Microbiology: Leptospira and Clostridium Species

Anaerobic Bacteria

• Anaerobic bacteria do not require oxygen to grow
• They vary in their tolerance to 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 prefer anaerobic environments
• Facultative anaerobes do not require oxygen but can utilize it if present

Oxygen Reduction

• Superoxide anion and hydrogen peroxide are toxic
• Strict aerobes and facultative anaerobes have superoxide dismutase and/or catalase to neutralize these toxic products

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
• Reducing agents (thioglycollate, cysteine, dithiothreitol) are included in anaerobic media

Normal Habitat

• Soil, water, animals
• Inside human body: oral cavity, upper respiratory tract (URT), intestinal tract, genitourinary tract, skin
• Facultative organisms use up oxygen in protected areas
• Reduce redox potential
• 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 the host's normal flora (NF) near mucosal surfaces (e.g., bacteremia, abscesses, gas gangrene, pneumonia)

Clues to Anaerobic Diseases

• Trauma (deep or puncture wounds)
• Animal or human bite
• Foul-smelling, gaseous discharge
• Necrotic tissue, vascular stasis
• Presence of sulfur granules (actinomycosis)
• Black or fluorescent red color
• Prior therapy with aminoglycosides
• Failure to grow on gram stain

Specimen Collection

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

Transport and Processing

• Inoculate media and immediately place in anaerobic environment
• Limit exposure to room air
• Do not allow specimens to dry out
• Do not refrigerate
• Minimize time at room temperature
• PRAS (Pre-reduced, Anaerobically Sterilized) media: anaerobically sterilized transport medium (e.g. Modified Cary-Blair or Amies), containing agar transport medium, rezasurin (oxygen indicator), and reducing substances
• Sterile container

Culture

• Specimens for anaerobic culture should be cultured aerobically on BAP, CHOC, MAC
• Anaerobes require Vitamin K, hemin, and yeast extract

Nonselective Media

• CDC or Anaerobic Blood Agar Plates (anaBAP): supports growth of obligate and facultative anaerobes
• Anaerobic Broth, Thioglycollate or chopped (cooked) meat: supports obligate and facultative anaerobes, anaerobes grow toward bottom (facultative grow throughout)

Anaerobic Incubation

• 35 to 37°C for 48 hours
• Nitrogen gas (80-90%)
• Hydrogen gas (5-10%) removes oxygen
• Carbon dioxide (5-10%)
• Some anaerobes are capnophilic

Anaerobic Systems

• Anaerobic jars
• Anaerobic bags
• Anaerobic chambers

Anaerobic Jars

• Technique involving a gas pak jar
• A catalyst of palladium pellets
• Generates H2 and CO2 when water is added
• Sealed, incubated at 35°C
• Use of a methylene blue or resazurin indicator: turns blue when oxidized, white when reduced (anaerobic)

Anaerobic Bags

• Commercially available
• Containers to hold 1-3 plates
• Contain an oxygen removal system
• Contain an indicator
• Using a gas-impermeable plastic bag

Anaerobic Chamber

• Optimal anaerobic system for incubating and examining media
• Contains a palladium catalyst, desiccant, and indicator
• Contains an anaerobic gas mixture

Culture Examination

• Examine bags and jars in the chamber at any time after keeping sealed for 48 hours
• Minimize oxygen exposure after incubation
• Process specimens quickly and return them to the anaerobic atmosphere

Anaerobic Identification Tests

• Presumptive ID
• Gram-stain key identification test
• Use colony morphology
• Rapid tests

Anaerobes Gram Stain RXN

• Categorizes anaerobes based on their gram stain reaction. (Gram positive rods, Gram positive cocci, Gram negative rods)

Rapid Identification Tests

• Catalase, motility, urea test
• Aerotolerance, fluorescence, disk tests
• Indole, lecithinase, naglar tests
• Lipase hydrolysis, reverse CAMP tests

