Clostridium Bacteria Quiz

Anaerobic Bacteria

• Anaerobic bacteria do not need oxygen to grow
• They vary in their ability to tolerate oxygen
• Obligate (strict) anaerobes: require anaerobic conditions for growth; oxygen is toxic and kills them
• Aerotolerant anaerobes: can grow in an atmosphere with oxygen but grow best in anaerobic environments
• Facultative anaerobes: do not require oxygen but will use it if available

Oxygen Reduction

• Superoxide anion and hydrogen peroxide are toxic to strict anaerobes
• Strict aerobes and facultative anaerobes possess enzymes superoxide dismutase and/or catalase to neutralize these toxins

Oxidation-Reduction (Redox) Potential

• Anaerobic bacteria require low redox potential; high redox environments contain oxygen
• Normal human tissue and aerobic culture media have high redox potential
• Thioglycollate, cysteine, and dithiothreitol are reducing agents included in anaerobic media

Normal Habitat

• Soil, water, animals
• Human normal flora (NF) includes oral cavity, upper respiratory tract (URT), intestinal tract, genitourinary tract, and skin
• Facultative organisms use oxygen in protected areas to reduce redox potential and inactivate harmful oxygen molecules

Diseases

• Exogenous infection: organisms, spores, or toxins enter the body through ingestion or trauma (e.g., tetanus, botulism)
• Endogenous infection: infections near mucosal surfaces of the host's NF (e.g., bacteremia, abscesses, gas gangrene, pneumonia)

Clues to Anaerobic Diseases

• Trauma (deep or puncture wounds)
• Animal or human bites
• Foul-smelling, gaseous discharge
• Necrotic tissue, vascular stasis
• Contains sulfur granules (actinomycosis)
• Black or fluorescent red color
• Previous therapy with aminoglycosides
• Failure to grow the organism on a Gram stain

Specimen Collection

• Appropriate specimens: best to aspirate with a needle and syringe. Examples include blood and sterile body fluids (CSF, bone marrow, cavity fluid), urine (suprapubic aspirate), abscesses, ulcers, and draining wounds; biopsy material
• Inappropriate specimens: site containing resident flora (oral, GI, GU). Examples include superficial skin sites, voided or catheterized urine, expectorated sputum, throat/nasopharyngeal swabs, bronchial washings, vaginal/cervical/urethral swabs, and stool/rectal swabs

Transport and Processing

• Inoculate media and immediately place in an anaerobic environment
• Limit exposure to room air
• Do not allow specimens to dry out
• Do not refrigerate
• Minimize time at room temperature
• PRAS media: prereduced, anaerobically sterilized transport medium. Includes agar transport medium (modified Cary-Blair or Amies), rezasurin (oxygen indicator), and reducing substances (to protect against oxygen and inhibit NF)

Culture

• Specimens for anaerobic culture should be cultured aerobically on blood agar plates (BAP), chocolate agar (CHOC), and MacConkey agar (MAC) to check aerotolerance
• Anaerobes require vitamin K, hemin, and yeast extract

Nonselective Media

• CDC or Anaerobic Blood Agar Plates (anaBAP): support the growth of obligate and facultative anaerobes
• Anaerobic Broth, Thioglycollate, or chopped/cooked meat: support all obligate and facultative anaerobes

Anaerobic-BAP

• Image of Clostridium perfringens grown on Thioglycollate & ANABAP

Thioglycollate Broth

• Image demonstrating growth of microbes in the broth, which is used in determining the oxygen requirements of microbes

Selective Media

• Phenylethyl alcohol blood agar (anaPEA): contains phenylethyl alcohol, supports Gram-negative (GN) and Gram-positive (GP) obligate anaerobes and GP facultative anaerobes, inhibits enteric Gram-negative rods (GNR)
• Colistin naladixic acid blood agar (anaCNA): supports GN and GP obligate anaerobes and supports GP facultative anaerobes, inhibits enteric GNR
• Bacteroides Bile-Esculin (BBE) agar: selective and differential for bile-tolerant organisms like B. fragilis
• Brucella blood agar: supports facultative and obligate anaerobes; best for Gram-negative bacteria

