Hard to Gram Stain (Mycobacteria, Chlamydia, Lyme Disease) and Microbiome

Mycobacterium

• Thin rods fail to stain by Gram

• Gram-positive wall structure

• Demonstrate acid fastness

• Non-motile

• Non-spore forming

• Obligate aerobes

• Cell wall 60% lipid

  • Mycolic acid & other lipids

  • Lipoarabinomannan (LAM) extends from cytoplasmic membrane to surface

    • Structurally and functionally analogous to GN LPS

• Lipid coat provides

  • Resistance to drying

  • Barrier to chemical agents (like Gram-negative LPS)

• Growth

  • Aerobic, specialized conditions

  • High CO2 and low pH

  • MTB very slow-growing, 3 to 6 weeks

Tuberculosis: Epidemiology

• 18th-19th century urbanization

  • Crowding, poor hygeine, poverty

    • Mortality 200-700 per 100,000 per year

• The “cause”

  • Constitutional weakness

  • Careless immoral living

• Consumption and culture

  • Chopin, Thoreau, Chekhov, the Brontes

  • Art & style

  • Verdi’s Violetta in La Traviata

• Transmitted by inhalation of coughed droplet nuclei

  • Cough generates thousands of bacteria

    • Dries while airborne and acan float well

    • <10 infecting dose

• Environmental survival enhances spread

  • Closed spaces, schools, jails, poor ventilation

  • Outdoor transmission less likely as susceptible to UV light

• HIV greatly increases risk of disease

• 20th & 21st centuries

  • Contained in developed world

  • 30,000 Deaths per week

  • Infects 1/3 of world

  • Threatened by multiresistance

Survival in Macrophages: Mycobacterium

• M. tuberculosis

  • Inhibits lysosome/phagosome fusion

  • Inhibits of phagosome acidification

  • Coat proteins on phagosomes to prevent lysosome delivery

• Inability to clear infection leads to granuloma formation

  • Granuloma forms in an attempt to wall off the infection from further dissemination

• Granulomas can necrotize and burst leading to inflammation and tissue damage resulting in coughing up blood

Tuberculosis: Pathogenesis

• Primary TB

  • 2-stage battle

  • Inhalation → lymphatics → Systemic dissemination → Primary disease (pulmonary)

  • <10% of immunocompetent people get the disease over their lifetime

• Latency (latent TB)

  • Prolonged dormant state

  • Reactivation (Reactivation TB)

    • Months to decades later

    • Usually never (<10%)

  • MTB widely distributed

    • In healed granulomas or solitary

  • Mechanism unknown

    • ?Oxygen, nutrient deprivation

    • ?Cell wall aids survival

• Lots of immunology involved in TB

• REACTIVATION

  • Mechanisms Unknown

    • Specific factors (genomic evidence)

    • Waning immunity

  • Lung upper lobes most common

    • Well aerated, high O2 tension

    • Cavity formation

• Injury

  • Progressive delayed-type hypersensitivity (DTH) destruction

    • Inflammatory reactions initiated by mononuclear leukocytes 48-72 hours post antigen exposure

  • Chronic inflammation

  • Caseous necrosis

  • Pore forming toxin is Esx-1

TB Clinical Aspects: Diagnosis

• Tuberculin skin test

  • Intradermal MTB proteins (Purified protein derivative)

    • >15 mm — 48-72 hrs

  • Demonstrates DTH

    • Resolved infection (including treated)

    • Latent infection

    • Active disease

    • Bacillus Calmette-Guerin (BCG) immunization (<15 mm)

    • Infection with other mycobacterial species (<15 mm)

• QuantiFERON - Gold test (Blood test)

  • Collect patient’s T cells

  • Detect release of MTB-specific IFN-gamma

    • Interferon: Immune signaling protein (typically for antiviral)

  • More specific for MTB than PPD

    • Not + after BCG immunization

    • Less frequently + with other mycobacteria

  • BUT - No better indicator of active TB than + skin test

• Direct Smears

  • Acid fast

  • Flurochrome

  • 65% sensitive (sputum & CSF concentrates)