Aerotolerance

• Determines if isolate is a strict anaerobe or facultative anaerobe.
• Incubate the suspected isolate in both aerobic and anaerobic environments
• Examing growth on both anaerobic and aerobic media to identify aerotolerance
• Growth on anaBAP only = strict anaerobe. Growth on anaBAP > growth on CHOC = aerotolerant anaerobes. Growth on both = facultative anaerobes

Fluorescence

• Fluorescent under ultraviolet light (366 nm)
• Appearance of red, orange, pink, or chartreuse

Antibiotic Disk Test

• Confirm gram stain
• Special potency disks
• Most GN are resistant to Vancomycin
• Most GP are sensitive to Vancomycin, Resistant to colistin
• Van S, Col R= Clostridia, Porphyromonas (Kan R)
• Van R = GN, not Porphyromonas

Disk Tests

• Sodium polyanethol sulfonate (SPS) disk used to identify anaerobic GPC. Peptostreptococcus sensitive to SPS.
• Nitrate disk used to identify nitrate reduction.
• Bile disk used to identify bile tolerant anaGNR (e.g., Bacteroides fragilis group)

Lecithinase

• Detect lecithinase and lipase activity
• Clostridium spp. identification
• Results show a white opaque zone surrounding growth if positive. No zone change if negative

Nagler Test

• Detect alpha toxin of C. perfringens
• Alpha toxin is a specific form of lecithinase produced by C. perfringens .
• Antiserum to alpha toxin neutralizes the activity of the alpha toxin produced by C. perfringens, not other clostridia.

Lipase

• Hydrolyzes triglycerides in egg yolk agar (EYA), producing glycerol and fatty acids
• Positive: oil-on-water surface or multicolored sheen in colony
• Negative: no sheen, normal colony morphology

Reverse CAMP

• Demonstrates alpha toxin synergy with beta hemolytic group of S.agalactiae on anaBAP resulting in an arrowhead shaped zone.

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 test media
• Commercial biochemical test systems & bacterial pre-existing enzyme minisystems

Gas Liquid Chromatography

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

Gene Sequencing

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

Clostridium Species

• Catalase negative
• Motile, except C. perfringens
• Anaerobic GPR
• Some gram-variable or GN
• Susceptible to vancomycin
• Spore forming, appearing as unstained refractile structures in gram stain
• Oval to round terminal to subterminal spores

Clostridium Species (cont.)

• Some species are aerotolerant
• Clostridia cause exogenous infections
• Gain access to body via ingestion and wounds
• Produce potent toxins causing tetanus, gas gangrene, botulism, food poisoning

Genera Differences

• Compares Clostridium, Bacillus, or Lactobacillus based on optimal growth conditions, sporulation, and catalase test

Clostridium perfringens

• Most common Clostridium spp.
• Found in undercooked meat, soil, water
• Gram positive boxcar-shaped rods
• Double zone of beta hemolysis on anaBAP
• Positive reverse CAMP, lecithinase, and Nagler tests
• Spore seldom observed

C. perfringens Clinical Significance

• Isolated from tissue infections and bacteremia
• Produces gas in infected tissue (myonecrosis, gas gangrene)
• Deep, penetrating wound or surgery
• Diabetic foot ulcers
• Food poisoning (ingested spores germinate in intestines, produce enterotoxin causing nausea, vomiting, diarrhea, abdominal pain).

Clostridium difficile

• May be normal flora (NF) of stool
• Antibiotic-associated diarrhea and pseudomembranous colitis
• Antibiotics upset intestinal ecosystem by killing indigenous NF
• Resistant C. difficile increase in number
• All toxigenic strains produce enterotoxin (toxin A) and cytotoxin (toxin B)

C. difficile Characteristics

• Yellow ground-glass colonies on cycloserine-cefoxitin-fructose agar (CCFA)
• Smells similar to horse manure
• Spore-forming GPR
• Fluorescent chartreuse color
• Test for toxin production is necessary