Other Selective Media

• Kanamycin-vancomycin laked blood (KVLB) agar: selects for Bacteroides and Prevotella; kanamycin inhibits most facultative GNR; vancomycin inhibits most GP; laked blood encourages Prevotella to produce brown-black pigments
• Cycloserine-cefoxitin-fructose agar (CCFA): selective and differential for Clostridium difficile; cycloserine & cefoxitin are antibiotics; fructose is a carbohydrate source; neutral red pH indicator changes from red in acidic conditions to yellow in alkaline conditions

Anaerobic Incubation

• 35 to 37°C for 48 hours
• Nitrogen gas (80–90%)
• Hydrogen gas (5–10%)
• Carbon dioxide (5–10%)

Anaerobic Systems

• Anaerobic jars
• Anaerobic bags
• Anaerobic chambers

Anaerobic Jars

• Jar technique (gas pak jar) - a classic principle of anaerobic culture
• Catalyst: palladium pellets
• Envelope: generates hydrogen (H₂) and carbon dioxide (CO₂) when water is added
• Sealing, incubation at 35°C
• Indicator: methylene blue or resazurin (blue when oxidized, white when reduced)

Anaerobic Bags

• Commercially available
• Hold 1-3 plates
• Contain oxygen removal system and indicator
• Gas-impermeable plastic bag

Anaerobic Chamber

• Optimal anaerobic incubation system
• Provides an oxygen-free environment for inoculation, incubation, and examination of media
• Contains palladium catalyst, desiccant, and indicator -anerobic gas mixture

Culture Examination

• Examination in the chamber at any time
• Bags and jars should be kept sealed for 48 hours
• Oxygen exposure is minimized after 48-hour incubation
• Examination and processing are done as quickly as possible, then returned to anaerobic atmosphere

Anaerobic Identification Tests

• Presumptive identification: Gram stain is a key identification test; colony morphology
• Rapid tests

Anaerobes Gram Stain RXN

• Diagram showing Gram-positive rods, Gram-positive cocci, non-spore-forming, spore-forming, and Gram-negative rods

Rapid Identification Tests

• Catalase test
• Motility test
• Urease test
• Indole
• Esculin hydrolysis
• Aerotolerance
• Fluorescence
• Disk tests
• Lecithinase
• Naglar test
• Lipase
• Reverse CAMP

Aerotolerance

• Determines if an isolate is a strict or facultative anaerobe
• Incubate the suspected isolate in both aerobic and anaerobic environments
• Check growth on anaBAP and anaerobically and CHOC aerobically in CO2
• Examine plates at 48 hours for growth

Fluorescence

• Fluorescent under ultraviolet light (366 nm)
• Red, orange, pink, chartreuse

Antibiotic Disk Test

• Confirming Gram stain
• Special potency disks (kanamycin, vancomycin, colistin)
• Most Gram-negative rods are resistant to vancomycin
• Most Gram-positive rods are susceptible to vancomycin and resistant to colistin
• Vancomycin is Susceptible & Colistin is Resistant = Clostridia, Porphyromonas (KanR)
• Vancomycin is Resistant = GN, not Porphyromonas (resistance of GN to vancomycin)*

Disk Tests

• Sodium polyanethol sulfonate (SPS) disk: identifies ana GPC; Peptostreptococcus are sensitive
• Nitrate disk: reduction of nitrate
• Bile disk: ability to grow in 20% bile; bile-tolerant anaGNR = Bacteroides fragilis group

Lecithinase

• Detecting lecithinase and lipase activity; Clostridium spp. Identification
• Enzyme cleaves lecithin
• Releases insoluble fat/opaque zone
• Positive reaction: white opaque zone in agar surrounding growth
• Negative reaction: no change in the agar surrounding growth

Nagler Test

• Detecting alpha toxin of C. perfringens on EYA
• Alpha toxin is a specific form of lecithinase produced by C. perfringens

Lipase

• Lipase hydrolyzes triglycerides in EYA to produce glycerol + free fatty acids
• Positive reaction: oil-on-water surface or multicolored sheen in the colony
• Negative reaction: no sheen observed in normal colony morphology

Reverse CAMP

• Alpha toxin of C. perfringens works synergistically with beta-hemolytic group of S. agalactiae to produce an area of hemolysis
• Test organism on anaBAP
• GBS perpendicular to test organism streak (close but not touching)
• Arrowhead-shaped zone = positive

Definitive Identification Tests

• Biochemical tests
• Commercial systems
• Gas-liquid chromatography
• Cellular fatty acid analysis
• 16S rRNA gene sequencing

