Microbiology Foodborne Pathogens Quiz

Anaerobic Bacteria

• Anaerobic bacteria do not require oxygen to grow
• Different types have varying tolerances to oxygen
• Obligate anaerobes require anaerobic conditions to grow, oxygen is toxic and kills them

Aerotolerant Anaerobes

• Can grow in atmospheres with oxygen, but grow best without oxygen
• These bacteria are not harmed by oxygen

Facultative Anaerobes

• Do not require oxygen, but use it if available
• They can survive with or without oxygen

Oxygen Reduction

• Superoxide anion and hydrogen peroxide are toxic to obligate anaerobes

• Strict aerobes and facultative anaerobes possess enzymes: superoxide dismutase/catalase to neutralize these toxic byproducts

Oxidation-Reduction (Redox) Potential

• Anaerobic bacteria need a low redox potential
• In high redox environments, oxygen is present, so these bacteria have a hard time surviving
• Normal human tissue and aerobic media have a high redox potential

Normal Habitat

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

Diseases

• Exogenous Infections: Organisms, spores, or toxins enter the body through ingestion or trauma (e.g., tetanus, botulism)
• Endogenous Infections: Infections involving host's NF near mucosal surfaces (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
• Presence of sulfur granules (actinomycosis)
• Black or fluorescent red color
• Previous therapy with ineffective aminoglycosides
• Failure to grow the organism when stained with a gram stain

Specimen Collection

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

Transport and Processing

• Inoculate media and immediately place in an anaerobic environment
• Limit exposure to room air
• Do not allow to dry out
• Do not refrigerate
• Minimize time at room temperature
• Use an anaerobic transport system (PRAS media): prereduced, anaerobically sterilized transport medium with: Agar transport medium (modified Cary-Blair or Amies) & Rezasurin (oxygen indicator) & Reducing substances
• An anaerobic chamber, or anaerobic bags can be used

Culture

• Specimens are cultured aerobically on BAP, CHOC and MAC plates first to determine if this organism is an aerotolerant anaerobe.
• Anaerobes require Vitamin K, hemin, and yeast extract to grow.

Nonselective Media

• CDC or Anaerobic Blood Agar Plates (anaBAP) support obligate and facultative anaerobes
• Anaerobic Broth, Thioglycollate, cut (cooked) meat support obligate and facultative anaerobes; anaerobes grow toward bottom (facultative grow throughout)

Anaerobic-BAP / Thioglycollate Broth

• Visual aids describing the growth pattern of different types of anaerobic organisms in respective media

Selective Media

• Phenylethyl alcohol blood agar (anaPEA) supports GN and GP obligate anaerobes; inhibits enteric GNRs
• Colistin nalidixic acid blood agar (anaCNA) supports GN and GP obligate anaerobes; inhibits enteric GNRs
• Bacteroides Bile-Esculin (BBE) agar: Selective / differential for esculin hydrolysis/ bile tolerant organisms including B. fragilis
• Kanamycin-vancomycin laked blood (KVLB) agar: selects for Bacteroides and Prevotella; kanamycin inhibits most facultative GNRs; vancomycin inhibits most GPRs; laked blood encourages Prevotella to produce brown-black pigments
• Cycloserine-cefoxitin-fructose agar (CCFA): selective/differential for Clostridium difficile; cycloserine & cefoxitin are antibiotics; fructose is a CHO source; neutral red (red in acid; yellow in alkaline); C. difficile causes alkaline (yellow) reactions when metabolizing proteins

Other Selective Media

• List of additional selective media for various anaerobic and facultative organisms. The different medias are tailored to different requirements concerning what compounds should inhibit or promote growth.

Anaerobic Incubation

• Maintaining a controlled anaerobic environment is critical for growing anaerobic organisms
• Usual incubation conditions include: temperatures of 35 to 37°C for 48 hours; 80-90% nitrogen gas; 5-10% hydrogen gas; and 5-10% carbon dioxide

Anaerobic Systems

• These systems include: jars, bags, and chambers to create and maintain anaerobic conditions

Anaerobic Jars

• Jar technique, or gas pak jar: An anaerobic jar utilizes a catalyst (palladium pellets) generating H₂ and CO₂ when water is added. To remove oxygen, a sealed chamber is incubated at 35°C. Indicators (Methylene blue or resazurin) change to white when reduced(anaerobic).

