From MicrobeWiki, the student-edited microbiology resource
A Microbial Biorealm page on the genus Burkholderia cepacia
Higher order taxa
Domain: Bacteria; Phylum: Proteobacteria; Class: Betaprotebacteria; Order: Burkholderiales; Family: Burkholderiaceae; Genus: Burkholderia
The Burkholderia cepacia complex consists of nine genomic species called genomovars: B. cepacia, B. multivorans, B. cenocepacia, B. vietnamiensis, B. stabilis, B. ambifaria, B. dolosa, B. anthina, and B. pyrrocinia.
Description and significance
Burkholderia cepacia was first described by Walter Burkholder of Cornell University in 1949 when he determined it to be the cause of bacterial rot of onion bulbs. It was originally named Pseudomonas cepacia and was later changed to its current name. Burkholderia cepacia refers to a complex of nine closely related species listed above. They are rod-shaped, free-living, motile Gram- negative bacteria ranging from 1.6- 3.2 μm. They have been found to possess multitrichous polar flagella as well as pili used for attachment. Burkholderia cepacia can be found in soil, water, and infected plants, animals, and humans (1). Aside from being a plant and human pathogen it has many significant agricultural uses. It is capable of breaking down toxic compounds found in pesticides and herbicides. It has also been noted to repress certain soil-borne pathogens and is being considered as an agent for promoting crop growth.
The appearance of Burkholderia cepacia species varies based on the strain and the culture medium used. Three media are currently being used to isolate the bacteria. They are the following: Pseudomonas cepacia agar (PCA), oxidation fermentation polymyxin bacitracin lactose agar (OFBL), and Burkholderia cepacia selective agar (BCA). The last medium has proved to be the most effective since it actually suppresses the growth of non-Burkholderia cepacia bacteria. Burkholderia cepacia bacteria will form visible pinpoint colonies within 24 hours, and the colonies appear to be smooth and somewhat elevated.
The replicon number and sizes vary from strain to strain in Burkholderia cepacia species. The largest replicon is found in strain N2P5 (9.3 Mb). Most species contain 2 to 4 large replicons, and many also contain smaller replicons as well. Many species contain plasmids, and all species have circular chromosomes. The Burkholderia cepacia type- strain ATCC 25416 (genomovar I) is 8.1 Mb in length and is known to have four circular replicons. Its largest replicon contains 4 rrn operons and the other two megabase- sized replicons contain a single rrn operon each. From this information it can be inferred that the organism has three chromosomes and one large plasmid. (6)
Cell structure and metabolism
Burkholderia cepacia is capable of growing on over 200 organic compounds. It is incredibly versatile in this regard. Of special interest is its ability to use the chlorinated aromatic compound 2,4,5- trichlorophenoxyacetic acid as a source of carbon and energy (4). This compound is found in many pesticides and herbicides. Some of Burkholderia cepacia’s cell structures include polar flagella used for motility and pili used in adhesion. Burkholderia cepacia complex species may express one of two flagellin types that differ in size. Type I is 55 kDa and type II is 45 kDa. Aside from motility, flagella have also been noted to function in adhesion, the production of biofilms, and in the production of an inflammatory response in an infected host. (2,5)
The Entner-Doudoroff pathway is an alternative pathway that breaks down glucose into pyruvate and releases ATP. It is utilized by many different bacteria and archaea. This pathway differs from glycolysis by utilizing different enzymes. Glucose is first converted into 6-phosphogluconate through the direct oxidative and phosphorylative pathways. Different enzymes, including 6-phosphogluconate (6PGA) dehydratase and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, then enter the pathway, converting one product to the next until pyruvate is formed. Burkholderia cepacia, or Pseudomonas cepacia, differs from most other pseudomonads in that it has higher levels of the enzyme 6-phospho-gluconate dehydrogenase (6PGAD). This enzyme is used in a pathway called the pentose shunt (another slower alternative to glycolysis). Thus, higher levels of this enzyme suggest that it has the potential to also utilize the pentose shunt. If one of the enzymes involved in the Entner-Doudoroff pathway was mutated, the pentose shunt would thus be utilized, resulting in a slower growth rate. However, in the event of enzyme mutation, the utilization of the pentose shunt is rarely seen due to a lack of growth. When an enzyme is mutated, it cannot convert one product to the next and the pathway is blocked at this point. This leads to a toxic accumulation of the unconverted product, for example 6PGA, and consequently, no growth occurs. One study, however, observed that certain strains of 6PGA dehydratase deficient mutants produced even more 6PGAD. This reflects the possibility that Entner-Doudoroff mutants may develop enough 6PGAD to overcome the toxic accumulation of 6GPA and utilize the pentose shunt. (15)
Resistance to penicillin
Pseudomonas cepacia possesses an inducible β-lactamase, encoded by the gene penA, which is what allows the organism to catabolize β-lactam compounds. This inducible β -lactamase activity is connected with P. cepacia’s greater resistance to β -lactam antibiotics. β-lactamase is a common product of many gram-negative bacteria, however, the activity of inducible penicillinase present in P. cepacia is linked with the ability of this species to hydrolyze penicillin and to use it as a source of carbon.
