Bacterial Classification: Study Guide (PDF)

Culture on chocolate agar or Thayer Martin medium and incubate at 37C for 24 hours 5-10% CO2 conditions (candle jar). The colonies are identified by morphology, gram stain (oxidase test, fermentation of glucose and maltose with acid only Diagnosis of N. meningitidis carriers: The specimen used is a...

Culture on chocolate agar or Thayer Martin medium and incubate at 37C for 24 hours 5-10% CO2 conditions (candle jar). The colonies are identified by morphology, gram stain (oxidase test, fermentation of glucose and maltose with acid only Diagnosis of N. meningitidis carriers: The specimen used is a nasopharyngeal swab (West swab) that is cultured on Thayer martin medium and incubate at 37C for 24 hours 5- 10% CO2 conditions.  Moraxella catarrhalis ( commensal neisseria ) It can grow at room temperature & on nutrient agar at 37˚c. Moraxella catarrhalis growing on chocolate agar after 24 hours of incubation Moraxella catarrhalis cause otitis media. Sinusitis and pneumonia Laboratory Diagnosis of Moraxella catarrhalis infection Specimen is ear discharge or sputum 1. Direct microscopy of smear stained with gram stain showing gram –ve diplococci. 2. Culture on nutrient agar 3. Colonies are dry, greyish - white, opaque. When colonies pushed with loop, they “scoot” across media 4. The colonies identified by biochemical reaction 5. No acid production from glucose, maltose, lactose and sucrose or (variable).  Commensal Neisseria The organism present in the throat, nasopharynx, nose, mouth and less frequently external genitalia They are mistaken for Neisseria meningitidis in examination of meningococcal carriers. Neisseria meningitidis Commensal Neisseria Colonies Non-pigmented Pigmented Growth Grow on chocolate agar Grow on nutrient agar Temperature 5-10% CO2 at 370C room temperature Arrangement intracellular and extracellular extracellular  Corynebacteria = club The genus Corynebaccteria include the Corynebacterium diphtheriae) and Diphtheroids Corynebacterium diphtheriae causing diphtheria Morphology: Gram-positive bacilli, Non motile, Non capsulated, Non-spore forming. The bacilli arranged in Chinese letters The organism is beaded due to presence of metachromatic granules (phosphate granules). The methylene blue is the only dye that stain bacilli and leave phosphate granule that appear beaded  Cultural characters C. diphtheriae are aerobic bacteria that grow best on loeffler’s serum at 37C , It can grow on blood agar. Virulence factor Virulence depends on production of exotoxin  Diphtheria Diphtheria has virtually disappeared in developed countries following mass immunization, but is still endemic in many regions of the world. Mode of transmission : Diphtheria is transmitted by oral droplets or physical contact. Diphtheria also transmitted by saliva The source of infection is case or carrier. Pathogenesis of C. diphtheriae The released exotoxin disseminated through blood and affecting heart, muscle, peripheral nerves, adrenals, kidneys Clinical picture of diphtheria Sign and symptoms: it is characterized by sore throat, low-grade fever, and the tonsils are covered with pseudomembrane. Any attempt to remove this membrane causes bleeding and Bull neck spread of toxins. respiratory obstruction and suffocation may occur. Low grade fever Bull neck Sore throat Difficulty in swallowing Severe myocarditis Diagnosis of diphtheria is clinically Laboratory diagnosis for confirmation Laboratory diagnosis of diphtheria case Diagnosis of diphtheria is clinically Laboratory diagnosis for confirmation Diagnosis of carrier: Throat or Nasal swabs are diagnosed as in a case. 1.Virulence tests to prove the toxigenicity of the isolated organism  Diphtheroids It is normal flora of the mucous membrane of the throat, skin and respiratory tract. Gram-positive bacilli, Non–spore-forming , Non motile arranged in angles or in palisade Diseases caused by Diphtheroid 1. Urinary tract infection 2. Bacteraemia 3. Osteomylitis  Tuberculosis Learning Outcomes By the end of the lecture the student will be able to: 1. Know morphology and growth characters of M. tuberculosis 2. Recognize the clinical form of tuberculosis 3. Describe the role of cell mediated immunity in tuberculosis 4. Analyse the tuberculin test results , Quanti-Feron test and it’s laboratory diagnosis of tuberculosis, prevention, and treatment.  