Anaerobic Bags

• Commercially available systems holding 1-3 plates, that contain an oxygen removal system and indicator. The gas-impermeable plastic bag is the primary component of the system.

Anaerobic Chamber

• An optimized anaerobic incubation system that provides an oxygen-free environment for inoculation, incubation, and inspection of media for growth. The chamber contains a palladium catalyst, desiccant, and an indicator and an anaerobic gas mixture

Culture Examination

• Cultures are examined in the specified anaerobic chamber at specified times.
• Maintaining anaerobic conditions from incubation to examination is required to avoid contaminations.

Anaerobic Identification Tests

• Presumptive identification using gram staining and colony morphology
• Rapid tests such as catalase, motility, urease, indole, and esculin hydrolysis assays.

Anaerobes Gram Stain RXN

• Flowchart for classifying anaerobic organisms based on their gram stain characteristics
• Includes spore-forming vs. non-spore-forming and gram positive/negative categories

Rapid Identification Tests

• Aerotolerance, fluorescence, disk tests, lecithinase, naglar test, lipase, reverse CAMP assays

Aerotolerance

• Determining if an isolate is a strict anaerobe, aerotolerant anaerobe, or facultative anaerobe
• Incubating specimens aerobically & anaerobically in parallel
• (i.e., anaBAP anaerobically; CHOC aerobically in CO2). The plates are examined in 48 hours

Fluorescence

• Organisms that fluoresce under UV light (366 nm)
• Demonstrating the use of UV light in analyzing organisms

Antibiotic Disk Test

• Confirming gram stain through special potency disks (kanamycin, colistin, vancomycin)
• Tests to distinguish organisms based on reaction/resistance to various antibiotics
• (e.g., Van S, Col R = Clostridia, Porphyromonas), (Van R = GN, not Porphyromonas)

Disk Tests

• Sodium polyanethol sulfonate (SPS) disk : Differentiating gpc based on susceptibility to sodium polyanethol sulfonate
• Nitrate disk: Nitrate is reduced in differing ways in the presence of reagents
• Bile disk: Determining whether an organism will grow in elevated bile concentrations (i.e., bile tolerant anaGNR = Bacteroides fragilis group)

Lecithinase

• Determining if the isolate produced a lecithinase
• Positive lecithinase reaction: Opaque, white zone surrounding the bacterial colonies on the egg yolk agar plate
• Negative lecithinase reaction: No change in the egg yolk agar surrounding bacterial colonies

Nagler Test

• Detecting the specific alpha toxin produced by C. perfringens through the use of antiserum
• A specific type of lecithinase produced by C. perfringens

Lipase

• Identifying bacteria capable of hydrolyzing triglycerides into glycerol and fatty acids
• Identifying positive/negative reaction based on whether an oil/water sheen is visible and the general morphology of each colony of bacteria

Reverse CAMP

• Alpha toxin of C. perfringens synergistically works with beta-hemolytic group of S. agalactiae causes hemolysis
• A positive test results in an arrowhead-shaped zone of hemolysis surrounding a perpendicular streak of C. perfringens

Definitive Identification Tests

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

Biochemical and Commercial Systems

• PRAS or non-PRAS biochemical media
• Commercial biochemical and pre-existing bacterial enzyme minisystems

Gas-Liquid Chromatography

• Analysis of cellular fatty acids & metabolic end products (volatile acids) to produce a characteristic pattern for identification purposes

Gene Sequencing

• 16S rRNA sequencing
• Extracting and amplifying ribosomal DNA (rRNA)
• Sequenced through PCR analysis to identify organisms

