Nonenteric, Gram negative bacteria of medical importance

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Non-enteric,Gram negative bacteria of medical
importance
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Neisseria and Branhamella spp.

• Neisseria spp. are Gram - cocci
• Seen in pairs (____________) with adjacent sides
  flattened

• Catalase positive, oxidase positive
• Obligate aerobes grow best in a ____________
• Pathogenic species are fastidious (will not grow
  on TSA), so biochemical tests should be performed
  in enriched media (enriched O/F tubes, CTA sugar
  broths)
• Branhamella (aka Moraxella) spp. are similar
• Technically rod shaped, but appear ____________
  in a Gram stain

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Habitat

• Neisseria spp. colonize or infect the
  ______________ _______________ of mammals
• Most Neisseria spp. are generally non-pathogenic,
  ______________________ of the mouth and throat
  (sometimes genital tract)
• They can cause disease as opportunistic pathogens
• N. meningitidis may colonize the mouth and throat
  in 10-25 of people, although causes
  meningitis
• Branhamella catarrhalis is an oxidase-positive,
  short, Gram negative rod, found as normal flora
  in the URT of humans
• Sometimes mistaken for ______________ spp.

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Disease - overview

• The only significant human pathogens are
• N. gonorrhoeae, the agent of gonorrhea
• N. meningitidis, a cause of acute bacterial
  meningitis
• N. gonorrhoeae infections have a high prevalence
  and ____________ mortality
• In contrast, N. meningitidis infections have a
  low prevalence and ____________ mortality
• Both species initially infect via the
  ____________ ______________ (conjunctiva, URT,
  genital tract)

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N. gonorrhoeae

• Aka gonococcus/gonococci/GC
• N. gonorrhoeae is spread almost exclusively by
  sexual contact
• Not toilet seats, towels, etc...
• The organisms do not survive drying and so
  _________________ contact is required for spread
• Gonococci infect the mucous membranes causing
  ________ or disseminated infections

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Gonorrhea

• Localized infections of the mucous membranes
• Urethritis and vaginitis typical gonorrhea
• ___________ will develop symptoms within 5 days,
  in contrast to only ______________________________
  
• Cervicitis, proctitis, pharyngitis, and
  conjunctivitis (neonatal infection occurs during
  birth)
• Disseminated infections occur either by
• Extension to adjacent organs pelvic
  inflammatory disease (in 10-15 of women),
  epididymitis
• Bacteremic spread skin lesions, septic
  arthritis, endocarditis, and meningitis

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Diagnosis of gonorrhea

• Gonorrhea cannot be diagnosed only on clinical
  grounds
• Men
• Gram stained smear of urethral exudate showing
  _________________, Gram negative diplococci is
  diagnostic
• While the Gram stain has a sensitivity of 95 for
  men, the sensitivity is only 50 for women
• Women, and men where a direct smear is not
  definitive
• Culturing on ______________ MTM containing
  antibiotics
• N. gonorrhoeae must be differentiated from other
  Neisseria spp.

Gonococci inside PMNs (neutrophils)
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N. meningitidis

• Identical cell morphology to N.
  gonorrhoeae

• Meningococci can be carried __________________ in
  the nasopharynx (10-25 of people) often
  transient
• Provides a reservoir for infection enhances host
  immunity
• Disease is more common in infants 3-12 months old
• ______________ occurs both sporadically (mainly
  groups B and C) and in epidemics (mainly group A)
  

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Meningitis

• Transmission is by respiratory droplets
• Bacteria attach to host cells, cross the mucosal
  barrier, and enter the _____________________
• Blood-borne bacteria enter the _______________
• Infection with N. meningitidis has two
  presentations
• Meningococcemia, characterized by skin lesions
• Meningitis (inflammation of the membranes
  covering the brain and spinal cord )
• Untreated, either presentation has a high
  mortality
• Diagnosis is by identifying N. meningitidis in
  blood, and ___________________________ (CSF)

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Classification of the Neisseria spp.