Mycobacteria The genus Mycobacteria comprises the acid-fast bacilli, aerobic , non-spore formers. They can not stain by simple stain due to cell wall of Mycobacterium tuberculosis contains a high amount of lipids, including mycolic acids, which form a waxy layer that makes it impermeable to most stains. Medically important species that infect humans are 1. Mycobacterium tuberculosis is the etiologic agent of pulmonary tuberculosis in humans. 2. Mycobacterium bovis is the etiologic agent of TB in cattle and humans. and the organism secreted in milk. The human infected by ingestion of milk from infected cattle and causes intestinal tuberculosis 3. M. leprae causing leprosy. 4. Atypical mycobacteria: it is nontuberculosis mycobacteria (NTM), worldwide distribution such as M. avium, M. kansasii, M. intracellulare that causes chronic pulmonary lung disease simulating tuberculosis in immunosuppressed patient. 5. Saprophytic mycobacteria : it is environmental acid- fast bacilli such as Mycobacteria smegmatis. They are not associated with human illness. Typical versus saprophytic or NTM mycobacteria Typical Saprophytic or NTM mycobacteria mycobacteria Slowly grower Rapid grower Incubation time Non-pigmented pigmented Colonies acid fast Acid fast Staining alcohol fast Non-tuberculosis Mycobacteria M. avium, M. kansasii, M. intracellulare ❑Opportunistic ❑It widespread in the environment ❑ it causes chronic pulmonary lung disease ❑No person to person transmission ❑Resistant to antituberculosis drugs  Mycobacterium tuberculosis Morphology Mycobacteria are thin straight or curved rods. They cannot be classified as either Gram-positive or Gram-negative. They are classified as acid-fast and alcohol- fast as demonstrated by Ziehl-Neelsen stain.They appear as thin pink rods single or in small groups. Mycobacterium tuberculosis is not stained by the Gram stain because the cell wall of contains a high amount of lipids, including mycolic acids, which form a waxy layer that makes it impermeable to most stains. Culture Characteristics: M. tuberculosis grows only on egg selective medium (Lowenstein-Jensen medium). It is an obligate aerobe but 6-8% CO2 enhances growth Slow growth rate 2-8 weeks. Virulence factor: the ability to survive intracellular inside macrophages Virulent bacilli grow on fluid media form serpentine cord factor in which hooked the acid fast bacilli together and arranged in chains Tubercle bacilli arranged in chain due cord factor Lowenstein-Jensen medium  Virulence factors The tubercle bacilli cell wall is rich in lipids, mycolic, waxes, phospholipids induce necrosis. The complex cell wall of ( lipid , polysaccharide, and proteins) responsible for the granuloma formation. Sulfolipids make the bacteria survive intracellular by prevent phagosome and lysosome fusion Mycolic acid ( lipid layer) make the Mycobacterium Resistant to acid Resistant to antibiotics Resistant to killing by complement Resistant to drying Resistant to Gram staining Resistant to drying and can survive for long periods in dried sputum They are sensitive to heat, UV light, alcohol and phenol They are destroyed by pasteurization Tuberculosis M. tuberculosis transmitted by air born which the bacilli carried by air. The disease has two clinical forms, Primary tuberculosis Reinfection tuberculosis Primary tuberculosis The site of initial infection is usually the lungs following inhalation of bacilli. They are engulfed by alveolar macrophages in which they replicate intracellularly with the development of delayed hypersensitivity after 6 weeks. The inflammatory lesion called (tubercles) characterized by granuloma formation consists of epithelial cells , lymphocytes and giant cell. Bacilli are disseminated by blood stream to many organs. These are known as secondary sites. Both primary and secondary sites of infection usually undergo fibrosis and calcification. Secondary tuberculosis ( latent tuberculosis) There are some organisms remain viable in a dormant form in these lesion. The patient have a latent infection. In 5-15 % of people , the primary lesions reactivate after months or years after primary infection. Laboratory identifications. Specimen: sputum not saliva. 3 morning sputum samples collected on three consecuative days should be examined as follows: 1. Direct smears are made from the specimen, and stained with Ziehl-Neelsen stain. 