Pseudomonas aeruginosa : Morphology,Diagnosis and Cultural characteristics

Pseudomonas aeruginosa : Morphology,Diagnosis and Cultural characteristics

Pseudomonas is a bacteria mostly saprophytic in nature, found in soil, water and other moist environments. It has emerged as an important cause of associated health care and opportunistic infections.Most Pseudomonas clinical isolates are resistant to many antibiotics. Pseudomonas aeruginosa is also a plant pathogen. Pseudomonas is a strict aerobic, motile Gram negative bacteria and belongs to the order Pseudomonadales, family Pseudomonadaceae and genus Pseudomonas.The family consists of about eight groups and 191 species, the species of which is Pseudomonas aeruginosa.In 1890, Walter Migula vaguely described these Gram negative motile organisms and named them Pseudomonas.Recently, it has been postulated that Pseudomonas may be the common nucleator of ice crystals in clouds, so the organism is important for the formation of snow and rain around the world.

Morphology of Pseudomonas

Pseudomonas is rod-shaped, slender (0.5 to 0.8 μm by 1.5 to 3.0 μm) Gram negative organism, motile by polar flagella, sometimes more than two flagella may be present.Some Pseudomonas strains, especially those isolated from cases of cystic fibrosis, are very mucoid and have a kind of pseudo capsule( glycocalyx) made of polysaccharides.Glycocalyx protects pseudomonas from host defence.

Gram negative bacilli Pseudomonas
Gram negative bacilli Pseudomonas

Cultural characteristic of Pseudomonas

Pseudomonas is a strict( obligatory) aerobe, but sometimes it can grow anaerobically if nitrates( NO3 act as a respiratory electron acceptor) are present in the medium. Pseudomonas can grow at wide temperature ranges; the optimum temperature is 37 ° C.It can grow in ordinary media such as nutrient agar and grows in almost all culture media routinely used in the bacteriology laboratory.Pseudomonas has been shown to grow in distilled water, and pseudomonas also produce large, opaque, flat colonies with irregular margins and distinctly fruity odor colonies.The color of growth depends on the type of pigments( listed below) produced by the organism.Water and soil isolates produce small, round colonies.Isolates from clinical specimens such as respiratory, urine, etc . Could produce mucoid colonies. The bacteria that form mucoid colonies are more virulent than others.

Those pigments produced by Pseudomonas are:

PigmentsColor
Fluorescent pigment pyoverdinGreenish yellow
Blue pigment pyocyaninBluish green
PyorubinRed
Pyomelanin Brown

Pseudomonas Biochemical Characteristics

Pseudomonas have oxidative metabolism. Since the organism is non- fermentative, acid is not produced from peptone water sugars.

Pseudomonas ‘ important biochemical characteristics include

The most common biochemical screening test used in the laboratory is the oxidase test.

Oxidase positive test
Oxidase positive test

Virulence and Pathogenicity of Pseudomonas auroginosa

Pseudomonas can infect any tissue, organ system, in an immunocompromised host.Pseudomonas usually can’t infect normal host.So, as compromised hosts are found in hospitals, Pseudomonas has emerged as a common cause of health- related or nosocomial or hospital- related infections.In addition, Pseudomonas causes many different organ system infections in humans.

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Pseudomonas aeruginosa produces exotoxin A, which is a virulence factor.This exotoxin inactivates ADP ribosylation eukaryotic elongation factor 2( EF 2) and thus interferes with protein synthesis leading to cell death.Aeruginosa also produces an exoenzyme ” Exo U,” which damages the cell membrane leading to membrane lysis and cell death.

It is reported that low levels of phosphate in the human intestines activate the symbiotic Pseudomonas to produce lethal toxins in the intestinal tract that can severely damage or kill the host.

The most important risk factor for Pseudomonas infections is the breakdown of host defense due to diseases or other factors. Pseudomonas is invasive and toxigenic. The following three steps involve infection:

  • bacterial attachment and colonization
  • local invasion
  • disseminated systemic disease.

