Burkholderia cepacia Pseudomonas cepacia burkholderia cepacia complex B. cepacia

1. BURKHOLDERIA CEPACIA
Prof. Abdelraouf Elmanama
by
Ahmed A. Abdallah

2. Intro
■ Burkholderia cepacia, formerly Pseudomonas cepacia is a motile gram negative bacillus
which is aerobic and glucose non-fermenting.
■ It proliferates under conditions of minimal nutrition and can survive in the presence of
certain disinfectants.
■ It has a wide distribution in the natural environment via water, soil, fruits, and vegetables.
■ In the last two decades, B. cepacia has emerged as a serious human pathogen causing fatal
necrotizing pneumonia and bacteremia, especially in patients with cystic fibrosis (CF) or
chronic granulomatous disease.
■ Cross-transmission, frequent pulmonary procedures, and central venous access facilitates
the nosocomial spread of this organism.
■ High transmissibility in the hospital setting, intrinsic resistance to many antibiotics and
association with a poor prognosis highlights the need for early detection and treatment of B.
cepacia infections
■ These bacteria also have contaminated many drug products and can create public health
concerns. Pharmaceutical products that are contaminated with B. cepacia may pose serious
consequences to vulnerable patients (e.g., compromised immune system).

3. Burkholderia cepacia
■ Burkholderia cepacia was known as Pseudomonas cepacia prior to 1992.
■ B. cepacia was discovered by Walter Burkholder in 1949 as the cause of onion
skin rot, and first described as a human pathogen in the 1950s.
■ It was first isolated in patients with cystic fibrosis (CF) in 1977, when it was
known as Pseudomonas cepacia.
■ In the 1980s, outbreaks of B. cepacia in individuals with CF were associated
with a 35% death rate.

4. Burkholderia cepacia
■ Similar to other opportunistic pathogens such as Pseudomonas aeruginosa,
Bcc strains do not normally infect healthy individuals but only those that are
immunocompromised.
■ Specific populations susceptible to infection include elderly people, young
children, cancer patients, pregnant women, and people with chronic illness.
■ However, B. cepacia occasionally causes illness in non-immunocompromised
patients.

5. Burkholderia cepacia
■ The most serious conditions caused by B. cepacia are pneumonia or patients
with impaired immune systems or chronic lung disease, particularly cystic
fibrosis (CF).
■ CF is the most common lethal inherited disease of Caucasian populations,
with pulmonary infections being the major cause of morbidity and mortality.
■ The severity of infection or colonization by B. cepacia may be different for
individual patients.
■ However, overall, pulmonary colonization reduces survival by 50%; about one
third to one-half of patients succumb to cepacia syndrome, a rapidly fatal
necrotizing pneumonia.

6. Burkholderia cepacia
■ Burkholderia is a genus composed of more than 60 organisms.
■ Many of which were formerly classed as Pseudomonas species (family:
Burkholderiaceae; phylum: Proteobacteria).
■ Within the Burkholderiaceae is the Burkholderia cepacia complex, a group of
some 20 organisms (the actual number is a matter of taxonomic debate,
falling between 17 and 20 species) subgrouped into nine genomovars (that is,
phylogenetically differentiable but phenotypically indistinguishable bacteria).
■ The BCC is a group of species that are so closely related that they can, for the
most part, only be differentiated using a combination of multiple molecular
diagnostic procedures.

7. overview of the Burkholderia
cepacia complex.

8. Burkholderia cepacia
■ Bcc are widely distributed in natural and man-made habitats.
■ Burkholderia cepacia is a motile aerobic oxidase positive Gram-negative
bacillus commonly found in liquid reservoirs and moist environments. The
cells are 0.5 to 1.0mm wide and 5mm in length.
■ They are common in nature and present in soils, plant rhizospheres,
water, and agriculture products.
■ Infections caused by Bcc have occurred worldwide, and accumulating
evidence implicates contaminated pharmaceuticals, cosmetics, disinfectants,
and preservative products as major sources of Bcc.

