Microbiology Finals PDF

MICROBIOLOGY 1 SMALL, PLEOMORPHIC, GRAM-NEGATIVE BACILLI o Haemophilus influenzae type b (Hib)– Topic Outline: cause of serious infections in humans and Haemophilus...

MICROBIOLOGY 1 SMALL, PLEOMORPHIC, GRAM-NEGATIVE BACILLI o Haemophilus influenzae type b (Hib)– Topic Outline: cause of serious infections in humans and Haemophilus leading cause of meningitis in unvaccinated Hacek (Aacek Group) children. Brucella (Bang's Bacillus) NON-TYPEABLE FORM Francisella Tularensis o Does not produce capsules and part of the Pasteurella (Zoonotic Bacteria) indigenous microbiota of the URT and Bordetella adheres to human epithelial cells Legionella o Second prevalent etiologic agent for otitis media with effusion (middle ear infection) HAEMOPHILUS after Streptococcus pneumoniae Greek word: haima and philos means “blood lover” o Other related infections: Conjunctivitis and Obligate parasites on the mucous membranes of sinusitis (localized infections) humans. Haemophilus parainfluenzae Normally inhabit the URT of humans except Commonly found as indigenous microbiota of the URT Haemophilus ducreyi of adults Fastidious, non-motile, capnophilic, and facultatively Haemophilus ducreyi anaerobic bacteria Not part of the indigenous human microbiota. Die rapidly in clinical specimen and very susceptible to Agent of chancroid which is highly communicable, drying and extreme temperatures. sexually transmitted, genital ulcer disease. Most species cannot grow on pure BAP Hallmarks of chancroid: Buboes or suppurative, Microscopy: enlarged, draining, inguinal lymph nodes. o Gram-negative, small, pleomorphic Culture: coccobacilli or rods o CAP- ·colonies are transparent, small, non- Biochemical test: mucoid, and tan or yellow. o (+) catalase Biochemical Test: o (+) oxidase except Haemophilus segnis o (-) Catalase & Oxidase Growth factors: DIFFERENTIAL CHARACTERISTICS OF o X (hemin) and V factors (NAD) HAEMOPHILUS SPECIES SPECIES DISTINGUISHING GROWTH ASSOCIATED Human pathogens: CHARACTERISTICS FACTOR INFECTION/DISEASE H.influenza H.ducreyi H. influenzae Mousy/bleach-like X, V Meningitis, epiglotitis, (Pfeiffer's bacillus) odor; non- haemolytic arthritis H.parainfuenzae H.paraphrophilus H. aegypticus Genetically related to X, V Pink eye conjunctivitis H.parahaemolyticus H.pittmaniae (Koch-Weeks H. influenzae bacillus) H.aegypticus H.segnis H. influenzae Non-typeable X, V Brazilian purpuric fever Haemophilus Influenzae biogroup aegypticus Main cause of meningitis in children H. haemolyticus B-haemolytic X, V Spreads from nasopharynx and the regional lymph H. ducreyi School or fish X Chancroid or soft chancre nodes to the blood and finally to the meninges H. Tan &dry colonies; B- V Pharyngitis parahaemolyticus haemolytic Very fastidious & can be rapidly killed by phagocytes H. parainfluenzae Fructose & maltose V Endocarditis Only member of the genus that produces IgA fermentation protease LABORATORY DIAGNOSIS Does not produce endotoxin SPECIMENS: MODE OF TRANSMISSION: person-to-person o CSF (droplets) o Sputum Culture: o genital lesion or ulcer o CAP- colonies are translucent, convex, tan- o joint fluid, vaginal swab colored & mucoid w/ a mousy or bleached-like o abscess drainage odor o conjunctival swab Principal Virulence Factor: o bronchial washing & blood o Polysaccharide capsule (serotype A to F) Haemophilus need an immediate transport and Other Virulence Factor: processing for their isolation o IgA protease, fimbriae & lipopolysaccharide For recovery of H. ducreyi, the ulcer should be Biochemical Test: cleansed with a sterile gauze that is pre-moistened w/ o Porphyrin (-) sterile phosphate-buffered saline and plating is Categories of Haemophilus influenzae preferred instead of a transport medium for the TYPEABLE FORM isolation of H. ducreyi. o Based on the capsular characteristics of H. GRAM STAIN influenzae o Haemophilus species resemble an o Encapsulated strains: A, B, C, D, E, and F "amorphous serous material" because of their pleomorphic appearances.  MICROBIOLOGY 1 o H. ducreyi have a "school-of-fish" Aggregatibacter actinomycetemcomitans arrangement FK as Actinobacillus actinomycetemcomitans CULTURE Only catalase-positive HACEK species o Culture Media: CAP, BAP, BHI & Cardiobactrium hominis thioglycollate Infects the aortic valve more freq. than the other o CAP is the preferred medium for HACEK species Haemophilus because it contains the X and V Shows "false Gram-positive" factors. Only indole-positive HACEK member PORPHYRIN TEST Eikenella corrodens o Detects the presence of enzymes that Corroding bacilli converts ALA into porphyrins. Assacharolytic like th especies of the genus Moraxella o Test for identifying the heme-producing Least common isolate of the HACEK group species of Haemophilus Kingella o Reagent: Kovac's reagent It has a tendency to resist decolorization SEROLOGICAL TEST K. kingae- most virulent specie o Serotype can be determined through the DIFFERENTIAL CHARACTERISTICS OF THE HACEK identification of the distinct capsular antigen GROUP o Neufeld-Quellung reaction is a rapid direct Species A. aphrophilus Oxidase v Glucose v Lactose v Maltose v Sucrose v identification of the capsular antigens of H. A. actinomycetemcomitans C. hominis + + + + + + + + + + influenzae. E. corrodens + + + + + K. kingae + + + + + GROWTH PATTERN OF HAEMOPHILUS SPECIES Haemophilus species grow best at 35-37degree BRUCELLA (BANG'S BACILLUS) cent., except H. ducreyi which grows at 35-degree Species of this genus are important human and animal cent. & in an environment with 5% to 10% CO2. pathogens. V factor-dependent -- H. influenzae, grow as Obligate aerobes and intracellular parasites. “satellites” on BAP around bacterial colonies and Non-motile, assacharolytic, and non-encapsulated. produce NAD like S. aureus Some species require an increased supply of CO2 for When Haemophilus are grown anaerobically, only growth. NAD & not hemin, is required. Localized in tissues that are rich in erythritol (placental H. aegyptus requires four days of incubation while H. tissue) & induces spontaneous abortion among ducreyi requires seven days. animals. No growth occurs on MacConkey agar. Preferred specimen for isolation: blood and bone SELECTIVE MEDIA FOR HAEMOPHILUS SPECIES marrow H. influenzae– horse’s blood-bacitracin agar for Microscopy: Small