A 23-year old female was admitted to burns ward of a hospital following 55% burns in a fire accident. After 13 days of admission, the wounds got infected and the exudate was bluish green.

What is your diagnosis?
It is a case of burns infection. Since the infection was acquired after hospitalization, it is a nosocomial infection. Presence of "blue pus" gives suspicion of Pseudomonas infection.

What is the specimen collected and how is the condition diagnosed with the aid of laboratory?
The surface of the infected wound is cleaned with saline and the exudate is collected with the help of a sterile cotton swab from the depth of the lesion. Two samples of pus may be collected; one for microscopy and the other for culture. Invasive burn wound sepsis is defined as the bacterial proliferation of 100,000 organisms per gram of tissue. To aid in diagnosis, obtaining burn wound biopsies with quantitative bacterial cultures is recommended. A bacterial count of greater than 10⁵ organisms per gram of tissue is diagnostic of a burn wound infection. A gram stained smear of the pus is made and cultured on Blood agar as well as on MacConkey's agar and incubated at 37^oC overnight. The growth is identified using biochemical reactions and antibiotic susceptibility test is formed. Pyocin typing may be performed to determine if it is the hospital strain.

What is your observation?
Gram stained smear of the pus shows gram negative bacilli along with pus cells. Culture on blood agar yield dark coloured flat irregular colonies with beta hemolysis. Non-lactose fermenting, irregular, flat colonies with bluish-green pigmentation on MacConkey's agar is seen. Pigmentation is better seen on Nutrient Agar. The colonies have a characteristic fruity or earthy odour. Gram stained smear of the colony shows  gram negative bacilli, hanging drop shows actively motile bacilli. Results of biochemical reactions include positive catalase test, positive oxidase test, negative indole test, negative urea hydrolysis, positive citrate utilization test. TSI agar shows alkaline slant/no change. None of the sugars is fermented. Fluorescence under ultraviolet light is helpful in identification of P. aeruginosa colonies.

What is your identification?
The organism isolated is Pseudomonas aeruginosa.

Which are the various pigments produced by this isolate?
Pseudomonas aeruginosa is known to produce several water soluble pigments; these are pyocyanin (blue), pyorubin (reddish-brown), pyomelanin (black), pyoverdin/fluorescein (green), which is fluorescent. Pyocyanin impairs the normal function of human nasal cilia, disrupts the respiratory epithelium and exerts a proinflammatory effect on phagocytes. Pyocyanin also interferes with the terminal electron transfer system by complexing with flavoproteins. Pyochelin, which is a derivative of pyocyanin, is a siderophore that is produced under low-iron conditions to sequester iron from the environment for growth of the pathogen. The pigments diffuse into the medium and render them coloured.

Which are the other infections produced by this isolate?
Pseudomonas aeruginosa is primarily a nosocomial pathogen and is a successful opportunistic pathogen. It is responsible many of the hospital acquired infections involving invasive procedure or prosthetic devices; such as iatrogenic meningitis, endopthalmitis following eye surgery, UTI following catheterization etc. Pseudomonas is also responsible for complication in cystic fibrosis (mucoid type), pneumonia, otitis externa, wound infections (especially in diabetics), dermatitis, soft tissue infections, bacteremia, osteomyelitis, joint infections, and gastrointestinal infections. Pseudomonal bacteremia produces distinctive skin lesions known as ecthyma gangrenosum. Green nail syndrome is a paronychial infection that can develop in individuals whose hands are frequently submerged in water. It is a common cause of hot tub or swimming pool folliculitis.

Which are its virulence factors?
Production of pili, exoenzyme S, exotoxin A, pyocyanin, lecithinase, collagenase, lipase, hemolysin, elastase, alkaline protease, phospholipase and leucocidin contributes to its virulence. Its outer membrane offers a natural permeability barrier to several antibiotics. It has intrinsic resistance to several antibiotics. Its tendency to colonize surfaces in a biofilm form makes the cells impervious to therapeutic concentrations antibiotics. It is tolerant to a wide variety of physical conditions, including temperature (up to 42^oC). It is resistant to high concentrations of salts, dyes and certain antiseptics (cetrimide).

How do you confirm that this isolate is nosocomial in origin?
Demonstrating by phenotypic or genotypic methods that isolate obtained from the patient is identical to the isolates obtained either from the hospital environment or hospital personnel confirms the nosocomial origin of the pathogen. Antibiogram, phage and pyocin typing are commonly used phenotypic typing methods.

What is the antibiotic susceptibility pattern of Pseudomonas?
Pseudomonas aeruginosa is resistant to multiple drugs, hospital strains are more so resistant. Resistance to antibiotics is due to production of extended spectrum beta lactamases or porin mutations. Carbenicillin and Piperacillin are anti-pseudomonal penicillins. Aminoglycosides such as Gentmicin or Amikacin is effective in many cases. Imipenem or meropenem is also found to be effective against many resistant strains. Silver sulfadiazine and mafenide acetate are useful in topical applications. Antibiotic susceptibility testing must be performed to select the appropriate antibiotic for treatment.