Medical Microbiology Lecture Notes PDF

Medical Microbiology  Haemophilus (Loving blood) The genus Haemophilus contains many species but H. influenzae is the most common. Other species of Haemophilus of clinical importance to immuno-competent humans are: ✓ H. ducreyi ✓ H. influenzae aegyptius ✓ H. parainfluenzae H. ducreyi: ca...

Medical Microbiology  Haemophilus (Loving blood) The genus Haemophilus contains many species but H. influenzae is the most common. Other species of Haemophilus of clinical importance to immuno-competent humans are: ✓ H. ducreyi ✓ H. influenzae aegyptius ✓ H. parainfluenzae H. ducreyi: causes chancroid an STD (soft sore) which is an ulcer on the external genitalia, lymph nodes are tender and enlarged. The H. ducreyi lesion (chancroid) is distinguished from a syphilitic lesion (chancre) in that it is a comparatively soft lesion. H. influenzae aegyptius: a biotype of H. influenzae. It is associated with purulent conjunctivitis and Brazilian purpuric fever (a life threatening childhood infection). H. parainfluenzae: a rare cause of pneumonia and endocarditis.  Haemophilus influenzae Morphology Gram negative, non-motile, facultative anaerobes pleomorphic small cocco-bacilli. H. influenzae can be grown on chocolate agar and requires hemin (factor X for synthesis of iron containing respiratory enzymes) and nicotinamide adenine dinucleotide (NAD+:factor V for oxidation-reduction processes) for growth which is enhanced by high CO2 concentration (5%). It does not grow on normal blood agar. ✓ H. influenzae which requires factor V and factor X ✓ H. parainfluenzae which requires factor V only ✓ H. ducreyi which requires factor X only. Serotypes Two major categories of H. influenzae were defined: 1. Encapsulated smooth strains were classified on the basis of their distinct capsular antigens. There are 6 generally recognized types of encapsulated H. influenzae: a-f. The type b capsule is composed of polyribitol phosphate (PRP). 2. Unencapsulated strains are termed nontypable (NTHi). Virulence factor: 1. IgA protease (degrade scretory IgA) 2. polysaccharide capsule (especially capsulated strains type b "Hib"). Pathogenesis Encapsulated type b, their capsule allows them to resist phagocytosis and complement-mediated lysis in the non- immune host. The unencapsulated strains are almost always less invasive, however they can produce an inflammatory response in humans which can lead to many symptoms. Hib infection initially causes A Runny Nose, Low Grade Fever Headache. Due to its invasive nature the organism enters the circulation and crosses the blood-brain barrier, resulting in a rapidly progressing meningitis (stiff neck), convulsions, coma and death. Type-b H. influenzae may also cause septic arthritis conjunctivitis, cellulitis, and epiglottitis. Non-typable strains of H. influenzae are the second commonest cause of otitis media in young children and eye infections in children. In adults, these organisms cause pneumonia, These organisms also cause acute or chronic sinusitis in individuals of all ages. Diagnosis ▪ Culture: Bacterial culture of H. influenzae is performed on Chocolate agar, plate with added X & V factors at 37°C in an enriched CO2 incubator. Blood agar growth is only achieved as a satellite phenomenon around other bacteria. H. influenzae will grow in the hemolytic zone of Staphylococcus aureus on Blood Agar plates. The hemolysis of cells by Staph. aureus releases nutrients vital to the growth of H. influenzae. ▪ Catalase and oxidase tests, both of which should be positive. ▪ Serological to differentiate between H. influenzae b and non-encapsulated species. ▪ Capsule swelling ▪ PCR assays have been proven to be more sensitive. Satellitism Treatment In severe cases cefotaxime and ceftriaxone are the elected antibiotics, and for the less severe cases ampicillin and sulbactam, cephalosporins of the second and third generation, or fluoroquinolones. Both H. ducreyi and H. influenzae aegyptius can be treated with erythromycin. Prevention A vaccine containing the capsular polysachride of Hib conjugated to diphtheria toxoid.  