Gram Negative Bacilli Notes

GRAM NEGATIVE BACILLI

A. NON-FERMENTATIVE GROUP

• Includes:
  • Fluorescent group of Pseudomonas
  • Non-Fluorescent group of Pseudomonas
  • Burkholderia group

PSEUDOMONAS GROUP

• Non-fermentative or oxidative group (K/K)
• Oxidase positive except for Stenotrophomonas maltophilia
• Examples:
  • Pseudomonas aeruginosa
  • Pseudomonas stutzeri
  • Pseudomonas fluorescens
  • Pseudomonas mendocina
  • Pseudomonas putida
  • Burkholderia cepacia
  • Burkholderia mallei
  • Burkholderia pseudomallei

FLOURESCENT GROUP: Pseudomonas aeruginosa

• Most frequently isolated non-fermenter
• General Characteristics:
  • Feathered edge/serrated and spreading colonies
  • Mostly beta hemolytic on blood agar plate
  • Motile by polar flagellum
  • Strict aerobe
  • Grows both at 35°C and 42°C
  • Grape-like or corn tortilla-like odor due to 2-aminoacetophenone production
  • Produces both pyocyanin and pyoverdin (fluorescein)
    • Pyocyanin (blue) - can be extracted using chloroform
    • Pyoverdin (Fluorescein, yellow green) - can be visualized using ultraviolet light.
• Biochemical Reactions:
  • Oxidase Positive
  • TSIA reaction: alkaline slant over alkaline butt
  • Gelatin hydrolysis positive
  • LOA reaction (- - +)
  • Acetamide utilization positive
  • Cetrimide test positive
• Conditions associated:
  • Severe wound infections in burn patients
  • Urinary tract infections, Nosocomial infections and Septicemia
  • Chronic lung infection in patients with cystic fibrosis
  • Destructive eye infections (keratitis, corneal ulcers) in contact lens wearers
  • Agent of ecthyema gangrenosum and swimmer’s ear
• Antibiotics:
  • Kanamycin resistant
  • Carbenicillin sensitive

FLOURESCENT GROUP: Other Fluorescent Pseudomonas members (P. fluorescens & P. putida)

• General Characteristics:
  • Produces pyoverdin but does not produce pyocyanin
  • Can grow at 4°C and can cause transfusion-associated septicemia
  • Grows at 35°C but not at 45°C
  • Motile by polar flagella
• Biochemical Reactions:
  • Cetrimide negative and Acetamide negative
  • Oxidase positive
• Antibiotics:
  • Resistant to carbenicillin
  • Sensitive to kanamycin

Pseudomonas fluorescens vs. Pseudomonas putida

NON-FLOURESCENT GROUP: Pseudomonas stutzeri

• Produce wrinkled, leathery, adherent colonies that can give off a light-yellow or brown pigment
• Can cause septicemia, pneumonia, endocarditis, postsurgical wound infections, septic arthritis, conjunctivitis and UTI

NON-FLOURESCENT GROUP: Pseudomonas mendocina

• Acetamide negative and 2-ketogluconate negative
• Non-proteolytic, Motile via polar flagellum
• Oxidase positive and Arginine dihydrolase positive

BURKHOLDERIA GROUP: Burkholderia mallei

• Causes glanders - a zoonotic infection primarily affecting horses, mules and donkeys
• Transmissible to humans

BURKHOLDERIA GROUP: Burkholderia pseudomallei

• Causes melioidosis (glander’s like disease) - aggressive granulomatous pulmonary disease
• Acquired through ingestion, inhalation, or inoculation of the organism
• Produces “Earthy” color and deep pink in the Ashdown medium, contains:
  1. Crystal violet and gentamicin as selective agents to suppress the growth of other bacteria
  2. 4% glycerol which is required by some strains of B. pseudomallei
  3. Neutral red which gives the colonies of Burkholderia pseudomallei a deep color pink

BURKHOLDERIA GROUP: Burkholderia cepacia

• Associated with pneumonia in patients with cystic fibrosis or chronic granulomatous disease

OTHER NON-FERMENTATIVE ORGANISMS: Acinobacter spp.

• Catalase positive, Oxidase negative organism that grows well on EMB and MacConkey
• Obligate aerobes, non-motile
• Important species include: Acinobacter baumannii (previously Acinobacter calcoaceticus var anitratus) and Acinobacter lwoffi (previously Acinobacter calcoaceticus var lwoffi)

OTHER NON-FERMENTATIVE ORGANISMS: Stenotrophomonas (Xanthomonas) maltophilia

• Oxidase negative and DNase positive
• Does not produce pyocyanin and pyoverdin
• Catalase positive and Esculin positive and Gelatin hydrolysis positive
• ONPG positive and Lysine decarboxylase positive
• Acetamide negative
• Resistant to carbenicillin but Sensitive to kanamycin

OTHER NON-FERMENTATIVE ORGANISMS: Kingella spp.