• Gram-negative diplococci, 0.6-1.0µm in diameter
• Catalase positive, ______________________
• Obligate aerobes, but grow best under reduced O2
  conditions candle jar
• N. gonorrhoeae grow on rich media, including MTM,
  but ___________________________ superoxol
  positive
• N. meningitidis has similar characteristics to
  N. gonorrhoeae, but can grow on TSA
  can also be differentiated with CTA sugar
  fermentations
• Branhamella spp. are similar not as fastidious
  and are ___________________________

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Haemophilus spp.

• Haemophilus spp. are small,
  ______________, Gram-negative
  coccobacilli
• Variable catalase, oxidase positive
• Facultative anaerobe, but prefers
  increased CO2 levels for optimal growth
• _____________________ and requires rich,
  blood-containing media for growth
• Some species require X and/or V factors which can
  be obtained from ______________________

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Habitat

• Many Haemophilus spp. are _________________ in
  humans and animals
• Human pathogens - H. influenzae, H.
  parainfluenzae
• There are a number of __________________ usually
  not associated with disease
• These are also found in the URT
• Haemophili are usually not found in the
  _______________________ and organisms spread
  directly among individuals

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H. influenzae

• First isolated in the 1889 influenza pandemic
• Thought to be the cause of influenza, hence the
  name
• The organism is typed into 6 types (a-f) on the
  basis of ______________________________
• H. influenzae ____________ was responsible for
  most of the serious diseases
• Main cause of meningitis in children between 6
  months and 5 years
• Occurs in 1-2 of healthy children, and these
  children act as a reservoir of infection
• Effective vaccine - type b capsular antigens

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H. influenzae disease

• Spread by respiratory droplets
• H. influenzae crosses the blood-brain barrier
• Resulting in meningitis may be fatal
• However, type b disease incidence is reduced due
  to _______________________
• H. influenzae of other types may still cause
  disease (more rarely)
• Non-typable strains of H. influenzae are the
  second most common cause of _____________________
• _______________________ is based on blood or
  cerebrospinal fluid (CSF) cultures

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Other Haemophilus spp. disease

• H. parainfluenzae
• Causes pneumonia or bacterial endocarditis
• H. ducreyi
• Cause of sexually transmitted genital ulcers
  aka chancroid
• H. aegyptius
• Causes a serious pediatric disease - Brazilian
  purpuric fever

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Classification of Haemophilus spp.

• Small, pleomorphic, Gram-negative coccobacilli
• Oxidase positive
• Prefers increased CO2 for optimal growth
• _________________ and requires rich,
  blood-containing media for growth
• H. influenzae requires ________________ factors
  catalase positive non-hemolytic
• H. haemolyticus requires X and V factors
  catalase positive hemolytic
• H. parainfluenzae requires only V factor
  catalase variable
• Sugar fermentation patterns will also
  differentiate the species

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Bordetella spp.

• Small (0.4x0.8µm), Gram negative coccobacillus
• Catalase positive, oxidase variable
• Obligate ________________
• Asaccharolytic
• ________________ (may take
  3-4 days to form colonies)
• Therefore, rich media containing blood are used
  for isolation (Bordet-Gengou medium)

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Habitat

• B. pertussis and B. parapertussis have only been
  isolated from the ___________ respiratory tract
• Carriage by healthy people has been observed, but
  these asymptomatic carriers do not appear to
  spread the disease
• B. bronchiseptica is carried by ____________
  ____________, including dogs, cats, guinea pigs,
  rabbits and swine

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Disease - overview

• The three common species of Bordetella, B.
  pertussis, B. parapertussis and B.
  bronchiseptica are respiratory pathogens of
  mammals
• These organisms do not survive well outside the
  host
• _____________ is almost always directly from
  person to person via respiratory droplets
  containing bacteria

• The bacteria colonize only ciliated cells of the
  respiratory mucosa
• Bordetella spp. cause disease by producing
  ______________ that impair ciliary function in
  the respiratory tract