Bacterial attachment and colonisation

Pseudomonas infection may be endogenous( may be from the intestines) or acquired from the outside( exogenous).Individuals outside the hospital may be colonized with Pseudomonas at different sites( 0- 24 percent). The adhesins are the pili of Pseudomonas aeruginosa, with which bacteria adhere to specific galactose or mannose or sialic acid receptors in the upper respiratory tract mucosal epithelial cells and others.The production of protease enzymes by bacteria breaks down fibronectin and exposes the pilus- specific receptors on the epithelial cell surface. Tissue injury caused by viral infection and other phenomena facilitates colonization by Pseudomonas( Opportunistic colonization).

Pseudomonas can also colonize by forming biofilm, which we will discuss later. Pseudomonas pili, mucoid polysaccharide, probably surface- bound exoenzyme S and possibly other cell surface adhesives help Pseudomonas colonize

Bacterial Invasion

Pseudomonas aeruginosa produces enzymes and toxins that break down barriers to host cells and damage them, resist phagocytosis, and host immune defenses.The polysaccharide slime and kind of false capsule produced by Pseudomonas effectively protects cells from antibodies, complementary deposition and phagocytic engulfment.Elastase and alkaline protease protease destroy the corneal substance and other supporting structures composed of fibrin and elastin, resulting in invasion and injury.Pseudomonas aeruginosa produces three other soluble proteins involved in invasion, a cytotoxin( mw 25 kDa) and two hemolysins.Cytotoxin is a pore- forming protein.It was originally called leukocidin because of its effect on neutrophils, but it appears to be cytotoxic to most eukaryotic cells.Of the two hemolysins, one is a phospholipase, and the other a lecithinase.They seem to act synergistically to break down lipids and lecithin.Cytotoxin and hemolysins contribute to invasion by their cytotoxic effects on neutrophils, lymphocytes and other eukaryotic cells.Pyocyanin, the pigmen, impairs the normal function of human nasal cilia and disrupts the respiratory epithelium. you can see that Pseudomonas has a varied armamentarium consisting of pilli, enzymes, capsules and pigments that help it to establish and produce harmful effects in human tissues.

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Bacterial dissemination

Pseudomonas can invade the bloodstream from the initial site of infection and is disseminated to different organs through blood.The factors that help bacteria to invade as described above help to invade the organs, tissues, wherever the bacteria reach.Bacterial endotoxin during septicemia can cause fever, hypotension and intravascular coagulation.

The Pseudomonas virulence / pathogenicity armamentarium includes:

Adhesins

  • Pili (N-methyl-phenylalanine pili)
  • Polysaccharide capsule (glycocalyx)
  • Slime

Invasins

  • Elastase Alkaline protease
  • Hemolysins (phospholipase and lecithinase)
  • Cytotoxin (leukocidin)
  • Pyocyanin

Toxins

  • Exoenzyme S
  • Exotoxin A
  • Lipopolysaccharide (LPS)

Antiphagocytic elements

False capsules, slime layer Lipo polysaccharide Biofilm formation

Biofilm formation

Like some other bacteria, Pseudomonas forms a biofilm that serves as a safe haven for bacteria. Biofilm is formed by quorum sensing. When a critical number of bacteria are reached, bacterial cells communicate with each other by forming certain molecules.Quorum sensing results in the expression of genes that help bacteria adapt to the environment, multiply, release virulence factors and produce enzymes, exotoxins, slime, glycocalyx, etc . This is the biofilm in which bacterial colonies are embedded.These bacteria are protected against host defense and antibiotics. Bacteria from this biofilm can invade, enter the bloodstream, and cause septicaemia. Pseudomonas aeruginosa biofilms lead to chronic opportunistic infections.

