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Transcript
Specific Bacteriology Learning Objectives (referenced to Murray’s textbook where relevant) 1. Bacterial Structure, Physiology & Classification (Chapters 2, 3, 4; Dr. Yother’s outline) A. Compare and contrast the properties of eukaryotes vs. prokaryotes. (p. 11, Fig. 3.1) Eukaryotes Prokaryotes Animal, plant and fungi Bacteria and blue green algae True nucleus Primitive nucleus 80s ribosome 70s ribosome DNA diploid strands Single circular haploid DNA Have organelles Lack nucleus and organelles Extrachromosomal DNA in organelles Extrachormosomal DNA in plasmids No cell wall (except fungi) Peptidoglycan cell wall (most) Sexual and asexual reproduction Asexual/binary fission Respiration via mitochrondria Respiration via cytoplasmic membrane Sterols usually present Sterols absent B. Compare and contrast cell wall components in gram-positive vs. gramnegative bacteria. (p. 12-24) Gram Positive: thick peptidoglycan layer containing wall teichoic acid (WTA) covalently linked to peptidoglycan and lipoteichoic acids (LTA) membrane anchored, not covalently linked Gram Negative: thin peptidoglycan layer, outer membrane containing lipopolysaccharide, phospholipids, and proteins. Periplasmic space between cytoplasmic and outer mem contains transport, degradative and cell wall synthetic proteins. Outer mem is joined to the cytoplasmic mem at adhesion points and is attached to peptidoglycan by lipoprotein links. C. Understand structure and function of each bacterial major ultrastructural component: chromosome, plasmid, ribosome, inner (cytoplasmic) membrane, outer membrane, mesosome, teichoic acid, peptidoglycan, lipopolysaccharide, capsule, pili, flagella, endospores. (p.12-22). Chromosome: single (haploid) double stranded circle not contained in nucleus but discrete area called nucleoid, no histones, no nucleosomes, 600-4500kb, ~1kb/gene Plasmid: smaller, circular extrachromosomal DNAs, usually in gram negative, usually not essential: provide selective advantage (antibiotic resistance, metabolic, virulence, conjugative), few to several hundred kb Ribosome: 30s + 50s subunits, form 70s ribosome, translation of mRNA to protein Cytoplasmic membrane: lipid bilayer structure, NO STERIODS (except mycoplasmas), responsible for electron transport, energy production, transport of metabolites, ion pumps and enzymes, photosynthesis, affected by antibacterials, detergents, polymyxins, ionophores Outer membrane: unique to gram-negative, stiff, maintains bacterial structure and is a permeability barrier to large molecules (>800Da) and hydrophobic molecules; asymmetric bilayer: inner phospholipids, outer LPS, proteins include transport and porins Mesosome: coiled cytoplasmic membrane, acts as an anchor to bind and pull apart daughter chromasome during cell division Teichoic acid: water soluble, anionic polymers of polyol phosphates, covalently linked to peptidoglycan and essential to cell viability, important in virulence, shed into media and host, can initiate host responses similar to endotoxin (gram +) Lipoteichoic acid: fatty acid and anchored in the cytoplasmic membrane, common surface antigens (gram +) Peptidoglycan: (murein) rigid layers surrounding cytoplasmic membrane, helps determine cell shape, NaG/NaM crosslinked LPS: amphiphatic (hydrophobic/philic ends) molecule, composes outer leaflet of outer membrane, ENDOTOXIN, activates B cells, release of IL-1, IL-6, TNF, causes fever and shock, shed into media and host, (gram -), structure: Lipid A---core polysaccharide-O Ag; polysaccharide varies with strain, 3-4 sugars/repeat up to 25 repeats, used for serotyping Capsule: loose polysaccharide or protein layer surrounding bacteria, also slime layer or glycocalyx, poorly antigenic and antiphagocytic, major virulence vactor, acts as a barrier and promotes adhesion to other bacteria and host tissue surfaces Pili: fimbriae, hairlike structures on the outside of bacteria; composed of pilin protein subunits, smaller in diameter, not coiled, usually arranged uniformly over entire surface, promote adherence, F pili or sex pili bind other bacteria and transfer chromosomes between, encoded by F plasmid Flagella: ropelike propellers composed of helically coiled protein subunits of flagellin, anchored in membranes through hook and basal body structures, driven by membrane potential. Provide motility, chemotaxis (swim and tumble); peritrichous-all around, polar-one end, bipolar-both ends Endospores: formed by gram POSITIVE, under harsh environmental conditions, convert from vegetative to dormant state, a dehydrated multishelled structure that protects and allows bacteria to exist in “suspended animation”, clostridium, bacillus D. Describe the procedure for Gram-stain and explain the purpose of each reagent. (p.12) Heat fix/dry to slide Stain with crystal violet (adheres to peptidoglycan), which is precipitated with iodine (acts as mordant by increasing the affinity of the dye for the cell) Unbound/excess stain removed with 95% alcohol/acetone-based decolorizer and water Red counterstain safranin added to stain decolorized cells Gram +: turn purple, stain trapped in thick peptidoglycan Gram -: thin peptidoglycan does not retain crystal violet, turn red E. Explain the structural differences of the mycobacterial cell wall from other bacteria that cause it to be “acidfast. (p. 18) Mycobacteria have peptidoglycan layer with a slightly different structure, which is intertwined with and covalently attached to an arabinogalactan polymer and surrounded by a waxlike lipid coat of mycolic acid (fatty acids), cord factor (glycolipid), waxD (glycolipid) and sulfolipids, which causes them to be “acid fast”. Responsible for virulence and is antiphagocytic. F. Explain the process by which peptidoglycan is synthesized. (p. 18-19) 1. Synthesized from prefabricated units constructed and activated for assembly and transport inside the cell. Glucosamine converted to MurNAc, activated. 2. At the membrane the units are attached to the bactoprenol (undecaprenol phosphate) conveyor belt and fabrication is completed. GlcNAc is added. 3. Unit is translocated to the outside, GlcNAc-MurNAc is attached to polysaccharide chain via transglycosylases. 4. Near membrane surface peptide is cross-linked via peptide bond exchange (transpeptidation) between free amine of amino acid in third position (lysine) and d-ala at fourth position, releasing terminal d-ala of precursor. Transpeptidases and carboxypeptidases catalyze reactions; these are penicillin-binding proteins (PBPs), targets for β-lactam antibiotics. G. Explain the process and purpose of spore formation and name the two main genera of bacterial pathogens that produce spores. (p. 22-24) -Depletion of nutrients triggers cascade: spore mRNA transcribed, other mRNA turned off. Dipicolinic acid produced, antibiotic and toxins are excreted. Chrom duplicated, one copy of DNA and cytoplasmic contents (core) surrounded by cytoplasmic mem, peptidoglycan and mem of septum. Two layers of mem and pep surrounded by the cortex, made of thin inner peptidoglylycan surrounding what used to be cytoplasmic mem, and loose outer pep layer. Cortex is surrounded by touch, keratin-like protein coat for protection. -Spore contains a complete copy of the chromosome, the bare minimum conc of essential prot and ribosomes, and a high conc of Ca bound to dipicolinic acid. It protects the genomic DNA from intense heat, radiation and attack by enzymes and chemical agents. -Bacillus (anthrax) and clostridium (tetanus and botulinum) H. Describe how mycoplasmas are unique from other bacteria and how these differences are responsible for their morphology and life cycle. (p. 18) Mycoplasmas have no peptidoglycan cell wall and incorporate steroids from the host into their membrane. I. Explain how the presence of a capsule is important as a bacterial virulence factor and provide examples of clinically important bacteria that are encapsulated. (p. 17) Capsules are unnecessary for growth of bacteria but important for survival in the host. It’s poorly antigenic and antiphagocytic, and is a major virulence factor. It blocks C3b deposition, if complement does attach it blocks phagocyte receptor binding. It acts as a barrier to toxic hydrophobic molecules, and promotes adherence. Streptococcus mutans’ capsule allows attachment to tooth enamel. Bacillus anthracis produces a polypeptide capsule, Streptococcus pneumoniae’s capsule acts as a major virulence factor. J. Understand the processes of DNA replication, mRNA transcription and translation and the steps involved in each. (p. 30-31) DNA replication: synthesized semiconservatively by DNA polymerase, using both strands, occurs at growing forks, proceeds bidirectionally. Leading strand synthesized continuously 5’-3’, lagging strand synthesized in okazaki fragments using RNA primers 5’-3’. DNA polymerases proofread, completes when forks meet 180 degrees from origin, topoisomerases release strain. Transcription: via DNA-dependent RNA pol, sigma factor binds promoter sequence and provides a docking site for RNA pol. Once bound, RNA synthesis proceeds with addition of ribonucleotides complementary to the sequence in DNA. Once gene or operon has been transcribed, RNA pol dissociates, produces mRNA. Translation: mRNA converted to protein; 3 nucleotides form codon, encodes a particular aa. tRNA contains anticodon, allows base pairing. Begins when ribosome binds start codon (AUG), contains A (aminoacyl) and P (peptidyl) sites for basepairing. tRNA matching second codon binds A site, aa forms peptide bond with aa in P site via transpeptidation, leaves tRNA in P site uncharged and is released. The ribosome moves down, transferring tRNA to P site, leaving A site for next tRNA. Continues until reaches termination codon. K. Describe the events that occur in each phase of bacterial growth. (p. 33, Fig. 4-11) When first added to new medium, bacteria require time to adapt to new environment, known as lag phase. Bacteria are actively metabolizing and preparing for active growth. During log or exponential phase, bacteria grow and divide with a doubling time characteristic of strain/conditions. When the culture runs out of metabolites or toxic substance builds up, metabolic activity and growth slows/stops and bacteria enter stationary phase. Finally, death phase occurs, an exponential decline and loss of viability. L. Explain the