Intro to Clinical Microbiology Flashcards
List the major disease-producing microorganisms
Major: viruses bacteria fungi protozoa helminthes
Other:
prions
ectoparasites
List and describe the bacterial cellular morphology types—shapes and groupings
Shapes o Round (coccus): 0.5-1.25 μm diameter o Rod (bacillus): 0.5-1.0 μm diameter o Helical (spirochete): like rods but can be 10x longer
Groupings Cocci: • Singular • Pairs (diplococcic) • Chains (streptococci) • Groups of four (Sarcinae, tetrads) • Clusters (staphylococci) Bacilli: • Short coccobacilli • Long filamentous rods • Pointed ends (fusiform) • Curved (vibrio) • Pleomorphic (vary in size and shape) Spirochetes: vary in length and number of helical turns
Explain how the Gram stain is used in clinical microbiology
• Determined by cell wall structure of organism
Steps:
o Crystal violet stain (stains all bacteria on slide)
o Fixed with iodine
o Washed with alcohol or alcohol/acetone (decolorizes Gram-negative cell walls)
o Add safranin (red counterstain; able to be taken up by Gram-negative bacteria)
Results:
- Gram-positive = purple/blue (Thicker peptidoglycan (murein) cell wall blocks decolorization step)
- Gram-negative = red/pink
Describe the 4 phases of the bacterial growth curve and the relationship of the log phase to antimicrobial therapy and susceptibility testing
1) Lag phase:
o Cells are adapting/replenishing/undergoing size increases
o Little or no cell division
o No increase in cell number
2) Exponential (log) phase:
o State of maximal growth and constant rate of division
o Generation time = time for 1 cell to become 2 cells
o Optimal time to perform metabolic testing, antibiotic susceptibility/resistance testing
o Optimal time for antibiotic effectiveness
3) Stationary phase:
o Nutrients are exhausted, waste products accumulate
o Leads to decreased growth rate
o Rate of new cells forming = rate of dying cells
4) Death phase (or period of decline)
o Does not always occur
o Number of nonviable cells outnumber viable ones
Discuss settings where bacteria do not conform to laboratory growth pattern
o This lab pattern = growth in broth under optimal conditions
Exceptions:
• Biofilms: different physiology, metabolism, gene expression, antibiotic resistance than free-living (planktonic) bacteria
• Latent infections: bacteria appear to be in persistent stationary phase
Describe the temperature requirements of bacteria; define and discuss the clinical relevance of the following terms: psychrophile, mesophile, and thermophile
• Temperature: both optimal temperature for growth and range of temperatures allowing survival
o Mesophiles: 20-45 °C
• Most human pathogens
• Typical lab culture condition is 35-37 °C
o Psychrophiles: < 20 °C
• problems in refrigerated food or blood products
o Thermophiles: 45-60 °C
• problems in food processing (canning)
o Stenothermophiles: > 60 °C
Define and discuss the clinical relevance of the following terms: aerobe, anaerobe, facultative anaerobe, microaerophile, and aerotolerant.
Aerobes
o Oxygen required for growth
o Possess enzymes
o Microaerophiles: require reduced levels of oxygen (2-10%) because have low levels of enzymes
o Capnophiles: growth enhanced by increased level of CO2 (5-10%)
Anaerobes o Oxygen inhibits growth o Do not possess enzymes o Common in GI tract and mouth o Aerotolerant: can tolerate short oxygen exposure
Facultative
o Grow with or without oxygen
o Common in GI tract
o Most human pathogens
Describe the oxygen-detoxifying enzymes
Superoxide dismutase
• Superoxide radical (O2-) → H2O2 + O2
Catalase
• H2O2 → H2O + O2
Peroxidase
• Breaks down H2O2
Describe the components of bacterial genetic material
Chromosome
• Circular, dsDNA
• Replication precedes cell division
Plasmid
• Extrachromosomal circular genetic element with replication origin
• Usually 5- 100 genes
• Can be passed during cell division or transferred between bacteria by conjugation or transformation
• Usually not essential but confers selective advantage
• Ex. Antibiotic resistance, virulence factors, etc.