9. Transmission
■ In epidemiological terms, the mode of interpersonal transmission for this
opportunistic pathogen primarily occurs through direct contact with other
people (e.g., a handshake), or through contact with body perspiration.
■ Although B. cepacia does not appear to survive on completely dry surfaces for
more than one week, it can survive for many months in water.
■ B. cepacia can use other routes of transmission including contact with hard
surfaces.
■ Perhaps most important to note is this microbe’s ability to remain viable
under harsh conditions (e.g., organic solvents, antiseptics, low nutrients, etc.)
for many months.

10. Resistance
■ Bcc are multi-drug resistant organisms.
■ The multi resistance of Bcc bacteria appears to result from various efflux
pumps that efficiently remove antibiotics from the cell, decreased contact of
antibiotics with the bacterial cell surface due to Bcc’s ability to form biofilms,
and changes in the cell envelope that reduce the permeability of the
membrane to the antibiotic.
■ B. cepacia is also resistant to many disinfectant cleansers and is unaffected by
many preservatives including Betadine.
■ Bcc are among the most antimicrobial agent–resistant organisms
encountered in the clinical laboratory.
■ Due to mucin-binding proteins, this species can form biofilms and
contaminate plastics, metals, water systems, hospital equipment, catheters,
and living tissue.

11. Resistance
■ Microbial biofilms develop when microorganisms adhere to a surface by
producing extracellular polymers that facilitate adhesion and provide a
structural matrix.
■ Once these cells attach and produce extracellular polysaccharides in the
biofilm, their rate of growth is influenced by flow rate, nutrient composition
of the medium, antimicrobial-drug concentration, and ambient temperature.
■ It is well established that microbial biofilms can impart physiological
resistance to antimicrobial treatment a thousand fold greater compared to
exposure to the same bacteria exposed as individual cells.

12. Resistance
■ Research indicates that B. cepacia strains exhibit random genetic changes that
can confer a high frequency of transmissibility, or given the right
epidemiologic circumstances, these altered genetic changes can benefit the
organism’s communal survival and pathogenicity.
■ Exchange of genes among different species for virulence factors might
increase adaptability and diminish the effectiveness of treatment and control.

13. Virulence
■ An enormous amount of research has been undertaken to define those
virulence factors, expressed by B. cepacia, which interact with the host, and
account for the greater morbidity and mortality.
■ Amongst virulence factors, endotoxin which has been clearly shown to have a
role in the pathogenesis of B. cepacia infection.
■ Lipopolysaccharide from clinical isolates has endotoxin activity and the
capacity to induce tumor necrosis factor (TNF-α) levels over nine times more
than endotoxin extracted from Pseudomonas aeruginosa.
■ This conforms to the evidence that CF infected B. cepacia patients, when
compared with P. aeruginosa colonized patients, have an up-regulated
inflammatory response when measuring plasma neutrophil elastase.

14. Virulence
■ Nitric oxide (NO) and hydrogen peroxide are important bactericidal mediators
in lung defense against B. cepacia.
■ Inducible NO synthetase is deficient in the bronchial epithelium of CF
patients.
■ This may contribute to their susceptibility to B. cepacia as a pulmonary
pathogen.

15. Complications
■ Bcc-contaminated products are most harmful to CF patients receiving lung
transplantations. The mortality rate is high.
- Mortality rates in the United Kingdom for the first year of infection were
reported at 50 –100%.
– Of the 11 patients with cystic fibrosis who were also infected with B.
cepacia complex, five died post-transplant because of progressive B.
cepacia related sepsis.
– All five patients were clinically unresponsive to cyclical antibiotics
and thoracostomy drainage.

16. Complications
- In Toronto, Canada
■ Of the 53 [transplant] recipients, 19 have died (15 of 28 [54%] B. cepacia
positive and 4 of 25 [16%] B. cepacia–negative).
■ One-year survival was 67% for B. cepacia–positive patients and 92% for
B. cepacia–negative patients.
- Another study in Liverpool, England
■ Thirty-seven patients had been colonized by epidemic B. cepacia and
these patients had four times the mortality of the remainder