Penicillinase is responsible for nearly eighty percent of the β -lactamase activity of the strain. In addition to penicillinase, a second β-lactamase has been identified; an enzyme with primarily cephalosporinase activities. Pseudomonas aeruginosa also produces a β-lactamase, however, the differences between these two species elicit different responses to the antibiotics. Pseudomonas cepacia infections are much less responsive to β-lactam antibiotic therapy than Pseudomonas aeruginosa infections in the same patient population. β-lactamase from P. cepacia 249 is the strain particularly associated with the ability to metabolize penicillin. This penicillinase was found to differ greatly from the ampC chromosomal β-lactamases associated with P. aeruginosa in terms of physical properties, substrate profile, and induction kinetics.
The role of the penA gene, which produces β-lactamase in P. cepacia, is primarily one of metabolism rather than resistance. While penA was also successfully expressed in E. coli, the other β-lactamase enzymes produced in P. cepacia are necessary for both the hydrolysis and metabolism of penicillin. (19)
Burkholderia cepacia complex species are soil-dwelling bacteria commonly found on plant roots. They are of significant environmental interest. Along with their antinematodal and antifungal properties, they can also degrade a large variety of toxic compounds. This makes them extremely useful in bioremediation, a practice using biological compounds to remove hazardous compounds and pollutants. Chlorinated phenols and phenoxyacetates are commonly used in agriculture as pesticides, herbicides, and preservatives. They compose a major group of recalcitrant environmental pollutants. Burkholderia cepacia has the ability to use these compounds as a source of carbon and energy, therefore breaking them down and removing them from the environment (4).
Burkholderia cepacia has long been known as both a plant and human pathogen. It was first discovered as a plant pathogen in 1949 when it was found to cause onion rot. In humans it is most well known for its infection in cystic fibrosis patients as well as patients with chronic granulomatous disease and other patients with compromised immune systems. All species within the Burkholderia cepacia complex have been isolated from cystic fibrosis patients, however genomovar III and Burkholderia multivorans seem to cause the majority of infections. (7) There is still much to be learned about the virulence factors used by Burkholderia cepacia. 3 known virulence factors that enhance its pathogenicity in epithelial cells include lipase, metalloproteases, and serine proteases. "Burkholderia cepacia" uses a type II pathway (under quorum sensing control) to secrete lipase. Although little is known as to how, lipase has been proven to play a role in the invasion of lung epithelial cells. Metalloproteases also play an important role in the virulence of "Burkholderia cepacia" in lung tissue. One specific metalloprotease, ZmpB, is proteolytically active against many proteins in the extracellular matrix of the lungs, including type IV collagen and fibronectin. ZmpB has also been proven effective against destroying key members of the immune system, showing that this virulence factor is directly related to the damage of lung tissue and generating a continued host response. With serine protease, just as lipase, has been shown to be a key factor in the intracellular invasion of lung tissue. However its major contribution to "Burkholderia cepacia" is its ability to allow the bacteria to use ferritin as an iron source. Ferritin has not been shown to be an iron source for any other pathogenic bacteria thus far, which gives "Burkholderia cepacia" a unique advantage over other bacteria (16). There is evidence that two species, Burkholderia cenocepacia (genomovar III)and the type strain Burkholderia cepacia (genomovar I) actually use quorum sensing to control the expression of their virulence factors, which means their pathogenicity is only expressed when a high enough population density exists to take over the host before it can mount a response. This was recorded of genomovar III in the lungs of cystic fibrosis patients and of genomovar I in onions (8). There is a high mortality rate in patients infected with Burkholderia cepacia. Many develop what is known as cepacia syndrome. This is a fatal necrotizing pneumonia that is currently untreatable.
