Med Micro

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Med Micro
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  1. Brightfield (light) Microscope
    stained bacteria and other microscopic organisms
  2. Darkfield Microscopy
    small objects (≤0.2 um) - Treponema pallidum and flagella
  3. Phase-Contrast Microscopy
    three-dimensional image of the organism
  4. Fluorescent Microscopy
    staining organisms with fluorescent dyes; colors vary depending on the fluorochrome selected
  5. Electron Microscopy
    • magnetic coils (rather than lenses) are used to direct a beam of electrons from a tungsten filament through a specimen and onto a screen
    • high magnification and resolution- individual viral particles
    • Transmission EM: electrons such as light pass directly through the specimen
    • Scanning EM: electrons bounce off specimen surface at an angle; three- dimensional
  6. Direct Examination
    • Wet mount: specimen mixed with water or saline; motile bacteria, stool concentrates for eggs and parasite larvae
    • Potassium hydroxide (10% KOH): detect fungal elements in sputum
    • Lactophenol cotton blue: stains fungal hyphae
    • India ink prep: for the detection of capsule (Cryptococcus neoformans)
  7. Differential Stains
    • Gram stain
    • Iron hematoxylin and trichrome stains (for trophozoites & cysts from stools)
    • Wright-Giemsa stain (for blood parasites)
  8. Acid-Fast Stains
    Kinyouns or Auramine-rhodamine (a fluorescent dye for Mycobacteri, Nocardi)
  9. Fluorescent Stains
    • Acridine orange fluorescent dye targets nucleic acid of dead and alive microorganisms
    • Fluorescent antibody stains
  10. Phenotype
    The observable characteristics of an organism's growth on or in the media.
  11. Nonselective Media
    • allows growth of many pathogens
    • Examples....
    • Sheep blood agar: Staphylococcus aureus, Streptococcus pneumonia, Klebsiella sp., etc
    • Chocolate agar: Haemophilus, some pathogenic Neisseria strains
    • Thioglycolate broth- enrichment broth for aerobes and anaerobes
  12. Selective media
    • Inhibitory substances (antibiotics) are added to suppress the growth of susceptible bacteria, but allow particular pathogen’s growth
    • Modified Thayer Martin- selects for N. gonorrhoeae and inhibits most other bacteria
    • Inhibitory mould agar- selects for fungi and inhibits bacteria
  13. Selective and Differential media
    • selects for specific bacteria and differentiates them
    • MacConkey agar: lactose, glucose, neutral red indicator, bile salts and crystal violet (inhibit GP bacteria); selects for enteric GNR and differentiates between lactose fermenters and nonlactose fermenters
  14. Specialized media
    • allows the growth of bacteria that require specific ingredients
    • Buffered charcoal yeast extract (BCYE) for Legionella
    • MacConkey sorbitol agar for recovery of Escherichia coli O157
  15. Cell Monolayer Culture
    cells grow as a single layer and can be maintained in the lab indefinitely
  16. Primary Cell Culture
    cells prepared from tissues/organs immediately before use
  17. Molecular Diagnosis
    • Advantages- identify difficult or slow growing organisms, strain determination (Mycobacteria tuberculosis, Chlamydia, N. gonorrhoeae, slow growing fungi)
    • Detection tools: PCR (increases the DNA/RNA required for analysis) and Probes (target specific nucleic acids in a strand of unknown DNA or RNA. Specific for genera, species, and strains)
  18. DNA Probe Analysis of Virus Infected Cell
  19. Gen-Probe
    • A rapid nucleic acid test that produces 400 results or more in 8 hrs
    • Used to diagnose disease and screen blood donations
    • Amplifies RNA or DNA a billion fold in 15-30 minutes Uses DNA probes tagged with chemiluminescent signal.
    • Culture required for Mycobacteria tuberculosis and Group B strep
    •  Patient specimen for N. gonorrhoeae, Chlamydia trachomatis, Group A strep, HIV
  20. Roche Molecular Diagnostics
    • PCR based testing and nucleic acid
    • hybridization
    • Identifies pathogens in clinical specimens: sputums and blood samples
    • 48 tests/run with results in 2.5 hours.
    • Cultures unnecessary
  21. Serology
    use of antibodies and antigens to determine the infecting agent, a primary or reinfection, and the course of the disease.
  22. Antigens
    foreign proteins and polysaccharides; cell wall, capsule, membrane proteins; many different antigenic sites are on a bacterium or virus.
  23. Epitope
    A specific region of an antigen that binds to an antibody. There can be more than one epitope per antigen, each binding to a specific antibody.
  24. Titer
    • amount of antibodies in a patient’s serum (quantitation of the antibody strength); the lowest dilution of the sample that retains a detectable activity
    • Diagnostic for an infection is a four-fold rise in titer pf antibodies; from acute to convalescent phase.
    • Serially dilute the serum two-fold and add a set amount of antigen; antibodies specific to the antigen will bind and precipitate; the last dilution with precipitate is the titer
    • Dilutions are 1:2, 1:4, 1:8. Titers are 2,4,
  25. Seroconversion
    During a primary infection, IgM is first produced.
  26. Anamnestic Respose
    When re-exposed to a previous infection, a booster.
  27. Detection Ag-Ab Complex
    • Direct precipitation techniques
    • Direct labeling the antibody with a radioactive,
    • fluorescent, or enzyme probe
    • Indirectly through measurement of an antibody-directed reaction, such as complement fixation
  28. Equivalence zone
    antibody cross-links the antigen into a complex that is too large to stay in solution (forming precipitation)
  29. Single radial immunodiffusion
    • detects and quantifies an antigen
    • Antibody in agar and antigen in a well and diffuses outward. A line of precipitate indicates a complex has formed. A line far from the well indicates a higher quantity of antigen than a line close to the well
  30. Ouchterlony immuno-double-diffusion technique
    • Determine the relatedness of different antigens
    • A central well and surrounding wells. Precipitation lines indicate an antibody-antigen reaction
  31. Analysis of Antigens and Antibodies by Immunoprecipitation
    http://micro.digitalproteus.com/pics/immunoprecipitation.jpg
  32. Immunohistology
    Detecting antigens on the cell surface or within the cell
  33. Direct immunofluorescence
    • Legionnaires’ disease: Legionella antigen complexs with
    • tagged antibodies
  34. Flow Cytometry
    The flow cytometer evaluates individual cell parameters as the cells flow past a laser beam at rates of more than 5000 per sec.
