HAEMOPHILUS INFLUENZAE: Short Pleomorphic Gram (-) Rods.
Bugs of Childhood
HAEMOPHILUS INFLUENZAE: Short Pleomorphic Gram (-) Rods.
Name-Derivation:
Haemophilus = blood-loving
Influenzae = discovered as part of flu epidemics.
Epidemiology / At Risk: Infantile Meningitis.
Manifestations: There are typable (encapsulated) and non-typable strains
TYPABLE: Haemophilus Type-B infections
Infantile Meningitis: #1 cause in kids older than 3 months.
Epiglottitis in kids 2 - 5, possibly with sepsis.
Cellulitis in kids.
Bacteremia: Can either be serum-sensitive (serum + complement lyse the bugs) or serum-resistant (serum + complement can't lyses the bug).
Serum sensitivity is conferred by the LOS coat. The higher the molecular weight of the LOS, the worse is the bacteremia.
High molecular weight LOS: Serum resistant.
Low molecular weight LOS: Serum sensitive.
NON-TYPABLE:
Otitis Media
Bronchitis and pneumonia
Processing:
Specimen: CSF from infants.
Stain: Gram (-) rods, but the poles tend to remain purple. Thus distinguishing with Strep Group-B can be a problem.
Culture: Both Smooth and rough colonies seen in culture.
ABSOLUTE GROWTH REQUIREMENTS: Both derived from RBC's.
Factor X: Hemin precursor.
Factor V: NAD, used for organism pyridine synthesis.
Chocolate Agar: Heat-lysed red blood cells.
Fildes's Agar: Enzymatically (rather than heat) lysed RBC's.
Satellite Growth: Put the bugs in blood agar with Staph Aureus, they will grow around the perimeter of the Staph. This is because the Staph Aureus lyses the RBC's and provides the needed nutrients for the Hib to grow around perimeter.
Identification:
TYPABLE: Types a thru f, but Type b is the only important one. Other strands are not typable.
QUELLUNG REACTION: Can be done on CSF to identify Hib. Add methylene blue to visualize: Positive test shows rod-shaped organisms with halo, due to swelling of the capsule.
Can use CIE, ELISA, or Latex Agglutination to look for residual antigens left in blood, CSF, or urine.
NON-TYPABLE: Pleomorphic appearance on stain, still with intensity at the poles.
Virulence:
Capsule: When present, it is the basis for typing.
Type-B is made out of poly-ribitol phosphate. We don't really make antibodies to it, thus it is more virulent. We do make antibodies after repeated exposure. Presence of Ab is AGE-DEPENDENT:
Infants younger than 3 months are protected by maternal IgG.
Infants older than 3 months have lost maternal IgG immunity, thus they become susceptible to Hib meningitis.
Immunity is re-acquired by age 10 due to cross-reactivity with Staph Aureus and E. Coli ribitol moieties.
Outer Membrane Proteins: Looking at them for a vaccine. Various ones are antiphagocytic and invasive.
P2, porin protein is most promising candidate.
Lipo-oligosaccharide (LOS): Differs from LPS in that it has less sugar. Bacteremia does not result in the serious endotoxic shock of E. COLI, for example.
The LOS coat inhibits movement of cilia in the airway and helps to establish infection.
Molecular weight of LOS determines serum-resistance in bacteremia.
IgA Protease: Allows Hib to establish in upper airway.
beta-Lactamase: Plasmid-mediated.
Host Immune Response: Antibodies are protective. IgG is the most protective.
Vaccine / Prevention: DPT Vaccine contains the Hib Conjugate -- Diphtheria Toxoid plus Poly-Ribitol Phosphate. 100% effective.
Vaccine administered at 2 months, 4 months, and 6 months.
PRP by itself can only be given to kids older than 2 years. PRP by itself is not as effective. The conjugate tends to elicit more of an IgG response (rather than IgM), which is what we want.
Treatment: Treated based on antibiotic susceptibility test results.
Penicillin-Resistant.
Chloramphenicol-Resistant, via Chloramphenicol Acetyl-Transferase (which inactivates the drug).
BORDETELLA PERTUSSIS: Pleomorphic Gram (-) Rods
Name-Derivation: Named after researcher
Epidemiology / At Risk: Highly communicable and REPORTABLE.