Biochemical and Commercial Systems

• PRAS or non-PRAS biochemical test media
• Commercial biochemical and preexisting bacterial enzymes minisystems

Gas Liquid Chromatography

• Gas-liquid chromatography of cellular fatty acids or metabolic end products (volatile acids)
• Produces a characteristic pattern

Gene Sequencing

• 16S rRNA gene sequencing
• Ribosomal DNA extracted, amplified, and sequenced via PCR

Clostridium Species

• Catalase: negative
• Motile: (except C. perfringens)
• Anaerobic GPR: some are gram variable or Gram-negative
• Susceptible to vancomycin
• Spore-forming: appear as unstained, refractile structures in the Gram stain; oval to round, terminal to subterminal

Clostridium Species (cont.)

• Aerotolerant
• Clostridia cause exogenous infections. Enter the body by ingestion or wounds
• Produce potent toxins (tetanus, gas gangrene, botulism, food poisoning)

Genera Differences

• | Test | Clostridium | Bacillus | Lactobacillus |
• | Optimal Growth Conditions | Anaerobic | Aerobic | Varies |
• | Sporulation Conditions | Anaerobic | Aerobic | No spores |
• | Catalase | Negative | Positive | Negative |

Clostridium perfringens

• Most common Clostridium spp.
• Mostly found in undercooked meat, soil, water
• Gram-positive, boxcar-shaped rods
• Double zone of beta hemolysis on anaBAP
• Positive reverse CAMP, lecithinase, Nagler test
• Spores are seldom observed

C. perfringens Clinical Significance

• Isolated from tissue infections and bacteremia
• Produces gas in infected tissue (myonecrosis), often from deep, penetrating wounds/surgery, or diabetic foot ulcers
• Causes food poisoning (ingested spores germinate and produce enterotoxin, causing nausea, vomiting, diarrhea, and abdominal pain)

Clostridium difficile

• May be a normal flora organism (NF) of stool
• Associated with antibiotic-associated diarrhea and pseudomembranous colitis
• Antibiotics upset the intestinal ecosystem by killing indigenous NF
• Resistant strains increase in number
• Toxigenic strains (toxin A and cytotoxin B) are associated with its effects

C. difficile Characteristics

• Yellow ground glass colonies on cycloserine-cefoxitin-fructose agar (CCFA)
• Smells like horse manure
• Spore-forming Gram-positive rods (GPR)
• Chartreuse fluorescence (sometimes)
• Test for toxin production

C. difficile Toxin Tests

• Cytotoxin test: measures the toxic effects of stool on human cells in culture; more sensitive, but takes 2-3 days to get the result
• Kit testing: determines if toxin A, toxin B, or glutamate dehydrogenase is present in stool; Enzyme-linked immunosorbent assay (ELISA)
• Molecular testing: under development

Clostridium botulinum

• Ingestion of botulism toxin from home-canned veggies/home-cured meat
• Infant botulism associated with honey
• Neuromuscular toxins cause paralysis or death
• Foodborne, wound, or infant botulism (lacking NF)

C. botulinum Diagnosis

• Clinically diagnosed from patient history and symptoms
• Isolation of organism or detection of toxin (in reference lab)

Clostridium tetani

• Found in soil and the intestinal tracts of animals
• Enters the body through a wound site
• Produces a potent neurotoxin (tetanospasmin)
• Causes severe muscle spasms in unimmunized individuals
• Tetanus (lockjaw syndrome)

C. tetani Diagnosis

• Clinically diagnosed

Terminal Spores

• Tennis Racquet Shaped (image)

C. septicum

• Aerotolerant
• Swarming colonies
• Subterminal spores
• Myonecrosis and bacteremia
• Associated with leukemia, lymphoma, and large bowel carcinoma

Other Species

• Cause bacteremia, intra-abdominal infections, and wound infections; myonecrosis

Non-Spore-Forming Anaerobic Gram-Positive Rods (GPR)

• Actinomyces spp: causes actinomycosis
• Bifidobacterium spp:
• Propionibacterium spp: commonly contaminated in a lab
• Eggerthella and Eubacterium spp:

Actinomyces

• Includes aerobic and anaerobic bacteria
• Nocardia: an aerobic Actinomyces, which causes mycetomas and nocardiosis
• A. israelii: most common anaerobe; causes actinomycosis
• Actinomyces inhabit human and animal mucous surfaces; they are not weakly acid fast, unlike Nocardia

