The Gram Negative Rods II - Pseudomonas, Legionella, Salmonella

Pseudomonas aeruginosa

• Overview

  • Among the most deadly of bacterial infection

  • Opportunistic infection at variable sites

  • Confined to the immunocompromised and debilitated

  • High level of resistance to antimicrobials

• Aerobic, oxidase positive

• Pigments (yellow, blue, rust, fluorescent)

  • Pyocyanin (blue) unique to P. aeruginosa

• Exopolysaccharide (alginate) polymer can form glycocalyx or biofilm

• Non spore forming

• Polar type IV pili

• Catalase positive

  • Breaks down hydrogen peroxide

• Colonies are oxidase positive

  • Has cytochrom oxidase and prefers aerobic (but can grow slowly anaerobically)

• Exotoxins

  • Exotoxin A or ExoA (can be induced by low iron levels)

    • ADP-ribosylates Elongation Factor-2 (same mechanism as in DT, but much different in manifestation)

  • Other enzymes (toxins)

    • Phospholipase, collegenase, elastase

P. aeruginosa Disease Epidemiology

• Extremely hardy-free living in water, soil, plants

• Survives at 42 C (High GC content)

• Throat, stool microbiota - up to 10%

• Colonizes patients with cystic fibrosis (CF)

• Contaminates medical environment

  • Respirators

  • Contact lens solutions

  • Medications

  • Even some disinfectants

P. aeruginosa Disease Pathogenesis

• Access via contamination of vulnerabe site, solution

• Immune compromise + antimicrobial resistance

• ExoA - no systemic action like DT

• Quorum-sensing

  • Bacteria secrete signal chemicals

  • ExoA produced when critical mass reached

• Cystic fibrosis (CF)

  • Alginate biofilm/glycocalyx (major virulence in CF)

    • Regulatory gene mutations allow overproduction of polymer

    • Limits access of complement, phagocytes, antimicrobials

    • Quorum-sensing activated by high ossmolarity of CF secretions

  • Mutations in the CFTR gene blocks CFTR protein function

    • Airways are dehydrated and cannot clear mucus

Biofilms

• Safety in numbers

• Van Leeuwenhoek credited for the discovery of microbial biofilms

• Alginate production triggered by quorum sensing

• Also full of DNA

• Alginate sticks to itself

• Prevents the need for adherence because the biofilm is sticky enough (mucoid)

• Immune factors can’t penetrate it

• Maintains population moisture

• Oxygen exchange?

P. aeruginosa Clinical Aspects

• Manifestations

  • Wound, burn, UTI, eye, hot tubbs, swimmers’ ear

  • Environmental contamination

  • Immunocompromised

  • Vascular → Necrotic lesions (ecythyma gangrenosum)

• Diagnosis

  • Culture

• Treatment

  • “Wild type” multiresistant to most antimicrobial classes

  • Outer membrane porins are relatively impermeable

  • Anti-pseudomonas drugs are prized (newer aminoglycosides, cephalosporings, etc.)

• PA exoproducts enhance MRSA biofilm resistance to vancomycin, the leading antibiotic against MRSA as well as other antibiotics

• PA changes SA metabolism which makes it more resistant (not a mutation but an adaptation)

Legionella pneumophila

• Overview

  • GNRs named for outbreak at American Legion convention

  • Commonly found in the environment

  • Prefer environment inside human macrophages or amoebas

• Staining

  • Stain poorly or not at all by common methods

    • Silver impregnation, immunoflorescence

  • Thin GNRs once isolated in culture (environmentally pleomorphic)

• Growth

  • Require L-cysteine, ferric ions

  • pH optimum 6.9

  • Aerobic

• Classification

  • 50 species - most do not cause human disease

  • L. pneumophila, multiple serogroups (16)

    • Serogroup 1 (Philadelphia strain) still most common

Legionnaires’ Disease Epidemiology

• 1976 Philadelphia Outbreak

  • The death toll in the outbreak of the mysterious respiratory disease in Philadelphia rose from 2 to 25 as medical detectives accelerated efforts today to seek a chemical or poison as the possible cause

• Legionella widely present in domestic and wild aquatic environments

  • Live in association with algae and inside amoebae

    • Protozoan Priming makes bacteria up to 100x more virulent

  • Dormant states occur in environment

• Facultative intracellular parasite

  • Capable of growing/replicating within amebae//macrophages

    • Survival out of macrophages

• Aerosol production allows transmission to humans by inhalation

  • Cooling towers

  • Grocery market mist sprayers

• Human-human transmission not demonstrated

• Attack rate <5% of those exposed

• Hospital Outbreaks

  • Immunocompromised patients

  • Faucets, showers, etc.