Manifestations:
WHOOPING COUGH: Bugs adhere to cilia up and down the airway. Systemic infection from a toxemia -- not a bacteremia. The organisms are non-invasive.
Incubation Stage: (7-10 days). Mild respiratory manifestations.
Catarrhal Stage: (1-2 weeks). More serious respiratory infections.
This is the best time to collect the bugs, when the bugs are in the greatest number.
Paroxysmal Stage: (2-4 weeks). Paroxysmal, rapid coughs, anoxia, followed by the "Whoop" sound to recover breath.
Intense lymphocytosis is found, unusual for bacterial infection -- more common with viral infection.
Vomiting
Convalescent Stage: (3-4 weeks or longer). Pneumonia, convulsions from anoxia, other complications.
Subconjunctival Hemorrhaging found in the eyes (red around iris) in babies who have had Whooping Cough.
Processing:
Specimen: Must use special Nasopharyngeal Swab, which contains no cotton. Cotton contains fatty acids that are lethal to the bug.
Specimen is immediately inoculated at the bedside, either into transport broth or on a plate.
Stain: Gram Stain is of no value.
Toluidine Blue stain used to visualize bugs. Look for small rods with bipolar intensity. Appearance is similar to Hemophilus.
Culture:
Bordet-Gangeau Plate required -- must notify laboratory.
Colonies become rough and lose virulence as they lose envelope, then can subculture for Gram stain.
Identification:
Obligate Aerobe
Direct Agglutination or Direct FA can be done on cultured bugs. It is only reliable on the culture -- not on the original specimen.
Virulence:
Cell-Associated:
Polysaccharide Capsule: Essential for virulence.
Filamentous Hemagglutinin (FHA): Filaments sticking out are adhesins to ciliated cells.
Also immunogenic (protective) and hemolytic.
Pertactin: Outer Membrane Protein, is an adhesin and is immunogenic. Antibody is protective, as it interrupts adhesion to cells.
Pertussis Toxin: AB-Toxin.
Structure and Mode of Action:
Fragment-B: Complex Hexamer, binds readily to ciliated cells.
Fragment-A: ADP-ribose Transferase, transfers to Gi subunit to block Gi ------> cAMP.
Effects of Pertussis via raised cAMP:
Causes Histamine sensitization which causes us to cough. Turns on a lot of IL-4 production, which causes Isotype switching to IgE. This also will lead to increased vascular permeability.
Lymphocytosis
Hypoglycemia due to activation of pancreatic beta-cells.
Toxin can also block some immune effector cells.
Adenylate Cyclase: Bug-derived, two isotypes.
70 kDa: Results in elevation of host cell cAMP.
100 kDa: Interferes with PMN phagocytosis and respiratory burst; hinders chemotaxis; hemolysin; impairs alveolar macrophage metabolism.
Other Toxins:
Heat-Labile Toxin: Released upon lysis of the bug. Causes dermonecrosis (or surface necrosis) of ciliated epithelial cells upon release. Remember that B. Pertussis is not invasive.
Heat-Stable Toxin: Endotoxin with Lipid-A, similar to LPS.
Also contains Lipid X which stimulates protective antibodies.
Tracheal Cytotoxin: A small peptidoglycan fragment. Ciliastatic.
Vaccine / Prevention: DPT vaccine, at 2, 4, and 6 months.
Initial shots: DPT. Pertussis component is methiolate-killed whole organisms.
Booster Shots: DTaP. At 15 months and preschool, we use an acellular vaccine for a booster of FHA + Pertussis Toxoid.
Side-Effect: Risk of 1/300,000+ of encephalopathy.
Treatment: Erythromycin.
CORYNEBACTERIUM DIPHTHERIAE: Gram (+) slender Rod.
Epidemiology / At Risk: Communicable. Person-person contact.
Manifestations: DIPHTHERIA primarily affects respiratory tract and skin.
Skin: Not systemic disease. Local cutaneous pseudomembranous necrosis of hands and feet. Host response contributed to damage.
Bull neck: Characteristic appearance found (similar to Mono), with enlargement of cervical lymph nodes.
Pseudomembranous Necrosis: Found in oropharynx in initial respiratory infection.
Systemic Toxemia: Diphtheria toxin affects three organs, in order:
Heart:
Nerves: Polyneuritis, anesthesia of palate
Kidney: Only late course.
