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Gram Positive Bacilli and AFB Flashcards

1
Q

Endospore Morphology

A
  • Ellipsoidal (oval)
  • cylindrical (rectangular)
    spherical
    swollen (bulging sporangium)
    position:
  • terminal (at the pole of the cell)
  • subterminal (par-central) (near the pole but not at the end)
  • central (middle of the cell)
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2
Q

Endospore Staining Characteristics

A

Staining properties:
- highly resistant to staining due to thick spore coat
- heat application (e.g. Schaeffer-Fulton method) enahnces stain uptake
- resistant to decolourisation, retaining primary stain (e.g. malachite green)

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3
Q

Acid-Fastness: Ziehl-Neelsen Stain

A

Target Organisms:
- Mycobacteria and related bacteria
Cell Wall Composition:
- high mycolic acid content -> waxy, hydrophobic barrier
Acid-Fastness Types:
- comple acid fast: retains stain after acid-alcohol wash
partial acid-fast: some decolourisation occurs

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4
Q

Bacillus: General Characteristics

A
  • aerobic, spore-forming, gram positive
  • three broad groups
    • bacillus cereus group
    • bacillus circulans group
    • bacillus subtilis group
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5
Q

Bacillus Habitat and Contamination

A
  • mostly saprophytic
  • ubiquitous in the environment
  • common culture contaminants, especially bacillus subtilis group
  • spore are resilient and widely distirbuted
  • can survive harsh conditions and resist sterilisation
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6
Q

Bacillus Anthracis - overview

A
  • first bacterium identified as a disease-causing agent
  • likely an obligate pathogen in humans and animals
  • endospore formation -> enables survival in harsh environments
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7
Q

Bacillus anthracis - clinical significance

A

anthrax forms:
- cutaneous (90-99%) entry via skin lesions
- intestinal - ingestion of infected meat
- pulmonary - inhalation of spores (high dose required)
Transmission:
- natural infection from infected animals or animal products (mainly herbivores)

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8
Q

Bacillus anthracis - identification

A

Genus level traits:
- gram positive bacilli
- aerobic
- spore-forming
- catalase positive
species-specific traits
- non-motile
non-haemolytic
- encapsulated
- tacky colonies (tease with loop)
- oxidase reaction
- some strains grown anaerobically
produces exotoxin -> causes tissue death

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9
Q

Bacillus cereus - clinical significance

A
  • causes foodborne illness -> two distinct syndromes
    1. Diarheal disease
  • toxin: heat-labile enterotoxin
  • sources: meat, vegetables, pasta, milk
  • onset: 8-16 hours after ingestion
    2. emetc syndrome
  • toxin: heat-stable enterotoxin
  • source: rice
  • onset: 1-5 hours after ingestion
    spore survive cooking and germinate in food
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10
Q

Listeria Monocytogenes - Clinical Significance

A
  • major food borne pathogen
  • can cause listeriosis, which is severe is pregnant women, newborns, elderly and immunocompromised individuals.
  • found in raw and processed meats, poultry, diary products, vegetables, and seafood.
  • animals and around 10% of humans can be carriers of listeria in their GI tract.
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11
Q

Listeria - Description and Morphology

A

cellular morphology:
- short, irregular rods with parallel sides and rounded ends, single or short chains.
Colonial morphology:
- small greyish-white colonies with narrow beta-haemolysis (may resemble group B streps)
Motility:
- tumbling motility at 28 degrees

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12
Q

Listeria - Identification

A

Gram stain:
- gram positive rods
Heamolysis:
- narrow beta haemolysis on BA
Catalase - positive
Motility - tumbling at 28-30 degrees
Aesculin hydrolysis: positive
CAMP test: positive (shovel shape)
cold enrichment: growth at 4 degrees
VP test: positive

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13
Q

Coryneforms - Description

A
  • around 90 species; around 50+ can cause infection, others found in bords/animals
  • facultative anaerobes, catalase pos (most), oxidase neg
  • non-motile and non-spore forming
  • not acid fast
  • only true corynebacterium have a “club” shape, others are irregular rods.
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14
Q

Corynebacterium - Clinical Significance

A
  • commensals in humans - found on skin, mucous membranes (oropharynx)
  • historically dismissed as contaminants but now linked to hospital infections
  • opportunistic infections, often forming biofilms and showing antibiotic resistance. pathogenic species include:
  • C.diphtheriae - respiratory or cutaneous
  • C.jeikeium - septicaemia in hospitalised patients
  • C.ulcerans and c. psuedotubculosis (diphtheria-like disease, zoonotic
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15
Q