Bordtella Bordetella pertussis causes pertussis or whooping cough in infants and young children. Bordtella pertussis Morphology and culture: B. pertussis is a Gram-negative, capsulated, non-motile aerobic coccobacillus of the genus Bordetella. It is a fastidious organism that does not grow on common laboratory media, oxidase positive but urease negative. It can be grown aerobically on special culture media at 37OC for 3- 5days. Colonies are grayish-white with a shiny covex surface “mercury drop” appearance on Bordet- Gengou agar Pertussis The bacteria spread by coughing and by nasal drops (droplet infection) Whooping cough is an acute infection of the respiratory system and characterized by a “whooping” sound when the person breathes in. Pathogenesis B. pertussis infects its host by colonizing lung epithelial cells. There is no blood invasion. The bacterium contains: 1-A surface protein, filamentous hemagglutinin (FHA) (agglutinogens): These are not exotoxins but are implicated in the binding of the organism to ciliated epithelial cells. 2-Tracheal cytotoxin: stops the cilia from beating. This prevents the cilia from clearing debris from the lungs, so the body responds by a coughing fit. In higher concentrations, it causes ciliated epithelial destruction. 3- Two exotoxins, known as: a. The pertussis toxin (PTx) (pertussigen): It contributes to bacterial binding to ciliated epithelial cells, increased mucus secretion b. Adenylate cyclase (CyaA): prevent phagocytes. 4. Dermonecrotic (heat-labile) toxin: It is a very strong vaso-constrictor causes necrosis of the tracheal tissue. 5. Lipopolysaccharide (LPS): They cause irreversible shock and cardiovascular collapse. Signs and symptoms The signs and symptoms are similar to a common cold, 3 stages of disease: 1.the catarrhal stage: runny nose, sneezing, mild cough, and low-grade fever. After a spell, they might make a “whooping” sound when breathing in or vomit. Adults have milder symptoms. 2.the paroxysmal stage: inspiratory sound characteristic of pertussis. prolonged and distressing inspiratory gasp (whoop). Hypoxia during prolonged attacks may lead to seizure, or coma. 3.convalescent stage: The cough episodes slowly decrease and recovery over 3-16 weeks Pneumonia, otitis media, and meningo-encephalitis are secondary complications. Diagnosis 1-Isolation of the organism is positive during the catarrhal and early paroxysomal phases from a nasopharyngeal or an oropharynx swab for Gram stain. 2- Growth on complex enriched media e.g.; Bordet-Gengou agar or which shows mercury-drop colonies. 3-Direct immunofluorescence to identify the organism in the smear. 4-PCR may be used. 5-Serologic detection of antibodies ELISA Treatment Antibiotic treatment can be effective during the catarrhal and early paroxysmal phases before virulence factors bounded to the corresponding cell receptors. Macrolides as erythromycin is the drug of choice for treatment and prophylaxis. Oxygen inhalation and sedation during paroxysms to avoid hypoxia and brain damage. Prophylaxis ▪ A heat killed vaccine from capsulated strains of B. pertussis in DPT vaccine. ▪ An acellular vaccine consisting of filamentous hemagglutinins and detoxified pertussigen Pertussis vaccine is part of the DTaP (diphtheria, tetanus, acellular pertussis) immunization: consisting of 5 antigens of inactivated pertussis toxin (pertussis toxoid).  Legionella Morphology and cultural characters Legionellae are facultative intracellular pathogens, which stain poorly as Gram negative, motile rods. It does not grow on conventional agar (as they are fastidious). L. pneumophila is cultured on medium that contains iron and cysteine which is vital for growth e.g. buffered charcoal yeast extract (BCYEα) agar. The organisms are slow growing requiring 3 to 7 days at 35 degrees. The organism is oxidase positive, catalase and do not ferment sugars. Pathogenesis Legionellae species requires to be phagocytosed into monocytes, the bacteria prevent phagosome-lysosome fusion and proceed to replicate until they lyse the phagosome and release of the bacteria. Humoral immunity has little effect. Clinical Presentation L. pneumophila resides in the environment in pools of stagnant water worldwide. It often infects hot water towers and air conditioning systems. Legionellae present as two distinct clinical diseases: -Legionnaires' disease, a typical pneumonia. community-acquired pneumonia and a nosocomial infection. - Pontiac Fever with with flu-like symptoms.  Diagnosis 1. Culture: pleural fluid or lung biopsy on BCYEα agar aerobically. 