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Bacteriology Review Session

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Title: Bacteriology Review Session


1
BacteriologyReview Session
2
Midterm Exam
  • Midterm Friday, October 13
  • 24 multiple choice questions (answer all)
  • Six true/false questions (answer all)
  • Answer two short answer questions (select from
    four)
  • Worth 100/400 points towards your final grade
  • There will be no make-up exams without a VERY
    good excuse and PRIOR notice

3
What you need to focus on
  • The general biology of the bacteria we studied in
    labs and clinical antimicrobial resistance
  • Review the summaries of each bacterial genus and
    the important points to remember slides
  • Know basic information about where the organisms
    are found (people/animals/environment), and
    location on the host (respiratory tract/gut/skin)
  • Is the organism a pathogen, normal flora or both?
  • What diseases does the organism cause?
  • You do NOT need to know details
  • What clinical material should you culture to
    isolate the organism?

4
What you need to focus on
  • How to grow the organisms
  • Is it fastidious?
  • Is it an anaerobe/microaerophile?
  • How to identify the organism
  • Review the methods used we used in the labs
  • You dont need to know the result for each
    organism for every possible test, but you need to
    know
  • How to perform the test (type of medium, reagents
    to add)
  • How the test works (pH change/growth)
  • Any special conditions that must be used/avoided
  • How to interpret the test (what is a
    positive/negative)

5
What you need to focus on
  • Identification of the organism
  • You should know key first stage tests for the
    genera we covered
  • Gram reaction, cell shape and arrangement
  • Aerobic/anaerobic growth
  • Catalase/oxidase
  • Spore formation
  • You should also known the results of organisms
    for key second stage tests
  • Hemolysis, lethicinase, mannitol salt agar, bile
    esculin agar, CAMP reaction, drug resistance,
    urease, X and V factors, lactose fermentation
    (MacConkey agar)

6
What you need to focus on
  • For antimicrobial resistance, you need to know in
    general terms
  • The mechanisms of antibiotic action
  • The mechanisms of antimicrobial resistance
  • The methods used to identify and measure
    antimicrobial resistance
  • Again, review the summary slides
  • I will not ask questions about specific
    antibiotics, such as their class or mode of
    action, etc...
  • Spelling counts make sure you know how to spell
    bacterial names

7
Things you do not need to focus on
  • You do NOT need to know any details about the
    pathogenesis of the organisms or the diseases
    they cause
  • However, you should know some general information
  • Clostridium tetani causes tetanus is not normal
    flora found in the soil
  • Corynebacterium diphtheriae causes diphtheria is
    not normal flora obligate human pathogen
  • Staphylococcus aureus causes food poisoning,
    abscesses is normal flora of the human
    nasopharynx (upper respiratory tract)

8
Things you do not need to focus on
  • I will not test you on any organism that the
    class (as a whole) did not study in the lab
    exercises
  • S. aureus, S. pyogenes, L. monocytogenes yes
  • C. ulcerans, S. hyicus no
  • For the Enterobacteriaceae, you need to
    concentrate on the big four
  • E. coli, Salmonella spp., Shigella spp., Yersinia
    spp.
  • You do not need to memorize any of the charts

9
Memory refreshers
10
Hemolysis
  • Hemolysis is measured on agar containing blood
    and is a result of lysis of the red blood cells
  • The most common medium used is sheep blood agar
  • There are two main types of hemolysis
  • Alpha hemolysis the zone around the colony is
    green
  • Beta hemolysis the zone around the colony is
    clear as a result of complete lysis of the red
    blood cells
  • Gamma hemolysis is actually NO hemolysis

11
Alpha hemolysis
  • This term is ONLY used for Streptococcus spp.
  • The term refers to a zone of green extending
    around the colony
  • It is due to reduction of the hemoglobin (red) to
    methemoglobin (green) in the medium surrounding
    the colony
  • Often referred to as partial or incomplete lysis
  • Brown or greenish discoloration under the colony,
    but NOT in a zone around the colony is not alpha
    hemolysis
  • This discoloration comes from non-specific damage
    of the blood cells due to bacterial growth, such
    as acid end products

12
Hemolysis
Alpha
Beta
Gamma
Comparison
13
Aerobic/anaerobic growth
  • Aerobe can grow in the presence of oxygen
  • Anaerobe cannot grow in the presence of oxygen
  • Obligate aerobe can only grow in the presence
    of oxygen
  • Obligate anaerobe can only grow in the absence
    of oxygen
  • Facultative anaerobe can grow in the presence
    or absence of oxygen

14
Aerobic/anaerobic growth
  • How much oxygen an organism prefers determines
    how we culture aerobes
  • More oxygen in air (regular incubator)
  • Less oxygen (microaerophile) candle jar
  • Anaerobic growth
  • No oxygen Brewers jar
  • There are two reasons to remember this
  • So that you grow your culture correctly
  • This is a characteristic that will help you
    identify the organism

15
Organism characteristics
  • I will test you on characteristics that are used
    to differentiate groups of organisms that have
    something in common
  • Share similar Gram stain characteristics
  • Gram positive cocci
  • Gram positive, spore-forming rods
  • Isolated from the same site on the host
  • Neisseria and Branhamella spp.
  • Staphylococcus and Streptococcus spp.