• Culture

  • Sputum treated & concentrated

  • Growth on agar plates - 3+ weeks

  • Automated methods (radiometric, fluorometric) - 15 days

  • Identification takes weeks more

  • Isolation required for susceptibility testing

• NAA

  • Commercial Xpert MTB/RIF system

    • Detects MTB & rifampin resistance

    • Less sensitive than culture but better than AFB smear

    • Used in high prevalence low resource countries

TB Clinical Aspects: Prevention

• Public Health Measures - USA

  • Routine PPDs or chest radiographs

  • Chemoprophylaxis of skin test converters

    • To prevent reactivation

    • Single drug (isoniazid) for 6-9 months

• BCG vaccine - worldwide

  • Live attenuated strain of M. bovis

  • Performance variable, particularly in adults

  • Converts PPD

  • New recombinant versions - virulence genes added

Chlamydia trachomatis

• Obligate Intracellular Bacteria

• Genital infection - like gonococcus

  • Trachoma → blindness

• Major cause of Sexually Transmitted Infection (STI)

• Small unicellular round (0.1-0.3 micrometer)

• Gram-negative (no peptidoglucan layer but has LPS)

• Shape is pleomorphic, neither cocci nor bacilli

• Metabolism

  • Protein synthesis

  • Must “borrow” energy sources from host cell

    • Amino acids

    • Nucelotides (ATP, NAD)

• Classification - 3 biovar groups (not serovar)

  • A-C : ocular epithelium → trachoma

  • D-K : urogenital —> urethritis, cervicitis, conjuncvitis

  • L1-L3 : lymphogranuloma venereum (LGV)

  • Biovar: differes physiologically and/or biochemically

  • Serovar: differe antigenically

Chlamydia trachomatis: Replication

• Elementary body

  • Compact, hardy, inert

  • Infectious

• Reticulate body

  • Lysosome fusion inhibition

  • Vesicle (inclusion) formation, fragile

  • RB → EB

  • Lysis, EB release

Chlamydia Epidemiology: Genital

• STD biovars D-K

  • Transmitted by contact with genital secretions

    • Pattern similar to gonorrhea

  • Urethritis, cervicitis

  • >4 million US cases/year

    • Higher proportion of men asymptomatic vs. GC

• LGV biovars L1-3

  • Minor skin breaks

  • Spread to regional lymph nodes

  • Tropical countries

Chlamydia Epidemiology: Eye

• Inclusion conjunctivitis

  • STD biovars

  • Transmission during labor : 30-50% risk

• Trachoma

  • Direct contamination of conjunctiva

    • Genital - during birth

    • Hand-eye

    • Repeated reinfection → chronic conjunctivitis

• Major cause of blindness worldwide

Chlamydia Pathogenesis

• EB has epithelial cell tropism

  • OMPs mediate attachment

  • Tropism varies with biovar

  • Enter by endocytosis, convert to RB ad replicate → inclusion

• Injury - LPS, inflammation, scarring

  • Eye - chronic follicular conjunctivitis

  • Acute genital - exudate (less acute than GC)

  • Chronic genital - salpingitis (PID)

  • LGV - inguinal adenopahty, drainage

Chlamydial Protease-like Activity Factor (CPAF)

• Injects serine protease, CPAF, into cytoplasm

• Cleaves pro-apoptotic BH3 proteins that activate Bim/Bod, Bad

• Inhibits cytochrome C release

• Prevents apoptosis

Chlamydia Clinical Aspects: Manifestations

• Trachoma

  • Corneal scaring over 15-20 years

• Genital

  • Discharge

  • Urethritis, cervicitis

  • Epididymitis, salpingitis (tube that carries sperm and fallopian tubes)

• Inclusion conjunctivitis

  • 1-2 weeks after birth

  • Watery then mucus discharge

Chlamydia Clinical Aspects: Diagnosis

• Microscopy - eye infections only

  • Requires epithelial cells (not pus)

• Culture, immunoassay - no more

• NAA RPR - for genital disease

  • Rapid, sensitive, specific

  • Urine

  • Combined with GC

Chlamydia Clinical Aspects

• Treatment

  • No beta lactams (no peptidoglycan)