coccobacilli that are arranged respiratory secretions of patients with cystic fibrosis. singly, in pairs which have a “sandy appearance” H. aegypticus– CAP with 1% IsoVitaleX or Vitox Culture: H. ducreyi– selective Nairobi biplate medium o BAP – colonies are small, convex, (combination of gonococcal agar and MHA with translucent, yellowish and non-haemolytic. horse’s blood and vancomycin) Species: o Brucella abortus HACEK (AACEK) GROUP o B. canis Group of fastidious Gram-negative bacteria includes o B. suis the following organisms: o B. melitensis o Aggregalibacter apbrophilus (formerly H. Most virulent species: aphrophilus) o B. melitensis o Aggregatibacter actinomycetemcomitans o B. suis o Cardiobacterium hominis Disease: o Eikenella corrodens E. Kingella species o Malta/Crimean/Mediterranean fever CHARACTERISTICS OF HACEK GROUP o Brucellosis (undulant fever) Part of the indigenous microbiota & are considered as UNDULANT FEVER opportunistic pathogens Characterized by normal temperatures in the morning Small, non-motile, cannot grown on MAC and then followed by high temperatures in the Fastidious and grow slowly on BAP & CAP & require afternoon and evening. 7-14 days of incubation PRIMARY ROUTES OF HUMAN INFECTIONS Cause slow & progressive bacterial endocarditis (BRUCELLOSIS) Utiltizes ALA A. Ingestion of unpasteurized and contaminated milk or Aggregatibacter aphrophilus cheese from infected animals. Foam-loving bacterium B. Inhalation of air around animal carcasses (aerosol Formerly known as Haemophilus aphrophilus infection) C. Penetration of ocular or oral mucosa.  MICROBIOLOGY 1 D. Direct inoculation into the bloodstream through Whooping cough (Pertussis) abrasions in the skin of needlestick injuries. Highly contagious, acute infection of upper respiratory LABORATORY DIAGNOSIS tract & primarily affects children. SPECIMENS: Mode of acquisition: Inhalation of infected droplets o Blood Incubation period: 7-14 days o Bone Marrow 3 STAGES OF WHOOPING COUGH: o Skin lesions 1. Catarrhal stage– highly communicable stage o Placental tissues characterized by mucous membrane inflammation Brucella species should be handled as a Biosafety and mild coughing with runny nose. Level 3 agent in a class III cabinet 2. Paroxysmal stage– associated with vomiting and GRAM STAIN “whooping” or hurried deep respiration that may last o Carbol fuchsin should be substituted for for six weeks. safranin O to improve the Gram Stain 3. Convalescent stage– in this stage symptoms slowly CULTURE decline, may last for six months after infection. o Culture: BAP, TSA & Castaneda's medium LABORATORY DIAGNOSIS o Isolates can be recovered after 7 days but SPECIMENS: may require prolonged incubation up to 30 o Nasopharyngeal swabs days. o Bronchoalvular lavage o Turbidity of the specimen is a normal GRAM STAIN occurrence o The use of two-minute safranin or 0.2% basic SEROLOGICAL TEST fuchsin as counterstain enhances its visibility. o Serum agglutination test CULTURE o Regan-Lowe agar BORDETELLA o Bordet-gengou potato infusion agar Obligately aerobic, fastidious, gram-negative o Modified Jones-Kendrick charcoal agar coccobacilli o Casamino acid broth. Non-carbohydrate fermenters and are non-motile SEROLOGIC TEST except for B. bronchiseptica o Bordetella species can be examined using Replicate on ciliated respiratory epithelial cells of Direct Fluorescent antibody (DFA) stains. humans NUCLEIC ACID TEST Mostly inactive in biochemical test systems o Polymerase chain reaction (PCR)- rapid Culture: test and considered as more sensitive test o Bordet-gengou agar– colonies are smooth, than cultures and DFA assays in identifying glistening and silver color bordetella species. Biochemical test: Differential Tests for Bordetella Species o (+) catalase, (-) indole Species MOTILITY TEST NITRATE REDUCTION OXIDASE TEST UREASE BAP Growth Growth factors: B. bronchiseptica B. parapertussis + + + + + + + + + + o Nicotinic acid, cysteine and methionine B. pertussis + + + + + Species: o B. pertussis FRANCISELLA TULARENSIS o B. parapertussis Category A select bioterrorism agent that requires o B. bronchiseptica to be processed according to biosafety level 3 o B. avium conditions. Bordetella Pertussis (Bordet-Gengou Bacillus) Very small, obligately aerobic, non-motile Ethiologic agent of whooping cough coccobacillus under the genus Francisella and Does not survive well outside the host transmitted by a vector. Culture: Considered as a potential bioterrorism weapon since it o Bordet-Gengou Agar– colonies are small easily spreads with a high mortality rate. and shiny and resembles mercury drops DISEASE: Tularemia (deer fly or rabbit fever) Growth inhibitors: Virulence Factor: Capsule o Fatty acids, metal ions, sulfides and Vector: Deer flies and ticks peroxides Reservoir: Cottontail rabbit Growth protectors: Microscopy: Gram-negative bacilli with bipolar o Charcoal, blood and starch staining. Principal virulence factor: Growth factors: Cysteine or cysteine and thiosulfate o Pertussis toxin (protein toxin) Biochemical Test: An agglutination titer of 1:40 is Preferred specimen for isolation: diagnostic o Nasopharyngeal swab  MICROBIOLOGY 1 LABORATORY DIAGNOSIS LEGIONELLA GRAM STAIN Only genus in the family Legionellaceae o Requires an acridine orange stain to visualize Fastidious, aerobic motile, and non-carbohydrate organisms that are obtained from a blood fermenter culture bottle. Primarily acquired through inhalation CULTURE Microscopy: o Culture Media: o Faintly staining, thin, gram-negative, ▪ CAP bacillary or coccobacillary in form. ▪ MTM Biochemical Test: ▪ Non-selective buffered charcoal o (+) catalase and gelatinase yeast extract (BCYE) o Weak positive in oxidase ▪ MHA Major Reservoir: ▪ TSB o Hot water system, cooling towers and o Growth is not enhanced by incubation at an evaporative condensers. increased carbon dioxide. Species: o Slowly growing organisms require 2-4 days o L. oneumophila for colony formation. o L. micdadei o It cannot grow on MAC. o L. bozemanii o L. dumoffii PASTEURELLA (ZOONOTIC BACTERIA) The distinguishing characteristics of Legionella Facultatively anaerobic and non-motile. species are as follows: Isolated from animal bites (mainly from cats) or scratch Can infect and multiply within some free-living wounds. amoebae, ciliated protozoa and biofilms. Species