C. difficile Toxin Tests

• Toxin test: Cytotoxin test: detects toxic effects of stool on human cells in culture
• More sensitive (requires 2-3 days), detects toxin B
• Kit testing: used to detect toxin A, toxin B, or glutamate dehydrogenase (not a virulence factor). Enzyme immunoassay is used

Clostridium botulinum

• Ingestion of botulinum toxin
• Home canned veggies, home cured meat
• Infant botulism associated with honey
• Neuormuscular toxins cause paralysis or death
• Foodborne botulism (ingested), wound botulism (inoculated), infant botulism (intestinal - infants lacking NF)

C. botulinum Diagnosis

• Diagnosed clinically (patient history and symptoms)
• Isolating the organism or detecting the toxin (reference lab)

Clostridium tetani

• Soil and intestinal tract of animals
• Enter body through wound site
• Produces potent neurotoxin (tetanospasmin)
• Severe muscle spasm in unimmunized individuals
• Tetanus (lockjaw)

C. tetani Diagnosis

• Diagnosed clinically

C. septicum

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

Other Species (cont.)

• Causes bacteremia
• Intra-abdominal infections, wound infections, myonecrosis

Non-Spore-Forming Anaerobic GPR

• Actinomyces spp.
• Bifidobacterium spp.
• Propionibacterium spp.
• Eggerthella and Eubacterium spp.
• All can cause actinomycosis

Actinomyces

• Actinomyces spp. includes aerobic and anaerobic bacteria
• Nocardia is an aerobic actinomyces
• Causes mycetomas and nocardiosis
• A. israelii is the most common anaerobe
• Causes actinomycosis.
• Inhabit human and animal mucosal surfaces
• Anaerobic actinomyces are not weakly acid fast like Nocardia spp.

Actinomyces Gram Stain

• Aerobic and anaerobic Actinomyces spp.
• GPR irregularly stained (beaded appearance).
• Coccid to filamentous appearance.
• Filaments branch

A. israelii Actinomycosis

• Actinomycosis
• Mainly A. israelii
• Usually in jaw
• Chronic, granulomatous infection
• Develops fistulae—drains pus with sulfur granules (colonies of bacteria)

A. israelii Cultures

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

Bifidobacterium

• Actinomyces-like morphology
• Gram-stain branched or bifurcated GPR
• Normal flora of intestines and oral cavity
• Rarely cause disease
• Can be the cause of actinomycosis

Propionibacterium

• Anaerobic diphtheroid-like GPR
• Resembles corynebacteria
• Normal flora of skin
• Most common anaerobe isolated in laboratory
• Contaminant in blood cultures and specimens
• Collected by penetrating the skin
• P. acnes linked to acne
• Catalase and indole positive
• May cause subacute bacterial endocarditis (SBE) and bacteremia

Eggerthella and Eubacterium spp.

• Eggerthella previously Eubacterium
• Anaerobic diphtheroid-like GPR
• No branching

Anaerobic GPC

• Peptostreptococcus
• GPC in chains
• P. anaerobius is susceptible to sodium polyanethol sulfonate (SPS) disk
• Other anaerobic cocci are resistant
• Peptococcus
• GPC in clusters

Anaerobic GNR

• Normal flora of mucous membranes
• Bacteroides
• Prevotella
• Prophyromonas
• Fusobacterium
• Veillonella

Bacteroides fragilis Group

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

Bacteroides fragilis Group (cont.)

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

Bacteroides ureolyticus Group

• Anaerobic GNR
• Bile-sensitive and bile-tolerant, non-pigmented organisms
• Forms pits in the agar
• Grows in formate and fumarate
• Resistant to vancomycin
• Sensitive to kanamycin and colistin

Prevotella

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

Fusobacterium

• Susceptible to kanamycin and colistin
• Vancomycin resistant
• Fluoresce chartreuse
• F. nucleatum – thin fusiform rods
• Bread-crumb, speckled colonies
• F. necrophorum
• Lipase positive

