VIBRIO & AEROMONAS Dr. Mohit Bhatia - PowerPoint Presentation

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VIBRIO & AEROMONAS

Dr. Mohit BhatiaAssistant ProfessorDepartment of MicrobiologyAIIMS Rishikesh

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Learning objectives

At the end of the session, students should be able toDescribe morphology and antigensDescribe Pathogenesis & Clinical featuresChoose appropriate lab diagnosis and interpret the resultsDescribe prevention and treatment

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General Properties of Vibrio

Curved Gram-negative bacilli Actively motile by single polar sheathed flagellumFermentative, strongly aerobic, oxidase positive

Non-sporing & non-capsulatedGrowth stimulated by salt (NaCl)Ubiquitously found worldwide in marine environments, surface waters, river & sewage

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VIBRIO CHOLERAE - CLASSIFICATION

Based on Salt RequirementNon-halophilic vibrios - grow without salt, 1% salt is optimum & cannot grow at higher concentrations

Examples V. Cholerae & V. mimicusHalophilic vibrios Cannot grow in absence of salt, grow at higher salt concentratin (7–10%)- Examples – V.parahaemolyticus, V. alginolyticus & V.vulnificus.

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VIBRIO CHOLERAE - CLASSIFICATION

Heiberg Classification (1934)Eight groups based on fermentation of three sugars

Mannose Arabinose Sucrose - V. cholerae was placed in Group I

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VIBRIO CHOLERAE – CLASSIFICATION

Gardner and Venkatraman Classification

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Gardner and Venkatraman Classification

O1 serogroupAgglutinated by O1 antiseraResponsible for all pandemics & most of the epidemics of cholera

Non-agglutinable (NAG) vibriosNot agglutinated by O1 antiserum Initially thought to be non-pathogenic (non-cholera vibrios –NCV)

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Gardner and Venkatraman Classification

O139 serogroupSince 1992 has caused several epidemics and outbreaks - coastal India & Bangladesh.Non O1/139 serogroups - occasional

sporadic outbreaks of diarrhoea & extra intestinal manifestations, but never epidemic cholera

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Differences between classical & El Tor V. cholerae

Biotypes of

V. cholerae O1Classical biotype El Tor biotypeβ hemolysis on sheep blood agar NegativePositiveChick erythrocyte agglutination

Negative

Positive

Polymyxin B (50 IU)

Sensitive

Resistant

Group IV phage susceptibility

Susceptible

Resistant

El Tor Phage V susceptibility

Resistant

Susceptible

VP (Voges Proskauer) test

Negative

Positive

CAMP test

Negative

Positive

Cholera toxin gene

CTX-1

CTX-2

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Serotypes of V. cholerae O1

Serotype

O antigen typesOgawaA,B InabaA,CHikojimaA,B,C

Ogawa

- most common serotype isolated followed by Inaba

During epidemics, shifting between serotypes can take place

Hikojima

- unstable transitional state, both Inaba and Ogawa antigens expressed

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Pathogenesis of Cholera

Transmission - ingestion of contaminated water or foodInfective dose: Acid-labile, High infective dose

Factors promoting transmission – hypochlorhydriaCrossing of protective layer of mucus – active motilitySecreting mucinase and other proteolytic enzymesSecreting hemagglutinin protease (cholera lectin)Adhesion and colonization- facilitated by a special type IV fimbria called toxin coregulated pilus (TCP)

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Mechanism of action of cholera toxin

Fragment B - binds to GM1 ganglioside receptors Fragment A2 - tethering A and B subunits together

Fragment A1 – active fragment - adenylate cyclase → cAMP

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Pathogenesis of Cholera

Increase in cyclic AMP - accumulation of sodium chloride in intestinal lumen  Water moves passively into the bowel lumen  accumulation of isotonic fluid (watery diarrhoea)

Loss of fluid and electrolytes shock (due to profound dehydration) and acidosis (due to loss of bicarbonate)

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Pathogenesis of Cholera

Gene for cholera toxin (CTX) - Pathogenicity island encoded filamentous bacteriophage (CTXf) - integrated as prophageOther genes for pathogenicity are clustered together

Biosynthesis of TCP, Accessory colonization factorsRegulator genesToxR gene regulates the expression of CT, TCP and other virulence factors

