MT 6320 Clinical Bacteriology Lecture Notes PDF

[MT 6320] CLINICAL BACTERIOLOGY (LEC/LAB) 3DMT | AY 2023-2024 UNIT #3.2: Haemophilus and Other Fastidious Bacteria Capsule (basis of serotyping)...

[MT 6320] CLINICAL BACTERIOLOGY (LEC/LAB) 3DMT | AY 2023-2024 UNIT #3.2: Haemophilus and Other Fastidious Bacteria Capsule (basis of serotyping) IgA Protease OUTLINE A. Family Pasteurellaceae Virulence Factors Fimbriae B. Haemophilus Outer membrane proteins and a. Haemophilus influenzae LPS b. Haemophilus ducreyi 6 Serotypes a, b, c, d, e, and f c. Haemophilus influenzae biogroup aegyptius d. Haemophilus aegyptius 8 BIOTYPES (BASED ON INDOLE, UREASE, AND ODC e. Laboratory Diagnosis C. Pasteurella REACTIONS a. Pasteurella multocida Meningitis and Septicemia Biotype I D. Family Brucellacaea Indole, Urease and ODC (+) a. Brucella Eye and RT infections E. Family Franciscellaceae Biotype II Biotype III: Indole (-), Urease (+) and a. Franciscella tularensis and III b. Franciscella ODC (-) F. Family Alcaligenaceae Biotype IV Ear and RT Infection a. Bordetella and V b. Bordetella pertussis c. Bordetella parapertussis Meningitis d. Bordetella bronchiseptica Capsulated Epiglottitis G. Family Legionellaceae Strains Causes a. Legionella Bacterial tracheitis b. Legionella pneumophila Non-typeable Sinusitis H. HACEK Group Non-capsulated Otitis media Bacteremia Conjunctivitis Pneumonia Rx: Ceftriaxone (3rd generation cephalosporin) or FAMILY Pasteurellaceae Cefotaxime Haemophilus Rifampin - for chemoprophylaxis Pasteurella ○ Also used to treat TB Haemophilus *Other Gram (-) are not encapsulated GENERALITIES *Children are usually given H. influenzae Type B (HIB) vaccine Gr(-) small, pleomorphic coccobacilli or bacilli Facultative Anaerobes Haemophilus ducreyi Non-motile organisms Ducrey's Bacillus Fastidious Organisms Strict human pathogen ○ Can be difficult to grow in a routine medium Causes Chancroid or Soft and require special nutrients and conditions Sore Most are Oxidase and Catalase positive ○ STD ○ Oxidase (+): dark purple ○ Genital Ulcer ○ Catalase (+): bubbling/effervescence Disease (penis, Nitrate reducers; Ferment CHO labia, vagina) ○ Nitrate → nitrite Painful Obligate Parasites lesion Most require X and V factors except H. aprophilus / ○ Presence of suppurative buboes A. aprophilus ○ Different from hard chancre caused by Satellite growth syphilis Inhabit the URT and oral cavity of humans except H. Rx: azithromycin, ceftriaxone, ciprofloxacin, ducreyi erythromycin Most are normal flora and opportunistic pathogens except encapsulated H. influenzae, and H. ducreyi Haemophilus influenzae biogroup aegyptius Or H. influenzae biogroup III Haemophilus influenzae Non-encapsulated Pfeiffer's Bacillus Causes pediatric conjunctivitis and Brazilian purpuric Major pathogen fever - Presence of fever and itchy rashes Name is a misnomer; frequently associated during the flu pandemic Haemophilus aegyptius May be recovered in upper RT Koch-Weeks Bacillus ○ Usually coughs, colds, coryza (runny nose) Resembles H. influenzae ○ Not debilitating but irritable biogroup aegyptius Causes pink-eye (highly contagious conjunctivitis) This bacteria causes sore eyes-like infection but more severe Haemophilus OPPORTUNISTIC PATHOGENS A. aprophilus: Endocarditis H. parainfluenzae: Endocarditis (mitral valve) H. parahaemolyticus: Pharyngitis PAGE 1 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 LABORATORY DIAGNOSIS SEROLOGY Specimens submitted: Blood, CSF, exudates, joint ID of Capsular Antigen fluids, upper and lower RT specimens, joint fluids, Neufeld Quellung Reaction vaginal swab, abscess drainage, swabs from ○ Gold standard method conjunctiva