Actinomyces Gram Stain

• Aerobic and anaerobic Actinomyces spp., which are gram-positive rods (GPR) that appear irregularly stained (beaded appearance)
• Coccid to filamentous
• Filaments branch

A. israelii Actinomycosis

• Mainly A. israelii
• Chronic, granulomatous infection in the jaw
• Forms fistulae that drain pus with sulfur granules (colonies of bacteria)

A. israelii Cultures

• Associated with "lumpy jaw"
• Pus and sulfur granules visible on anaBAP
• Grows slowly (7–9 days)
• Molar-tooth colonies
• Anaerobic branching GPR

Bifidobacterium

• Actinomyces-like morphology
• Gram-stained, branched, or bifurcated GPR
• Found in the intestines and oral cavity
• Rarely causes disease
• Possible cause of actinomycosis

Propionibacterium

• Anaerobic diphtheroid-like Gram-positive rods (GPR) resembling Corynebacteria
• Normal flora of skin
• Common contaminant in lab specimens
• P. acnes: link to acne
• Catalase and indole-positive
• May cause subacute bacterial endocarditis (SBE) and bacteremia

Eggerthella and Eubacterium spp.

• Eggerthella or Eubacterium: anaerobic diphtheroid-like GPR; no branching

Anaerobic Gram-Positive Cocci (GPC)

• Peptostreptococcus spp: GPC in chains; P. anaerobius are susceptible to sodium polyanethol sulfonate (SPS) disks, but all other anaerobic cocci are resistant)
• Peptococcus spp: GPC in clusters

Anaerobic Gram-Negative Rods (GNR)

• Bacteroides spp:
• Prevotella spp:
• Prophyromonas spp:
• Fusobacterium spp:
• Veillonella spp:

Bacteroides fragilis Group

• Anaerobic GNR
• Normal flora of the GI tract
• B. fragilis is the most common
• Intra-abdominal infections, bacteremia, and soft tissue infections
• B. thetaiotaomicron: second most common

Bacteroides fragilis Group (cont.)

• Bile resistant
• Resistant to kanamycin, vancomycin, and colistin disks
• Growth on KVLB agar and Bacteroides Bile-Esculin (BBE) agar
• Growth = bile tolerance
• Brown to black colonies = esculin hydrolysis

Bacteroides ureolyticus Group

• Anaerobic GNR
• Bile sensitive and bile-tolerant nonpigmented organisms
• Some organisms pit the agar
• Growth in formate and fumarate
• Vancomycin-resistant
• Sensitive to kanamycin and colistin

Prevotella

• Anaerobic GNR
• Bile sensitive
• Resistant to kanamycin and vancomycin
• Growth on KVLB agar, but not on BBE
• Some produce protoporphyrin
• Dark pigmented colonies
• Colonies fluoresce a “brick red” under UV light.

Porphyromonas

• Anaerobic GNR
• Require hemin and vitamin K
• Resistant to kanamycin and colistin
• Sensitive to bile and vancomycin
• No growth on KVLB
• Pigmented colonies and fluoresce “brick red”

Fusobacterium

• Susceptible to kanamycin and colistin
• Vancomycin-resistant
• Fluoresce chartreuse
• F. nucleatum: thin fusiform rods, bread-crumb and speckled colonies, some can cause anaerobic infections
• F. necrophorum: lipase positive

Mobiluncus

• Associated with bacterial vaginosis (BV), pelvic inflammatory disease (PID), and abdominal infections
• Curved bacilli
• Variable Gram stain
• Motile
• Catalase and indole negative
• Inhibited by vancomycin

Selective Anaerobic GNR ID

• Diagram showing a flowchart for identifying anaerobic GNR based on susceptibility to kanamycin/colistin, vancomycin, bile sensitivity, and related tests

Veillonella

• Only commonly encountered anaerobic GNC
• Sensitive to kanamycin and colistin
• Resistant to vancomycin
• Smallest gram-negative cocci

Gram-Positive Rods

• Non-spore-forming GPR
• Non-spore-forming branching GPR
• Spore-forming non-branching GPR

Non-Spore-Forming GPR

• Corynebacterium and Coryneforms: includes C. diphtheriae, C. jeikeium, C. urealyticum, C. pseudodiphtheriticum, C. striatum, C. xerosis
• Listeria:
• Erysipelothrix:
• Acranobacterium:
• Lactobacillus:
• Gardnerella:

Corynebacterium

• Normal skin and mucous membrane flora, often considered contaminants
• Diphtheroids or coryneforms (“club-shaped”)
• Gram-positive rods (GPR); pleomorphic, non-spore forming; stain looks like “Chinese letters” or palisades.
• Facultative anaerobe
• Small gamma colonies on SBA
• Catalase positive
• Nonmotile
• Frequent contaminants

C. diphtheriae

• Significant pathogen
• Virulence factor is Diphtheria toxin
• Blocks protein synthesis in strains infected with a bacteriophage carrying the tox gene
• Destroys host cells.
• Can affect heart and nervous system and be lethal
• Two forms of the disease
  • Cutaneous (non-healing ulcer)
  • Respiratory (Diphtheria)

Diphtheria infection

• URT infection (tonsils, pharynx)
• Bacteria multiply and release toxin
• Tissue necrosis
• Exudate inflammation→ pseudomembrane
• Necrotic epithelial cells, WBCs, fibrin, bacteria
• Suffocation

C. diphtheriae Treatment and Prevention

• Treated with antitoxin
• Prevented by immunization (DIP/TET)

C. diphtheriae Lab Diagnosis

• Pleomorphic GPR
• Darker staining area of the cell
• Metachromatic granules (visible with methylene blue)
• Pockets of inorganic phosphates or nutrient reserves

C. diphtheriae Media

• SBA: small zone of beta-hemolysis
• Serum or blood-containing media: enhances the development of metachromatic granules
• Loeffler media:
• Pai agar: egg-based agar
• Tinsdale agar (cystine-tellurite blood agar) : selective and differential; Potassium tellurite inhibits non-coryneforms; tellurite reduction produces brown or black colonies; cystinase activity = halo around the colony

Elek Test

• Immunodiffusion test for toxin production
• Strip of filter paper saturated with diphtheria antitoxin, imbedded in agar medium
• Isolate, positive, and negative controls streaked onto agar parallel to each other and perpendicular to filter paper Incubate for 1–2 days
• Line of precipitate forms in agar when toxin interacts with antitoxin (arc of identity)

Corynebacterium spp.

• Normal skin flora
• Most isolates are contaminants
• C.jeikeium: associated with catheters and prosthetic devices; can cause diphtheroid prosthetic valve endocarditis
• C. urealyticum: usual urinary pathogen

Other Non-spore-forming GPR

• Rothia dentocariosa: oral NF; causing endocarditis and wound infections
• Filamentous or coryneform groups
• Undesignated CDC coryneform groups
• Opportunistic or nosocomial infections

Listeria monocytogenes

• Vaginal and intestinal NF in humans
• Clinical infections due to ingestion (foodborne) of contaminated food
• Virulence factors (Listeriolysin O)
• Listeriosis (sepsis) in neonates and pregnant women, which has a 50% fatality rate
• Predisposition for CNS infections, particularly immuncompromised individuals

L. monocytogenes Cultures

• Specimens include CSF, blood, and amniotic fluid
• Facultative anaerobes
• Prefers increased CO₂
• Grow on routine media (BAP, CHOC)
• Colonies resemble S. agalactiae
• Cold enrichment (growth at 4°C)

L. monocytogenes ID

• Gram-positive rods (GPR)
• Non-spore forming
• Small beta colonies on BAP
• Hemolysis sometimes hidden under the colonies
• Catalase-positive
• Bile-esculin positive
• Motile at 25°C

L. monocytogenes Motility

• Tumbling motility at 25°C, not at 35°C
• One tube incubated at 35°C, other at room temperature (RT)
• Tumbling motility on broth, wet mount (in broth incubated at 25°C)
• Umbrella pattern at 25°C but not at 35°C; semisolid agar motility
• Positive CAMP test distinguishes L. monocytogenes from other Listeria spp.
• Blocks vs. arrowhead

Listeria Differentiation

• Catalase: positive
• Esculin hydrolysis: positive
• Motility: tumbling
• β-hemolysis: +
• 6.5% NaCl: +
• Differentiates from Corynebacterium, S. agalactiae, and Enterococcus