DIFFERENTIAL: Vincent's Disease can be confused with this disease. Vincent's Disease is pseudomembranous pharyngitis, and is caused by poor oral hygiene.
Vincent's will show halitosis and signs of gingivitis (swelling, bleeding).
Stain with Vincent's disease will show a whole mess of bacteria -- both Gram (+) and Gram (-), while Diphtheria will show only slender Gram (+) rods.
Processing:
Specimen: The Pseudomembrane must be cut with a surgical scissors -- not by tugging or pulling, as that will spread infection.
Stain:
Methylene Blue: Pleomorphic slender rods, with Ernst-Babes bodies (granules) stained purple-red.
Culture: Must culture for toxin. Not every strand produces toxin (which is phage-mediated).
Blood-Tellurite Agar: Special medium selective for Corynebacterium and Staph. Either bug will give rise to grey black colonies. Then do a Gram stain to differentiate the two.
Coagulated Serum (Loeffler's) Agar: Can be used to determine presence of toxin. Supports both the bug and the toxin. Positive test will show creamy colonies.
Identification:
TOXIN ID: Two alternatives.
Precipitin Reaction: Take agar from above that is not completely hard. Apply filter paper containing antitoxin. Streak isolates of known positive and negatives across it. Look for Ab-Ag precipitation.
Guinea Pig Rxn:
Innoculate at Site A
Administer antitoxin 5 hours later.
Reinject at Site B 5 minutes later (antitoxin having been administered)
Positive test occurs when only slight reddening occurs at Site B, while Site A undergoes necrosis.
Virulence:
Diphtheria Toxin:
The Tox Gene encodes for this toxin and is carried in a lysogenized beta-Phage. Iron Regulation:
C. Diphtheriae codes for a gene called the Aporepressor. Aporepressor complexes with Iron (Fe+2) to successfully bind the beta-phage tox operon ------> inhibit toxin production.
Absence of iron ------> disinhibition ------> toxin production ensues.
AB-Toxin: Antitoxin works against Fragment-B to prevent binding. Once the toxin is inside the cell, the cell is doomed.
Fragment-B binds to heart, nerve, and kidney cells.
Fragment-A is an ADP-Ribose Transferase which binds to a His residue on Elongation Factor 2 (EL-2) ------> EL-2 can no longer accept amino acids from tRNA ------> inhibit protein synthesis ------> eventual cell death.
Lipase: Important in cutaneous lesions.
Neuraminidase: Acts on respiratory tract mucosa.
Siderophore: As long as iron levels are high, toxin level remains low. Unfortunately, inflammation and fibrinous exudate cut off iron supply.
Cornymycolic Acid: Cell wall glycolipids, contribute to invasiveness.
K-Antigens: Heat-labile, able to induce hypersensitivity.
O-Antigens: Heat-Stable polysaccharides. Hypersensitivity.
Host Immune Response:
Vaccine / Prevention: DPT Vaccine. Huge success story.
Children: Formyl-inactivated toxoid. Alum is added as an adjuvant.
The additional antigens trapped in with the alum can be a source of hypersensitivity.
Adults: Same vaccine, but ammonium is used as the adjuvant. This eliminates some possible contaminating antigens that can cause hypersensitivity.
SHICK-TEST: Used to determine (1) whether or not your are susceptible to Diphtheriae (and need the vaccine) and (2) whether or not you will show hypersensitivity (and thus would need the special vaccine)
TOXIN
Used to check for presence of antibody
TOXOID
Used to check for hypersensitivity
REPORT
36 hrs 120 hrs 36 hrs 120 hrs
Neg Pos
erythema and inflammation found
Neg Neg Shick-Positive: Patient is susceptible to Diphtheria toxin, and can be immunized with regular vaccine.
Neg Neg Neg Neg Shick-Negative: Antibodies to Diphtheria toxin were present. Patient doesn't need vaccine.
Neg Neg Pos Neg Hypersensitive: Patient is hypersensitive to toxoid. He is Shick-negative and doesn't currently need a vaccine, but would need the adult vaccine if one is ever needed in the future.
Neg Pos Pos Neg Cross-Reaction: Patient is both susceptible to diphtheria toxin, and hypersensitive. Adult vaccine is required.