difference between oxidation and fermentation and give examples of bacteria which are “fermenters”, “oxidizers” or “asaccharolytics”. (p. 28-29; lab syllabus) Oxidation converts pyruvic acid to water and CO2 via the TCA cycle in the presence of oxygen. This allows the organism to generate 3 moles of ATP/NADH, and 2 moles of ATP/FADH2, and 38 ATP/glucose. Carried out by obligate aerobes and facultative anaerobes. “Oxidizers”: Staphylococcus, Enterococcus, Neisseria, Helicobacter, Fermentation converts pyruvic acid to various end products in the absence of oxygen. Organic molecules, rather than oxygen, are used as electron acceptors to recycle the NADH produced during glycolysis. This produces 2 ATP/glucose. An anaerobic process carried out by obligate and facultative anaerobes. “Fermenters”: Streptococcus, Lactobacillus, enteric bacteria (E. coli, Salmonella), Clostridium. “Asaccharolytics”: use substrates other than sugars for metabolic energy, unable to oxidize/ferment carbs; Campylobacter, B. pertussis, L. pneumonphila M. Explain the importance of determining genetic relatedness of bacteria in epidemiology and infection control. (lecture outline) Serological classifications become important in epidemiology. Bacteria are examined to determine what isolates are causing the most disease. For example, the Streptococcus pneumoniae (causes pneumonia) vaccine is directed against the polysaccharide capsule. There are more than 90 different serological capsules that that bacterium can produce. When vaccines are being developed, it is important to know that those vaccines are directed to that capsule. It is also important to know what serotypes are actually most encountered in disease because those are the ones that we want to use in making our vaccine and antiserum to that capsule. In serological classification, you are usually looking at surface antigenscapsules, O Ag (LPS), flagella, polysaccharides, proteins, and pili. Not as important in trying to identify an isolate, but important in epidemiology. N. Explain the differences, advantages, and disadvantages among phenotypic, analytic, and genotypic classification of bacteria and provide examples of each approach. (p. 8;lecture outline) Phenotypic classification: microscopic and macroscopic morphologies used to identify bacteria. Because many orgs can appear very similar, morphologic characteristics provide a tentative id of the org and are used to select more discriminating classification methods. Most common methods consist of measuring presence or absence of specific biochemical markers (ferment specific carbs, use different compounds as source of carbon, presence of specific enzymes, biotyping), antigens can be used in serotyping, antibiogram patterns (analysis of antibiotic susceptibility) and phage typing. Analytic classification: used to classify bacteria at the genus, species, or subspecies level; includes chromatographic pattern of cell wall mycolic acids, analysis of lipids in the entire cell, analysis of whole cell proteins and cellular enzymes. Accurate and reproducible, but labor intensive and instrumentation is expensive; used primarily in reference labs. Genotypic classification: the most precise method for classifying bacteria by analyzing their genetic material. Initially ratio of G:C used, now DNA hybridization, is used for rapid detection and identification, and can identify organisms without growing them. Nucleic acid sequence analysis, plasmid analysis, ribotyping, etc are also used, usually to classify organisms at the subspecies level for epidemiologic investigation, now simplified to point that clinical labs use them in day-to-day practice. O. Explain the specific advantages and reasons that characterization of rRNA is a useful means for determining genetic relatedness of bacteria. (lecture outline) rRNA is associated with the ribosome, it is critical for protein synthesis, it binds the Shine-Delgarno sequence/initiation sequence in mRNA, it must have a secondary structure (base pairs with itself), changes in critical areas are likely detrimental, and DNA that encodes the rRNA is highly conserved among bacteria of common ancestry = high sensitivity, phylogenetic trees are based on rRNA sequences. Gram – are not as closely related as Gram +, rRNA sequence validates and shows that mycoplamsa/mycobacteria belong in Gram +. 2. Bacterial Pathogenesis (Chapters 9, 15, 19; Dr. Briles’ outline) A. Explain the differences between microbial colonization and infection and give examples of each process. (p. 83) Organisms that colonize humans, whether transiently or permanently, do not interfere with normal body functions. In contrast, disease/infection occurs when the interaction between microbe and human leads to a pathogenic process characterized by damage to the human host. Organisms that colonize humans include normal microbial flora such as S. aureus, E. coli, C. albicans. Organisms that cause infections include N.gonorrhoeae, M. tuberculosis. B. Understand the differences between strict pathogens and opportunistic pathogens; be able to give specific examples of each and describe host conditions that are favorable for opportunistic infection. (p.83) Strict pathogens: are organisms always associated with disease, such as M. tuberculosis, N. gonorrhoeae. Opportunistic pathogens: are organisms that are typically members of the patient’s normal flora that do not produce disease in their normal setting but establish disease when they are introduced into unprotected sites (blood, tissues), such as S. aureus, E.coli, C.albicans. If a patient’s immune system is defective, that patient is more susceptible to disease caused by opportunistic pathogens. C. Describe which anatomic locations in the human body contain normal flora versus those locations which are normally sterile and the major types of bacteria that comprise the normal flora in each of these sites. (p. 84-86) Normal flora occurs in the mouth, oroharnx, and nasopharynx (Peptostreptococcus, Actinomyces), the ear (Staphylcoccus), the eye (Haemophilus), the esophagus, stomach (H. pylori), small intestine (anaerobes such as Peptostreptococcus, Porphyromonas), and in very great numbers in the large intestine (Bifidobacterium, Eubacterium, Bacterioides,Enterococcus). The anterior urethra (Lactobacillli, streptoand staphylococci) and the vagina (staphylococci, streptococci, and Enterobacteriaceae) and the skin (Staph, Candida) contain normal flora, although the skin is a hostile environment that does not support survival of most organisms. Normally sterile locations include the lower respiratory tract (larynx, trachea, bronchioles and lower airways), although transient colonization may occur, the urinary bladder may also be transiently colonized, the ureters are generally sterile, and so is the cervix, D. Describe the beneficial roles of normal flora in the host-microorganism ecological relationship. (p. 83-87) The normal commensal population of microbes participates in the metabolism of food products, provides essential growth factors, protects against infections with highly virulent microorganisms, and stimulates the immune response. Some produce vitamins, such as Vitamin K, which aids in blood clotting. E. Explain how prolonged hospitalization or antibiotic therapy can affect the composition of normal flora. (p.83) The microbial flora in and on the human body is in a continual state of flux, throughout the life of a human being the microbial population continues to change. Changes in health can drastically disrupt the delicate balance that is maintained among the heterogeneous organisms coexisting within us. Hospitalization can lead to replacement of normally avirulent organisms in the oropharynx with gram negative rods (Klebsiella, Pseudomonas) that can invade the lungs and cause pneumonia. The indigenous bacteria present in the intestines restrict the growth of Clostridium difficile in the GI tract. In the presence of antibiotics, this indigenous flora is eliminated, and C.difficile is able to proliferate and produce diarrheal disease and colitis. F. Describe the clinical manifestations of endotoxin shock and mechanisms responsible for these manifestations (p. 197; Figure 19-2) At low concentrations, endotoxin stimulates the mounting of protective responses, such as fever, vasodilation, and the activation of immune and inflammatory responses. However, endotoxin levels in the bloods of patients with gram negative bacterial sepsis (bacteria in the blood) can be very high and the response to these acan be overpowering, resulting in shock and possibly death. High concentrations of endotoxin can also activate the alternative pathway of complement; promote high fever, hypotension, and shock, produced by vasodilatation and capillary leakage; and disseminate intravascular coagulation stemming from activation of blood coagulation pathways. The high fever, petechiae, and potential symptoms of shock (from increased vascular permeability) assoc with N. meningitides can be related to the large amount of endotoxin released during infection. Endotoxin-Mediated Toxicity: Fever, leukopenia, followed by leukocytosis, activation of complement, thrombocytopenia, DIC, decreased peripheral circulation and perfusion to major organs, shock and death. G. Describe the similarities and differences between exotoxins and endotoxins, including structure, mechanism of action, targets, and sources. (p. 196-197) Exotoxin: proteins that can be produced by gram + or gram – bacteria and include cytolytic enzymes and receptor-binding proteins that alter a function or kill the cell. In many cases the toxin gene is encoded by a plasmid or lysogenic phage. Many are dimeric with A and B subunits. The B portion binds to a specific cell surface receptor, and the A subunit is transferred into the interior of the cell where injury is induced. The tissues targeted are very defined and limited. (Secreted molecules). Endotoxin: LPS, produced by gram – bacteria, a powerful activator of acute-phase and inflammatory reactions. The lipid A portion is responsible for the endotoxin activity, released during infection, binds to specific receptors on macrophages, B cells and stimulates production and release of acute-phase cytokines, like IL-1, IL-6, and TNF-α. H. Describe 3 mechanisms by which exotoxins work and provide examples of bacterial diseases that are caused by each of them. (p. 198-199) C. diphtheriae