Transposon
• Genetic element contained on chromosome or plasmid
• Usually 1-10 genes (often including antibiotic resistance gene)
• Does not replicate independently
• Can move/jump from one site on DNA to another site on same DNA or to a different DNA molecule
List the mechanisms for changing a bacterial genome
1) Mutation
2) Genetic transfer
- transformation
- transduction
- conjugation
3) Genetic recombination
Describe the process of bacterial transformation
Mediated by free (naked) DNA from lysed organisms
• Uptake of free DNA
• Recombination with donor DNA and recipient DNA
Limited number of bacteria naturally competent for transformation
o Streptococcus pneumonia
o Haemophilus influenzae
o Neisseria
Describe the process of bacterial transduction
Mediated by bacterial virus (bacteriophage)
• Injects DNA or RNA into host bacterium
• Bacteria cell fills with new phages
• Lysis → release of new phages
2 types: 1) Generalized transduction • Mediated by lytic phage • Any portion of degraded bacterial DNA may be “mistakenly” packaged into assembling phage = transferred to another bacterium • A random error in packaging
2) Specialized transduction
• Mediated by temperate or lysogenic phage
• During lysogeny: temperate phage DNA integrates into bacterial chromosome at specific sites as a prophage (a latent viral infection that enables replication of the phage along with the chromosome)
• Certain conditions may cause excision of the integrated lysogenic prophage and initiation of a lytic cycle
• Imprecise excision → carries bacterial DNA sequences adjacent to site of integration = transferred to another bacterium
• Lysogenic conversion: if bacterial genes enable a nonvirulent organism to pick up virulence factors
Describe the process of bacterial conjugation
Mediated by transfer apparatus (cell-to-cell contact)
• Gram-negative bacteria: a sex pilus encoded by a fertility (F) plasmid
• Gram-positive bacteria: donor and recipient cells clump together via adhesions
• Transient cytoplasmic bridge forms through which plasmid or chromosomal DNA may transfer (donor → recipient)
• Can occur between related and unrelated bacteria
• Clinical importance: allows for passage of plasmids carrying virulence factors (including genes called R factors that encode for antibiotic resistance)
Discuss the clinical implications of lysogenic conversion.
• Occurs when phage infection and lysogenic prophage integration changes the phenotype of the host bacterium
Ex. Prophage may carry a gene encoding a virulence factor:
o Diphtheria toxin: only expressed by corynebacterium diphtheria strains lysogenized by the β prophage carrying the DT gene
o Cholera toxin: only expressed by vibrio cholera strains lysogenized by the CTX prophage carrying the CT gene
List and discuss the points to consider when doing a history and physical exam for an infectious disease
History
o Timing and nature of fevers
o Contact with others who are ill
o Predisposing factors (diabetes, immunosuppression, COPD, etc.)
o History of recent or recurrent infection
o Travel history
o Animal contacts
o Recent or current antimicrobial therapy
Exam
o Temperature
o Search for localized or generalized lymphadenopathy
o Skin (trauma, ulcers, line sites, rashes)
o Exam of each organ system
Discuss the terms sensitivity and specificity as they relate to laboratory tests
Sensitivity:
o Probability of a positive test result given the presence of disease
o How good is the test at detecting infection in those who have the disease?
o A sensitive test will rarely miss someone who has the disease (few false negatives)
o Screening tests should be highly sensitive (ex. VDRL for syphilis or ELISA for HIV) because they pick up all the true positives, as well as some false positives
Specificity:
o Probability of a negative test result given the absence of disease
o How good is the test at calling uninfected people negative?
