Pseudomonas

Pseudomonas in general

• Large, diverse genus within the gammaproteobacteria

  • Same group as Escherichia, Salmonella, and Vibrio

• Gram negative rods, frequently flagellated

• Found in huge range of environments and climes

  • Water, soil, air

  • Reportedly the most common nucleator of ice crystals in clouds

• Includes plant pathogens (P. syringea) soil buds (P. putida) and plant commensals (P. fluorescens)

• By and large do not cause disease in humans

• Pseudomonas species are frequently encountered as contaminants in the clinical laboratory

Pseudomonas species as opportunistic Pathogens

• A tiny fraction of Pseudomonas species are human opportunistic pathogens

  • Opportunistic pathogens typically do not cause disease in healthy individuals

    • Normally commensal or not found in the microbiota

  • Infect individuals with a weakened immune system or altered microbiota

    • Exploit vulnerable individuals for their own gain

• Some Pseudomonads fall into this category

  • P. aeruginosa

  • P. luteola

  • P. plecoglossicida

  • P. stutzeri
    

P. aeruginosa General Identification

• Long, narrow GNR

• Oxidase +

  • Differentiates from Enterobacteriaceae

• Grows well at 42°C (optimally at 37°C)

  • Differentiates from other Pseudomonas

• Characteristics:

  • Non-lactose fermenter

  • Non-spore forming

  • Beta hemolytic

  • Aerobic (obligate)

  • Motile (single, polar flagellum)

“Give aways” : Pigmentation and Smell

• Characteristic Smell

  • Grape or corn chips

• “Metallic sheen” on blood agar

• Pigment production

  • Pigments serves as redox-active virulence factor, iron sequestration, and quorum sensing

  • Pyocyanin (blue, unique to P. aeruginosa) and pyoverdin (yellow-green) are typically expressed together = greenish color

  • Sometimes pyorubin (red-brown) or pyomelin (dark brown) dominate

    • Different strains = different colors

• Pyoverdin fluorescence under UV light

P. aeruginosa is everywhere!

• “Ubiquitous” organism

• Can grow in water with minimal nutrients

• Has been isolated from 

  • Soil

  • Water

  • Animals

  • Plants

  • Sewage

  • Human skin
    

Classic Diseases Caused by P. aeruginosa

• Opportunistic cause of acute and chronic infections

• In healthy individuals:

  • “Hot tub folliculitis”

    • Aka P. aeruginosa folliculitis

    • Superficial infection of hair follicle root

    • Inoculation from contaminated water (frequently hot tubs or swimming pools)

    • Usually clears on its own, can treat with antibiotics

  • “Swimmers ear”

    • Aka Otitis Externa

    • Superficial infection of external ear canal skin

    • Inoculation from contaminated water

      • If waxing coating of ear is cut = easy entry

    • Topical antibiotics indicated

Classic diseases caused by P. aeruginosa

• In sick/elderly/immunocompromised individuals:

  • Urinary tract infections

  • Pneumonia (typically ventilator associated)

• In burn patients and uncontrolled diabetes

  • Wound infections

• In individuals with Cystic Fibrosis

  • Chronic pulmonary infection

  • Organism-disease association → “P. aeruginosa causes chronic pulmonary infection in CF patients.”

Cystic Fibrosis (CF)

• CF is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) protein

• Modulates flow of water and chloride out of cells into secretions

• Mutations results in inefficient pumping, causing thick mucinous secretions

• Major symptoms:

  • Pulmonary infections

  • Pancreatic dysfunction → poor nutrition

• 1:25,000 live births (in Caucasians)

• The most common, “fatal” genetic disease in the United States

• The most common inherited disease in United States Caucasians

Pathophysiology of Cystic Fibrosis Lung Disease

• Thick mucus builds up in airway →

• Colonized by bacteria

  • Causes chronic infection

  • Causes chronic inflammation →

• Causes progressive lung infection decline

• Baseline vs. “exacerbation”

  • An exacerbation means worsening of symptoms from baseline

  • Exacerbation = acute worsening of chronic disease

  • “A pulmonary exacerbation is an acute worsening of respiratory symptoms and lung function from a patient's baseline state due to increased infection and inflammation.

CF Patient Management

• Treatments typically include:

  • Antibiotic therapy to keep bacterial burdens in check

    • Inhaled vs. systemic antibiotics

  • Pulmonary hygiene

• In some cases “curative” measure can be employed

  • Lung transplant ← not ideal

  • CFTR modulator therapy ← pill (recent)

• Otherwise managed as a chronic disease

P. aeruginosa and Cystic Fibrosis (CF)

• Predominant lung pathogen in CF is P. aeruginosa

  • Infects > 80% of patients 18 years and older

• Major effector of chronic airway inflammatory response, correlated with poor clinical outcomes

• Individuals are typically colonized early in life

  • Presumably from the environment

• Typically the same infection P. aeruginosa strain persists in the lung for years or decades

  • Undergoes adaptive changes over time to better thrive in the CF lung

Study of P. aeruginosa Population Structure in CF Lung

• When organisms are spatially isolated, they can undergo genetic diversification and specialization

• The human lung is composed of physically discrete lobes

• What happens in CF?

This team took the lungs of CF patients undergoing lung transplant (3X:

• dissected them

• cultured out ~200 P. aeruginosa isolates per geographical region of the lung (~1,200 total per patient)

• confirmed isolate clonality by genomic fingerprinting

• subjected isolates from different regions to biochemical characterization

Lineages from different regions have distinct antibiotic resistance and metabolic profiles.

• Despite being descended from the same initial clones, isolates from different regions of the lung are phenotypically distinct!