toxin binds to a groth factor receptor precursor, inactivates elongation factor-2 which prevents protein synthesis by ribosomes, causing cell death. V. cholerae toxin binds a ganglioside receptor, activates adenylate cyclase which increases cAMP levels, causing loss of cell nutrients and secretory diarrhea. C. tetani toxin binds a ganglioside receptor on the post-synaptic side of a nerve/muscle synapse, blocking inhibitory transmitter release, causing continuous stimulation by excitatory transmitter resulting in spastic paralysis. C. botulinum binds a ganglioside on the presynaptic side, blocking release of Ach from vesicles, blocking muscle stimulation resulting in flaccid paralysis. I. Explain how bacteria can circumvent destruction by the host immune system in order to effectively colonize humans and produce disease. (p. 198-201; Box 19-3) Encapsulation (capsule shields the bacteria from immune and phagocytic responses, protects from destruction within phagolysosome), antigenic mimicry, antigenic masking (immunoglobulin G-binding protein, protein A), antigenic shift (vary structure of surface antigens), production of antiimmunoglobulin proteases (degrade IgA), destruction of phagocyte, inhibition of chemotaxis (degrade C5a), phagocytosis (inhibit via capsule or M protein), phagolysosome fusion (block, prevent release of enzymes), resistance to lysosomal enzymes (catalase), and intracellular replication (grow and hide from immune response, disseminate throughout body). S.aureus esapes host defenses by walling off the site of infection via production of coagulase, which promotes conversion of fibrinogen to fibrin to produce a clotlike barrier. M. tuberculosis survives by promoting the development of a granuloma, within which viable bacteria may reside for the life of the infected person. J. Describe 3 mechanisms by which certain bacteria can circumvent phagocytic killing after ingestion by host phagocytes and provide an example of a bacterial species that utilizes each mechanism. (p. 200; Table 19-4) Inhibition of phagolysosome fusion – Legionella, Mycobacteium tuberculosis, Chlamydia. Prevents contact with its bactericidal contents. Resistance to lysosomal enzymes – Salmonella typhimurium, coxiella, Ehrlichia, Mycobacterium leprae, Leishmania. Catalase makes myeloperoxidase less effective. Adaptation to cytoplasmic replication – Listeria, Franciscella, Rickettsia. Escape lysosome and grow in cytoplasm. K. Explain the differences between active and passive immunization. (p. 159) Active immunization occurs when an immune response is stimulated because of challenge with an immunogen, such as exposure to an infectious agent (natural immunization) or through exposure to microbes or their antigens in vaccines. Passive immunization occurs when the injection of purified antibody or antibody-containing serum provides rapid, temporary protection or treatment, also newborns receive natural passive immunization from maternal Ig that crosses the placenta or is present in the mother’s milk. L. Explain why a polyvalent polysaccharide-conjugate vaccine is used to immunize infants against invasive pneumococcal disease whereas a polyvalent vaccine alone is used in adults at risk for invasive pneumococcal disease. (p. 161-162) Polysaccharides are generally poor immunogens (T-independent antigens). The immunogenicity of polysaccharides can be enhanced by chemical linkage to a protein carrier, creating a conjugate vaccine, which can be administered to infants and children. Polysaccharide vaccines are less immunogenic and should be administered only to children older than 2 years. (old objectives state that children don’t make Ab to polysaccharide). M. Describe the different mechanisms by which host resistance to infection by extracellular bacteria versus intracellular bacteria occurs. (lecture outline p. 7) Extracellular bacteria replicate outsie of cells and must avoid being killed by phagocytes or complement (ex: Staph) Intracellular bacteria replicate inside cells and must avoid being killed by phagocytosis and antibacterial properties of lysosomes (ex: TB, typhoid fever) 3. Bacterial Genetics (Chapter 5; Dr. Steyn’s outline) A. Describe differences between the bacterial and human genomes, including size, composition, arrangement, presence of extrachromosomal elements, numbers of chromosomes. (p. 35) The chromosome of a typical bacterium is a single, double-stranded circular molecule containing approx. 5 million base pairs, an approximate length of 1.3 mm (~1000x the diameter of the cell). The smallest bacterial chromosomes (from mycoplasma) are approximately ¼ this size. In comparison, humans have two copies of 23 chromosomes, which represent 2.9 x 10^9 base pairs 990mm in length. Eukaryotes usually have two distinct copies of each chromosome (diploid), bacteria usually only have one copy (haploid). Because bacteria only have one copy, alteration/mutation will have a more obvious effect. Structure of the bacterial chromosome is maintained by polyamines, such as spermine and spermidine, rather than histones. Bacteria may also contain extrachromosomal elements, such as plasmids or bacteriophages. These elements are independent of the bacterial chromosome and in most cases can be transmitted from one cell to another. B. Explain three different mechanisms of transfer of genetic information between bacterial cells: transduction, transformation, and conjugation. (p. 41-44) Transduction: the transfer of genetic information from one bacterium to another by bacteriophages, which pick up fragments of DNA and package them into bacteriophage particles. The DNA is delivered to infected cells and becomes incorporaed into the bacterial genomes. Can be specialized, if they transfer particular genes, or generalized if selection of the sequences is random. Transformation: the process by which bacteria take up fragments of naked DNA and incorporate them into their genomes, results in acquisition of new genetic markers from exogenous or foreign DNA. Both gram + and gram – can take up and stably maintain exogenous DNA (said to be competent), competence develops toward the end of logarithmic growth, some time before a population enters the stationary phase. Most bacteria do not exhibit a natural ability for DNA uptake, but chemical methods and electroporation can be used to indroduce DNA. Conjugation: is the mating or quasisexual exchange of genetic information from one bacterium (the donor) to another bacterium (the recipient). Occurs with most, if not all, eubacteria, usually between members of the same or related species but has been demonstrated between prokaryotes and cells from plants, animals, and fungi. Results in the one way transfer of DNA through the sex pilus. Mating type (sex) depends on presence (male) or absence (female of the F plasmid, which carries all the genes necessary for it’s own transfer, inducing the ability to make sex pili and initiate DNA synthesis at the transfer origin of the plasmid. Single stranded DNA is transferred, recirculizes, and the complementary strand is synthesized. Integration of the F plasmid into host DNA results in transfer of part of the plasmid sequence and a portion of the bacterial chromosome. **Transposition: Once inside a cell, a transposon, a jumping gene, can jump between different DNA molecules (plasmid to plasmid or plasmid to chromosome), or within a single genome. Transposons contain genetic information necessary for their own transfer, and may contain genes for resistance against antibiotics. They can insert into and inactivate genes, sometimes resulting in cell death. 4. Antimicrobial Agents, Chemotherapy and Resistance (Chapter 20; Dr. Waites’ outline) A. Describe the differences between antibiotics, antiseptics, and disinfectants. (lecture outline) Antibiotic – an agent that can be naturally occurring, partially or completely synthetic that selectively inhibits the growth of microorganisms at low concentrations (penicillin) Antiseptic – an agent used to inhibit or eliminate microbes on the skin or other living tissue (alcohol, iodine, chlorhexidine) Disinfectant – an agent used to destroy microbes on inanimate objects (phenols, formaldehyde, chlorine bleach) B. Recognize the generic names of all antibiotics and groups discussed in the lectures and be able to classify them according to mechanism of action. (lecture outline; Table 20.1), C. Describe the targets in the bacterial cell where antibiotics act to inhibit growth and how the various drugs work at each site (e.g., cell wall, cell membrane, ribosome, DNA replication, etc.) (p. 204) D. Relate specific antibiotics (or classes of antibiotics) to their major therapeutic applications and toxicities. (lecture outline) Beta-lactams: act on cell wall to inhibit growth Penicillin – binds active site of transpeptidase enzyme (also called PBPs, penicillin binding proteins) that cross-links peptidoglycan by joining D-ala and gly; mimics D-alanyl-D-ala that would normally bind; irreversibly inhibits transpeptidase; broad spectrum, effective against gram – bacteria. Cephalosporins – same mechanisms of action as penicillin but have a wider antibacterial spectrum, are resistant to beta lactamases and have improved pharmacokinetic properties. Monobactams – narrow spectrum active only against aerobic, gram-negative bacteria. Carbapenems – broad spectrum antibiotics active against virtually all groups of organisms. Non-beta lactams acting on cell wall Cycloserine – inhibits synthesis of D-alanyl-D-ala within cell wall mucopeptide inhibiting cross linking of peptidoglycan, used to treat mycobacterial infections. Glycopeptides (vancomycin) – binds D-ala-D-ala moiety of precursor subunit blocking transpeptidation (blocks cross linking) used against growing gram + bacteria. Bacitracin – complexes with lipid carrier that transports peptidoglycan precursors from cytoplasm to cell membrane (inhibits cytoplamic membrane and movement of peptidoglycan precursors), used for treatment of skin infections caused by gram +, particularly Staph and group A strep. Isonazid (INH) – inhibits fatty acid and lipid components of mycolic acid synthesis in mycobacteria, bactericidal against actively replicating mycobacteria. Agents acting on bacterial ribosome Streptogramins – bind 50s, prevent peptide elongation and premature release from ribosome, effective against staphylococci, streptococci, and E. faecium. Oxazolidinones – bind 50s and prevent formation of initiation complex, active against all staphylococci, streptococci and enterococci. Aminoglycosides – bind 30s, inactivate initiation complex, misread mRNA genetic code and prematurely release peptide, used to treat serious infections caused by many gram – rods and some gram + orgs, anaerobes are resistant. Chloramphenicol - bind 50s, prevent peptide bond formation, broad spectrum not commonly used in US because it disrupts protein synthesis in human bone marrow cells and can cause blood dyscrasias. Clindamycin – bind 50s, prevent peptide bond formation, active against staphylococci and anaerobic gram – rods, generally inactive against aerobic gram -. Macrolides – bind 50s, prevent release of deacylated tRNA preventing peptide elongation, bacteriostatic with a broad spectrum of activity, most gram – are resistant. Tetracyclines – prevent attachment of tRNA-AA to 30s, broad spectrum, effective against Chlamydia, mycoplasma, rickettsia and other selected gram + and gram -. Agents interfering with Cell Membranes Polymyxins – cyclic polypeptides derived from B acillus polymyxa, insert into bacterial membranes like detergents, increasing cell permeability and eventual cell death. Used for localized infections, against gram – rods. Daptomycin – disrupts cell membrane function, binds the membrane and causes rapid depolarization resulting in a loss of membrane potential leading to inhibition of DNA, RNA, and protein synthesis. Active against gram + only. Agents interfering with DNA Replication Rifampin – binds DNA-dependent RNA pol, prevents mRNA transcription, bactericidal for Mycobacterium tuberculosis and active against aerobic gram + cocci, including staph and strep. Metronidazole – intermediate metabolites damage DNA in anaerobes, effective in treatment of amebiasis, giardiasis and serious anaerobic infections. Fluoroquinolones – bind to DNA gyrase and Topoisomerase IV, inhibiting DNA rep, excellent activity against gram + and gram – bacteria. Antimetabolites Sulfonamide – inhibits dihydropteroate synthase, disrupts folilc acid synthesis, active against broad range of gram + and gram – such as Nocardia, Chlamydia, and some protozoa. Trimethoprim – inhibits dihydrofolate reductase, synergy with sulfonamide (used together). E. Be able to list and describe desirable properties and requirements of antibiotics useful in treating microbial infections in the human host. (lecture outline) Selective toxicity, water soluble, bactericidal, high serum levels achieved for several hours, broad spectrum, minimal effect on normal flora, low potential for inducing resistance, minimal side effects/toxicity. F. Be able to list and describe 5 mechanisms of antimicrobial drug resistance and give specific examples of each. (lecture outline) Decreased permeability – Aminoglycosides, resistance caused by inhibited transport of the antibiotic into the bacterial cell. Active Efflux – Erythromycin, actively exporting the antibiotic out of the cell. Altered Target – Fluoroquinolones, mutation of genes for binding sites (DNA gyrase and topoisomerase) Metabolic Bypass – Trimeth/sulfa, bacterial strains develop alternative enzyme systems to synthesize folic acid Enzyme inactivation – Penicillin, bacteria produce β-lactamases that inactivate the beta-lactam antibiotics. G. Understand the difference between innate and acquired antibiotic resistance and give examples of each. (lecture outline) Innate – Primary resistance, the natural resistance bacteria possess to some antimicrobials. For example, organisms may be naturally impermeable to some antibiotics due to their cell structure. Organisms with innate resistance are often of low virulence, but, because they are resistant to many agents, they persist in the environment. Acquired – An organism can acquire resistance to an antimicrobial to which it was previously sensitive. This can be due to chance mutation in the genetic material of the cell, or the acquisition of resistance genes from other drug resistant cells. H. Understand the concept of clonal spread as it relates to transmission of antimicrobial resistant bacteria. (lecture outline) Old outline: Clonal spread refers to the fact that bacteria can mutate and become resistant to certain drugs, and a person with a drug-resistant strain of bacteria can spread it to others. I. Understand differences between bactericidal and bacteriostatic drugs and how this relates to drug choice in specific clinical settings. (lecture outline) Bactericidal – level of antimicrobial activity that kills the organism. Bacteriostatic – level of antimicrobial activity that inhibits growth of an organism. Old Objectives: In most infections, bacteriostatic drugs are used because preventing the bacteria from dividing is generally enough to stop the infection. Caution must be taken when prescribing combination therapy because a drug such as penicillin, which can act only when the bacteria is dividing, used in combination with tetracycline, a bacteriostatic drug which stops bacteria from dividing, would be antagonistic. J. Explain the rationale for antibiotic prophylaxis and combination therapy. (lecture outline) Antimicrobial prophylaxis can prevent endocarditis following dental work, provide protection for contacts of meningococcal meningitis, prevent opportunistic infections in AIDS, recurrent UTIs and post-surgical infections. Combination therapy (old outline) provides a broader spectrum for infection of unclear etiology, polymicrobial infections, prevents emergence of resistance (mycobacterium), infections difficult to eradicate (immunosuppressed host), and synergy (drugs enhance effects of other drugs). K. Know major characteristics of bacteria that favor development of drug resistance. (lecture outline) - Intrinsic resistance to some drugs - Ability to exchange genetic information - Ability to survive adverse environmental conditions - Easily colonize, infect, and transmit - Reservoirs in body L. Explain the reasons why drug resistance in bacteria is increasing in the hospital and community and its consequences to the health care system. (lecture outline) Resistance increases in hospitals due to greater severity of illness, immunocompromised patients, new devices and procedures, ineffective infection and control practices, increased use of antimicrobial prophylaxis, and empiric polymicrobial antimicrobial therapy. Its consequences are prolonged illness/hospitalization, increased mortality, inappropriate therapy, more expensive/toxic therapy, more lab tests, and the spread of infectious organisms that may have no effective treatment and that may be impossible to eradicate from the hospital. M. Discuss significance of emerging drug-resistant bacteria in the hospital and community including: S. aureus, E. faecium, S. pneumoniae, ESBL-producing E. coli and Klebsiella spp., and P. aeruginosa. (lecture outline) 5. Anaerobic Bacteria (Chapters 40, 41, 42; Dr. Moser’s outline) A. Describe 2 enzyme systems that are lacking in strict anaerobes and why the lack of these enzymes renders oxygen toxic to them (lecture outline) Superoxide dismutase (SOD) and catalase, detoxify high energy radicals such as hydrogen peroxide produced from oxygen metabolism that otherwise kill the cell. Cytochrome systems normally turn radicals into water and atmospheric oxygen via these enzymes. B. Describe the anatomic sites where anaerobes are normal flora. (p. 83-87) Colon (over 90% are anaerobes), mouth, vagina, skin. C. Learn clinical characteristics, responsible organism(s), and mechanisms of disease for these conditions caused as described in the lecture and assigned reading: Periodontitis – chronic, progressive receding of the gums due to the toxins being produced by the bacteria that are hiding in crypts. No matter how good the teeth are, eventually the gums are gone and they can’t hold their roots into the bone. Caused by Acinetobacter spp. Brain and lung abscess - if you have poor dentition and your teeth are loose or you get your teeth cleaned, activity can cause a showering of oral bacteria. That puts you at risk if, for example, you have a defective heart valve, you could get endocarditis. If it’s chronic, it may get to the brain and cause an abscess. Brain abscesses usually due to gram + rods, cocci, and a few gram -: Bacteriodes, Prevotella, and Porphyromona. Aspiration pneumonia, the classic example is an alcoholic who passes out and they regurgitate stomach acid and contents and they aspirate. They get oral flora and foreign body material from the stomach and acid. All of that destroys tissue and once you get destroyed tissue, anaerobes are happy because they have a place to hide from oxygen. They will cause abscesses and bacteremias as well. Often due to Actinomyces. Vincent’s Angina – acute dental infection, all gram negative. Treponema are very thin and corkscrew-like. Fusobacterium are long and fusiform. The short fat rods are probably Prevotella/Bacteroides. There is overgrowth of these organisms that are normally there, but because of poor oral hygiene have multiplied and cause damage. Gas gangrene - a very active infection. In the Civil War, people got wounded in the trenches and they lost their arms or legs due to this infection. There are a number of toxins, but the most important one in gas gangrene is Phospholipase C, also called alpha-toxin or lethal toxin. It takes your tissue and makes hydrogen gas and CO2 out of it. That’s why it has gas in the tissue. Gangrene means dead tissue and gas is in the dead tissue, so you can poke it and it feels like packing material. It has about 12 possible toxins that do lots of things. Basically, it chews up your tissue. Often caused by C. perfringens. Lumpy Jaw - Actinomycosis. The face becomes asymmetric; normal face on one side and greatly swollen on the left side. Looking down at the root of the crown and looking at the x-ray, you see the roots and the bone are being eaten away. An abscess forms and is quite painful. People will come in to have it looked at and if you leave it alone they make these draining sinuses. This will come out the side of the jaw. If it’s opened up and put some gauze on it, the gauze soaks up the fluid and you can capture the sulphur granules and diagnose without having to go into the abscess inside, but not everyone waits this long. This is the classic description of Actinomyces israelli infection. Tetanus - step on a