While infection in patients with cystic fibrosis is most common and extremely detrimental, Burkholderia cepacia can also infect non-cystic fibrosis patients, even non-immunocompromised patients. B. cepacia has been associated with cutaneous foot lesions in military personnel, a malady known as ’swamp foot.’ B. cepacia isolates have been obtained from catheters, wounds, sputum, urine, etc. It can even infect hospital soaps, dextrose solutions, and much more. This means that those individuals working in centers that care for Burkholderia cepacia infected patients are not necessarily safe. Because cross-infection is so prevalent it is important that infected patients be segregated from non-infected patients (13).
The severity of Burkholderia cepacia infection is due in part to its formation of biofilms, which are commonly resistant to antibiotics. The initiation of a biofilm is dependent upon the organism's pili and flagella used for attachment as well as through lateral movement which enlarges the colony. The maturation of the biofilm has been found to depend on the production of the exopolysaccharide cepacian which is known to stabilize the three-dimensional architecture of the biofilm (10). The beginning stages of infection have also been linked to the scavenging of iron. Burkholderia cepacia produces the following four iron-binding siderophores: salicylic acid, ornibactin, pyochelin, and cepabactin. Burkholderia cepacia is also capable of crossing the epithelial barrier by somehow inhibiting the body's natural pulmonary defense mechanisms. Lipopolysaccharides have also been shown to induce an inflammatory response in the lungs of infected patients, allowing infection to become increasingly necrotizing (14).
Research has suggested that B. cepacia’s binding site is a molecule called, GalNAc_1-4Gal, that is found in glycolipids. These glycolipids are part of the mucous that is found in humans. This offers a possible explanation for the increased susceptibility of CF patients as their mucocilliary clearance mechanisms are impaired. The increased mucin in the lungs of CF patients allows the persistence of the B. cepacia bacteria. One study even found a correlation between strains that have a greater affinity for mucin and more persistant infections. (18) Certain strains of B. cepacia has been show in several studies to possess pili. These pili, or fimbriae, are easily associated with adhesive properties of bacteria. New research has attributed some of the virulence of B. cepacia to the binding of its peritrichous fimbriae to cells within the human respiratory tract. The fimbriae adhere to a protein, cytokeratin 13, that is found on the surface of normal human bronchial cells. Since these cells of the upper respiratory tract present CK 13 they can act as receptors that bind B. cepacia. It has also be suggested that inflammation causes an increase in CK 13 and thus facilitates growth and proliferation of the bacteria. (18)
Application to Biotechnology
Burkholderia cepacia does produce antinematodal and antimicrobial compounds. These compounds protect plants from soil-borne pathogens, and are being proposed for use in place of environmentally harmful pesticides and herbicides. Some of these compounds have been identified as pyrrolnitrin which exerts an inhibitory effect on the electron transport chain (11), pyoluteolin, and the siderophore cepabactin. The production of volatile ammonia has also been found to control soil pathogens (12).