16
Staphylococcus spp.
  • Gram positive cocci in clusters (grapes)
  • Catalase positive
  • Grow well under anaerobic conditions
  • Grow on gt7.5 NaCl (all grow on MSA)
  • S. aureus is positive for coagulase usually
    hemolytic on SBA ferments mannitol on MSA
  • S. epidermidis does not produce coagulase, is
    non-hemolytic and does not ferment mannitol on
    MSA
  • S. saprophyticus does not produce coagulase and
    is resistant to novobiocin
  • S. aureus is normal flora of the moist skin and
    URT
  • S. epidermidis is normal flora of drier skin

17
Streptococcus spp.
  • Gram positive cocci in chains or pairs, not
    clusters
  • Catalase negative not salt tolerant
  • Some grow poorly under aerobic or anaerobic
    conditions grow best in a candle jar fastidious
  • Hemolysis is often used to identify the
    streptococci
  • S. pyogenes is beta hemolytic and bacitracin
    sensitive usually not normal flora of URT
  • S. agalactiae has variable hemolysis and is CAMP
    and hippurate positive normal vaginal flora
  • S. pneumoniae is alpha hemolytic and displays
    optochin sensitivity may be normal flora of the
    URT in some people

18
Enterococcus spp.
  • Gram positive cocci, often in pairs
  • Catalase negative
  • Grow well under anaerobic conditions grow well
    in ambient air
  • Are not fastidious (compare to streptococci)
  • Salt tolerant (compare to streptococci/staphylococ
    ci)
  • Hemolysis is variable (alpha or none)
  • Is not used in classification
  • Grow on bile and hydrolyze esculin
  • Normal flora of the gut cause nosocomial
    infections

19
Bacillus spp.
  • Large, spore-forming, Gram positive rods
  • Catalase positive, oxidase negative
  • Some species are obligate aerobes some are
    facultative anaerobes (never obligate anaerobes)
  • B. cereus is motile, hemolytic on SBA and
    produces lethicinase
  • B. subtilis displays variable motility and
    hemolysis, but is lethicinase negative
  • Enzymatic activity and sugar fermentation
    patterns will further distinguish the species
  • Found in the soil B. cereus causes food poisoning

20
Clostridium spp.
  • Large, spore-forming, Gram positive rods
  • Spore position can distinguish between species
  • Obligate anaerobes
  • Catalase negative, oxidase negative
  • C. perfringens is hemolytic (double zone),
    non-motile, lethicinase positive and can ferment
    sugars
  • C. tetani has characteristic terminal spores
    (tennis racquet), is hemolytic, motile,
    lethicinase negative and asaccharolytic (does not
    ferment any sugars)
  • Found in the soil C. perfringens also normal
    flora of the gut

21
Corynebacteria spp.
  • Small, pleomorphic, club-shaped rods often form
    palisades aka Chinese letters
  • Non-motile, non-sporeforming
  • Catalase positive, oxidase negative
  • Facultative anaerobes
  • These characteristics will distinguish this genus
    from most other Gram positive rods
  • C. diphtheriae is urease negative and nitrate
    positive not normal flora
  • Sugar fermentation patterns will distinguish
    species

22
Listeria spp.
  • Small, non-sporeforming, Gram positive rods
    (compare size with the spore-formers)
  • Motile at room temperature (25oC), less at 37oC
  • Catalase positive, oxidase negative
  • Facultative anaerobes grow at lt15oC
    (psychrophilic)
  • L. monocytogenes is hemolytic L. innocua is not
  • The CAMP test using S. aureus and R. equi can
    distinguish between the hemolytic Listeria spp.
  • Found in the environment feces associated with
    unprocessed food

23
Neisseria spp.
  • Gram-negative diplococci
  • Catalase positive, oxidase positive
  • Obligate aerobes, but grow best under reduced O2
    conditions candle jar
  • N. gonorrhoeae are fastidious and only grow on
    chocolate agar superoxol positive not normal
    flora
  • N. gonorrhoeae will grow on Modified Thayer
    Martin agar
  • Non-pathogenic Neisseria spp. (e.g., N. sicca) do
    not grow on MTM are negative with superoxol

24
Branhamella spp.
  • Gram-negative coccobacilli
  • Catalase positive, oxidase positive
  • Non-fastidious (grow on TSA)
  • DNase positive (compare with Neisseria spp.)
  • Normal flora, but can be confused with Neisseria
    spp.