nail, get a splinter, some kind of gross contamination of a wound and the organisms spores in the environment are now in your tissue and they grow and produce this toxin called tetanospasm. Tetanospasm blocks the inhibitory neurotransmitters, the gammaaminobutyric acid and glycine. That causes the inability to relax the muscle, so you get constant firing of muscles. This is lockjaw. It’s called “sardonic smile,” because the muscles are clamped and you can’t undo them. Caused by Clostridia, gram + spores. Botulism - aka food poisoning. It’s mostly pre-formed toxin that we ingest. This organism lives in the soil and it can come from poor handling of foods, improper canning. Bacterial spores are harder to kill than cells, so there has to be higher temperature and pressure and it takes a period of time to kill them all off. If canning isn’t done properly and all the spores aren’t killed then the organism grows in the anaerobic environment. This toxin is extremely toxic; it does not take much of it. This toxin blocks Ach and you don’t get firing of the muscles and you get flaccid paralysis. It’s the opposite of tetanus. This is a problem as it progresses because the diaphragm is a muscle and it quits firing. That’s why most of the treatment is support and can include ventilators to maintain the patient until they recover. An exception to it being pre-formed is infant botulism. Infants don’t have a GI flora established yet, they’re not born with one, and so over time they pick up bacteria that colonize the GI tract and that protects against introduction of different organisms. If during that period of time, they’re exposed to Clostridium botulinum and the classic way this happens is by giving them honey. Honey is environmental and it can have spores from the organism and you get production in the GI tract. This still occurs occasionally and it’s the opposite of the usual ingestion of pre-formed toxin from food. Caused by Clostridium botulinum. Pseudomembranous colitis - C. difficile colitis, antibiotic associated. Patients present with diarrhea, fever, and abdominal pain. D. Understand how laboratory diagnosis of the above anaerobic infections is achieved. Gram stain: may be helpful in establishment of a mixed infection or the presence of clostridia in wounds Culture: sample collection and transport are critical, require complex medium supplemented with hemin, vitamin K and/or blood, should include media containing antibiotics (aminoglycoside) to suppress facultative anaerobes, incubation and work up perfomed in CO in nitrogen/hydrogen mix. Anaerobic infections generally have a foul smelling discharge, are located close to a mucosal surface, cause gas in tissue, have abscess formation. E. Explain differences between gaseous requirements for bacterial growth, including microaerophiles, capnophiles, obligate anaerobes, obligate aerobes, facultative anaerobes, aerotolerant anaerobe. (p. 25; lecture outline) Aerobic: require oxygen as electron acceptor (respires) Microaerophilic: require oxygen in reduced quantity, can grow without Capnophilic: require carbon dioxide Obligate anaerobes: cannot survive in presence of oxygen, always ferments Obligate aerobes: cannot survive without oxygen, cannot ferment Facultative anaerobes: can grow with or without oxygen, can respire or ferment Aerotolerant anaerobe: grows with or without oxygen, always ferments Important concepts related to specific groups of bacteria Chapters: 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 43, 44, 45, 46, 47; Lecture outlines from Drs. Benjamin and Waites) Listed below are specific examples of important concepts related to major bacterial groups and species and their diseases that should be learned in addition to the characteristics of individual bacteria. A. Gram-positive cocci and bacilli 1. How do Protein A and the P-V leucocidin aid Staphyloccoccus aureus in causing disease? (lecture outline) Protein A coats the surface of most S. aureus and inhibits antibodymediated clearance by binding IgG (1,2,4) Fc receptors. Binding the Fc region of IgG inhibits the Fab region from binding and opsonizing the bacteria. It can also bind antibodies and form immune complexes with the subsequent consumption of complement. P-V leukocidin toxin lyses white blood cells. Cell lysis by these toxins is mediated by pore formation with subsequent increased permeability to cations and osmotic instability. 2. What advantage does the presence of coagulase confer on Staphylococcus aureus?(lecture outline) S. aureus has two forms of coagulase: bound and free. Coagulase bound to the cell wall can directly convert fibrinogen to insoluble fibrin and cause the staph to clump. Cell-free coagulase does the same thing by reacting with globulin plasma factor (coagulase reacting factor) to form staphylothorombin (thrombin-like). This factor catalyzes the conversion of fibrinogen to insoluble fibrin. Coagulase may cause the formation of a fibrin layer around a staph abscess, localizing the infection and protecting it from phagocytosis. 3. Explain the concept of a “superantigen” and how the staphylococcal toxic shock toxin initiates tissue damage in the Toxic Shock Syndrome. (p. 224) Superantigens belong to a class of polypeptides that bind MHC II on macrophages, which interact with T-cell receptors, leading to non-specific proliferation of T-cells and release of cytokines with subsequent tissue damage. Examples: exfoliative toxin A, enterotoxins, TSST-1. In TSS, Toxic shock syndrome toxin-1 stimulates the T-cells (as above) and causes leakage or cellular destruction of endothelial cells. 4. How does catalase assist organisms such as staphylococci resist destruction by the host immune response? (lecture outline) Hydrogen peroxide can accumulate during bacterial metabolism or after phagocytosis. All staph produce catalase, which catalyzes the conversion of hydrogen peroxide to water and oxygen. 5. Compare and contrast the virulence factors and diseases caused by Staphylococcus aureus versus the coagulase-negative staphylococci. (p. 228233) S. aureus: Virulence factors: Structural components - capsule, peptidoglycan, teichoic acid, Protein A, cytoplasmic membrane Toxins – Cytotoxins, exfoliative toxins, enterotoxins, TSST-1 Enzymes – coagulase, catalase, hyaluronidase, fibrinolysin, lipases, nucleases, penicillinase Diseases: Toxin-mediated: food poisoning, TSS, scalded skin syndrome Pyogenic: impetigo, folliculitis, furuncles, carbuncles, wound infections Other systemic disease (associated with bacteremia): pneumonia, empyema, septic arthritis, osteomyelitis, acute endocarditis, catheter-related bacteremia Coagulase-negative staph: Virulence factors: Everything except coagulase? Diseases: Endocarditis, Wound infections, UTI, catheter and shunt infections, prosthetic device infections 6. Acute glomerulonephritis and rheumatic heart disease are both autoimmune sequelae of Group A streptococcal infections. Explain their similarities and differences with respect to immune mechanisms involved and clinical manifestations. (p. 244-245) These are both nonsuppurative (non-pus forming) streptococcal diseases. Rheumatic fever is a complication of S. pyogenes disease, characterized by inflammatory changes of the heart, joints, blood vessesl and subcutaneous tissue. Rheumatic fever is associated with strep pharyngitis and not cutaneous strep infections. It is most common in young children. There is no specific diagnostic test and diagnosis is made based on evidence of a recent S. pyogenes infection. Acute glomerulonephritis is characterized by inflammation of the glomeruli with edema, hypertension, hematuria and proteinuria. Specific nephritogenic strains of Group A streptococci are associated with the disease and there are both pyodermal and a pharyngitis associated strains. Diagnosis is made based on evidence of a recent S. pyogenes infection. Children usually recover uneventfully, but loss of renal function has occurred in adults. 7. How can a recent Group A streptococcal infection be diagnosed in someone who has already taken antibiotics? (p. 242) S. pyogenes release the virulence factors streptolysin O and streptolysin S. Streptolysin S is non-immunogenci whereas antibodies against streptolysin O are readily formed in infected people. The anti-ASO test is useful for documenting recent Group A infections. Antibodies against streptokinase can be used as a marker for infection. Antibodies against DNase B are a marker for cutaneous S. pyogenes infection because these infections do not cause formation of antibodies against streptolysin O because strep O is inhibited by cholesterol in skin lipid. 8. Explain the role of bacteriophage lysogeny in pathogenesis of some streptococcal infections. (p. 243) Scarlet fever is a complication of streptococcal pharyngitis that occurs when the infecting strain is lysogenized by a temperate bacteriophage (does not cause immediate lysis) that stimulates the production of pyrogenic exotoxin. 9. Describe the pathogenesis of streptococcal necrotizing fasciitis. (p. 244) Lay term: “flesh-eating bacteria” Occurs deep in the subcutaneous tissue and spreads along the fascia. Characterized by destruction of fat and muscle. The organism is introduced through a break in the skin. Initially, there is cellulitis, after which bullae form and gangrene and systemic symptoms develop. Systemic toxicity, multiorgan failure and death are hallmarks. Unlike cellulitis, which can be treated with antibiotics, fascitis requires surgical removal of the infected tissue. subcutaneous air is classically described in necrotizing fasciitis 10. Explain the basis for the Lancefield groups and how they are used to classify the streptococci? (lecture outline) Lancefield groups differentiate beta-hemolytic organisms based on groupspecific antigens that are typically cell wall carbohydrates. These antigens can be readily detected with immunologic assays and are used to identify strep pathogens. For example, S. pyogenes is group A. Most, but not all, alpha-hemolytic and nonhemolytic strep do not possess the group-specific antigens. The scheme is used to identify a few species of strep: A, B, C, F, G. 11. Explain why the Enterococcus is especially well-suited to be a nosocomial pathogen. (p. 260-262) Enterococcus is part of the normal flora of the GI tract and is Inherently resistant to common antibiotics (cephalosporin) or have acquired resistance genes (vancomycin). Thus patients being treated with broadspectrum antibiotics (and are typically already ill) are highly susceptible to infection. Cell wall structure typical of gram-+ bacteria allows them to survive on environmental surfaces for prolonged periods. 