2. Decontamination and concentration of sputum by NaOH to kill all bacteria except TB and the deposit is cultured on L-J medium and incubated at 35-37 oC. Growth appears up to 8 weeks. M. Tuberculosis required selective media for isolation because of it is slow grower and the rapid grower organism consume all nutrients in the media that lead to no growth the specimen is contaminated by many types of flora 3. Rapid diagnosis can be done by A. Detection of DNA in the patient ’s specimens by PCR B. QuantiFERON TB test: it depend on measuring of interferon gamma released when the blood of tuberculosis patient or latent infection is mixed with specific protein derived from tubercle bacilli such as ESAT-6 or CFP or specific tuberculosis antigen C. BACTEC system D. Indicator tube Ziehl-Neelsen stain experiment Type of stain: differential stain it is used to differentiate between acid fast bacilli and non- acid fast bacilli Ziehl-Neelsen stain Bacterial culture Culture on Lowenstein –Jensen medium ( egg enriched medium) Type of medium : selective ( solidification, no agar) Components of medium : malachite green dye to inhibit bacteria other than TB Growth on medium : dry and buff yellow Other type of medium : Middlebrook’s broth Culture on fluid media ( rapid diagnosis) Radiometric fluid medium of BACTEC TB system Non-radiometric medium  Rapid diagnosis Culture on fluid media by C. BACTEC culture system D. Mycobacteria indicator tube Culture on media containing C Labeled plamitic acid. The growing bacteria utilize the palmitic acid and released radioactive CO2 detected by machine culture on fluid media containing fluorescence sensor, when Mycobacteria consume O2 produce fluorescence that detected E. Nucleic acid detection by UV. GeneXpert ( real time PCR) Automated diagnostic test Bacillus group This genus includes large aerobic, Gram positive , spore-forming bacilli that have - Pathogenic : Bacillus anthracis, which causes diseases mainly in animals, but it can affect man and lead to anthrax - Saprophytic : Anthracoids ( which are widely spread in nature ( water, soil, and air). Some strains of Anthracoids are pathogenic such as B. cereus cause food poisoning Bacillus cereus is Gram positive bacilli , non-capsulated, motile, spore forming Laboratory identification of anthrax Specimens: sputum , skin exudates and blood Direct microscopic examination 1.Gram stained smear: Gram positive aerobic bacilli with square ends arranged in chains (bamboo stick appearance) and surrounded by unstained halo capsule, 2.Direct smear stained with polychrome methylene blue; for demonstration of polypeptide capsule: the organism appears blue while the capsule purple or pink. The specimen are culture on : Blood agar: non-haemolytic reaction with Medusa head colonies Gelatin medium produce inverted fire tree appearance (due to slow gelatin liquefiers) maximum liquefication on the surface than at the bottom  Identification of Bacillus anthracis Gram stained film of B. anthracis in Gram stain film of Bacillus anthracis culture. Bamboo stick or square shape showing blue bacilli surrounded by a with central spores pink capsule (MacFaydean reaction of polychrome methylene blue stain) Culture of Bacillus anthracis on blood agar shows non-haemolytic reaction with Medusa head colonies Inverted fire tree on gelatin medium Laboratory identification of Anthracoids Anthracoids are frequently laboratory contaminants on culture media Specimen for isolation of Anthracoids bench , skin , and shoe swab Culture of swab on : ▪ Nutrient agar: incubated aerobically for 37 C. Anthracoid produce large- white colonies, with a rough surface and irregular fimbriated edge. ▪ Blood agar: β-haemolytic colonies. The colonies are further identified by: Microscopy: Gram stained film: Gram positive bacilli with square ends arranged in chains and contain spores, which appear as unstained oval and central spaces Biochemical reactions: Gelatin liquefication test appear rapidly. Culture of saprophytic Anthracoids Culture of saprophytic Anthracoid on blood on N. agar agar showing B- haemolysis Central spores of B. anthracis Other Bacillus species Bacillus stearothermophilus It used as biological indicator to test efficiency of autoclave vial containing bacteria are involved with article being autoclaved and are subsequently incubated at 37 C. If the color changed indicate bacterial growth. Anaerobic non spore forming bacteria Learning Outcomes (LOs) By the end of