.

a. Transmission EM: BEST for thin sections of specimens.
b. Scanning EM (SEM): BEST for whole cells and cells attached to surfaces.

Specimen Preparation and Staining

Specimens MUST be prepared and then stained to be seen under light microscope because refractive index of MOST organisms is clear. Basic dyes (e.g., crystal violet, methylene blue, safranin) are positively charged and combine with negatively charged cell constituents. Acidic dyes (e.g., eosin, nigrosin, basic fuchsin) possess negative charge and combine with positively charged cell components.

A. Specimens MUST be heat fixed to slide for bright field microscopy so that cells adhere and are NOT washed off during staining procedures.
B. Simple staining: employs single stain, such as methylene blue or crystal violet; useful ONLY to distinguish cellular morphology.
C. Differential stains: employ two different-colored stains to indicate cellular morphology and distinguish between groups of organisms and/or cellular structures.

1. Gram stain: separates ALL bacteria into two groups: gram-positive (purple-blue) cells or gram-negative (red-pink) cells; stain reflects differences in cell wall structure.

a. MOST important staining procedure in microbiology, first step in bacterial identification.
b. Procedure has four steps:

(1) Crystal violet (purple) is primary stain.
(2) Iodine is mordant that helps to “fix” primary stain to cells.
(3) Alcohol is wash that decolorizes ONLY gram-negative cells.
(4) Safranin (red) is counterstain that enables observation of colorless gram-negative cells.

2. Acid-fast stain: BEST used to differentiate mycobacteria, causative agents of tuberculosis, which appear red, from other bacteria, which appear blue.

a. Primary stain MUST be heated into cell and is NOT washed out, even with acid alcohol, because of hydrophobic waxy nature of cell wall; thus the stain is acid “fast.”
b. Procedure has three steps:

(1) Carbolfuchsin (red) is primary stain heated into cell.
(2) Wash with 3% HCl in alcohol that decolorizes non-acid-fast cells.
(3) Methylene blue is counterstain that enables observation of the non-acid-fast cells.

Bacterial (Prokaryotic) Structure and Function

Small bacteria possess variety of shapes and sizes and internal or external structures that aid in survival and are useful in identification of different bacteria. Survival is KEY to involvement with infectious processes.

A. Cellular shape:

1. Coccus (plural, cocci), spherical:

a. Diplococci (pairs): Streptococcus pneumoniae.
b. Chains of cocci: Streptococcus mutans, Streptococcus pyogenes.
c. Random clusters: Staphylococcus aureus.

2. Bacillus (plural, bacilli), cylindrical rods:

a. Large rods: Bacillus subtilis, Clostridium botulinum.
b. Small rods: Escherichia coli, Salmonella, Shigella.
c. Chains aligned end to end: Streptobacillus moniliformis.
d. Fusiform types (threadlike): rods with pointed ends.
e. Filamentous types (threadlike): branching bacillus rods.

3. Spirillum (plural, spirilla) (spirochetes), cylindrical with an amplitude (spiral with axial fibrils):

a. Leptospira, Treponema, Borrelia.
b. Vibrios have a comma shape: Vibrio cholerae.

4. Palisade arrangement (“snapping division”): Corynebacterium diphtheriae.
5. Pleomorphic (lack a defined cell shape): Mycoplasma pneumoniae.

B. Cellular ultrastructure:

1. Internal cellular structures:

a. Chromosomal DNA: circular and haploid; possesses MOST cellular genes and genetic information.
b. Plasmids: autonomously self-replicating, small, circular DNA molecules, may be transferred from cell to cell and may contain genes for resistance to multiple antibiotics; NOT present in all bacteria.
c. Ribosomes: composed of 30S and 50S subunits, which make up 70S ribosomal RNA required for protein synthesis, distributed throughout the cytoplasm.
d. Inclusion bodies: noted in some bacteria; consist of energy storage molecules, such as poly-beta-hydroxybutyrate, that form granules or refractive bodies.

2. Cytoplasmic membrane encompasses cytoplasm:

a. Composed of lipids, proteins, ions; provides hydrophobic matrix; exists as bilayer.
b. Provides permeability barrier and support for transporting proteins, as well as electron transport system.

