Oxidative GramNegative Fermenters

1
Oxidative Gram-Negative Fermenters

• Dr. John R. Warren
• Department of Pathology
• Northwestern University
• Feinberg School of Medicine
• June 2007

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Characteristics of the Fermentative Oxidative
Gram-Negative Bacilli

• Fermentative for glucose (acidification of the
  deep in triple sugar iron agar slants)
• Positive cytochrome oxidase

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Most Important Fermentative Oxidative
Gram-Negative Bacilli

• Vibrio cholerae
• (family Vibrionaceae)
• Aeromonas hydrophila
• (family Aeromonadaceae)
• Plesiomonas shigelloides
• (family Enterobacteriacae1)
• 1Oxidative-positive P. shigelloides clusters with
  oxidative-negative Proteus by 16S rRNA sequence,
  and the family Enterobacteriaceae is undergoing
  revision to include cytochrome-oxidase positive
  organisms.

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Vibrio cholerae Natural Habitats

• Brackish water, marine and coastal environments
• Associated with mollusks and crustaceans
• Freshwater lakes and rivers, birds, and herbivors
  geographically distant from coastal regions
  (density of V. cholerae decreases with water
  temperatures below 20oC)

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Vibrio cholerae Modes of Infection

• Ingestion of contaminated water and food
  (especially shell fish)
• Human carriers are rare

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Vibrio cholerae Types of Infectious Disease

• Secretory diarrhea with rice water stools and
  dehydration
• 60-75 of individuals with small intestinal
  colonization are without diarrhea

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Pathophysiology of Cholera

• Small intestinal colonization by large numbers of
  Vibrio cholerae
• Bacterial mucinase allows penetration of cholera
  bacilli of small intestinal mucin with attachment
  to epithelial surface
• Adherence of cholera bacilli to intestinal
  epithelial cells by Tcp (Toxin Co-regulated) pili

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Pathophysiology of Cholera

• Synthesis and secretion by attached bacilli of
  cholera enterotoxin, an A-B exotoxin with
  adenosine diphospho-
• ribosylating activity
• Stimulation by cholera enterotoxin of mucosal
  adenylate cyclase activity with increased cAMP
  inducing hypersecretion of chloride, potassium,
  and bicarbonate

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Pathophysiology of Cholera

• Obligatory loss of water due to Cl, K, and
  HCO3 hypersecretion causes a watery diarrhea
• Somatic antigens O1 and O139 are associated with
  enterotoxin production and serve as markers for
  strains of Vibrio cholerae causing epidemic and
  endemic outbreaks of cholera (other 150
  serogroups usually non-toxigenic)

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Cholera

• V. cholerae serogroup O1 has two biotypes El Tor
  and classical
• El Tor responsible for virtually all cases of
  cholera in the world, classical cases only in
  Bangladesh
• El Tor Voges-Proskauer , ß-hemolytic, inhibited
  by polymyxin B
• Classical serogroup Voges-Proskauer ,
  non-hemolytic, not inhibited by polymyxin B
• V. cholerae serogroup 139 in 1992 caused cholera
  epidemic in Madras, India, since then reported in
  11 countries

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Other Infectious Diseases Due to Vibrio

• Non-01 V. cholerae, V. mimicus self-limiting
  gastroenteritis, sepsis, wound infection
• V. parahaemolyticus gastroenteritis following
  consumption of contaminated shellfish (most
  common species in US)
• V. vulnificus bacteremia and wound infection
  following consumption or handling of contaminated
  shellfish in cirrhosis (high mortality)
• V. alginolyticus wound and ear infections
  following exposure to sea water

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Vibrio cholerae Microbiological Properties

• Curved or comma-shaped gram-negative rods
  (Kommabacillus)
• Fermentation of glucose and sucrose (acid/acid
  reaction with TSI)
• Growth on 5 sheep blood and chocolate agar in
  carbon dioxide or ambient air
• Growth on MacConkey and eosin methylene blue agar
  as a non-lactose fermenter

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Thiosulfate Citrate Bile Salt Sucrose (TCBS) Agar

• TCBS agar selective for Vibrio by presence of
  bile salts (B) inhibitory for gram-positive
  organisms, and an alkaline pH (8.6) that enhances
  recovery of Vibrio
• TCBS agar differential for Vibrio by presence of
  sodium thiosulfate (T) and ferric citrate (C) for
  H2S production (Vibrio negative for H2S) and
  sucrose for Vibrio fermentation

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Reaction of Vibrio Species on TCBS Agar

• The pH indicators bromthymol blue and thymol blue
  turn yellow at an acid pH
• Sucrose fermenters form yellow colonies on TCBS
  agar Vibrio cholerae, V. alginolyticus, V.
  vulfinicus (variable)
• Sucrose non-fermenters form green colonies
  Vibrio parahaemolyticus, V. mimicus

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Identification of Vibrio Species

• Growth in Group 11 Group 62
• No NaCl
• 1 NaCl
  
• 1V. cholerae and V. mimicus
• 2V. parahaemolyticus, V. vulnificus, V.
  alginolyticus

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Identification of Group 1 Vibrio Species1

• Sucrose VP
• V. cholerae2
• V. mimicus
• 1V. cholerae and V. mimicus only two members
• 2Serogrouping with O1 and O139 antiserum

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Identification of Group 6 Vibrio Species1

• Suc Cit ONPG VP Cellobiose
• V. para.2
  
• V. vulnif. v v v
  
• V. algin.
  
