Vibrios
- Briefly describe Vibrios spp.
- The name Vibrio was derived by Filippo pacini from isolated microorganisms he called vibrions from cholera patients in 1854. . Vibrio is derived from the characteristic vibratory motility (vibrare, meaning to vibrate)
- Vibrio is a gram negative comma-shaped bacilli (S-shaped or spiral forms may be seen for 2 or more cells lying end to end)
- They are **highly motile (**darting motility) with a single polar flagellum (monotrichious) with a sheath that is appreciated in dark field staining.
- They are non-spore forming and most strains are unencapsulated.
- They are facultative anaerobes and they reduce Nitrates to nitrites.
- Vibrios are oxidase positive and are able to ferment sugars (sucrose and glucose but not lactose, thus yielding and A/A reaction on TSI)
- Vibro possesses both O and H antigens. O antigens differ between groups giving rise to different serotypes.
- Vibrio are habitants of salty water (seafood, vegetables cleaned with contaminated water)
- Vibrio has 32 species but 12 are important for human infections.
- Important strains are:
- *Vibrio cholerae (*O1 and non-O1)
- Vibrio parahemolyticus
- Vibrio vulnificus.
- Briefly describe Vibrio cholerae (Comma bacillus)
- Vibrio cholerae is also known as the comma bacillus.
- Serological classification was suggested by Gardner and Venkatrama in 1935. More than 200 serotypes have been classified broadly into 2 groups based on whether they agglutinate with antisera to the O1 group. ****
- Vibrio cholerae O1 = cholera vibrios, agglutinable vibrios.
- VIbrio cholerae non-O1 = non-cholera vibrios, nonagglutinating vibrios.
- It is further Classified into 138 serotypes (02 to O139)
- Only O139 causes cholera in humans. ****
- O1 and O139 (Bengal, 1992) are associated with epidemics.
- Non-O1/Non-O139 groups are Non-pathogenic and some cause sporadic diarrhea.
- O1 is further grouped into biotypes based on its biochemical characteristics, geographical distribution and susceptibility to different phages.
- **Classical O1 (**Bangladesh) and **EL Tor O1 (**worldwide, associated with infections in the recent past)
- O139 has similar characteristics to El Tor.
- Each biotype is further divided into serotypes. ****
- Ogawa (O antigens A and B)
- Inaba (O antigens A and C)
- Hikojima (O antigens A, B, C)
- O1 is unencapsulated while O139 is encapsulated
- Describe the taxonomic classification of Vibrio cholerae
- Serogroups: Contains 200 serotypes classified into 2 groups based on whether they agglutinate with antisera to O1 group
- Vibrio cholerae O1 (Cholera vibrios, Agglutinable vibrios)
- Vibrio cholerae non-O1 (Non-cholera vibrios, Non-agglutinable vibrios) classified into 138 serotypes O2-139, O130 causes cholera in humans and is encapsulated
- Epidemic cholera
- V. cholerae O1, V, cholerae O139
- Biotypes: O1 is further classified into biotypes based on biochemical characteristics, geographical distribution and susceptibility to different phages
- Classical biotype O1 (Bangladesh)
- El Tor biotype O1 (Similar to O139)
- Serotypes: Classical and El tor biotypes are further divided into serotypes according to the structure of the O antigen
- Ogawa (O antigens A and B)
- Inaba (O antigens A and C)
- Hikojima (O antigens A, B, C)
- Serogroups: Contains 200 serotypes classified into 2 groups based on whether they agglutinate with antisera to O1 group
- Describe the Epidemiology of cholera
- Cholera occurs as an endemic, epidemic, or pandemic disease.
- There have been 7 major pandemics since 1817, caused by V.cholera O1 biotype El Tor that began in 1961 (Asia),1970s and 1980s (spread to Africa, Europe, and Oceania), and in **1991 (**spread to Peru, South and Central America).
- The other 6 were caused by classical biotypes.
- 2nd epidemic strain was isolated in 1992 India (V.cholera O139 Bengal)
- There are 3-5 million cases and 120,000 deaths worldwide each year.
- Most recent epidermis include:
- 2004( Bangladesh)
- 2008-2009 (Zimbabwe)
- 2010 (Haiti).
- How is vibrio transmitted?
- Vibrio infections mostly occur from **consumption of raw or undercooked shellfish (**Since they require salt to grow, they are associated with ocean-sourced seafood. Vibrio can multiply freely in water and bacterial levels in contaminated water increase during warm months.)
- Most common route of spread is via consumption of contaminated water.
- Direct person-person spread is rare because the Infectious Dose (ID) of vibrio is high.
- Reservoirs for Vibrio cholera include humans and water.
