• Hemophilus influenzae also called ‘ Pfeiffer bacillus ‘/ influenza bacillus

Morphology & Taxonomy

• Gram -ve coccobacillus
• Non motile
• Non sporing
• Cells from older cultures exhibit pleomorphism
• Capsulated strains are pathogenic
• Classification
  • By Pitman
  • 6 capsular types ‘ a to f ‘
  • Based on capsular polysaccharide
  • 95% of H. influenzae isolates from acute invasive disease belongs to type ‘ b ‘
  • Non capsulated strains are termed – ‘ non typable ‘ since they lack capsular polysaccharide

Culture

• Aerobic & facultative anaerobe
• Fastidious growth requirements
  • X factor, V factor ( present in blood ) are essential for growth
  • X factor – heme and its enzymes, Heat stable
  • V factor – NAD/NADP, Heat labile, synthesized by some fungi & bacteria ( S. aureus ) ; exhibits satellitism – satellite colonies of H. influenzae develop around S. aureus

• Chocolate agar – when Blood agar is heated, factor V is released from RBCs

• Blood agar – streaked with S. aureus
• Nutrient agar – with X & V factors
• Levinthal medium – prepared by boiling & filtering the mixture of blood agar & nutrient broth
  • Capsulated strains form distinct iridiescence
• Fildes agar
  • Best for isolation of H. influenzae

Epidemiology

• Exclusively human pathogen
• Transmission – droplets / direct contact with secretion or fomites
• Colonisation of upper respiratory tract is common but such strains are usually uncapsulated
• Immunity is type specific

Pathogenesis

• Virulence factor – Type b polysaccharide capsule
• Disease due to invasion & hematogenous spread
• Persistent non typable H. influenzae colonisation of lower airway contributes to airway inflammation in adults with COPD – a Hallmark feature

Clinical features

• Meningitis – most serious
  • Primarily affects children < 2 yrs
  • Fever & altered CNS function – most common presentation
  • Most common complication – subdural effusion
• Epiglottitis / croup
  • Life threatening – tracheostomy is often necessary
  • Cellulitis of epiglottis & Supra epiglottic tissue
  • occurs in older children & also in adults
• Cellulitis
  • In young children
  • Most common location – head / neck
  • Associated with bacteremia
• Pneumonia
  • In infants
  • More likely to involve pleura also
• Other – osteomyelitis, Septic arthritis, orbital cellulitis, pericarditis, endophthalmitis, UTI, abscesses, bacteremia

Lab diagnosis

• Microscopy – pleomorphic Gram -ve, bacilli
• Direct antigen detection –
  • By rapid latex agglutination test
  • capsular polysaccharide in CSF & urine
• Culture

Treatment

• Cephalosporins – ceftriaxone or cefotaxime
• In Hib meningitis – glucocorticoids reduces neurological sequelae

Prevention

• Vaccination
  • Three conjugate vaccines
  • Only for Hib
• Chemoprophylaxis
  • Oral Rifampicin

Courtesy of featured image :- CDC/ Sarah Bailey Cutchin

• Etiological agent of Whooping cough/ pertussis

Morphology & Taxonomy

• Gram -ve coccobacillus
• Capsulated
• Non motile
• Non sporing
• ‘Thumb print’ appearance – in culture films
• Bipolar metachromatic granules stained by toulidine blue
• Species include
  • B. pertussis – causes Whooping cough
  • B. parapertussis –
    • infrequent cause of Whooping cough ;
    • differentiated from the former by the
      1. ability to grow on nutrient agar with production of brown diffusible pigment
      2. More rapid growth on charcoal agar
      3. Strong agglutination with parapertussis antisera
      4. Less severe disease
  • B. bronchiseptica
    • Motile with peritrichate flagella
    • Small proportion of whooping cough

Culture

• Obligate aerobe
• Complex media necessary for primary isolation
• Bordet Gengou glycerine potato blood Agar or Regan Lowe media – blood neutralises inhibitory materials
  • Bisected pearls/ mercury drops appearance
  • Colonies surrounded by hazy zone of hemolysis
  • Confluent growth – aluminium paint appearance
• Charcoal blood agar – charcoal absorbs inhibitory materials
• Transported by Casamino acid solution, modified Stuart medium, Mischulow charcoal agar

