GRAM NEGATIVE RODS

 Gram negative rods

 3 genera:
o Vibrio
o Aeromonas
o Pseudomonas

Genus Vibrio
 General Characteristics
o Gram-negative, comma-shaped bacilli
o Non-spore forming, non-capsulated
o Metabolism is both respiratory and fermentative
o Oxidase (+)
o Motile – single polar flagellum
 Species
o V. Cholera
o V. Parahaemolyticus

Vibrio cholerae
General characteristics  Komma bacillus
 Most important human pathogen
 Short gram-negative rod (0.5u x 1.5u to 3.0u)
 Curved/comma-shaped
 On stained smear, appear characteristically lying parallel to one another (fish in the stream
arrangement)
 Motile – single, thick polar flagellum
o Scintillating or darting motility
Antigenic Structure O / Somatic Antigen
 Major Ag for serologic typing of V. cholerae

H / Flagellar Antigen
 Most commonly shared by all vibrios

2 biotypes
El Tor V. cholerae
Voges-Proskawer + -
Agglutination to chicken RBC + _
Sensitivity to 50u Polymyxin B S S
Chlorephage sensitivity S S
 Both biotypes are responsible for classic epidemic cholera and are divided into 3 antigenic
subtypes
o Ogawa – A,B
o Inaba – A, C
o Hikojima – A, B, C

1st Group
 V. Cholerae O1 – Classic epidemic cholera
 Caused by organisms that agglutinate in the antisera
 Directed against the O1 antigen and produce disease primary by means of a specific enterotoxin
o O1 antigen is present in the lipopolysaccharide layer of most organisms.
o It is specifically found on Lipid A, as an O side chain
o Specific enterotoxin – determinant of pathogenicity

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 2nd Group
 Atypical or nontoxigenic V. cholerae O1
 Biochemically similar to the O1 organisms and type with O1 antisera
 Do not produce enterotoxin

3rd Group
 Non-O1 V. Cholera
 Does not agglutinate in O1 antisera
 Referred to as nonagglutinating vibrios (NAG) or noncholera vibrios (NCV)
Determinants of Pathogenicity Cholera Enterotoxin
 Major pathogenic factor that act on the cells of the SI
 Heat labile
 2 major subunits
o Subunit A – responsible for the biologic activity of the organism

Stimulate adenylyl cyclase activity of the epithelial cell,

converting ATP to cAMP
↓
Overproduction of intracellular cAMP
↓
Hypersecretion of H2O and Cl
↓
Inhibition of Na+ absorption
↓
Watery diarrhea – dehydration leading to hemoconcentration, anuria
and hypovolemic shock d/t massive loss of water and electrolytes

o Subunit B – responsible for the cellular binding of the toxin to the intestinal
epithelial cell (brush border)

Invasiveness of the Organism

 Ability to adhere/penetrate to the intestinal mucosa and attached to the microvilli of the brush
border of epithelial cells
Biochemical Characteristics  Facultative anaerobe
o Metabolism is both respiratory and fermentative
 Oxidase (+), Urease (-), H2S (-)
o Possesses lysine and ornithine decarboxylase , and reduces nitrate to nitrite
 Non-lactose fermenter, but ferments Sucrose, Maltose, Glucose
 Grows best at alkaline pH (extremely basophilic)
 Extremely sensitive to acidic pH → kills organisms
 Susceptible to heat, drying and to common chemical disinfectants

V. cholerae V. parahaemolyticus
Oxidase + +
Urease -
H2S -
Lactose - _
Sucrose + _
Maltose +
Glucose +
Growth on 2 % NaCl - +

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 Culture Characteristics  Optimum growth temperature is 18—37°C
 Grows best on simple ordinary culture media

Meat Extract Agar (MEA)

 Translucent colony with iridescent green → red brown color viewed on oblique light