3. Cell wall surrounds the cytoplasmic membrane, which provides cellular shape and rigidity.

a. Cell wall: prevents cell from lysing because of increased internal osmotic pressure.
b. Periplasmic space: region between cytoplasmic membrane and cell wall; contains various enzymes and proteins associated with transport.

4. Cell surface structures external to the cell wall:

a. Capsule: generally composed of polysaccharides; thick, tightly bound structure found in some; when present, may be antigenic and may protect against phagocytosis and desiccation.
b. Slime layer: composed of polypeptides; exists as relatively thin layer over the cell surface of some; loosely bound and in some instances MUST be present to provide virulence in bacteria.
c. Glycocalyx: surrounds the surface of some; composed of a macromolecular network of primarily polysaccharide molecules; provides cellular adhesion to surfaces such as tooth enamel (e.g., Streptococcus mutans).
d. Flagella: long (as long as 70 μm), thin protein (flagellin) filaments extending from the cell surface; provide bacterial motility by turning in a circular motion.

(1) Allow movement toward or away from chemical substances (chemotaxis).
(2) Bacteria without flagella MUST depend on brownian motion (random activity by van der Waals forces) for movement.

e. Pili (singular, pilus) and fimbriae (singular, fimbria): short, proteinaceous, hairlike structures that extend from the cell surface; associated with gram-negative bacteria.

(1) Pili: involved with the cell-to-cell transfer of genetic material between bacteria.
(2) Fimbriae: provide cell-to-cell adhesion (e.g., pellicle formation, clumping together of cells).

f. Endospore: large, generally spherical structure that forms within the cytoplasmic interior of the cell; one spore is formed per cell and one bacterium is produced per spore upon germination.

(1) Spore coat: contains large amounts of dipicolinic acid and divalent cations (Ca++).
(2) Spores: means of survival and NOT reproduction; formation is induced by deteriorating growth conditions.

(a) Spores resist high temperatures, desiccation, ultraviolet light, and disinfectants and are major reason for elaborate and harsh sterilization procedures; prevalent throughout environment and may remain viable for thousands of years (the Dick Clark of the microbial world).
(b) Bacillus (aerobic) and Clostridium (anaerobic) are the two major genera of spore-forming bacteria.

C. Chemical differences in cell wall structure between gram-positive and gram-negative bacteria:

1. Peptidoglycan (murine) is present in BOTH gram-positive and gram-negative cells:

a. Three-dimensional macromolecular net that surrounds the cytoplasmic membrane.
b. Composed of a polysaccharide backbone (repeating N-acetyl glucosamine/N-acetyl muramic acid subunits) to which amino acid chains are attached (to muramic acid residues).
c. Amino acid chains are further cross-linked to amino acid chains on adjacent peptidoglycan molecules by additional amino acid residues, thus providing three-dimensional structure.

(1) Gram-positive bacteria possess thick, highly cross-linked peptidoglycan layers because of high internal osmotic pressures; stains purple (e.g., Streptococcus and Actinomyces species; susceptible to antibiotic cillins and sporins).
(2) Gram-negative bacteria possess thin, sparsely cross-linked peptidoglycan layers because of relatively low internal osmotic pressures with lipopolysaccharides and lipoproteins; stay red-pink (e.g., Escherichia coli, Salmonella, Neisseria gonorrhoeae).

2. Gram-positive wall macromolecules:

a. Teichoic acid: polymers of repeating glycerol phosphate and ribitol phosphate residues attached to peptidoglycan; may serve as antigenic determinants or receptor sites for bacterial viruses.
b. Lipoteichoic acids: structures similar to teichoic acid, but with diglyceride residue(s) on one end that provide an anchor into the cytoplasmic membrane.
c. Various polysaccharides may bind to peptidoglycan.

3. Gram-negative wall macromolecules: lipid-enriched outer membrane is formed externally to the peptidoglycan layer by the presence of lipopolysaccharides and lipoproteins.

a. Lipopolysaccharides (LPS) (endotoxins): complex lipid; molecule linked to a polysaccharide chain oriented on the outer surface of the peptidoglycan, with polysaccharide chains extending into the environment.

(1) Antigenic and may serve as receptor sites for bacterial viruses.
(2) Important NOT only in systemic infections but also in initiation and progression of periodontal disease.

b. Lipoproteins: lipid and extended amino acid chains oriented in a manner similar to that of LPS.
c. Porins: globular hollow proteins that form pores in the lipid-rich outer membrane.