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Whooping cough

• B. pertussis causes _________________ aka
  pertussis
• Incubation period lasts 1-2 weeks
• Catarrhal phase lasts 1-2 weeks
• Characterized by progressive cough
• Paroxysmal phase lasts 2-4 weeks
• Characterized by severe, _________________ cough
  episodes
• Convalescent phase lasts 3-12 weeks
• Continuous decline of the cough before the
  patient returns to normal
• Serious, sometimes fatal complications
• Effective vaccine whole killed cells

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Other Bordetella spp.

• B. parapertussis
• Causes a milder form of pertussis
• B. bronchiseptica
• Causes respiratory disease in animals
• - Kennel cough in dogs
• Generally ____________________, although it can
  cause disease in severely immuno-compromised
  people (after close animal contact)

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Classification of Bordetella spp.

• Small (0.4x0.8µm), Gram negative ____________
• Catalase positive, ________________________
• Do not ferment carbohydrates ______________
• Slow growing (3-4 days)
• B. pertussis is nitrate and urease negative, and
  oxidase positive
• B. parapertussis is nitrate negative, urease
  positive and oxidase negative
• B. bronchiseptica is nitrate, urease and oxidase
  positive

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Pasteurella spp.

• Small (0.3-1.0x1.5?m), _______________, Gram
  negative coccobacilli or rods
• Catalase positive, oxidase positive
• Facultative anaerobes
• O/F
• Most species do not grow on
  MacConkey agar
• Compare with enteric bacteria
• P. multocida produces a __________
• Mucoid colonies

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Habitat

• Pasteurella spp. can cause disease in many animal
  species, either as primary or _________________
  pathogens
• Some Pasteurella spp. are commensals in the
  nasopharynxes of dogs, cats and birds
• Humans usually become infected by handling
  ____________ either infected livestock or from
  a dog or cat bite
• Pasteurella spp. are not commensal in humans
• It is unknown if Pasteurella spp. live in the
  environment

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P. multocida

• The most widely studied member of the genus
• Four ____________ types (A, B, D, and E)
• Capsular serotypes are specific to disease
• A1, A3 and A4 causes fowl cholera in
  chickens
• B2 and E2 cause hemorrhagic septicemia in cattle
• A causes bronchopneumonia in feedlot cattle
• D causes atrophic rhinitis in pigs
• Human disease usually involves abscesses and/or
  septicemia (often from animal bites)
• __________ is by ___________ of the infected site

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Other Pasteurella spp.

• P. hemolytica (now Mannheimia hemolytica)
• Commensals of the URT and digestive tracts of
  cattle, sheep, chickens and turkeys
• Also a cause of pneumonia (shipping fever) in
  feedlot cattle sometimes P. multocida can be
  involved

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Classification of Pasteurella spp.

• Small (0.3-1.0x1.5?m), ________________, Gram
  negative coccobacilli or rods
• Catalase positive, ____________________
• Facultative anaerobes
• P. multocida is _____________, does not grow on
  MacConkey agar
• P. hemolytica is weakly hemolytic and grows on
  MacConkey agar
• Ornithine decarboxylase, urease and sugar
  fermentations can further distinguish the species

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Tests for entericGram negative rods
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Selective and differential media

• There are a number of media that have developed
  for selection and _____________ of enteric
  bacteria
• MacConkey (MAC) Agar
• Hektoen Enteric (HE) Agar
• Eosin Methylene Blue (EMB) Agar
• Most of these are designed to inhibit ________
  ____________ organisms (selective) and use sugar
  fermentation and H2S production to differentiate
  between bacteria (differential)

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MacConkey Agar

• Inhibits the growth of Gram positive bacteria
  with bile salts
• _____________ and neutral red, a pH indicator,
  are added to differentiate the lactose positive
  coliforms from the potentially pathogenic lactose
  non-fermenters
• When lactose is fermented, acidic end products
  lower the pH, and the resulting colonial growth
  is _____________
• If the organism does not ferment lactose, the pH
  will not decrease, and the colonies will be
  _____________