grow well on BAP and CAP but most species Can be isolated from lakes, rivers, hot springs and cannot grow on MAC. mud. Virulence Factor: Can tolerate up to 3mg/L of chlorine and thus resist o Endotoxin & Capsule water disinfection treatment. Microscopy: Cannot grow on routine primary plated media like BAP o Small, straight, Gram-negative bacilli with a Legionella pneumophila "safety-pin" appearance It is the most commonly isolated human pathogen Culture: in the genus Legionella o BAP & CAP- colonies are gray and non- It is the agent of Legionnaire's disease and Pontiac haemolytic fever. Biochemical Test: It invades the bronchoalveolar macrophage, which is o Oxidase, catalase & indole (+) a facultative intracellular pathogen. o Weak glucose fermenter It is isolated in air-conditioned units, cooling towers, Species: humidifiers, and nebulizers. o P. multocida Serogroups: o P. stomatis o 1 to 7 o P. dagmatis o Serogroups associated to the o P. bettyae Legionnaire's disease: 1,4 and 6 o P. canis Preferred medium: Pasteurella multocida o BCYE with L-cysteine that is buffered to Most commonly isolated species in humans pH 6.9 Common isolate in dog and cat bite infections. Culture: Culture has a characteristic “mushroom smell” o BCYE- Colonies are blue-green and Grows only on BAP and susceptible to penicillin. glistening and have a convex elevation. Biochemical Test: PRIMARY CLINICAL MANIFESTATIONS OF Legionella o (+) Oxidase, OD, indole & urease pneumophila o (-) ONPG LEGIONNAIRE'S DISEASE Pasteurella bettyae o Also known as legionellosis which is a febrile Isolated from amniotic fluid, blood, and urogenital and pneumonic illness. specimens from humans. o Direct physical contact does not spread the Can be sexually transmitted. infection. Glucose & lactose fermenter. PONTIAC FEVER Can grow on MAC. o It is a non-fatal respiratory infection that Biochemical Test: resembles an allergic disease, but exhibits o (+) Catalase the symptoms of pneumonia. o Variable indole production of oxidase WOUND ABSCESS & ENCEPHALITIS  MICROBIOLOGY 1 LABORATORY DIAGNOSIS Preferred Specimens: o Sputum & bronchoalveolar lavage Other Specimens: o Urine o Pleural fluid o Blood o Transbronchial lung biopsy materials STAINING o Microscopy: Faint staining and usually undetectable through Gram staining CULTURE o Most important test for the Legionella species. o Selective medium: ▪ BCYE with L-cysteine, ferric salt, and a-ketoglutarate SEROLOGIC TEST o Indirect fluorescent antibody (IFA) test o Direct fluorescent test (DFA) RAPID METHODS o DNA test (PCR) o Urine antigen test  MICROBIOLOGY 1 MYCOBACTERIA Mycobacterium tuberculosis (Koch bacillus) TOPIC OUTLINE: Microscopy: slender, beaded rods w/ X, V, Y and L MYCOBACTERIA formation MYCOBACTERIUM TUBERCULOSIS COMPLEX Culture: Slow growing, buff in color raised and dry (MTC) o cauliflower colonies NONTUBERCULOUS MYCOBATERIA (NTM) ▪ Rough colonies exhibit 'cording NONTUBERCULOUS MYCOBATERIA (NTM) Biochemical tests: catalase and niacin (+); reduced LABORATORY DIAGNOSIS nitrate to nitrite. It is inhibited by nitroimidazopyran (NAP) MYCOBACTERIA Virulence factor: cord factor Are non-motile, non-spore forming and Specific gravity: 0.79 - 1.07 nonencapsulated and acid fast bacili. Mycobacterium bovis Very thin, slightly curved or straight rods (0.2-0.6 3 1- It produces TB in cattle, dogs, cats, swine, parrots and 10 mm) humans. Are strictly aerobic; catalase (+) It attenuated strain is used for vaccination of new born. o Produces Much's Granules (inlusion bodies) Culture: slow growing, small, granular, rounded, white Closely related to: Nocardis & Rhodococcus colonies and non-pigmented. Cell wall contains: N - glycolylmuramic acid and has Biochemical tests: (-) niacin and do not reduce very high lipid content. nitrate no growth in presence of T2H. Resists decolorization w/ acid - ethanol (acid fastness) Mycobacterium bovis BCG o Resistant to heat, cold and drying Bacillus Calmette–Guérin (BCG) is a live attenuated Most species associated with diseases grow slowly (2- strain of Mycobacterium bovis currently used as the 6 weeks incubation) only vaccine against TB. o Rapidly growing mycobacteria grow: 2-3 Mycobacterium africanum days It is associated with human cases in tropical Africa. o Some species requires 5-10% CO2 Detection of this bacteria requires used spoligotyping Microscopy: (spacer oligotyping). o Slender, slightly curved or straight Gram (+) Mycobacterium canettii rod It is smooth strain of M. tuberculosis. o Tendency to clump Biochemical test: (+) niacin and nitrate o Gram neutral / Gram ghosts It grows rapidly than M. tuberculosis High lipid content of the cell wall: The first human isolate was from cervical lymph o Does not take up dye w/ increased staining node; also isolated from an AIDS patient with time or application heat. mesenteric tuberculosis. Incomplete staining: Beaded appearance Mycobacterium microti o Irregular uptake of stain Found in rodents, guinea pigs, rabbits, cats, llamas, Culture: and meerkats. o Smooth and soft or a rough friable Usually fails to grow in culture. appearance - egg based media. It has been isolated from TB patients in both pH requirement: immunocompetent and immunocompromised o 6.5-6.8 (culture media) individuals. Generation time: PATHOGENESIS o >12 hours - grow slowly because of their TUBERCULOSIS hydrophobic cell surface It is a disease of the respiratory tract. It is a chronic granulomatous infection which is transmitted by the inhalation of infected droplets by TWO GROUPS: means of coughing sneezing or talking. o MYCOBACTERIUM TUBERCULOSIS Mode of acquisition: COMPLEX (MTC) o Airborne droplet of nuclei, 1-5 um enter the o NONTUBERCULOUS MYCOBACTERIA respiratory tract of an exposed individual and (NTM) are deposited in the lung alveoli. Signs and symptoms: MYCOBACTERIUM TUBERCULOSIS COMPLEX (MTC) o Low-grade fever, Night sweats, Fatigue, Mycobacterium tuberculosis (Koch bacillus) Anorexia, and weight loss Mycobacterium bovis o Reactivation occurs when there is an Mycobacterium bovis BCG alteration or a diminution of the cellular Mycobacterium africanum immune system. Mycobacterium canettii If the disease has been chronic and fibrotic, loss of Mycobcaterium microti lung volume and calcifications will be demonstrated. Malnutrition, with or without other factors such as  