17. Burkholderia
Cepacia Selective Agar (BCSA)
■ The slower growing Burkholderia cepacia can be missed on conventional media such as
blood or MacConkey Agar due to overgrowth caused by other faster growing
organisms found in the respiratory tract of CF patients such as Klebsiella species,
Pseudomonas aeruginosa and Staphylococcus species.
■ This may lead to the infection being missed or wrongly diagnosed.
■ The BCSA medium contains peptones and sugars that supply nutrients for the growth
of Burkholderia cepacia and other microorganisms.
■ All the bacteria in the BCC described to date are capable of growing on this medium.
■ Crystal violet and bile salts are added to inhibit growth of Gram-positive cocci.
■ Antimicrobials such as ticarcillin and polymyxin B used to inhibit the growth of other
Gram-negative bacilli

18. Technique and results of BCSA
■ Take a routine respiratory sample from the patient e.g. sputa, deep pharyngeal swabs or
bronchial washings, Specimens may also include blood.
■ Streak onto Burkholderia cepacia Medium and incubate at 37°C for 48 to 72 hours.
■ Examine after 48 hours for sage green colonies and the medium turning from straw-green
to bright pink.
■ All colonies should be further identified and confirmed. Re-incubate for a further 24 hours if
necessary.
■ Typical colonies of Burkholderia cepacia are circular, and entire.
■ Burkholderia cepacia colonies are typically translucent and rough.
■ Suspect colonies on BCSA medium generally appear greenish-brown with a yellowish halo
or white with a yellowish-pink halo (phenol red color indicator).
■ This method is presumptive and requires confirmation of the identification of the suspect
colonies.

19. Burkholderia
Cepacia Selective Agar
(BCSA)

20. PCR-based identification

21. PCR-based identification
■ Genomovars is a term commonly used within the genera Burkholderia and
Agrobacterium to denote strains which are phylogenetically differentiable, but are
phenotypically indistinguishable.
■ The group of B. cepacia complex (BCC) organisms consists of nine genomovars
associated with different levels of virulence and patient-to-patient transmissibility.
■ To reduce the probability of BCC spreading among patients with CF, a reliable early
test that detects small quantities of the bacteria in clinical samples is needed.
■ Conventional microbiological diagnostics of the BCC based on the results of culture
and subsequent biochemical identification is insufficient because of the potential risk
of misidentification or false negativity.
■ Also, Conventional methods are not able to reliably distinguish the genomovars of the
BCC

22. PCR-based identification
■ Two target genes are commonly used for BCC analysis: the 16S rRNA gene and
the recA gene.
■ Although sequence variation in the 16S rRNA gene is generally useful for
differentiating bacterial species, among Bcc species there is limited sequence diversity
in this gene.
■ The variation within the rRNA operon is obviously too small to separate all members of
the B. cepacia complex.
■ Due to this discriminatory limitation, a novel PCR-based identification assay based on
the recA gene developed.
■ The recA gene sequence variations are a useful means of differentiating genomovars,
and have become one of the main Bcc identification method.
■ The recA gene shows 94 to 95% similarity between the different genomovars, and
typically 98 to 99% similarity can be found within the genomovars.

23. Prevention
■ The best way to prevent the spread of B. cepacia, and all infections, is to clean your hands
often. This includes washing hands with soap and water or using an alcohol-based hand rub.
■ Health care workers should follow specific infection control precautions.
■ Patients and health care workers should clean their hands often.

24. Sources
■ Mahenthiralingam, E., Urban, T. A., & Goldberg, J. B. (2005). The multifarious,
multireplicon Burkholderia cepacia complex. Nature Reviews Microbiology,
3(2), 144–156. doi:10.1038/nrmicro1085.
■ Coenye, T., Vandamme, P., Govan, J. R. W., & LiPuma, J. J. (2001). Taxonomy
and Identification of the Burkholderia cepacia Complex. Journal of Clinical
Microbiology, 39(10), 3427–3436. doi:10.1128/jcm.39.10.3427-3436.2001.
■ Torbeck, L., Raccasi, D., Guilfoyle, D. E., Friedman, R. L., & Hussong, D.
(2011). Burkholderia cepacia: This Decision Is Overdue. PDA Journal of
Pharmaceutical Science and Technology, 65(5), 535–
543. doi:10.5731/pdajpst.2011.00793.
■ A.M. Jones, M.E. Dodd, A.K. Webb. (2001) Burkholderia cepacia: current
clinical issues, environmental controversies and ethical dilemmas. European
Respiratory Journal 2001 17: 295-301.