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Hektoen Enteric (HE) Agar

• Hektoen Enteric Agar is a moderately ____________
  and differential medium used for the isolation of
  Salmonella and Shigella spp.
• This medium contains ________ to inhibit Gram
  positive organisms and non-enteric Gram negative
  bacteria
• The medium contains lactose, sucrose, and salacin
  and the dyes bromthymol blue and acid fuchsin,
  which allows differentiation due to the colony
  and medium colors
• The medium can also be used to detect H2S
  production due to the addition of sodium
  thiosulfate and ferric ammonium citrate

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Hektoen Enteric (HE) Agar

• Gram negative, lactose fermenters produce yellow
  to salmon-pink colonies (e.g., Enterobacter
  aerogenes and E. coli)
• Salmonella spp. (lactose non-fermenter) produce
  blue-green colonies, which may have a _____
  coloration or black center due to
  __________________
• Shigella spp. (lactose non-fermenter) colonies
  are raised, green, and moist

E. coli
Enterobacter spp.
Salmonella spp.
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Eosin Methylene Blue Agar

• Selective and differential agar for members of
  the Enterobacteriaceae
• Contains methylene blue and eosin yellow dyes to
  inhibit the growth of Gram positive bacteria, and
  lactose
• Lactose fermentation produces acid
• Most lactose fermenters have pink colonies
• E. coli forms a methylene blue eosinate complex
  which has __________________
• Organisms which do not ferment lactose will be
  colorless, taking on the color of the medium

Enterobacter cloacae
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Triple sugar iron (TSI) slant

• Differentiates enteric bacteria on the ability to
  ferment carbohydrates and produce
  ____________________
• The medium contains peptone, lactose, sucrose, a
  small amount of glucose, ferrous sulfate, and
  phenol red
• If an organism can ferment any of the three
  sugars present, all the medium (slant and butt)
  will turn __________ (acid)
• If an organism can only ferment glucose, the
  small amount of glucose in the medium is used in
  the first 10 hours
• Then peptone utilization produces ammonia, that
  turns the pH in the aerobic area of the slant
  ___________ (alkaline)
• The butt (more anaerobic), will not revert to an
  alkaline state, and will remain yellow (e.g.,
  Salmonella and Shigella)

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Triple sugar iron (TSI) slant

• If sulfate is reduced, H2S is produced which
  reacts with iron to form iron sulfide which is
  black
• The black color may mask the red/yellow changes
• However, H2S production can only occur in an
  acidic environment (some fermentation must have
  occurred)
• To interpret the TSI test, four parameters are
  observed

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Triple sugar iron (TSI) slant

• Read the test as slant color/ butt color/gas
  production/ H2S production
• A Uninoculated B No change/alkaline/-/- C
  Alkaline/acid// D Acid/acid/-/ E
  Acid/acid//-

A B C D E
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ONPG test

• Lactose utilization requires a several enzymes,
  including beta-galactosidase
• Ortho-nitrophenyl-ß-D-galactopyranoside (ONPG) is
  an analog of lactose that can be cleaved by
  beta-galactosidase
• ONPG is ___________, but one of the cleavage
  products, o-nitrophenol is ________________
• To perform this test, the organism must be grown
  in the presence of lactose, to induce
  beta-galactosidase expression

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ONPG test

• The organism to be tested is inoculated into
  0.2ml of saline
• An ONPG disk is aseptically added and the culture
  incubated at 37oC for up to 4 hours
• Any yellow color on the disk or the saline is
  positive for the presence of beta-galactosidase,
  which indicates the ability to ______________

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Indole production

• Tryptophan can be metabolized by the enzyme
  tryptophanase
• Only a few bacteria express tryptophanase, so
  indole production is a useful diagnostic tool
• When tryptophan is broken down, __________ is
  produced, along with pyruvic acid and ammonia
• Indole is detected with _____________ reagent
  (para-dimethylaminobenzaldehyde in alcohol)

