Objectives By the end of this lecture the student must be:  A) Identify the genus Haemophilus & Bordetella  B) describe the chemical tests for this genus C) Differentiate between different sps. D) List and match the symptoms, diagnosis and treatment for different sps.

Haemophilus General characteristics: Belonging to the Pasteurellaceae Non intestinal pleomorphic Gram negative coccobacilli Microaerophilic Non motile, Non spore forming Usually capsulated Fastidious – Dependent on the growth promoting substances X (haematin) and V (NAD) which present in blood Stellatism = Growth of Heamophilus on a culture of Staph. aureus. Oxidase and catalase positive 2

Differentiation of Species Hemolysis Growth Factor XV H. influenzae -++ H. aegyptius -++ H. ducreyi -+- H. parainfluenzae +-+ 3

H. influenzae Opportunistic pathogen Exclusively human pathogen Not pathogenic for animals Diseases due to H. influenzae considered under 2 groups –invasive and non invasive 1.Nonencapsulated strains (Non invasive) Part of normal flora of the respiratory tract Colonize in nasopharynx of 80% of healthy individuals Causes pneumonia, otitis media and sinusitis 4

H. influenzae 2.Encapsulated strains (Invasive) Typing based on capsule polysaccharide a → f Type b is the most pathogenic & colonized in 5% Polyribose-ribitol phosphate (PRP) capsule (type b) Penetrate nasopharynx and invade blood directly Causes meningitis, pneumonia, bacteremia 95% of invasive disease caused by type b Vaccination with Hib conjugate vaccine Given IM at 2,4,6 month & poster dose at month 5

Virulence factors of H. influenzae Polysaccharide capsule – Antiphagocytic and major pathogenesis factor Fimbriae the adherence to human mucosal cells LPS- lipid A -major role in non capsule strains All virulent strains produce neuraminidase and IgA protease Many respiratory pathogens, including H. influenzae, S. pneumoniae and P. aeruginosa, express neuraminidases – Bacterial neuraminidase facilitates mucosal infection by participating in biofilm production No exotoxins 6

H. aegypticus Cause of acute mucopurulent conjunctivitis (Pinkeye) The disease is common in Egypt It is spread very easily, especially among children Pinkeye is transmitted mechanically by common towels or by flies Diagnosis is made from the conjunctival discharge 7

H. ducreyi This is the cause of chancroid (Soft chancre) which is venereal disease Chancroid is characterized by painful genital ulcers This organism needs only X factor Diagnosis is made from the discharge of the ulcer 8

Diagnosis of Haemophilus Specimen: CSF, blood, sputum Diagnosis is confirmed when the organism is isolated from a sterile body site (Blood & CSF) Diagnosis is not confirmed when it is isolated from nasopharyx or sputum Stain: Gram negative coccobacilli Culture: On chocolate agar: A 24 h colony of H. influenzae is larger than that observed on blood agar On nutrient agar plate with added X & V factors at 37°C in an enriched CO 2 incubator 9

Diagnosis of Haemophilus Satellitism: Blood agar contains much X factor and little V factor S. aureus produces V factor A lawn of test bacteria is plated onto a blood agar plate S. aureus is placed on plate & the culture is incubated H. influenzae will grow in the hemolytic zone of S. aureus The hemolysis of cells by S. aureus releases nutrients H. influenzae will not grow outside the hemolytic zone of S. aureus due to the lack of nutrients in these areas Capsule swelling: Specific antiserum added to a slide of the organism allows swelling of the bacterial capsule thus permitting rapid diagnosis of H. influenzae in sputum stds-genital-ulcer 10

Bordetella Small capsulated Gram negative coccobacillus Strict aerobe, oxidative, oxidase variable? Three important species 1. B. pertussis causes WHOOPING COUGH (Pertussis) Strictly human pathogen: infant & young children 2. B. parapertussis –causes mild form of whooping cough 3.B. bronchoseptica Widespread in animals (dog) & causes kennel cough Rarely causes respiratory or wound infection in humans 11

B. pertussis Pathogenesis 1.Respiratory Colonization Attaches to ciliated epithelium of URT VIA a.Filamentous hemagglutinin (FHA) – Protein on pili – Antibodies against FHA inhibit colonization – Fimbriae are NOT involved b.Pertussis Toxin ◦ Do not invade underlying lung tissues ◦ Decreased cilia activity & epithelial cell death occur ◦ 7-10 days, NO symptoms ◦ Positive cultures toward the end of this stage 12

Toxin-mediated disease 1.Pertussis toxin Responsible for adherence ↑Adenylate cyclase  ↑ intracellular cAMP This causes cellular dysfunction Inhibition of host phagocyte oxidative responses and the inhibition of natural killer cell activity 2.Dermonecrotic toxin (lethal toxin) Released upon cell lysis causing strong vasoconstriction Results in  O 2  convulsion 3.Tracheal cytotoxin Prevents ciliated epithelial cells from beating 4. LPS –endotoxin -irritation and damage of cell 13

Clinical significance Pertussis is Toxin-mediated disease Very contagious & can cause serious illness―especially in babies too young MODE OF TRANSMISSION : droplet inhalation 1. INCUBATION PERIOD :1-2 weeks Organism multiply & start to liberate toxins Pertussis lasts for  6 weeks and appear in 4 stages 3. CATARRHAL STAGE occurs - rhinorrhea, mild cough, sneezing & fever whereby a lot of organisms are spread through respiratory secretions This last ~ 1-2 weeks 14

4. PAROXYSMAL STAGE that lasts 2-4 weeks Patient has rapid, consecutive cough with a rapid intake of air between coughs (has a whooping sound السعال الديكي ) – Ciliary action of Respiratory Tract has been compromised – Mucous has accumulated – Patient is trying to cough up mucous accumulations – Coughs are strong enough to break ribs ! 5.Finally there is a CONVALESCENT STAGE during which symptoms gradually subside This can last for months 15

Diagnosis Specimen is taken during PAROXYSMAL STAGE – Nasopharyngeal swabs – Cough plate method Highly fastidious & requires special media+additional nutrients for growth+absorbents to remove toxic substances found in complex media e.g. fatty acids Culture on Bordet-Gengou medium Contains glycerol, potato infusion, albumin (binds fatty acids), and up to 50% defibrinated SRBCs Charcoal agar +10% horse blood ± Cephalexin 3-7 days for growth, hemolytic colonies A characteristic pearl like colonies are observed Direct fluorescent antibody testing, PCR, Slide agglutination 16

Differentiation of Bordetella species Species Growth on Urease Oxidase? Motility Sheep blood agar and MacConkey Bordet- Gengou agar B. pertussis B. parapertussis ++-- B. bronchiseptica

Prevention 1.Pertussis Whole cell vaccine Merthiolate-killed bacterial cell suspension Part of the DTP vaccine P in DTP for Pertussis 2. Pertussis Acellular vaccines Consist of 1–5 purified, detoxified toxins & adhesins Introduced in developed regions ∼ 15 years ago to replace Killed vaccines Has fewer side effects than the whole cell vaccine Diphtheria-Tetanus-Pertussis vaccine using acellular pertussis is known as DTaP 2, 4, 6, months and 4-6 years of age. 18