Phadebact ○ Swabs: Cotton, Rayon, Dacron, Calcium Alginate ○ Transport Media: Amie's with Charcoal; Modified Stuart's CSF is one of the most dangerous and contagious specimen GRAM STAIN H. influenzae: Gr(-) small coccobacilli or small rods H. influenzae biotype aegyptius: Long slender rods H. ducreyi: School of fish ○ Arrangement of bacteria appears in groups H. aprophilus: Very short coccobacilli ISOLATION Very fastidious Immediate and prompt transport to ensure recovery ○ Direct plating is preferred Pasteurella multocida Gr (-) coccobacilli, may appear as ovoid, filamentous, Preferred isolation medium rod shaped 35-37C Facultative Anaerobe 24 hour incubation Non-motile CAP Increased CO2 Catalase and Oxidase (+) Addition of bacitracin is preferred Glucose Fermenter, Nitrate Reducer Enriched with 1% IsoVitalex Indole (+) For primary isolation Staph Streak Satellite growth of Haemophilus Virulence Endotoxin, Capsule Nairobi Biplate For H. ducreyi Factor Clear medium with X and V factors Shipping Fever among cattle, RT Disease Encapsulated H. influenzae infections, wound infections ○ Iridescent colonies Odor Mushroom Smell/Musty Odor Levinthal Agar Non-encapsulated H. influenzae Oral Cavity of domestic dogs, cats, ○ Transparent , bluish, Reservoir pigs, horses, cattle noniridescent ○ Not usually seen in human H. influenzae Non-hemolytic Odor On SBA ○ Mousy/bleach like odor Unable to grow in MacConkey Bipolar "safety pin appearance" X AND V FACTOR REQUIREMENT Staining Oblong-shaped bacteria Use of impregnated disk or strips Haemophilus Quad Plate Disadvantage: Carry-over; misidentification PORPHYRIN TEST Differentiate heme producing species Test for X factor Dependence (+) Result: Red Fluorescence FAMILY Brucellaceae Bang's Bacillus Gr (-) bacilli/ coccobacilli; "sand appearance" Facultative Intracellular Pathogens Non-motile Non-encapsulated Strictly Aerobic Catalase and Oxidase (+) PAGE 2 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 FAMILY Franciscellaceae Brucella Franciscella tularensis Urease (+); Nitrate Reducers F. tularensis subsp tularensis - (Jellison Type A) Asaccharolytic causes the most severe disease (USA) Able to grow in SBA and CAP F. tularensis subsp holartica (in Eurasia and N. Classified as a Category B Select Biological Agents by America) CDC F. tularensis subsp mediasiatica Zoonosis - More common in animals than in humans F. tularensis subsp novicida Animals: Induces spontaneous abortions Humans: Franciscella ○ Systemic Disease: Brucellosis/ Undulant Gr (-) short bacilli, coccobacilli Fever/ Malta Fever / Bang's Disease/ Gibraltar Strictly aerobic Fever/ Mediterranean Fever Encapsulated, Non-motile ○ Systemic Disease affecting the RES, bone; Catalase (+) long term sequelae occurs Oxidase (-) & Urease (-) Long-term sequelae: defined as Ferments glucose, maltose, mannose persistent symptoms or signs ≥ 6 Facultative Intracellular pathogen and Highly Invasive months after acute COVID-19 Fastidious and requires cysteine, cysteine, thiosulfate infection. Zoonosis Classified as a CDC Category A Select Biological Occurs 1-4 weeks after exposure Agent Acute Phase Non specific S/S (Fever, malaise, Highly infectious and has low infective dose anorexia, myalgia etc) Francisella philomiragia Occurs within a year of exposure Causes waterborne tularemia Subchronic / S/S undulant fever, arthritis, Less virulent Undulant Phase inflammation of testes and epididymis MOT Contact with salty water Occurs 1 year after of exposure Capsule, LPS, pathogenecity islands Virulence Chronic Phase S/S depression, arthritis, chronic Highly infectious and has low infective Factor fatigue syndrome dose Causes Rabbit