Gram Negative Cocci and Rods

Neisseria

• Aerobic Gram-negative diplococci

• N. meningitidis & N. gonorrhoeae are closely related

• Outer membrane, OMPs, LPS (LOS of lipooligosaccharide is a variant)

• Pili

• Oxidase positive GNC = Neisseria

Neisseria gonorrhoeae

• Gonococcus - gonorrhea

  • Genital pathogen - never considered microbiota

  • Disease is primarily localized at or adjacent to genital mucosa

  • Direct extension to adjacent structures (fallopian tube) is a complication

  • Bloodstream invasion is uncommon

  • Causes 2nd most common STD in US

    • 468,000 reported, 820,000 suspected cases

    • Worldwide: >100 million new cases/year

  • Often a co-pathogen with Chlamydia

• 60-90% of infections are asymptomatic transmission

• Mild symptoms in women often undetected

  • PID (pelvic inflammatory disease), ectopic pregnancy, sterility

• Ngo enhances HIV transmission

• Vaccine development - a gaping hole

• Antibiotic resistance outstrips pace of drug development - EXTREMELY Competent

Gonococcus

• Growth

  • Fragile - drying, pH, temperature

  • Fastidious aerobe (complex nutritional requirement)

    • Cannot live outside the body

• Structure

  • No capsule, no serotyping system

  • Pili - multiple antigenic types

  • Outer membrane proteins (OMP)

    • OMPs (PorA, PorB)

    • OPAs (opacity-associated proteins)

Gonorrhea Epidemiology

• Women

  • Urethritis

  • Cervicitis

  • Salpingitis (PID)

• Men

  • Urethritis

  • Epididymitis

• Gonococcus

  • Strict human pathogen

    • Asymptomatic “carriers'“

  • Sexual transmission

    • Mucosal contact

    • Sexual intercourse carries 20-50% attack rate

Gonorrhea Pathogenesis

• Attachment

  • Microvilli of non-ciliated cells

• Pili

  • Epithelium

  • Sperm

  • Fallopian tube

• OPAs

  • Epithelium

  • Gonococcal cell-cell

• Invasion

  • Endocytosis

  • OMPs mediate

    • Porins are OMPs NOT exotoxins

  • Submucosa

    • Sticky packets

    • Opas

    • ? Infectious unit

• Evasion

  • No capsule but… many Neisseria species fo have capsules and they AREN’T pathogenic to humans

  • Antigenic variation

• Process

  • Pili attach to surface microvilli

  • Endocytotic vacuole

  • Escape to submucosa

  • Spread locally

  • Bacteremia uncommon

Gonorrhea Clinical Aspects

• Gram Smear

  • Men - Diagnostic (CDC)

• Women

  • Less sensitive

  • Less specific due to other flora

• Culture - specialized medium

  • Genital

    • Men - Urethra

    • Women - cervix (+ anal)

  • Non-genital - throat, notify lab

  • Blood (but rare)

  • NAA-PCR

    • Sensitivity & specificity comparable to culture

    • Packaged with Chlamydia

Gonorrhea Treatment/Resistance

• Altered Target

  • MIC 0.1 mcg/ml → 4.0 mcg/ml

• Enzymatic Inactivation

  • E. coli beta-lactamase - Vietnam

  • MIC >64 mcg/ml

• Now 3rd generation cephalosporins

Gonorrhea Prevention

• Block Transmission - (condoms, foams)

• Ophthalmia neonatorum

  • Erythromycin ointment

• Case Tracing

  • Find asymptomatic patients

  • No serologic test

• No vaccine in sight

Enterobacteriaceae

• “Large” Gram-negative rods/coccobacilli

• Biochemical tests

• Antigenic structure

  • O = cell wall LPS

    • A repetitive glycan polymer

    • O antigen

    • >160 in Escherichia coli alone

  • K = surface polysaccharide capsule

  • H = flagellar protein

• Oxidase negative

• Opportunistic Pathogens

  • Symptoms

    • Urinary tract infection

    • Wounds

    • Soft tissue infections

    • Pneumonia

    • Meningitis

  • Pathogens

    • Escherichia

    • Klebsiella

    • Enterobacter

    • Proteus

    • Serratia

    • Providencia

    • Yersinia

• Enteric Pathogens

  • Symptoms

    • Watery diarrhea

    • Dysentery

    • Enteric fever

  • Pathogens

    • Shigella

    • Salmonella

    • Escherichia

    • Yersinia

• Nonpathogenic

Enterobacteriaceae Epidemiology

• Human microbiota

• Animal flora (and disease)