Differentiating Corynebacterium and Diphtheria

A
  • C. diphtheriae is the main pathogenic species
  • effective immunisation has made diphtheria rare, but still endemic in some regions
    Two forms of diphtheria:
  • upperrespiratory tract illness
  • cutaneous form
  • toxigenic strains causes severe systemic effect
  • other diphtheria-causing species: C. ulcerans, C.pseudotuberculosis (zoonotic)
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16
Q

Cutaneous vs. Respiratory Diphtheria

A

Cutaneous Diphtheria:
- more common in tropical areas and poor hygience conditions
- affects skin rather the pharynx
- caused by superinfection of skin wounds (e.g. burns) with C.diphtheriae
- typically non-toxigenic strains
- false membrane forms over the wound
- slow healing and insensitive to touch
Respiratory Diphtheria
- C.diphtheriae replicated and secretes toxin
- causes epithelial damage, inflammation and necrosis
- exudative membrane forms over tonsils and pharynx, leading to respiratory obstruction
- membrane removal releases more toxin, spreading to kidneys, heart and CNS.

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17
Q

C. diphtheriae - isolation and identification

A
  • rarely detected, lab expertise declining, so reference labs used
    Best swab samples:
  • beneath membrane for suspected cases
  • nasopharyngeal swabs for carrier detection
    culturing methods:
  • swabs plated on BA and tellurite agar
  • potassium tellurite inhibits normal flora of the upper respiratory tract
  • 5% CO2 atmosphere for initial isolation
  • if delay in inoculation, enrich in blood/plasma broth before plating
  • PCR test detects diphtheria toxin genes.
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18
Q

C.diphtheriae - Morphology

A

Colonial morphology (BA)
- small, greyish-white colonies
- variable B-hameolysis
- may require a lipid source for growth
- needs selective and differential agar for identification
Cellular morphology
- not acid fast and does not branch
- club-shaped rods: slightly curves with wider ends
- singles, pairs, palisades (snapping division)

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19
Q

Reverse CAMP Test - overview

A
  • also called CAMP inhibition test
  • detects inhibition of S.auereus B-heamolysis on sheep RBCs
    Method:
  • test organism streaked at a right angle to S.aureus
  • incubate overnight
    Positive result:
  • dark triangle (no haemolysis) due to phospholipase D breaking down S. auereus heamolysins.
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20
Q

Reverse CAMP Test - Significance

A
  • standard CAMP test differentiates S. agalactiae
  • reverse CAMP test differentiates C. perfringens from other clostridium species.
    works because:
  • C.perfringens’ alplha toxin (phospholipase) interacts with CAMP factor.
  • creates synergistic “bow tie” haemolysis
21
Q

Clostridium spp. - General feautures

A
  • gram positive
  • spore forming (heat resistant)
  • anaerobic or aerotolerant
  • motile, catalase negative, not acid-fast
  • metronidazole-sensitive
  • some ferment sugars
22
Q

Clostridium spp. - classification

A
  • one of the largest and most diverse bacterial genera
  • over 130 species identified
  • due for reclassification
  • up to 5 new genera proposed based on 16s rRNA homology
23
Q

Clostridium spp. - clinical significance

A

exogenous infections:
- gas gangrene - c. perfringens Type A
- intoxication - c. perfringens Type A
- botulism - c. botulinum (affects PNS)
- tetanus - c. tetani (affects CNS)
- enteritis necroticans (pig bel ) - c. perfringens Type C
endogenous infection:
- hospital acquired diarrhea - c. difficile (linked to broad spectrum antibiotics)
- septicemia (often iatrogenic)
- intra-abdominal infections
- pelvic inflammatory disease (PID), intrauterine infections
- abscesses (post surgery or medical intervention)