D. Mycobacterial (acid-fast) cell walls differ from those of typical gram-positive and gram-negative cells:

1. Waxes (wax D) and mycolic acids cover peptidoglycan layer; creates hydrophobic surface layer correlated with staining reaction of the bacteria (e.g., causative agent of tuberculosis).
2. Basis for the formulations of many disinfectants is whether they can break down this strong cell wall (see later discussion in chapter).

Bacterial Growth and Nutrition

Bacterial growth is the increase in the numbers of bacterial cells by binary fission. Growth is influenced by the physical environment and the availability of essential nutrients. This is important NOT only in pathogenesis of systemic infections but also with development of infectious oral disease (periodontal disease and caries) and its MAIN etiological factor, dental biofilm (dental plaque).

A. Bacterial growth:

1. Generation time: amount of time it takes a single cell to divide into two cells (∼30 minutes for a typical bacterium growing under ideal conditions).
a. Cell numbers expressed as log to the base 10 (e.g., 1 × 107 = 10 million cells) for convenience because of the large number of bacterial cells that may be attained under ideal growth conditions.
b. Measured by visually observing turbidity of a culture, increase in optical density, and increase in protein concentration, or by direct cell counts in a Coulter counter.
2. Bacterial growth curve has four phases:

a. Lag phase: on inoculation into a new medium, cells do NOT immediately divide; cells swell, begin metabolic preparation for growth in the new medium, and use ATP.
b. Exponential (logarithmic, log) phase: cells divide and generation time is at a minimum; ALL cells are viable (alive) and use nutrients; Gram stain is MOST reliable during this phase; antibiotics and infection control agents such as disinfectants are MOST effective.
c. Stationary phase: number of dying cells equals number of dividing cells (constant population numbers); endospore formation may begin; nutrients are being depleted (used up) and toxic metabolic products are increasing.
d. Death (decline) phase: cells begin dying exponentially; now the nonviable cells outnumber the viable ones and nutrients are depleted.

B. Nutritional requirements for bacterial growth:

1. Include liquid water, inorganic elements, carbon, energy source for growth and reproduction.
2. Bacteria may be physiologically classified based on how they derive their carbon and/or energy (sorry, they do not drink coffee to keep them going like some of you are doing right now!).

a. Heterotrophs: obtain carbon from an organic compound (e.g., glucose).
b. Autotrophs: obtain carbon from inorganic source (e.g., CO2).
c. Phototrophs: derive energy from light.
d. Chemotrophs: derive energy from the oxidation of chemical compounds.

3. Bacteria may be further subdivided based on the carbon plus the energy source:

a. Photolithotrophs: obtain energy from light and carbon from CO2.
b. Photoorganotrophs: obtain energy from light and carbon from organic compounds.
c. Chemolithotrophs: obtain energy from inorganic compounds (e.g., sulfur, iron) and carbon from inorganic CO2.
d. Chemoorganotrophs: obtain BOTH energy and carbon from organic compounds, often from the same molecule (e.g., glucose).
e. Saprophytes: grow on dead organic matter.
f. Parasites: grow on living organic matter.

C. Media for bacterial growth:

1. May be liquid or solidified by addition of 1.5% agar agar (obtained from kelp); inert to bacterial metabolism.
2. May be identified as one of several media based on its composition:

a. Defined medium: exact concentration and exact composition of all compounds added are known.
b. Complex medium: exact concentration of components added is known; exact composition of all constituents is NOT known.
c. Selective medium: agents are added that inhibit growth of one group of bacteria but NOT another.
d. Differential medium: distinguishes between different organisms.
e. Differential and selective medium: contains components of BOTH selective and differential mediums.
f. Enriched media: for fastidious (difficult to culture) organisms; contain such complex components as serum and red blood cells.
g. Reducing media and special techniques are necessary for the cultivation of anaerobic organisms that CANNOT be grown in the presence of oxygen.

(1) Reducing agents such as thioglycollate, hydrogen sulfide, cysteine are added to remove oxygen from the medium.
(2) Air may be replaced with nitrogen, hydrogen, and/or carbon dioxide in special jars; hydrogen generator and palladium catalyst used to remove oxygen; employed for cultivating organisms on agar agar plates.