• Environment (water + carbon source)

• Adapted to mammals, reptiles, birds

• Adapted to humans

  • Salmonella ser. Typhi

Escherichia coli

• O/H/K serotypes

  • O157:H7

  • O111:H12:K1

• Pili

  • Type 1 (common)

    • Bind to mannose

  • Specialized

    • CFA - enterocyte (intestinal cell)

    • BFP - enterocyte

    • P - uroepithelium

E. coli Exotoxins

• α hemolysin - pore forming

  • Works like streptolysin O (which bacteria?)

• Shiga toxin (Stx)

  • A/B toxin

  • B binds surface glycolipid

  • A modifies ribosomal RNA

  • Blocks protein synthesis (similar to ricin)

• Enterotoxins

  • Labile toxin (LT)

    • cAMP like cholera toxin

    • Less potent

  • Stable toxin (ST)

    • cGMP

    • Mechanism still like cholera toxin

E. coli: UTI (UPEC)

• Opportunistic Infection

• E. coli gains access to normally sterile bladder

• Disease ranges from simple bladder infection (cystitis) to renal pelvis and kidney infection (pyelonephristis)

  • Frequency, dysuria

  • Fever

  • Flank pain, bacteremia

  • Endotoxic shcok

E. coli UTI Epidemiology

• Among the most frequent of all infections

  • Women in sexually active years

  • Linked to perineal-intestinal flora

• Structural defects of urinary tract

  • Men - prostatic hypertrophy → obstruction

• >90% of UTIs are E. coli

E. coli UTI Pathogenesis

• Adherence

  • Type 1 pili — 90%

  • P pili — gal-gal receptor (upper tract uroepithelium)

    • Stool 20%

    • Cystitis 40-50%

    • Pyelonephritis >70%

• Perineal Colonization

  • Type 1 pili

• Displacment

  • Minor trauma

  • Intercourse

  • Cathreters, obstruction

• Uroepithelial Adherence

  • Type 1 pili

  • P (gal-gal) pilli — upper tract

• Other Factors

  • Motility — upper tract

  • Superficial invasion — urine flushing

• Injury

  • α-hemolysin

  • LPS endotoxin

  • Bacteremia

E. coli Diarrhea

• Nursery Outbreaks - list of “enteropathogenic” serotypes

• Traveler’s diarrhea

  • Vacations to developing countries

  • Military maneuvers

• Foodborne Outbreaks

  • Hamburger

  • Spinach, sprouts

  • Unpasteurized juices

• Pathogens

  • Enterotoxigenic E. coli (ETEC)

  • Enteropathogenic E. coli (EPEC)

  • Enterohemorrhagic E. coli (EHEC)

  • Enteroaggregative E. coli (EAEC)

  • Enteroinvasive E. coli (EIEC)

EHEC Diarrhea Epidemiology

• Animal source (cattle)

• Foodborne

• Low infecting dose - 200 bacteria

• Outbreaks, industrialized countries - O157:H7

• Sources

  • Hamburger meat - cooked rare

  • Milk & fruit drinks - unpasteurized

  • Vegetables

  • Sprouts

• Bloody diarrhea

• 10% hemolytic uremic (HUS) syndrome (5% mortality)

EHEC Dysentery Pathogenesis

• O157:H7

• A/E lesions - secretion

  • Attaching and effacing

  • Large intestine

• Attacks large intestine

• A/E lesions - secretion Cytoskeleton disruption

• Stx production - hemolytic uremic syndrome (HUS)

E. coli Clinical Aspects

• Manifestations - Opportunistic

  • UTI

    • Cystits - frequency, dysuria

    • Pyelonephritis fever, flank pain

  • Wounds, pneumonia, etc.