Clostridium Species

• This group of bacteria include catalase-negative, motile (except C. perfringens), anaerobic gram-positive rods (GPR), and some gram-variable or gram-negative
• Gram positive bacteria that are susceptible to vancomycin
• Produce endospores (i.e., they appear as unstained refractile structures in gram stain and are oval to round, terminal to subterminal)
• Aerotolerant, cause exogenous infections via ingestion or wounds (e.g., tetanus, gas gangrene, botulism, food poisoning)
• Produce potent toxins

Genera Differences

• Chart comparing Clostridium, Bacillus, and Lactobacillus in terms of optimal growth conditions, sporulation conditions, and catalase test results

Clostridium perfringens

• Most common Clostridium species; found in undercooked meat, soil, water
• Gram-positive, boxcar-shaped rods producing a double zone of beta hemolysis
• Positive reverse CAMP, lecithinase, and Nagler reactions
• Spore production is seldom observed or not common

C. perfringens Clinical Significance

• Isolated from tissue infections and bacteremia
• Produces gas in infected tissue (Myonecrosis; Gas gangrene)
• Deep penetrating wounds or surgical procedures
• Diabetic foot ulcers

C. perfringens Clinical Significance (Food poisoning/gravy)

• Food poisoning comes from meat, meat products, or gravy
• Ingested spores that germinate in intestines & cause enterotoxin
• Symptoms such as nausea, vomiting, diarrhea, and abdominal pain

Clostridium difficile

• May be a normal flora member of the stool
• Antibiotic-associated diarrhea and pseudomembranous colitis
• C. difficile increases in number when antibiotic use disrupts the natural intestinal ecosystem.
• 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 like horse manure
• Spore-forming GPR
• Chartreuse fluorescence
• Must be tested for toxin production

C. difficile Toxin Tests

• Cytotoxin test: Evaluating the toxic effects of stool on cultured human cells
• Kit testing: Determines if toxin A, toxin B, or glutamate dehydrogenase (not a virulence factor) is present in the stool; using an enzyme immunoassay
• Molecular testing: Still under development

Clostridium botulinum

• Botulism toxin ingestion from home-canned vegetables, home-cured meats
• Infant botulism associated with honey
• Neuormuscular toxins cause paralysis or death
• Foodborne botulism, wound botulism, infant botulism (lacking NF)

C. botulinum Diagnosis

• Diagnosed clinically based on symptoms
• Isolating the organism or detecting toxin requires a reference laboratory

Clostridium tetani

• Found in soil and intestinal tracts of animals
• Enters the body through a wound site
• Produces the potent neurotoxin (tetanospasmin) causing severe muscle spasm in unimmunized individuals
• Can cause tetanus (lockjaw).

C. tetani Diagnosis

• Usually diagnosed clinically

Terminal Spores

• Tennis-racquet-shaped

C. septicum

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

Non-Spore-Forming Anaerobic GPR

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

Actinomyces

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

Actinomyces Gram Stain

• Aero- and anaerobic Actinomyces spp.
• GPR irregularly stained (beaded appearance)
• Coccoid to filamentous
• Filaments branch

A. israelii Actinomycosis

• Can cause actinomycosis
• Forms granulomatous infection usually in the jaw
• Develops fistulae & drains pus with sulfur granules

A. israelii Cultures

• Associated with "lumpy jaw"
• Pus and sulfur granules observed in anaBAP
• Growth is slow, taking 7-9days
• Colony morphology resembles molar teeth
• Anaerobic, branching GPR

Bifidobacterium

• Actinomyces-like morphology
• Gram stain is branched or bifurcated GPR
• Normal flora member of the intestines and oral cavity
• Rarely cause disease, but can cause actinomycosis

Propionibacterium

• Anaerobic diphtheroid like GPR
• Resembles corynebacteria
• Normal flora member of skin
• Most common anaerobe isolated in the lab as a contaminant in blood cultures & specimens collected from the skin penetration
• P. acnes linked to acne
• Catalase and indole positive
• May cause subacute bacterial endocarditis (SBE) & 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

• Bacteroides, Prevotella, Prophyromonas, Fusoacterium, Veillonella (Normal flora member of the mucous membrane)

Bacteroides fragilis Group

• Anaerobic GNR
• Bacteroides fragilis is the most common
• Causes intra-abdominal infections, bacteremia, & soft tissue infections
• B. thetaiotaomicron is the second most common