MICROBIOLOGY 1 alcoholism, homelessness, incarceration, Are present in the environment (soil and water) and immunosuppression, and AIDS, can contribute greatly sometimes colonize healthy Individuals skin, to the progression to active TB. respiratory and GI tracts. CLINICAL DIAGNOSIS Chronic pulmonary disease resembling TB is the usual PRIMARYTUBERCULOSIS clinical presentation associated with these organisms. Is usually limited to detection of a positive tubercullin AIDS has contributed significantly to the incidence and test using purified protein derivative (PPD). awareness of NTM disease. CASEOUS NECROSIS Infections caused by these bacteria are not considered GRANULOMA transmissible from person to person. Is an organization of lymphocytes, macrophages, giant Mycobacterium avium complex (MAC) cells, fibroblasts and capillaries. The most common NTM causing tuberculosis POTT'S DISEASE Can be isolated from sputum, blood and aspirates of It is also known as the tuberculosis spondylitis or bone marrow. skeletal TB of the spine. These organisms are environment saprophytes, and A grave form of tuberculosis caused by the invasion of present in soil, -water and house dust. M. tuberculosis Are important pathogens in immunocompromised and MILARY TB immunocompetent populations (AIDS patients). Is an extrapulmonary TB which refers to the seeding Species: of many organs outside the pulmonary tree with AFB o M. avium through hematogenous spread. o M. Intracellulare It occurs shortly after primary pulmonary disease but o M. avium subsp. avium can take place anywhere in the course of acute or o M. avium subsp. paratuberculosis chronic TB. o M. avlum subsp. silvaticum ("wood pigeon Common sites spread: spleen, liver, lungs, bone bacillus") marrow, kidney, adrenal gland and eyes. Culture: MULTIDRUG-RESISTANT MYCOBACTERIUM o opaque, glossy, white, colony morphology or TUBERCULOSIS (MDR-TB) produce a smaller translucent colony This strain develops when only one antimycobacterial morphology. agent is used or if the patient is on multidrug therapy Reservoir: Natural water and falls to complete the course of medication. Portal of entry: It is usually acquired by spontaneous mutation as a o Respiratory tract and GIT result of the inappropriate use of antimicrobial agents Microscopy: to treat M. tuberculosis and the lack of patient o pleomorphic, short, coccobacillary without compliance. beading stain (+) with PAS Primary MDR -TB Biochemical tests: Without previous history of TB disease and resistance o (+) heat-stable catalase and (+) growth on to at least Isoniazid and rifampicin (anti-TB drugs). media with T2H Extensively drug Resistant TB (XDR-TB) M. avium- is a cause of disease in poulty and swine. Resistance to isoniazid and rifampicin + M. avium subsp. paratuberculosis fluoroquinolone + at least one of three injectable Is the causative agent of Johne's disease second-line anti-TB drugs (aminoglycosides, (Inflammatory bowel disease) in cattle, sheep, and amikacin, kanamycin or capreomycin). goats. Also isolated from bowel mucosa of patients with NONTUBERCULOUS MYCOBATERIA (NTM) Crohn's disease Runyon's Classification Color Produced Growth Rate Species May take 3-18 months for primary isolation ("very slow Photochromogens (Group 1) Not pigmented unless exposed to light. 10-21 days M. kansasil M. asiaticum grower") a. grows in the dark b. grows in the light - cream or buff - orange/yellow M. marinum M. simiae Growth factor: mycobactin Scotochromogens (Group II) Pigmented both in the dark and light. 10-21 days M. szulgai M. gordonae Mycobacterium kansasii (Yellow bacillus) - yellow to orange colonies M. scrofulaceum M. flavescens It is second to M. avium as the cause of NTM lung Nonphotochromogens Non-pigmented both 10-21 days M. xenopi M. avium complex disease (chronic cavitary pulmonary lesions) In (Group III) In the dark and light. M. ulcerans M. terrae complex humans. M. gastri M. haemophilum It is not considered contagious from person to Rapid Growers Pigment variation 3-7 days M. fortuitum M. chelonae person. M. smegmatis M. phlei Microscopy: M. abscessus M. mucogenicum o long rods with distinct cross banding Nontuberculous Mycobateria (NTM) SLOW ("shepherd's crook); some cording GROWERS Culture: A.K.A anonymous, atypical, unclassified, o smooth to rough colonies with dark centers unknown,tuberculoid, MOTT and waxy edges-M8 7H10 agar  MICROBIOLOGY 1 photochromogenic colonies-" dark red branching filaments = cornmeal-glycerol agar crystals of 10-8-carotene" "bird's nest" appearance with sticklike Biochemical tests: projections (young colonies) o (+) Tween 80 hydrolysis in 3 days and It can be either nonphotochromogen or pyrazinamidase; strong nitrate reduction scotochromogen (bright yellow colonies). Mycobacterium marinum Growth temperature: Causes diseases of the fish and has been isolated o 42°C from an aquarium. Biochemical tests: It is the causative agent of "swimming pool o (+) heat stable catalase, pyrazinamidase and granuloma"- a red or blue-red subcutaneous nodule arysulfatase on the elbow, knee, toe or finger Mycobacterium terrae-complex A cutaneous infection in human which occur when Also known as M. terrae - "radish bacillus" traumatized skin came in contact with inadequately It normally saprophytic and rarely causes human chlorinated freshwater or saltwater. infections. Microscopy: Species: o Moderate to long rods with cross-barring o M. terrae Culture: o M. triviale o Smooth to rough and wrinkled deep blue o M. nonchromogenicum colonies (photochromogenic) Microscopy: Growth temperature: o Short to medium coccobacilli o 28-32°C (preferred low temperature) Culture: Biochemical tests: o rough and dry colonies (M. triviale) o (+) Tween 80 hydrolysis, urease and o smooth colonies (M.terrae) pyrazinamidase o smooth to rough and white to buff colonies (M. Natural reservoir: fresh water and salt water. nonchromogenicum) Mycobacterium ulcerans Biochemical tests: 3rd most common Mycobacterium species after M. o (+) Tween 80 hydrolysis and heat-stable tuberculosis and M. leprae. catalase reduction of nitrate A rare cause of Buruli ulcer, a painless nodule under M. triviale- (+) growth in 5% NaCl the skin after previous trauma. Organism M. kansasii Epidemiology Infection more common in Type of infection Chronic pulmonary disease Microscopy: white