D. Factors that influence microbial growth:

1. Oxygen requirements for growth and reproduction:

a. Aerobes (need O2): require and use oxygen.
b. Anaerobes (NO O2): grow in absence of oxygen; exposure to oxygen may be toxic.
c. Facultative anaerobes (okay O2 or NO O2): use oxygen or grow in absence oxygen.
d. Microaerophiles (low O2): require and use oxygen but in low concentrations.

2. Hydrogen ion concentration (pH): MOST bacteria grow optimally near neutrality, pH 7.0 (see Chapter 3, Anatomy, Biochemistry, and Physiology).

a. Acidophiles: grow ONLY at acidic values, ∼0 to 4.0 pH.
b. Alkalophiles: grow ONLY in alkaline values, > 9.0 pH.
c. Aciduric: tolerate an acid environment (e.g., Streptococcus mutans).
d. Acidogenic: produce weak acids by fermentation (e.g., Streptococcus mutans).
3. Osmotic pressure: MOST bacteria grow optimally at salt concentrations near salinity (0.9%).

E. Microbial growth relationships:

1. May be beneficial, harmful, or benign metabolic interactions between microorganism and host; interactions (Table 8-1) (just like the cast of “Friends”).
2. Involve growing on or within other organisms (hosts), including humans.

F. Bacterial genetics: microorganisms vary in the composition of their DNA and RNA and in the way their genetic codes are expressed, similar to the cells of the human body.

1. Genetic transfer between bacteria:

a. Involves the passage of DNA from a donor bacterial cell to a recipient cell.
b. Spontaneous mutations occur infrequently in a population, but recipient bacteria may instantly acquire new genes, which allows changes to the genetic constitution and capabilities of the organism.
c. Incoming DNA may be degraded by nucleases of the recipient or may be exchanged and integrated with existing sequences in the recipient DNA by recombination.

2. Types of transfer between bacteria:

a. Transformation: gene transfer mediated by free “naked” DNA.
b. Transduction: gene transfer mediated by bacteriophages (bacterial viruses).
c. Conjugation: plasmid-mediated gene transfer, which requires cell-to-cell contact.

3. Significance of conjugation and gene transfer: NOT only can these plasmids become donated and spread like an infection throughout a bacterial population, but they may be transferred between different genera of bacteria and between species of the same genus.

Table 8-1 Microbial interactions

Term Microorganism Host
Symbiosis Benefits Benefits
Commensalism Benefits Benign
Parasitism Benefits Benign or harmed
Pathogenesis Benefits Harmed

INFECTIOUS DISEASES

Microorganisms generally MUST breach host defense barriers to initiate disease-causing reactions and transfer to new hosts, whether it involves bacteria or viruses. EXCEPTION is transmission by ingestion of preformed microbial (bacterial) toxin that can cause toxemia (poison in the bloodstream) (see later discussion). There are many overlaps between the routes of transmission of infectious materials, since the overwhelming goal of the microorganism is to survive at ANY means; there are also nonspecfic and specific host defenses.

Each route of transmission is discussed here with the MOST common examples of infectious disease that have practical association with major risks for exposure in the dental setting. Risk factors for NOT achieving oral health are also listed when involved. Infection control protocol and standard precautions to prevent transmission are discussed later.

A. Nonspecific host defenses: constitute first-line barriers and reactions against invading microorganisms:

1. Physical defenses of skin cells and hair.
2. Chemical defenses:

a. Acidic pH present in stomach (acid secretion), vagina (microbial flora), and skin (lipids).
b. Microbe-digesting enzymes at mucosal sites (e.g., lysozyme), including oral mucosa.
c. Blood proteins termed “acute phase” proteins, which harm microbes by being deposited on their surface and facilitating clearance by phagocytosis.

3. Biological defenses:

a. Phagocytic cells, which internalize and degrade potential pathogens.
b. Complement: series of proteins that damage microbial membranes and facilitate clearance by phagocytosis.

B. Specific host defenses: immune system recognizes and responds to microbes and microbial toxins (discussed later).
C. Common ways for infection transmission:

1. Contact:

a. Direct contact with other infected host(s), which occurs during patient care; especially involves hands, such as NOT washing after restroom use and between patients.
b. Nondirect contact: occurs with a contaminated intermediate object, usually inanimate, e.g., contaminated instruments or gloves that are NOT changed between patients.