Epidemiology

• Highly communicable
• Attack rates 80-100% among unimmunized household contacts
• Worldwide distribution
• Cyclical outbreaks every 3-5 yrs
• An important cause of infant mortality & morbidity in developing nations
• Can affect people of all ages
• Decreased incidence due to improved vaccine coverage

Pathogenesis

• Incubation period 7-10 days
• Both humoral & cell mediated immunity play a role
• Virulence factors – agglutinogens, pertusis toxin, filmentous hemagglutinin, lipopolysaccharide
• Infection initiated by attachment of organism to ciliated epithelial cells of nasopharynx, mediated by adhesins

Clinical features

• Obligate human parasite
• Prolonged coughing illness with clinical features varying with age
• Most often seen in preschool & school age children
• 3 stages
  1. Catarrhal stage – indistinguishable from common cold
  2. Paroxysmal stage – after 1-2 wks
     • violent spasms of continuous cough followed by rapid inspiration against closed glottis – characteristic whoop
     • post tussive vomiting is frequent, with expulsion of mucus plug at the end
     • During spasm, neck-vein distention, bulging eyes, protrusion of tongue, cyanosis
     • May be precipitated by noise, eating, physical contact
     • Frequency 5-10/ day usually
     • Often worsen at night
     • Fever uncommon, suggests bacterial superinfection ( usually by capsulated organisms )
  3. Convalescent stage – after 2-4 wks, gradual resolution of coughing episodes
• In adolescents & adults – more often atypical presentation
• Complications :-
  • Pressure effects – subconjunctival hemorrhages, subcutaneous emphysema, hernias, petechiae
  • Respiratory – bronchopneumonia, lung collapse
  • CNS – convulsions, coma
• Differential diagnosis – Respiratory syncitial virus,Adenovirus, Mycoplasma pneumoniae, Chlamydia pneumoniae, ACE inhibitors, reactive airway disease, GERD

Lab diagnosis

• Microscopy – demonstrating bacilli by fluorescent antibody technique

• Culture – gold standard
• PCR
• Serology

Treatment

• Antibiotics – macrolides , cotrimaxazole ( alternative )
  • Don’t alter clinical course substantially unless given in catarrhal phase
• Supportive care
• Infection control measures

Prevention

• Chemoprophylaxis
  • For household contacts
  • Erythromycin, Clarithromycin, Azithromycin, Cotrimaxazole
• Immunisation
  • Mainstay of prevention is active immunization
    1. Whole cell killed vaccine
    2. Acellular pertusis vaccine

Courtesy of featured image : CDC/ CDC-Antibiotic Resistance Coordination and Strategy Unit

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• Normal inhabitants of upper respiratory tract
• Most prevalent organisms in Pneumonia & Otitis media in children

Morphology & Taxonomy

• Gram +ve
• Diplococci –
  • lanceolate shaped/ flame shaped
  • Slightly elongated cocci, one end rounded & other end pointed
  • Broad ends in apposition
• Differentiating features from other streptococcus
  1. Its morphology – diplococci
  2. Bile solubility
  3. Optochin sensitivity
  4. Specific polysaccharide capsule
• Non motile
• Non sporing
• Readily stained by aniline dyes
• Capsule seen by India ink preparation

Culture

• Grow only in enriched media
• Aerobes, facultative anaerobes
• Growth improved by 5-10% CO2
• Blood agar
  • After 18 hrs – small dome shaped glistening colonies with surrounding green area of α-hemolysis
  • On further incubation – flat colonies with raised edges – doughnut / draughtsman / carrom coin appearance
  • In anaerobic conditions – colonies surrounded by beta hemolysis due to oxygen labile hemolysin O

• Liquid media
  • Uniform turbidity
  • Autolysis of cocci due to intracellular enzymes
• Optochin sensitivity
  • Sensitive to optochin – ethyl hydrocuprein