Treatment: Use antitoxin to collect up free toxin in blood. Erythromycin.
HAEMOPHILUS INFLUENZAE: Short Pleomorphic Gram (-) Rods.
Name-Derivation:
Haemophilus = blood-loving
Influenzae = discovered as part of flu epidemics.
Epidemiology / At Risk: Infantile Meningitis.
Manifestations: There are typable (encapsulated) and non-typable strains
TYPABLE: Haemophilus Type-B infections
Infantile Meningitis: #1 cause in kids older than 3 months.
Epiglottitis in kids 2 - 5, possibly with sepsis.
Cellulitis in kids.
Bacteremia: Can either be serum-sensitive (serum + complement lyse the bugs) or serum-resistant (serum + complement can't lyses the bug).
Serum sensitivity is conferred by the LOS coat. The higher the molecular weight of the LOS, the worse is the bacteremia.
High molecular weight LOS: Serum resistant.
Low molecular weight LOS: Serum sensitive.
NON-TYPABLE:
Otitis Media
Bronchitis and pneumonia
Processing:
Specimen: CSF from infants.
Stain: Gram (-) rods, but the poles tend to remain purple. Thus distinguishing with Strep Group-B can be a problem.
Culture: Both Smooth and rough colonies seen in culture.
ABSOLUTE GROWTH REQUIREMENTS: Both derived from RBC's.
Factor X: Hemin precursor.
Factor V: NAD, used for organism pyridine synthesis.
Chocolate Agar: Heat-lysed red blood cells.
Fildes's Agar: Enzymatically (rather than heat) lysed RBC's.
Satellite Growth: Put the bugs in blood agar with Staph Aureus, they will grow around the perimeter of the Staph. This is because the Staph Aureus lyses the RBC's and provides the needed nutrients for the Hib to grow around perimeter.
Identification:
TYPABLE: Types a thru f, but Type b is the only important one. Other strands are not typable.
QUELLUNG REACTION: Can be done on CSF to identify Hib. Add methylene blue to visualize: Positive test shows rod-shaped organisms with halo, due to swelling of the capsule.
Can use CIE, ELISA, or Latex Agglutination to look for residual antigens left in blood, CSF, or urine.
NON-TYPABLE: Pleomorphic appearance on stain, still with intensity at the poles.
Virulence:
Capsule: When present, it is the basis for typing.
Type-B is made out of poly-ribitol phosphate. We don't really make antibodies to it, thus it is more virulent. We do make antibodies after repeated exposure. Presence of Ab is AGE-DEPENDENT:
Infants younger than 3 months are protected by maternal IgG.
Infants older than 3 months have lost maternal IgG immunity, thus they become susceptible to Hib meningitis.
Immunity is re-acquired by age 10 due to cross-reactivity with Staph Aureus and E. Coli ribitol moieties.
Outer Membrane Proteins: Looking at them for a vaccine. Various ones are antiphagocytic and invasive.
P2, porin protein is most promising candidate.
Lipo-oligosaccharide (LOS): Differs from LPS in that it has less sugar. Bacteremia does not result in the serious endotoxic shock of E. COLI, for example.
The LOS coat inhibits movement of cilia in the airway and helps to establish infection.
Molecular weight of LOS determines serum-resistance in bacteremia.
IgA Protease: Allows Hib to establish in upper airway.
beta-Lactamase: Plasmid-mediated.
Host Immune Response: Antibodies are protective. IgG is the most protective.
Vaccine / Prevention: DPT Vaccine contains the Hib Conjugate -- Diphtheria Toxoid plus Poly-Ribitol Phosphate. 100% effective.
Vaccine administered at 2 months, 4 months, and 6 months.
PRP by itself can only be given to kids older than 2 years. PRP by itself is not as effective. The conjugate tends to elicit more of an IgG response (rather than IgM), which is what we want.
Treatment: Treated based on antibiotic susceptibility test results.
Penicillin-Resistant.
Chloramphenicol-Resistant, via Chloramphenicol Acetyl-Transferase (which inactivates the drug).
BORDETELLA PERTUSSIS: Pleomorphic Gram (-) Rods
Name-Derivation: Named after researcher
Epidemiology / At Risk: Highly communicable and REPORTABLE.
