18 -chapter_20_-_curved_gnb
TRANSCRIPT
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Chapter 20 CURVED GRAM-NEGATIVE
BACILLI
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Describe the gram stain morphology of Vibrio species.
Discuss the clinical significance of Vibrio cholerae .
List the selective media for the isolation of Vibrio species and describe the different colony morphologies observed on the media.
Discuss the mode of transmission of Vibrio cholerae .
Identify Vibrio cholerae based on biochemical reactions.
Explain the role of the vibrio toxin the pathogenesis of cholera infections.
Differentiate the species of Vibrio species discussed in this unit based on colony morphology on Thiosulfate Citrate Bile Salts Sucrose (TCBS) and clinical symptoms of patient.
OBJECTIVES
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VIBRIO SPECIES
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More than 30 recognized species
Approximately 12 species have been associated with human disease
Persists in the environment:
Grows in saltwater, freshwater
Causes human disease because it can adapt to colonize intestinal tract if ingested
GENERAL CHARACTERISTICS
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PROGRESSION OF INFECTION
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Gram-negative, curved rod
Motile single polar flagellum
“Darting” motility
“Shooting star” motility
MICROSCOPIC CHARACTERISTICS
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Most are:
Indole positive
Urea negative
Nitrate positive
Most are oxidase positive
Posses the vibriostatic compound O/129
GENERAL BIOCHEMICAL CHARACTERISTICS
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Media Grow quite readily on various routine media
Use of Thiosulfate-Citrate-Bile Salts-sucrose agar (TCBS) has enhanced recovery
Alkaline peptone water - enrichment broth
Conditions for growth Facultative anaerobes
Cultures should be incubated at 35°C in air or CO2
MEDIA AND GROWTH
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On TCBS agar:
Sucrose fermenters are yellow
V. cholera and V. alginolyticus
Non-sucrose fermenters are green
V. parahaemolyticus
On BAP and chocolate agar:
Most are iridescent with a greenish hue
COLONY MORPHOLOGY
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VIBRIO CHOLERAE
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Mostly found in aquatic environments
Transmitted:
Fecal-oral route
Contact with contaminated water
Ingestion of contaminated shellfish or other seafood
EPIDEMIOLOGY
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Production of Cholera toxin or choleragen
Provokes an accumulation of cyclic AMP in the cell membrane which causes mucosal cells to hypersecrete electrolytes and water into the lumen of the GI tract which results in watery diarrhea and fluid loss = rice -water stools
Such a rapid fluid and electrolyte loss leads to severe dehydration, hypovolemic shock, metabolic acidosis and death in just a matter of hours
VIRULENCE FACTORS
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Etiologic agent of cholera
Cholera is mostly seen in Asia and South America
Causes both intestinal and extraintestinal infections
Infections are acquired by ingesting contaminated food or water
Characteristic “rice-water” stools
CLINICAL SIGNIFICANCE
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Contaminated water enters stomach
Bacterial cells adhere to gastric and intestinal mucosal epithelial cells
Enterotoxin is produced Increase in cyclic AMP - prevents the re-absorption of
Na+ ions and the excretion of sodium bicarbonate and potassium
Causes water to leave the epithelial cells into intestinal lumen causing "rice water" stools
It may lead to death in matter of hours
EPIDEMIOLOGY
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Stool specimens are preferred - must be transported in Cary-Blair medium
Rectal swabs acceptable
SPECIMEN MANAGEMENT
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Will grow on BAP, Choc, and MAC (non -lactose fermenter on MAC)
Alkaline peptone water broth for enrichment
Thiosulfate Citrate Bile Salts Sucrose (TCBS) - selective and differential media (yellow or green colonies) Yellow - ferment sucrose
Green - do not ferment sucrose
MEDIA FOR ISOLATION
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COLONY MORPHOLOGY
Vibrio cholerae - BAP
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COLONY MORPHOLOGY
Vibrio cholerae - MAC
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COLONY MORPHOLOGY
Vibrio cholerae - TCBS
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Oxidase positive
Sucrose positive (yellow on TCBS)
Lactose negative (clear on MAC)
Lysine positive
Ornithine positive
Arginine negative
String test positive – 0.5% deoxycholate
BIOCHEMICAL CHARACTERISTICS
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String test
Principle:
Addition of 0.5% sodium deoxycholate causes most Vibrio spp . to lyse and release DNA, which can be pulled up into a string with a inoculating loop
BIOCHEMICAL CHARACTERISTICS
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Therapy - fluid and electrolyte replacement
Antimicrobial therapy will shorten the course of the disease and may decrease bacterial excretion
Tetracycline is the drug of choice
TREATMENT
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VIBRIO ALGINOLYTICUS
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Least pathogenic for humans and least isolated
Wound, ears, or eyes after trauma and sea water exposure
CLINICAL SIGNIFICANCE
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Strict halophile = requires added NaCl for optimal growth