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Other virulence factors

Zona occludens toxin - disrupts the tight junctions between mucosal cellsAccessory cholera enterotoxin- phage packaging and secretion

Vero cell toxin - analogous to Shigella toxinAccessory colonization factors - adhesion & colonizationSiderophore - required for iron acquisitionBacterial endotoxin(LPS) - does not contribute to the pathogenesis of cholera. Immunogenic – included in killed vaccines

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Clinical Manifestations of Cholera

1. Asymptomatic infection (75% of cases)2. Mild diarrhoea or cholera (20% of cases)3. Sudden onset of explosive and life-threatening diarrhoea (cholera gravis – 5%)IP - 24 to 48 hours

Watery diarrhoea - sudden onset of painless watery diarrhoeaRice water stool- watery with mucus flakes & inoffensive odourVomiting may be present but fever is usually absent

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RICE WATER STOOLS

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Complications of cholera

Body weight loss by

Symptoms<5%Increased thirstAt 5–10%Postural hypotension, WeaknessTachycardia, Decreased skin turgorAt >10%Renal failure (due to acute tubular necrosis) and fluid loss result in-Oliguria

Weak or absent pulses

Sunken eyes

Sunken fontanelles in infants

Wrinkled ("washerwoman") skin

Somnolence and coma

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Epidemiology - History of Pandemics

Cholera can occur—sporadic, limited outbreaks, endemic, epidemic or pandemicTill 19th century – confined to its home land (West Bengal & Bangladesh)1817 -1923 – 6 pandemics originating from Bengal – Classical Vibrio

1923 – 1961 – Restricted to homeland

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Epidemiology - History of Pandemics

1961 – 7th Pandemic – differed from previous pandemicsOnly pandemic that originated outside India, i.e. From

Caused by El Tor biotypeMilder cholera with more carrier rate Rapid spread of El Tor, involving the entire globe including some unusual parts

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CHOLERA OUTBREAK IN LONDON- LEGACY OF JOHN SNOW

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O139 (Bengal strain)

Isolated first from Chennai in 1992O139 – Not agglutinated by any of the antisera available at that time (O1 to O138)Bengal strain - spread rapidly along the coastal region of Bay of BengalDerivative of O1 El Tor – differs in having a

distinct LPS & capsulated Invasive  bacteremia and extra intestinal manifestationsNo cross protection between O1 and O139By 1994 - O1 El Tor replaced O139

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Current Situation - World

Cholera is a notifiable disease, often under reportedAnnual cases >1.3-4 million Annual deaths - 21 000 to 1.4 Lakh

Majority of cases are due to O1 El Tor

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Current Situation - India

Situation has greatly changedWest Bengal is no longer the home land, all states affectedBoth morbidity and mortality have greatly reduced

National Institute of Cholera and Enteric Diseases (NICED), Kolkata - National reference Center for cholera in India

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Epidemiological Determinants

Reservoirs - Humans the only reservoirSource - asymptomatic cases or carriersIncubatory carriers

- less common, as cholera has a short incubation periodConvalescent carriers: 2–3 weeks after recoveryContact or healthy carriers: infection from subclinical cases  shed bacilli for <10 days Chronic carriers - El Tor has more carrier rate than classicalCholera season- high temperatures, heavy rainfall & flooding

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Epidemiological Determinants

Factors promoting transmission - poor sanitation, poverty, overcrowding, population mobilityFactors determining severity disease:

Lack of pre-existing immunity Blood group - ‘O’ greater risk ; AB - least riskMalnutrition, People with low immunity - during epidemics - childrenHabitat- sea water and brackish estuaries

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Epidemiological Determinants

Persistence of V. Cholerae Epidemics - maintained by carriers & subclinical casesInter epidemic period - maintained in sea water

ResistanceAcid-labile but stable to alkali Heat-labile but stable to refrigerationEasily killed by drying and sunshine & disinfectantsBiotype El Tor more resistant than classical

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Laboratory Diagnosis

Specimens: Freshly collected watery stool - casesRectal swab - convalescent patients or carriers

Transport/Holding MediaVenkatraman-Ramakrishnan (VR) medium - crude sea salt & peptone water (pH 8.6–8.8) Alkaline salt transport medium - VR medium + boric acid, NaOH and KCl (pH 9.2) Cary-Blair medium - buffered solution of sodium chloride, sodium thioglycollate, disodium phosphate and calcium chloride (pH 8.4) Autoclaved sea water