Bacteroides fragilis Group

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

Bacteroides ureolyticus Group

• Anaerobic GNR
• Bile sensitive and bile-tolerant, non-pigmented organisms
• Some GNRs pit the agar
• Growth in formate and fumarate are observed
• Resistant to vancomycin
• Sensitive to kanamycin and colistin

Prevotella

• Anaerobic GNR
• Bile susceptible
• Resistant to kanamycin & vancomycin
• Growth on KVLB, but not BBE
• Some produce protoporphyrin pigmenting colonies
• Colonies fluoresce a “brick red” under UV light

Fusobacterium

• Susceptible to kanamycin and colistin
• Vancomycin-resistant
• F. nucleatum: Thin fusiform rods; bread-crumb, speckled colonies
• F. necrophorum: Lipase positive

Mobiluncus

• Associated with bacterial vaginosis, pelvic inflammatory disease, & abdominal infections
• Curved bacilli
• Gram variable
• Motile, catalase, and indole-negative
• Inhibited by vancomycin

Selective Anaerobic GNR ID

• Flowchart to aid in identifying gram-negative anaerobic rods

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

• Normal skin and mucous membrane flora
• Called diphtheroids or coryneforms, are club-shaped
• Gram positive rods (GPR), are pleomorphic (variable in size), non-spore forming
• Gram stain: looks like "Chinese letters" (V, L, Y formation) or palisades (line up side by side)
• 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
• Blocks protein synthesis, destroys host cells
• Absorbed in heart and nervous system; lethal at 130 ng/kg of body weight (150 lb person = 0.3 ounces)
• Causes cutaneous (non-healing ulcer) and respiratory (diphtheria) diseases

Diphtheria

• URT infection (tonsils, pharynx)
• Bacteria multiply, release toxin
• Tissue necrosis
• Exudate (inflammation → pseudomembrane)
• Necrotic epithelial cells, WBCs, fibrin, bacteria involved in suffocation
• Treatment with antitoxin
• Prevention 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 (enhances development of metachromatic granules)
• Pai agar (egg-based agar)
• Tinsdale agar (cystine-tellurite blood agar): Selective (potassium tellurite inhibits non-coryneform bacteria); Differential (tellurite reduction produces brown or black colonies; cystinase activity = halo around colony)
• Elek Test: Immunodiffusion test for toxin production

Corynebacterium spp.

• Normal skin flora
• May contaminate cultures
• C. jeikeium: devices/prosthetic valve endocarditis
• C. urealyticum: Urinary pathogen

Other Non-spore-forming GPR

• Rothia dentocariosa: oral NF, endocarditis, and wound infections
• Filamentous or coryneform groups
• Undesignated CDC coryneforms groups: opportunistic or nosocomial infections

Listeria monocytogenes

• Vaginal and intestinal NF in humans
• Clinical infections via contaminated food
• Virulence Factors: Hemolysis (listeriolysin O), others

Listeria monocytogenes

• Listeriosis (sepsis)
• Neonates (50% mortality)
• Pregnant women (spontaneous abortion/stillbirth)
• Immunocompromised individuals
• CNS predilection

L. monocytogenes Cultures

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

L. monocytogenes ID

• GPR & non-spore forming
• Small beta colonies on BAP
• Hemolysis may be hidden under colonies
• Catalase positive
• Bile-esculin positive
• Motile at 25°C

L. monocytogenes Motility

• Tumbling motility at 25°C but not 35°C
• Results in umbrella-like growth patterns
• Positive CAMP test differentiates L. monocytogenes from other Listeria spp.
• Block vs. arrowhead pattern

Differentiation of Listeria

• Table comparing L. monocytogenes, Corynebacterium spp., S. agalactiae, and Enterococcus spp. based on catalase, esculin hydrolysis, motility, β-hemolysis, and 6.5% NaCl results

Erysipelothrix rhusiopathiae

• Reservoir: domestic swine
• Occupational risk to butchers, vets, and fishermen
• Causes erysiploid (red skin lesion)
• Can disseminate into bacteremia/endocarditis in humans

