Bacteriology Review Session

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BacteriologyReview Session
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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

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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?

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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)

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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)

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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

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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)

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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

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Memory refreshers
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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

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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

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Hemolysis
Alpha
Beta
Gamma
Comparison
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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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.

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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

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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

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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

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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

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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

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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

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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

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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

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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

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Other Enterobacteriaceae
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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

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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

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Clinical antimicrobial resistance

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

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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