• 1Supplementation of broth with 1 NaCl
  (halophilic species) V. para.V.
  parahaemolyticus, V. vulnif.V. vulnificus, V.
  algin.V. alginolyticus. Group 6
  characteristically cytochrome oxidase , nitrate
  , inositol , arginine , lysine
• 280 arabinose (V. vulfinicus 0 , V.
  alginolyticus 1 )

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Aeromonas hydrophila Natural Habitats

• Fresh and polluted water, and confluent
  interfaces of fresh with salt water
• Associated with warm and cold blooded animals
  (frogs, fish, reptiles, and humans)

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Aeromonas hydrophila Modes of Infection

• Exposure of disrupted skin or mucosa to
  contaminated water
• Ingestion of contaminated food or water

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Aeromonas hydrophila Types of Infectious Disease

• Secretory diarrhea (stool cultures most common
  source of A. hydrophila)
• Wound cellulitis (traumatic exposure to
  contaminated water and soil, especially lower
  extremities) (wound cultures second most common
  source of A. hydrophila)
• Bacteremia (associated with hepatobiliary disease)

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Aeromonas hydrophila Microbiological Properties

• Straight gram-negative rods
• Fermentation of glucose and sucrose (acid/acid
  reaction with TSI)
• Growth on MacConkey and eosin methylene blue
  agars as a non-lactose fermenter
• Growth on 5 sheep blood or chocolate agar in
  carbon dioxide or ambient air

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Aeromonas hydrophila Microbiological Properties

• ?-hemolysis on sheep blood agar
• Positive for Voges-Proskauer, esculin hydrolysis,
  arabinose, and mannitol, negative for ornithine
  and inositol (opposite to results with
  Plesiomonas shigelloides)

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Aeromonas Species Identification

• ßhem VP Esc Ara Lac Lys
• A. hydrophila
  
• A. veronii1
  /3
• A. caviae2
  
• 1Causes traumatic wound infection.
• 2Associated with watery diarrhea.
• 3Biovariant sobria , biovariant veronii .

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Plesiomonas shigelloides Natural Habitats

• Warm fresh water especially in tropical countries
• Wide range of animal hosts including fish,
  shellfish, toads, snakes, monkeys, dogs, cats,
  goats, pigs, poultry, and cattle

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Plesiomonas shigelloides Modes of Infection

• Ingestion of contaminated water, raw seafood, or
  reptiles

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Plesiomonas shigelloides Types of Infectious
Disease

• Mild watery or dysentery-like diarrhea
• Bacteremia (rare)

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Plesiomonas shigelloides Microbiological
Properties

• Straight gram-negative rods
• Fermentation of glucose, no fermentation of
  lactose or sucrose (alkaline/acid with TSI slant,
  growth on MacConkey and eosin methylene blue
  agars as non-lactose fermenter)
• Growth on 5 sheep blood and chocolate agar in
  carbon dioxide or ambient air

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Plesiomonas shigelloides Microbiological
Properties

• No hemolysis on sheep blood agar
• Positive for lysine, ornithine, and arginine (a
  combination not found in any Vibrio or Aeromonas
  species)
• Positive for ornithine and inositol, negative for
  Voges-Proskauer, esculin hydrolysis, arabinose,
  and mannitol (opposite to results with Aeromonas
  hydrophila)

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Vibrio cholerae, Aeromonas hydrophila,
Plesiomonas shigelloides

• Lys Orn Arg Ino TCBS1 O/1292
• V. chol.3
  
• A. hydr.3
  
• P. shig.
  
• 1Growth on TCBS agar.
• 2Susceptibility of growth to the vibriocidal
  compound O/129
• 3El Tor biotype of V. cholerae O1 and Aeromonas
  hydrophila ß-hemolytic on sheep blood agar

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Recommended Reading

• Winn, W., Jr., Allen, S., Janda, W.,
• Koneman, E., Procop, G.., Schreckenberger,
• P., Woods, G.
• Konemans Color Atlas and Textbook of
• Diagnostic Microbiology, Sixth Edition,
• Lippincott Williams Wilkins, 2006
• Chapter 8. Curved Gram-Negative Bacilli and
  Oxidase-Positive Fermenters Campylobacteraceae
  and Vibrionaceae.
• Part II The Families Vibrionaceae and
  Aeromonadaceae

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Recommended Reading

• Murray, P., Baron, E., Jorgensen, J., Landry,
• M. Pfaller, M.
• Manual of Clinical Microbiology, 9th
• Edition, ASM Press, 2007
• Abbott, S.L. Chapter 45. Klebsiella,
  Enterobacter, Citrobacter, Serratia, Plesiomonas,
  and Other Enterobacteriaceae
• Horneman, A.J., Ali, A., and Abbott, S.L.
  Chapter 46. Aeromonas.
• Abbott, S.L., Janda, J.M., Johnson, J.A., and
  Farmer, J.J., III. Chapter 47. Vibrio and
  Related Organisms.