Erysipelothrix rhusiopathiae

• GPR
• Catalase negative
• Alpha or gamma hemolysis
• Nonmotile
• H₂S positive in TSI

Culture

• Skin biopsies, blood
• Routine media (BAP, CHOC, CNA, PEA)
• Small colonies visible after 48 hours

Arcanobacterium haemolyticum

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

Lactobacillus

• Normal vaginal flora
• Produce lactic acid
• Reduce vaginal pH
• Inhibits growth of other organisms
• Rarely causes disease (e.g., L. acidophilus probiotic), may assist in inhibiting other species

Lactobacillus

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

Gardnerella vaginalis

• Pleomorphic coccobacilli
• Cell wall with GPR characteristics, stains gram-variable
• Non-motile
• Facultative anaerobe
• NF in 50-70% women
• Associated with bacterial vaginosis

Bacterial Vaginosis (BV)

• Change in NF (lack of lactobacillus)
• Increase in pH, allowing G. vaginalis growth
• Foul-smelling discharge
• Untreated leads to PID and UTIs

Clue Cells

• Squamous cells with bacteria clustered at the edges

Cultures

• Appropriate for extravaginal sites
• Grows on CHOC, BAP, but 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
• Gordonia
• Tsukamurella
• Rhodococcus equi

Nocardia spp.

• Found in soil
• Usually infects immunocompromised individuals
• May cause pulmonary or cutaneous diseases

Nocardia spp.

• Pulmonary: N. asteroides complex, pneumonia → abscess → necrosis
• Cutaneous: N. brasiliensis, abscess → invasive, draining pus with sulfur granules
• Aerobic GPR morphologically similar to fungi
• Form beaded, branching filaments on gram stain
• Weakly acid-fast

Nocardia spp.

• Growth is slow, with colonies described as waxy, chalky, or crumbly
• Can be identified using acid-fast staining

Nocardia ID

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

Other Actinomycetes

• Tropheryma whipplei: Whipple Disease, intracellular pathogen; diarrhea, weight loss, malabsorption
• Streptomyces, Actinomadura, Gordonia, Tsukamurella, Rhodococcus equi

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
• Found in nature, metabolically diverse (lab contaminants)
• B. anthracis, B. cereus: Important pathogens

Bacillus

• Large aerobic GPR
• Spore-formers
• Spores appear as holes in gram stain
• Catalase positive (B. cereus is beta-hemolytic and motile; B. anthracis isn't)

Bacillus

• Catalase positive
• Aerobic spore formation distinguishes from Clostridia
• Growth on SBA and PEA
• Does not grow on enteric agars; is a GPR

B. anthracis

• Causes anthrax (usually in herbivores)
• Bioterrorism agent; virulence factors include glutamic acid capsule, protective antigen, edema factor, and lethal factor

B. anthracis

• Clinical significance includes cutaneous, pulmonary, and gastrointestinal forms
• Cutaneous: pimple → vesicles → erythematous ring → necrotic lesion (black eschar)
• Pulmonary (woolsorter's disease): flu-like symptoms progressing to respiratory distress, coma, death
• Ingestion: pain, nausea, vomiting
• Gram stain: very long chains ("bamboo shoots"), square-ended
• Colonies: non-motile, non-hemolytic, large, gray, flat, irregular edges, medusa head

B. anthracis Identification

• Aerobic/anaerobic
• Spore-forming GPR
• Nonhemolytic on SBA
• Nonmotile
• Catalase-positive
• Requires a biological safety hood

B. cereus

• Beta hemolytic and motile; distinguishes it from B. anthracis
• Produces enterotoxin
• Causes food poisoning
• Self-limiting

Identification of GPR

• Flowchart summarizing the identification of gram-positive rods based on spore formation, catalase reaction, motility, bile-esculin reaction, and other specific characteristics

Spirochetes

• Borrelia, Leptospira, Treponema: Long, slender spiral-shaped, motile, not seen in gram stain, require silver stains

