Fastidious Bacteria: Hemophilus, Bordetella, Brucella PDF

Fastidious bacteria Hemophilus Bordetella Brucella Dr. Ebtisam Salem  Fastidious: are difficult to grow in the laboratory because they have complex or restricted nutritional and/or environmental requirement. Parvobacteriaceae= small bacteria Haemophilus Species H...

Fastidious bacteria Hemophilus Bordetella Brucella Dr. Ebtisam Salem  Fastidious: are difficult to grow in the laboratory because they have complex or restricted nutritional and/or environmental requirement. Parvobacteriaceae= small bacteria Haemophilus Species H. influenzae H. para influenza H. aphrophilus H. para aphrophilus H. duceryi H.egypticus Haemophilus Small, pleomorphic, gram-negative rods or coccobacilli Blood loving Non-motile Mostly oxidase +ve Facultative anaerobes, fermentative Fastidious, require enriched media for isolation. Most species require X and/or V factor for growth Grow better in CO2 –enriched condition  Haemophilus influenzae It was first isolated by Richard Pfeiffer during the influenza pandemic of 1890. Secondary invader First living organism to have its genome fully sequenced. Richard Pfeiffer The growth of Haemophilus requires 1. Haemin (X factor for “unknown factor”), required for the synthesis of cytochrome C and other iron containing respiratory enzymes. 2. Nicotinamide adenine dinucleotide NAD(P); also called V factor for “vitamin”). It is essential for oxidation – reduction processes in cell metabolism. both factors are present in blood-enriched media, sheep blood agar must be gently heated at ( 70-80c˚) for a few minutes to destroy the inhibitors of V factor( NADase). Capsule The surface of many, but not all, strains of H. influenzae is covered with a polysaccharide capsule, and six antigenic serotypes (a - f) H. influenzae type b is clinically most virulent with polyribitol phosphate [PRP] in capsule  Antibodies directed against the capsule greatly stimulate bacterial phagocytosis and complement-mediated bactericidal activity. These antibodies develop because of 1. Natural infection 2. Vaccination with purified PRP 3. Passive transfer of maternal antibodies. Epidemiology H. influenzae is isolated exclusively from humans, predominantly from the respiratory tract. No animal or environmental reservoir. Non capsulated stains present in nasopharynx or throat of 25-80% normal people, capsulated strains( half type b) in 5-10%. H. influenzae type b was the most common serotype that caused systemic disease( more than 95% of all invasive Haemophilus infections(Before vaccine) After vaccine, other serotypes of encapsulated bacteria and nonencapsulated strains has become more common H. influenzae type b remains the most significant pediatric pathogen in many countries of the world. It is estimated that 3 million cases of serious disease and up to 700,000 fatalities occur in children each year worldwide. Pathogenesis Transmitted via respiratory droplets, or direct contact with contaminated secretions It enters the body through the upper respiratory tract, resulting in either asymptomatic colonization or may cause infection such as sinusitis, otitis media or pneumonia. The organism produces IgA protease that degrades the secretary IgA which facilitate its attachment to the respiratory mucosa. Encapsulated organisms can penetrate the epithelium of the nasopharynx and invade blood capillaries directly, and reaches the meninges  The presence antiphagocytic polysaccharide capsule is a major factor in virulence. Nontypable strains are less invasive, but they, as well as typable strains, induce an inflammatory response that causes disease No exotoxins are produced. Outbreaks of type b infection occur, especially in nurseries and child care centers Virulence factors 1. Pili and nonpilus adhesins mediate colonization of the oropharynx with H. influenzae. 2. Cell wall components of the bacteria (e.g., lipopolysaccharide and a low-molecular-weight glycopeptide) impair ciliary function, leading to damage of the respiratory epithelium. 3. The major virulence factor in H. influenzae type b is the antiphagocytic polysaccharide capsule, which contains ribose, ribitol, and phosphate (polyribitol phosphate [PRP]). 