  • Gram-negative sepsis

    • Bacteremia

    • LPS endotoxin

  • EHEC - bloody diarrhea, 3-10 days

    • 10% develop HUS (5% mortality)

• Diagnosis

  • Opportunistic

    • UTI

      • Gram stain, unspurn

      • Quantitative cuture

    • Wounds, blood, etc. - culture

  • Enteric

    • Screening agar - sorbitol

      • O157:H7 fails to ferment

      • Confirm serologically

  • PCR - ETEC, EPEC, EHEC, EIEC, EAEC

• Treatment

  • UPEC UTI - antibiotics guided by susceptibility testing

  • Diarrhea

    • Support rehydration - antimotility agents contraindicated for EHEC

    • Antibiotic contraindicated for EHEC - release of Stx

    • EHEC → hemodialysis, hempheresis

• Prevention

  • Travel - “Don’t drink the water”, cooked foods

    • Chemoprophylaxis - high risk persons

  • EHEC

    • Hamburger cooked through

    • Irradiation of meats, pasteurized juices

Yersinia pestis

• Member of the Enterobacteriaceae

• Pleomorphic (shape shifter) with bipolar staining

• Adhesin similar to invasins of Shigella

• Yersinia outer membrane proteins (Yops)

• Non-motile

• Non spore forming

• Facultative anerobe

• Facultative intracellular

• Catalase

• Virulence plasmids

  • F1 antigen - protein capsule

  • Multiple enzymes

Yersinia pestis- Virulence factors

• F1 antigen — Prevents phagocytosis

• LPS — Not recognized by TLR (lacks O antigen)

• Type III secretion system — Injects Yops

• Yop effector proteins — Disrupting actin cytoskeleton of phagocytes

• Adhesins — Adhesion

• YadA — Adhesion

• Yap — Adhesion

• Coagulase — Converts (soluble) fibrinogen to (insoluble) fibrin

• Ymt — Murine toxin

• Invasins

• AIL Proteins — Adhesion, invasion, aggregation, Yop delivery

• PsapH6 — Binds Fc receptor human IgG

• Pla protease — Invasion, dissolves fibrin, digest laminin

Plague Epidemiology

• Black Death - Middle Ages

  • 25M deaths 1346-50

  • Pandemics into 20th Century

• Disease of Rodents

  • Rats, prarie dogs

  • Transmitted by flea bite

    • Fleas leave dead host

  • Humans “step into” the cycle

• Flea bite leads to infection lymph nodes called bubo

• Bubonic plague

  • 50-75% bacteremia

  • 5% bacteremic pneumonia

• Pneumonic plague spreads human to human

• Sylvatic plague

  • Rodent-rodent in the wild

  • Direct human contact—prarie dog/flea

  • Plains of western US

    • Arizona (4 corners)

    • US 1-17/year, 40 in 1985

    • 2015 - 14 cases, 4 deaths

• Urban plague

  • Rodents migrate to cities - famine, cold

  • Rats are the dominant rodent in cities

    • Rat-flea-rat transmission

    • Fleas leave dead rat seeking another host

    • Might find a human instead

  • Human flea contact usually occult

  • Bubonic → pneumonic

  • Madagascar

    • 400 cases/yr

    • 2017 - 1800 cases, 127 deaths

• FLEA BIT → BUBONIC

• BUBONIC → BACTEREMIA → PNEUMONIC

Plague: Pathogenesis

• Two Sets of Virulence Factors

  • In the Flea (20-28 C)

    • Y. pestis multiplication

      • Proventriculus blockage
        

    • Coagulase, biofilm

    • Flea regurgitates bacteria into bite wound

  • In humans (35-37 C)

    • F1 capsule - antiphagocytic

    • Yops - type III secretion system

    • Synthesizes LPS not recognised by TLRs

    • Plasminogen activator (Pla) - facilitates spread

Plague Clinical Aspects

• Manifestations

  • Bubonic plague

    • Bubo - 2 to 7 days from flea bite

      • Fever, local pain

    • Untreated 50-70% die in bacteremic shock

  • Pneumonic plague

    • 2 to 3 days from respiratory exposure

    • Cough, sputum, dyspnea, cyanosis = black death

    • 100% fatal untreated (2-3 days)

• Diagnosis

  • Smears, culture

  • DFA in reference laboratories

  • F1 antigen detection test used by WHO in field

• Treatment

  • Streptomycin (gentamicin) ± doxyxycline

  • Bubonic plague - <10% mortality

  • Pneumonic plague - 10-20% mortality

• Prevention

  • Doxycycline chemoprophylaxis

  • No vaccine

  • Stay away from dead animals

Plague 5,000 Years Ago

• Evolutionary biologists & anthropologists 2015

  • Y. pestis genome detected in Eurasion Bronze Age teeth

  • Human virulence genes present

  • Flea virulence genes absent

  • 3,000 years later

    • Endemic disease → Epidemic disease

    • Flea genes present