Saturday, 3 January 2015

Isolation of Burkhoderia cepacia from sputum specimen

Burkhoderia cepacia is a Gram negative rod named after the man who discovered it in 1949 – Walter Burkholder. B. cepacia causes pneumonia in immunocompromised patients. It is usually isolated from cystic fibrosis patients but can also be seen in patients with bronchiestasis and chronic obstructive pulmonary disease (COPD). Like Stenotrophomonas maltophilia, B. cepacia was previously in genus Pseudomonas before it was re-classified.
B. cepacia can be isolated in the microbiology laboratory from sputum specimen or blood. However, this organism was isolated from sputum specimen from a patient with COPD clinical details as shown here.
Micro-organisms present in a sputum sample are usually distributed unevenly in the sample which is viscous due to mucus or pus. Dilution of the sputum sample helps to remove the normal flora present and retains the significant organisms. This is because the number of organisms causing an infection should be significantly higher than the number of contaminating organisms. Sputasol (Oxoid Ltd, Basingstoke, UK; containing 100µg/ml dithiothreitol) was used to homogenise the sputum sample by adding equal volume of 0.1% solution of Sputasol to the sample and vortexing for approximately 10 seconds and leaving for 15 minutes at room temperature. This is followed by gentle vortexing to assist homogenisation. 10µl of the homogenised sputum is then diluted in 5ml of sterile water. 1µl of this dilution is used to inoculate the media plates. Please note that all clinical samples containing or may contain the hazard group III organisms should be handled within the appropriate facilities and working practices which include processing them in a class I safety cabinet. This microbiology laboratory is equipped with Class I safety cabinet in the Category 3 laboratory section. Class I safety cabinets are designed with an open-front negative pressure cabinet which helps to protect the laboratory staff or worker from hazardous aerosols by extracting air through a High efficiency particulate absorption (HEPA) filter.
The organism was grown on B. cepacia selective agar (CEP PP0160 E&O Laboratories, UK), however it can also grow on blood agar. This selective medium contains bile salts and crystal violet as selective agents and additional agents such as Polymixin B and Ticarcillin to help inhibit most Pseudomonas species. B. cepacia appears as light green colonies and turns the selective agar pink after incubation at 37oC for 40-48 hours incubation then further incubation at 30oC for 5 days).
After incubation, the isolate was identified using Biomerieux Vitek MS - Matrix Assisted Laser Desorption Ionization Time-of-Flight (VMS MALDI-TOF). This is an automated microbial identification system that uses innovative mass spectrometry technology to give results in minutes.
Biomerieux Vitek 2 automated analyser was used for the susceptibility testing. the sensitivity pattern was typical of B. cepacia with resistance to most antibiotics tested. This isolate was only sensitive to co-trimoxazole (Trimethoprim/Sulfamethoxazole), intermediate to Ceftazidime, Meropenem, Chloramphenicol and resistant to Amikacin, Aztreonam, Cefotaxime, Gentamicin, Ciprofloxacin, Colistin, Imipenem, Tazocin (Piperacillin/Tazobactam), Ticarcillin/Clavulanic acid, Tigecycline, Tetracycline and Tobramycin.

Figure 1 shows the Gram stain from B. cepacia as Gram Negative rods.

Figure 2 shows the change in the colour of B. cepacia selective medium before and after the growth of organism.

Figure 3 shows a closer look at the colonies of B. cepacia on the selective medium.

Figure 4 shows a closer look at the colonies of B. cepacia on the blood agar medium.

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