Selective Differential CM – colonies from fecal matter

 Tellurite Taurocholate Gelatin Agar (TTGA)
o Gray, flattened opaque zone around colony
 Thiosulfate Citrate Bile Sucrose (TCBS)
o Large, smooth, flat yellow colonies with opaque center and transparent periphery
 MacConkey’s Agar
o Most strains grow luxuriantly
o On short incubation – colorless colonies
o Prolonged incubation – pink colored colonies, opaque and ruggated stain with age
Clinical Features Cholera
 Food and waterborne disease
 Human carrier serves as source of new cases of cholera
 Man only host infection
 Acquired fecal – oral route (contaminated food/H2O)
 Incubation period 2-3 days
 Serious disease characterized by:
o Rice water stool – sudden onset of voluminous, non-bloody, non odorous watery diarrhea
containing flakes of mucous; hallmark of the disease
 Fluid loss – severe 13-20 L/day
 Patient severely dehydrated:
o Eyes – sunken, lips – dry and crackling
o Skin – loss turgor, hands – washerwoman
 No fever, in untreated cases, 60% die
 High attack rate in children
 Organism is non – invasive (remains localized GIT)
Laboratory Diagnosis Direct Microscopy of stool specimen
 Observe for comma-shaped bacilli
 Inoculation into Alkaline Peptone Broth and incubated for 6 hours and examined under darkfield
microscopy to demonstrate darting motility

Stool Culture – MEA/TCBS incubated for 2—3 days

Direct Fluorescent Antibody Test

Treatment & Prevention  Prompt replacement of fluid and electrolytes
 Tetracycline – DOC
 Doxycycline
 If resistant: use other IV regimens
 Prevention – Sanitation
o Maintenance of adequate sewage treatment
o H2O purification
o Prompt detection and treatment of cases
o Cholera vaccine – no significant protection

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Vibrio parahaemolyticus
General characteristics  Marine or saltwater organism
 Found in estuaries throughout the world
 Major cause of gastroenteritis involving seafood
 Closely related to V. cholerae
Determinants of Pathogenicity Kanagawa Phenomenon
 Hemolysis on Wagatsuma agar is used to identify pathogenic strains
 Most strains produce diarrhea due to heat stable hemolysin (Kanagawa hemolysin) which is both
cytotoxic and cardiotoxic
 Can lyse human RBS – Kanagawa Hemolysis Test
Biochemical Characteristics  Extremely halophilic
 Oxidase (+)
 Non-lactose and non-sucrose fermenter
Culture Characteristics  TCBS - Large, smooth, green colonies
Clinical Features Gastroenteritis / Foodpoisoning
 Self-limiting disease acquired through the ingestion of raw or undercooked seafood (shellfish,
oysters)
 Incubation period 12-24 hours
 Clinical manifestations
o NAV
o Explosive watery diarrhea (without blood and mucus)
o Headache
o Fever (not common)
o Clinical manifestations last for 3—5 days
Laboratory Diagnosis  Specimen – Rectal swab
 Cary-Blaire/Aimes – Transport medium
 TCBS & Alkaline Peptone Broth
Treatment & Prevention  Chloramphenicol
 Kanamycin
 Tetracycline

GENUS CAMPYLOBACTER, HELICOBACTER AND PSEUDOMONAS

Genus Campylobacter
Species  Incidence is worldwide
o Campylobacter jejuni  Closely related to V. cholerae morphologically and physiologically
o Campylobacter coli  Most important human pathogen associated with disease production
 Gram (-) spirally curved or seagull-wing shaped rod
 Motile – single polar flagellum
o Darting motility (like V. cholerae)
Biochemical Characteristics  Asaccharolytic (non-CHO fermenter)
 Microaerophilic
 Fastidious
 Grows best at 42—43°C
 Susceptible to gastric acid (low pH)
Genus Campylobacter Genus Helicobacter Genus Pseudomonas
Oxidase + + +
Urease - + -
Catalase + + +
H2S + - -
Lactose - - -
Sucrose - - -
Maltose - - -
Glucose - + +
Can oxidize glucose to acetate

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 Determinants of Pathogenicity Heat Labile Enterotoxin
 Acts in the same manner as choleragin toxin
*See pathogenesis of V. cholerae
Culture Characteristics  Slow growing and fastidious
 Skirrous Agar
o Composed of : Vancomycin, Polymyxin, Trimethoprim
 Campy-BAP Agar
o Incubated with 10% CO2, 5% O2, 85% N2
o Colorless convex colonies, watery and spreading
Clinical Features Enterocolitis
 One of the most common causes of diarrhea in pediatric patients in the US (infectious type)
 Self-limiting disease; lasts less than 7 days with a mean average of 3—5 days
 Causes gastroenteritis, producing inflammation of the intestine with ulceration of the GIT mucosa
(d/t secretion of gastric juices)
 Reservoir: Normal GIT flora of many wild and domestic animals (dogs)
 MOT: Ingestion of raw undercooked meat
 Incubation period: 1—7 days
 Characterized by:
o Acute crampy abdominal pain
o Vomiting
o Fever
o Malaise
o Diarrhea – bloody with pus
Laboratory Diagnosis Gram Staining – demonstrate seagull-wing morphology