12. What unique features of Bacillus anthracis help to differentiate from other Bacillus species in the clinical laboratory? (p. 267) Identification is based on microscopic (gram -, nonmotile rods) and colonial morphology (non-hemolytic, adhering). Confirmed by demonstrating capsule and either lysis with gamma phage or positive DFA test for specific capsular polysaccharide. 13. Discuss the features of Bacillus anthracis that make it such a logical organism for bioterrorism attacks. (p. 265-269) Spores can be grown in culture with low CO2 and are long-lived outside a host. Low infectious dose. Spores can be treated to minimize clumping so they reach the lower airway. Inhalation anthrax is usually fatal. 14. Describe the epidemiology and pathogenesis of meningitis due to Listeria monocytogenes, Escherichia coli, Streptococcus agalactiae, Neisseria meningitidis, and Haemophilus influenzae and emphasize the differences and similarities among them. Listeria monocytogenes: - facultative, intracellular pathogen that grows in macrophages, epithelial cells and fibroblasts and can avoid antibody-mediated clearance - causes meningitis in neonates, elderly, pregnant women, and patients with defects in cellular, but not in humoral immunity - acquired by ingestion of contaminated food, it may be asymptomatic in healthy adults the organism can cross the placenta and cause spontaneous abortion and stillbirth E. coli: - normal flora of GI - neonatal meninigitis, not caused by endogenous bacteria - approximately 75% of E. coli strains possess the K1 capsular antigen - this serogroup is also commonly present in the GI tracts of pregnant womand newborn infants - Neisseria meningitidis: - purulent inflammation of meninges associated with headache, meningeal signs, and fever - high mortality rate unless promptly treated with effective antibiotics - approx. 0.6 cases per 100,000 population reported in 2004 - very young children may have only nonspecific signs such as fever or vomiting - incidence of neurologic sequelae is low, with hearing deficits and arthritis most commonly reported Haemophilus influenzae: - used to be a common cause of pediatric meningitis, but not anymore due to wide use of conjugated vaccines - dz in non-immunized patients results from bacteremic spread of the organisms from the nasopharynx and cannot be differentiated clinically from other causes of bacterial meningitis - initial presentation is 1-to-3 day history of mild upper respiratory dz after which the typical signs of meningitis appear Streptococcus agalactiae: - carried in GI tract of pregnant women as normal flora - if acquired by baby at delivery, can cause severe pheumonia and severe meningitis - most common cause of septicemia and meningitis in newborns - approximately 60% of infants born to colonized mothers become colonized with their mother’s organisms - risk factors for neonatal colonization—premature delivery, prolonged membrane rupture, intrapartum fever - serotypes most commonly assoc. with neonatal dz are Ia, III, and V. - serotypes Ia and V are the most common in adult dz - colonization of neonate can occur in utero, at birth, or during the first few months of life - risk of adult dz greater in pregnant women than in men and non-pregnant women B. Gram-negative bacilli 1. Compare and contrast diarrheal diseases caused by Salmonella, Shigella, Campylobacter, Staphylococcus aureus, Bacillus cereus, Escherichia coli, Yersinia,and Vibrio species with respect to epidemiology, modes of transmission, mechanism (infection vs. intoxication), and invasiveness. Epidemiology Transmission Mechanism Invasiveness Disease Salmonella S. typhi and S. Contaminated Low Limited to Gastroenteritis, paratyphi only food; fecalinfectious GI tract septicemia, infect humans oral spread in dose for S. enteric fever, children typhi asymptomatic Shigella Humans are the Fecal-oral Highly Limited to Bacterial only host infectious, colon dysentery, S. epithelium Gastroenteritis dysentery (shigellosis) produces Shiga toxin Campylobact Zoonotic Contaminated Infection; C. jejuni Acute enteritis er infection food and usually damages lg. with diarrhea water self-limited intestine mucosa S. Aureus Protein food Preformed Infection is Watery enterotoxin short-lived diarrhea ~24 hrs B. cereus Starchy food, Preformed Watery spores enterotoxin diarrhea E. coli Traveler’s diarrhea Yersinia Zoonotic Flea bite or Infection Resists Bubonic infection direct contact phagocytosis plague (Y. with infected and spreads pestis); tissue gastroenteritis Vibrio Serotype O1 Contaminated Cholera Limited to Cholera starts causes water; High toxin, GI with watery pandemics infectious causes epithelium diarrhea, can dose, killed secretion progress to by stomach of hypovolemic acid electrolytes shock; and water gastroenteritis Salmonella- most infections from contaminated food, direct fecal-oral spread in children(grows black on XLD agar- shows H2S). Salmonella really invades our cells. It causes a profuse diarrhea. Salmonella causes a few million cases of food borne illness each year in the US. Most of it is not so severe that it is identified. There are 2400 serotypes, but there are only 2 of those that cause 40-50% of all salmonella outbreaks- enterica Typhimurium, and enterica enteritidis are the most common. You have vomiting and perfuse diarrhea for 8-48 hours after you start developing symptoms and this then resolves in 7-10 days. The source of salmonella is usually our meat animals or people. The way to prevent this organism is to cook it. (Don’t cut chicken and salad on the same cutting board or use the same knife!) Shigella- causes bacillary dysentery, a high volume, watery diarrhea. It invades the colon epithelium and can be treated with Ampicillin or Bactrim. Humans are the only normal host. 200 organisms is an infectious dose, which is a very low amount in comparison to most bacterial infections. It can be transmitted by the 4Fs: feces, fingers, food, flies, and water. It can be controlled by washing your hands and other sanitary measures in society. Common sites of this disease are nursing homes, day cares, wars, and cruise ships. It causes 15% of the pediatric diarrhea in the US. Campylobacter- It causes a bloody diarrhea. It is more common and less severe than Salmonella. It is carried by food animals and pets. Most human infections are from contaminated food and water sources. Treatment consists of rehydration. You can prevent this organism through cleanliness and cooking your food. Staph aureus- causes watery diarrhea. It is a preformed enterotoxin that is heat stable. It is in foods such as ham, cream-filled cakes, and potato salad. It’s toxins infect kittens and non-human primates. The effects of this organism usually last less than 24 hours. Bacillus cereus- is a preformed enterotoxin that is heat stable. The diarrhea produced is profuse and watery. Several of the symptoms are much like staph. The difference is that the foods are typically a starchy food instead of a protein rich food. Those spores will germinate after it is cooked, grow in the rice, and then refry the rice and kill the organisms again, but the toxin will stay around and cause disease. A large number of spores germinate in vivo. E. coli- causes secretory diarrhea or watery diarrhea. Features of this diarrhea include stimulation of net intestinal secretion, no morphological damage, and no impairment of Na- dependent solute absorption. Species of E.coli are responsible for traveler’s diarrhea and infantile diarrhea. Eneterovasive E. coli (EIEC) causes bacillary disentry. The most common outbreak strain in food borne illness is EHEC, or Enterohemorrhagic E. coli. The diarrhea present is usually mild. Yersinia- is an enterobacteriaceae but it is not food borne. It causes a systemic disease much like salmonella typhi. It causes the bubonic and pneumonic plague and is contracted by flea infected bites. It causes septicemia and often death. Pneumonic plague is passed from person to person coughing up the organisms. The plague in 14th century Europe killed 25 million people and was often of the pneumonic type. Bubonic plague can result in 75% mortality with a few days; the pneumonic form can result in greater than 90% mortality within 24 hours. Control of rat populations concurrent with elimination of their fleas prevents spread of the plague to humans. Decontamination of water and milk prevents gastroenteritis. Treatment of the plague must be rapid and aggressive. Y. pestis is generally susceptible to streptomycin and chloramphenicol but concomitant therapy is sometimes recommended. Treatment of Y. enterocolitica infections usually involves the use of ampicillin or tetracycline. Vibrio- Toxins produced in vivo. It produces a profuse watery diarrhea. It is spread by contaminated water. Stomach acid offers protection. An infectious dose is 107 CFU. It has a 1-5 day incubation period. Rehydration by fluids is sufficient treatment. 2. Describe 4 characteristics that can be used to define the family Enterobacteriaceae (p. 323) gram -, facultative anaerobe, oxidase negative, ferment lactose, resistance to bile salts, all have enterobacterial common antigen, nonspore forming, reduce nitrate, catalase positive, either nonmotile or motile with peritrichous flagella, some have capsule 3. Which property of Helicobacter pylori enables it to reside in the stomach at pH of 2 and how does this relate to the ability of the organism induce an inflammatory reaction? (p. 352) Produces urease, which neutralizes gastric acid with ammonia. Heat shock protein enhances the expression of urease. Acid-inhibitory protein induces hypochlorhydria by blocking acid secretion from parietal cells. 4. Which virulence factor of Pseudomonas aeruginosa contributes to its pathogenesis in burn patients? (p. 359) The capsule anchors cells to epithelia, protects the organism from phagocytosis and antibiotic activity and suppresses neutrophil activity. Moist surface of the burn and the lack of a neutrophilic response to tissue invasion predispose patients to infection. Topical antibiotics are ineffective. Other outline: Exotoxin A: disrupts protein synthesis by blocking peptide chain elongation in eukaryotes. Contributes to dermatonecrosis in burn wounds and is immunosuppressive. 