Systemic Bacteriology

1. 1 Hemophilus

2. Introduction :
Group of small , non motile, non sporing, Pleomorphic Gram negative coccobacilli,
oxidase positive and capsulated.
Hemo – Blood, Philos – Loving ( Greek word).
It requires special growth accessory factors present in the blood – X and V factor
Species :
1. Haemophilus influenzae
2. Haemophilus ducreyi
3. Haemophilus aphrophilus
4. Haemophilus aegyptius
5. Haemophilus parainflunzae
6. Haemophilus haemolyticus.
2 Hemophilus

3. 3 Hemophilus

4. H. Influenzae
Morphology :
 Small, Gram negative coccobacilli or rods, non motile,
 Nonsporing, exhibiting considerable pleomophism.
 In sputum – clusters of coccobacillary form.
 In CSF- long bacillary and filamentous forms are predominant.
 It is difficult to stain. Using loffers methylene blue 5- 10 minutes.
4 Hemophilus

5. Cultural Characteristics :
Aerobic and facultative anaerobic. Optimum temp – 37˚C.
1.X- Factor(Haemin, haematin)- heat stable protoporphyrin IX,
Haemin or some other iron containing porphyrin.
Synthesis of respiratory enzymes
Cytochrome C , Catalase, Peroxidases, Cytochrome
oxidases.
Not required when H.influenzae grows anaerobically.
5 Hemophilus

6. 2. V- Factor – it is a coenzyme, Nicotinamide adenine
dinucleotide (NAD), NAD Phosphate.
Heat labile, acts as hydrogen acceptor, Produced by some animals,
plant cells and bacteria ex. Staph aureus
Blood agar – growth is scanty because, X – available in medium and
V factor is intracellular( inside the RBCs).
Colonies- small , translucent, non hemolytic.
Chocolate agar- grows well, lysis RBCs relaeasing excess Vfactor
and inactivating the NADase .
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7. Statellistism:
It is observed that, H.influenzae can grow blood agar if the source
of V factor is provided.
Procedure :
S.aureus – streaked across the blood agar plate perpendicularly to
H.influenzae streak line
Incubate overnight
Observation :
V factor released from S.aureus . So, it forms large colonies near
to S.aureus streak line and size of colonies gradually decreases
away from S.aureus streak line.
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8. S.aureus
streak line
statellitism
Size of colony
decreases
gradually
Growth on
Chocolate agar
Growth on Blood agar
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9. Antigenic Structure
3 major surface antigens
1. Capsular polysaccharide
2. Outer membrane proteins(OMP)
3. Lipo oligosaccharides(LOS).
Capsular polysaccharide:
Major antigenic determinant
Based on capsular polysaccharisde , it is typed into 6 serotypes ( a to f).
Of these H.influenzae type b is more virulent among all other serotypes.
Hib Capsule – pentose sugars polyribosyl phosphate ( ribose and rabitol). Instead of
hexoses and hexosamines.
It is strongly immunogenic, induces IgG, IgM, IgA antibodies – bactericidal, opsonic and
protective
PRP antigen – used for vaccination.
9 Hemophilus

10. Outer membrane protein (OMP): Shows considerable variation.
Hib classified into atleast 13 subtypes.
Lipo oligosaccharides- antigenically complex.
OMP and LOS –sub typing may be used as epidemiological value.
Virulence Factor :
Capsular Polysaccharide- Inhibits phagocytocysis
Endotoxin –induces hosts inflammatory responses
OMP- adhesion and invasion of host tissues.
IgA1 Proteases- Inactivate IgA present on the mucosal surfaces.
Pilli and other adhesion proteins – helps in colonization on epithelial cells.
10 Hemophilus

11. Pathogenesis:
Resides in upper respiratory tract and spread by Respiratory droplets.
H.influenzae causes invasive and non invasive infections.
Invasive infection caused by capsulated strain, spread through blood causing
meningitis, bacteremia, pneumonia, arthritis etc.
whereas Non invasive caused by non capsulated strain. Spread by local
invasion causing secondary or superadded infections.
Non invasive present in nasopharynx or throat of 25-80 % of the healthy
individuals.
Whereas Invasive strains only 5-10%.
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12. Clinical Manifestations
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13. Meningitis:
More common in children less than 3 years of age,
Fatality rate is upto 90% - untreated .
Signs - Fever
Headache
Stiff neck
Nausea with or without vomiting
Increased sensitivity to light (photophobia)
Altered mental status (confusion).
Subdural effusion – Seizures or
hemiparesis
13 Hemophilus

14. Pneumonia :
non-invasive : Bacteremia or pleural fluid (fluid surrounding the lungs) infection
doesn’t occurring at the same time.
When there is pneumonia with either bacteremia or pleural fluid infection
occurring at the same time, it is considered invasive.
Older children and adult – lobar pneumonia- capsulated strain.
Bronchopneumonia – secondary infection, non capsulated strains.
Symptoms:
Fever and chills
Cough
Shortness of breath or difficulty breathing
Sweating
Chest pain
Headache
Muscle pain or aches
Excessive tiredness
14 Hemophilus

