بسم الله الرحمن الرحيم

Assistant professor of microbiology & immunology Wafaa khalil zaki Assistant professor of microbiology & immunology

Intended learning outcomes By the end of the lecture students should be able to: 1- Recognize morphology ,cultural characteristics of the Haemophilus & Bordetella group 2- Understand pathogenesis and virulence factors of pathogenic members and its relation to clinical disease 3- Recognize clinical spectrum, microbiological diagnosis prevention and treatment of them

Haemophilus

It is a group of short Gram negative pleomrphic bacteria Require enriched media for isolation obligate parasite that colonize human and animal tissue . Most important pathogens : H. influenza - H. ducreyi –H. aegypticus H. parainfluenzae (normal flora)

H. influenza Some strains posses polysaccharide capsule Six antigenic types for capsulated strains (a-f) …….type b is the most virulent Immunity is due to anti-capsular antibodies .

Virulence factors Polysaccharide capsule with polyribitol phosphate is the major virulent factor Also produce IgA specific protease>>>>mucosal colonization

Clinical spectrum Transmitted from person to person by respiratory route Invasive infection : Meningitis is the most common manifestation in children caused by H. influenza type b (Hib) It can also cause epiglottitis , septic arthritis , cellulites & pneumonia

Non - invasive infections : Caused by non capsulated strains in the form of otitis media in children(second to ??) sinusitis and bronchitis in all age groups Pneumonia with other underlying pulmonary infection

Laboratory diagnosis specimen >>>>> Microscopy

Direct & rapid detection Detection of capsular antigen by slide agglutination with specific antisera (Mainly on supernatant of CSF) Direct or indirect immuno- fluorescence PCR

Cultivation and identification Cannot grow on ordinary media , need growth factor X( Hemin) & V (NAD) , culture is best done on freshly prepared chocolate agar with X & V factors in presence of 5-10% CO2 for 1-2 days

H. influenzae grows well around colonies of S. aurous ……… why? This phenomenon is called satellitism. Colonies are identified by : Gram stained smear Detection of capsular polysaccharide by ELISA or Latex agglutination

Detection of capsular antigen

Prevention and control 1- H. influenza type b (Hib) conjugate vaccine ,,, consists of capsular polysaccharide of H. influenza type b conjugated with carrier protein Can be given to infants at 2, 4,6 months and booster dose at 15- 18 month cases

Chemoprophylaxis: Rifampin for close contact to meningitis It used because it reaches high bactericidal concentrations in mucosal secretions & decreases respiratory carriage of the organism, thereby reducing transmission.

Treatment due to common resistance to ampicillin Cefotaxime or ceftriaxone in meningitis and epiglottitis Amoxacillin- clavulanic , azithromycine and second generation cephalsporins for non invasive infections

Haemophilus ducreyi Short Gram negative pleomorphic bacilli ….. Need 5-10% Co2 and factor X only for growth . Causes sexually transmitted disease called soft sore or “chancroid “

Clinical Disease: It causes a sexually transmitted disease called "soft sore" or "chancroid" in the form of a painful soft ulcer on the external genitalia with enlarged tender draining lymph nodes

PCR for detection of organism in clinical specimen Diagnosis PCR for detection of organism in clinical specimen Isolation & identification of organism from the lesion Treatment : Ceftriaxone I.M Or oral azithromycine

H. aegyptius Was called Koch – Weeks bacillus now it is subgroup of H. influenza Can cause highly communicable purulent conjunctivitis Diagnosed by PCR from clinical specimen Treated by ampicillin + chloramphinicol

Gardenella vaginalis Normally present in female genitourinary tract. They are Gram variable facultative anaerobes. Clinical disease : Causes bacterial vaginosis (non-specific vaginitis). This is a polymicrobial infection where certain organisms (as Mobiluncus spp, G. vaginalis, Bacteroides & peptococci) overgrow the lactobacilli of the normal vaginal flora. These result in an increase in vaginal pH. Charactrized by Vaginal discharge with a foul (rotten-fish) smell.

Diagnosis: Specimen: Vaginal exudates or swab (high vaginal or end cervical) 1-Vaginal pH>4.5 2-Amin or Whiff test: addition of a drop of 10% KOH on vaginal secretion produces a fishy odor.

3-Saline wet film examined by high power lens shows characteristic “clue cells” 4-Gram stained film shows Gram –ve or variable bacili & clue cells (squamous epithelial cells coated by a a large number of Gram variable rods). 5- PCR Tretment: Metronidazole

2- Bordetella para pertussis causes whooping cough-like disease. Bordetellae Classification: Most important human pathogens include: 1-B. Pertussis: causes whooping cough. 2- Bordetella para pertussis causes whooping cough-like disease.

The Main virulence factors include: Pertussis toxin: it is the major toxin produced by B.pertussis that stimulates adenylate cyclase resulting in increases cAMP that inhibits normal cellular signaling. It causes adherence of the organism to the tracheal epithelium. The toxin causes serval systmeic effects, among wich is marked lymphocytosis

Tracheal cytotoxin :Inhibits ciliary movement and regeneration of damaged cells. Adenylate cyclase toxin: increases cAMP in leucocytes resulting in decreased chemotaxis and phagocytosis. Dermonecrotic toxin Endotoxin Fimbrial haemagglutinin: Capsule

Epidemiology: Mode of transmission: droplets from a case specifically during the catarrhal stage. B.Pertussis is highly contagious.

Pathogenesis: B.Pertussis infects tracheo-bronchioles. Adhesion and multiplication of the organisms on ciliated epithelial surface of trachea and bronchi interfere with the ciliary action. Liberation of different toxins causes irritation of surface epithelia leading to coughing.