the lecture the student will be able to: 1. To know what is anaerobic bacteria 2. To recognize the classification anaerobic bacteria 3. To know the medically important anaerobic bacteria and it is laboratory diagnosis  Oxygen requirements of bacteria aerobes anaerobes facultative aerotolerant microaerophilic Anaerobic bacteria that grow only in complete absence of oxygen. Obligate aerobes : these can grow only in the presence of free O2 Facultative anaerobes : These bacteria can grow in the presence of O2 and also grow when deprive of it Microaerophilic : these organisms grow best in the presence of low amount of O2 Specimen collection 1-Avoid contamination with normal flora 2-Blood sample cultured on anaerobic blood culture bottle 3-Collection of samples by needle aspiration It should be freshly prepared It should be used within 2 weeks of preparation It supplemented with special nutrients It contains reducing agents To Heat or steam in water bath at 80C for 2min. when used to expel any dissolved oxygen Robertson’s cooked meat media Anaerobic classification Non- spore forming anaerobes Spore forming anaerobes Gram negative Gram positive Clostridium spp bacilli bacilli Bacteroides Lactobacillus Actinomyces  Anaerobic non-spore forming Gram negative bacilli Gram negative, Intraabdominal infections Post operative wound infection short bacilli, non- following abdominal operation motile non spore Puerperal sepsis forming, Preiodonitis pleomorphic with Lung abscess terminal or central swellings, with or without vacuoles. They are normal inhabitants of The most important the bowel, vagina infections are: and oral cavity Prof. Haneya Anani  Anaerobic non-spore forming Gram positive bacilli Characters : They are Gram- positive bacilli, non-motile Disease of arranged in chains, Lactobacilli: It found in oral cavity, intestine, Dental caries vagina, milk & milk products. Benefits of lactobacilli: Acidogenic Aciduric Protective low pH in normal adult female Used as probiotic Prof. Haneya Anani  Actinomyces Spider colonies Actinomyces is a genus of Gram-positive filamanteous branching bacilli. non-spore forming Some species are anaerobic, while others are microaerophilic. The important species is Actinomyces israelii It cause actinomycosis : abcess The organism form mycelial masses that protrude from the sinus and are known as sulphur granules Sulphur granules visible in yellow color Sulphur granules are crushed between 2 slides to prepare Gram stain film that show gram positive mycelia Culture Sulphur granules are cultured on blood agar for10 days. Spider colonies are identified Gram positive branching bacilli  Anaerobic spore forming bacteria Clostridium Clostridia are gram-positive spore forming anaerobic bacteria Natural habitat: –Intestinal tract of human and animals –saprophytic in soil and water Medically important Colstridia: Cl. tetani causing tetanus Cl. perfringens causing gas gangrene and food poisoning Cl. botulinum causing botulism Cl. difficile causing antibiotics associated diarrhea 10/1/2024 dr. haneya anani 92  Tetanus Causative organism :Clostridium tetani Morphology: Gram positive bacilli Anaerobic Gram stained film demonstrating Motile drum stick spores of Cl. tetani Non capsulated Have terminal spores – drumstick shape. Habitat Soil and intestinal tract of human and animals Culture characters Cl. tetani grows in cooked meat medium and produce thin film (when grow on blood agar ) The organism produce exotoxin 10/1/2024 dr. haneya anani 93 Laboratory diagnosis Specimen – wound exudate Direct smear shows Gram-positive bacilli with drum- stick appearance Culture: culture the specimen on Robertson cooked meat medium , incubated overnight at 37 °C then subculture on blood agar and incubated anaerobically at 37 °C Cl. tetani produce thin film or swarming ; they are α- hemolytic followed by β-haemolysis The colonies identified by motility test ( positive ) Slowly gelatin liquefication 10/1/2024 dr. haneya anani 94 Clostridium perfringens Morphology: Gram positive bacilli Anaerobic Non-motile Non capsulated They have subterminal spores It is normal commensal in human intestine Culture characters Cl. perfringens grows blood agar produce β- haemolysis Disease : gas gangrene 10/1/2024 dr. haneya anani 95 Laboratory diagnosis of gas gangrene lacerated wound swab is cultured on : Robertson cooked meat medium , incubated over night at 37 °C and