  35. Enzyme-linked immunosorbent assays (ELISA)
    • Antigen immobilized on a plastic surface, bead, or filter to capture and separate the specific antibody from other antibodies in a patient's serum.
    • Anti-human antibody with a covalently linked enzyme (e.g., horseradish peroxidase, alkaline phosphatase, β- galactosidase) then detects the affixed patient antibody.
    • Crytococcal meningitis, Giardiasis, Rotavirus gastroenteritis, etc.
    • Can also be used to quantitate the soluble antigen in a patient's sample
  36. Enzyme Immunoassays
    • for quantitation of antibody or antigen.
  37. Western Blot Analysis
    • Proteins of an infectious agent are separated by electrophoresis according to their molecular weight or charge are transferred (blotted) onto a filter paper (nitrocellulose, nylon).
    • When exposed to a patient's serum, the immobilized proteins capture virus-specific antibody and are visualized with an enzyme-conjugated antihuman antibody.
    • Western blot analysis is used to confirm ELISA results in patients suspected to be infected with the human immunodeficiency virus (HIV).
  38. Western Blot Analysis
  39. Complement Fixation Test
    • A standard but technically difficult serologic test
    • Complement is added to the Ag-Ab mixture
    • Antibody-antigen complexes bind, activate, and fix (use up) the complement. The residual complement is then assayed through the lysis of red blood cells coated with antibody.
    • RBC lysis indicates no antibody-antigen complex formed
    • No RBC lysis indicates the complement was used for the
    • antigen-antibody complex.
    • Diagnosis of fungal, respiratory virus, arbovirus, and Q fever infections
  40. Antibody Inhibition Assay
    • Use of the specificity of an antibody to prevent infection (neutralization) or other activity (hemagglutination inhibition), identify the strain of the infecting agent (virus), quantitate antibody responses to a specific strain of virus
    • Hemagglutination inhibition is used to distinguish different strains of influenza A
  41. Latex Agglutination Assay
    • Antibodies or antigens are coupled with latex beads. Add serum or a suspension of an organism. Visible agglutination occurs or not.
    • Crytococcal meningitis, Staphylococcus aureus
    • Virus-specific antibody causes latex particles coated with viral antigens to clump. Antibody- coated latex particles are used to detect soluble viral antigen
  42. Red Azo Dye Protosil
    1935 - protected mice against systemic streptococcal infection.
  43. Alexander Fleming
    1928 - noted mold Penicillium prevented the multiplication of staphylococci.
  44. Antibacterial spectrum
    Range of antimicrobial activity (Broad vs Narrow)
  45. Bacteriostatic activity
    • Level of antimicrobial activity that inhibits the growth of an organism
    • Minimum inhibitory concentration (MIC)
    • The lowest concentration that inhibits the growth of the
    • organism
  46. Bactericidal activity
    • Level of antimicrobial activity that kills an organism
    • Minimum bactericidal concentration (MBC)
    • The lowest concentration that kills 99.9% of the population
  47. Antibiotic synergism
    Combinations of antibiotics enhanced bactericidal activity
  48. Antibiotic antagonism
    One antibiotic interferes with the activity of the other
  49. Antibiotic Combinations
    • (1) Broaden the antibacterial spectrum
    • Empirical therapy
    • Treatment of polymicrobial infections
    • (2) Prevent the emergence of resistant organisms during therapy
    • (3) Achieve a synergistic killing effect
  50. Basic Mechanisms of Antibiotic Action
    • Disruption of Cell Wall
    • Inhibition of Protein Synthesis
    • Inhibition of Nucleic Acid Synthesis
    • Antimetabolite
  51. Inhibition of Cell Wall Synthesis
    • The most common mechanism
    • Beta-lactam antibiotics (e.g., penicillins, cephalosporins, cephamycins, carbapenems, monobactams, Beta-lactamase inhibitors)
    • Share a common Beta-lactam ring structure
  52. Beta-Lactam Antibiotics
    • Building of peptidoglycan chains catalyzed by specific enzymes (serine proteases) - Transpeptidases, Transglycosylases & Carboxypeptidases
    • These regulatory enzymes also called penicillin- binding proteins (PBPs)
  53. penicillin- binding proteins (PBPs)
    • Bound by Beta-lactam antibiotics
    • Bactericidal agents
    • Antibiotic binds to specific PBPs in the bacterial cell wall
    • • Inhibits formation of cross-links between peptidoglycan chains
    • • Activatesautolysinsthatdegradethecellwall,resultinginbacterial
    • cell death
  54. Resistant to Beta-lactam Antibiotics
    • (1) Prevention of the interaction between the antibiotic and the target PBP
    • - Gram (-) outer membrane
    • (2) Modification of the binding of the antibiotic to the
    • PBP
    • - (1) Overproduction of PBP
    • - (2) Acquisition of a new PBP through gene transfer (e.g.,
    • methicillin resistance in Staphylococcus aureus)
    • - (3) Modification of an existing PBP through mutation:
    • recombination (e.g., penicillin resistance in Streptococcus pneumoniae) or a point mutation (penicillin resistance in Enterococcus faecium).