Mobiluncus

• Associated with BV, PID, and abdominal infections
• Curved bacilli
• Gram-variable
• Motile, catalase and indole negative
• Inhibited by vancomycin

Selective Anaerobic GNR ID

• Diagram depicting identification of anaerobic GNRs based on tests like kanamycin/colistin susceptibility, vancomycin susceptibility, bile sensitivity
• Various species fall into different branches indicating their distinctive reactions

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
• Listeria
• Erysipelothrix
• Acranobacterium
• Lactobacillus
• Gardnerella

Corynebacterium

• C. diphtheriae
• C. jeikeium
• C. urealyticum
• C. pseudodiphtheriticum
• C. striatum
• C. xerosis

Corynebacterium (cont.)

• Normal skin and mucous membrane flora
• Called diphtheroids or coryneforms
• Club-shaped
• Gram-positive pleomorphic non-spore forming
• Gram stain appears like Chinese letters (V, L, Y formation) or palisades (lines up side by side)

Corynebacterium (cont.)

• Facultative anaerobic
• Small gamma colonies on SBA
• Catalase positive
• Nonmotile
• Frequent contaminants.

C. diphtheriae

• Significant pathogen
• Virulence factor is diphtheria toxin
• In strains infected with bacteriophage carrying tox gene
• Exotoxin blocks protein synthesis
• Destroys host cells
• May be absorbed, damaging heart and nervous system
• Lethal at 130 ng/kg of body weight
• 150 lb person = ~0.3 ounces

C. diphtheriae (cont.)

• Two forms of disease
• Cutaneous (non-healing ulcer)
• Respiratory (diphtheria)

Diphtheria

• URT infection (tonsils, pharynx)
• Bacteria multiply, release toxin
• Tissue necrosis
• Exudate: inflammation → pseudomembrane
• Necrotic epthithelial 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 cell
• Metachromatic granules
• Methylene blue stain
• Pockets of inorganic phosphates or nutrient reserves.

C. diphtheriae Media

• SBA - small zone of beta hemolysis
• Serum or blood containing media
• Loeffler medium
• enhances development of metachromatic granules
• Pai agar – or egg based agar
• Tinsdale agar (cystine-tellurite blood agar): Selective
• Potassium tellurite inhibits non-coryneform bacteria
• Differential
• Tellurite reduction produces brown/black colonies
• Cystinase activity = halo around colony

Elek Test

• Immunodiffusion test for diphtheria toxin production and confirmation
• A strip of filter paper saturated with diphtheria anti-toxin is imbedded into agar medium
• Isolate, positive, and negative controls are streaked on agar parallel to each other and perpendicular to the 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: catheters or prosthetic devices
• Diphtheroid prosthetic valve endocarditis
• C. urealyticum: urinary pathogen

Other Non-spore-forming GPR

• Rothia dentocariosa: oral normal flora, endocarditis, wound infections
• Filamentous or coryneform groups
• Undesignated CDC Coryneform groups: opportunistic or nosocomial infections

Listeria monocytogenes

• Vaginal and intestinal normal flora (NF) in humans
• Clinical infections due to ingestion of contaminated food
• Virulence factors: Listeriolysin O, Others
• Listeriosis (sepsis)
• Neonates
• Fatality rate ~50% in pregnant women
• Can cause spontaneous abortion and stillbirth
• Immunocompromised
• Predisposition to CNS

L. monocytogenes Cultures

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

L. monocytogenes ID

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

L. monocytogenes Motility

• Tumbling motility at 25°C, but not at 35°C
• Motility test using a single tube incubated at 35°C and another at RT
• Motility on wet-mount broth is a tumbling pattern at 25°C, not 35°C
• Umbrella pattern at 25°C, not 35°C in semisolid agar motility test
• Positive CAMP test distinguishes L. monocytogenes from other Listeria spp.

Differentiation of Listeria

• Table showing differentiation of Listeria spp. from Corynebacterium spp., S. agalactiae, and Enterococcus sp. based on catalase, esculin hydrolysis, motility, β-hemolysis and 6.5% NaCl tests.