then subculture on : Neomycin blood agar , incubated under anaerobic condition for 2- 3 days at 37 °C Clostridium perfringens produce β- haemolysis The colonies identified by Gram stained film shows Gram positive bacilli with subterminal spores The colonies identified by Biochemical identification: Motility is negative Catalase negative Indole negative Gelatin liquefication : Slowly gelatin liquefier Stormy clot formation with litmus milk medium Culture on egg yolk medium shows opaque zones Rapid diagnostic test : Naglar’s reaction is positive Isolation of Clostridium perfringens Anaerobic jar β- haemolysis of Clostridium perfringens  β-haemolysis on blood agar Lecithinase activity of Clostridium perfringens showing opaque zones on egg Gram positive bacilli with yolk agar subterminal spores Rapid diagnostic test Nagler’s reaction shows Opacity around streak on right. Antibody against α-toxin inhibits activity around left streak. Stormy clot formation Lactose IV) Identification Fermentation Acid Gas Stormy clot Coagulation milk protein Clot Stormy clot reaction by Clostridium perfringens on litmus milk Laboratory identification of Listeria & Haemophilus Learning Outcomes 1. Know the disease caused by Listeria & Haemophilus 2. Describe the laboratory identification 3. Interpret the results of case study Prof. Haneya Anani 101 Listeria monocytogenes Morphology & Culture ❑Gram positive bacilli arranged in short chain ❑Non- capsulated ❑ Motile ❑ Non- spore forming ❑Listeria is cold-tolerant and can grow in Gram positive bacilli refrigerated drink and food Prof. Haneya Anani 102 Listeria monocytogenes cause Neonatal listeriosis (neonatal meningitis) Adult listeriosis: meningitis and gastroenteritis Laboratory identifications : Blood culture CSF culture Grows well on blood agar; colonies produce a narrow zone of hemolysis similar to Group B Streptococcus The colonies identified by Direct smear of specimen shows gram positive bacilli Catalase test : positive They have tumbling motility at 25 C; "umbrella" type Prof. Haneya Anani 103 Differentiating Characteristics between L. monocytogenes and Diphtheroids Species Catalase Hemolysis Motility Esculin hydrolysis L. monocytogenes Beta hemolytic Diphtheroids Variable Prof. Haneya Anani 104  Haemophilus influenzae Normal habitat Present in the nasopharynx 75% of healthy children and adult, H. influenza type b (Hib) 3-4% Haemophilus species. Haemophilus influenza : meningitis Haemophilus parainfluenzae : urethritis Haemophilus aegyptius (associated with eye infection Haemophilus ducreyi causes chancroid (sexually transmitted disease) Haemophilus influenzae Pathogenicity : H. influenza is the most common etiologic agent of acute bacterial meningitis young children Haemophilus “ Loves Heme” Cell morphology: H. influenzae are Gram negative coccobacilli that are non motile, non spore forming and have polysaccharide capsule.  Haemophilus influenzae Culture and growth characters: H. influenzae is fastidious requires (factor X) and factor V) for growth chocolate agar is a good medium which provide both X and V factors. X factor : hemin V factor: nicotinamide adenine dinucleotide Grow best at 35-37 C , pH 7.6 & 5% CO2 is necessary for growth. Aerobic or facultative anaerobic It does not grow on normal blood agar. Laboratory diagnosis Specimens are pus, sputum or CSF. 1-Gram staining of CSF commonly reveals pleomorphic, gram-negative coccobacilli 2- Direct detection of H. influenza Type b capsule in specimens either by the o Capsular antigen may be detected in CSF or other body fluids using latex agglutination, ELISA o Detection of capsular polysaccharides (Quellung reaction) Culture; on chocolate agar at in 5% CO2. Colonies are identified by their morphology References 1. Jawetz, Melnick, & Adelbery’s Medical Microbiology. Authors: Geo F. Brooks; Janet S. Butel; Stephen A. Morse. Chapter 7. 27th International ed. McJraw-Hill Education 2. Bauman, Robert, Microbiology with diseases by body system , (2018), 5th ed., Pearson. 3. Keith Struthers. Clinical Microbiology. © 2017 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business. International Standard Book Number-13: 978-1-49878689-8 (Pack – Book and E book) 4.Baliey & Scott’s diagnostic microbiology, fifteenth edition. ISBN: 978-0-323- 68105-6. Copyright © 2022 by Elsevier, Inc. International Standard Book Number: 978-0-323-68105-6 Prof. Haneya Anani 109