    • (3) Hydrolysis of the antibiotic by Beta-lactamases
  55. Beta-Lactamase
    • Enzyme hydrolyze Beta-lactam ring
    • More than 200 different Beta-lactamases
    • Extended-spectrum Beta-lactamases (ESBLs) encoded on plasmids that can be transferred from organism to organism
    • Specific for penicillins (penicillinases), cephalosporins (cephalosporinases), and carbapenems (carbapenemases; metallo-Beta-lactamases)
    • Classes A-D
    • Plasmid or chromosomal
  56. Penicillins
    • Natural penicillins [benzylpenicillin (penicillin G), phenoxymethyl penicillin
    • (penicillin V)]
    • Penicillinase-resistant penicillins [methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin]
    • Broad-spectrum penicillins [aminopenicillins (ampicillin, amoxicillin, carbenicillin, ticarcillin); ureidopenicillins (piperacillin)]
    • Beta-Lactam with Beta-lactamase inhibitor [ampicillin-sulbactam, amoxicillin-clavulanate, ticarcillin- clavulanate, piperacillin-tazobactam]
  57. Cephalosporins and Cephamycin
    • Cephalosporins (Beta-lactam antibiotics) derived from 7-aminocephalosporanic acid
    • Beta-lactam ring is fused with a dihydrothiazine ring
    • - Same mechanism of action as the penicillins
    • - Wider antibacterial spectrum
    • - Resistant to many Beta-lactamases
    • - Improved pharmacokinetic properties: longer half-life
    • Cephamycins has oxygen in place of sulfur in the dihydrothiazine ring
  58. Selected Examples of Cephalosporins and Cephamycins
    • Narrow spectrum (cephalexin, cephalothin, cefazolin, cephapirin, cephradine)
    • Expanded-spectrum cephalosporins (cefaclor, cefuroxime)
    • Expanded-spectrum cephamycins (cefotetan, cefoxitin)
    • Broad spectrum (cefixime, cefotaxime, ceftriaxone, ceftazidime
    • Extended spectrum (cefepime, cefpirome)
  59. Other Beta-Lactam Antibiotics
    • Carbapenems (e.g., imipenem, meropenem, ertapenem)
    • Broad-spectrum antibiotics
    • Active against virtually all groups of organisms, with only a few exceptions
    • - Resistance has been reported for all oxacillin-resistant staphylococci, selected Enterobacteriaceae and Pseudomonas, and other gram-negative rod
    • Monobactams (e.g., aztreonam).
    • Narrow-spectrum antibiotics
    • Active only against aerobic, gram-negative bacteria
  60. Polypeptides/Bacitracin
    • Bacillus licheniformis
    • Topically applied products (creams, ointments, sprays)
    • to trea skin infections caused by Gram (+)  Gram(-) are resistant  Inhibits cell wall synthesis
    • - Interfering with dephosphorylation and the recycling of the lipid carrier
    • • Transportingpeptidoglycanprecursors-cytoplasmicmembrane-cell wall)
    • May damage cytoplasmic membrane and inhibit transcription
    • Resistance caused by failure to penetrate into the bacterial cell
  61. Polypeptides/ Polymyxins
    • Bacillus polymyxa
    • Cyclic polypeptides insert into bacterial membranes
    • like detergents
    • Interacting with lipopolysaccharides and the phospholipids in the outer membrane
    • Increased cell permeability and eventual cell death
    • Most active against gram (-) rods
    • Polymyxins B and E (colistin) cause serious
    • nephrotoxicity
    • - External treatment of localized infections ( external otitis, eye infections, skin infections)
  62. Inhibition of 30S
    • Aminoglycosides (streptomycin, kanamycin, gentamicin, tobramycin, amikacin)
    • Aminocyclitol (spectinomycin)
    • Tetracyclines
  63. Inhibition of 50S
    • Inhibition of 50S
    • Oxazolidinones (linezolid)
    • Chloramphenicol
    • Macrolides (Erythromycin)
    • Lincosamides (Clindamycin)
  64. Aminoglycoside Antibiotics
    • Amino sugars linked to an aminocyclitol ring (through glycosidic bonds)
    • Free NH, and OH groups bind to specific ribosomal proteins
    • Bactericidal
    • Commonly used to treat
    • serious G(-) infections
    • Aerobic, energy-dependent penetration
    • Ex: Gentamicin, Tobramycin, Amikacin
  65. Resistance to Aminoglycosides
    • (1) Mutation of the ribosomal binding site
    • (2) Decreased uptake of the antibiotic into the bacterial cell
    • (3) Increased expulsion of the antibiotic from the cell
    • (4) Enzymatic modification of the antibiotic (most
    • common) on the amino and hydroxyl groups
    • - Phosphotransferases, Adenyltransferases, Acetyltransferases
  66. Inhibition of Nucleic Acid Synthesis: Quinolone
    • Synthetic chemotherapeutic agents
    • Inhibit bacterial DNA topoisomerase type II (gyrase;
    • G(-)) or type IV (G(+))
    • Nalidixic acid: 1st used in clinical practice
    • Fluoroquinolones (ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin)
    • Resistance
    • - Chromosomal mutations in DNA gyrase and topoisomerase
    • type IV
    • - Decreased drug uptake (mutations inmembrane permeability
    • regulatory genes)
    • - Overexpression of efflux pumps that actively eliminate the drug
  67. Inhibition of Nucleic Acid Synthesis: Rifampin
    • Binds to DNA-dependent RNA polymerase and inhibits the initiation of RNA synthesis
    • Bactericidal for Mycobacterium tuberculosis
    • Very active against aerobic G(+) cocci
    • G(-) are resistant intrinsically: decreased uptake of the hydrophobic semisynthetic antibiotic
  68. Antimetabolites
    • Mammalians do not synthesize folic acid (required as a vitamin)
    • Certain microbes synthesize own folic acid
    • Sulfonamides compete with p-aminobenzoic acid
    • Trimethoprim inhibit dihydrofolate reductase (di- to tetrahydrofolate)