2. Serology by detection of IgM or rising titer of IgG by ELISA. 3. PCR tests Treatment - Erythromycin alone or in combination with rifampin is the treatment of choice. - If erythromycin cannot be used, doxycycline is recommended. Prevention By eliminating aerosols from water sources, and reducing the incidence of legionella in hospital water supplies by using temperature and hyperchlorination.  Francisella tularensis ▪ Francisella tularensis is a small, Gram-negative, non- motile, encapsulated, pleomorphic coccobacillus. ▪ It is a facultative intracellular parasite. ▪ Care must be taken in handling the sample because of the low infectious dose. As few as 10 - 50 bacilli will cause disease in humans if inhaled or introduced intradermally, whereas a very large inoculum (~108 organisms) is required for the oral route.  Tularemia (rabbit fever) F. tularensis is the causative agent of tularemia a zoonotic disease. Its primary reservoirs are rabbits, hares, and ticks. Man acquires tularemia via insect bites (ticks primarily, deer flies, mites, blackflies, or mosquitoes) or by handling infected animal tissues. A small skin papule usually develops at the site of entry. Ulceration occurs together with fever, chills, malaise, fatigue, and usually lymphadenopathy. Bacteremia usually occurs. Pathogenesis The capsule of the organism renders it resistant to phagocytosis. Symptoms are due to cell-mediated hypersensitivity Prevention and treatment Streptomycin is the drug of choice. A live attenuated organism vaccine is available.  Pasteurella Pasteurella multocida Animal pathogen (zoonotic infection), specially mice and rabbit in human causing 1. Local infection (cat scratch, animal bite). 2. Systemic infection (bacteraemia and meningitis). 3. Respiratory infection. Treatment Self-limiting but successful treatment with aminoglycoside, penicillin or cephalosporin  Gardinerella vaginalis Morphology Gram negative rods, non-capsulted and non motile Pathogenicty The organism may present as a commensal in the vagina but it may cause bacterial vaginosis (non specific vaginitis). The patient suffers from vaginal grayish white discharge with fishy odor. Laboratory diagnosis 1. The discharge usually gives (fishy) odour after addition of KOH drop. 2. microscopic analysis 3. culture on blood agar.. beta haemolysis. Treatment Metronidazole  Bacteroides Bacteroides are Gram-negative capsulated coccobacilli. Bacteroides is the most common cause of serious anaerobic infections, e.g. sepsis, peritonitis and abscess ✓ B. fragilis ✓ B. melanogenicus Pathogenesis Because Bacteroides species are part of the normal flora, infections are of endogenous type. Infection arises from a break in a mucosal surface, and is not communicable. Predisposing factors for infection are ▪ Trauma, ▪ Necrosis, ▪ Impaired Blood Supply And ▪ The presence of facultative anaerobes (E. coli) which utilize O2, and allow the growth of anaerobes. Bacteroides causes a variety of infections such as sepsis, peritonitis and local abscess at the site of mucosal break, metaststic abscesses by haematogenous spread to distant organs, or lung abscess by aspiration of the oral types. Laboratory diagnosis ▪ Specimen: pus or blood collected in a transporting medium to the laboratory as oxygen or dryness kills the microorganism. ▪ Stain: Gram-negtaive capsulated coccobacilli. ▪ Culture: Blood agar containing kanamycin (to inhibit unwanted organisms), anaerobically, colony is smooth, translucent and non haemolytic: ✓ B. fragilis is stimulated by bile and hemin. ✓ B. melanogenicus is stimulated by vit.K and hemin. ▪ Serology: indirect immunofluorescent anticapsular antibodies Prevention Cephalosporins before abdominal and pelvic operations. Treatment Metronidazole and surgical intervention to drain pus. Aminoglycosides are frequently combined to teat faculatative Gram negative rods in the mixed infections.  Acid Fast Bacteria Classification of Mycobacteria Microbiologic characteristics ▪ Mycobacteria are obligating aerobic and nonmotile bacteria. ▪ Mycobacteria do not contain endospores or capsules. ▪ While mycobacteria do not seem to fit the Gram-positive category from an empirical standpoint (i.e., in general, they do not retain the crystal violet stain well), they are classified as an acid-fast Gram-positive bacterium (resist decolourization with acid as 20% H2SO4) due to their lack of an outer cell membrane. ▪ A characteristic cell wall, thicker than in many other bacteria, which is hydrophobic, waxy, and rich in mycolic acids/mycolates.  