  • Azithromycin, doxycycline, fluoroquinolones

• Prevention

  • Public health, case tracing

Spirochetes

• Examples

  • Treponema pallidum - Syphilis

  • Leptospira interrogans - Leptospirosis

  • Borrelia recurrentis - Relapsing Fever

  • Borrelia burgdorferi - Lyme Disease

• Structure

  • Fibrils wound around peptidoglycan

  • Covered with bilayer sheath similar to outer membrane

  • Lengths between 3 and 500 μm

  • Corkscrew spirals, close spirals with hooked ends, loose spirals

• Staining

  • Some too thin or take strains poorly - use dark field

  • Others visible on routine blood smears (Borrelia)

• Reservoir

  • Most are zoonotic or venereal

• Motile

  • By flexion of fibrils

Borrelia burgdorferi - Lyme DIsease

• Epidemiology

  • Complex tick-mouse-deer cycle

  • Deer essential for tick maturations (no deer, no disease)

  • Humans infected by tick bite in wooded area (we step into life cycle)

  • Eastern USA

  • Fastest-growing vector-borne infection in the US

  • 30,000 cases of Lyme disease are reported to CDC per year

  • Arizona : 8-12/yr

Lyme Disease Pathogenesis

• Infecting dose is <20 organisms

• Some OMPs undering antigenic variation

• OMP class called outer surface proteins (Osp)

  • Expression regulated by temperature, pH

  • Tick - OspA

  • Tick Feeding

    • Producing OspA transitions to OspC for human transmission

  • OspC protects from macrophage ingestion and antibodies

• Cell wall peptidoglycan, outer membrane

  • Joint deposits → arthritis

• Modulation of immune responses

• No LPS

Lyme Disease Clinical Aspects

• Manifestations

  • Primary Infection

    • Erythema migrans (EM) rash ((a migrating rash))

    • Bullseye rash

    • Fever, arthritis, fatigue

    • Secondary skin lesions

  • Chronic infection

    • Relapsing arthritis, neuritis, carditis, autoimmune

• Treatment

  • Doxycycline, amoxicillin, cefuroxime

  • Response slow (2-3 weeks)

  • There is an antibody response, but target unknown

• Prevention

  • Clothing, tick removal in endemic areas

  • Cehmoprophylaxis - endemic area

  • Prophylaxis of doxycycline post tick bite

  • LYMErix

    • Directed at antigen in tick (OspA)

    • Was between 76 and 92% effective

    • Reports of adverse reactions (data not supported by FDA)

      • About the same time a certain Lancet article came out (and was soon retracted)

    • Lyme disease increased and doses went from1.5 million in 1999 to 10,000 in 2002

    • GSK discontinued

  • Prevent mice from getting infected through a mouse targetted vaccine

Lyme Disease Diagnosis

• Laboratory

  • Culture - Not Practical

  • Serologic Tests

    • Enzyme Immunoassay + Immunoblot for antigen

    • Lack sensitivity & specificity

    • Improving

• Caution

  • Chronic Lume diagnosis is difficult

  • Even harder outside primary geographic area (AZ)

Microbiome

• All Host-Associated Microbes Are Symbionts (With Variation)

  • Commensals: Colonize host and receive benefits from host

  • Mutualist: Its symbiosis with host benefits both

  • Pathobiont: Can colonize as a commensal to mutualist, but environment can drive towards pathogenic

  • Pathogens: Only interaction with host is pathogenic one

• Microbiome changes by location

• Microbiome changes over time

• The microbiome is defined as all the bacteria, viruses, fungi, archaea, and eukaryotes that inhabit the human body

• The microbiome is connected to everything

• Colonization resistance (Protection against pathogens)

  • Neisseria vs Neisseria

  • E. coli vs Salmonella

  • Fecal transplant vs. C diff

  • Tribal Warfare

    • Killing of N. gonorrhoeae by commensals through a DNA-dependent mechanism

16s Sequencing

• Used to determine the microbial composition

• rRNA = ribosomal RNA

• 16s rRNA is the component of the 30S small subunit of a prokaryotic ribosome