    • Trimethoprim + Sulfamethoxazole: Synergistic inhibition of folic acid synthesis
  69. Virulence Factors in Neisseria gonorrhoeae
    • Pilin (attachment)
    • Por protein (protein I; porin; prevent phagolysosome
    • fusion)
    • Opa protein (protein II; opacity; firm attachment)
    • Rmp protein (protein III; reduction-modifiable protein; protection from antibodies)
    • Transferrin-binding proteins (acquisition of iron)
    • Lactoferrin-binding proteins (acquisition of iron)
    • Hemoglobin-binding proteins (acquisition of iron)
    • LOS Lipooligosaccharide (inflammation, TNF-α)
    • IgA1 protease
    • Beta-Lactamase
  70. Epidemiology (Gonorrhea)
    • Humans (only natural hosts)
    • The major reservoir: asymptomatically infected person asymptomatic, particularly in women
    • no other known reservoir
    • Second (only to chlamydia) STD in US (~360,000 new
    • infections in 2006)
    • Sexual contact
    • 1st contact risk: 50% female, 20% male
    • Peak incidence age: 15 to 24 years
    • Disseminated disease: deficiencies in late components (C5-C8) of complement (6000X higher risk)
  71. Gonorrhea
    purulent discharge (urethra, cervix, epididymis, prostate, anus) after incubation period (2-5 day)
  72. Disseminated infections
    spread from genitourinary tract through blood to skin or joints; pustular rash with erythematous base and suppurative arthritis
  73. Ophthalmia neonatorum
    Ophthalmia neonatorum: purulent ocular infection acquired by neonate at birth
  74. Gonorrhea Genital infections
    • Genital infections
    • Men
    • - Urethra (purulent urethral discharge and dysuria)
    • - Most (95%) have acute symptoms
    • - Complications (rare): epididymitis, prostatitis, and periurethral abscesses Women
    • - Cervix (endocervical columnar epithelial cells)
    • - Symptoms: vaginal discharge, dysuria, and abdominal pain
    • - Ascending genital infections (10% to 20%): salpingitis,
    • tuboovarian abscesses, and pelvic inflammatory disease
  75. Pelvic Inflammatory Disease (PID)
    • A generic term for inflammation of the female uterus, fallopian tubes, and/or ovaries
    • Progresses to scar formation with adhesions o nearby tissues and organs
    • Viral, fungal, parasitic, though most often bacterial infections
    • In US, > million women/month are affected (teenagers 15-19 yr, new mothers 20-24 yr)
    • Causes over 100,000 women to become infertile in the US each year
    • One of the most serious complications of sexually transmitted diseases
  76. Gonococcemia
    • Disseminated infections with septicemia and infection of skin and joints
    • 1% to 3% of infected women
    • Fever; migratory arthralgias; suppurative arthritis in the wrists, knees, and ankles
    • Pustular rash over the extremities
    • A leading cause of purulent arthritis in adults
  77. Other N. gonorrhoeae Syndromes
    • Purulent conjunctivitis (newborns: ophthalmia neonatorum)
    • Perihepatitis (Fitz- Hugh-Curtis syndrome)
    • Anorectal gonorrhea (homosexual men)
    • Pharyngitis
  78. Laboratory Diagnosis
    Gonorrhea
    • Microscopy
    • - Gram stain
    • - •sensitive and specific for men with purulent urethritis
    • - •insensitivity in asymptomatic men and in both symptomatic and asymptomatic women
    • Culture
    • - Avoiddryingandcoldtemperatures
    • - Inoculated onto both selective media (modified Thayer-Martin medium) and nonselective media (chocolate blood agar)
    • Identification
    • - Preliminary: oxidase-positive, gram-negative diplococci; grow on chocolate blood agar or selective media
    • - Definitive: Glucose positive
    • Nucleic Acid Amplification
  79. Treatment
    Gonorrhea
    • No more Penicillin
    • β-lactamase production (plasmid-mediated)
    • Chromosomally mediated changes in penicillin-binding proteins and in cell wall permeability (resistance to tetracyclines, erythromycin, and aminoglycosides)
    • Fluoroquinolone (ciprofloxacin)- 1993 Ceftriaxone- 2007
  80. Prevention
    Gonorrhea
    • No effective vaccine
    • Lack of protective immunity
    • - Antigenic diversity
    • - Antibodies detected to pili antigens, Por proteins, and LOS
    • Chemoprophylaxis- ineffective
    • Except in the protection of ophthalmia neonatorum (1% silver nitrate, 1% tetracycline, or 0.5% erythromycin eye ointments)
  81. Gonococcus Summary
    • Gram-negative cocci
    • Cause gonorrhea which can lead to pelvic
    • inflammatory disease (PID) in woman and epididymitis in men
    • Can be carried asymptomatically by both men and women
    • Exhibit phase variation and antigenic variation
    • Can cause disseminated infections (serum-resistant strains)
    • Infection are treated with antibiotics (widespread penicillin- resistance)
  82. A Meningococcal Case
    • A previously healthy 18-year-old man who presented to a local emergency department with the acute onset of fever and headache. His temperature was elevated (40 C), he was tachycardic (pulse of 140 per minute), and hypotensive (blood pressure 70/40 mm Hg).
    • Petechiae were noted over his chest.
    • Neisseria meningitidis was recovered in the patient's
    • blood and CSF cultures. Despite the prompt administration of antibiotics and other support measures, the patient's condition rapidly deteriorated, and he died 12 hours after arrival in the hospital. This patient illustrates the rapid progression of meningococcal disease, even in healthy young adults.