• Subsets of isolates subjected to whole genome sequencing

• In isolated geographical regions of the lung, different genetic sub-lineages evolve over time

Huge amounts of evolution occur during CF colonization!

• 10 to 600 times the amount of yearly genetic divergence seen at baseline for P. aeruginosa

1. P. aeruginosa infects CF lung

2. P. aeruginosa populations become isolated

3. Isolated populations evolve independently and differ functionally

• Implications:

  • Is CF sputum a useful diagnostic specimen?

  • “One-size fit all” Antibiotic therapy valid?

Virulence Factors

• Virulence factors are genes expressed by organisms which contribute to their pathogenicity:

  • Colonization (attachement)

  • Evasion of the host’s immune response

  • Inhibition of the host’s immune response

  • Entry into and exit out of cells

  • Obtain nutrition from the host

• P. aeruginosa has a lot of these systems . . .

  • Iron sequestration (pigments!)

  • Exotoxins (release and scavenge material from human cells)

  • Proteases (tissue destruction & disruption of the epithelial barrier, tissue invasion)

  • Biofilm formation

  • Mucoid phenotype

Factor 1: Biofilm Formation 

• A biofilm is a multicellular collection of bacteria which form a 3-dimensional structure on a surface

• Held together by a matrix which is composed of secreted polysaccharides (alginate) > proteins > bacterial debris (DNA and protein)

• Predominant form of bacteria in the environment

  • May be mono- or multi- species
    

Stages of Biofilm Formation

1. Initial reversible attachment of free swimming microorganisms to surface

2. Same as step 1

3. Permanent chemical attachment, single layer

4. Same as step 3

5. Matrix productionGrowth of biofilm

6. Growth of biofilm

7. Mature biofilm with seeding/dispersal of biofilm clumps

8. Same as step 8

Competitive advantages in infection

• Favorable for bacterial persistence! → because. . .

• Increased resistance to host mediated killing

  • Inhibits complement activation, antibody opsonization

  • Decreases phagocytosis and killing

• Enhanced adherence

• Increased antibiotic resistance

Biofilms are inherently antibiotic resistant

• Same P. aeruginosa strain was grown as planktonic (-P) vs. biofilm (-B)

• In some studies up to a 1,000X increase in resistance observed with biofilm formation!

• Makes them extremely difficult to eradicate

Mechanisms of resistance in biofilms

1. Bacteria in biofilms undergo profound changes in gene expression

   1. Changes in gene expression influence metabolism and/or antibiotic resistance directly

2. The center core of biofilms are nutrient limited and thus metabolically inactive

   1. Provides a reservoir of dormant cells

   2. Re-seed / repopulate the biofilm

• Biofilms behave as a “superorganism”

Factor 2: “Mucoid” phenotype

• Persistent P. aeruginosa infections in CF nearly always progress to a mucoid phenotype

  • Important adaptive change in chronic infection

  • Associated with worse clinical outcomes

• Caused by overproduction of alginate (major polysaccharide in biofilms)

• Makes a capsule around the bacteria

Consequences of a Mucoid Phenotype

• “Protected microcolonies” = small numbers of bacteria have the same benefits as larger numbers of organisms that are organized into mature biofilms (i.e., step 8)

  • Bind some antibiotics

  • Protects organisms against host response

Genomics - The P. aeruginosa Genome

• PAO1 genome was sequenced in 2000, was the first Pseudomonad

• ~6.3 Mbp (largest bacterial genome sequenced at the time)

• ~5500 genes

• Huge number of catabolism, transport, efflux, and chemotaxis genes

• Give it lots of flexibility, antibiotic resistance potential

• Now, lots of different strains have been sequenced

• What about their genomic diversity??

Genetic differences among bacterial strains are greater than genetic differences among humans

• Single nucleotide polymorphisms (SNPs)

• Can change the coding sequence of genes, alter protein function

• SNPs are a large contributor to human (and bacterial) genetic variation

• Entire genes can be variably present or absent in different bacterial strains from the same species

The Concept of a Bacterial “Pan-genome:”

As you sequence more genomes from a species:

• Some genes are always found in that organism

  • “Core genome”

  • Genes that are essential to that species

  • Define what the organism really is, genetically

• Some genes are variably found in that organism

  • “Accessory genome”

  • Genes that may offer some advantage in some situations (antibiotic resistance, metabolic genes, etc.)

  • Define what an organism can do

• The Pan-genome comprises both the core and the entire accessory genome for the entire species.

  • Defines the entire possible genetic content of a bacterial species

  • The pan-genome is far larger than the genomic content of any single strain.

    • Ex. International Space Station

    • Accessory genome = bells and whistles

    • Core genome = life support functions

    • Pan-Genome = all of it (core + accessory)

Pan-genome of P. aeruginosa

Studies of large numbers of P. aeruginosa genomes (500-1,200)

• Accessory genome is ~13-49% of total gene content in any given strain

• Several hundred new accessory genes discovered with each new strain sequenced!

• Up to HALF of all genes are unique to the genome examined (i.e., “unique,” not seen in any other P. aeruginosa examined)

• Antibiotic resistance genes, metabolic genes, and virulence islands make up a big fraction of the accessory genome

In Summary

• P. aeruginosa is a ubiquitous opportunistic pathogen

  • Usually does not cause serious disease in healthy people but can be deadly immunocompromised or sick individuals

  • Has particular significance in Cystic Fibrosis

• Has a number of adaptive traits that enable it to successfully colonize and infect hosts

• Has a flexible and expansive pan-genome which aids its environmental and pathogenic success

• It is a worthy enemy