males Natural reservoir is tap water Extrapulmonary diseases such as cervical lymphadenitis o Moderately long rods without beading Aerosols are involved in transmission and cutaneous disease Culture: M. marinum Natural reservoirs: freshwater and saltwater Cutaneous disease Bacteremia o Smooth and rough nonpigmented colonies Transmission is by contact with contaminated water and (6-12 eeks incubation) organism entry by means of trauma or small breaks in the skin Biochemical test: Associated with aquatic o (+) heat-stable catalase activity fishing usually involving M. scrofulaceum Raw milk, soil, water, dairy Cervical adenitis in children Growth temperature: products Bacteremia o 30-33°C Pulmonary disease Skin infections M. gordonae Tap water, water, soil Infections only in Mycobacterium gordonae (Tap water bacillus) immunocompromised hosts M. avium complex Environmental sources, Patients without AIDS: A problem in the preparation of bacteriologic smears. including natural waters and pulmonary infections in soil patients with preexisting It rarely causes infection in humans. pulmonary disease Cervical lymphadenitis Culture: Disseminated disease* in immunocompromised patients o smooth, yellow-orange colonies who are HIV-negative Patients with AIDS: (scotochromogen) disseminated disease M. xenopi Water, especially hot water Primarily pulmonary infections Biochemical tests: taps in hospitals in adults Believed to be transmitted in Less common, o (+) Tween 80 hydrolysis and heat-stable aerosols extrapulmonary infections (bone, lymph nodes, sinus catalase; (-) nitrate reduction tract) and disseminated disease Growth temperature: M. ulcerans Stagnant tropical waters Indolent cutaneous and Also harbored in an aquatic subcutaneous infections o 22" to 37°C insect’s salivary glands (African Buruli ulcer or Infections occur in tropical or Australian Bairnsdale ulcer) Mycobacterium xenopi temperate climates M. haemophilium Unknown Disseminated disease Recovered from hot and cold-water taps, especially Cutaneous infections immunocompromised adults in water storage tanks of hospitals, and from birds. Mild and limited skin infections in preadolescence or early First isolated from an African toad. adolescence Cervical lymphadenitis in It causes pulmonary infection in adults; a potential Mycobacterium terrae- Trauma and respiratory routes children Tenosynovitis and pulmonary pathogen. complex May be found in aquatic environments disease Microscopy: o long and filamentous rods Culture: small colonies with dense centers and filamentous edges- MB 7H10 agar round colonies with  MICROBIOLOGY 1 CHARACTERISTICS OF OTHER NTM Mycobacterium chelonae Species Microscopy Culture Biochemical Test Distinguishing Characteristis It mostly associated with disseminated cutaneous M. asiaticum Acid-fast coccoid cells Dysgonic, smooth and pigmented (+) high level of heat-stable Saphrophytes; rarely cause infections in immunocompromised patients. colonies (Photochromogen) catalase human infections It is also associated with infections of the skin, lungs M. celatum Large nonpigmented Grows best at 35°C and bone. M. genavense colonies Dysgonic (+) semi- Fastidious It exhibits more resistance to antimicrobial agents than colonies; requires an extended quantitative and heat-stable mycobacteria M. fortuitum. incubation (6-8 catalase It does not grow weeks) on routine Culture: (+) mycobacterial Pyrazinamidase media o Rough or smooth nonpigmented colonies; and urease It has been does not produce extensive flamentous recovered from BACTEC culture branching colonies. M. Strongly acid- Rough to smooth Requires haemophilium fast; short or nonpigmented hemoglobin or Biochemical tests: curved without colonies hemin for growth beding, in o (+) 3-day Arysulfatase test and growth on clusters or Media: cords CAP, MHA with Mac Conkey agar without crystal violet; 5% Flides enrichment, LJ without nitrate reduction. with 2% ferric ammonium citrate Mycobacterium abscessus M. malmoense Short Nonpigmented (+) Tween 80 Growth at 22°C coccobacilli w/o smooth, glistening hydrolysis, heat- require 7-12 Reservoir: tap water cross-bands and opaque stable catalase weeks of colonies with and incubation Related infections/disease: chronic lung disease, dense center pyrazinamidase M. Medium to long Light yellow to (+) high level otitis media. scrofulaceum rod deep orange heat-stable smooth colonies with dense catalse and urease Biochemical tests: centers (scotochromogen) o (+) 3-day Arysulfatase test and growth on M. simiae Short coccobacilli Yellow smooth colonies (+) niacin and high level heat- It is one of the very few NTM Mac Conkey agar without crystal violet; (photochromogen) stable catalase that produces niacin without nitrate reduction. M. szulgai Medium to long rods with cross- Yellow to orange smooth and rough Mycobacterium smegmatis Group barring colonies (scotochromogen) These organisms are causes of pulmonary, skin and soft-tissue and bone infections. Species: M. Nontuberculous Mycobateria (NTM) RAPID smegmatis sensu strict, M. goodle and M. wolinskyi GROWERS Microscopy: Are considered potentially pathogenic. o Long and tapered or short rods are curved, Are found in soil, rivers, municipal water supplies, branching or y-shaped forms marine and terrestrial life forms. Culture: Organisms gain entry into the host by inoculation into o Rough, wrinkled or coarsely folded smooth or the skin and subcutaneous tissues during trauma, rough with dense centers - MB 7H10 agar injections, surgery or animal contact. pigmentation is rare or late - nonpigmented to Microscopy: pink colonies o Weakly Gram-positive rods resembling Biochemical tests: diptheroids. o (-) 3-day Arylsulfatase test; Cultures: o (+) nitrate reduction and iron uptake; o Colonies appear on solid media in 7 days or o (+) growth on Mac Conkey agar without CV less. and media containing 5% NaCI Mycobacterium fortuitum LABORATORY DIAGNOSIS It is the most common rapidly growing mycobacteria The procedures should be done in class II or III associated with localized cutaneous infections - skin biological safety cabinet (BSC) and soft tissue infections. The area which the specimens and cultures are It has been isolated from water, soil, and dust. processed should have negative air pressures in relation to other areas - the airflow should be from