- What are the virulence factors for Vibrio cholerae
- Pili: adhesion
- Mucinases (Proteases): dissolve protective glycoprotein coating epithelial cells
- High motility: via monotrichious flagella
- Siderophore: iron sequestration
- Neuraminidase: what does it do?
- Accessory colonization factors: adherence
- Cholera toxin: Gene carried on a single-stranded DNA bacteriophage called CTX introduced by horizontal gene transfer. Encodes the genes for the 2 subunits of cholera toxin: ctxA and ctxB)
- Briefly describe the pathogenesis of cholera
- Transmission: Consumption of raw or undercooked shellfish, consumption of contaminated water, rare person-person spread, human and water are reservoirs
- Virulence factors: PilI, Mucinase (Protease), Monotrichious flagella, Siderophore, Neuraminidase, Accessory colonization factors (adhesin), Cholera toxin – Remember to list their functions in the exam
- A large infectious dose (10^3 – 10^6) is required when ingested in H20, food requires 10^2-10^4
- Susceptible individuals: On antacids, Achlorhydria, After meals
- Inoculation after consuming contaminated sources
- Mucinase facilitates bypass of the mucus layer of the stomach
- Pili facilitate adherence
- Vibrios multiply and release Cholera toxin
- MOA of cholera toxin: Gene carried on a single-stranded DNA bacteriophage (CTX coding ctxA and ctxB subunits – B binds to receptor, A activates Gs via ADP ribosylation, stimulating AC and cAMP causing Cl- secretion via CFTR and H2O secretion at the gut and osmotic diarrhoea. Blocks absorption of sodium and chloride
- Massive rice-water diarrhea, losing water and electrolytes
- What are the clinical features and complications of cholera
- Incubation: short (hours)
- Clinical features
- Rice-watery diarrhea
- Dehydration (loss of skin turgor)
- Complications
- Hypovolemic shock
- Acute tubular necrosis
- Metabolic acidosis
- Arrythmia
- Hypoglycemia
- Leg cramps
- Briefly describe the laboratory features of Cholera
- Specimen: stool or rectal swabs in Cary Blair transport medium (kept at room temperature, liquid stool is refrigerated) ***(Note that Aimes media is used to transport genital specimen i.e. Neisseria gonorrhea, Haemophilus ducreyi)
- Wet prep: Darting motility appears like “shooting stars” or “swarms of gnats” in dark-field or phase contrast microscopy
- Specificity: demonstrated by inhibiting motility with specific antisera
- Gram stain of stools: Sheets of curved gram negative rods, parallel rows (described by Koch as “fish in stream” appearance. Frequently pleomorphic in old cultures)
- Culture
- Alkaline peptone water: To select for alkaline-tolerant vibrios
- TCBS: Yellow colonies, Sucrose fermenter
- TTGA (Monsur’s media): Grey colonies with dark centre, clear halo around colonies (seen best with tranillumination, caused by gelatinae productio
- Biochemical tests
- TSI: A/A, no gas, no H2S
- Oxidase: Oxidase positive (distinguish from enterobacteriacea which are mostly oxidase negative)
- Indole: Positive
- Reduce nitrated to nitrites
- Cholera red reaction: Positive
- Methyl red: positive
- Catalase: positive
- Urease: negative
- String test: Incomplete
- Serotyping: for V.cholerae O1 and O139 with specific antisera
- Monoclonal antibody-based tests: detect O1 & O139
- PCR: for toxigenic strains + ELISA or Latex agglutination assay
- Rapid dipstick immunochromatographic – detects V.cholerae O1 and O139 in fecal specimen
- Tests to demonstrate toxin
- Rabbit ileal loop test
- cAMP estimation
- Histological changes i.e. adrenal tumor cells
- Serological tests
- Vibriocidal Abs react to cell surface LPS
- Peak 7 days after infection: retrospective confirmation of diagnosis of V.cholerae infection (not useful in the acute phase of management)
- Day 1-5: acute
- 7-21: convalescent
- 4 fold increase between acute and convalascent
- 4 fold decrease between early and late (2 months) convalescent sera is considered diagnostic
- Abs to CT: rise 1-4 weeks after infection and remain elevated, may also be diagnostic
- Useful in epidemiologic surveys
- Referring to Cholera, briefly describe the skin-bluing test (reaction) and Blue death
- The skin-bluing test is done on rabbits.
- Cholera toxin is injected into skin and Evans Blue dye is injected intravenously.
- Cholera toxin increases the skin’s permeability for the dye it to become blue.
- Blue death (Blue terror or Black cholera) is a historic description of cholera because of its cyanotic reaction (due to the extreme fluid loss that happens)
- The skin-bluing test is done on rabbits.