Fever, deerfly fever, LABORATORY DIAGNOSIS Disease lemming fever, water rat trapper's fever or Tularemia Blood Reservoir Hosts Rabbits, and other rodents Specimen Bone Marrow (If suspected for meningitis) Flu-like symptoms, lymph node enlargement, dissemination into blood Gram Stain Substitute Safranin with Carbol Fuchsin stream; pneumonia, conjunctivitis B. abortus Requires niacin for growth ○ Ulceroglandular Brucella Medium, Castaneda Bottle, S/S ○ Pneumonic Septi-Chek, W medium, CAP, SBA; Culture Media ○ Oculoglandular Thayer Martin or Martin Lewis ○ Typhoidal (contaminated specimens) ○ Oropharyngeal Detection of Febrile Agglutinins ○ Glandular Serology ○ Gold standard Sx: Paired Sera LABORATORY DIAGNOSIS Rose Bengal Test Specimens: Lymph node biopsy, scrapings from Other Tests Indirect Coomb’s Test ulcers, sputum, blood EIA Difficult to Gram Stain; Fluorescent stains such as acridine orange - "faint bipolar staining" Culture: BCYE, CAP, Mueller Hinton Broth, TSB ○ Supplemented with cysteine ○ Slow growers; visible growth after 48 hours; plates checked for 14 days ○ Colonies: gray-white, raised colonies, smooth appearance Serology: DFA, immunohistologic staining, slide agglutination test; ELISA Foshay test PAGE 3 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 FAMILY Alcaligenaceae Bordetella Generalities Gram (-) short bacilli / coccobacilli Obligate Aerobes Catalase and Oxidase (+) except B. parapertussis Non fermenters Most are non motile except B. bronchiseptica (peritrichous), B. avium, B. hinzii, B. ansorpii Some species are Zoonotic; also infects animals such as dogs, birds Most are non fastidious except B. pertussis Requires protective substances such as charcoal, blood, starch B. pertussis Bordet Gengou Bacillus Capsulated Most clinically significant species No reservoir host; only humans are infected Causes Whooping cough or pertussis ○ Highly contagious acute infection of the URT ○ Affects children - Younger than 6 months old FAMILY Legionellaceae ○ Severe infection: masikip, dry, at parang Legionella hinihila yung hininga pag umuubo Gram (-) filamentous bacilli ○ S/S Aerobic Catarrhal Stage - most infectious Catalase and Oxidase (+) Paroxysmal Stage Gelatinase (+) Convalescent Stage Motile ○ MOT: Inhalation/ Aerosol Facultative intracellular pathogens Fastidious: requires Cysteine VIRULENCE FACTORS Slow growers - 3-5 days Filamentous hemagglutinin and pertactin Able to grow at a wide range temperatures Pertussis Toxin - exotoxin; modifies host proteins by Asaccharolytic ADP ribosyl transferase; stimulate adenylate cyclase Do not grow in primary media such as SBA Adenylate cyclase toxin Organisms found in natural and artificial water sources Tracheal cytotoxin Most species produce fluorescence after exposure to Capsule UV light (366nm) Rx: Erythromycin L. micdadei Causes Pittsburgh pneumonia B. parapertussis L. bozemanii Causes Wiga agent pneumonia Similar to B. pertussis L. dumoffi Causes pneumonia Causes a less severe respiratory tract infection Non-toxigenic Legionella pneumophila Virulence Factor: endotoxin Most clinically significant species Bordetella bronchiseptica Virulence Factors: Ability to survive intracellularly, proteolytic enxymes Reservoir: dogs and rabbits Causes Legionnaire's Disease and Pontiac Fever Causes Kennel Cough among animals ○ MOT: Inhalation/Airborne spread Humans: Septicemia or Meningitis ○ Legionnaire's Disease (Legionellosis) Virulence Factor: Endotoxin Febrile pneumonic illness LABORATORY DIAGNOSIS Patient has pneumonia and high grade fever which can Nasopharyngeal Aspirates or Swabs, only be treated with a high Specimens Bronchoalveolar Lavage dose of