Erysipelothrix rhusiopathiae

• Domestic swine are the major reservoir
• Occupational hazard for butchers, vets, and fishers
• Causes erysipeloid (red skin lesion)
• Can disseminate into humans by way of blood (bacteremia and endocarditis)
• Gram-positive rods (GPR)*
• Catalase negative*
• Alpha or gamma hemolysis, nonmotile*
• H₂S positive in TSI*

Arcanobacterium haemolyticum

• Formerly Corynebacterium: A. haemolyticum, A. pyogenes, and A. bernardiae are significant pathogens and can cause pharyngitis
• Faculative anaerobe
• Beta hemolysis (inhibits S. aureushemolysis)
• Catalase negative
• Reverse CAMP positive

Lactobacillus

• Normal vaginal flora
• Produce lactic acid
• Reduce vaginal pH
• Inhibit the growth of other organisms
• Rarely cause disease
• L. acidophilus: probiotic
• Non-spore-forming Gram-positive rods (GPR)
• Medium to long rods
• Aerotolerant anaerobes
• Catalase negative
• Nonmotile

Gardnerella vaginalis

• Pleomorphic coccobacilli
• Cell wall has Gram-positive characteristics
• Variable Gram stain
• Nonmotile
• Facultative anaerobe
• Normal flora in 50–70% of women
• Associated with bacterial vaginosis (BV)

Bacterial Vaginosis (BV)

• Change in normal flora (lactobacillus)
• pH increase favors G. vaginalis growth
• Foul-smelling discharge (amine odor)
• Can lead to pelvic inflammatory disease (PID) and UTIs
• Clue cells (squamous cells with bacteria clustered at their edges) on a wet prep or Gram stain

Cultures (extravaginal sites)

• Grow on CHOC and BAP, not MAC
• Small pleomorphic Gram-variable coccobacilli
• Catalase and oxidase negative
• Hippurate hydrolysis positive

Non-Spore-Forming Branching GPR (Aerobic Actinomycetes)

• Nocardia spp: found in soil; usually infects immunocompromised individuals; pulmonary or cutaneous disease
• Tropheryma whipplei: Whipple disease; intracellular pathogen, diarrhea, weight loss, malabsorption, diagnosis by PCR or 16S rRNA
• Streptomyces, Actinomadura, Gordonia, Tsukamurella, Rhodococcus equi

Nocardia spp.

• Found in soil
• Pulmonary and cutaneous diseases
• May infect immunocompromised individuals
• N. asteroides complex, N. brasiliensis: disseminated infection that manifests as pneumonia, abscesses, and invasive disease, often with draining pus containing sulfur granules

Nocardia spp.: Microscopy

• Aerobic Gram-positive rods (GPR) morphologically similar to fungi in branching filamentous or beaded forms
• Often weakly acid fast

Nocardia spp. Colonies

• Grow slowly (over one week) on nonselective media
• Waxy, chalky, and crumbly colonies

Nocardia ID

• Acid-fast (Ziehl-Neelsen or Kinyoun stain) to reveal filamentous, branching organisms.
• Staining with carbolfuchsin, decolorizing with acid-alcohol, then counterstaining with methylene blue
• Reddish purple filaments are positive, blue staining is negative with the acid-fast stain

Other Actinomycetes

• Streptomyces, Actinomadura, Gordonia, Tsukamurella, and Rhodococcus equi

Spore-Forming Non-Branching GPR

• Bacillus is spore-forming, non-branching bacilli
• Spores are protective and metabolically inactive; the organism increases its survival and resistance to biocides in nature due to a thick outer wall
• Bacillus anthracis: found in nature; causes anthrax, a bioterrorism agent; has virulence factors (encapsulated with glutamic acid, protective antigen, edema factor, and lethal factor); causes cutaneous, inhalational, and gastrointestinal diseases, and is characterized by its large aerobic Gram-positive rod (GPR), spore-forming status, nonhemolysis, and its nonmotile characteristic upon Gram stain
• Bacillus cereus: beta-hemolytic and motile; causes food poisoning; colony morphology is frosted glass-like

Identification of GPR

• Flowchart showing identification criteria and characteristics based on presence or absence of spores, catalase, motility, bile-esculin, and lipase test results

Spirochetes

• Borrelia, Leptospira, Treponema: long, slender, spiral-shaped, motile - can't be seen with Gram stain
• Require silver stains
• Seen with darkfield or phase contrast microscopy