Diseases and Infections caused by Pseudomonas spp

Pseudomonas aeruginosa is the most common cause of burn injuries and external otitis( outer ear infection). As previously stated, Pseudomonas can infect any tissue / organ system in the immunocompromised host. The disease produced will depend on the infected organ system.Pseudomonas colonized medical equipment, forms biofilms and causes chronic opportunistic infections. Pseudomonas can be present in healthy hosts as commensal and does not cause any disease.

The following are diseases and infections caused by Pseudomonas

Respiratory infectionsPneumonia in neutropenic cancer patients undergoing chemotherapy; diffuse bronchopneumonia; infection in cystic fibrosis patients
Bacteremia and septicemiaPseudomonas causes hospital acquired Gram Negative bacteremia in immunocompromised patients and in severe burns. Pseudomonas accounts for 25 percent of hospital acquired BSI caused by Gram negative bacilli.
Ear infectionsPseudomonas usually causes external otitis and ” swimmer’s ear.”
Central nervous system infectionsPseudomonas can invade meninges from nearby structures such as external ear / paranasal sinuses after an invasive procedure or trauma to the head.
Urinary tract infectionIt usually causes hospital- acquired UTI related to urinary tract catheterization, instrumentation, or surgery. Pseudomonas aeruginosa is the third leading cause( 12 percent) of all hospital acquired UTIs.
EndocarditisPseudomonas bacteremia may lead to infection of damaged heart valves and prothesized heart valves.
Bone and joint infectionsPseudomonas bacteremia may lead to bone and joint infection by direct inoculation. May cause chronic contiguous osteomyelitis from direct bone inoculation. Pseudomonas also causes osteochondritis after foot puncture wounds.
Gastrointestinal infectionsAny part of the gastrointestinal system can be infected with Pseudomonas in an immunocompromised host. The diseases produced include diarrhoea, gastroenteritis, perirectal infections. Pseudomonas can sometimes produce enterocolitis necrotizing. It is an important cause of antibiotic diarrhea.
Skin and soft tissue infections, including wound infections, pyoderma and dermatitisAny part of the skin and soft tissue affected by trauma, burn injury, bad hygiene can be infected with Pseudomonas. The infections caused include folliculitis, acne vulgaris and abscesses.
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How Pseudomonas resist Drug

Pseudomonas is heat sensitive and is killed in one hour at 55 ° C.Pseudomonas is inherently resistant to many antibiotics, common disinfectants and can gently grow in bottles of antiseptic solutions.The resistance to antibiotics is based on different mechanisms such as multidrug efflux pumps; antibiotic resistance chromosomal genes( mexAB, mexXY, etc.) and the low permeability of the bacterial false capsule in biofilms.Pseudomonas also acquires drug resistance through mutations that can be spontaneous and also drug- induced. Pseudomonas is sensitive to aminoglycosides( amikacin and gentamicin); cephalosporins( ceftazidime and cefotaxime); fluoroquinolones( ciprofloxacin, pefloxacin) and penicillins such as piperacillin, ticarcillin) and colistin.Localized infections may be treated with topical colistin, polymyxin B.

How to diagnose infections caused by Pseudomonas

  1. Collect the relevant sample. This will depend on the tissue / system affected. Specimens can be pus, urine, blood, CSF, tissue, etc.;
  2. Carry out gram staining expected observation will be Gram negative bacilli and pus cells are seen;
  3. Cultivate the specimen on Blood agar and MacConkey agar plates. Incubate at 37 ° C overnight;
  4. Examine bacterial growth: Examine colonial type, pigment production, odor and do oxidase test from MacConkey agar. Pale colonies on MaCconkey Agar, fruity odour, pigment( greenish, brownish) and oxidase positive test means that growth is probably Pseudomonas;
  5. Confirm the OF test and the Arginine dihydrolase test
  6. Conduct an antibiotic susceptibility test using the Kirby Bauer disc diffusion method.
  7. Read the results of the inhibition zones and label as sensitive, resistant or intermediate sensitivity taking into account the zone size
Pseudomonas aeruginosa on Nutrient agar
Pseudomonas aeruginosa on Nutrient agar

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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