2. Droplet (airborne): travel on dust particles or respiratory droplets that may become aerosolized (sneeze, cough, laugh, or exhale); increased with aerosol production generated in dental practice, e.g., ultrasonic use.
3. Vehicle: transmitted to host by contaminated items, e.g., food, water, drugs, devices, equipment; includes fecal contamination of food, water, or hands.
4. Many infections are transmitted from insects or other living carriers (vector borne).

D. MORE common infection transmission routes:

1. MOST common route is via respiratory system, oral mucosa, eyes (do not ever touch your eyes at workplace).

a. Facilitated by the absence of protective skin cells.
b. Potential access to various body cells (e.g., epithelial cells, lymphocytes, macrophages).

2. Second MOST common route is fecal-oral (see handwashing discussion).
3. Skin protection can be breached by cuts or abrasions of unprotected skin, e.g., reschedule patients with open infected lesions until healed.
4. Other infections are transmitted by sexual mode; protection afforded by intact skin is bypassed.
5. Perinatal route from the mother to the fetus and/or newborn (congenital infections).

Bacterial Infections

Many infections are bacterial in origin, but many have vaccinations developed against them or are sensitive to antibiotics and other infection control procedures. However, resistant strains are now a developing problem in healthcare. See later discussion on antibiotics.

• See Chapters 6, General and Oral Pathology: oral infections, infectious peptic ulcers; 9, Pharmacology: antibiotic premedication patient; 17, Community Oral Health: epidemiology.
A. Group A Streptococcus (e.g., “strep throat”) infection: caused by S. pyogenes transmitted by ANY route, but most commonly from infected person (or carrier) via respiratory droplets or skin contact; nasal discharge is IMPORTANT source.

1. Pathogenesis: site dependent, with rapid spreading; attachment to pharyngeal epithelium in the case of sore throat, with disease ranging from local skin infections to fatal bacteremia and toxic shock syndrome.
2. Diagnosis: strep throat, pharyngitis, tonsillitis, redness and edema of mucous membranes, purulent exudate, fever, enlarged cervical lymph nodes; isolation of streptococci in smears or cultures; rapid detection of group A streptococcal antigen from throat swabs is available; may have symptoms of rheumatic fever (see below), including carditis, polyarthritis, subcutaneous nodules.
3. Prevention: unavailable.
4. Treatment: penicillin (Pen-Vee K) for 10 days.
5. Epidemiology of rheumatic fever: in a few cases, untreated strep throat may progress to rheumatic fever; mechanism responsible is antigenic cross-reaction with heart tissue (see later discussion of infective endocarditis), then immune response becomes directed against heart antigens (rheumatic heart disease).

B. Tuberculosis (TB): caused by Mycobacterium tuberculosis transmitted by respiratory route (droplets produced by coughing).

1. Pathogenesis: chronic infection spreads throughout the body via lymphatics and blood, with growth of bacteria in monocytes and intracellular growth, which stimulates T-cell lymphocytic response and results in cell-mediated hypersensitivity and associated tissue damage, causing fibrosis and the calcification of granulomas in the lungs and possibly liver and kidneys.
2. Symptoms: fever, night sweats, chills, malaise, cough, lymphadenopathy of cervical and submandibular lymph nodes; weight loss is common.

a. With lung, coughing and spitting blood.
b. With CNS: may include meningitis.
c. Oral: ulcer on tongue (common and highly infectious lesion).

3. Diagnosis: by fever, fatigue, and specific organ involvement; examination of smears and culture; tuberculin (“TB”) skin testing using PPD (purified protein derivative) reveals positive cell-mediated response to antigens.
4. Prevention: bacille Calmette-Guérin (BCG) vaccination, NOT recommended because of questionable efficacy, also now renders tuberculin skin test false positive; avoidance of close contact with infectious persons is first defense; long-term therapy with antibiotics is second defense.

a. Can be transmitted from patient to patient if infection control procedures such as standard precautions are NOT followed.
b. Avoid elective dental care for patients with active TB; includes first 2 weeks of therapy or until tests are negative.