• Includes: Kingella denitrificans, Kingella kingae, and Kingella oralis
• Oxidase positive, catalase negative, indole positive and ferments glucose

B. MISCELLANEOUS GROUP

VIBRIO SPECIES

• Facultative anaerobes, that are motile via monotrichous
• Grows on Thiosulfate-citrate-bile salts-sucrose medium
• Halophilic organisms except for: V. mimicus and V. cholera
• Oxidase positive and can be classified based on sucrose fermentation

MISCELLANEOUS ORGANISMS: Vibrio Cholerae

• Vibrio cholera 01 strain is the causative agent of cholera (also known as Asiatic cholera or epidemic cholera)
• Produces choleragen toxin which
• Biogroups of Vibrio cholerae 01

MISCELLANEOUS ORGANISMS:

Vibrio vulnificus

• Lactose positive Vibrio
• Infection is associated with septicemia and wound infections in patients handling seafoods or working in marine environment with pre-existing liver disease
  Vibrio mimicus
• Nonhalophilic, sucrose non-fermenter
• Least pathogenic among Vibrio and most infrequently isolated
• Wound and ear infections associated with marine environment
  Vibrio alginolyticus
• Gast roenter itis and ear infections associated with mar ine environment

MISCELLANEOUS ORGANISMS: Vibrio parahaemolyticus

• Wound, gastroenteritis and diarrheal infection (number one cause of summer diarrhea in Japan)
• Ingestion of contaminated seafood or direct skin contact with contaminated water
• Kanagawa phenomenon - phenomenon in which strains isolated from human hosts (clinical isolates) are hemolytic on blood agar plates, while those isolated from nonhuman sources are not hemolytic
• Hemolysis was due to the presence of Kanagawa toxin, a thermostable hemolysin
• The hemolysin can be detected by its ability to lyse human RBCs in a special high salt mannitol medium (Wagatsuma agar)

MISCELLANEOUS ORGANISMS: Campylobacter spp.

• Causes gastroenteritis and diarrhea
• Grows at 42°C and exhibits "wings of seagull" appearance
• Isolated using Campy's agar or Skirrow's medium
• Oxidase positive
• Motile by polar flagellum; exhibit darting motility

MISCELLANEOUS ORGANISMS:

Helicobacter pylori

• Associated with peptic ulcers
• Motile by 4-6 polar flagella
• Strongly urease positive, microaerophilic organism
  Aeromonas hydrophila
• Agent of red leg in frogs, causes diarrhea in humans
• Oxidase positive; lipase positive, DNAse positive, gelatin hydrolysis positive
  Plesiomonas shigelloides
• Now a member of Enterobacteriaceae; oxidase positive
• Can cause diarrhea

HAEMOPHILUS GROUP

• Non-motile; Non-spore-forming bacilli
• Facultative anaerobes; Mostly oxidase and catalase positive
• Haemophilus means "blood loving"
• Does not grow on sheep blood agar; grows well on chocolate agar plate
• Exhibits Satellitism - Can be demonstrated using the Staphylococcal Streak
  1. Streak the Haemophilus culture heavily on the sheep blood agar plate. Use a swab if necessary.
  2. Make a single narrow streak of Staphylococcus aureus through the area were Haemophilus was streaked.
  3. Incubate at 35-37°C under capnophilic environment for 18-24 hours.
• Satellitism appears as the colonies of Haemophilus grow adjacent to (or satellite around) the Staphylococcus aureus colonies.
• Staphylococcus aureus synthesizes NAD and since S. aureus is usually beta hemolytic, hemin (which is also required by some Haemophilus) is released for the RBC.

HAEMOPHILUS GROUP: X and V factors

• Haemophilus can be classified according to factor X and V requirement
  • X factor = hemin; heat-stable factor - released after degradation of hemoglobin (during hemolysis)
  • V factor = coenzyme I/NAD (nicotinamide adenine nucleotide); heat-labile factor
• Porphyrin test is an alternative method for differentiating the heme-producing species of Haemophilus
• Performed in agar or broth oror on a disk
• Detects the ability of the organism to convert delta-aminolevulinic acid (ALA) into porphyrins or porphobilinogen,which are intermediates for the synthesis of X factor
• After incubation at 35°C for 4 hours, porphobilinogen is detected using the Kovac's reagent
  • Positive for porphobilinogen is red color forms in the aqueous layer
  • Positive for Porphyrins if there is reddish-orange fluorescence under ultraviolet light (Wood's lamp at 360nm)