4. The lipopolysaccharide lipid A component induces meningeal inflammation 5. Immunoglobulin IgA1 proteases are produced by H. influenzae (both encapsulated and non-encapsulated strains) and may facilitate colonization of the organisms on mucosal surfaces by interfering with humoral immunity. Clinical Diseases Invasive infection Meningitis Epiglotitis Pneumonia Cellulitis Septic arthritis Osteomyelitis Non-invasive infection Otitis media Sinusitis Acute exacerbation of COPD conjunctivitis The risk of invasive disease (meningitis and epiglottitis) is significantly greater in patients with No anti-PRP antibodies Depletion of complement Undergone splenectomy 3vacc ? Immunosuppression Hypogammaglobuliemia. Note: Children around 1 year: due to decrease in maternal IgG, and inability of the child to form sufficient anticapsular antibodies until the age of 2 years. Meningitis H. influenzae type b was the most common cause of pediatric meningitis, Peak incidence around 1 year, (3 to 18 mo ) More in Winter months Increased risk in Household contacts of a case < 5ys,esp 60 ys Case fatality rate is high Laboratory Diagnosis Specimen : CSF , sputum , blood, joint or pleural fluid Meningitis: Both blood and CSF Epiglottitis, cellulitis,and arthritis: Blood cultures Specimens should not be collected from the posterior pharynx in patients with suspected epiglottitis because the procedure may stimulate coughing and obstruct the airway. Microscopy Sensitive and specific. Detected in more than 80% of CSF specimens from patients with untreated Haemophilus meningitis. Gram stains of Haemophilus influenzae( CFS, Sputum) Antigen Detection Rapid latex agglutination test Rapid and sensitive Detect less than 1 ng/ml of PRP capsular Ag in clinical specimen(CSF and urine) Only H. influenzae type b Useful when specimen taken after antibiotic treatment , but (Some serotypes of S. pneumoniae and E.coli may share similar Ag) Culture Chocolate agar ( if overheated, V factor is destroyed) Incubated in aerobic atmosphere enriched with 5-10% CO2 1- 2mm, smooth, opaque colonies after 24 hrs “mousy” or “bleach-like” odor Blood culture Delayed Not supplemented with optimum concentrations of X and V factors and inhibitors of V factor. Grow better in anaerobically do not require X factor for growth. Identification by: 1.Culture on NA with V, X , XV factors H. influenzae requires both X and V factors H. Parainfluenzae requires V factor alone H. ducreyi require X factor alone Hemolysis X V H. influenzae - + + H. parainfluenzae + - + H. ducreyi - + - H. aegyptius - + + The Quellung reaction, Capsule swelling test: Antibodies bind to the capsule and allow them to be visulaized under microscope, if the reaction is positive, the capsule becomes opaque and appears to enlarge H.influenza type b Satellite phenomenon Growing around colonies of S. aureus on BA lysing RBCs in the medium and releasing X factor and excreting V factor. The colonies are much smaller because the V factor inhibitors present in blood are not inactivated. PCR techniques are used to identify Hemophilus species in clinical specimens and as confirmatory test on isolate. Capsular type of H. influenzae isolates is determined by slide agglutination or PCR Treatment MR in pts with untreated meningitis or epiglottitis approaches 100%. Serious infections as meningitis&acute epiglottitis are treated with 3rd generation cephalosporins: Ceftriaxone in AB of 1st choice why? Bacteriocidal for H.influenzae and achieve good concentrations in the meninges and cerebral tissues and is highly effective. Less severe infections, such as sinusitis and otitis, can be treated with oral antibiotics such as: amoxicillin ,azithromycin, clarithromycin Prevention and Control Vaccination Purified capsular PRP conjugated (protein carrier ) vaccine Why conjugated? Only for H. influenzae type b Given at 2, 3 and 4 months( in Libya 2,4,6 penta) Remarkably successful in reducing the incidences of H. influenzae type b disease and colonization. Recommended in children and adults with splenic dysfuction bec they are at increased risk of invasive Hib infection. Impact of Vaccination on incidence of invasive Hib disease Antibiotic prophylaxis Rifampin orally once daily for 4 days eradicates H.influenzae carriage Household members of someone with a serious Hib infection at increased risk of Hib based on age (