Borrelia

• Contains several species
• Transmitted by ticks or lice
• 3–10 spirals/organism
• Most cause relapsing fever
• B. burgdorferi: causes Lyme disease

Borrelia recurrentis

• Causes relapsing fever with repeated febrile episodes
• Caused by antigenic variation to evade immune systems
• Episode ends when immune system responds to new antigen makeup
• Different types of relapsing fever include epidemic and endemic

Relapsing Fever Lab Tests

• Microscopic examination of blood during febrile periods (Giemsa or the Wright's stain is used to assess this)
• Can be cultured in Kelly medium.
• Serology is not as useful due to antigenic variation

Borrelia burgdorferi

• Lyme disease
• Originally from northeast USA, still spreading
• Transmitted by Ixodes tick (deer or mouse ticks)

Lyme Disease Lab Tests

• Serologic tests are the most common and fastest (antibody detection tests and western blot confirmation)
• Direct microscopic examination of skin or blood
• Cultures are typically negative
• Kelly medium is a difficult culture media. .

Leptospira

• L. biflexa: nonpathogenic
• L. interrogans: animal pathogen passed to humans through contaminated water (with contaminated animal urine), occupational hazard for vets, farmers, and sewer workers

Leptospira interrogans

• Spirals with hooked ends
• Tightly coiled

Leptospirosis

• Zoonotic (animal) infection
• Spirochetes infect animals' kidneys
• Leptospires enter humans through skin or intact mucosa
• Clinical manifestations range from asymptomatic to severe; incubation period of 3–30 days
• Possible symptoms include nonspecific influenza-like symptoms, severe systemic disease, Weil's disease (CSF+), and potentially liver (hepatic), kidney (renal), and intravascular diseases

Leptospirosis Lab Tests

• Blood and CSF (first week); urine (later)
• Direct examination: darkfield microscopy or direct fluorescent antibody (DFA) of blood, CSF, or urine
• Cultures (Fletcher's semi-solid media, incubate at 30°C in the dark for 6 weeks)
• Darkfield microscopy from media
• Serology (ELISA assay)

Treponema

• 4–14 spirals/organism
• Four pathogenic organisms
• T. pallidum subsp. pallidum: venereal syphilis
• T. pallidum subsp. pertenue: yaws
• T. pallidum subsp. endemicum: endemic syphilis
• T. pallidum subsp. carateum: pinta

Treponema pallidum subspecies pallidum

• Venereal syphilis
• Great imitator
• Variety of clinical presentations
• Transmitted sexually or by active, non-genital lesions
• Can cross the placenta (congenital syphilis)
• Three stages: primary, secondary, and tertiary

Primary Syphilis

• Few days to months after organism acquisition
• Firm chancre at inoculation site
• Infectious lesion
• Painless

Secondary Syphilis

• 2–12 weeks after primary lesion
• Organisms disseminate throughout the body
• Fever, headache, lymphadenopathy
• Secondary, infectious lesions on skin and mucous membranes
• Widespread unusual skin rashes (may include palms and soles)

Tertiary or Late Syphilis

• 1/3 of untreated cases develop tertiary syphilis years after initial infection
• Not infectious
• Many body sites affected, with gummas (granulomatous lesions)
• CNS: neurosyphilis (deafness, blindness, partial paralysis, disturbances in gait and mental function)
• Cardiovascular lesions: syphilitic aortitis

Early Onset Congenital Syphilis

• Mother has early syphilis
• Treponemes cross placenta
• Infection of fetus, high mortality rates
• Manifests in many body systems such as skin and mucous membranes; anemia; hepatosplenomegaly; and meningitis, and bone lesions.

Late Onset Congenital Syphilis

• Mother has chronic, untreated syphilis

• 2 years old

• Symptoms such as blindness, deafness, mental retardation, bone and tooth deformities.

Serological Laboratory Diagnosis

• Nontreponemal tests: screening. VDRL, and RPR
• Treponemal tests: confirmation. EIA (enzyme immunoassay); TPPA (Treponema pallidum particle agglutination) and FTA-ABS (fluorescent treponemal antibody absorption)

Nontreponemal Tests

• Detects reagin or reaginic antibodies against treponemal lipids and cardiolipin-lecithin
• Sensitive but not specific
  • False positives common in Lyme disease, certain viral infections, autoimmune diseases, or pregnancy.