  83. Meningitis
    • ’Meninges' - thin membrane, or lining surrounds the brain
    • Meningitis - infection of this lining
    • Affects people of all ages, most common in pre-school children
    • Caused by several different viruses or bacteria
    • Viral meningitis, less severe and resolves without specific treatment
    • Bacterial meningitis, quite severe and may result in brain damage, hearing loss, or learning disability
  84. Signs and Symptoms of Meningitis
    • High fever, headache, and stiff neck are common symptoms- over the age of 2 years (several hours- 1 to 2 days)
    • Other - nausea, vomiting, discomfort looking into bright lights, confusion, and sleepiness
    •  In newborns and small infants, may absent classic symptoms, only appear slow or inactive, or be irritable, have vomiting, or be feeding poorly
    •  As the disease progresses, patients of any age may have seizures
  85. Bacterial Meningitis
    • Bacterial menigitis, an infection of the membranes (meninges) and cerebrospinal fluid (CSF) surrounding the brain and spinal cord
    • A major cause of death and disability world-wide
    • The etiology of bacterial meningitis varies by age group and region of the world
    • Beyond the perinatal period, three organisms, transmitted from person to person through the exchange of respiratory secretions, are responsible for most cases of bacterial meningitis: Neisseria meningitidis Haemophilus influenzae Streptococcus pneumoniae
    • Neisseria meningitidis, a leading cause of bacterial menigitis and sepsis in children and young adults in the United States, causes both sporadic disease and outbreaks
  86. Neisseria meningitidis
    Physiology and Structure
    • Gram-negative diplococci with fastidious growth requirements
    • Grows best at 35° to 37°C in a humid atmosphere
    • Oxidase and catalase positive; acid produced from maltose and glucose oxidatively
    • Outer surface antigens include polysaccharide capsule, pili, and lipooligosaccharides (LOS)
  87. Virulence
    Neisseria meningitidis
    • Pili- bind to specific pili receptors for nasopharynx colonization
    • Capsule- protects bacteria from antibody- mediated phagocytosis
    • Bacteria can survive intracellular killing in the absence of humoral immunity
    • Endotoxin mediates most clinical manifestations
  88. Clinical Case
    N. gonorrhoeae
    • Upon returning home to Nebraska, a 28 year old man who had vacationed in Thailand presented to the local emergency room (ER) complaining of painful urination and a purulent discharge from his penis.
    • Urethral specimens were collected for detection of Neisseria gonorrhoeae and Chlamydia trachomatis using nucleic acid amplification tests (NAATs) and the patient was empirically given a single 500-mg dose of ciprofloxacin orally and a single 1-g dose of azithromycin.
    • Subsequently, the NAATs were positive for N. gonorrhoeae, but negative for C. trachomatis.
  89. Family Neisseriaceae
    • Genera of medical interest: Neisseria, Eikenella, and Kingella
    • Eikenella corrodens and Kingella kingae (opportunistic pathogens) colonize the human oropharynx
  90. Neisseria
    • 10 species found in humans
    • Neisseria gonorrhoeae and Neisseria meningitidis, strictly
    • human pathogens
    • The remaining Neisseria species:
    • • limited virulence • compromised patients • oropharynx and nasopharynx (anogenital) mucosal membranes
  91. Characteristics of Neisseria
    • Aerobic, Gram (-) cocci (0.6 to 1.0 um)
    • Diplococci resembling coffee beans (with adjacent sides flattened)
    • Not motile
    • No endospores
    • All are oxidase positive
    • Acid produced by oxidation of carbohydrates
    • - N. gonorrhoeae - glucose - N. meningitidis - both glucose
    • and maltose
  92. Growth Requirement
    Neisseria
    • Nonpathogenic species of Neisseria grow on nutrient agar at 35° to 37°C.
    • N. meningitidis has variable growth on nutrient agar
    • N. gonorrhoeae is a fastidious organism
    • - Complex media for growth
    • - Sensitive to dry conditions or fatty acids-neutralized by adding soluble starch
    • - All strains require cystine for growth
    • - A humid atmosphere supplemented with carbon dioxide (5% CO2)
  93. Cell Structure
    Neisseria
    • Typical of gram-negative bacteria
    • Outer surface of N. gonorrhoeae is not covered with a true carbohydrate capsule, with a capsule- like negative charge surface
    • Serogrouping based on antigenic differences in the polysaccharide capsule of N. meningitidis
    • - 13 serogroups (A, B, C, D, H, I, K, L, W-135, X, Y, Z, 29E)
    • - most infections caused by A, B, C, Y, and W135
  94. Virulence Factors
    Neisseria
    • Horizontal DNA exchange
    • Phase and antigenic structural variation
    • Capsular switching
    • Molecular mimicry
    • - Polysialic acid capsule of serogroup B meningococci is identical to structures on the human neural cell adhesion molecule (N-CAM)
    • -LOS (lacto-N-neotetrose) mimics the carbohydrate moieties of human glycosphigolipids
    • Blebbing and autolysis
    • - Frequent vesiculation (blebbing) of the outer membrane-rapid initiation of the inflammatory cascades of sepsis
    • - Naturalautolysisinstationarygrowthphase-DNArelease-genetic transformation
  95. Epidemiology
    Neisseria
    • Humans (only natural hosts)
    • Person-to-person spread (aerosolization of RT secretions)
    • Highest incidence < 5 years, institutionalized people, and patients with late complement deficiencies
    • Disease occurs worldwide, most commonly in the dry, cold months of the year
    • - Meningitis and meningococcemia - serogroups B and C
    • - Pneumonia- serogroups Y and W135
    • - Serogroups A and W135 - underdeveloped countries
  96. Meningitis
    Purulent inflammation of meninges associated with headache, meningeal signs, and fever; high mortality rate unless promptly treated with effective antibiotics
  97. Meningococcemia
    Disseminated infection characterized by thrombosis of small blood vessels and multiorgan involvement; small, petechial skin lesions coalesce into larger hemorrhagic lesions
  98. Pneumonia
    Milder form of meningococcal disease characterized by bronchopneumonia in patients with underlying pulmonary disease
  99. Diagnosis
    Neisseriaceae
    • Gram stain of CSF is sensitive and specific
    • - of limited value for blood specimens (too few organisms are generally present, except in overwhelming sepsis.
    • Culture is definitive (fastidious and dies rapidly when exposed to cold or dry conditions)
    • Antigen tests- insensitive and nonspecific.