Obstruction of terminal bronchioles with mucus plugs lead to diminished oxygenation and convulsions. Secondary invaders (Staphylococci or H. influenzae) may cause bacterial pneumonia.

Clinical Picture: It causes whooping cough. The incubation period is 2 weeks. the disease is characterized by . 1-Cataral Stage 2-Paroxymal Stage 3-convalescent Stage

1-Catarrhal stage: mild coughing and sneezing during which the patient is highly infectious. 2- Paroxysmal stage of severe successive attacks of cough, followed by deep inspiration (characteristic whoop). 3- Convalescent stage: chronic cough which may last for weeks.

Complications: cyanosis, convulsions, subconjunctival hemorrhage, vomiting and secondary bacterial infection

Laboratory Diagnosis: Specimen: A saline nasal wash (preferred) per=-nasal swab to collect the nasopharyngeal secretions. 1-PCR: and culture are done when cases present <3 weeks. 2.Isolation and Identification:

. Specimen is inoculated onto charcoal blood agar (preferably) or Bordet-gengou medium (potato-glycerol-blood agar+Pencillin). After incubation for 3-5 Days aerobically, at 37C, the growing colonies further identification of the organism depends on:

Microscopic examination of a gram stained film: Capsulated short gram-negative bacilli. I.F. test and slide agglutination. Blood cultures will always be negative. 3.ELISA: Single high IgG reading is helpful in diagnosis (>3weeks) 4. Complete blood count: marked lymphoctyosis (caused by Pertussis toxin).

Prevention and Control: Treatment: 1. Azithromycin or clarithromycin. 2. Symptomatic treatment: Oxygen inhalation, sedatives, bronchodilators, etc. Prevention and Control: 1. Whole-cell killed pertussis vaccine: It is a killed vaccine given usually with diphtheria and tetanus toxoids (DPT vaccine) at 2,4,6 months of age.

Encephalitis may occur. 2. Sub-unit vaccines, called “acellular” vaccines are used in several countries. They are safer with decreased side effects. 3.Prophylactic administration of erythromycin for 5 days for exposed person .

How many antigenic types had H How many antigenic types had H. influenza, and what is the most virulent type ? What are the main virulance factors of B. Pertussis

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Mycoplasmas

The smallest (150-250nm), free living organisms. They are prokaryotic cells that resemble Gram negative bacteria. They are bounded by a single trilaminar membrane containing sterol but lack a cell wall . 

-They are not destroyed by lysozymes They are pleomorphic in shape. They stain poorly with Gram stain, but stain well with Giemsa’s stain or leishman stain. They are not sensitive to antibiotics that act on cell wall e.g. -lactam antibiotics. -They are not destroyed by lysozymes

The important species for humans are: Mycoplasma(M.). pneumoniae. M. hominis. M. genetilium. Ureaplasma urealyticum.

Mycoplasma pneumoniae M. pneumoniae, a pathogen only for human Transmitted by respiratory droplets and causes atypical pneumonia. Common cause of community acquired pneumonia (5-15 years age) It has only one serotype and there is no antigenic variation among strains.

Pathogenesis and Immune Response In the lung, the organism is rod-shaped with a tapered tip containing specific proteins that serve as the point of attachment to mucosal ciliated epithelial cells. The respiratory mucosa is not invaded, but ciliary motion is inhibited and the mucosa is damaged by producing hydrogen peroxide and superoxide that leads to epithelial necrosis.

Humoral immunity (IgG and IgA) controls infection by: 1-Complement mediated lysis, 2- Anti-attachment and opsonization by polymorphs.

3-Protection correlates with the acquisition of serum IgG against M 3-Protection correlates with the acquisition of serum IgG against M. pneumoniae.  4- This acquired immunity explains the decline in incidence after childhood. Cell mediated immunity is not protective.

Cold agglutinins are autoantibodies to M. pneumoniae surface glycolipids cross react with human erythrocytes which cause: *agglutination of RBCs at 4C. *hemolytic anemia during infection .

Clinical Presentation 1-Atypical pneumonia; pneumonia, tracheobronchitis, pharyngitis or otitis media. 2-Extrapulmonary e.g. joint, skin, CNS, liver, myocardium and hemolytic anemia.

Laboratory Diagnosis 1. Direct Diagnosis: Specimen: Sputum. Culture: (heart infusion peptone broth) with added 5-10% CO2. They can grow on fluid -Agar- Biphasic media. They are microaerophilic Grow slowly and require at least one week to form minute microscopic visible colonies .

The colonies are identified by: Morphology: The colonies have characteristic fried egg appearance with a central zone embedded in the agar. Giemsa’s stain: A block of medium containing colonies is removed, fixed to a glass slide, stained, and examined microscopically (red colour).

The organism is very fastidious, laboratory diagnosis relies on non-culture techniques for identification e.g: Direct Ag detection by- (I.F) and (EIA) Nucleic acid detection by PCR.

 2. Indirect (Serological) methods for diagnosis: Detection of specific antibodies by: ELIZA, and indirect I.F. CFT. Latex agglutination.

Treatment : Erythromycin and tetracyclines.  Long acting macrolides and quinolones are alternative agents for resistant strains.

M. hominis & U. urealyticum Found in the genitourinary tract of asymptomatic individuals. Infants are often colonized at birth via vaginal tract. Adults are infected via sexual transmission.

Clinical manifestations M. hominis cause nongonococcal urethritis, pelvic inflammatory disease in females and pyelonephritis.

U. urealyticum 1-nongonococcal urethritis in males 2- premature rupture of membranes in pregnant females 3- chronic lung disease in premature neonates and 4-high incidence of isolation from CSF of low birth weight newborns. Produce urease enzyme which is the base of Urease activity tests used in diagnosis

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