Erysipelothrix rhusiopathiae

• Domestic swine major reservoir
• Occupational hazard (butchers, vets, fishermen)
• Causes erysiploid (red skin lesion) in animals and humans
• Disseminate to bacteremia and endocarditis

Erysipelothrix rhusiopathiae (cont.)

• GPR
• Catalase negative
• Alpha or gamma hemolysis
• Nonmotile
• H2S positive in TSI (Triple Sugar Iron Agar)

Cultures

• Specimens: skin biopsies, blood
• Grow on routine media (BAP, CHOC, CNA, PEA)
• Small colonies (48 hours)

Arcanobacterium haemolyticum

• Formerly Corynebacterium
• Significant: A. haemolyticum, A. pyogenes, and A. bernardiae
• Causes pharyngitis
• Facultative anaerobe
• Beta hemolysis
• Catalase negative
• Reverse CAMP positive
• Inhibits S. aureus hemolysis

Reverse CAMP

• A. haemolyticum and S. aureus are streaked perpendicular to each other on anaBAP. A zone of hemolysis appears if the organism produces the factor in the presence of S. agalactiae.

Lactobacillus

• Normal vaginal flora
• Produce lactic acid
• Reduce vaginal pH
• Inhibits growth of other organisms
• Rarely causes disease
• L. acidophilus probiotic, used as a probiotic.

Lactobacillus(cont.)

• Non-spore-forming GPR
• Medium to long rods
• Aerotolerant anaerobes
• Catalase negative
• Nonmotile

Gardnerella vaginalis

• Pleomorphic coccobacilli
• Cell wall with gram-variable characteristics. Gram-variable
• Nonmotile
• Facultative anaerobe
• Normal flora (NF) in 50–70% women
• Linked to bacterial vaginosis (BV)

Bacterial Vaginosis (BV)

• Change in NF (lactobacillus)
• Increase in pH allows growth of G. vaginalis
• Foul-smelling discharge with amine odor
• Untreated can lead to pelvic inflammatory disease (PID) and urinary tract infections (UTIs)
• Culture not recommended.
• Perform wet prep or gram stain
• Clue cells: squamous cells with bacteria clustered at edges; used for diagnosis of BV.

Cultures (Vaginal)

• Appropriate for extravaginal sites
• Grow on CHOC, BAP, not MAC
• Small pleomorphic gram-variable coccobacilli
• Catalase and oxidase negative
• Hippurate hydrolysis positive

Non-Spore-Forming Branching GPR (Aerobic Actinomycetes)

• Nocardia spp.
• Tropheryma whipplei
• Streptomyces
• Actinomadura
• Gordona
• Tsukamurella
• Rhodococcus equi

Nocardia spp.

• Found in soil
• Infects immunocompromised: pulmonary and cutaneous
• Pulmonary: N. asteroides complex
• Dissemination → abscess → necrosis
• Cutaneous (mycetomas): N. brasiliensis
• Draining pus with sulfur granules

Nocardia spp.(cont.)

• Aerobic GPR morphologically similar to fungus
• Form beaded, branching filaments on gram stain
• Weakly acid fast

Nocardia spp. (cont.)

• Grow slowly (1-week or more) on nonselective media
• Waxy, chalky, crumbly colonies
• Can be identified by acid fast staining.

Nocardia ID

• Filamentous, branching organism
• Acid-fast stains (Ziehl-Neelsen or Kinyoun)
• Primary stain carbolfuchsin
• Rinsed and decolorized with acid
• Counterstained with methylene blue
• Reddish purple filaments are positive, blue is negative
• Requires modified acid-fast stain

Other Actinomycetes

• Tropheryma whipplei (causes Whipple's disease)
• Intracellular pathogen
• Uniformly fatal if untreated
• Diarrhea, weight loss, malabsorption
• Identify using PCR or 16S rRNA

Spore-Forming Non-Branching GPR

• Bacillus
• Spore-forming, non-branching bacilli
• Spores
• Protective, metabolically inactive
• Increase survival in nature
• Resistant to biocides
• Thick outer wall

Bacillus

• Found in nature
• Metabolically diverse
• Lab contaminants
• B. anthracis and B. cereus are two most important pathogens
• Important to rule out in case of infections

Bacillus (cont.)