  100. Treatment/Prevention/Control
    Neisseria
    • Breast-feeding infants- passive immunity (first 6 months)
    • Treatment - penicillin (drug of choice), chloramphenico,
    • ceftriaxone, and cefotaxime
    • Chemoprophylaxis -rifampin, ciprofloxacin, or ceftriaxone
    • A polyvalent polysaccharide-protein conjugate vaccine
    • effective against serogroups A, C, Y, and W135 was licensed in the United States in 2005 (11-18 yrs and other high risk)
    • Immunoprophylaxis- only for serogroups A, C, Y, and W135; no effective vaccine is available for serogroup B
  101. Neisseria meningitidis (the meningococcus)
    Summary
    • Gram-negative cocci
    • Cause septicemia and meningitis
    • Heavily encapulated
    • Hemolysin for dissemination in the bloodstream, leading to disseminated intravascular coagulations
    • Vaccine with capsular polysaccharide, except for group B strains
  102. Neisseria sicca and Neisseria mucosa
    • Commensal organisms in the oropharynx
    • Implicated in isolated cases of meningitis, osteomyelitis, endocarditis, bronchopulmonary infections, acute otitis media, and acute sinusitis
  103. Eikenella corrodens
    • Human bite wounds: traumatic (e.g., bite, fistfight injury) introduction of oral organisms into deep tissue
    • Subacute endocarditis: infection of endocardium characterized by gradual onset of low grade fevers, night sweats, and chills
  104. Kingella kingae
    Subacute endocarditis: As with E. corrodens
  105. Case Study
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    • A 22-year-old female schoolteacher was brought to the emergency room after a 2-day history of headache and fever. On the day of admission, the patient had failed to come to school and could not be reached by telephone. When notified of this fact, the patient's mother went to her daughter's apartment, where she found her daughter in bed, confused and highly agitated. The patient was rushed to the local hospital, where she was comatose on arrival.
    •  Purpuric skin lesions were present on her trunk and arms. Analysis of her CSF revealed the presence of 380 cells/mm3 (93% polymorphonuclear leukocytes), a protein concentration of 220 mg/dL, and a glucose concentration of 32 mg/dL.
    •  Gram stain of CSF showed many gram-negative diplococci, and the same organisms were isolated from blood and CSF. The patient died despite prompt initiation of therapy with penicillin.
  106. Case Study
    S. aureus
    • An 18-year-old man fell on his knee while playing basketball. The knee was painful, but the overlying skin was unbroken. The knee was swollen and remained painful the next day, so he was taken to the local emergency department.
    • Clear fluid was aspirated from the knee, and the physician prescribed symptomatic treatment. Two days later, the swelling returned, the pain increased, and erythema developed over the knee.
    • Because the patient also felt systemically ill and had an oral temperature of 38.8°C, he returned to the emergency department.
    • Aspiration of the knee yielded cloudy fluid, and cultures of the fluid and blood were positive for S.aureus.
  107. Staphylococcus
    • Spherical shape; Gram (+) reaction; No endospores
    • Catalase catabolizes hydrogen peroxide into water and oxygen gas (bubbles)
    • Aerobic catalase-positive genera (Staphylococcus, Micrococcus, Kocuria, Kytococcus, Alloiococcus)
    • Aerobic catalase-negative genera (Streptococcus, Enterococcus)
    • Anaerobic gram-positive cocci
    • 0.5 to 1.5 um in diameter, nonmotile, facultatively anaerobic (grow both aerobically and anaerobically)
    • Grow in high salt (10% sodium chloride)
    • Grow at 18°C to 40°C  On skin and mucous membranes (humans)
    • Genus currently consists of 35 species and 17
    • subspecies
    • Many are found on humans
    • S. aureus is catalase (+) AND coagulase (+; fibrinogen- fibrin- clot)
  108. Types of Staph
    • S. aureus; aureus, golden (golden or yellow)
    • S. epidermidis; epidermidis, outer skin (of the epidermis or outer skin)
    • S. lugdunensis; Lugdunum, Latin name for Lyon, France (location of first isolate)
    • S. saprophyticus; sapros, putrid; phyton, plant (saprophytic or growing on dead tissues)
    • S. haemolyticus; lyse RBC
  109. S. aureus Virulence Factors
    • Pathology of staphylococcal infections depends on
    • 1. Production of surface proteins mediating adherence
    • to host tissues
    • - Capsule and slime layer
    • - Teichoic acid
    • - Protein A
    • - Bound coagulase (clumping factor)
    • - Surface adhesion proteins: MSCRAMM (microbial surface components recognizing adhesive matrix molecules)
    • 2. Elaboration of extracellular proteins (specific toxins and hydrolytic enzymes)
  110. T oxins
    Staph
    • Cytotoxins (α, β, δ, γ, leukocidin)
    • Exfoliative toxins (ET A, ETB)
    • - Serine proteases
    • - Staphylococcal scalded skin syndrome (SSSS)
    • Enterotoxins (A-E, G-I)
    • - Superantigens
    • - Heat-stable at 100°C for 30 minutes
    • - Resistant to hydrolysis by gastric and jejunal enzymes
    • Toxic shock syndrome toxin-1
    • - Superantigens
    • - Heat-, proteolysis-resistant, chromos. mediated exotoxin
    • - Penetrate mucosal barrier
  111. Enzymes
    • Coagulase
    • Catalase
    • Hyaluronidase
    • - Hydrolyzes hyaluronic acids in connective tissue,
    • - Promoting the spread of staphylococci in tissue
    • - Fibrinolysin (staphylokinase)
    • - Lipases
    • - Nucleases
    • - Penicillinase
  112. Epidemiology
    Staphylococcus
    • NF on human skin and mucosal surfaces
    • Survive on dry surfaces for long periods
    • Person-to-person spread through direct contact or exposure to contaminated fomites
    • Risk factors
    • Patients at risk