Epidemiology

• Normal commensal in upper respiratory tract
• Transmission by droplets

Pathogenesis

• Virulence factors
  • Capsule – acidic, hydrophilic, protects from phagocytosis, capsular polysaccharide also called ‘Specific soluble substance'( SSS ), adapts to external environment
    • Also shows Quelling reaction – capsule swells on addition of antisera & methylene blue for visualisation
  • Pneumolysin – secreted cytotoxin, causes cytolysis of cells & tissues
  • Pneumococcal H inhibitor – impedes formation of C3 convertase in complement
  • Pneumococcal surface protein C – binds to factor H & accelerate breakdown of C3
  • Pili help in binding of bacteria to cells

Clinical features

• Asymptomatic/ mild symptoms in initial infection when young
• Spread through blood to distant sites or locally to mucosal surfaces, direct spread to CNS is rare but can occur in skull base fractures
• Non invasive – otitis media
• Invasive – Bacteremic pneumonia, Meningitis
• Pneumonia – mild to life threatening disease
  • Associated with viral upper respiratory tract infection
  • Abrupt onset of cough & dyspnea, rusty sputum
  • Along with fever, shaking chills, myalgias
  • Pleuritic chest pain & significant dyspnea – indicates involvement of parietal pleura
  • High degree of suspicion required
  • Empyema – most common focal complication
• Meningitis
  • Pyogenic
  • Primary or secondary from otitis media, Bacteremia, mastoiditis
  • One of the Most common etiologies of meningitis in children
  • Severe generalized gradual onset headache, fever, nausea
  • CNS symptoms – Stiff neck, photophobia, seizures, confusion along with altered consciousness, Brady cardia, hypertension ( Increased Intracranial pressure )
  • Brudzinski sign, Kernig sign +ve in some adult patients
• Osteomyelitis
• Septic arthritis
• Endocarditis
• Pericarditis
• Peritonitis
• Sinusitis & otitis media – acute, severe pain, fever, deafness, tinnitus
  • Red swollen Tympanic membrane with reduced movement

Lab diagnosis

• Microscopy – gram +ve diplococci
• Culture
• Biochemical tests
  • Catalase & oxidase -ve
  • Hiss serum sugars – fermentation
  • Bile solubility – constant property of Pneumococcus
• Antigen detection – SSS detection in CSF by precipitation
• Biomarkers – CRP
• Mouse inoculation
• Molecular methods

Treatment

• Parenteral penicillin G – drug of choice for susceptible strains
• Macrolides, cephalosporins
• Menigitis – Vancomycin + cefotaxime/ ceftriaxone ; if allergic to ß-lactams, rifampin can be replaced
• Other invasive infections – Penicillin G ; cefotaxime/ ceftriaxone
• For OP management – Amoxicillin/ Levofloxocin
• Acute otitis media – Amoxicillin

Prevention

• Capsular polysaccharide vaccines
  • PPSV23 – 23 valent, T cell independent
• Polysaccharide protein conjugate vaccines
  • PCV – 2 vaccines containing 10 & 13 serotypes available

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• Vibrio – derived from characteristic vibrating motility
• V. cholera – causes cholera, first isolated by Koch

Morphology & Taxonomy

• Gram -ve
• Curved bacilli – comma shaped
• Actively motile – single polar flagellum – darting motility
• Fish in stream appearance
• Swarm of gnats appearance – when actively motile
• Classification
  • Heiberg – 6 groups based on fermentation of mannose, sucrose, arabinose
  • Serological classification ( by Gardner & Venkatraman )
    1. Grp A – cholera vibrios,
       • have common flagellar(H) antigen
       • Subdivided into O1 & non-O1 serovars
       • O1 consists of classical & El Tor biotypes, both are classified into 3 serotypes – ogawa, inaba, hikojima
       • Non-O1 or non-agglutinable(NAG) vibrios classified upto 139 serotypes
    2. Grp B – heterogeneous vibrios
  • By requirement of NaCl
    1. Halophilic – V. alginolyticus, V. vulnificus, V. parahaemolyticus
    2. Non halophilic – V. cholera