antibiotic Transport at Room Temperature One common cause of community Amies with charcoal acquired pneumonia Transport Swab expressed in 1% casein Dissemination can occur Media hydrolysate - Not routinely used More serious type/form of DFA Stain - Preferred pneumonia and more contagious Staining (+) Small, coccobacilli with intense Mild flu-like yellow-green fluorescence Risk: immunocompromised, heavy smokers, elderly Charcoal horse agar, Bordet Gengou Pontiac Fever Potato Infusion Agar, Regan Lowe Agar, ○ Non-pneumonic form Culture some may be able to grow in BAP or ○ Shorter incubation period MAC ○ Flu-like illness Molecular Methods LABORATORY DIAGNOSIS Serology Respiratory Tract Specimens ○ Sputum Specimen ○ BAL ○ Bronchial washings Gram Stain Staining ○ Faint Staining Gr (-) bacilli PAGE 4 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 ○ Substitute Safranin with Carbol fuchsin Modified Kinyoun Method ○ L. micdadei Silver Staining; Giemsa Culture BYCE with Cysteine Ground Glass Appearance Colonies DFA Procedure Rapid Methods Urine Antigen Test DNA Detection Serology IFAT HACEK Group Causes Endocarditis ○ Haemophlius ○ Aggregatibacter (Acinetobacter actinomycetemcomitans) ○ Cardiobacterium hominis ○ Eikenella corrodens ○ Kingella Gr(-) bacilli; non-motile Fastidious and Capnophilic organisms Opportunists and infect immunocompromised host Slower or Poorer growing Causes Subacute Bacterial Endocarditis Not common bacteria PAGE 5  [MT 6320] CLINICAL BACTERIOLOGY (LEC/LAB) 3DMT | AY 2023-2024 UNIT #3.3: Aerobic Gram-Positive Bacilli Gram stain of bacillus OUTLINE A. Bacillus a. Bacillus anthracis b. Bacillus cereus B. Corynebacterium a. Corynebacterium diphtheriae C. Listeria monocytogenes D. Erysipelothrix rhusiopathiae E. Actinomycetes F. Nocardia sp. Spore stained with Malachite green BACILLUS SPORE-FORMING NONBRANCHING CATALASE POSITIVE BACILLI G(+) sporeforming bacilli Aerobic or facultative anaerobes Catalase (+) Motile SBA Common laboratory contaminant LABORATORY DIAGNOSIS Bacillus anthracis Specimen B. anthracis: blood, lung tissue, CSF VIRULENCE FACTORS Glutamic acid capsule SBA, PEA Protein exotoxin: Protein antigen, edema factor, lethal Colonies: nonhemolytic, gray, raised Culture factor w/irregular curled margin & whirling projections “medusa head” Causative agent of Anthrax ○ Associated w/ cattle, hides, goat hairs Bamboo fishing poles appearance with ○ Occupational hazard (Woolsorter’s disease endospores and Ragpicker’s disease) ○ Spores are located at the center ○ Humans: get infected by direct contact Microscopy Square ends GS: G(+) w/infected animals: fibers, wool, hair G(V): violet rods Fulton Schaeffer: red-veg cells; FORMS OF ANTHRAX green-spore Most common but least severe form S/S: lesion forms on the site of Medusa head Tenacious consistency inoculation that develops into black Cutaneous eschar MOT: Wounds are contaminated with spores Woolsorter’s disease or Ragpicker’s disease S/S: Initial, mild fever, malaise, Inhalation/ fatigue Pulmonary Sudden severe phase: respiratory distress; more severe can lead to death S/S: Abdominal pain, nausea, anorexia Gastrointestinal Higher fatality compared to cutaneous MOT: Ingestion of spores Associated with drug users Injectional “Skin popping” IDENTIFICATION anthrax MOT: Direct injection of spores into Catalase (+) tissues Nonhemolytic Nonmotile Cutaneous anthrax skin infection ○ Wet mount preparation / hanging drop method ○ SIM Transparent (nonmotile) Turbid (motile) Encapsulated ○ India ink stain Direct Fluorescent Antibody Assay Nucleic Acid Amplification Test AST: Penicillin; Gentamycin, Erythromycin, chloramphenicol PAGE 1 