Microscopy: clean areas (corridors) into less clean areas o Long and tapered to short, thick rods (mycobacteriology lab). (pleomorphic) have tendency to decolorize All potentially infectious specimens should be tightly and appear partially AFB (old cultures) covered when they are outside the BSC. Culture: SPECIMEN COLLECTION AND PROCESSING o Rough or smooth multilobate nonpigmented a) Sputum colonies- U medium branching Preferred specimen: early morning sputum (3 o Filamentous colonies with aerial hyphae- MB consecutive days) 7H11 agar; Required volume: 5-10 mL aerosol-induced sputum o (+) growth on Mac Conkey agar without b) Gastric lavage crystal violet Uses Levine collection tube Advantage: for senile, non-ambulatory patients and Biochemical tests: children younger than 3 yrs old o (+) 3-day Arylsulfatase test and nitrate reduction  MICROBIOLOGY 1 Procedure: 3 specimens should be collected within 3 o Voided urine days after overnight o Autopsy tissue Required volume: 20-25 ml of gastric secretions o Abdominal fluid Should be processed within 4 hrs of collection or o Any contaminated fluid neutralized w/ sodium carbonate to pH 7.0- Specimens that require decontamination and prolonged exposure to gastric acid kills mycobacteria. digestion: c) Urine o Sputum Preferred specimen: first morning midstream urine (3 o Gastric washing consecutive days) o BAL (branchoalveolar lavage) Required volume: 15 ml or entire volume of voided o Bronchial washing urine o Transtracheal aspirate May be collected through an indwelling catheter with a Specimens do not require decontamination: needle and syringe o CSF d) Bronchoscopy specimens o Synovial fluid Specimens: bronchoalveolar lavage (BAL), bronchial o Biopsy tissue from deep organs washings and transbrochial biopsy REAGENTS It is the specimen of choice for detecting NTM 1) 2-4% NAOH e) Fecal specimens o Is the most common decontamination agent To identify patients who who may be at risk of (2mL sputum + 2ml NaOH). developing M. avium-complex disease, especially o It serves as both a decontaminant and those with AIDS digestant. Stool specimens should be submitted without a 2) 2.5% Oxalic acid preservative o For decontamination of sputum specimens If processing will be delayed, the specimen should be containing Gram-negative rods like frozen at -20 Celsius Pseudomonas (cystic fibrosis). f) Body fluids- pleural, pericardial, peritoneal, joint o Sputum specimens treated with this agent aspirate and CSF can be inoculated into broth culture. Required volume: CSF- 2 mL; for exudates and 3) Zephiran-trisodium PO4 (Z-TSP) pericardial and synovial fluids; 10-15 mL for o A decontamination-digestion reagent. abdominal and chest fluids o Zephiran is an effective decontaminant with Pleural, pericardial and peritoneal fluids- collected little bactericidal effect on the tubercle bacilli. in sterile anticoagulant (EDTA or heparin) o TSP liquefies sputum rapidly but requires a g) Blood long exposure time to decontaminate the It is collected to diagnose individuals with AIDS specimen. Culture medium: BACTEC 13A vial o Phosphate buffer- results in greater isolation h) Wound, skin lesion aspirates and tissue of mycobacteria For wound and skin aspirates, the specimen are o Advantage: for specimens containing large transferred in liquid medium. numbers of bacteria. If the tissue specimens cannot be processed 4) 4.1% Cetyl-Pyridium Chloride immediately, 10-15 mL of sterile saline should be o Prolonged shelf life of sputum for 8 days (for added to prevent dehydration transport). DECONTAMINATION AND DIGESTION 5) N-Acetyl-L-Cysteine (NALC)-NOOH These procedures are performed to kill all o Both decontamination and digestion agents. contaminating organisms (non-mycobacteria) and to o NALC is also known as dithiothreitol, a dissolve mucous substances. digestion agent. Dissolving (liquefying) the mucin prior to inoculation STAINING onto culture medium enables the mycobacteria to use Mycobacteria possess cell walls that contain mycolic the nutrients of the medium. acid, which are long chain, multiple cross-linked fatty The high lipid content in the cell wall of mycobacteria acid. makes them less susceptible to the killing action of Acid fastness is affected by age of colonies, medium various chemical. for growth and exposure to UV light. Factors affecting the action of the Rapidly growing species appear to be acid fast decontaminating agent: variable. o concentration of the chemical agent If at least 2 of the first three sputum direct smears are o exposure/contact time positive, then 3 specimens are often sufficient to o temperature confirm a diagnosis. CSF, urine, and bone marrow specimes- concentrated specimens are used and heat- fixed for Specimens that require decontamination: 15 mins o Sputum  MICROBIOLOGY 1 Distinct acid-fast organisms: Composed of vitamins, salts, cofactors, glycerol, o Mycobacterium malachite green, agar, oleic acid, bovine albumin, o Cryptosporidium glucose and beef catalase. o Isospora. Excessive heating and light exposure of agar-based Partially acid-fast organisms: media result in the release of formaldehyde, which is o Nocardia toxic to mycobacteria o Gordonia A. Middlebrook 7H10-11 o Rhodococcus Produce positive cultures in 3-4 weeks o Tsukamurella Contains 0.1% casein hydrolysate (MB7H11) o Legionella micdadel B. Mitchison's medium 7H11 AFB Staining Methods: Contains polymyxin V, amphotericin B, carbenicillin a) Zehl-Neelsen /Hot Stain Procedure and trimethoprim lactate b) Kinyoun/Cold Stain Procedure- preferably for B) Liquid/Broth Media tissues Use is recommended standard practice for c) Auramine-Rhodamine - sensitive, reliable and mycobacteriology laboratories. specific Increased recovery of mycobacteria and decreased o Auramine is more sensitive than carbol time to detection compared to solid media. fuchsin AFB are examined at 250x and 400x More easily contaminated than solid and the addition magnification. of antimicrobials is required. o Auramine-stained smears may be restained Average time for growth detection of slowly growing by the Ziehl-Neelsen or Kinyoun method. mycobacteria is 12-16 days. o (+) result: bright yellow-orange bacilli against Some fastidious mycobacteria grow only in liquid a dark background media. False Negative Acid-Fast Smear: Shelf life is long; can be stored at room temperature. 