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Direct Microscopy

Gram-staining of mucus flakes of faeces - short curved comma-shaped gram-negative rods, arranged in parallel rows - fish in stream appearance

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Laboratory Diagnosis

Motility testing (hanging drop) - darting motility /shooting star/ swarming gnats motilityMotility testing after adding H-antisera - V.Cholerae

becomes non-motile when a drop of the watery stool specimen is added with flagellar (H) antiserum

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Culture

Nutrient agar - Translucent coloniesPeptone water-

Uniform turbidity with surface pellicle - strongly aerobicBlood agar – Hemodigestion Optimum Temp 37°COptimum pH 8.2 (range 7.4–9.6)NaCl (0.5–1%) stimulates growth

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Culture Medium

Enrichment broths - incubated for 4–6 hours  subculture onto selective medium

Alkaline peptone water (APW)- peptone, NaCl in distilled water (pH 8.6)Monsur’s taurocholate tellurite peptone water (pH 9.0)Both can also be used as transport media.

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Selective media

Alkaline bile salt agar (pH 8.2): glistening, oil drop, translucent coloniesMonsur’s gelatin taurocholate trypticase tellurite agar (pH 8.5)- translucent colonies with a greyish black Center and a turbid halo

MacConkey agar: Late lactose fermentation Mildly selective, also supports Shigella and Salmonella.

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Culture Media

TCBS agar: Thiosulfate, citrate, bile salts (as inhibitor), sucrosepH of 8.6yellow coloured colonies

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Biochemical Tests

Catalase and oxidase positiveIndole test

—positiveCitrate test—variableUrease test—negativeTriple sugar iron medium— A/A, gas absent, H2S absentMR (methyl red) test—positiveVP (Voges-Proskauer) test—positive for El Tor, negative for classical biotype

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Biochemical Tests

Nitrate reduction test is positiveCholera red reaction: Positive

Sugar fermentation test: Ferments glucose, sucrose and mannitol with production of acid but no gas. String test: colony mixed with a drop of 0.5% sodium deoxycholate on a slideSalt tolerance test: to differentiate from halophilic vibriosBiotyping

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Biochemical Tests

Decarboxylase tests:Vibrio utilizes lysine and ornithine Aeromonas utilizes only argininePlesiomonas utilizes all, i.e. lysine, arginine and ornithine

Susceptible to O/129 (vibriostatic agent): Vibrio species are susceptible to 10 μg of O/129 disk while Aeromonas and Plesiomonas are resistant

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Biochemical Tests

Serogrouping - Species identification confirmed by agglutination with V. cholerae polyvalent O antisera.Using group-specific antisera. First the colony is tested with O1 antisera → If found negative, then tested with O139 antisera

SerotypingSerotypeOgawa antiseraInaba antiseraOgawa+-Inaba-+

Hikojima

+

+

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Treatment of Cholera

Fluid replacement: most importantMild to moderate fluid loss: oral rehydration solution (ORS)

Severe cases: Intravenous fluid replacement with Ringer’s lactate (or normal saline)  ORSAntibiotics have a minor role as the pathogenesis is toxin mediatedDrug of choice: Macrolides (Erythromycin or Azithromycin)Alternatives - Doxycycline, tetracycline or ciprofloxacin

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Prevention

General MeasuresSafe water, sanitary disposal of faecesProper food sanitationPrompt outbreak investigation and steps to reduce transmissionNotification

Heath educationChemoprophylaxis - Tetracycline - Household contacts, only during epidemics

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Prevention – Oral Vaccines

Killed Whole-cell vaccine:1. Whole-cell (WC) vaccine - (classical and El Tor, Inaba and Ogawa)2. Whole-cell recombinant B subunit cholera vaccine (WC/rBS) (Dukoral) – same WC vaccine + recombinant cholera toxin B subunit

Schedule: Two doses at 7 days gap (> 2 years)Protection is short lived. Children are better protectedUsed during epidemics and outbreaks but not during inter epidemic period

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Prevention – Oral Vaccines

Oral live attenuated vaccines (OCV) – Under trial. Use mutant strains lacking gene encoding cholera toxinV. cholerae O1 - CVD 103-HgR, Peru-15 and