o A specific test will rarely misclassify someone without the disease as infected (few false positives)
o Confirmatory tests should be highly specific (FTA-ABS fro syphilis or Western Blot for HIV) because they rule out the false positives
List 4 microscopic methods to diagnose infectious processes
- Gram stain
- Acid-fast stain (AKA Ziehl-Neelsen stain)
- KOH preparations
- Wet preparations
Gram Stain
Uses:
• If bacteria present in normally sterile fluids (CSF, urine)
• Which morphologic group is present, so gives empiric antibiotic selection
• Can be diagnostic for certain clinical specimens (Gram-negative diplococci inside PMNs from urethral swab in symptomatic male = gonorrhea)
Less useful when samples are from sites with normal microbiota (can’t tell pathogens from normal organisms)
o So it is important to know which organisms to expect in a site (and which would be considered pathogenic)
Acid-Fast Stain
(AKA Ziehl-Neelsen stain)
o To confirm a diagnosis of TB
o Identifies mycobacteria in sputum specimen
o Mycobacteria = have lipids and mycolic acids in cell walls that prevent decolorization
o Instead = retain initial red carbol-fuchsin stain (other bacteria are decolorized and pick up blue counterstain)
KOH preparations
o To look for yeast and other fungal forms
o Ex. Skin infections from fungal dermatophyte
Process:
• Skin scraping
• Place specimen on glass slide and treat with 10% KOH
• Dissolves host cells and bacteria but fungal elements remain
• Examine under microscope for yeast cells or hyphal forms
Wet preparations
o Rapid test for microorganisms
o Liquid sample (urine, vaginal secretions) placed on glass slide and examined
o Organism must be present in high numbers (>10^4 organisms/ml) to visualize
Describe the use of culture in identifying the etiologic agents causing infectious diseases, including their advantages and limitations
Able to describe: How many organisms • Few • Moderate • Heavy • Inoculate plates in standardized way to obtain individual colonies • Can make semi-quantitative estimate of amounts based on which quadrant growth occurs Size of colonies • Colony = progeny of one organism; used for identification and antibiotic susceptibility testing Pigment (yellow vs white) Concise vs swarming pattern Hemolysis (change in RBCs) Selective and differential media Enzyme testing Susceptibility testing
Describe the use of antigen detection in identifying the etiologic agents causing infectious diseases, including their advantages and limitations
Antigen Response tests
Advantages:
• Faster diagnosis (hours)
• Greater sensitivity
• Antigens or nucleic acids can be detected even after treatment with antibiotic and negative culture results
• Can identify organisms difficult/impossible to grow in culture
Disadvantages:
• Inability to do further tests
• Need to perform separate tests for each suspected pathogen
Direct Fluorescent antibody (DFA) test
• Test specimen with monoclonal antibody tagged with fluorescein
• See fluorescence under UV microscope
Molecular methods
Type of hemolysis
(change in RBCs)
• Alpha hemolysis: green color around colonies due to incomplete lysis of the sheep RBCs in media
• Beta hemolysis: clear area around colonies due to complete lysis of RBCs
• Gamma hemolysis: no change in media (no hemolysis)
Selective vs differential media in cultures
Selective media = used to inhibit growth of normal microbiota; selects for growth of groups of organisms
• MacConkey agar: has bile salts, inhibits Gram-positive cocci but allows Gram-negative rods to grow
• PEA or CNA plates: inhibit Gram-negative rods but allow Gram-positive cocci to grow; useful when trying to recover staph- or streptococci from specimens with Gram-negative rods
Differential media = distinguish groups of organisms based on specific biochemical reactions
• MacConkey agar: differentiates Gram-negative rods based on organism’s ability to ferment lactose → changes pH of media → colony turns pink
Enzyme testing in cultures
Catalase test: if has catalase, can break H2O2 → H2O + O2
• See bubbles on plate
• Quick/easy way to differentiate staph from strep
o All staph = catalase positive (bubbles)
o All strep = catalase negative (no bubbles)
Bacitracin Test: used to ID Group A beta-hemolytic streptococcus (streptococcus pyogenes)
• Paper disk with bacitracin placed on BAP inoculated with beta-strep
• If strep is inhibited by bacitracin (clear area around disk) = group A
• If no zone of inhibition = all other groups resistant to bacitracin
Types of susceptibility testing in cultures
Broth dilution
• Antibiotic is serially diluted
• Organism is added to each tube and incubated overnight
• Examine tubes for visible growth or turbidity
• Minimal Inhibitory concentration = lowest concentration of drug that inhibits growth
Microtiter plate dilution
• 96-well microtiter plate with freeze-dried dilutions of several antibiotics
• Inoculate with organism and incubate overnight
• Well with lowest concentration of each drug that inhibits visible growth = MIC
Disk diffusion (Kirby-Bauer)
• Paper disks with different antibiotics are placed on agar plate inoculated with organism
• Incubation
• Measure diameters of zones of inhibition
• Zone sizes and MIC values have been correlated with each drug
• Results reported: susceptible (sensitive), intermediate, or resistant to antibiotic
Agar dilution (E strip susceptibility test)
• Plastic strips with drug in descending gradient placed on inoculated plate
• Incubated
• Point where growth intersects strip = MIC
Automated systems
• Uses plastic cards that can test up to 22 antimicrobials at multiple concentrations
• Re-hydrate wells with organism
• Place cards in reader controlled by computer
• Reads optical density every 15 minutes
• Generates antimicrobial profile when sufficient growth is detected
• Can produced SAME DAY results (not need overnight incubation)