25
Haemophilus spp.
  • Small, pleomorphic, Gram-negative coccobacilli
  • Oxidase positive
  • Facultative anaerobe, but prefers increased CO2
    levels for optimal growth (candle jar)
  • Fastidious, requires blood-containing media for
    growth (or chocolate agar for X and V factors)
  • H. influenzae requires both X and V factors not
    usually normal flora causes meningitis
  • H. parainfluenzae requires only V factor
  • Sugar fermentation patterns also differentiate
    species

26
Bordetella spp.
  • Small, Gram negative coccobacilli
  • Catalase positive variable oxidase
  • Obligate aerobes grow well in a candle jar
  • Asaccharolytic (do not ferment sugars)
  • Fastidious and require rich media for optimal
    growth
  • B. pertussis is nitrate and urease negative, and
    oxidase positive causes whooping cough
  • B. parapertussis is nitrate negative, urease
    positive and oxidase negative
  • Both of these species are found only in humans
    asymptomatic carriage possible

27
Pasteurella spp.
  • Small, non-motile, Gram negative coccobacilli or
    rods
  • Catalase positive, oxidase positive
  • Facultative anaerobes
  • P. multocida is non-hemolytic, does not grow on
    MacConkey agar
  • Ornithine decarboxylase, urease and sugar
    fermentations can further distinguish the species
  • Normal flora is some animals usually not found
    as normal flora in humans

28
Enterobacteriaceae
  • Members of the Enterobacteriaceae are
  • Gram negative rods
  • Oxidase negative
  • Facultative anaerobes
  • Ferment glucose
  • If motile, have peritrichous flagella
  • If it does have ALL these characteristics, it is
    NOT a member of the Enterobacteriaceae
  • The Enterobacteriaceae can be isolated from the
    gastrointestinal tract, and some are also found
    in the environment in water, soil and on
    vegetation

29
Non-Enterobacteriaceae
  • Many other bacteria are also associated with the
    gastrointestinal tract and/or may cause diarrhea
  • However, these genera are NOT members of the
    Enterobacteriaceae
  • Bacteroides obligate anaerobe
  • Campylobacter asaccharolytic (does not ferment
    glucose)
  • Clostridium Gram positive, obligate anaerobe
  • Enterococcus Gram positive cocci
  • Pseudomonas O/F- (oxidative respiration only)
  • Vibrio oxidase positive

30
Escherichia coli
  • Motile does not produces H2S citrate negative
  • Lactose fermenter
  • Pink (red) colonies on MacConkey agar
  • Metallic green colonies on EMB agar
  • Pink-orange colonies on HE agar
  • Pathotypes are identified by specific PCR tests
  • Normal flora of many mammals and birds

31
Shigella spp.
  • Non-motile does not produce H2S citrate
    negative
  • Lactose non-fermenter
  • Yellow/colorless colonies on MacConkey agar
  • Colorless colonies on EMB agar
  • Green colonies on HE agar (no black center)
  • Species are differentiated by sugar fermentations
  • Severe, bloody diarrhea is the hallmark of
    dysentery
  • Humans (and primates) are the only mammals
    infected

32
Salmonella spp.
  • Motile produces H2S citrate variable
  • Lactose non-fermenter
  • Yellow/colorless colonies on MacConkey agar
  • Colorless colonies on EMB agar
  • Blue-green colonies with black centers on HE agar
  • Serotyping at a reference lab must be performed
    if an absolute ID is required
  • Normal flora in birds and reptiles not usually
    found in uninfected people

33
Yersinia spp.
  • Unique bipolar (safety pin) staining
  • Y. enterocolitica is motile at 27oC, but not 37oC
  • Does not produce H2S citrate negative
  • Lactose non-fermenter
  • Yellow/colorless colonies on MacConkey agar
  • Does not grow well/at all on EMB agar
  • Does not grow well/at all on HE agar
  • Normal flora in animals not usually found in
    uninfected people

34
Other Enterobacteriaceae
35
Enterobacteriaceae
  • Not all Enterobacteriaceae are human commensals
  • Some are obligate, human, enteric pathogens
  • Shigella, Salmonella, Yersinia spp., toxigenic E.
    coli
  • Cause infections associated with diarrhea
  • Some are commensals in animals
  • Acquired via ingestion of fecally contaminated
    food or water
  • Many others cause nosocomial infections in either
    debilitated people or via contaminated IV
    solutions, wound contamination, catheterization,
    etc

36
Clinical antimicrobial resistance
  • Antimicrobial agents (antibiotics) are natural
    substances produced by soil microorganisms
  • Protect against nutritional competition by other
    bacteria
  • Antimicrobial agents specifically target
    bacterial structures and/or physiological process
  • Cell wall biosynthesis
  • Plasma membrane integrity
  • Ribosomal function (protein synthesis)
  • Nucleic acid synthesis
  • Metabolic processes

37
Clinical antimicrobial resistance
  • Antimicrobial resistance can be
  • Intrinsic
  • Phenotypic
  • Acquired by mutation
  • Acquired by DNA exchange
  • Improper use of antimicrobial agents selects for
    resistance

38
Clinical antimicrobial resistance
  • Susceptible and resistant breakpoints are based
    on the therapeutic concentration of the
    antibiotic AND the organism
  • Standardized methods improve test reliability
  • Methods for identification and measurement of
    antimicrobial resistance
  • Kirby-Bauer disk diffusion
  • MICs in broth or agar
  • E-test strips
  • Automated systems Vitek 2
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