• Large aerobic GPR
• Spore-formers
• Spores appear as holes on gram stain.
• Beta hemolytic, except for B. anthracis
• Catalase positive

Bacillus (cont.)

• Catalase positive and aerobic spore formation distinguishes them from Clostridia
• Growth on SBA and PEA
• Does not grow on enteric agars, it is GPR

B. anthracis

• Causes anthrax, usually in herbivores
• Bioterrorism agent
• Virulence factors: Glutamic acid capsule, Protective antigen, Edema factor, Lethal factor

B. anthracis (cont.)

• Three forms: cutaneous, pulmonary, gastrointestinal
• Cutaneous anthrax: pimple → vesicles → erythematous ring → necrotic lesion (black eschar)
• Inhalation anthrax (Woolsorter's disease): flu-like phase → respiratory distress → coma → death
• GI anthrax: ingestion → pain, nausea, vomiting
• Gram stain: very long chains, “bamboo shoots”, square ended
• Colonies: large, gray, flat, irregular edges, medusa head colonies
• Identification: aerobic/anaerobic, spore-forming GPR, nonhemolytic on SBA, nonmotile, catalase-positive
• Biological safety hood for handling

B. cereus

• B. cereus is beta hemolytic and motile.
• Distinguishes it from B. anthracis
• Produces enterotoxin
• Causes food poisoning
• Self-limiting, resolves without intervention

Identification of GPR

• Flowchart for identification of Gram-positive rods based on spore presence, catalase test result, motility, bile susceptibility and esculin hydrolysis tests

Spirochetes

• Borrelia, Leptospira, Treponema
• Long, slender, spiral-shaped
• Motile
• Differ in number and tightness of spirals
• Not seen in gram stain
• Require silver stains
• Seen in wet preparations by darkfield or phase contrast microscopy

Borrelia

• Contains several species of spirochetes
• Transmitted by arthropods (ticks & lice)
• 3 to 10 spirals/organism
• Often causes relapsing fever
• B. burgdorferi causes Lyme disease

Borrelia recurrentis

• Causes relapsing fever
• Repeated febrile episodes of spirochetemia
• Cyclic relapses caused by antigenic variation
• Illness ends when immune system responds to new antigen makeup
• Epidemic relapsing fever (louse-borne)
• Endemic relapsing fever (tick-borne)

Relapsing Fever Lab Tests

• Microscopic examination of blood during febrile periods
• Giemsa or Wright's stain preferred method (organisms are seen in blood).
• Can be cultured in Kelly medium (rare)
• Serology (antigenic variation makes this impractical)

Borrelia burgdorferi

• Lyme borreliosis/Lyme disease
• Originally in the Northeast (spreading)
• Transmitted by Ixodes ticks (deer or mouse ticks)

Lyme Disease Lab Tests

• Serologic tests: most common and fastest (antibody detection tests with western blot confirmation)
• Direct microscopic examination of skin or blood (usually negative)
• Cultures: labor intensive (Kelly medium)

Leptospira

• L. biflexa: nonpathogenic
• L. interrogans: animal pathogen passed to humans via water contaminated with animal urine
• Occupational hazard for vets, farmers, sewer workers

Leptospira interrogans

• Spiral with hooked ends
• Tightly coiled

Leptospirosis

• Zoonotic infection
• from dogs, rats, cattle.
• Spirochetes infect kidneys of animals→urine→water indirect pathway
• Leptospires enter humans via breaks in skin or intact mucosa

Leptospirosis (cont.)

• Clinical manifestations: range from asymptomatic to severe illness
• Incubation period: 3–30 days
• Symptoms onset abrupt; nonspecific influenza-like symptoms

Leptospirosis (cont.)

