Week 2 Flashcards
Koch’s Postulates
- The suspected pathogenic organism should be present in all cases of the disease and absent from healthy animals
- The suspected organism should be grown in pure culture
- Cells from a pure culture of the suspected organism should cause disease in a healthy animal
- The organism should be reisolated and shown to be the same as the original
GENERAL CHARACTERISTICS Vibrio
- Gram-negative, straight or curved, short rods, may be comma- shaped on initial isolation
- motile by single thick sheathed polar flagellum, some strains produce multiple lateral flagella when grown on solid media
- do not form spores
- aerobic and facultatively anaerobic, capable of both respiratory and fermentative metabolism
- all species produce oxidase, except Vibrio metschnikovii; all catalase positive
- Fermentative
- all species grow in media containing increased NaCl concentrations
- species require NaCl for growth (halophilic vibrios), except Vibrio cholerae and Vibrio mimicus (non-halophilic vibrios)
- sensitive to drying, exposure to sunlight, development of acid pH
- easily inhibited by normal intestinal flora or contaminating organisms
- natural inhabitants of brackish and salt water
- some Vibrio spp. pathogenic for marine animals
- human disease associated with ingestion of contaminated water or consumption of contaminated shellfish or seafood
VIRULENCE FACTORS - VIBRIO CHOLERAE
- Adherence
−virulent strains establish themselves in intestinal tract by attaching to intestinal lining
−virulent organisms penetrate intestinal mucus and attach to microvilli at brush border of epithelial cells - Cholera toxin (CT)
− major pathogenic factor produced by Vibrio cholerae O1 and
some non-O1 Vibrio cholerae (e.g., O139 Bengal)
− potent extracellular enterotoxin, also called choleragen, acts on cells of small intestine
− closely related to heat-labile enterotoxin (LT) of Escherichia coli
in structure and function
− complex protein molecule, composed of 2 subunits: - subunit A (responsible for enzymatic activity)
- subunit B (responsible for binding to receptor)
The cholera toxin genes, ctxA and ctxB, are encoded on a lysogenic phage, CTXФ
The cholera toxin is an AB5 subunit toxin whereby the B subunits bind to GM1 gangliosides on host cells
The enzymatic activity of cholera toxin is mediated by the A subunit which is an ADP-ribosyltransferase
Cholera toxin is primarily responsible for the secretory diarrhoea that accompanies cholera disease
Mode of Action of Cholera Toxin
The B subunits of the AB5
cholera toxin bind to the receptor, the GM1 ganglioside. The A subunit dissociates from the
B subunits and is taken up into the cell.
The A1 portion ADP-ribosylates the α subunit of a host cell G-protein which then becomes active.
The activated G-protein stimulates an adenylate cyclase to produce cAMP. Elevated levels of cAMP affect the normal function of the ion channel CFTR (cystic fibrosis
transmembrane conductance regulator) causing chloride ion efflux
CLINICAL SIGNIFICANCE - VIBRIO
CHOLERAE
- infections: asymptomatic, mild, severe
- large numbers of organisms shed in carrier’s faeces, which contaminate water and food supplies
- 2 types of carriers:
− convalescent carrier - one recovering from disease
- under 50 years of age
- sheds organism for several months to 1 year
− chronic carrier - harbours organisms in gallbladder
- over 50 years of age
- sheds organism intermittently over period of years when natural purging results from other intestinal disorder
- transmission by faecal-oral route
- fluid losses in severe cases 15L to 20L per day
- stool watery, odourless, resembling “rice water”
- hypovolemic shock and metabolic acidosis result from fluid loss
- eyes and cheeks appear sunken, skin turgor diminished
COLLECTION, TRANSPORT, AND STORAGE
OF SPECIMENS Vibrio
- for diarrhoeal disease, stool specimen collected in acute stage of illness before antibiotics given - specimen of choice