1. Overzealous decontamination Incubation with additional CO2 is not required. 2. Loss from concentration techniques smear 1. BACTEC 12B (MB7h12) and BACTEC 13 B (MB 3. Organisms obscured by a too thick 7H13) 4. Over-decolorizing of the smear Improves the isolation rate of mycobacteria and 5. Poor counterstaining reduces the recovery time. 6. Lack of observer proficiency in reading stains These liquid media are part of the BACTEC 460TB False Positive Acid-Fast Smear: System, an automated radiometric method for 1. Changes in the cell wall detection of mycobacteria. 2. Insufficient decolorization Requires daily agitation to enhance growth; should be 3. Laboratory contamination read within 4 days of inoculation 4. Delayed processing Growth indicator: releases of CO2 5. Overgrowth of other bacteria A. BACTEC 12B (MB 7H12) CULTURE MEDIA Specimen volume: 0.5 mL A) Solid Media Growth enhancer: polyoxyethylene stearate Cultures are incubated at 35 Celsius in the dark with Antimicrobial agents added: 5-10% CO2 o polymyxin B Grow best at 30-32 Celsius o nalidixic acid o M. marinum o trimethoprim o M. hemophilum o azlocillin o M. ulcerans Grow best at 42 C B. BACTEC 13 B (MB 7H13) o M. xenopi It improves the isolation rate of mycobacteria and Malachite green is an inhibitory agent of reduces the recovery time compared to conventional nonmycobacteria isolation media 1. Egg-based media Same components with BACTEC 12B + SPS + Are composed of fresh whole eggs, potato flour, polysorbate 80 glycerol, milk, potato and malachite green. Advantage: for culturing large volume of blood and Shelf-life: one year bone marrow Lowenstein Jensen- most common used Specimen volume: 5mL Petragnani medium- for recovery of mycobacteria from heavily contaminatetd specimen 2. BBL Mycobacteria growth indicator tubes o contains 0.052 g/dL of malachite green 3. Middlebrook 7H9 broth Wallenstein medium- for M. avium complex Nonselective liquid medium used for subculturing 2. Serum Agar-based media (Transparent media) stock strains  MICROBIOLOGY 1 4. Septi-Chek AFB 2. Niacin (nicotinic acid) Test Biphasic media: for rapid growth and identification of It is the most commonly used biochemical test for the mycobacteria; 6-8 weeks incubation; does not require identification of Mb and other slow growing radioactivity and CO2 incubator mycobacteria. Pigment Production This test detects the deficiency of the enzyme (niacin o M. szulgai- scotochromogenic at 35*C and connecting enzyme) that converts free niacin to niacin photochromogenic at 25*c non pigmented at ribonucleotide. 25-30°C Niacin plays a role in the redox reaction during Virulence Test mycobacterial metabolism. o Serpentine cord formation It should be performed only from cultures on U that are o Tuberculin test (PPD) 3 weeks old and show 50 colonies. ▪ It detects a patient's cell-mediated It should not be performed on scotochromogen or immune response to the bacterial NTM rapid growers. antigens in a type IV hypersensitivity Reagents: cyanogen bromide + aniline reagent reaction. (+) result: yellow color ▪ It does not differentiate active o M. tuberculosis, M. simiae disease from infection. 3. Nitrate Reduction Test (broth method) ▪ PPD (antigen)- a protein extracted The enzyme nitroreductase converts nitrates to and purified from the cell wall of nitrites. culture- grown M. tuberculosis The results are influenced by age of the colonies, ▪ A reactive (positive) result indicates temperature, pH and enzyme inhibitors. past exposure to M. tuberculosis. Test organisms are incubated in 2 mL of sodium nitrate a. Mantoux Test (0.1mL) at 37C for 2 hours. (+) result: erythema and induration (10mm) or Reagents: sulfanilamide + N- napthylenediamine hardening around the site of Injection dihydrochloride + HCI A positive result indicates M. tuberculosis (10mm Indicator reagent: zinc (detects nitrate) induration). (+) result: red color 45mm (height of the 5. Tween 80 Hydrolysis bubbles) It is useful for separating species of High level Heat-stable catalase positive: nonphotochromogens and scotochromogens. o M. aslaticum It detects the ability of lipase (mycobacteria) to o M. scrofulaceum hydrolyze Tween 80 into oleic acid and o M. simiae polyoxyethylated sorbitol in 24 hours, 5 days or Heat-stable catalase positive: 10days. o M. ulcerans pH indicator: neutral red (amber color)- bound to o M. gordonae Tween 80 o M. xenopi (+) result in 3 days: pink color o M. terrae complex o M. kansasii o M. genavense (+) result: pink color o M. malmoense o M. asiaticum, M. gastri, M. triviale, M. Heat-stable catalase negative: marinum, M. malmoense, M. scrofulaceum, o Mtb complex M. flavescens and M. terrae complex o M. gastri 6. Tellurite Reduction o M. hemophilum It detects the ability of M. avium complex to reduce o M. marinum tellurite in 3-4 days. Isoniazid-resistant strains may not produce catalase AIl NTM rapid growers also reduce tellurite in 3 days. at all. Reagent: potassium tellurite (colorless)  MICROBIOLOGY 1 (+) result: black metallic (tellurium) It is mostly found in the Schwann cells that surround 7. Pyrazinamidase Test peripheral nerve axons and in mononuclear This enzyme hydrolyzes pyrazinamide to pyrazinoic phagocytes. acid and ammonia in 4 days. Microscopy: Reagent: ferrous ammonlum sulfate o Rod shaped exhibiting "cigar pocket/pocket (+) result: red pigment fence" arrangement. o M. kansasil, M. marinum and M. xenopi Culture: 8. Iron Uptake o No growth in artificial culture media (+) growth It detects the ability of mycobacteria to convert ferric in living tissues like the armadillo and ammonium citrate to an iron oxide. footpads of mice It is useful in distinguishing M. chelonae (negative Optimal growth: 30°C reaction) from other rapid growing NTM (positive It may not be detected in skin scrapings (tuberculoid reaction). form). Reagent: 20% ferric ammonium citrate History (+) result: rusty brown colonies (egg-based medlum) Oldest disease known to mankind o M. smegmatis group Leper- Greek word -scaly 9. Urease Test Confused with psoriasis, elephantitis and pellagra It is helpful in distinguishing M. scrofulaceum 1873- Hansen of Norway discovered M. leprae (positive) from M. gordonae (negative) 1943- sulphone drugs used in the treatment Reagent: 4 mL urea broth incubated at 37°C Epidemiological determinants (+) result: pink color in 3 days Agent: Caused by M. leprae o M. scrofulaceum, Mtb, M. kansasii and M. Source of Infection marinum All patients with "active leprosy" must be considered 10. NAP Inhibition Test infectious NAP (p-nitroacethylamino-B-hydroxypropiophenone) Man is the only source and host is a precursor in the synthesis of chloramphenicol. Portal of Exit NAP selectively Inhibits the Mb complex. Nose is a major portal of exit Specimen: colonies from agar or broth (BACTEC M. leprae are discharged in the nasal mucosa medium) Can also exit through ulcerated or broken skin (+) result: 20% increase in growth index (GI) - Mtb Infectivity complex Highly infectious but of low pathogenicity 11. Sodium Chloride Tolerance Test Can be rendered non-infectious by treatment of 3 Reagent: 5% NaCl added into egg-based media weeks (+) result: growth on the media Local application of rifampicin can destroy bacilli o M. triviale, M. flavescens and most NTM rapid within 8 days growers Host Factors Nucleic Acid Test for M. tuberculosis Age 1. Nucleic Acid Hybridization Test Infection can take place at any time depending upon Is the most rapid test for Identification of common the opportunity for exposure. mycobacterial species. Incidence rates peak between 10 and 20 years of age Required Inoculum: single colony (1 mm diameter) and then fall. 2. Restriction Enzyme Analysis A high prevalence of infection among children means 3. Automated DNA Sequencing that the disease is active and spreading. 4. Direct Nuclelc Acid Amplification test Sex It is designed to detect Mb complex bacilli directly from Incidence and prevalence higher in males than in patient specimens females IX. Chromatographic Analysis (TLC, HPLC and GC) Migration PLC identifies mycobacterial colonies in 2-4 hours. Mostly a rural problem Sequencing hypervariable regions of the 16s Due to migration, it is causing a problem in urban ribosomal RNA gene areas. Tuberculosis Stearic Acid- a fatty acid that can be Environmental Factors extracted from the cell wall of mycobacteria and then Humidity favors survival of M. leprae detected by GC/MS in clinical aspects containing few Can remain viable in dried nasal secretion at least 9 mycobacteria; the presence of this substance in CF is days thought to be diagnostic of TB meningitis. In moist soil at room temp. for 46 days Mycobacterium leprae (Hansen's bacillus) Modes of transmission Is a non-cultivatable NTM (not cultivated in vitro) Droplet infection It invades peripheral nerve and skin cells, and Aerosols containing M. leprae becomes obligately intracellular parasite.  MICROBIOLOGY 1 Contact transmission o The number of solid -staining cells/100 total Person to person by close contact (direct or indirect) bacilli is reported as the morphologic index Other routes (MI). Insect vectors o Solid staining cells - are those with dense, Tattooing needles uniform staining of the entire bacillus with Incubation period even sides and rounded ends in which the 3-5 years or more length of the bacillus is at least 5x the width of Early reaction the organism. Known as Fernandez reaction o The BI and Ml aid in determining the progress Inflammatory reaction seen in 24-48hrs of the disease. Tends to disappear in 3-4 days Serological tests If the redness is more then 10mm at the end of 48hrs o Fluorescent leprosy antibody absorption test then the test is considered to be positive o DNA amplification Late reaction o ELISA Known as Mitsuda Biochemical test Reaction becomes apparent in 7-8 days o Heat Stable Catalase test (result: negative) Maximum in 3-4weeks Inoculation of specimen to the mouse footpad is If there is a nodule more than 5mm in diameter then the definitive test for M. leprae. test is positive o Specimen: biopsy material Classification of Leprosy o (+) result: the development of the disease Patients can be classified into three groups, each with (Hansen's disease) slightly different signs and symptoms: 1. Paucibacillary (PB), or tuberculoid, Hansen’s disease o is characterized by one or a few hypopigmented or hyperpigmented skin macules that exhibit loss of sensation (anesthesia) due to infection of the peripheral nerves supplying the region. 2. Multibacillary (MB), or lepromatous, Hansen’s disease o is characterized by generalized or diffuse involvement of the skin, a thickening of the peripheral nerves under microscopic examination, and has the potential to involve other organs, the eyes, nose, testes, and bone. Lepromatous leprosy o Numerous poorly defined lesions o Symmetrical distribution. o Positive smear test. o Muscle weakness or paralysis (especially in the hands and feet) Enlarged nerves (especially those around the elbow and knee and in the sides of the neck) Eye problems that may lead to blindness (when facial nerves are affected) 3. Borderline, or dimorphous, Hansen’s disease o is the most common form. When compared to tuberculoid or lepromatous forms, it is of intermediate severity. The skin lesions seem to be of the tuberculoid type, but are more numerous, and may be found anywhere on the body. Peripheral nerves are affected as well, with ensuing weakness and anesthesia. Acid-fast staining - biopsy specimen o The number of organisms is reported as the bacteriologic index (BI).  MICROBIOLOGY 1 ANAEROBIC BACTERIA Peptostreptococci, Oral, sinus and dental infections Porphyromonas WHAT ARE ANAEROBIC BACTERIA? Fusobacterium Actinomyces Aspiration pneumonia and "ANAEROBES"= organisms that do not require B. fragilis pleuropulmonary infections F. nucleatum oxygen to obtain energy or to grow. Peptostreptococci These are bacteria that do not live or grow when Porphyromonas oxygen is present. INDICATORS OF ANAEROBIC BACTERIA Anaerobes are found in soil, in freshwater and Foul odor upon opening an anaerobic jar or bag (such saltwater sediments. as the C. difficile, Fusobacterium and Porphyromonas) "Exogenous Anaerobes"= anaerobes that exists Presence of sulfur granules (such as the Actinomyces outside of the bodies of animals and the infection they spp., Propionibacterium spp., Eubacterium nodatum) cause is termed as exogenous infections (usually Brick-red Fluorescence (such as Prevotella or caused by gram-positive, spore-forming bacilli Porphyromonas) belonging to the genus Clostridium) Absence of superoxide dismutase (SOD) "Endogenous Anaer