- Describe the principal and components of Thiosulphate citrate bile salt sucrose (TCBS) agar)
- yellow colonies, sucrose fermenter
- Sucrose: fermentation produces acid turning pH indicator blue/green → yellow
- Bile salt: inhibit growth of other bacteria
- Na thiosulphate: source of sulphur, blackening, not important in cholera
- Thymol blue + bromothyl blue: indicators
- pH: Alkaline
- Preferred because it is easy to prepare and colonies are well presented
- Positive for cholera = yellow colonies, sucrose fermenter
- Describe the principal and components of Taurocholate tellurite gelatine agar (TTGA/ Monsur’s media)
- Monsur’s media is used for the isolation and selective identification of Vibrios from specimen.
- Positive for cholera = grey colonies with dark centre, clear halo around colonies (seen best with transillumination, caused by gelatinase production)
- Describe the principal of the cholera red reaction
- Few drops of concentrated sulphuric acid is added to peptone water for 24H at 37*C giving a red colour as the organism produces nitrosos indole
- Positive for cholera = red colour
- How is cholera treated
- Prompt ORS administration (up to 6L in 1st day for moderate dehydration. Severe dehydration given rapid IVFs isotonic solution (Ringer’s lactate) 20mL/Kg to a max of 2L every 5-30 min (improve HR returns to a normal range) Dextrose containing solutions should not be used for volume repletion
- Antibiotics reduce volume of diarrhea, duration of vibrio excretion to about 1 day, volume of rehydration fluids needed, reduced treatement expense. Tetracycline, doxycycline, FQs, Erythromycin. Pregnant women and children (Erythromycin standard choice in children, 2nd choice is Azithromycin), TMP-SMX and furazolidone
- Mass antibiotic administration is not recommended (no proven effect on spread)
- Antibiotic prophylaxis can be given to household contacts of patients
- How is cholera prevented and controlled
- Chilling seafood to <5C (41F)
- cooking seafood to at least 65C (149F)
- Hand-washing with soap,
- safe food preparation + storage,
- safe disposal of the feces of children
- Oral inactivated cholera vaccine (OCV): Endemic areas, humanitarian crises, during outbreaks, additional to other prevention/control strategies, Should not disrupt provision of other high priority health interventions for control/prevention ***Require 2 doses for full protection. Sanchol and Euvichole for mass vaccination campaigns, protect for 3 y
- Dukoral: all individuals > 2 y, travellers, protection for 2y
- Shanchol
- Euvichol-plus
- Vaxchora (US) – single dose
- Briefly describe Non-O1, Non-O139 Vibrio Cholerae
- Non-O1/139 Vibrio cholerae are transmitted by consuming raw, improperly cooked, or cooked and re-contaminated sea food i.e. shellfish.
- Rarely transmitted through wounds exposed to water containing V.cholerae. ****
- Few strains produce Cholera Toxin.
- Produce heat-stable enterotoxin (NAG-ST) bearing a close resemblance to heat-stable enterotoxin of ETEC.
- Possess genes for a type III secretion system.
- Produce extracellular products (cytolysisn, hemolysisn) and are heavily encapsulated.
- They cause gastroenteritis (ranging from mild to severe disease, no fever or bloody changes, Infective dose is suspected to be > 1 million)
- Sepsis occurs in immunocompromised and individuals with liver disease.
- Cause wound infections.
- Differentiated from O1 and O139 by serotyping.
- Treatment is supportive. Antimicrobials include tetracycline, ciprofloxacin and 3rd gen cephalosporins
- Non-O1/139 Vibrio cholerae are transmitted by consuming raw, improperly cooked, or cooked and re-contaminated sea food i.e. shellfish.
- Briefly describe Vibrio parahemolyticus
- Parahemolyticus is a halophilic (salt-requiring) organism with a single sheathed monotrichous flagellum and thin peritrichous flagellae.
- He grows in alkaline solution, 8-10% NaCl solution and is transmitted by ingesting undercooked seafood (oysters and improper food handling)
- It is associated with the Kanagawa phenomenon (Named after the prefecture in Japan where it was discovered,
- Virulent human strains produce B-hemolysis on human BA while non-human strains are non-hemolytic when cultured on human BA)
- They are frequent cause of seafood-associated Gastroenteritis.
- Incubation period is 24 hours with Diarrhoea, Nausea, Vomiting and abdominal crapms +/- fever.
- It is self-limited and lasts for 3 days.
- Severe disease is rare and is seen in immunocompromised individuals.
- It less commonly causes wound infections and septicemia.
