Oxidase Positive Curved Gram Negative Rods: Vibrio, Aeromonas, Plesiomonas, Campylobacter, and Helicobacter Study Notes

Overview of Oxidase Positive, Curved Gram Negative Rods

• General Morphology: Curved, Gram-negative rods.

• Key Biochemical Feature: Oxidase positive.

• Organisms Investigated:

  • Vibrionaceae: Vibrio, Aeromonas, Plesiomonas.

  • Campylobacteraceae: Campylobacter, Helicobacter.

Vibrionaceae Characteristics

• General Traits:

  • Oxidase: Positive.

  • Glucose: Fermenters.

  • Motility: Positive.

• Environmental Habitat: Associated with fresh or salt water environments.

• Clinical Significance: Known to cause gastroenteritis, particularly after the consumption of improperly cooked seafood.

Vibrio Species: General Profile

• Environmental Requirements and Habitat:

  • All species are associated with either fresh or salt water.

  • Most species are halophilic, meaning they require increased salt concentrations for growth.

• Infection Source: Closely associated with shellfish, specifically oysters, clams, and mussels.

• Medically Important Species:

  • V. cholerae

  • V. parahemolyticus

  • V. vulnificus

  • V. mimicus

  • V. alginolyticus

Laboratory Isolation of Vibrio

• Collection and Transport:

  • Testing is typically performed only on request.

  • Stool specimens are the primary source.

  • Cary-Blair transport media: Used for transport to the lab. The organism is extremely sensitive to drying and changes in pH.

  • Specimens must be collected before the administration of antimicrobials.

• Selective Media: TCBS Agar (Thiosulfate Citrate Bile Salt Sucrose Agar):

  • Sulfur source: Thiosulfate.

  • $H_2S$ indicator: Ferric ammonium citrate.

  • Carbohydrate source: Sucrose.

  • Selective agent: Oxgall (bile salt) inhibits Gram-positives and most Gram-negatives.

  • pH indicator: Bromthymol blue (Acid = Yellow, Alkaline = Blue/green).

• Growth Patterns: Vibrio will grow on Blood Agar Plate (BAP) and MacConkey Agar (MAC), but will not grow on XLD or HE agar.

Vibrio cholerae

• Disease Presentation:

  • Causes cholera, characterized by severe watery diarrhea known as ’rice water’ stools (watery fluid containing mucus flecks).

  • Fluid loss can reach up to $20\,L$ per day.

  • Humans are the only known host for the organism.

• Epidemiology:

  • Causes epidemics and pandemics.

  • Present in the Gulf Coast states of the US (Texas, Louisiana, Florida).

• Pathogenesis:

  • Cholera toxin: An enterotoxin that disrupts the function of the intestinal mucosa.

  • Mechanism: Toxin attaches to the intestinal mucosa, activating cyclic adenosine monophosphate (cAMP). Activated cAMP leads to the hypersecretion of water and electrolytes.

  • Clinical Consequences: Loss of up to $1\,L$ of fluid per hour, leading to metabolic acidosis, decreased K+ (potassium), shock, and potentially death.

• Transmission:

  • Exposure to fresh and salt water.

  • Consumption of contaminated seafood or food cooked in contaminated water.

  • Fecal-oral transmission occurs in areas with poor sanitation.

• Treatment:

  • Fluid replacement is critical.

  • Antibiotics such as Tetracycline or Doxycycline may shorten disease duration.

  • Alternative treatments for resistant strains: Chloramphenicol, Ampicillin, or Bactrim.

Laboratory Identification of V. cholerae

• Isolation Strategy:

  • Alkaline peptone water: Used for stool transport. The increased pH suppresses normal flora growth, while increased salt enhances Vibrio growth. Subculture to TCBS agar at 18-24 hours.

  • Appearance on TCBS: Yellow colonies (Sucrose positive).

• Biochemical and Microscopic Tests:

  • Gram stain: Curved, comma-shaped Gram-negative rods.

  • TSI: A/A (Acid/Acid).

  • Lysine decarboxylase: Positive.

  • Ornithine decarboxylase: Positive.

  • Indole: Positive.

  • NaCl Requirement: Will grow in $1\%\,NaCl$.

  • Motility: ’Shooting star’ motility due to a single, polar flagellum.

  • String test: Organism is suspended in one drop of $0.5\%\,sodium\,desoxycholate$. A positive result is the formation of a viscous string.

• Serogroups and Biotypes:

  • Classification is based on the somatic O antigen.

  • O1 Serogroup: Associated with severe pandemics; divided into three serotypes: Inaba, Ogawa, and Hikojima.

  • O139 Serogroup: Part of the pandemic group.

  • Non-O1 Serogroups: Cause extra-intestinal wound infections.

  • Biotypes of O1: Classical and El Tor (the latter is more severe).

  • Confirmation is performed at state labs.

• Direct Detection: Latex agglutination or ELISA testing exists but is not widely used in the US due to rare occurrence.

Other Medically Significant Vibrio Species

• Vibrio parahemolyticus:

  • Causes a mild, self-limiting gastroenteritis (2-3 days) due to an exotoxin.

  • Endemic in Japan and the Chesapeake Bay area; associated with oysters, clams, shrimp, and crabs.

  • TCBS: Blue-green colonies (Sucrose negative).

  • TSI: K/A (Alkaline/Acid).

  • H2S: Negative.

  • Other: Halophilic; Ornithine decarboxylase negative.

• Vibrio vulnificus:

  • Causes wound infections from contaminated water (occupational risk for fishermen/sailors).

  • Causes septicemia in patients with pre-existing liver disease after consuming raw oysters (50% mortality rate even with treatment).

  • TCBS: Blue-green colonies (Sucrose negative).

  • Other: Halophilic.