5. Treatment: antibiotics (isoniazid [INH, Nydrazid], rifampin [Rifadin, Rimactane], pyrazinamide [PZA], or ethambutol [Myambutol]); requires long course (6 to 9 months) to eliminate disease; additional drugs if antibiotic resistance.
6. Epidemiology: close contact and massive exposure enhance transmission rate; immunosuppression, such as with acquired immunodeficiency syndrome (AIDS), leads to enhanced susceptibility.

a. Increased risk for healthcare personnel, including dental healthcare personnel.
b. Patients with human immunodeficiency virus (HIV)-positive history and undiagnosed pulmonary infection SHOULD be suspected of having TB.
c. Poor compliance has resulted in emergence of multidrug-resistant strains (MDR-TB), especially with HIV infection.

C. Actinomyces infection: caused by Actinomyces israelii (MOST common) and A. naeslundii, gram-positive, rod-shaped, anaerobic or facultatively anaerobic; part of normal oral flora.

1. Pathogenesis: opportunistic infection (see later discussion in this chapter) that follows injury, introducing contaminated debris into tissues in immunocompromised; with facial swelling (“lumpy jaw”) and abscess formation in soft tissues becomes fibrotic (“wooden”) and can then discharge tangled masses of bacteria (“sulfur granules”) (Figure 8-1).
2. Treatment: antibiotics such as penicillin (Pen-Vee K) or amoxicillin (Amoxil, Larotid, Polymox) for 6 months to year; surgery if extensive.

D. Meningitis: inflammation of the membranes (meninges) of brain and spinal cord; MAINLY caused by group B streptococci, Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Streptococcus pneumoniae (but also caused by enteroviruses, mumps virus, fungi, parasites), with transmission by contact with another individual or other outside source or as result of invasion by microorganisms normally present (normal flora).

1. Pathogenesis: occurs after microbial invasion of CNS.
2. Diagnosis:

a. Clinical, including fever, irritation, headache, neurological signs.
b. Lab analysis of cerebrospinal fluid (CSF) for cells, elevated protein level, and depressed glucose level.
c. Lab identification of bacteria present in CSF is critical for rapid institution of antibiotic therapy.

3. Prevention:

a. Vaccines for several causative microbes (e.g., H. influenzae, S. pneumoniae, N. meningitidis).
b. Prophylactic use of antibiotics for close contacts.

4. Treatment:

a. Prompt use of appropriate antibiotics, antifungals, or antiparasitics.
b. Antiviral therapy when involving herpes simplex virus.

5. Epidemiology:

a. Occurs in all age groups; neonatal meningitis (group B streptococci or E. coli) has HIGH fatality rate.
b. Use of H. influenzae vaccine has greatly reduced incidence of childhood form caused by H. influenzae.
c. AIDS patients are at risk for meningitis caused by Cryptococcus neoformans.

E. Legionnaire’s disease: caused by gram-negative bacillus; Legionella pneumophila is MAIN species transmitted to humans by way of infected water supply, primarily by aerosol.

1. Pathogenesis: infection of lower respiratory tract; results in toxic effects on lung cells and stimulation of inflammatory responses; exacerbates tissue damage.
2. Diagnosis: lab culture, detection of bacterial antigen and seroconversion.
3. Prevention: decontamination of contaminated water supplies by heating or chlorination.
4. Treatment: antibiotic erythromycin (Robimycin, E-Mycin, E.E.S.) for symptomatic infections.
5. Epidemiology: bacterium common in aquatic systems; dissemination by aerosols (e.g., from evaporative condensers, cooling towers, nebulizers) plays important role in outbreaks; immunosuppression appears to play a role in susceptibility; risk factors are greater for healthcare facilities.

F. Infective (bacterial) endocarditis (IE): inflammation of lining of heart, MAINLY from infected valves (especially mitral) that may lead to massive sepsis (blood infection), with all signs of infection (mainly fever); ends in fatality.

1. Pathogenesis: presence of artificial (prosthetic) valves is MAJOR risk factor, with or without heart transplant; valves need replacement because of congenital disease, IE, rheumatic heart disease, or drugs (phen-fen).

a. Bacteremia (bacteria in the blood) from invasive procedures, e.g., surgery, dentistry, intravascular medical or drug abuse devices, that allow entry of infectious agents.
b. Caused by Staphylococcus aureus (MOST common) or Streptococcus viridans; however, other microorganisms can be involved; thus name is now “infective” instead of bacterial.
c. Risk factors besides valvular complication: past history of IE, cancer, diabetes mellitus (DM), corticosteroid use, IV drug use, alcoholism, renal failure, systemic lupus erythematosus (SLE).