24
Q

Clostridium spp. identification

A
  • gram positive rods in young cultures
  • detects heat-resistant spores
  • anaerobic culture required for growth
  • catalse negative
    sugar fermentation varies by species
25
Clostridium Perfringens - Isolation and Culture
Selective media for anaerobes: - phenyl ethyl alcohol (PEA) blood agar - colistin/nalidixic acid BA - Thioglycolate or cooked meat broth Subculture for identification - gram stain: observe spore shape and location - EYA: - Lipase - positive -> pearly layer on colony surface - Lecithinase positive --> white precipitation in agar - BA - examine haemolysis pattern
26
Clostridium perfringens - Identification
- double zone haemolysis on BA - colonies can spread on media - Lipase-negative - lecithinase positive - spores are oval, subterminal - Nagler reaction: - detects alpha toxin (phospholipase activity) - enzyme breaks down lecithin --> tissue damage - activity inhibited by anti-alpha toxin
27
Clostridium Spp. - Lipase and Lecithinase Activity
Lipase - hydrolyses fats --> produces iridescen sheen on agar Lecithinase (Nagler reaction on EYA) - breaks down lecithin --> forms insoluble diglyceride - causes tissue damage - positive in C. perfringens (white precipitate in agar) - inhibited by anti-alpha toxin.
28
Clostridium Sporogenes - Identification
- common clostridium species - part of the gut microbiome - single zon heamolysis with rhizoid margin - colonies can spread - lipase-positive (produces iridescent sheen) - lecithinase negative - spore are oval, subterminal - Nagler reaction negative
29
Erysipelothrix - Description
Cellular Morphology - long filamentous rods - short single or paired rods Colonial Morphology - cells from smooth colonies: - short rods/coccobacilli - cells from rough colonies: long filamentous rods (may over decolourise) - growth may take up to 3 days - pin-prick colonies, small, smooth or rough, transparent - may be a- or y-heamolytic
30
Erysipelothrix - Clinical Significance
Clinically significant species: Erysipelothrix rhusiopathiae - zoonotic infection: transmitted via contact with animals, their products, or wastes. - high risk occupations: butchers, vets, meat processors, farmers, poultry workers, fish handlers. Forms of Human Infection: Erysipeloid – mild inflammatory skin infection Diffuse cutaneous form Systemic infection – rare but severe; includes septicaemia and endocarditis (40% mortality with therapy)
31
Erysipelothrix - Identification Tests
- Gram-positive rod, may appear Gram-variable - Catalase-negative (Listeria is catalase-positive) - Non-motile (Listeria is motile at 28°C) - Slowly produces H₂S on TSI agar (helps differentiate from Lactobacillus) - One of the few Gram-positive bacteria that produce H₂S
32
Mycobacteriaceae - Description
- slightly curves or straight, thin rods (may branch) - high lipid content (mycolic acids) - prevents gram staining - requires acid fast stain - acid fast - resists decolourisation by acid/alcohol (ETOH) Other Characteristics: Aerobic Non-spore forming Non-motile Some species produce yellow pigment Slow-growing (generation time 2–20 hours) Optimal growth: 30–45°C M. leprae cannot be cultivated on artificial media
33
Mycobacteriaceae - Classification
Grouped into: - TM (Tuberculosis Mycobacteria) & NTM (Non-Tuberculous Mycobacteria) MTB Complex (Slow Growers): - M. tuberculosis, M. bovis, M. microti, M. africanum MAC (Mycobacterium Avium Complex - Slow Growers): - M. avium, M. intracellulare MFC (Mycobacterium Fortuitum Complex - Rapid Growers): - M. fortuitum, M. chelonae - M. leprae: Non-cultivable on artificial media - New species identified that do not fit these groups
34
Mycobacteriaceae - Habitat
Diverse environmental presence: - Saprophytes (live on decaying organic matter) - Opportunistic pathogens - Obligate parasites Common sources: - Soil - Water - Infected tissues of humans and animals
35
Mycobacteriaceae - Clinical Significance
MTB Complex (M. tuberculosis, M. bovis, etc.) M. tuberculosis: - Discovered by Robert Koch in 1886 - Resembles fungi in behavior (hence the name "Myco") - Transmission: Airborne particles from an infected person Pathogenesis: - Inhaled into lungs - Engulfed by alveolar macrophages - May survive, spread, and lead to latent or active infection
36
Tuberculosis - Overview