15. Epiglottitis:
Cellulitis of epiglottis and supraglotic tissues.
It is seen in older children (2-7 ) years old ,
untreated cases may be fatal within hours, Lead to acute airway
obstruction .
15 Hemophilus

16. Less common ,
Cellulitis of neck and head region
Osteomyelitis , Septic arthritis
Pericarditis
Orbital cellulitis, Endophtalmitis
Urinary tract infection
Bacteremia without an identifiable focus.
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17. Non invasive :
Non-tyable strains because they do not have capsules.
Contagious spread, usually affects adults.
Manifestations include,
1. Childhood otitis media
2. Exacerbations of Chronic Obstructive Pulmonary Disease ( COPD)
3. Pneumonia
4. Puerperal sepsis and neonatal bacteremia
5. Sinusitis .
17 Hemophilus

18. Laboratory Diagnosis:
Sample : blood, CSF, Throat Swabs, Sputum, Pus, Aspirates from joint, middle
ears or sinuses.
Storage - highly sensitive to temperature, Should never Refrigerator.
Direct Detection
Gram Staining – Pleomorphic gram negative cocco bacilli
Capsule Detection (Quelling reaction)- drop of CSF + type b antiserum+
Methylene blue
Observed under microscope .
Antigen Detection – Latex agglutination
Coagglutination- S.aureus coated with type b antigen
18 Hemophilus

19. Culture :
1. Blood Agar – Scanty Growth.
Statellitism – colonies of H.influenzae seen in adjacent to S.aureus streak line .
2. Cholocate Agar - Grows well , lysis RBCs releasing excess V factor and
inactivating the NADase .
3. Fildes Agar and Levinthal’s Agar – Transparent media , Produces
iridescent colonies
Biochemicals
Catalase – Positive
Oxidase – Positive
Ferments- Glucose and xylose.
19 Hemophilus

20. Disk Test – X and V requirements
Based on the growth surrounding X,V and XV discs on the medium, detect the
species of Hemophilus.
Growth surrounding X disc – H.ducreyi, H.aphrophilus.
Growth surrounding V disc – H. parainfluenzae, H.parahaemolyticus, H.
paraphrophilus.
Growth surrounding XV disc – H.influenzae, H. haemolyticus, H. aeyptius.
20 Hemophilus

21. Treatment and prophylaxis
Susceptible – Sulphonamides, Trimethoprim, Ampicillin, Chloramphenicol,
Ciprofloxacin, Cefroxime, Cefotaxime, Ceftazidime.
Prophylaxis
Hib Conjugate Vaccine – Polyribosyl Ribitol
Phosphate (PRP) used for Vaccination.
Capsular antigens are poor immunogenic to
children.
Conjugated with adjuvant ( Dipheria toxoid,
Tetanus toxoid, and N.meningitidis OMP.
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22. Doses of Hib vaccine are usually recommended at these ages:
First Dose: 2 months of age
Second Dose: 4 months of age
Third Dose: 6 months of age (if needed, depending on brand of
vaccine)
Final/Booster Dose: 12-15 months of age
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23. H. ducreyi
Etiological agent of Chancroid or Soft Chancre.
It is transmitted sexually characterized by,
Painful genital Ulceration that bleeds easily,
No inflammation of the surrounding skin
Enlarged, Tender inguinal lymph nodes(Bubo).
Incubation Period : 4-7 days
No immunity , but Hypersensitivity may develop.
23 Hemophilus

24. Epidemiology
Genital ulcers in developing countries,
Transmission – Heterosexual
Male to Female ratio – 3:1 to 25:1
Chancroid and HIV - Chancroid increases efficiency of both
transmission and degree of susceptibility to HIV infection.
24 Hemophilus

25. Lab Diagnosis
Samples : Exudate or swab from edge of the ulcer
lymph node aspirates
Direct Microscopy :
Pleomorphic gram negative cocco bacilli, occurs in groups or in parallel of
chains.
Frequently take bipolar staining.
Arrangement described as “ School of Fish or rail road track”
appearance
25 Hemophilus

26. Culture :
Opt temp : 35˚C - 2-8 Days, or 10% CO2 , high humidity
Medium :It require factor X (hemin) , factor V (NAD) not required.
Primary isolation is difficult
Rabbit blood agar or chocolate agar enriched with 1% isovitalex or
fetal calf serum and adding vancomycin as a selective agent.
It also grown on Chorioallantoic membrane of the chick embryo.
Colony morphology : small, grey, translucent, 1-2 mm in size in 2-3 days.
Treatment : Azithromycin (1 g oral, single dose)- Drug of choice
Ceftriaxone, Ciprofloxacin or Eryhromycin- Alternative drug
26 Hemophilus