Darkfield microscopy – motility

Culture on special media

 Skirrous or Campy-BAP incubated microaerophilically at 42°C for 72 hours
Treatment and Prevention  Erythromycin
 Tetracycline – allergic to PCN
 Cholramphenicol
 Ciprofloxacin – alternate drug
 Prevention – proper hygiene; no vaccine available

Genus Helicobacter
Specie: Helicobacter pylori  Isolated in the gastric tissue of stomachs among patients with ulcers
 Associated with gastritis, gastric, and duodenal ulcer
 Does not invade gastric tissue but is found in association with gastric mucous secreting cells
 Now classified by WHO as a type 1 carcinogen
 Curved or spiral-shaped gram (-) organism
 Motile, 4—6 lophotrichous flagella
 Found in the antrum and fundus of the stomach
Biochemical Characteristics  Microaerophilic
 Killed at an acid pH <7.0
Determinants of Pathogenicity Ability to adhere to gastric epithelium
 By the production of urease and mucinase
 Urease – neutralize the stomach acid on its migration to the lining epithelium of the stomach
 Mucinase – enhance penetration to the mucous layer of the GIT

Cytotoxin
 Induces intracellular vacuolation of cultured cells
 Frequently associated with peptic ulcer and gastritis

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 Culture Characteristics Skirrous Agar
 incubate for 7 days at 37°C
 Small, circular, translucent colonies
Clinical Features Anthral Gastritis / Chronic Gastritis
 Mode of transmission: Unknown, but oral-oral, fecaloral are possible routes of transmission
(contaminated food & H2O)
 Characterized by: Epigastric pain, Anorexia, Nausea and Vomiting
Laboratory Diagnosis  Skirrous Agar – see culture characteristics
 Histologic Examination of biopsied gastric tissue
 Warthin-Starry Stain – direct detection from biopsied specimen
 Urease Breath Test – radioactive urease swallowed
 Serological – EIA
Treatment and Prevention  Triple Therapy: Bismuth + Metronidazole + Tetracycline

Genus Pseudomonas
Specie: P. aeruginosa  Most frequently isolated human pathogen
 Major agent of nosocomial infections
 Leading cause of death in patients with cystic fibrosis, neoplastic diseases, severe burns
 Most species do not infect humans
 Some are important opportunistic pathogens (infect individuals with impaired host defense)
 Human infection is severe and difficult to treat
 Slender gram (-) rod, arranged singly or in short chains
 Motile – single polar flagellum
 Nonsporing, noncapsulated
 Piliated and most strains have a mucoid slime layer
Biochemical Characteristics  Biochemically inactive
 Strictly obligate aerobe
 Grows best at 37°C
 Optimum pH of 7.2—7.4 (normal body pH)
Determinants of Pathogenicity  Colonization to appropriate site using pili and slime
 Hemolysin
o Phospholipase and glycolipids
o Invasiveness of organism
 Production of extracellular enzyme
o Protease and Lipase
o Help in the formation of hemorrhagic skin lesions
 Exotoxin - Inhibits CHON synthesis
 Elastase - Digests elastin of the arterial wall causing tissue necrosis
Culture Characteristics  Can grow on ordinary culture medium, helps with identification
 Blood agar – beta-hemolytic
 Pseudomonas agar – special medium to enhance pigment production
o Pyocyanin – bluish-green diffusible phenazine pigment which is soluble in H2O and chloroform
o Pyoverdin – greenish-yellow fluorescent pigment; soluble in H2O, but not in chloroform
 MacConkey’s Agar – colorless nonlactose fermenting colonies
 Nutrient Agar – large, smooth, convex, grayish-blue colonies that emit fruity aromatic odor.
Resistant to chemical disinfectant and destroyed by boiling
Clinical Features  Meningitis
 Extensive burns
 UTIs following catheterization
 Eye, ear and wound infections
Laboratory Diagnosis  Microscopic examination – morphologic determination
 Culture (if positive proceed to antibiotic susceptibility test)
 Antibiotic susceptibility test – obligatory bc of multiple antibiotic resistant organisms
Treatment and Prevention Aminoglycosides: Amikacin, Gentamycin, Tobramycin
Prevention: Isolation, Hyperimmune gamma globulin to decrease mortality rate, Vaccination
(Pseudogen, heptovalent vaccine)

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