Culture

• Aerobic
• Optimum temperature 37°C
• pH optimum 8.2
• 0.5-1 % NaCl required for optimal growth
• Ordinary media
  • Nutrient agar – moist, translucent, round discs
  • MacConkey agar – colourless but red coloured on prolonged incubation – late lactose fermentor
  • Blood agar – intially zone of greening, cleared later due to hemodigestion
  • Gelatin stab culture – funnel shaped/ turnip shaped liquefaction in 3 days
  • Peptone water – growth in 6 hrs
• Special media
  • Holding/ Transportation media – VR ( Venkatraman Ramakrishnan ) media, Cary Blair media – also for Salmonella & Shigella , autoclaved sea water
  • Enrichment media – alkaline peptone water, Monsur taurocholate tellurite peptone water
  • Plating media – Alkaline bile salt agar, Monsur Gelatin taurocholate tellurite trypticase agar, Thiosulphate citrate bile salt sucrose ( TCBS ) medium

• ‘String test’ – to identify Vibrio colonies, growth is mixed with Sodium deoxycholate in saline – mucoid string formation when loop is drawn slowly ( +ve Test )

Epidemiology

• No animal reservoir
• Natural habitat – coastal salt water, brackish waters
• Mostly pediatric disease in endemic areas
• Burden greatest during “cholera seasons” – high temperature, high rainfall, flooding
• Blood group O – greatest risk of severe disease if infected, Blood group AB – least risk
• 7 global pandemics occured – El tor biotype displaced endemic classical type in 7th pandemic
• Asymptomatic carriers may be present

Pathogenesis

• Toxin mediated disease
• Virulence factors – Cholera toxin, Toxin coregulated pilus, other factors ( regulated by ToxR protein )
• Environmental factors, bacterial response to density of bacterial population ( quorum sensing ) modulate virulence
• Cholera toxin – A & B subunits
  • A subunit – enzymatic, causes intracellular accumulation of cAMP
  • B subunit – pentameric binding moeity, facilitates attachment of A subunit
• In intestine, cAMP inhibits absorptive Na+ transport system & activates secretary Cl- transport
• Thus NaCl accumulates in lumen & passive movement of water into the lumen results in watery diarrhea
• Other effects of Cholera toxin – increase skin permeability ( permeability factor ), skin bluing test ( when injected intradermally along with pontamine blue I.v, injected site becomes blue ) – used for detection of toxin

Clinical features

• Incubation period 1-2 days
• Asymptomatic / mild diarrhea in some patients
• Sudden onset explosive & life threatening diarrhea – cholera gravis
• Painless watery diarrhea, vomitings
• Hypovolemic shock ( cause of death in Cholera )
• Fever absent
• Muscle cramps due to electrolyte disturbances
• Stool – Rice watery stool
  • Non bilious
  • Gray, slightly cloudy with mucus flecks
  • No blood
  • Somewhat Fishy odour
• Symptoms depend on water volume lost
  • < 5% of body wt lost – thirst
  • 5-10% lost – postural hypotension, weakness, tachycardia, decreased skin turgor
  • > 10% lost – oliguria, weak pulse, sunken eyes, washerwoman/ wrinkled skin, somnolence, coma
• Complications develop from effects of volume & electrolyte disturbances – renal failure, acute tubular necrosis

Lab diagnosis

• Microscopy – identification of V. cholerae in stool
• Culture
• Slide agglutination tests
• Biochemical tests
  • Ferments carbohydrates but no gas
  • Ferment glucose, mannitol, maltose, mannose
  • Indole formed, nitrates reduced – contributes to cholera red reaction ( reddish pink colour on adding conc. H2SO4 to 24 hr peptone water culture )
  • Catalase +ve, oxidase +ve
• Testing of water samples
• Lab data usually indicates
  • Elevated hematocrit
  • Mild neutrophilia
  • Elevated blood urea nitrogen & creatinine – prerenal azotemia
  • Normal Na+, K+, Cl-
  • Reduced bicarbonate
  • Elevated anion gap

Treatment

• Fluid & electrolyte replacement – ORS is effective ( hexose- Na+ cotransport is intact ), ringer lactate with potassium supplements
  • Total fluid deficit in severe dehydration can be replaced in 3-4 hrs safely – half in first hr
• Oral tetracycline, ciprofloxacin

Prevention

• Provision of safe water
• Safe disposal of feces
• Improved nutition
• Safe food preparation & storage practices
• Vaccines
  1. Parenteral – killed suspension of V. cholera, protection <60%, no local immunity
  2. Oral – killed whole cell & live oral

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