Burkholderia cepacia is an important organism in the current research on biodiesel fuel. Vegetable oils have attracted much attention as a potential renewable source for the production of an alternative for petroleum based diesel fuel. The reason we are looking for an alternative energy source is because of the harmful exhaust gases from petroleum diesel engines, and vegetable oils can be converted into a substance called biodiesel. Methyl and ethyl esters of fatty acids, more commonly known as biodiesel, are nontoxic and biodegradable, which are perfect reasons why biodiesel is a great substitute for petroleum diesel. In this experiment, Noureddini et al. used the transesterification of soybean oil with methanol, ethanol, and lipases from various microorganisms to determine which microorganism would yield the highest amount of alkyl esters. Alkyl esters are another name for the molecules that are commonly referred to as biodiesel. They determined that the lipase from Burkholderia cepacia showed the greatest methanol resistance among all the tested lipases from the various microorganisms, and it also exhibited the highest catalytic activity toward the transesterification reaction, which actually creates the biodiesel. The next key step in this experiment was to be able to isolate and immobilize the lipase in order to be able to use it as needed for the transesterification process. For the immobilization of lipase from Burkholderia cepacia, they used a phylloscilicate sol-gel matrix, which is routinely used to immobilize enzymes from organisms. In this process, phylloscilicate clay, saturated with sodium ions, was suspended in water and then exchanged with alkyl ammonium ions by the addition cetyltrimethyl ammonium chloride. This mixture was then used in the entrapment of Burkholderia cepacia with tetramethoxysilane (TMOS) as the polymerization precursor. The immobilized enzyme is then ready to be used to create the biodiesel fuel through the transesterification process. It was also determined that the immobilized lipase was much more active than the actual free lipase, as well as being more stable when subjected to repeated use. In conclusion, Burkholderia cepacia is an effective bioagent in the creation of biodiesel in order to help eliminate harmful waste being released into the atmosphere by petroleum diesel. (17)
The most current research on Burkholderia cepacia includes its use in the agricultural industry to protect plants from pathogens. Burkholderia cepacia can colonize the roots of many plants where it produces compounds that protect against soil-borne pathogens. In absence of these pathogens, plant growth has been noted to improve (9).
Another area of interest for Burkholderia cepacia is in bioremediation because of its ability to metabolize certain toxic and hazardous compounds. This includes 2,4,5- trichlorophenoxyacetic acid, which it the main component of Agent Orange (9).
Current research on Burkholderia cepacia also includes its pathogenicity in immunocompromised patients. Over the last decade or two, Burkholderia cepacia has become an increasingly dangerous threat to cystic fibrosis patients, other immmunocompromised patients, and personnel caring for the infected. Its large genome makes it a strong and versatile microorganism. It is resistant to many antibiotics, and infected patients do not seem to respond to treatment. The eradication of Burkholderia cepacia as a human pathogen will become increasingly important in the years to come (13).
The possibilities of this organism are very exciting as stated in the following quote: "What better microbial challenge to unite agricultural and medical microbiologists than an organism that reduces an onion to a macerated pulp, protects other crops from bacterial and fungal disease, devastates the health and social life of cystic fibrosis patients, and not only is resistant to the most famous of antibiotics, penicillin, but can use it as a nutrient!" (J. R. W. Govan, 1998)
Although these are exciting possibilities, little is known as to how the addition of this species into an open environment would affect its pathogenicity. Care must be taken because Burkholderia cepacia is also a very serious human pathogen.