    • Infections found worldwide and generally with no seasonal prevalence
  113. Diseases
    • Toxin-mediated diseases
    • - Food poisoning, toxic shock syndrome, and scalded skin syndrome
    • Pyogenic diseases
    • - Impetigo, folliculitis, furuncles, carbuncles, and wound infections
    • Other systemic diseases (frequently associated with bacteremia)
    • - Pneumonia (typically after viral respiratory infection), empyema (complication of pneumonia or surgical intervention), septic arthritis, osteomyelitis, acute endocarditis, and catheter-related bacteremia
  114. Clinical Summaries
    S. aureus
    • Toxin-Mediated Diseases
    • • Scalded skin syndrome • Food poisoning • Toxic shock
    • Suppurative Infections
    • • Impetigo • Folliculitis • Furuncles or boils • Carbuncles • Bacteremia and endocarditis • Pneumonia and empyema • Osteomyelitis • Septic arthritis
  115. Patients at Risk
    Staphylococcus
    • Infants (scalded skin syndrome)
    • Young children with poor personal hygiene (impetigo and other cutaneous infections)
    • Menstruating women (toxic shock syndrome)
    • Patients with intravascular catheters (bacteremia and
    • endocarditis) or shunts (meningitis)
    • Patients with compromised pulmonary function or an antecedent viral respiratory infection (pneumonia)
  116. Diagnosis
    Staphylococcus
    • Microscopy
    • Culture: BAP
    • Biochemical: 7.5% NaCl, Mannitol, coagulase, protein A
    • Serology: Aby to teichoic acids
    • Fluorescent in situ hybridization (FISH): detects from clinical samples
    • Pulsed-field gel electrophoresis (PFGE): genomic DNA analysis
    • Antibiograms, biotyping, phage typing
  117. Resistance
    Staphylococcus
    • Semisynthetic penicillins resistant to β-lactamase hydrolysis (e.g., methicillin, nafcillin, oxacillin, dicloxacillin)
    • Currently, 30% to 50% of the strains of S. aureus and more than 50% of the coagulase-negative staphylococci are resistant to semisynthetic penicillins
    • MRSA: Methicillin-resistant S. aureus
    • Acquisition of a gene (mecA) that codes for a novel
    • penicillin-binding protein, PBP2’
    • Expression of PBP2' renders the bacteria resistant to all penicillin, cephalosporin, and carbapenem antibiotics
  118. Treatment/Control/Prevention
    Staphylococcus
    • Antibiotics of choice are oxacillin (or other penicillinase-resistant penicillin), or vancomycin for oxacillin-resistant strains
    • The focus of infection (e.g., abscess) must be identified and drained
    • Treatment is symptomatic for patients with food poisoning
    • Proper cleansing of wounds and use of disinfectant help prevent infections
    • Thorough hand washing and covering of exposed skin helps medical personnel prevent infection or spread to other patients
  119. National MRSA Education Initiative
    • The goal of the National MRSA Education Initiative is to help Americans better recognize and prevent MRSA skin infections.
    • MRSA is methicillin-resistant Staphylococcus aureus, a potentially dangerous type of staph bacteria that is resistant to certain antibiotics and may cause skin and other infections.
    • Recent data show that Americans visit the doctor approximately 12 million times each year for suspected Staph or MRSA skin infection.

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  120. pharyngitis? strep throat?
    • A school teacher who can't talk and is suffering mightily with a sore throat. He was well until two nights ago. His symptoms include 102o C fever, difficulty breathing due to enlarged tonsils, enlarged cervical lymph nodes. Two school kids in his class had similar episodes. He has no cough, and is otherwise in good health.
    • A look at his throat shows markedly enlarged and reddened tonsils, plus,
    • a creamy exudate on their surface.
    • Throat culture is requested for lab diagnosis.
    •  What is the most likely source of this infection
    • (pharyngitis, strep throat)?
  121. Streptococcus
    • Gram-positive cocci typically arranged in pairs or chains
    • Facultative anaerobes; some are capnophilic
    • Catalase-negative
    • Ferment carbohydrates
    • Blood- or serum-enriched media
  122. Classification
    Streptococcus
    • (1) Serologic properties
    • - Lancefield groupings (originally A to W) of cell wall carbohydrates
    • (2) Hemolytic patterns
    • - Complete (beta [β]) hemolysis
    • - Incomplete (alpha [α]) hemolysis
    • - No (gamma [γ]) hemolysis
    • (3) Biochemical (physiologic) properties
  123. β−Hemolytic Streptococci
    • S. pyogenes - Small colonies with a large zone of hemolysis
    • S. agalactiae - Large colonies with a small zone of hemolysis
  124. Streptococcus pyogenes
    • Gram-positive cocci arranged in pairs and long chains
    • β-Hemolytic; more viruluent strains with capsule
    • Facultative anaerobe
    • Catalase negative; PYR positive; bacitracin susceptible (important identification tests)
    • Group-specific carbohydrate (A antigen) and type- specific antigens (M and T proteins) in cell wall
    • Produce streptolysin O and DNase B
    • - Antibodies against these antigens [ASO, anti-DNase B] are clinically important
    • Virulence factors
  125. Virulence Factors of
    Streptococcus pyogenes
    • Capsule
    • Lipoteichoic acid Binds to epithelial cells
    • M protein: Adhesin-mediates internalization; antiphagocytic;
    • degrades C3b
    • M-like proteins: Binds Ig M and G and β2-macroglobulin (protease inhibitor); antiphagocytic
    • F protein: Mediates adherence to epithelial cells and internalization
    • Pyrogenic exotoxins
    • Streptolysin S & Streptolysin O
    • Streptokinase
    • DNase
    • C5a peptidase
  126. S. pyogenes (group A) Diseases
    Suppurative infections.