VDRL and RPR

• Examples of nontreponemal tests Results are demonstrated through image.

Treponemal Tests

• Detects treponemal specific antibodies
• Remain positive after treatment
• Examples include EIA, TPPA, and FTA-ABS

Direct Microscopic Exam

• Darkfield microscopy in a fluid sample from the lesion, requiring patience and experience to identify the corkscrew motility
• Oral lesions are not appropriate samples
• Non-pathogenic treponemes may be present

Other Pathogenic Treponemes

• Spread through direct contact with contaminated drinking/eating utensils
• May exhibit primary, secondary, or tertiary disease stages
• T. endemicum*: endemic syphilis (non-venereal); Middle East/hot, arid areas
• T. pertenue*: yaws (skin and bones); occurs in humid tropical areas
• T. carateum*: pinta (skin); occurs in Central and South America.

Mycobacteria

• Aerobic: needing increased levels of CO2 for growth.
• Non-spore forming
• Non-motile
• Cultures held for 6 weeks before a negative result
• Slim Gram variable rods
• High lipid content (mycolic acid) in cell wall: leading to an inability for effective Gram staining of the organism.

Mycobacteria Gram Stain

• Poor gram stain; cell wall lipids interfere with penetration of crystal violet and safranin
• No organism
• Beaded GPR
• Ghost cells

Mycobacteria Safety

• High incidence of positive tuberculosis skin test (PPD) is observed in mycobacteriology labs
• Lab is separate; non-recirculating ventilation, negative air pressure, and biosafety cabinets are essential for mycobacteria safety

Mycobacteria Species

• Major pathogens (M. tuberculosis complex: (most common) M. bovis, M. africanum, M. canettii, M. microti)
• Nontuberculous mycobacteria (NTM) or Mycobacterium other than tubercule (MOTT) - Hansen's disease (leprosy)

Specimen Collection

• Sputum, bronchial washing, gastric contents, urine, stool, tissue: required
• Sputum: morning specimen; 3 consecutive mornings; 5–10 mL minimum volume, refrigerated overnight
• Gastric aspirates and washings: for young children (<3) to produce sputum; need neutralization with sodium bicarbonate if refrigerated overnight; mycobacteria can get damaged by stomach acid
• Urine: neutralization with sodium bicarbonate; refrigerate overnight
• Stool: AIDS patients often get M. avium screen
• Inappropriate specimens: Swabs: not enough material, and mycobacteria may not dislodge; 24-hour pooled specimens (sputum or urine) may not be appropriate due to contamination and/or inhibition

Digestion and Decontamination

• Specimen digestion frees the mycobacteria from protein clumps by using sodium N-acetyl-L-cysteine (NALC) as a mucolytic agent
• Contaminant removal (NF): using NALC to release mycobacteria from clumps of proteins, then using NaOH for the decontamination of NF
• Decontamination timed to kill contaminants without killing the mycobacteria

Specimen Processing

• Concentration to increase the number of organisms per mL of specimen using centrifugation; 20-min. time period, 3000 rpm
• Make the AFB smear and inoculate liquid and solid media with the sediment
• Include single liquid and solid AFB media cultures

Staining for AFB Smear

• Ziehl-Neelsen - “hot” stain (using heat)
• Kinyoun stain - “cold” stain (using detergent/phenol)
• Primary stain is carbolfuchsin
• Rinse and decolorize with strong acid
• Counterstain with methylene blue
• Reddish filaments (acid fast) are positive, blue staining is negative

Staining for Acid-Fast Bacilli

• Fluorochrome stains use auramine or auramine-rhodamine which penetrates mycobacteria cells due to lipids;
• Phenol is included to enhance penetration
• Examination with a fluorescence microscope for improved sensitivity
• Results show bright yellow-orange bacilli for positive cultures

AFB Culture Media

• Solid media: egg-based (e.g., Lowenstein-Jensen) or agar-based (e.g., Middlebrook)
• Liquid media: Tween 80 or instrumentation broth

Solid Egg-Based Media

• Lowenstein-Jensen (L-J): 60% egg in nutrient base, malachite green to inhibit Gram-positive organisms and may

Test your knowledge on Clostridium species and their associated conditions. This quiz covers various aspects including diagnosis, characteristics, and clinical manifestations of infections caused by these bacteria. Assess your understanding of key topics related to C. perfringens, C. difficile, and C. botulinum.