Manifestations:
WHOOPING COUGH: Bugs adhere to cilia up and down the airway. Systemic infection from a toxemia -- not a bacteremia. The organisms are non-invasive.
Incubation Stage: (7-10 days). Mild respiratory manifestations.
Catarrhal Stage: (1-2 weeks). More serious respiratory infections.
This is the best time to collect the bugs, when the bugs are in the greatest number.
Paroxysmal Stage: (2-4 weeks). Paroxysmal, rapid coughs, anoxia, followed by the "Whoop" sound to recover breath.
Intense lymphocytosis is found, unusual for bacterial infection -- more common with viral infection.
Vomiting
Convalescent Stage: (3-4 weeks or longer). Pneumonia, convulsions from anoxia, other complications.
Subconjunctival Hemorrhaging found in the eyes (red around iris) in babies who have had Whooping Cough.
Processing:
Specimen: Must use special Nasopharyngeal Swab, which contains no cotton. Cotton contains fatty acids that are lethal to the bug.
Specimen is immediately inoculated at the bedside, either into transport broth or on a plate.
Stain: Gram Stain is of no value.
Toluidine Blue stain used to visualize bugs. Look for small rods with bipolar intensity. Appearance is similar to Hemophilus.
Culture:
Bordet-Gangeau Plate required -- must notify laboratory.
Colonies become rough and lose virulence as they lose envelope, then can subculture for Gram stain.
Identification:
Obligate Aerobe
Direct Agglutination or Direct FA can be done on cultured bugs. It is only reliable on the culture -- not on the original specimen.
Virulence:
Cell-Associated:
Polysaccharide Capsule: Essential for virulence.
Filamentous Hemagglutinin (FHA): Filaments sticking out are adhesins to ciliated cells.
Also immunogenic (protective) and hemolytic.
Pertactin: Outer Membrane Protein, is an adhesin and is immunogenic. Antibody is protective, as it interrupts adhesion to cells.
Pertussis Toxin: AB-Toxin.
Structure and Mode of Action:
Fragment-B: Complex Hexamer, binds readily to ciliated cells.
Fragment-A: ADP-ribose Transferase, transfers to Gi subunit to block Gi ------> cAMP.
Effects of Pertussis via raised cAMP:
Causes Histamine sensitization which causes us to cough. Turns on a lot of IL-4 production, which causes Isotype switching to IgE. This also will lead to increased vascular permeability.
Lymphocytosis
Hypoglycemia due to activation of pancreatic beta-cells.
Toxin can also block some immune effector cells.
Adenylate Cyclase: Bug-derived, two isotypes.
70 kDa: Results in elevation of host cell cAMP.
100 kDa: Interferes with PMN phagocytosis and respiratory burst; hinders chemotaxis; hemolysin; impairs alveolar macrophage metabolism.
Other Toxins:
Heat-Labile Toxin: Released upon lysis of the bug. Causes dermonecrosis (or surface necrosis) of ciliated epithelial cells upon release. Remember that B. Pertussis is not invasive.
Heat-Stable Toxin: Endotoxin with Lipid-A, similar to LPS.
Also contains Lipid X which stimulates protective antibodies.
Tracheal Cytotoxin: A small peptidoglycan fragment. Ciliastatic.
Vaccine / Prevention: DPT vaccine, at 2, 4, and 6 months.
Initial shots: DPT. Pertussis component is methiolate-killed whole organisms.
Booster Shots: DTaP. At 15 months and preschool, we use an acellular vaccine for a booster of FHA + Pertussis Toxoid.
Side-Effect: Risk of 1/300,000+ of encephalopathy.
Treatment: Erythromycin.
CORYNEBACTERIUM DIPHTHERIAE: Gram (+) slender Rod.
Epidemiology / At Risk: Communicable. Person-person contact.
Manifestations: DIPHTHERIA primarily affects respiratory tract and skin.
Skin: Not systemic disease. Local cutaneous pseudomembranous necrosis of hands and feet. Host response contributed to damage.
Bull neck: Characteristic appearance found (similar to Mono), with enlargement of cervical lymph nodes.
Pseudomembranous Necrosis: Found in oropharynx in initial respiratory infection.
Systemic Toxemia: Diphtheria toxin affects three organs, in order:
Heart:
Nerves: Polyneuritis, anesthesia of palate
Kidney: Only late course.