Mycobacterium tuberculosis (Tubercle bacilli, tuberculosis bacteria, TB) Can be stained by Zeihl-Neelsen stain and appear as thin rods arranged singly or in small groups. Culture characteristics They grow on egg-enriched media such as Lowenstein-Jensen (L-J) medium. They grow very slowly; no growth appears before 2-4 weeks incubation at 37oC because the generation time is 12-18 hours. Biochemical activities and resistance They are relatively resistant to acids, alkali and dryness. They can survive in dried expectorated sputum, which is a factor in their transmission by aerosol. They are killed by pasteurization of milk.  Tuberculosis Tuberculosis is caused by Mycobacterium tuberculosis complex (MTC) ✓ M. tuberculosis (human type) transmitted by droplets ✓ M. bovis (bovine type) transmitted by ingestion of milk from infected cattle and causes intestinal tuberculosis. Mycobacterial cell wall: 1-outer lipids 2-mycolic acid 3-polysaccharides (arabinogalactan) 4-peptidoglycan 5-plasma membrane 6-lipoarabinomannan (LAM) 7-phosphatidylinositol mannoside 8-cell wall skeleton Antigenic characters The cell wall contains several antigenic fractions: 1. Lipoid fraction or cord factor: found only in virulent bacilli which grow in a characteristic cord-like pattern which damages mitochondria. 2. A lipid fraction which is largely bound to proteins and polysaccharides. It is partly responsible for acid- fastness and for most cellular and tissue reactions to the bacilli. 3. A protein fraction causes delayed hypersensitivity. the antigens in the purified protein derivatives (PPD) 4. A polysaccharide fraction of unknown biological significance. Pathogenesis of tuberculosis ▪ The organism does not produce invasive enzymes or toxins. ▪ M. tuberculosis infects the lung, and is distributed systemically within macrophages and survives intracellularly. Inhibition of phagosome-lysosome fusion and resistance to lysosomal enzymes play a role. ▪ It then multiplies within the cellular vacuole or phagosome. Lead to the production of a substance that interfere with fusion of phagosome and lysosome and inhibit generation of H2O2 Primary infection (tuberculosis, PTB) Most commonly occurs in the lungs causing an initial exudative lesion (acute inflammatory response called the Ghon’s complex characterized by: A. An acute exudative lesion which involves the regional lymph nodes. B. The local lymph nodes caseate and usually calcify. C. The primary lesion usually heals leaving the person immune and hypersensitive i.e. tuberculin positive. Cell mediated immunity helps to limit the primary infection by the formation of granuloma. In most people the primary lesion is self-limiting by formation of fibrin around the granuloma with central caseation necrosis and such a lesion is called a “tubercle”. Mycobacteria within the tubercle remain dormant but viable and can be released later in life. Reinfection (post primary) type or post-primary tuberculosis (PPTB)=secondary stage This may be endogenous due to reactivation of tubercle bacilli that have survived in the primary lesion or exogenous from the environment. Koch’s phenomenon When a guinea pig is injected subcutaneously with virulent tubercle bacilli, a nodule forms after 2 weeks, ulcerates, the draining lymph nodes enlarge and caseate. When the same animal is injected later with tubercle bacilli in another part of the body, there is rapid necrosis at the site of injection due to hypersensitivity, but the ulcer heals rapidly and regional lymph nodes are not involved (due to immunity). So immunity and hypersensitivity are the features of cell mediated immunity to tuberculosis. The relation between infection, allergy and immunity called Koch’s phenomena. Clinical findings ▪ Fever, fatigue, night sweats and weight loss are common. ▪ Pulmonary tuberculosis causes chronic cough and spitting of blood. Military tuberculosis: Multiple disseminated lesions and a high fatality rate. Diagnosis I. Direct methods Specimens: Sputum (3 successive morning samples) is examined for diagnosis of pulmonary tuberculosis, urine samples (3 successive morning samples or 24 hr urine) for renal tuberculosis. ▪ The presence of acid fast bacilli gives presumptive diagnosis. ▪ Subsequently, when cultured, M. tuberculosis will grow very slowly after several weeks. It grows on : o Conventional cultures containing egg L-J medium. o Rapid radiometric culture method (BACTEC TB system) o Mycobacteria growth indicator tube (MGIT) II. Indirect method Tuberculin test: Skin testing for delayed hypersensitivity with tuberculin (PPD). The test is done by intradermal injection of PPD of the tubercle bacilli, containing 1-10 IU. Treatment Mycobacterial infections are notoriously difficult to treat due to: ▪ Their cell wall, which is neither truly Gram negative nor positive. ▪ Their unique cell wall, they can survive long exposure to acids, alkalis, detergents, oxidative bursts, lysis by complement, and many antibiotics. Tuberculosis is usually treated for extensive time periods (9 months or longer) since: ▪ The organism grows slowly and may become dormant (metabolically inactive). ▪ Weak penetration of the drug because of waxy cell wall of the bacilli. ▪ Most bacilli are intracellular. ▪ The caseous material interferes with the drug. By using two or more antibiotics (including rifampin and isoniazid) due to: ▪ The possibility of resistance developing during this extended time is minimized. ▪ To reduce the drug toxicity. The first line of treatment drugs are; isoniazid (INH), rifampicin, ethambutol and pyrazinamide. In infections with multiple drug resistance; second line drugs should be used. These include streptomycin, para- amino salicylic acid, ethionamide, cycloserine, capreomycin, amikacin , kanamycin and ciprofloxacin. BCG vaccine (Bacillus de Calmette et Guerin, an attenuated strain of M. bovis) immunize against M. tuberculosis and M. bovis. It stimulates CMI  Mycobacterium leprae (leprosy bacteria, LB) The causative agent of leprosy (Hansen's Disease) a chronic disease which affects mainly the mucous membrane of the nose, the skin and nerve fibers, often leading to disfigurement. The organism infects the skin, because of its low temperature. Cultural characters The organism does not grow in culture media or cell culture. However, it grows well in the armadillo (which has a low body temperature). Animal inoculation Injection of material containing lepra into foot-pads of mice induces a characteristic lesion. Pathogenesis Infection may be acquired by prolonged contact with patients with lepromatous leprosy, who discharge M. leprae in large numbers in nasal secretions and from skin lesions. The incubation period between 2 and 10 years and the onset is insidious.The organism replicates intracellularly within skin histocytes, endothelial cells, and nerve cells. Leprosy has two major clinical forms: Lepromatous leprosy: The course is progressive and malignant. It is characterized by skin nodules and symmetric nerve affection. Acid fast bacilli are abundant in skin lesions with bacteraremia due to immunosuppression and lepromine test is negative. Tuberculoid" leprosy: The course is benign and non-progressive with macular skin lesions and asymmetric nerve involvement. There are few organisms due to control by active CMI. The lepromin test is positive and the skin infiltrated with T helper cells. Differences between clinical forms of leprosy Diagnosis Clinical picture. M. leprae has been identified on the basis of acid-fast stains. Staining with Z-N Acid fast bacilli in bunches intracellularly is diagnostic. Take 6 smears, slit skin smears (SSS): o 2 from the skin of ear lobes. o 2 from the skin over elbow joints. o 2 from the skin over knee joints. Bacterial index (BI): the relation between the number of the bacilli in both the skin and the nasal smear to know the type of leprosy. Morphological index(MI): the relation between the viable bacilli which stain evenly, clearly and solid from the nonviable one which unevenly, poorly stained and fragmented. This MI is used to follow up treatment. Lepromin The test is done by intradermal injection of heated exudates from lepromatous nodules or from armadillo lesions. A positive test indicates the presence of CMI due to past or recent exposure. The test may be negative in severe cases of lepromatous leprosy. Treatment Dapson, rifampicin Prevention No vaccine is available. Chemoprophylaxis with dapson for exposed children. Isolation of lepromatous patients