    • Pharyngitis (strep throat)
    • Scarlet fever
    • Pyoderma
    • Erysipelas
    • Cellulitis
    • Necrotizing fasciitis
    • Streptococcal toxic shock syndrome
    • Other suppurative diseases (puerperal sepsis, lymphangitis, pneumonia)
  127. S. pyogenes (group A) Diseases
    Nonsuppurative Infections
    • Rheumatic fever
    • Characterized by inflammatory changes of the heart (pancarditis), joints (arthralgias to arthritis), blood vessels, and subcutaneous tissues
    • Specific M types (types 1, 3, 5, 6, and 18)
    • Incidence (US): > 10,000 cases/yr in 1961 to 112 cases in 1994 ; Estimated 100 cases per 100,000 children per year in developing countries
    • Acute glomerulonephritis
    • Acute inflammation of the renal glomeruli with edema, hypertension, hematuria, and proteinuria
    • Specific nephritogenic strains of group A streptococci (pharyngeal and pyodermal strains)
  128. Epidemiology
    Streptococcus
    • Asymptomatic colonization in URT and transient colonization of skin
    • Ubiquitous; Survive on dry surfaces
    • Person-to-person spread
    • Higher risk individuals, most children
    • - 5 to 15 years old (pharyngitis) - 2 to 5 years w/ poor personal hygiene (pyoderma)
    • - Children with severe streptococcal disease (rheumatic fever, glomerulonephritis)
    • Seasonal incidences of specific diseases
  129. Diagnosis
    Streptococcus
    • Microscopy
    • Direct antigen tests (streptococcal pharyngitis)
    • Culture is highly sensitive
    • ID by negative catalase, positive PYR reaction, susceptibility to bacitracin, and presence of group A antigen
    • ASO test (confirming rheumatic fever and acute glomerulonephritis)
    • Anti-DNase B test (suspected acute glomerulonephritis)
  130. Treatment, Control, and Prevention
    Streptococcus
    • Penicillin is drug of choice (erythromycin or oral cephalosporin)
    • Treat oropharyngeal carriage
    • Starting antibiotic therapy within 10 days in patients with pharyngitis
    • - Prevents rheumatic fever
    • Antibiotic prophylaxis before procedures
    • - Prevent bacteremias leading to endocarditis
  131. Other β Hemolytic Streptococci Disease
    • Group C Streptococcus (S. anginosus, small-colony species and S. dysgalactiae, large-
    • colony species)
    • Abscess formation in deep tissues (Associated with S. anginosus group)
    • Pharyngitis (Associated with S. dysgalactiae, Resembles S. pyogenes infections. Complicated with acute glomerulonephritis)
  132. Streptococcus agalactiae (Group B) Diseases
    • Early-onset neonatal disease
    • - Within 7 days of birth; pneumonia, meningitis, and sepsis
    • Late-onset neonatal disease
    • - More than a week after birth; bacteremia with meningitis
    • Infections in pregnant women
    • - Urinary tract infections (most often)
    • - Bacteremia and disseminated complications may occur
    • Infections in other adult patients
    • - Most common diseases include bacteremia, pneumonia, bone and joint infections, and skin and soft-tissue infections
  133. Streptococcus pneumoniae Disease
    • Isolated independently by Pasteur and Steinberg more
    • than 100 years ago
    • Pneumococcal disease
    • - A leading cause of morbidity and mortality
    • - Pneumonia • Acute onset with severe chills and sustained fever;productive cough with blood-tinged sputum; lobar consolidation
    • - Meningitis • Severe infection involving the meninges with headache, fever, and
    • sepsis; high mortality and severe neurologic deficits in survivors
    • - Bacteremia
  134. Viridans Group of Streptococci
    • A heterogeneous collection of α-hemolytic and nonhemolytic streptococci.
    • Group name is derived from viridis (Latin for "green"), a reflection of the fact that many of these bacteria produce a green pigment on blood agar media.
    • More than 30 species and subspecies have been identifiedinto five subgroups: Anginosus, Mitis, Mutans, Salivarius, Bovis
  135. Viridans Streptococci Diseases
    • Abscess formation in deep tissues (Associated with S. anginosus group)
    • Septicemia in neutropenic patients (Associated with S. mitis group)
    • Subacute endocarditis (Associated with S. gordonii, S. mutans, S. mitis, S. oralis, and S. sanguis)
    • Dental caries (Associated with S. mutans and S. sobrinus)
    • Malignancies of gastrointestinal tract (Associated with S. bovis)
  136. Enterococcus and Other Gram- Positive Cocci
    • Previously classified as group D streptococci
    • In 1984, reclassified into the new genus Enterococcus
    • (29 species)
    • Colonizes the gastrointestinal tracts of humans and animals
    • The most commonly isolated, clinically important species
    • - Enterococcus faecalis
    • - Enterococcus faecium
    • Common colonizers of the human intestinal tract
    • - Enterococcus gallinarum and Enterococcus casseliflavus
    • - Misidentified as vancomycin-resistant E. faecium
  137. Enterococcus Infections
    • Disease (Urinary tract infections, Wound infections (particularly intraabdominal and usually
    • polymicrobic), Bacteremia and Endocarditis)
    • Antibiotic resistance is becoming increasingly common (particularly E. faecium)
    • Diagnosis (Catalase negative , PYR positive, Resistant to bile and optochin)
  138. Case Study
    Streptococcus
    • A 72-year-old man was admitted to the hospital because of a fever that had risen as high as 40。 C, myalgias, and respiratory complaints. The clinical diagnosis of influenza was confirmed by the laboratory isolation of influenza virus from respiratory secretions.
    • This patient's hospitalization was complicated by the development of pneumonia caused by oxacillin-resistant Staphylococcus aureus that was treated with a 2-week course of vancomycin. Declining pulmonary function necessitated the use of a ventilator, which led to the development of a secondary infection with Klebsiella pneumoniae. Ceftazidime (a cephalosporin) and gentamicin were added to the patient's treatment.
    • After 4 weeks of hospitalization, the patient became septic. E. faecium resistant to vancomycin, gentamicin, and ampicillin was cultured from three blood specimens.

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