• Vibrio mimicus:

  • Gastroenteritis associated with raw oysters or contaminated water.

  • TCBS: Blue-green colonies.

  • Biochemicals: Similar to V. cholerae but does not require salt to grow.

• Vibrio alginolyticus:

  • Causes superficial wound infections, infections of soft tissues, ears, and eyes; may cause septicemia.

  • Associated with salt water and swimming.

  • TCBS: Yellow colonies (Sucrose positive).

  • Other: Halophilic.

Vibrio Summary Table

Aeromonas Species

• Natural Habitat: Fresh and salt water, sink drains/traps, seafood, and meats.

• Infection Hosts: Commonly causes infections in cold-blooded animals; can cause nosocomial human infections.

• Species Groups: Aeromonas hydrophila group includes A. hydrophila (most common), A. caviae, and A. veronii.

• Pathogenicity:

  • Cellulitis and wound infections after water exposure.

  • Gastroenteritis resembling cholera.

  • Septicemia in patients with liver disease.

  • Miscellaneous: UTIs and ear infections.

• Isolation and Identification:

  • MAC: Lactose fermenter (resembles E. coli).

  • BAP: Most are $\beta$ hemolytic.

  • CIN Agar: Clear colonies with pink centers (’bull’s eye’ colonies). Optimum growth at $28^\circ C$.

  • Enhanced Recovery: BAP supplemented with $20\,\mu g\,ampicillin$ or alkaline peptone medium.

  • Biochemicals: Oxidase positive (must test all LF/$\beta$ hemolytic organisms), Glucose fermenter, Indole (+), Esculin hydrolysis (+), Lysine decarboxylase (+), Ornithine decarboxylase (+), DNase (+).

Plesiomonas shigelloides

• Habitat: Fresh and salt water, soil; infects cold-blooded animals.

• Epidemiology: Seen in tropical and under-developed countries; transmitted via contaminated drinking water.

• Cinical Disease:

  • Gastroenteritis with watery diarrhea (lasts 2-4 weeks) due to an enteropathogenic toxin similar to Shiga toxin.

  • Extra-intestinal: Bacteremia and meningitis.

• Identification:

  • BAP: Non-hemolytic; MAC: NLF.

  • Selective Media: Inositol brilliant green bile salts agar (IBB). P. shigelloides appears as white to pink colonies.

  • Biochemicals: Oxidase positive, Glucose fermenter, Inositol (+).

  • Amino Acids: Lysine, Ornithine, and Arginine are all positive.

• Treatment: Antibiotics like Tetracycline, Chloramphenicol, Bactrim, Quinolones, Imipenem, Aminoglycosides, and Cephalosporins. Typically resistant to Penicillins ($\beta$-lactam producers).

Campylobacter and Helicobacter General Characteristics

• Gram Stain: Curved, Gram-negative rods; often described as ’seagull-shaped’; staining is faint.

• Environment: Microaerophilic (requires $5\%\,O_2$ and $5-10\%\,CO_2$). This is achieved via the CampyPak evacuation system.

• Biochemicals: Oxidase positive, Motility positive (’darting motility’ via a single polar flagellum), Asaccharolytic.

Campylobacter Species

• Epidemiology: Worldwide distribution in poultry, cattle, and pigs.

• Infection Dose: Low dose required for infection (approx. 500 organisms).

• Clinical Species:

  • C. jejuni: Most common cause of bacterial gastroenteritis.

  • C. coli

  • C. fetus

• Diseases:

  • Gastroenteritis: Similar to Shigella; organism invades intestinal submucosa. Characterized by bloody diarrhea with neutrophils, abdominal pain, headache, myalgias, and nausea.

  • Extra-intestinal disease: Primarily in immunocompromised patients (febrile disease with bacteremia caused by C. fetus).

  • Periodontal disease.

• Transmission: Fecal-oral route; undercooked poultry/beef; unpasteurized milk; contaminated water or apple cider.

• Culture:

  • Media: Routine media (BAP/CHOC) grow slower. Gray to pinkish colonies may ’trail’ along streak lines.

  • Selective Choice: Campy CVA: Contains Cefoperazone (inhibits GNR/cocci), Vancomycin (inhibits GPC/GPB), and Amphotericin B (inhibits yeasts).

  • Incubation: Incubate at $42^\circ C$ under microaerophilic conditions for 48-72 hours.

• Identification:

  • Oxidase (+), Catalase (+), Darting motility (+).

  • Campy test: Detects both viable and non-viable organisms (useful since the organism is difficult to grow).

• Speciation Differentiation Table:

• Treatment: Usually self-limiting; relapses are common and can be treated with Erythromycin or Ciprofloxacin.

Helicobacter pylori

• Description: Formerly known as Campylobacter pylori.

• Primary Habitat: Inhabits the stomach mucosa.

• Clinical Significance: Most common cause of peptic ulcers and stomach cancers.

• Acid Survival: Produces large amounts of urease, which hydrolyzes urea to produce ammonia, neutralizing stomach acid.

• Diagnostic Techniques:

  • CLO test: Tissue biopsy of an ulcer is placed on a test cartridge. Urease hydrolyzes urea to produce a pink color within 2 hours.

  • Urea breath test: Patient ingests carbon-labeled urea. If urease is present, it is hydrolyzed to ammonia and $^{13}CO_2$. The exhaled $^{13}CO_2$ is measured by infrared spectrophotometer.

  • Other Methods: EIA for stool (expensive), PCR, and culture. Culture uses Skirrow’s media, CHOC, or Brucella agar with $5\%\,sheep\,blood$; incubate microaerophilically at $35^\circ C$ for up to 7 days.

• Treatment: Requires a three-drug regimen: Bismuth salts, Metronidazole, and either Amoxicillin or Tetracycline.