27. H.aeyypticus :
Also called Koch- Weeks Bacillus , closely resembles non capsulated
H.influenzae biotype III
Differs from the later in having more predilection for conjunctiva , not
occurring as pharyngeal carriers.
Epidemiology:
It is world wide in distribution , Common in tropical and sub tropical regions.
May occur in epidemic form.
causes highly contagious form of conjunctivitis (Pink eye )
27 Hemophilus

28. Clinical Manifestation :
Purulent contagious conjunctivitis ( Egyptian ophthalmia)
Brazilian Purpuric fever – Fulminant condition characterized by
fever, purpura, hypotension, and shock.
28 Hemophilus

29. Culture :
Requires both X and V factor
Do not ferment xylose
Hemagglutination with Guinea pig RBC at 4 ˚C
Slower growth than H. influenzae
29 Hemophilus

30. Other hemophilus spp :
1. H.parainfluenziae:
commensal - mouth and throat
occasionally ,it can be opportunistic pathogen
causes – Endocarditis
conjunctivitis
Bronchopulmunary infections
It requires only factor V not factor X
Ferments sucrose, not xylose.
2. H.haemolyticus and H. parahemolyticus:
Commensal – throat ( Both spp), Mouth( H.parahaemolyticus only )
Beta hemolytic is produced in sheep or Ox blood agar , when incubated
aerobically
H. parahemolyticus- rare case of Endocarditis
H. parahemolyticus- require only factor V
H.haemolyticus- require both factor X and V
30 Hemophilus

31. H. aphrophilus and H.paraphrophilus:
Capnophilic , require 5-10% CO2 – Opt growth
Commensal – mouth .
Ocasionally cause endocarditis , head and neck infections,
invasive bone and joint infections.
H.aphrophilus – require only factor X, whereas
H. paraphrophilus require only factor V.
31 Hemophilus

32. 32 Hemophilus

33. HACEK Group :
Group of highly fastidious , slow-growing, capnophilic, Gram negative bacteria
Commensal in oral cavity
Occasionally cause local infections of mouth and systemic infection such as
endocarditis
Species
Haemophilus Species ( H.aphrophilus,H.paraaphrophilus,H.parainfluenzae)
Aggregatibacter ( Formely Actinobacillus) actinomycetemcomitans
Cardiobacterium hominis
Ekinella corrodens
Kingella kingae
33 Hemophilus

34. Aggregatibacter actinomycetemcomitans :
Formely called Actinobacillus actinomycetemcomitans
Common member of these group causing endocarditis
Also isolated from soft tissue infection and abscesses associated with
Actinomycetes israeli
Rarely, it causes peridonitis, brain abcesses, meningitis and endophtalmitis
Lab Diagnosis :
Small, non motile, gram negative coccobacilli
Grows on blood agar supplemented with 5% defribinated horse blood.
Catalase and nitrate- positive, Indole- negative, citrate- negative, urease-
negative, DCA- negative
Ferments- glucose, galactose and maltose
34 Hemophilus

35. Cardiobacterium hominis
Frequently affects Aortic value.
Association with Arterial embolization, immune complex glomerulonephritis and
arthritis
Lab diagnosis :
Non motile, non capsulated, pleomorphic , gram negative bacillus.
Grows on blood agar under 3-5% CO2 and high humidity
Ferments wide range of sugars,
Indole- positive, Oxidase- positive, Catalase – negative, Nitrate- negative
35 Hemophilus

36. Ekinella corrodens:
It is a small slender, non capsulated gram negative bacteria.
Apart from endocarditis, it occasionally causes skin and soft tissue
infection.
Lab diagnosis :
It lacks flagella , but shows twitching or jerky motility, due to
contraction of fimbriae.
Name corrodens- pitting or corroded colonies on blood agar
Oxidase- positive, catalase- positive
Lysine and ornithine decarboxylase – postive
Indole , citrate, urease- negative
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37. Kingella kingae
It causes infections in bones, joints and tendons
Non –motile, gram negative exists as coccobacilli and diplococcal forms.
Oxidase – positive, catalase- negative.
Ekinella corrodens
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38. HACEK Endocarditis
It accounts for only 3% of endocarditis cases
Typically has an subacute course
Pre-existing valvular defects or those undergoing dental procedures.
Aortic and mitral value are most commonly affected.
La diagnosis :
Highly fastidious and require special media
Capnophilic, growth is opt in presence of 5-10% CO2
Incubation upto 30days may required.
Bactec- reduced the detection time – 1 week
PCR- more sensitive and rapid.
38 Hemophilus

39. Treatment :
Ceftriaxone(2g/day)- drug of choice except Ekinella corrodens
where ampicillin is indicated.
Beta lactam producers - Quinolones
Duration : Antibiotics are given’
4 weeks – native value endocarditis.
6 weeks – Prosthetic value endocarditis.
39 Hemophilus