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Indole production

• The yellowish Kovacs reagent reacts with indole
  in the presence of O2, producing a _________
  color on the surface
• This test can be performed in broth or in an MOI
  agar deep
• Tests for Motility, Ornithine decarboxylase and
  Indole

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Methyl red (MR) and V-P tests

• Used to identify bacteria that produce stable
  acid end products by means of mixed acid
  fermentation of glucose
• The medium used for this test contains peptone,
  glucose, and a buffer
• Following inoculation, the culture is incubated
  for _______
• The sample is split into two tubes (two broths
  can be used)
• One is for the MR test, the other is for the V-P
  test
• For the MR test, the pH indicator methyl red is
  added
• A red color is read as positive (pH lt4.4)
• A yellow color (pH gt6.0) is ___________________
• An orange color (ph 4.5-6.0) usually requires
  further incubation

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Methyl red (MR) and V-P tests

• Methyl red is added to the culture to detect a
  _____________ change
• Acid (red) is positive
• Neutral (yellow) is negative
• Orange (pH 4.5-6.0) usually means that the
  bacteria needed a longer incubation time to
  complete the reaction (presumptive ________)

Uninoculated
Negative
Positive
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Voges-Proskauer (________) test

• This test determines the ability of organisms to
  produce acetoin as an end product from glucose
  fermentation
• The growth medium contains peptone, glucose and a
  buffer
• As acetoin is produced only at the end stages of
  fermentation, after inoculation, the culture
  should be incubated for at least 48 hours
• Reagents are added to detect the acetoin produced
  (5 a-naphthol and 40 KOH)
• The reaction should be checked after 15 and 60
  minutes

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Voges-Proskauer (V-P) test

• Following incubation, add 12 drops of
  a-naphthol and 6 drops of KOH
• If a ________ is formed, acetoin in detected,
  which is a positive result
• In a _________ reaction, the reagents will change
  the broth to a darker yellow

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Simmons citrate agar

• Bacteria that utilize _________ as their sole
  source of carbon for growth and metabolism also
  utilize ammonium salts as their sole nitrogen
  source
• In this test we actual measure ammonium
  dihydrogen phosphate utilization
• The medium contains citrate, ammonium dihydrogen
  phosphate and bromthymol blue
• The ammonium salts are broken down to
  ____________, an alkaline product that turns the
  pH indicator a ___________ color
• Due to the lack of nutrients, organisms which do
  not utilize citrate may not grow well on this
  medium

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Simmons citrate agar

• Uninoculated Simmons citrate agar has a neutral
  pH, so is a green color
• Ammonium dihydrogen phosphate utilization (which
  coincides with citrate utilization) resulting in
  release of ammonia, changing the indicator to
  Prussian blue (positive)
• If ammonium dihydrogen phosphate is not utilized,
  
• The pH indictor remains ______ or green-yellow
  (negative)

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Phenylalanine deaminase test

• This test medium is used to identify bacteria
  possessing the enzyme phenylalanine deaminase
• It is used to distinguish _____________,
  Morganella and Providencia spp. (positive) from
  other enteric bacteria (negative)
• The medium contains the amino acid ____________
• Phenylalanine deaminase removes the amine group
  (NH2) from phenylalanine, releasing free
  ____________ (NH3) and phenylpyruvic acid
• The phenylpyruvic acid can be detected by adding
  an oxidizing reagent such as ferric chloride to
  the incubated tube

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Phenylalanine deaminase test

• Add 5 drops of 10 ferric chloride to the tube,
  allowing it to run over the slant
• If phenylpyruvic acid is present, a _____ color
  will be detected (positive)
• A yellow color (no color change) is a _________
  result
• The green color fades, so the test should be read
  ____________ (less than 1-2 minutes)

Negative no color change Positive green color