2. Diagnosis: repeated blood cultures.
3. Treatment: early intervention is KEY; prolonged bactericidal antimicrobial therapy, parenterally in high dosages (see discussion below on MRSA).
4. Prevention: antibiotic premedication (prophylaxis) before invasive procedures in high-risk patients, excellent oral hygiene, monitoring past history of disease (high risk for recurrence).

G. Prosthetic (artificial) joint infection: caused by Staphylococcus aureus or S. epidermidis (NOT viridans group streptococci), the major oral bacterial species involed.

1. Pathogenesis: bacteremias from invasive procedures cause hematogenous seeding of total joint implants (ALL joint surfaces are replaced), BOTH in early postoperative period and for 2 years (critical period) following implantation or continually for persons at risk such those with comorbidities and immunocompromised health (e.g., diabetes mellitus, systemic lupus erythematosus).
2. Diagnosis: redness, warmth, and inflammation around affected area, which may be stiff, drain pus, lose range of motion; radiographs and magnetic resonance imaging (MRI) scans, as well as blood samples and aspirated fluid for culturing.
3. Prevention: antibiotic premedication before invasive procedures in high-risk patients, excellent oral hygiene, monitoring past history of disease (high risk for recurrence).
4. Treatment: early intervention is KEY; prolonged bactericidal antimicrobial therapy, administered parenterally in high dosages (see discussion below on MRSA).

H. Methicillin-resistant Staphylococcus aureus (MRSA) infections: caused by gram-positive S. aureus (“staph”) bacteria transmitted by contact.

1. Pathogenesis: deep wound infection in older adults, chronically ill, persons with weakened immune systems; leads to serious skin and soft tissue infections, pneumonia.
2. Diagnosis: small red bumps that resemble pimples, boils, or spider bites, quickly turning into deep, painful abscesses that require surgical draining; tissue sample, nasal secretions, detection of organism’s DNA.
3. Prevention: isolation of infections in healthcare settings, standard precautions for healthcare personnel with emphasis on washing hands, basic hygiene practices after sports, special precautions with immunocompromised, testing if suspected, CORRECT use of basic antibiotics.
4. Treatment: antibiotic vancomycin and others already proved effective against particular strains, such as gentamicin (Garamycin); resistant to all β-lactam antibiotics (penicillins [Pen-Vee K], amoxicillin [Amoxil], cephalosporins [Keflex]).
5. Epidemiology: now community acquired (CA-MRSA), NOT only in healthcare setting (i.e., hospital).

I. Osteomyelitis: infection of bone and bone marrow, usually caused by pyogenic bacteria or mycobacteria.

1. Subclassified on basis of causative organism, route, duration, anatomical location; Staphylococcus aureus MOST common.
2. Pathogenesis: microorganisms may be disseminated to bone hematogenously (i.e., via the bloodstream).

a. Spread contiguously to bone from local areas of infection, such as cellulitis, or are introduced by penetrating trauma, including such iatrogenic causes as joint replacement, internal fixation of fractures, or endodontically treated teeth.
b. Leukocytes release enzymes that lyse bone while trying to heal area; pus spreads into the bone’s blood vessels, impairing the flow, and areas of devitalized infected bone, known as sequestra, form the basis of a chronic infection.
c. The body commonly tries to create new bone around area of necrosis; new bone is an involucrum.

3. Treatment: prolonged antibiotic therapy via IV.

image

Figure 8-1 Facial abscess caused by infection with Actinomyces israelii.

CLINICAL STUDY

Age 42 YRS SCENARIO
Sex image Male image Female The patient presents at a walk-in medical and dental clinic with weight loss, fever, oral sore. A sputum smear shows the presence of acid-fast bacilli. A chest radiograph shows evidence of pulmonary lesions. Two days later, a test the patient took on his skin is determined to be positive; however, he says he has never received any vaccinations since he was a baby.
Height 5′10″
Weight 140 LBS
BP 98/67
Chief Complaint “I am coughing all the time now.”
Medical History
Serious car accident 5 years ago
Smokes cigars when he can get them
Used to abuse alcohol

Current Medications None Social History
Homeless for the last 5 years
Past work as a taxi driver

1. Does the patient pose a risk to healthcare personnel, and if so, what precautions could be taken?
2. What oral lesion may be present in his mouth?
3. What is the significance of the patient’s positive skin test?
4. How would therapy for condition be handled?