Yellow colonies on TCBS
MEDIA AND GROWTH
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COLONY MORPHOLOGY
Vibrio alginolyticus - BAP
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COLONY MORPHOLOGY
Vibrio alginolyticus - TCBS
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Sucrose positive
BIOCHEMICAL CHARACTERISTICS
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VIBRIO PARAHAEMOLYTICUS
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Acute gastroenteritis associated with the ingestion of raw contaminated seafood, particularly oysters
Usually mild but can be fatal
Endemic in Japan; #1 cause of “summer diarrhea”
Usually self-limiting disease
Rare extraintestinal pathogen
CLINICAL SIGNIFICANCE
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COLONY MORPHOLOGY
Vibrio parahemolyticus - BAP
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COLONY MORPHOLOGY
Vibrio parahemolyticus - TCBS
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Sucrose negative
Beta-hemolytic on blood agar
BIOCHEMICAL CHARACTERISTICS
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VIBRIO VULNIFICUS
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Second most pathogenic
Causes wound infections:
Wound infections – after exposure to marine animals or marine environment
Wound infections may lead to septicemia
Primary septicemia – 24 hours after ingestion of raw oysters
CLINICAL SIGNIFICANCE
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COLONY MORPHOLOGY
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COLONY MORPHOLOGY
Vibrio vulnificus - green or yellow on TCBS Agar
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Sucrose variable
BIOCHEMICAL CHARACTERISTICS
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CAMPYLOBACTER SPECIES & HELICOBACTER PYLORI
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Discuss the clinical significance of Campylobacter and Helicobacter pylori .
Summarize specimen collection and transport procedures for Campylobacter and Helicobacter .
Select appropriate culture media and incubation conditions for Campylobacter cultures.
Compare the methods for obtaining microaerobic conditions.
Describe the microscopic morphology of Campylobacter and Helicobacter .
Differentiate species of Campylobacter based on biochemical identification.
Explain the principle and procedure of the CLO test and breath tests for the detection of Helicobacter .
OBJECTIVES
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CAMPYLOBACTER SPECIES
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Gastroenteritis - Campylobacter spp. is one of the leading causes of bacterial diarrhea worldwide
Septicemia
CLINICAL SIGNIFICANCE
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Transmitted via contaminated food, milk, or water
EPIDEMIOLOGY
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Stool and rectal swabs - if specimen cannot be cultured within 4 hours of collection, place in Cary -Blair transport medium
Blood
SPECIMEN MANAGEMENT
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Optimal temperature for growth is 42 ̊C
Microaerophilic (5-10% oxygen)
Capnophilic (8-10% CO2)
Needs at least 48-72 hours for growth
GROWTH CONDITIONS
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Curved gram negative rods
Comma shapes, "S" shapes and gull wing forms are seen
Organisms may occur in short or occasionally long chains
Oxidase positive
Non-sporeforming
Cells have a single polar unsheathed flagellum at one or both ends = “darting motility”
MICROSCOPIC CHARACTERISTICS
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Media
Campy BAP (most commonly used)
Butzler Medium
Skirrow medium
Campy-Thio for enrichment
Examine at 24, 48 and 72 hours
MEDIA FOR ISOLATION
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Colonies smooth
Convex
Translucent
May be flat and watery with irregular edges
They may be pinpoint to spreading over large areas of the plate
COLONY MORPHOLOGY
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COLONY MORPHOLOGY
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Catalase positive
Oxidase positive
Darting motility
BIOCHEMICAL CHARACTERISTICS
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C. coli Hippurate negative Resistant to cephalothin Susceptible to nalidixic acid
C. jejuni
Hippurate positive Resistant to cephalothin Susceptible to nalidxic acid
BIOCHEMICAL CHARACTERISTICS
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Antimicrobic therapy is not recommended in most cases of diarrhea
Supportive care and fluid replacement
When needed:
Erythromycin is the drug of choice
Systemic infections are treated with Gentamycin
TREATMENT
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HELICOBACTER PYLORI
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Found only on mucus-secreting epithelial cells of the stomach
Causative agent of active chronic gastritis (type B gastritis) = Peptic ulcer disease
CLINICAL SIGNIFICANCE
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Gram negative
Small curved (gull wing and U shapes)
Slightly plump bacilli
MICROSCOPIC MORPHOLOGY
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Stool cultures are not often done for detection of Helicobacter pylori
Specimens/test of choice include:
Invasive (tissue biopsy)
CLO rapid urease test
Non-invasive
Urea breath test
Serological detection of IgG
SPECIMENS
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Oxidase positive
Catalase positive
Urease positive
BIOCHEMICAL CHARACTERISTICS
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