Pseudomonas aeruginosa: Infections, Pathogenesis and Lab Diagnosis

Pseudomonas aeruginosa is a gram-negative rod. Pseudomonas aeruginosa can resist high concentrations of salt, dyes, weak antiseptics, and many commonly used antibiotics. Most pseudomonads known to cause disease in humans are associated with opportunistic infections.  Pseudomonas aeruginosa and Pseudomonas maltophila are mostly responsible for disease conditions.  

This opportunistic pathogen may infect virtually any tissue. Infection is facilitated by the presence of an underlying disease. It is a major threat to hospitalized patients, particularly those with serious underlying diseases such as cancer and burns (burning causes breakdown of nonspecific host defenses).  

Sites of infection by P. aeruginosa

1. Central nervous system infections
2. Localized infections of the ear and sinus
3. Skin and musculoskeletal tissues, burn wounds, surgical wounds
4. Respiratory tract: chronic infections in cystic fibrosis patients, acute pneumonia in other patients
5. Bacteremia
6. Endocarditis
7. Urinary tract infections

Mortality in P. aeruginosa infection

Infections with P. aeruginosa are associated with a high mortality rate. This is because of the combination of

1. Bacterial resistance to antibiotics
2. Weakened host defenses
3. Production of extracellular bacterial enzymes and toxins.

Pathogenesis of P. aeruginosa

Pseudomonas aeruginosa produces many factors that may contribute to its virulence. Some of them are

1. Hemolysins: Glycolipid hemolysin may play a role in P. aeruginosa pulmonary infections.
2. Extracellular polysaccharides: They may impede phagocytosis and impair the diffusion of antibiotics and thus facilitate colonization and persistence. These mucoid strains usually are isolated only from patients with cystic fibrosis.
3. Pigments: Pyocyanin (a phenazine pigment) and fluorescein are two common pigments produced by P. aeruginosa. Pyocyanin retards the growth of some other bacteria and thus may facilitate colonization by P. aeruginosa.
4. Extracellular protease: May play role in the formation of hemorrhagic lesions and tissue destruction.

Pseudomonas species are a group of aerobic, non-spore-forming, Gram-negative rods found in the environment and soil. Only a few species are pathogenic, of which Pseudomonas aeruginosa is the most common. Pseudomonas aeruginosa is an opportunistic pathogen and causes urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections, and a variety of systemic infections, mostly in immunosuppressed patients.

Due to its ability to survive under a variety of environmental conditions, it easily colonizes surgical instruments, catheter tubes, respiratory ventilators and is transmitted from patient to patient in healthcare facilities leading to nosocomial infections.

Laboratory Diagnosis

In the laboratory, P.aeruginosa can be isolated from any sample (urine, pus, blood, ear swabs, tissue biopsies, body fluids, etc.)
They grow well on standard broth and solid media such as blood agar, chocolate agar, and MacConkey agar, which are recommended to isolate Pseudomonas species from clinical specimens. Selective agar-containing inhibitors such as cetrimide can also be used for isolation and presumptive identification.

Colony morphology:

• On MacConkey agar colonies of P.aeruginosa are flat, 2-3 mm, smooth, non-lactose fermenting colonies with irregular margin (leafy margin) and slightly pigmented (greenish pigmentation).
• On Blood agar: It is documented that, P. aeruginosa isolates may produce three colony types on blood agar depending on the source of isolation.
  • Natural isolates from soil or water typically produce a small, rough colony.
  • Clinical samples, yield one or another of two smooth colony types. On blood agar, Pseudomonas appear as give large colonies with metallic sheen, mucoid, rough, or pigmented (pyocyanin), and often β-hemolytic
    • One type has a fried-egg appearance which is large, smooth, with flat edges and an elevated appearance.
    • Another type, frequently obtained from respiratory and urinary tract secretions, has a mucoid appearance, which is attributed to the production of alginate slime.

• Cetrimide Agar is used as a selective medium for the isolation of Pseudomonas aeruginosa from pus, sputum and drains, etc. Pseudomonas aeruginosa produces yellow-green to blue colonies and fluoresces under UV light.

Gram stain

Pseudomonas aeruginosa is a Gram-negative rod measuring 0.5 to 0.8 µm by 1.5 to 3.0 µm.

Biochemical characteristics

1. Pseudomonas aeruginosa is often preliminarily identified by its typical odor in vitro. The smell is described as grape-like, tortilla-like, or “Philadelphus coronarius-like” (production of aminoacetophenone).
2. Catalase-positive
3. Rapid oxidase-positive within 10 seconds (exception P. luteola and P. oryzihabitans)
4. Motile by means of one or more polar flagella.
5. It is not an active fermenter of carbohydrates and produces acid, but no gas, in glucose and is lactose-negative. Alkaline slant/alkaline deep (K/K) reaction in TSI or KIA agar.
6. Few strains of P. aeruginosa secret a variety of pigments, including pyocyanin (blue-green), pyoverdine (yellow-green and fluorescent), pyomelanin (brown to black), and pyorubin (red-brown).
7. Strict aerobic respiratory metabolism with oxygen but in some cases, nitrate has been used as an alternative that allows anaerobic growth.
8. Can grow in temperatures up to 42°C.The combination of pyocyanin production and the ability to grow at 42°C is sufficient to distinguish P.aeruginosa from other Pseudomonas spp. (e.g., P.fluorescens, P.putida, P.stutzeri, P.putrefaciens).

All strains of P.aeruginosa may not produce Pyocyanin.

Biochemical Test	Result
Catalase Test	Positive
Oxidase Test	Positive
Motility	Motile with one or more polar flagella
Lactose fermentation	Positive
Glucose Fermentation	Positive
Other carbohydrate fermentation	Non-fermentatibe
Indole Test	Negative
VP Test	Negative
MR Test	Negative
Citrate test	Positive
Urease test	Negative
Nitrate Reduction	Positive
H2S Production	Negative
Pigment production	Positive

Distinguishing Characteristics from Other Species

Species	Pyocyanin production	Fluorescein Production	Growth at 42°C	Arginine dehydrolase	Lysine decarboxylase	Gelatin liquefaction	Aesculin hydrolysis	Lactose utilization (oxidative)	Maltose utilization (oxidative)	Reduction of nitrate to nitrite
P.aeruginosa	+	+	+	+	–	+	–	–	–	+
P.fluorescens	–	+	–	+	–	+	–	–	–	–
P.putida	–	+	–	+	–	–	–	–	–	–
P.maltophila	–	–	+	–	+	+	+	–	+	–
P.stutzeri	–	–	+	–	–	–	–	–	+	+
P.cepacia	–	–	+	–	+	+	+	+	+	–
P.pseudomallei	–	–	+	+	–	+	+	+	+	+
P.mallei	–	–	+	+	–	+	*	*	+/-	+

References and further readings

• Public Health England. (2015). Identification of Pseudomonas species and other Non-Glucose Fermenters. UK Standards for Microbiology Investigations. ID 17 Issue 3. https://www.gov.uk/uk-standards-for-microbiology-investigations-smi-quality-and-consistency-in-clinical-laboratories 
• Mackie and McCartney “Practical medical Microbiology”21st edition, Churchill Livingstone
• Image source: https://www.microbiologyinpictures.com/