1. Close contact and heavy exposure enhance tuberculosis (TB) transmission by respiratory droplets, so healthcare personnel are at greater risk. Limiting droplet exposure by wearing facemasks helps reduce the risk of acquiring TB. Also, using a high- to intermediate-level disinfectant against the microorganism (tuberculocidal) on surfaces that are not covered with disposables will help prevent transmission, as will other standard precautions. However, the patient should not be treated differently from other patients; standard precautions should be strictly followed for all patients. Unless it is necessary, such as with emergency care, elective dental care should wait until he is on drug therapy and no longer contagious. This includes the first 2 weeks of drug therapy or until tests are negative.
2. Common oral sign of secondary TB is a tongue ulcer with irregular border, which is a highly infectious lesion. Confirmed by biopsy and culturing, it resolves after treatment.
3. Positive TB test indicates active immunity (with T-cell lymphocytes and cell-mediated immunity [CMI]) to tubercle bacillus, Mycobacterium tuberculosis. Based on the symptoms and results of the other lab tests and no other past medical history of vaccination, active infection with TB is likely.
4. Therapy would consist of immediate antibiotic therapy and monitoring of compliance with drug therapy, which consists of pyrazinamide (PZA) combined with INH (Nydrazid) and rifampin (Rifadin, Rimactane). Ethambutol (Myambutol) is being added to the usual three-drug therapy regimen. Therapy requires long course (6 to 9 months) to eliminate disease; additional drugs may be added if antibiotic resistance develops. Compliance is very important for eradication of the infection but will be difficult for this patient to maintain in his present homeless situation.

Bacterial Toxins

Some bacteria may cause disease in humans because of toxic (poisonous) properties present in protein molecules (toxins), which they carry and release, producing a toxemia. Disease from toxin exposure may NOT require bacterial replication in the body, because in some cases exposure to the toxin alone is sufficient.

A. Enterohemorrhagic Escherichia coli infection: from toxin-producing enteric gram-negative rod, transmitted via contaminated food by fecal-oral route (food poisoning).

1. Pathogenesis: production of toxins, which cause hemorrhagic colitis and hemolytic-uremic syndrome (HUS, with kidney failure, hemolytic anemia, thrombocytopenia); NOT usually the harmless strains that are part of normal gut flora, producing vitamin K or preventing establishment of pathogenic bacteria within intestine.
2. Diagnosis: strain-specific immunological detection and growth in culture.
3. Prevention: thoroughly cooking ground beef, which is MOST common source.
4. Treatment: rapid administration of antibiotics, reconstitution of fluid and electrolyte imbalances, treatment of disseminated intravascular coagulation.
5. Epidemiology: fatal cases may occur; children and debilitated are MOST susceptible.

B. Staphylococcal food poisoning: toxin (enterotoxin):from half of Staphylococcus aureus strains.

1. Pathogenesis: toxin action on gut nerves, stimulates vomiting and diarrhea.
2. Diagnosis: by symptoms and epidemiology, may be assayed.
3. Prevention: complicated by normal presence of S. aureus on skin and mucous membranes; use of antiseptics and good personal hygiene may help control spread to food; antibiotic therapy for carriers has limited use because of emergence of drug resistance and need for long-term therapy; heating of contaminated food may be sufficient to destroy the bacteria, although toxin remains active.
4. Treatment: symptomatic because of short incubation period (1 to 8 hours) after exposure to toxin.

C. Whooping cough (pertussis): toxin from Bordetella pertussis, gram-negative bacterium transmitted in respiratory droplets.

1. Pathogenesis: colonization of ciliated cells in the respiratory mucosa, followed by production of bacterial toxins; MOST likely play a role in cell damage.
2. Diagnosis: lab culture, demonstration of specific IgA antibodies, molecular detection of DNA.
3. Prevention: vaccination with whole-cell vaccines, effective but occasionally toxic; LESS toxicity with acellular vaccines.
4. Treatment: antibiotic erythromycin (Robimycin, E-Mycin, E.E.S.) reduces infectious period to 5 to 10 days but has LITTLE effect on the disease.
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Jan 1, 2015 | Posted by in Dental Hygiene | Comments Off on 8: Microbiology and Immunology

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