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 CORYNEBACTERIUM NONSPOREFORMING NONBRANCHING CATALASE POSITIVE BACILLI G(+) bacilli that are pleomorphic ○ May appear club-shaped, Chinese character, picket fence ○ Diphtheroid appearance: club-shaped, coryneform Facultative anaerobe, catalase (+) Non-motile, non-encapsulated, non-sporeforming A - Catalase Positive C - India Ink stain Widely distributed in the environment: soil, water B - nonmotile D - String of pearl String of pearl reaction Gram stain of B. anthracistaken from a medium containing Corynebacterium diphtheriae 0.05 –0.5 units of Penicillin. (1000x) Klebs Loeffler’s Bacillus VIRULENCE FACTORS Bacillus cereus Most virulent sp (toxin-producing) “Fried rice poisoning” Diphtheria toxin (tox gene) – lysogenic B-phage Common cause of food poisoning and opportunistic Infects humans – Diphtheria infection Spores: resist pasteurization CLINICAL INFECTIONS SBA – aerobic Isolated from human nasopharynx; not a normal flora Beta-hemolytic MOT: person to person exposure to respiratory Motile droplets, direct contact to cutaneous lesions Found in the environment: soil, water Nonspecific S/S Mild sore throat, Low-grade fever, Morphologically and metaboillically same with B. body malaise anthracis, but grown aerobically in SBA ○ Abdominal pain, cough, difficulty in breathing, fatigue Drugs: Penicillin, Erythromycin Respiratory diphtheria & cutaneous diphtheria DIPHTHERIA FORMS Acute, contagious Site of infection: tonsils, pharynx ○ Often accompanied with high grade fever Production of toxin Respiratory form ○ Leading to respiratory obstruction ○ Toxin can damage heart, 2 FORMS OF FOOD POISONING kidneys peripheral nerves Diarrhea Emeti ○ Death due to cardiac failure Ingestion of meat Common in the tropics products, poultry, Ingestion of fried Systemic complications are less Implicated food vegetables, sauces, or boiled rice Cutaneous form common pasta Nonhealing ulcers Incubation period Longer (8-16 hrs) Shorter (1-6 hrs) ○ Specially if patient is diabetic Diarrhea More common Less common Vomiting Less common More common Response when fed Diarrhea Vomiting to Rhesus monkey Eye infection Meningitis Nongastrointestinal infections Treatment: ○ Vancomycin ○ Clindamycin PAGE 2 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 LABORATORY DIAGNOSIS Nasopharyngeal and throat swabs, Specimen skin/wound lesions Highly pleomorphic Presence of Babes-Ernst granules / metachromatic granules / volutin granules ○ Presence of nutrient reserves Most frequently isolated sp GS: G(+) pleomorphic bacilli from humans Stained by methylene blue C. amycolatum Normal flora skin –metachromatic stain Associated w/ prosthetic joint infection Microscopy Associated w/ nosocomial infections Most common cause of C. jeikeium Corynbacterium-associated prosthetic valve endocarditis in adults Produces diphtheria-like toxin Dsc: humans: diphtheria-like Methylene blue stain C. ulcerans infection CAP, CTBA, Tinsdalemedium Cattle: mastitis (mammary gland) C. xerosis Opportunistic infection C. urealyticum Associated w/ UTI Culture Rare cause of lymphadenitis in C. pseudotuberculosis humans following contact w/ infected livestock Normal flora skin, mucus C. psudodiphtheriticum membrane Cause RTI, endocarditis Colony types of Corynebacterium diphtheriae Listeria monocytogenes NONSPOREFORMING NONBRANCHING CATALASE POSITIVE BACILLI G(+) coccobacillus in singles, chains, palisade Aerobic, Facultative anaerobe Catalase (+), motile Recovered from environment: soil, water, sewage, fecal matter, animal products like raw milk Isolated from crustaceans, flies, ticks, domestic animals TEST FOR TOXIGENICITY Disease: Listeriosis ELISA MOT: ingestion of contaminated meat, poultry, Immunochromatographic assay coleslaw PCR ○ Contaminated meat has putrid odor and change in meat’s color Infection of neonates, pregnant, immunocompromised ○ Pregnant S/S: flu-like illness leading to spontaneous abortion and stillborn Elek Test ○ Fetus: granulomatosis infantiseptica ○ Newborn: meningitis, sepsis ○ Immunocompromised: CNS infection, endocarditis VIRULENCE FACTOR Elek plate for Demonstration of Toxin Listeriolysin O Dismutase Scheck’s Toxigenicity Test Catalase Phospholipase C Test Results to swelling Superoxide Protein p60 PAGE 3 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 LABORATORY DIAGNOSIS Erysipelothrix rhusiopathiae Specimen Blood, CSF, swab of lesions NONSPOREFORMING May be mistaken for group NONBRANCHING GS CATALASE NEGATIVE BACILLI B-Streptococcus, Corynebacterium BAP, CAP, nutrient agar, and broth BHI, G(+) bacilli thioglycolate Catalase (-) ○ 30 –35C Nonspore forming Culture Pleomorphic ○ 4C (cold enrichment) ○ Colonies: small, round, translucent Aerobic/facultative anaerobe ○ Beta hemolytic Veterinary infection and occupational hazard (meat, Inverted Christmas tree or umbrella poultry, fish handling) SIM Scratches pattern at room temperature Wet mount Tumbling motility Cuts and skin Arrow head-shaped zone of (+) CAMP CLINICAL INFECTIONS beta-hemolysis at the junction of the two Reaction Disease: Erysipeloid organisms ○ Localized skin infection ○ Painful lesions seen on hands or fingers PATHOGENICITY TEST Ocular test of Anton Rabbit/guinea pig – installation of culture on Erysipeloid conjunctival sac (+) result: Keratoconjunctivitis within 24hrs LABORATORY DIAGNOSIS Tissue biopsy or aspirates from skin Specimen lesions Microscopy Thin, G(+), V-form Culture BAP, CAP, nutrient broth Catalase (-), nitrate (-), VP (-), urease (-) Nonmotile, nonhemolytic Identification H2S positive Test tube brush growth on gelatin at 22C Inverted tree or umbrella Tumbling motility pattern motility Actinomycetes Branching or Partially Acid Fast Aerobic Actinomycetes Species: Nocardia, Streptomyces, Actinomadura, CAMP TEST Rhodococcus, Gordonia, Tsukamurella, Trophery mawhipplei ○ Trophery mawhipplei Causative agent of whipple disease - rare bacterial infection that affects joints and digestive system Nocardia sp. G(+) bacilli Conventional CAMP test with L. monocytogenes showing block Thin branching filaments (beads) hemolysis on the junction of the organism Staphylococcus “Partially acid-fast” aureus. 48-72 hours growth - weeks Species of Nocardia: Depending on the culture conditions, L. ○ N.asteroides monocytogenes can resemble Streptococcus when ○ N.brasiliensis found in the coccoid form and Corynebacterium when ○ N.caviae the bacillus forms prevail. Soil, water, humans Organisms are not usually seen on CSF smear. Catalase (+) Sabouraud’s Dextrose Agar (SDA) 10% CO2 ○ Bumpy, velvety, yellow-orange colonies PAGE 4 MT 6320 (LEC/LAB) 3DMT | AY 2023-2024 CLINICAL INFECTIONS Inhalation / airborne Pulmonary Nocardia asteroids complex Infection Chronic, confluent bronchopneumonia Inoculation of the skin or subcutaneous tissue Chronic granulomatous of the skin, Cutaneous subcutaneous tissue Infection On outdoor activities Nocardia brasiliensis Actinomycotic mycetoma / eumycotic mycetoma LABORATORY DIAGNOSIS May be mistaken for Gram(+) cocci; Beads so not touch each other Microscopy Filamentous branching isolate –partially Acid-fast Wet mount Presence of granules on cutaneous tissue Grows on common nonselective medium Culture Grow at 22C –37C May take longer incubation period Resistant to Penicillin Treatment Susceptible to Sulfonamides PAGE 5