1. Miller SC, LiPuma JJ, Parke JL “Culture based and Non-Growth Dependent Detection of the Burkholderia cepacia complex in Soil Environments” Applied Environmental Microbiology, August 2002
2. Barbara A. Hales, J. Alun W. Morgan, C. Anthony Hart, Craig Winstanley “Variation in Flagellin Genes and Proteins in Burkholderia cepacia”
3. P. Wigley, N. F. Burton “Multiple Chromosomes in Burkholderia cepacia and Burkholderia gladioli and their distribution in clinical and environmental strains of B. cepacia” Journal of Applied Microbiology, Volume 88 Issue 5
4. Department of Microbiology and Immunology, College of Medicine, University of Illinois; “Genes for 2,4,5- Trichlorophenoxyacetic Acid Metabolism in B. cepacia AC1100"
5. Teresa A. Uirban, Adam Griffith, Anastasia M. Torok, Mark E. Smolkin, Jane L. Burns, Joanna B. Goldberg. Infect. Immun. 2004 September. Contribution of Burkholderia cenocepacia Flagella to Infectivity and Inflammation
6. Rodly PD, Romling U, Tummler B. “A Physical genome map of the Burkholderia cepacia type strain” Mol. Microbiology 1995; 17:57-67
7. Andrew McDowell, Eshwar Mahenthiralingam, John E. Moore, Kerstin E. A. Dunbar, A Kevin Webb. “PCR-Based Detection and Identification of Burkholderia cepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients” J Clin Microbiol. 2001 December
8. Shawn Lewenza, Barbara Conway, E. P. Greenberg, Pamela A, Sokol. “Quorum Sensing in Burkholderia cepacia: Identification of the LuxRI Homologs CepRI” Journal of Bacteriology 1999 February 181(3): 748-756
9. Peter A. R. Vandamme "Burkholderia cepacia: Pandora's Box Redefined" BCCM. Edition 9-01. Article 2
10. Monica V. Cuaha, Silvia A. Sousa, Jorge H. Leitao, Leonilde M. Moreira, Paula A. Videira, Isabel Sa-Correia "Studies on the Involvement of the Exopolysaccharide produced by Cystic Fibrosis- Associated Isolates of Burkholderia cepacia Complex in Biofilm Formation and in Persistance of Respiratory Infection" Journal of Microbiology. July 2004. p. 3052-3058. Vol. 42. No.7
11. El-Banna, Winkelmann "Pyrrolnitrin from Burkholderia cepacia: antibiotic activity against fungi and novel activities against streptomycetes" Journal of Applied Microbiology 85 (1), 69–78.
12. Mansour Baligh, Martin A. Delgado, Kenneth E. Conway "Evaluation of Burkholderia cepacia Strains: Root Colonization of Catharanthus roseus and In-Vitro Inhibition of Selected Soil-Borne Fungal Pathogens" Feb. 15,1999 Dept. of Entomology and Plant Pathology
13. A. M. Jones, M. E. Dodd, A. K. Webb "Burkholderia cepacia: current clinical issues, environmental controversies, and ethical dilemnas" European Respiratory Journal. 2001; 17:295-301
14. Georg Maschmeyer, Ulf B. Gobel "217 Strenotrophonomonas maltophila and Burkholderia cepacia Principles and Practices of Infectious Disease pp. 2615-2620
15. Allenza, P., and Lessie, T.G. "Burkholderia cepacia mutants blocked in the Entner-Doudoroff pathway" Journal of Bacteriology, 1982. Volume 150. p.1340-1347.
16. Callaghan, M., and McClean, S. "Burkholderia cepacia complex: epithelial cell-pathogen confrontations and potential for therapeutic intervention" Journal of Medical Microbiology, 2009. Volume 58. p. 1-12.
17. Noureddini, H., Gao, X., and Philanka, R. S. “Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil” Bioresource Technology, 2004. p. 769-777.
18. Mohr, Christian D., Christine A. Herfst, and Miaden Tomich. "Cellular Aspects of Burkholderia Cepacia Infection." Microbes and Infection 3 (2001): 425-35. Science Direct. Web. 27 Apr. 2010.
19. Prince, Alice, Mark S. Wood, Grace Soong Cacalano, and Nai Xing Chin. "Isolation and Characterization of a Penicillinase from Pseudomonas cepacia 249" Antimicrobial Agents and Chemotherapy, 1988 p. 838-843.
Edited by Heather McClendon, student of Rachel Larsen and Kit Pogliano
Edited by Marco Almeda and Akadia Kachaochana, students of M Glogowskiat Loyola University
Edited by Allison McGraw and Brett Voigt, students of M Glogowskiat Loyola University
Editied by William Martin and Justine Holleman, students of M Glogowski at Loyola University
Edited by Natali Rutiaga and Mehreen Syed, students of M Glogowski at Loyola University