5. Discuss the relative organism load needed to cause diarrheal disease due to Escherichia coli, Shilgella spp., Salmonella spp. and Vibrio cholera. (lecture outline) E. coli: not listed in the hand out. But, from lab and other sources, the number has to be relatively high since it is part of our normal flora. I think the number is somewhere around 10^6. Also could be opportunistic. Shigella spp.: 200 organisms. However, there are approximately 10^8 colony forming units/gram (CFU/g) in infected stool samples. Salmonella spp.: Low dose needed for S. typhi Vibrio cholerae: 10^7 CFU. Stomach acid will kill most of it. 6. Explain the difference between osmotic and secretory diarrhea. (lecture outline) Osmotic diarrhea occurs when too much water is drawn into the bowels. Occurs with maldigestion, lactose intolerance and laxatives. Secretory diarrhea occurs when there is an increase in the active secretion or an inhibition of absorption. There is little to no structural damage. Fasting will not control diarrhea. E.g. cholera. 7. What is the hemolytic-Uremic Syndrome and how is it related to bacterial infection?(lecture outline) Hemolytic-uremic syndrome is characterized by microangiopathic hemolytic anemia, acute renal failure and thrombocytopenia. It is one of the most common causes of sudden, short-term kidney failure in children. Most cases of HUS occur after an infection of the digestive system by E. coli. Some people have contracted HUS after swimming in pools or lakes contaminated with feces. C. Fastidious bacteria 1. Describe the process by which Mycoplasma pneumoniae attaches to the respiratory epithelium and produces pneumonia. (lecture outline) P1 is an adhesion protein that ineracts specifically with sialated glycoprotein receptors at the base of cilia on the epithelial cell surface. Ciliostasis occurs, and then the cilia and the epithelial cells are destroyed. This interferes with the clearance of the airways and permits the lower airway to be contaminated with microbes. This leads to a persistent cough. 2. Explain the differences in clinical manifestations, epidemiology, natural history, and pathogenesis of pneumonia caused by Streptococcus pneumoniae, Staphylococcus aureus, Klebsiella pneumoniae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,Haemophilus influenzae, and Legionella pneumophila. (C2page9) BUG Streptococcus pneumonia Clinical Manifestations -onset abrupt -severe shaking chill and sustained high fever -often preceeded by symptoms of viral resp. tract infection 1 to 3 days before onset -chest pain, blood-tinged sputum -often lobar pheumonia, associated with aspiration Epidemiology -common inhabitant of throat and nasopharynx in healthy people -common cause of bacterial pneumonia -500,000 cases in US annually Natural History Pathogenesis -endogenous oral organisms are aspirated into the lower airways Staphylococcus aureus -radiograph reveals patchy infiltrates with abscesses Clinical presentation NOT unique -seen in very young and elderly and patients with underlying or recent pulmonary disease -severe form of necrotizing pneumonia with septic shock and high mortality Klebsiella pheumoniae -thick non-purulent blood sputum -25-50% mortality -community-acquired primary lobar pneumonia Mycoplasma pheumonia -atypical interstitial “walking” pheumonia -clinically similar to the other pheumonias -all ages affected, but more common in younger persons -reinfection common—no protective immunity Chlamydophila pheumoniae -frequently asymptomatic -acute lower respiratory illness, pharyngitis, sinusitis -persistent cough and malaise, but normally does NOT require hospitalization -similar to mycoplasma Most infectious occur in adults Diagnosis difficult Infections transmitted person-toperson by respiratory secretions -NO animal reservoir identified -inflammation and consolidation of the lungs observed primarily in the elderly with underlying chronic pulmonary dz -commonly colonize pts. Who have chronic pulmonary dz -frequently assoc. with exacerbation of bronchitis and frank pneumonia -respiratory aerosol dissemination -2-10 day incubation period -requires antibiotic therapy -15-20% mortality rate; higher if diagnosis delayed -multilobar -Legionnaire’s Dz -severe pneumonia -epidemic, sporacid -no person-to-person spread -epidemic dz in late summer and autumn -there is normally an underlying pulmonary dz Haemophilus influenzae Haemophilus influenzae (cont’d) Legionella pheumophila -consolidation and abscess formation in lungs -can occur by aspiration or from the hematogenous spre of the organism from distan sites Predisposing factors: -hospitalization -respirator -increased age -alcoholism -Diabetes Mellitus 1980s labs testing antibody on people with respiratory infection were getting lots of positive tests for Chlamydia psittaci, but they hadn’t been around birds, so this is when they realized it was another type of Chlamydia (now called Chlamydophila pheumoniae) American Legion Conference, disease spread through AC ducts and water supply (possibly) and infected many Legionnaires, hence the name. -necrosis and abscess formation -cytadherence: P1 and other proteins -altered macromolecular synthesis -induction of inflammation -human pathogen ??? -typically caused by nontypeable strains -endogenous infection by normal flora -antiphagocytic capsule -endotoxin damages resp. epithelium leading to bacter spread -IgA protease -organism can survive in moist environments for a lo time, at high temps. And in presence of disefectant like chlorine -organism proliferates in water reservoirs during war months 3. Discuss the impact of the Hib vaccine on epidemiology and spectrum of disease caused by Haemophilus influenzae in adults and children. (lecture outline) H. influenza type b (Hib) was a leading cause of meningitis and pneumonia in children under five years old, causing an estimated 20,000 cases a year in the early 1980s. Since routine vaccination, the incidence of Hib disease has declined by greater than 99%. Infections in adults have also decreased (herd immunity) because prior to vaccination some children carried Hib in their nasal passages for extended times before they cleared the infection. 4. Explain why Haemophilus influenzae must be supplied with X and V factors in order to grow on trypticase soy agar. (lecture outline; p. 372) H. influenzae require specific growth factors that are released from lysed blood cells: Intracellular heme (X factor) and NAD (V factor). These factors are not generally present in standard growth media. They will grow on chocolate agar unless it has been overheated, destroying V factor. 5. Explain why Bordetella pertussis infections have increased in adolescents and adults in the United States over recent years. (lecture outline) B. pertussis is a human disease. Once considered a pediatric disease, there has been an increase in older children and adults, due to waning immunity and the selection of strains that are not recognized by the current vaccines. 6. Compare and contrast the diseases of diphtheria and pertussis, including their epidemiology, pathogenesis, and prevention. (lecture outline) Pertussis: aka. whooping cough Epidemiology: human is the only host; children less than one year old are at greatest risk, but increasing in adults; spread by person to person contact Pathogenesis: Bacteria attach to ciliated epithelial cells, proliferate and produce localized tissue damage and systemic toxicity. Produce pertussis toxin that leads to increased cAMP levels and subsequent increased respiratory secretions and mucus. Prevention: vaccination of inactivated pertussis toxin and a bacterial component; given over a number of years up to age 7 Diptheria: Epidemiology: asymptomatic carriers and unvaccinated hosts; humans only host, carried in nasopharynx and on skin; spread person to person by droplets and skin contact; highest risk are children and immunosuppressed Pathogenesis: exotoxin (classic A-B exotoxin) secreted at the site of infection stops host cell protein synthesis. Organism does not need to enter bloodstream to cause systemic symptoms. Prevention: immunize with diphtheria toxoid. Given to all children, booster every 10 years. 7. Explain why the initial outbreak of legionellosis in 1976 was so difficult to characterize initially. (lecture outline) The Legionella bacterium was first identified in the summer of 1976 during the 58th annual convention of the American Legion, which was held at the Bellevue-Stratford Hotel in Philadelphia. The presentation of affected persons ranged from mild flulike symptoms to multisystem organ failure. Of the 182 people infected, 29 died. A bacterium that would later be named L pneumophila was isolated from different organ tissues of guinea pigs inoculated with lung tissue samples from 4 fatal cases. Several problems were encountered when researchers first tried to classify this outbreak. First, the bacteria are difficult to culture. It would not grow on standard media, and a buffered charcoal yeast extract agar was developed to culture it. Also, characterization was hindered due to the lack of person to person contraction. The source was not known since none of the affected persons had symptoms prior to arriving at the hotel. It was later discovered that Legionella is water-born and was actually aerosolized in the air-conditioning system. 8. Describe 5 methods for laboratory diagnosis of legionellosis and discuss the advantages and disadvantages of each. (p. 394) 1. microscopy: direct fluorescent antibody (DFA) test. Fluorescein labeled antibody against legionella. Low sensitivity 2. buffered charcoal yeast extract agar: most sensitive method 3. urinary antigen test: high sensitivity for L. pneumophila but low for other species. Antigens persist in the urine of infected people, lasting up to 2 months. 4.nucleic acid amplification: highly specific and sensitive. Good for respiratory secretions. May cause false-negatives 5. indirect fluorescent antibody: increase in titer may be detected in up to 40% of patients in the first week of disease, but it may take 6 months for some. High titers can persist and cannot be used as an indication of active infection. Poor sensitivity. 