DIFFERENTIAL: Vincent's Disease can be confused with this disease. Vincent's Disease is pseudomembranous pharyngitis, and is caused by poor oral hygiene.
Vincent's will show halitosis and signs of gingivitis (swelling, bleeding).
Stain with Vincent's disease will show a whole mess of bacteria -- both Gram (+) and Gram (-), while Diphtheria will show only slender Gram (+) rods.
Processing:
Specimen: The Pseudomembrane must be cut with a surgical scissors -- not by tugging or pulling, as that will spread infection.
Stain:
Methylene Blue: Pleomorphic slender rods, with Ernst-Babes bodies (granules) stained purple-red.
Culture: Must culture for toxin. Not every strand produces toxin (which is phage-mediated).
Blood-Tellurite Agar: Special medium selective for Corynebacterium and Staph. Either bug will give rise to grey black colonies. Then do a Gram stain to differentiate the two.
Coagulated Serum (Loeffler's) Agar: Can be used to determine presence of toxin. Supports both the bug and the toxin. Positive test will show creamy colonies.
Identification:
TOXIN ID: Two alternatives.
Precipitin Reaction: Take agar from above that is not completely hard. Apply filter paper containing antitoxin. Streak isolates of known positive and negatives across it. Look for Ab-Ag precipitation.
Guinea Pig Rxn:
Innoculate at Site A
Administer antitoxin 5 hours later.
Reinject at Site B 5 minutes later (antitoxin having been administered)
Positive test occurs when only slight reddening occurs at Site B, while Site A undergoes necrosis.
Virulence:
Diphtheria Toxin:
The Tox Gene encodes for this toxin and is carried in a lysogenized beta-Phage. Iron Regulation:
C. Diphtheriae codes for a gene called the Aporepressor. Aporepressor complexes with Iron (Fe+2) to successfully bind the beta-phage tox operon ------> inhibit toxin production.
Absence of iron ------> disinhibition ------> toxin production ensues.
AB-Toxin: Antitoxin works against Fragment-B to prevent binding. Once the toxin is inside the cell, the cell is doomed.
Fragment-B binds to heart, nerve, and kidney cells.
Fragment-A is an ADP-Ribose Transferase which binds to a His residue on Elongation Factor 2 (EL-2) ------> EL-2 can no longer accept amino acids from tRNA ------> inhibit protein synthesis ------> eventual cell death.
Lipase: Important in cutaneous lesions.
Neuraminidase: Acts on respiratory tract mucosa.
Siderophore: As long as iron levels are high, toxin level remains low. Unfortunately, inflammation and fibrinous exudate cut off iron supply.
Cornymycolic Acid: Cell wall glycolipids, contribute to invasiveness.
K-Antigens: Heat-labile, able to induce hypersensitivity.
O-Antigens: Heat-Stable polysaccharides. Hypersensitivity.
Host Immune Response:
Vaccine / Prevention: DPT Vaccine. Huge success story.
Children: Formyl-inactivated toxoid. Alum is added as an adjuvant.
The additional antigens trapped in with the alum can be a source of hypersensitivity.
Adults: Same vaccine, but ammonium is used as the adjuvant. This eliminates some possible contaminating antigens that can cause hypersensitivity.
SHICK-TEST: Used to determine (1) whether or not your are susceptible to Diphtheriae (and need the vaccine) and (2) whether or not you will show hypersensitivity (and thus would need the special vaccine)
TOXIN
Used to check for presence of antibody
TOXOID
Used to check for hypersensitivity
REPORT
36 hrs 120 hrs 36 hrs 120 hrs
Neg Pos
erythema and inflammation found
Neg Neg Shick-Positive: Patient is susceptible to Diphtheria toxin, and can be immunized with regular vaccine.
Neg Neg Neg Neg Shick-Negative: Antibodies to Diphtheria toxin were present. Patient doesn't need vaccine.
Neg Neg Pos Neg Hypersensitive: Patient is hypersensitive to toxoid. He is Shick-negative and doesn't currently need a vaccine, but would need the adult vaccine if one is ever needed in the future.
Neg Pos Pos Neg Cross-Reaction: Patient is both susceptible to diphtheria toxin, and hypersensitive. Adult vaccine is required.
Treatment: Use antitoxin to collect up free toxin in blood. Erythromycin.