- Production of thermostable direct hemolysin (Vp-TDH, Kanagawa hemolysisn) is responsible for B-hemolysis on a modified BA (Wagatsuma agar)
- Kanagawa-positive strains produce diarrhea in volunteers
- Kanagawa-negative strains failed to do so in 15 volunteers.
- Deletion of the Vp-TDH gene causes loss of enterotoxic activity.
- Laboratory features
- Specimen: stool, wound swabs, blood.
- Culture: BA hemolysis, TCBS blue-green colour (does not ferment sucrose)
- Oxidase positive
- Positive Kanagawa reaction.
- Accurace of biochemical tests is improved with addition of a few drops of 10% NaCl to conventional biochemicals prior to inoculation.
- Treatment is by fluid replacement and antimicrobial (Doxycycline, ciprofloxacin e.t.c)
- Briefly describe Vibrio vulnificus
- Vulnificus is known as terror of the deep sea (due to the severe fulminant infection it causes).
- It inhabits salty waters (halophilic), found in most coastal waters, primarily in estuaries where the tide flows in to a river causing fresh and salt water to mix,
- It is assocaited with plankton, shellfish and finfish.
- It is encapsulated and is considered the most serious pathogenic vibrio in industrialized nations.
- Most severe infection is in patients with:
- Hepatic disease
- Hematopoietic disease
- Chronic Kidney Disease
- Those receiving immunosuppresive drugs.
- Biotypes
- Biotype 1 (all human infections)
- Biotype 2 (primarily eel pathogens)
- **Biotype 3 (**a hybrid of 1 and 2, associated with tilapia-associated wound infections in Israel).
- Has fimbriae, polysaccharide capsule, hemolysisns, proteases, collagenases, mucinases, esterases, chondroitinases, hyaluronidases, DNAses and Siderophores.
- Clinical features:
- Gastroenteritis when consumed, incubation of around 38 hours, disease progression is rapid.
- Primary wound infection presents as initial swelling, erythema and pain at the wound site, followed by development of vesicles or bullae and eventual tissue necrosis +/ systemic signs of fever and chills.
- Morality ranges 20-30% for those with primary wound infection.
- Primary septicemia after Gastroenteritis or rapidly progressive wound infection is characterized by sudden onset of fever and chills, Vomiting, Diarrhoea and abdominal pain.
- 70% have distinctive bulbous skin lesions with tissue necrosis and mortality is more than 50%.
- Laboratory features
- Speciment: stool, pus, skin lesions, blood.
- Direct gram stain reveals GN bacterium.
- BA: no hemolysis, routinely used for wound cultures. TCBS: typical blue-gree colonies.
- Oxidase positive. No sucrose fermenting but LF (Other vibrios are NLFs).
- Treatment is by management of symptms. Antimicrobial include (doxycycline, ciprofloxacin, minocycline, imipenem, 3rd gen cephalosporins) and wound debridement. P
- revention in high-risk individuals is by avoiding raw seafood and chilling to <5 C etc.
- Give a summary of the growth characteristics of Vibrio spp.
- Oxygen requirements: Grows best under aerobic conditions, Scanty and slow growth unfer anerobic conditions
- Temperature requirements: 37*C
- pH requirement: alkaline medium, pH 8.2
- Haplophilic: NaCl concentration of 0.5-1%, >5% is inhibitory, Vibrio cholerae can grow in the absence of salt
- Media:
- Non-selective: growth on NA, MAC, BA, Gelatin agar
- Special media (transport, enrichment, selective): TCBS, TTGA, Alkaline peptone water
Aeromonas
- Briefly describe Aeromonas
- Aeromonas are aquatic organisms.
- Main species are:
- Aeromonas hydrophila
- Aeromonas sobria
- Aeromonas caviae.
- It causes diarrhea and soft-tissue infections.
- Diarrhea is more common in summer months when water concentrations of aeromonads are increased. Also associated with outbreaks.
- Diarrhea is watery and self-limiting +/- more severe +/- chronic colitis.
- Septicemia occurs in immunocompromised individuals.
- Wound infections in healthy people and soft tissue infection in those with water exposure.
- Rarely causes:
- nosocomial bacteremia
- peritonitis
- meningitis
- eye and bone joint infection.
- Laboratory features
- Facultative anerobic GNR
- B-hemolytic on BA
- ferments CHOs to produce acid and gas.
- Variable LFs on MAC.
- TCBS yellow colonies.
- In Vitro clinical improvement with FQs, Co-trimoxazole and Aminoglycosides (except streptomycin) Resistance to carbapenems (chromosomal carbapenemases) and ampicillin
- How is Aeromonas differentiated from Vibrios
- Motile in distilled water (vibrios are immobilised) ****
- String test
- Hydrolyzes aesculin
- Oxidase positive.