- culturing of vomitus may be productive
- Cary-Blair transport medium recommended
CULTURE MEDIA Vibrio
Enrichment media
* Alkaline-peptone water (APW)
− contains: 1% peptone, 1% NaCl, pH 8.6
− high pH suppresses growth of commensal intestinal bacteria and allows multiplication of Vibrio cholerae
− recommended when low concentrations of organisms in specimen expected
Selective media
* Thiosulphate-Citrate-Bile salts-Sucrose (TCBS) agar medium
− contains: peptone base agar, yeast extract, bile salts, citrate, sucrose, ferric citrate, sodium thiosulphate
− pH 8.6, bromothymol blue indicator
Non-selective media
* all vibrios grow on non-selective media such as 5% sheep blood agar
ISOLATION PROCEDURES Vibrio
- growth enhanced by adding 1% NaCl to medium
- colonies: smooth, convex, creamy in consistency, grey-white, with entire margins; rough colonies adhere to agar
Direct examination Vibrio
- Enzyme-Linked Immunosorbent Assay (ELISA)
− used for detection of cholera toxin from samples in cholera outbreak - PCR assays
− used for detection of cholera toxin A subunit (ctxA) gene from food, water, patient samples
IDENTIFICATION Vibrio
- Colony morphology
- Gram stain of colony
- Oxidase Test
− for separating vibrios from Enterobacteriaceae
− all Vibrio spp. positive except Vibrio metschnikovii - String Test
− suspension of suspected Vibrio spp. colony in 0.5% sodium deoxycholate in saline, on glass slide, stirred with loop
− Vibrio spp. lyse , released DNA pulled up into string with loop - Nitrate reduction Test
− all Vibrio spp. positive except Vibrio metschnikovii - Salt requirement and tolerance tests
- MALDI-TOF MS
TREATMENT Vibrio cholerae
- prompt replacement of fluid and electrolytes normally reverses patient’s condition within hours
- susceptible to: tetracycline, chloramphenicol, gentamicin, nalidixic acid, quinolones
GENERAL CHARACTERISTICS Campylobater
- Gram-negative, slender, curved, S-shaped, seagull-winged, or long spiral rods; may be coccoid in older cultures
- motile by means of single polar unsheathed flagellum at one or both ends
- distinctive cork-screw-darting type of motility observed with phase-contrast or dark-field microscopy
- do not form spores
- microaerophilic
require low oxygen tension 3% to 15% for growth - capnophilic
require increased CO2 level 3% to 5% for growth - oxidase and catalase positive
- unable to use sugars either oxidatively or fermentatively
VIRULENCE FACTORS Campylobacter
- motility provided by flagella
−allows organism to penetrate mucus layer covering gut
surface
−enhances attachment - heat-labile enterotoxin
− protein, structurally and immunologically related to cholera
enterotoxin
−causes secretory diarrhoea by stimulating adenylate cyclase
activity in intestinal mucosa and disrupting normal ion
transport in enterocytes
CLINICAL SIGNIFICANCE -
CAMPYLOBACTER JEJUNI SUBSP. JEJUNI
- most important human pathogen among campylobacters
- causative agent of Campylobacter enteritis
- highest incidence in infants and young children, 2nd peak in young adults
- most human infections result from contact with poultry, cattle, raw milk, surface water, pets
- faecal-oral route plays role in human infections
Acute enteritis
* enteritis symptoms include:
−cramps, abdominal pain
− diarrhoea (may vary from loose stools to massive watery stools or stool
containing blood and inflammatory cells)
−chills, fever
* tenesmus
Extraintestinal infections are rare but include:
− bacteraemia (occurs in less than 1% of patients)
− meningitis
−cholecystitis
− urinary tract infections
−septic arthritis
−inflammatory proctitis (in male homosexuals)
GUILLAIN-BARRÉ SYNDROME (GBS)
- autoimmune disorder of peripheral nervous system
- most common cause of acute flaccid paralysis since eradication of polio
- characterised by weakness, usually symmetrical, evolving over period of several days or more
- Campylobacter infection - single most identifiable antecedent infection associated with development of GBS