• Severe systemic disease
• Weil's disease – CSF positive
• Renal failure
• Hepatic failure
• Icteric leptospirosis
• Intravascular disease

Leptospirosis Lab Tests

• Blood & CSF (first week), urine (after that).
• Direct examination (darkfield microscopy or DFA)
• Cultures (Fletcher's semi-solid media)
• Incubate in the dark at 30°C for 6 weeks + darkfield microscopy from media
• Serology (ELISA assay)

Treponema

• 4 to 14 spirals/organism.
• Four pathogenic organisms:
  • T. pallidum subsp. pallidum
  • T. pallidum subsp. pertenue
  • T. pallidum subsp. endemicum
  • T. pallidum subsp. carateum

Treponema pallidum subspecies pallidum

• Venereal syphilis
• Great imitator; variety of clinical presentations
• Transmitted sexually or via nongenital lesions (mouth or skin).
• Can cross the placenta (congenital syphilis)
• Three stages (primary, secondary, tertiary)

Primary Syphilis

• Few days to months after organism acquired
• Firm chancre forms at inoculation site
• Lesion contains many spirochetes and is highly infectious
• Painless

Secondary Syphilis

• 2-12 weeks after primary lesions
• Organisms disseminate throughout body
• Fever, lymphadenopathy, headache
• Secondary skin and mucous membrane lesions
• Widespread unusual skin rashes
• May include palms and soles

Tertiary or Late Syphilis

• 1/3 of untreated develop tertiary syphilis years after initial infection
• Not infectious
• Affects many body sites
• Gummas (granulomatous lesions) in skin, liver, bones
• CNS: neurosyphilis
  • deafness, blindness, partial paralysis
  • shuffling walking gait & mental disturbances
  • Cardiovascular lesions: syphilitic aortitis

Early Onset Congenital Syphilis

• Mother has early syphilis
• Treponemes cross placenta.
• Infected fetus often dies
• Affects many body systems
• Manifestations include skin and mucous membrane lesions, anemia, hepatosplenomegaly, meningitis, bone lesions

Late Onset Congenital Syphilis

• Mother has chronic, untreated syphilis
• Symptoms in >2-year-old
• Blindness, deafness, mental retardation
• Bone and tooth deformities
• Prevented by screening of pregnant women and treatment if necessary

Serologic Laboratory Diagnosis

• Nontreponemal tests (screening): Venereal Disease Research Laboratory (VDRL), Rapid Plasma Reagin (RPR)
• Treponemal tests (confirmation): Enzyme Immunoassay (EIA), Treponema pallidum particle agglutination assay (TPPA), Fluorescent treponemal antibody absorption (FTA-ABS)

Nontreponemal Tests

• Detects antibodies against cardiolipin-lecithin formed against lipids from infection
• Sensitive but not specific
• False positive occurs in Lyme disease, certain viral infections, autoimmune disease, pregnancy
• Screen, monitor therapy (titers), detect reinfection

VDRL and RPR

• Results indicating positive or negative status based on the test

Treponemal Tests

• Detects treponemal-specific antibodies
• Confirmation of infection
• Remains positive after treatment
• Enzyme immunoassay (EIA): used.
• Treponema pallidum particle agglutination assay (ΤΡΡΑ); used
• Previously, FTA-ABS used

TPPA

• Positive control and Negative control results
• Interpretation based on the result.

Direct microscopic exam

• Darkfield microscopy requires patience and experience; examines fluid from lesions looking for corkscrew motility.
• Oral lesions not appropriate
• Nonpathogenic treponemes may be present

Other Pathogenic Treponemes

• Spread through direct contact or contaminated utensils
• May exhibit primary, secondary, or tertiary syphilis; tertiary is rare.
• Not usually sexually transmitted
• No congenital infections.

Other Pathogenic Treponemes (cont.)