Borrelia

• Several species of spirochetes
• Transmitted by arthropods (ticks and lice)
• 3 to 10 spirals per organism
• Most cause relapsing fever
• B. burgdorferi causes Lyme disease

Borrelia recurrentis

• Causes relapsing fever (repeated febrile episodes of spirochetemia)
• Cyclic relapses caused by antigenic variation
• Episode ends when immune system responds

Relapsing Fever Lab Tests

• Microscopic examination of blood during febrile periods (Giemsa or Wright's stain)
• Culture in Kelly medium (rare)
• Serology (inadequate due to antigenic variation)

Borrelia burgdorferi

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

Three Stages of Lyme Disease

• Stage 1: Erythema chronicum migrans (ECM), lymphoadenopathy, flu-like symptoms
• Stage 2: Dissemination, fever, bone and joint pain, splenomegaly, malaise
• Stage 3: Late or persistent infection; months to years; chronic arthritis, neurological defects, cerebrovascular lesions

Lyme Disease Lab Tests

• Serologic tests (most common & fastest): antibody detection tests (immunoassays)
• Direct microscopic examination of skin or blood (usually negative & labor intensive)
• Cultures are labor intensive & use Kelly medium

Leptospira

• L. biflexa: Nonpathogenic
• L. interrogans: Pathogenic
• Transmitted to humans via contaminated water with animal urine
• Occupational hazard to vets, farmers, sewer workers

Leptospira interrogans

• Spirals with hooked ends
• Tightly coiled

Leptospirosis

• Zoonotic infection
• Spirochetes infect kidneys & enter urine → contaminated water
• Leptospires enter through breaks in skin or intact mucosa
• Symptoms range from asymptomatic to severe

Leptospirosis

• Clinical manifestation range from asymptomatic to severe
• Incubation period: 3-30 days
• Abrupt onset of influenza like symptoms & nonspecific symptoms
• Severe systemic diseases. Weil's disease (CSF positive); renal failure; hepatic failure; icteric leptospirosis; intravascular disease

Leptospirosis Lab Tests

• Blood & CSF (1st week); urine (later)
• Darkfield microscopy or DFA
• Cultures (Fletcher's semisolid media, 30°C dark, 6 weeks)
• Darkfield microscopy in media
• Serology (ELISA)

Treponema

• 4 to 14 spirals/organism
• 4 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 non-genital lesions (mouth or skin)
• Can cross placenta (congenital syphilis)
• Three stages (primary, secondary, tertiary)

Primary Syphilis

• Few days to months after organism acquisition
• Painless firm chancre (lesion) at inoculation site
• Highly infectious due to numerous spirochetes present

Secondary Syphilis

• 2-12 weeks after primary lesion
• Organisms disseminate throughout the body
• Fever, lymphoadenopathy, headache
• Secondary infectious lesions on skin and mucous membranes
• Widespread 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 (gummas, skin, liver, bones)
• CNS (neurosyphilis): deafness, blindness, partial paralysis, shuffling gait, mental disturbances
• Cardiovascular lesions (syphilitic aortitis)

Early Onset Congenital Syphilis

• Mother has early syphilis, treponemes cross placenta
• Severe infection; infected fetus often dies from congenital infection
• Affects many body systems, with skin and mucous membrane lesions, anemia, hepatosplenomegaly, meningitis, & bone lesions

Late Onset Congenital Syphilis

• Mother has chronic, untreated syphilis
• Symptoms in children > 2 years old: blindness, deafness, mental retardation, bone/tooth deformities
• Prevention by screening pregnant women and necessary treatment

Serological Laboratory Diagnosis

• Nontreponemal tests (screening): VDRL, RPR
• Treponemal tests (confirmation): EIA, TPPA, FTA-ABS

Nontreponemal Tests

• Detects antibodies (reagin/reaginic antibodies) against treponemal lipids (e.g., cardiolipin-lecithin)
• Sensitive but not specific; can have false positives from Lyme, viral infections, autoimmune diseases, and pregnancy
• Monitor therapy & detect reinfection

