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Burkholderia Cepacia Agar Base Preparation, Composition, Principle

Burkholderia cepacia can be viewed as an opportunity-based bacterium that is associated with nosocomial illnesses caused by contamination of medical equipment and...

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This article writter by MN Editors on February 07, 2022

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Burkholderia Cepacia Agar Base Preparation
Burkholderia Cepacia Agar Base Preparation

Burkholderia cepacia can be viewed as an opportunity-based bacterium that is associated with nosocomial illnesses caused by contamination of medical equipment and disinfectants. However, the highest risk group is CF patients. People with cystic fibrosis are at an increased risk of infection. those with the infection, if not treated are prone to rapid declines of lung functions, frequent bacteremia and even death due to lung dysfunction.

Henry, Campbell, LiPuma and Speert devised a new selective medium in 1996 that demonstrated better selectivity and quicker growth of Burkholderia cepacia when compared to PC (Pseudomonas cepacia) Agar and OFPBL (oxidative-fermentative, polymyxin, bacitracin, lactose) Agar. Henry et al. demonstrated that the use of BCSA resulted in less false positives, as well as faster and more effective separation from Burkholderia colonies.

BCSA is a rich source of nutrients which include pancreatic digest of casein as well as yeast extract and two sources of carbohydrates such as sucrose and lactose. A majority Burkholderia cepacia isolates can ferment both sucrose and lactose. the acid products cause the medium to change from yellow to orange because of the presence of the indicator for pH called the phenol red. 

The higher selectiveness of the medium owes itself to four primary components: crystal violet polymyxin, vancomycin and gentamicin. Similar to MacConkey Agar, violet is added to deter Grampositive organisms, particularly staphylococci. Crystal violet’s limited ability to inhibit enterococci calls for the addition of vancomycin, an extremely potent glycopeptide with excellent bactericidal properties against enterococci as well as a variety of other organisms that are gram-positive. The two compounds work together to kill and inhibit the growth of many Gramnegative aerobic bacilli, including Pseudomonas.

Intend to use

Burkholderia Cepacia Agar Base is an effective medium to isolate Burkholderia cepacia from respiratory secretions of patients suffering from cystic fibrosis, as well as other clinical samples.

Principle of Burkholderia Cepacia Agar

Burkholderia Cepacia Agar inspired by PC medium, that was initially developed by Gilligan. The medium was found superior to MacConkey Agar in the development in B. Cepacia . It is also able to be more specific to B. cepacia via the addition of crystal violet, bile salts and antibiotics. The antibiotics that are included include Polymyxin B, Gentamycin, Ticarcillin in the form of freeze-dried supplement (FD). Peptone and yeast extracts within the media provide vitamin B source, nitrogenous as well as other vital nutrients. Bile salts and crystal violet, and antimicrobial agents are utilized to treat a variety of ailments. Crystal violet and bile sodium are able to inhibit gram-positive cocci such as Enterococci as well as Staphylococci. These antibiotics (FD) specifically ticarcillin polymixin B, and gentamycin block Gramnegative bacteria. B. cepacia can metabolize Pyruvate, resulting in alkaline end-products. These end products increase your medium’s pH. The indicator phenol red alters colour from pink orange to pink in alkaline pH.

Composition of Burkholderia Cepacia Agar

IngredientsGms / Litre
Peptone5g
Yeast extract4g
Sodium pyruvate7g
Potassium dihydrogen phosphate4.4g
Disodium hydrogen phosphate1.4g
Bile salts1.5g
Ammonium sulphate1g
Magnesium sulphate0.2g
Ammonium ferrous sulphate0.010g
Phenol red0.020g
Crystal violet0.001g
Agar12g

Final pH ( at 25°C) 6.2±0.2

Preparation of Burkholderia Cepacia Agar

  1. Take a suspension of 18.26 grams into 500 milliliters of distillate water.
  2. Then heat to boiling, allowing the medium until the medium is completely dissolving.
  3. Sterilize using autoclaving at 15lbs pressurization (121degC) over 15 mins.
  4. Cool to 50°C, then include the rehydrated contents of one vial of Burkholderia Selective Supplement.
  5. Mix well before pouring into sterilized Petri plates.

Interpretation of Results

Most often, Burkholderia cepacia colonies appear as greenish-brown colonies surrounded by yellow halos. The medium’s yellow color indicates the presence of carbohydrate and could not occur in all Burkholderia cepacia strains. Any growth can be considered a positive result , and additional tests for serology and biochemistry must be conducted on isolated colonies of pure culture to ensure be able to identify them completely.

Other microorganisms, including Enterococcus fasciculus and Burkholderia pickettii could thrive on BCSA. The high specificity of this medium might warrant parallel plating of samples onto another less selective medium to determine the presence of other pathogens (Stenotrophomonas maltophilia, which is a pathogen of nosocomial origin that is prevalent in CF sufferers, can be blocked by BCSA). The large amount of polymyxin present in the medium can hinder the growth of many molds and yeasts .

OrganismsRecoveryRemarks
Burkholderia cepacia ≥50%Greenish–brown colonies with yellow halo
Burkholderia cepacia ≥50%Greenish–brown colonies with yellow halo
Burkholderia cenocepacia ≥50%White colonies
Burkholderia multivorans ≥50%White colonies
Staphylococcus aureusinhibitedw. selective supplement
Pseudomonas aeruginosaInhibited (partial to complete)w. selective supplement

Quality Control

After testing for the correct pH color, depth, and the following microorganisms are utilized to evaluate the performance of the final medium.

OrganismExpected Results
Burkholderia cepacia
ATCC 25416
Growth, Greenish-brown with yellow halo
Pseudomonas aeruginosa
ATCC 27853
Complete inhibition

Storage and Shelf Life

Burkholderia Cepacia Selective Agar should be kept out of direct light between 4 and 8degC. The medium side must be placed on top to prevent the growth of moisture on the agar’s surface. This medium is shelf-life of 8 weeks after the date of manufacturing.

Limitation

  • The isolats of B. the cepacia-related complex result in an insipid, weak-positive the oxidase reaction.
  • This formulation is not an agent that is selective against filamentous fungi or yeast.
  • BCSA is a useful tool to perform quantitative tests for the identification from B. cepacia complex.
  • First time isolates of B.cepacia complex from the blood of a CF patient must be sent to a reference laboratory for confirmation of identification. It is the United States Cystic Fibrosis Foundation has set up a reference lab located at the University of Michigan for this reason.
  • Additional tests that are required to confirm the identity that belong to the B. cepacia include at a minimum maltose and lactose oxidation the decarboxylase of lysine, and ONPG.
  • Although this medium is extremely selective against Gram-negative organisms and some rods with gram-negative morphology has been seen. Particularly, Burkholderia gladioli and Chryseobacterium indologenes could be found to show slow growth. Positive reactions that result in the oxidation of maltose as well as lactose are a way to differentiate B. Cepacia and B. Gladioli. Indole reactions that are positive is a method to differentiate C. indologenes (B. cepacia complex is negative).9
  • Stenotrophomonas maltophilia (colistin resistant strains) have been found to multiply on BCSA. A positive reaction to DNase Agar after time of 72 hours incubation could be used to determine the difference the two strains: S. maltophilia (positive) and B. cepacia complex (negative).

References

  • https://www.rapidmicrobiology.com/news/burkholderia-cepacia-selective-agar-supplement-acc-to-the-new-usp
  • https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/808/084/39573dat.pdf
  • https://www.dalynn.com/dyn/ck_assets/files/tech/PB95.pdf
  • https://himedialabs.com/TD/M1640.pdf
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