• T. endemicum: Endemic syphilis (non-venereal); Middle East/hot, arid areas
• T. pertenue: Yaws (skin and bones), Humid, tropical areas
• T. carateum: Pinta (skin), Central and South America
• Diagnose based on clinical manifestations and patient history.

Mycobacteria

• Aerobic, require increased levels of CO2
• Non-spore forming
• Non-motile
• Cultures held for 6 weeks before negative result obtained
• Slim gram variable rods
• High lipid content (mycolic acid) in cell wall
• Don't gram stain well.

Mycobacteria Gram Stain

• Gram stain poorly (if at all), as lipids from the complex cell wall interfere with staining penetration.
• No organisms
• Beaded GPR
• Ghost cells present.

Mycobacteria Safety

• Incidence of positive tuberculosis skin test (PPD) is 3X higher in mycobacteriology
• Separate lab, noncirculating ventilation, and negative air pressure.
• Work done in biosafety cabinet
• Airborne hazard: Wear gloves and gowns.
• BSL2 for processing specimens
• BSL3 for working with cultured organisms
• Specimens double sealed during centrifugation

Mycobacteria Species

• Major pathogens: M. tuberculosis complex
• Nontuberculous mycobacteria (NTM): M. leprae, Hansen's disease (leprosy)

Specimen Collection

• Sputum, bronchial washing, gastric contents, urine, stool, and tissue
• First morning, on 3 consecutive mornings
• 5–10 ml sputum minimum volume, refrigerate overnight.
• If sputum can't be produced, bronchoscopy, collect BAL, or biopsy.

Specimen Collection (cont.)

• Gastric Aspirates and Washings
• Young patients less than 3 years can't produce sputum
• Young children swallow sputum, mycobacteria damaged by stomach acid
• Neutralize gastric aspirates and urine with sodium bicarbonate if refrigerated overnight
• Stool: HIV patients screened for M. avium screen

Inappropriate Specimens

• Swabs: not enough material, mycobacteria don't dislodge
• 24-hour pooled sputum/urine: Contamination/interference

Digestion and Decontamination

• Specimens from sterile sites (CSF) may be concentrated and directly inoculated.
• Specimens with NF and mucous require digestion (e.g. N-acetyl-L-cysteine (NALC)).
• Decontamination with NaOH is used.
• NALC-Sodium Hydroxide mixture used in digestion-decontamination processes

Specimen Processing

• Digestion frees mycobacteria from clumps of protein (use of NALC - mucolytic agent)
• Decontamination is included to remove non-mycobacterial factors. Timer is used to optimize destruction of contaminants with minimal effects on mycobacteria.
• Concentration: increase the number of organisms per ml
• Centrifuge decontaminated specimen for 20 min at 3000 rpm.
• Make AFB smear
• Inoculate media with sediment.
• Include one liquid and one solid medium

Staining for AFB smear

• Ziehl Neelsen (hot stain)
• Kinyoun (cold stain)
• Primary is carbolfuchsin
• Rinse and decolorize with strong acid
• Counterstain with methylene blue
• Reddish filaments are positive; blue is negative (acid fast).

Staining for AFB smear (cont.)

• Heat or phenol forces special dye into mycobacterial cells
• High lipid content resists destaining with acid
• Organisms not decolorized = acid fast
• Scan at least 300 oil immersion fields

Acid-Fast Stain Carbolfuchsin

• Acid-fast stain result of AFB.

Staining for Acid-Fast Bacilli

• Fluorochrome stains (auramine or auramine-rhodamine), which contain phenol to increase stain penetration.
• Examined using a fluorescence microscope.

Auramine Stain

• Fluorescent bright yellow-orange stained smear of AFB is a result of using the Auramine stain process.

AFB Culture

This quiz covers essential topics related to the transmission, clinical manifestations, and laboratory identification of Leptospira interrogans and Clostridium species. Test your knowledge on the risks, growth conditions, and distinguishing features of these bacteria. Perfect for students or professionals in microbiology and infectious diseases.