9. What are the consequences of having the smallest genome of any known freeliving human pathogen on laboratory diagnosis of Mycoplasma genitalium infections? (lecture outline) The small genome of M. genitalium requires an enriched medium (SP4 agar) and a lot of time in order to grow in a laboratory. As a result, PCR and serology (for IgM) are used more commonly to identify the presence of the bug. D. Spirochetes and Rickettsiae 1. Explain the differences between obligate intracellular bacteria and facultative intracellular bacteria and give examples of each. (lecture outline) Obligate intracellular bacteria cannot make their own ATP and rely on the host machinery. They frequently cause chronic disease. Example: Rickettsiae and Chlamydiae Facultative intracellular bacteria have developed the ability to survive and grow within professional phagocytes frequently giving rise to chronic and/or chronic disease. Example: tuberculosis, Salmonella typhimurium, Listeria monocytogenes. 2. Describe the life cycle of Borrelia burgdorferi, the agent of Lyme disease. (lecture outline) For Lyme disease to exist in an area, at least three closely interrelated elements must be present in nature: the Lyme disease bacteria, Borrelia burgdorferi, ticks that can transmit them, and mammals (such as mice and deer) to provide food for the ticks in their various life stages. The tick life cycle consists of three distinctive stages: larvae, nymphs, and adults. A blood meal is required for ticks to molt from the larvae stage to the nymph stage and from the nymph stage to the adult stage. The tick larvae and nymphs typically become infected with Borrelia burgdorferi when they feed on infected small animals, particularly the white-footed mouse. The bacteria remain in the tick as it changes from larva to nymph or from nymph to adult. Infected nymphs and adult ticks then bite and transmit Borrelia burgdorferi to other small rodents, other animals, and humans, all in the course of their normal feeding behavior. Adult ticks preferentially feed on the white-tailed deer, which thereby becomes an important reservoir in regions of infestation. The tick life cycle takes two years to complete. 3. Does Lyme Disease occur endemically in Alabama? Explain the reason for your answer. (lecture outline) No, Lyme disease does not occur endemically in Alabama because it’s transmitted by Ixodes ticks, which are not native to Alabama. 4. Explain why laboratory diagnosis of Lyme Disease is complex and difficult. (p. 436) B. burgdorferi are present in very small amounts in clinical specimens from tissues and body fluids of patients, so microscopic examination and even nuceic acid amplification have low sensitivity. Cultures are rarely done because media is NOT readily available and organisms grow slowly on them. 5. What is the difference between a “non-treponemal test” and a “treponemal test” and how can each be used most effectively in diagnosis of syphilis? (p. 431432) Non-treponemal tests (eg: VDRL or RPR) are biologically nonspecific and they measure IgG and IgM antibodies developed against lipids released from damaged cells during the early stage of disease and present on the cell surface of treponemes. Positive reactions with these tests develop late during the first phase of disease; the findings are negative in many patients who initially have chancres. However, serological results are positive within 3 months in all patients and stay positive in untreated patients with secondary syphilis. Successful treatment of primary or secondary syphilis leads to reduced titers measured in the nontreponemal tests and thus can be used to monitor the effectiveness of therapy. Treponemal tests: Treponemal tests are specific antibody tests used to confirm a positive reaction with the VDRL or RPR test. Results of these tests usually remain positive for the life of the person who has syphilis. These tests are influenced less by therapy than the nontreponemal tests 6. Explain how the life cycle of Rickettsia rickettsii is responsible for clinical manifestations of Rocky Mountain Spotted Fever. (lecture outline) Rickettsia rickettsii are carried in the fluids of arthropods like ticks. When the tick bites you, the bacteria will invade the endothelium. The organisms multiply in the endothelium of small vessels causing inflammation, which leads to vasculitis. The cells swell, become necrotic and thrombose the vessel, leaving a bruised looking spot at the site of infection. This is the rash that spreads with RMSF. 7. Describe the procedures that are necessary in order to cultivate rickettsiae in vitro and explain whether or not this is a worthwhile method for diagnosis of rickettsial diseases.(p. 452) Rickettsiae will grow in tissue culture or embryonated eggs. They require a culture to grow in because they are obligate intracellular bacteria. They are hazardous to work with (infectious) so they are typically diagnosed using microscopy, serological procedures (Ab presence) and PCR. 8. Describe 3 different human diseases for which ticks are a vector. Rocky Mt. Spotted Fever: Rickettsia rickettsii Monocystic ehrlichiosis: Ehrlichia chafeensis Granulocytic ehrlichiosis: Anaplasma phagocytophilum Lyme Disease: Borrelia burgdorferi F. Neisseriae and Chlamydiae 1. Describe the unique life cycle of chlamydiae and the roles of the elementary body and reticulate body in infectivity. (lecture outline) Chlamydia is an obligate intracellular organism (it doesn’t have the ability to make its own ATP) Life Cycle: 1. infectious elementary body taken up into cytoplasm of host cell>>>grows and becomes metabolically active inside of phagosome 2. transformational change of elementary body to reticulate body 3. reticulate body grows and forms more elementary bodies 4. reticulate body condenses back to elementary body 5. elementary bodes released from cell to infect other cells. Reticulate body: replicating form of Chlamydia trachomatis; non-infectious, but makes more elementary bodies which are infectious 2. Give an explanation for the failure to develop an effective vaccine against Neisseria gonorrhoeae. (p. 319) There is high antigenic diversity/variation in the variable region of the pilin protein, to which antibodies are made. Antibodies against this region protect against a homologous strain, but not a heterologous strain. This results in no permanent immunity following infection and the failure to develop an effective vaccine. 3. Describe the diagnostic method of choice for chlamydial urogenital infections and explain why it is preferred over other methods. (p. 469) Nucleic acid amplification tests (NAATs) are highly sensitive, very specific and can be done using voided urine from a patient with urethritis. Car must be taken to avoid contamination and inhibitors (urine). Cell staining is insensitive, culture can be easily contaminated and has low sensitivity and antigen detection assays are unreliable. G. Mycobacteria 1. How do the methods of laboratory diagnosis for mycobacteria differ from those used for the common gram-positive cocci? (p. 305-308) 1. Mycobacteria are detected by the acid-fast stain. Stain with carbofuchsin or fluorescent dye, decolorize with acid-alcohol solution and counterstain. The gram stain uses an acetone alcohol solution to decolorize. 2. Specific species are identified with amplified nucleic acid probes. 3. Culture on solid agar, egg-based or broth-based media 4. Growth properties and colonial morphology 5. Definitive identification based on: production of niacin and reduction of nitrate, characteristic cell wall lipids, species-specific molecular probes, sequencing the 16S rRNA gene 2. Give a logical argument why the BCG vaccine is not used routinely in the United States. (p. 301) BCG immunization results in a positive skin test for prolonged periods, making it more difficult to determine who has active infection. BCG is not used in the USA because TB is rare. Vaccine is effective when given to young children, but not in adults with latent infection. BCG cannot be given to immunocompromised patients. 3. Is tuberculosis increasing or decreasing in the United States? Explain your answer. (p 301; lecture outline) Decreasing due to genetics and increased standard of living. 4. Explain why persons with HIV/AIDS are especially prone to develop disease due to mycobacteria. (p. 301) From book: The likelihood that infection will progress to active disease is a function of both the infectious dose and the patient’s immune competence. Active disease develops within one year in ~10% of patients with HIV and a low CD4 count, compared to a 10% risk during the lifetime of a patient that does not have HIV. In patients with HIV, disease appears before the onset of other opportunistic infections, is twice as likely to spread to extrapulmonary sites and can rapidly progress to death. From other outline: Because of low immune system; However, the vaccine they are given prevents the primary disease of TB, so stops the infection. But they are a carrier and can transmit it to other people. 5. A 30 year-old dentist in apparent good health who is exposed to a patient with tuberculosis develops a positive PPD skin test. Explain the most appropriate course of action that should be taken. First need positive skin test. Prophylaxis regimen for exposure: 1. INH daily for 9 months (*this is the only one listed in the notes) 2. rifampin daily for 4 months 3. rifampin and pyrazinamide daily fro 2 months Patients exposed to drug-resistant TB receive a longer course of antibiotics. 6. Compare and contrast the pathogenesis and clinical manifestations of tuberculoid and lepromatous leprosy. (lecture outline) M. leprae: aka Hansen’s disease, humans and armadillos are natural host, transmitted by inhalation or skin contact, incubation period is 3 months to 3 years Diagnosis: does not grow on artificial media; AFB stain of nasal secretions; lepromin skin test Tuberculoid: Non-progressive; intact cell-mediated response to M. leprae; organisms rare in tissue; organisms grow in nerves and cooler parts of the body and make granulomas; cutaneous loss of sensation from nerve damage due to cell-mediated immunity People lose body parts because they can’t feel them and they damage them. Lepromatous: Depressed cell-mediated immunity response specific for M. leprae; bacteremia with localization in nerves and skin; high number of organisms in macrophages; loss of nerve function, leonine facies (low immune response); other organs involved (testes, spleen, liver)