HAEMOPHILUS GROUP: Species

• Haemophilus aphrophilus and Haemophilus paraphrophilus have been reclassified into a single species: Aggregatibacter aphrophilus. "Aphrophilus" means "foam loving" - due to its requirement for carbon dioxide
• Major Haemophilus species: Haemophilus influenzae
  • Also known as Pfeiffer's bacilli, most frequently encountered serotype is the Serotype b
  • Encapsulated strains are pathogenic
  • Leading cause of epiglottitis and meningitis
  • Main cause of meningitis in children <5 years old
  • Virulence factors include: capsule, IgA proteases, fimbriae, lipopolysaccharide

HAEMOPHILUS GROUP: Haemophilus aegyptius and Haemophilus influenzae biogroup aegyptius

• Haemophilus aegyptius is also known as Koch-Week's bacilli and was previously known as Haemophilus influenzae biotype III
• Both can cause pink eye conjunctivitis
• Haemophilus influenzae biogroup aegyptius causes Brazilian purpuric fever (BPF)
  Haemophilus ducreyi
• Causes sexually transmitted genital ulcer disease
• Soft chancres or chancroid
• "schools of fish" in Gram stain of genital ulcers

HAEMOPHILUS GROUP: Factor Requirements

MISCELLANEOUS ORGANISMS: Legionella pneumophila

• Agent of Legionnaire's disease (febrile disease with pneumonia) or Pontiac fever (febrile disease without pulmonary involvement)
• Isolated in air conditioning towers and heating systems
• Stains poorly with Gram stain; stained using siiver impregnation (Dieterie)
• Medium for culture: Buffered Charcoal Yeast Extract Agar (BCYE)
• Laboratory diagnosis:
  1. Isolation using BCYE
  2. Urine antigen detection - rapid method
  3. Direct fluorescent antibody
  4. Serology

BORDETELLA SPECIES

• Includes: B. pertussis, B. parapertussis, B. bronchiseptica
  Bordetella pertussis
• The agent of whooping cough or pertussis
• Pertussis is a highly communicable diseases that can be prevented by vaccine (DPТ)
• Infection has three phases: catarrhal, paroxysmal, convalescent
  1. Catarrhal phase - symptoms are insidious and nonspecific
  2. Paroxysmal phase - sudden onset of severe, repetitive coughing followed by the characteristic "whoop" at the end of the coughing spell
  3. Convalescent phase - decrease in frequency and severity of the cough spells
• Ideal specimen: nasopharyngeal swab
• General Characteristics of B. pertussis:
  • Regan Lowe (charcoal cephalexin agar) or Bordet Gengou (potato blood glycerol agar)
  • Bordetella pertussis produces mercury droplet colonies in Regan-Lowe (preferred medium)
  • Fastidious, does not grow in BAP and Mac
  • Non-motile, oxidase positive; urease negative

MISCELLANEOUS ORGANISMS:

Bordetella parapertussis

• Causes a disease similar to pertussis but with milder symptoms
  Bordetella bronchiseptica
• Respiratory pathogen to animals

BORDETELLA SPECIES: Differentiation

MISCELLANEOUS ORGANISMS: Brucella spp.

• Small, nonmotile, aerobic, gram-negative coccobacilli or short rods
• Culture medium: biphasic medium (Castañeda medium)
• Agents of Brucellosis, undulant fever, Malta fever
• Organisms are normal flora of the urinary tract and gastrointestinal tracts of sheep/goat (B. melitensis), cattles (B. abortus), pigs/swine (B. suis), and dogs (B. canis)
• General Characteristics of Brucellae group:
  1. Oxidase positive, catalase positive
  2. Nitrate reduction positive, and urease positive within 2 hours
  3. Nonmotile coccobacilli that can grow in sheep blood agar

MISCELLANEOUS ORGANISMS: Brucella spp. Differentiation

MISCELLANEOUS ORGANISMS:

Pasturella multocida

• Associated with animal bite and scratches
• Previously classified under Yersinia species
• Bipolar staining (safety pin appearance when poles of the cells are more intensely stained)
• Produces musty odor (odor of mushrooms)
• Non-motile, facultative anaerobic coccobacilli
• Produces grayish colonies in sheep blood agar and chocolate agar but does not usually grow in MacConkey agar
  Francicella tularensis
• Causes tularemia (also known as rabbit, or deerfly or lemming fever, rat trapper's disease)
• Acquired through ingestion, inhalation, arthropod bite or contact with infected tissues
• Requires cysteine, cystine or thiosulfate for growth
• Culture Medium: Blood-Cystine-Glucose-Agar (BCGA)

HACEK GROUP

• Group of capnophilic organisms associated with endocarditis
• Includes: Haemophilus aphrophilus, Aggregatibacter (Actinobacillus) mycetemcomitans, Cardiobacterium hominis, Eikenella corrodens, Kingella denitrificans