VDRL and RPR

• Visual aids describing the different results of the VDRL and RPR tests

Treponemal Tests

• Detects treponemal specific antibodies
• Remain positive after treatment
• Enzyme immunoassay (EIA)
• Treponema pallidum particle agglutination assay (TPPA)
• Previously FTA-ABS (fluorescent treponemal antibody absorption)

TPPA

• Positive control wells, negative control wells

Direct Microscopic Exam

• Darkfield microscopy (fluid from lesions); patience and experience
• Look for corkscrew motility
• Oral lesions are not ideal due to prevalence of nonpathogenic treponema

Other Pathogenic Treponemes

• Spread via direct contact or utensils, with contaminated drinking/eating utensils
• May exhibit primary, secondary, or tertiary disease; Tertiary syphilis and congenital syphilis are rare
• T. endemicum (Endemic syphilis in Middle East/arid areas), T. pertenue (Yaws in humid/tropical areas), T. carateum (Pinta in Central and South America)

Mycobacteria

• Aerobic; require increased CO₂
• Non-spore forming
• Non-motile
• Cultures held for 6-weeks before considered negative
• Slm gram-variable rods
• High lipid content (mycolic acid) in cell wall, so they do not gram stain well

Mycobacteria Gram Stain

• Gram stain poorly due to cell wall lipids
• Crystal violet and safranin do not properly penetrate
• Resulting stains: absence of organisms, beaded gram-positive rods, ghost cells

Mycobacteria Safety

• High incidence of positive tuberculin skin tests
• Labs should be separate, nonrecirculating ventilation, negative air pressure, and work within a biosafety cabinet

Mycobacteria Safety

• Airborne hazard
• BSL-2 for specimen processing (gloves and gowns); BSL-3 for cultured organisms
• Specimens double sealed during centrifugation

Mycobacteria Species

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

Specimen Collection

• Sputum, bronchial washings, gastric contents, urine, stool, and tissue samples
• First morning sputum on 3 consecutive mornings
• 5-10 ml sputum minimum; refrigerate overnight
• If sputum can't be obtained, proceed to bronchoscopy or biopsy
• Gastric aspirates and washings; young patients (cannot produce sputum); young children (swallow sputum); neutralize with sodium bicarbonate; mycobacteria damaged by stomach acid
• Stool specimens: AIDS patients get M. avium screen; Neutralize gastric aspirates/urine with sodium bicarbonate (refrigerate overnight)

Inappropriate Specimens

• Insufficient material for identification
• Contamination from other organisms
• Interference from prolonged exposure (e.g., urine) to specimens interfering with growth of mycobacteria

Digestion and Decontamination

• Specimens from sterile sites (CSF): concentrate for inoculation
• Specimens contain NF and mucous membranes: Digestion with N-acetyl-L-cysteine (NALC), decontamination with NaOH, concentration

Specimen Processing

• Digestion: frees mycobacteria from protein clumps using NALC (mucolytic agent)
• Decontamination: removes NF
• May use NALC, timed and controlled to remove contaminants with minimal effects on mycobacteria
• Concentration: increases the number of organisms per mL using centrifugation

Staining for AFB Smear

• Ziehl-Neelsen (hot stain, heat): stains acid-fast bacilli (AFB)
• Kinyoun (cold stain, detergent/phenol): stains acid-fast bacilli (AFB)
• Primary stain: carbolfuchsin; rinse and decolorize with acid; counterstain with methylene blue
• Reddish filaments = AFB (acid-fast positive); blue filaments are negative

Staining for AFB Smear

• Heat/phenol forces special dye into mycobacterial cells
• High lipid content resists decolorization = acid fastness
• Scan at least 300 oil immersion fields before reporting a specimen as negative
• Any number of AFB bacteria on the slide is significant

Acid-Fast Stain Carbolfuchsin

• Microscopic image of acid-fast stain, positively stained acid-fast bacilli

Test your knowledge on foodborne pathogens and microscopy techniques in microbiology. This quiz covers various aspects, including enterotoxin production, spirochetes observation, motility characteristics, and mycobacterial behaviors. Challenge yourself with these important microbiological concepts!