Chapter 20 CURVED GRAM-NEGATIVE

BACILLI

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

VIBRIO SPECIES

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

PROGRESSION OF INFECTION

Gram-negative, curved rod

Motile single polar flagellum

“Darting” motility

“Shooting star” motility

MICROSCOPIC CHARACTERISTICS

Most are:

Indole positive

Urea negative

Nitrate positive

Most are oxidase positive

Posses the vibriostatic compound O/129

GENERAL BIOCHEMICAL CHARACTERISTICS

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

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

VIBRIO CHOLERAE

Mostly found in aquatic environments

Transmitted:

Fecal-oral route

Contact with contaminated water

Ingestion of contaminated shellfish or other seafood

EPIDEMIOLOGY

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

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

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

Stool specimens are preferred - must be transported in Cary-Blair medium

Rectal swabs acceptable

SPECIMEN MANAGEMENT

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

COLONY MORPHOLOGY

Vibrio cholerae - BAP

COLONY MORPHOLOGY

Vibrio cholerae - MAC

COLONY MORPHOLOGY

Vibrio cholerae - TCBS

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

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

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

VIBRIO ALGINOLYTICUS

Least pathogenic for humans and least isolated

Wound, ears, or eyes after trauma and sea water exposure

CLINICAL SIGNIFICANCE

Strict halophile = requires added NaCl for optimal growth

Yellow colonies on TCBS

MEDIA AND GROWTH

COLONY MORPHOLOGY

Vibrio alginolyticus - BAP

COLONY MORPHOLOGY

Vibrio alginolyticus - TCBS

Sucrose positive

BIOCHEMICAL CHARACTERISTICS

VIBRIO PARAHAEMOLYTICUS

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

COLONY MORPHOLOGY

Vibrio parahemolyticus - BAP

COLONY MORPHOLOGY

Vibrio parahemolyticus - TCBS

Sucrose negative

Beta-hemolytic on blood agar

BIOCHEMICAL CHARACTERISTICS

VIBRIO VULNIFICUS

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

COLONY MORPHOLOGY

COLONY MORPHOLOGY

Vibrio vulnificus - green or yellow on TCBS Agar

Sucrose variable

BIOCHEMICAL CHARACTERISTICS

CAMPYLOBACTER SPECIES & HELICOBACTER PYLORI

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

CAMPYLOBACTER SPECIES

Gastroenteritis - Campylobacter spp. is one of the leading causes of bacterial diarrhea worldwide

Septicemia

CLINICAL SIGNIFICANCE

Transmitted via contaminated food, milk, or water

EPIDEMIOLOGY

Stool and rectal swabs - if specimen cannot be cultured within 4 hours of collection, place in Cary -Blair transport medium

Blood

SPECIMEN MANAGEMENT

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

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

Media

Campy BAP (most commonly used)

Butzler Medium

Skirrow medium

Campy-Thio for enrichment

Examine at 24, 48 and 72 hours

MEDIA FOR ISOLATION

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

COLONY MORPHOLOGY

Catalase positive

Oxidase positive

Darting motility

BIOCHEMICAL CHARACTERISTICS

C. coli Hippurate negative Resistant to cephalothin Susceptible to nalidixic acid

C. jejuni

Hippurate positive Resistant to cephalothin Susceptible to nalidxic acid

BIOCHEMICAL CHARACTERISTICS

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

HELICOBACTER PYLORI

Found only on mucus-secreting epithelial cells of the stomach

Causative agent of active chronic gastritis (type B gastritis) = Peptic ulcer disease

CLINICAL SIGNIFICANCE

Gram negative

Small curved (gull wing and U shapes)

Slightly plump bacilli

MICROSCOPIC MORPHOLOGY

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

Oxidase positive

Catalase positive

Urease positive

BIOCHEMICAL CHARACTERISTICS

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