- other MTB complex members can cause TB in humans and animals M.bovis - infects cattle, humans, other mammals and birds - human TB from M.bovis isindistinguishable from M.tuberculosis disease M.bovis BCG (Bacillus of Calmette-Guérin) - basis of TB vaccine - attenuated through repeated subculture M.africanum: - causes TB in africa - may not be a separate species M.microti: rodent TB pathogen
37
Tuberculosis - Host Factors & Pathogenesis
Clinical Outcome depends on host factors reactivation risk factors: - age - time since initial infection - underlying disease (HIV/AIDS major factor) - very high mortality in immunocompromised patients - cell mediated immunity (CMI) controls infection - chronic pro-inflammatory cytokine activation -> wasting symptoms - multidrug resistant TB (MDR-TB) is a major global concern.
38
Tuberculosis - Disease Forms
Pulmonary TB (PTB): - Affects lungs only - Extrapulmonary TB (EPTB): - Targets other organs, including: - Pleura - Lymph nodes - Abdomen - Genitourinary tract - Skin - Joints & bones - Meninges (CNS TB)
39
Tuberculosis - Social Determinants
- Poverty - Stress - Poor housing - Malnutrition - Air pollution - Crowded living conditions - Travel to high TB-endemic countries - Pregnancy & childbirth - Alcohol misuse
40
Tuberculosis - Risk Factors for Active Disease
HIV/AIDS Diabetes mellitus Close contact with active PTB cases Immunosuppressive therapy Genetic predisposition Malnutrition Excessive alcohol consumption Active smoking Congenital immunodeficiencies, including: Chronic granulomatous disease Common variable immunodeficiency
41
Tuberculosis - Diagnosis
Pulmonary TB (PTB): Clinical signs & symptoms Chest X-ray Sputum analysis: Microscopy Culture Genomic testing Extrapulmonary TB (EPTB): Clinical signs & symptoms Histology Latent TB: T-cell response tests: Mantoux/tuberculin skin test (not reliable in vaccinated individuals) Interferon gamma release assay (IGRA)
42
Tuberculosis - Isolation & Identification
Nosocomial infections are a major concern (especially in developing countries) Infective dose: <10 bacilli Sputum is the most dangerous sample due to aerosol risk Plating precautions: Incinerate loops (avoid Bunsen flame) Microscopy: Presence of acid-fast bacilli (AFB) with typical morphology Staining patterns may indicate treatment success (e.g., increased beading = higher death rate of bacilli)
43
Tuberculosis - Culture Methods
Requires long-term culture on nutritious media Sample decontamination: NaOH treatment to kill non-mycobacteria Necessary for sputum samples (fast-growing contaminants may overgrow TB) Neutralization → Centrifugation → Culture → Ziehl-Neelsen (ZN) stain Early morning gastric aspirate (GA) used for young children (swallowed sputum) Three samples tested per patient for confirmation
44
Tuberculosis - Culture Media & Drug Testing
Egg-based & malachite green media: Lowenstein-Jensen (LJ) medium (solid media, used in slopes) Middlebrook agar (clear, allows early detection) Liquid broth culture for drug susceptibility testing
45
Tuberculosis - Rapid Culture Methods
Mycobacterium Growth Indicator Tube (MGIT): Detects oxygen consumption during metabolism Fluorescence quenched when O2 is present, but released when used BACTEC & MGIT use highly selective Middlebrook-based broth with: PANTA: Polymyxin B, Amphotericin B, Nalidixic Acid, Trimethoprim, Azlocillin
46
Leprosy (Hansen’s Disease) - Overview
Caused by: Mycobacterium leprae Obligate human pathogen (cannot survive outside a host) Cannot be artificially cultured Grown in footpads of armadillos for research Reservoir: Likely humans, but also found in nine-banded armadillos Transmission: Prolonged close contact with multibacillary individuals
47
Leprosy - Clinical Spectrum
Paucibacillary (Tuberculoid) Leprosy: Occurs when CMI (cell-mediated immunity) is adequate Symptoms: Anesthetic (numb) skin discoloration Localized nerve damage Fewer bacteria present Lepromatous Leprosy: Occurs when CMI is inadequate Symptoms: Extensive bacilli spread Peripheral neuropathy Tissue damage, secondary infections Severe disfigurement
48
Leprosy - Isolation & Identification
Microscopy is the only diagnostic method Clinical presentation is often pathognomonic Split-skin smears from 3 sites used to confirm disease type: Multibacillary leprosy (widespread bacilli) Paucibacillary leprosy (localized patches) Sample collection sites: Ear lobes Back of arms Nasal septum

MI3021 - 2025 (6 decks)

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