V. cholerae 638 for classical and/or El Tor biotypesV. cholerae O139 – CVD-112 and Bengal-15CVD 103-HgR vaccine (Orochol) contains a live attenuated strain derived from reference strain 569 B (classical, O1, Inaba), given as single dose. Its protection starts after 8 days

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Non O1/O139 V. Cholerae

Biochemically resemble V. cholerae O1/O139, but do not agglutinate with O1 or O139 antisera.Gastroenteritis: Sea food consumption (raw oysters)

Stool – watery/partly formed & bloody/ mucoidAbdominal cramps, nausea, vomiting and fever Never cause epidemic cholera. Treatment is same as that of choleraExtra intestinal manifestations: Otitis media, wound infection & bacteremiaOccupational or recreational exposure to seawaterTetracycline, ciprofloxacin and third generation cephalosporins

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HALOPHILIC VIBRIOS

Can withstand higher salt concentration (>6%) in contrast to V. cholerae, which can tolerate up to 6%Widespread in marine environmentsCases tend to occur during late summer and early rain fall, when the bacterial counts are highest in the water

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Vibrio parahaemolyticus

Clinical ManifestationsFood-borne gastroenteritis – MC presentationFollowing ingestion of raw or uncooked sea food (e.g. oyster)

Watery diarrhoea or rarely asExtra intestinal manifestations - rare, wound infection, otitis and sepsis.

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Pathogenesis

Virulence factors:Polysaccharide capsuleHaemolysin (thermo-stable)Urease enzymeSecretion systems in cell wall - directly inject toxic bacterial proteins into host cells.

Serotype: Most infections are caused due to serotypes O3:K6, O4:K68, and O1:K-untypable

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Laboratory Diagnosis

Similar to that of V. Cholerae with some distinct properties:Morphology- capsulated

, bipolar staining in fresh isolates and pleomorphism in older culturesMotile by peritrichous flagellaTCBS- green colonies (sucrose no fermenter)Kanagawa phenomenon - β haemolysis on Wagatsuma agarSwarming on blood agarUrease test is positive in few strainsSalt tolerance test - resist maximum of 8% NaCl

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Treatment V. parahaemolyticus

Mostly self-limiting Treatment is same as that of cholera

Indications for antibiotic use: Severe gastroenteritis or extra intestinal manifestations associated with underlying diseases, such as diabetes, pre-existing liver disease, iron overload states, or immunosuppression

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Vibrio vulnificus

Rare but most severe infectionClinical ManifestationsPrimary sepsis - underlying liver disease and iron overload or rarely in renal insufficiency and immunosuppression.

2. Primary wound infection - painful erythematous swelling or cellulitis or even vesicular, bullous or necrotic lesions- Generally affects people without underlying disease

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Laboratory Diagnosis

Blood cultureBiochemical Reactions:- Ferments lactose [the only lactose fermenting

Vibrio]Arginine is not dehydrolyzedTreatment Vibrio vulnificusEarly antibiotic institution- tetracycline, fluoroquinolones, and third-generation cephalosporins Wound debridement & general supportive care

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Vibrio alginolyticus

Occasional cause eye, ear and wound infectionsRarely otitis externa, otitis media and conjunctivitis have been reported, bacteremia in immunocompromised hostsMost salt tolerant Vibrio

(>10%)Usually self-limitingSevere infections - tetracycline and drainage

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AEROMONAS

Earlier placed in the family Vibrionaceae; now assigned to a separate family, AeromonadaceaePathogenicity:Tissue adherence mediated by adhesions such as S-layer and fimbriae

Capsular polysaccharideExotoxins - Aerolysin, phospholipases, haemolysins, enterotoxin and cytotoxin similar to Shiga toxinEndotoxin or LPS

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Clinical manifestations

>85% human infections caused by A. hydrophila, A. caviae and A.veroniiGastroenteritis and peritonitisMusculoskeletal & wound infections

Bacteraemia in immunocompromisedRespiratory tract infectionsHaemolytic uremic syndrome (HUS) - enterotoxin similar to Shiga-S toxin

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Laboratory diagnosis

Motile with single polar flagellum MacConkey agar— non-lactose fermenting coloniesOxidase and catalase positive

Decarboxylase test —utilizes only arginineGrowth is not stimulated by NaCl.Genotypic classificationTreatment Ciprofloxacin and levofloxacinAlternatives - cotrimoxazole and cefepime

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