  • Biofilms, dormant nutrient-restricted organisms

  • Institutional disinfection

    • Old pipes, scale, etc.

    • Hot water, chlorine

    • Silver, copper ions

• The Flint Crisis (It’s in the water)

  • Flint, Michigan is known for having the third worst Legionella pneumophila outbreak in United States History

    • 87 infected, 12 dead

  • The bacteria was found in kitchen faucets, showers, air conditioning units and hot water heaters

  • Likely underdiagnosed at they used antigen test to diagnose, but this was serotype 6, not 1

Legionnaires’ Disease Pathogenesis

• Alveolar macrophage

  • Attachment - flagella, pili,, other proteins

  • Legionella containing vacuole (LCV)

    • Ribosomes, mitochondria, ER

    • Inhibits lysosomal fusion

  • M-phage death, enzymes, injury, spread

  • Process similar in amoebas

Legionnaires’ Clinical Aspects

• Manifestations

  • Severe toxic pneumonia

  • Dry cough, scant sputum

  • 10-50% mortality

  • Confined to lung

• Pontiac fever

  • Self-limiting febrile illness

  • Probably endotoxin or immune reaction

• Despite treatment, 5-30% of patients will die of complications with Legionnaire’s Disease in a given year (~18,000 infected annually in US)

  • Many patients complete treatment in long intensive care units

  • Persistence of fatigue (75%), neurologic symptoms (66%) and neuromuscular symptoms (63%) in survivors

• Diagnosis

  • High quality specimen - aspirate, BAL, biopsy

    • Direct fluorescent antibody (DFA) - 50%

    • Culture - special medium (BCYE) 2-5 days

      • L-cysteine, ferric ions

      • Low pH (6.9)

    • Antigen test but only for Serotype 1

    • NAA

• Treatment

  • Beta-lactamase producer

  • Erythromycin - original outbreak

  • Also - azithromycin, fluoroquinolones

• Prevention

  • Environmental control

Salmonella

• Biochemical Rxns

• Antigenic Structure

  • O, H, K

• Phase variation of H

  • O:1, 2, 12, H:i, 1, 2

• S. enterica

  • Serotypes → 1000

  • Typhi, Typhimurium, Enteritidis, etc.

Salmonellosis Epidemiology

• Fecal-oral transmission

  • Relatively high infecting dose — 10^4-10^6

  • #2 foodborne infection to Norovirus #1

• Food and water - “food poisoning” (gastroenteritis)

  • S. ser. Typhi - strictly human (others adapted to animals)

    • Convalescent cases (during recovery)

    • Chronic carriers - “Typhoid Mary”

    • Sanitation system failures (more a problem of modern food delivery services)

  • Other serotypes - animal sources

    • Poultry, exotic pets, raw milk, eggs

    • Picnics, restaurants, Thanksgiving turkey, etc.

    • Multistate outbreaks

Salmonellosis Pathogenesis

• Salmonella serotypes

  • Surface adhesin(s) react with a host cell receptor

    • Stimuates localized filamentous actin cytoskeletal rearrangement (membrane rfuffling)

  • Type III secretion system inject multiple effector proteins into host cells

  • Invasion

    • Local

    • Bloodstream

    • Variable with Serotype

Enteric (Typhoid) Fever Pathogenesis

• Salmonella ser. Typhi

  • Capsular Vi (virulence) antigen lowers infecting dose to ~10^4 organisms

  • Invasion

  • Survival in macrophage

    • Inhibit oxidative burst

  • Seeds bloodstream, other organs

Salmonella Manifestations

• Gastroenteritis

  • Nausea, vomiting, diarrhea 24-48 hours after ingestion

  • Can lead to…

    • Bacteremia

      • Can lead to…

        • Enteric (Typhoid) Fever

Typhoid Fever

• Fever - stepwise, lasting weeks

• Bacteremia starts disease

  • Bowel reseeded

  • Other organs

• Hemorrhage and perforations

Salmonella Diagnosis

• Routine Stool Culture

  • Selective medium (Hektoen) is routine

• Blood Culture

  • Salmonella Serotypes - Variable

  • Salmonella ser. Typhi

    • Primary diagnostic test

    • Stool culture may be negative

• NAA-PCR

  • Stool panel includes Salmonella, Shigella, Campylobacter, E. coli (ETEC…) and others

Salmonella Treatment and Prevention

• Salmonella gastroenteritis

  • Self-limiting, antibiotics may prolong carrier state

  • Children - Lower threshold to Rx

• Typhoid fever

  • Antibiotics Essential - Resistance requires susceptibility testing

  • Prevention

    • Don’t leave food out

    • Typhoid fever vaccine for Vi polysaccharide (needs boosters)