Burkholderia pseudomallei
Encyclopedia
Burkholderia pseudomallei (also known as Pseudomonas pseudomallei) is a Gram-negative
, bipolar, aerobic
, motile rod-shaped bacterium. It infects humans and animals and causes the disease melioidosis
. It is also capable of infecting plants.
B. pseudomallei measures 2-5 μm in length and 0.4-0.8 μm in diameter and are capable of self-propulsion using flagellae. The bacteria can grow in a number of artificial nutrient environments, especially betaine- and arginine
-containing.
in vitro
, optimal proliferation temperature is reported around 40°C in pH
-neutral or slightly acidic environments (pH 6.8–7.0). The majority of strains are capable of fermentation
of sugar
s without gas formation (most importantly, glucose
and galactose
, older cultures are reported to also metabolize maltose
and starch
). Bacteria produce both exo-
and endo-
toxins. The role of the toxins identified in the process of melioidosis symptom development has not been fully elucidated.
, EMB
, etc.). Ashdown's medium
(or Burkholderia cepacia medium) may be used for selective isolation.
Cultures typically become positive in 24 to 48 hours (this rapid growth rate differentiates the organism from B. mallei
, which typically takes a minimum of 72 hours to grow). Colonies are wrinkled, have a metallic appearance, and possess an earthy odour. On Gram staining
, the organism is a Gram-negative
rod with a characteristic "safety pin" appearance (bipolar staining). On sensitivity testing, the organism appears highly resistant (it is innately resistant to a large number of antibiotics including colistin
and gentamicin
) and that again differentiates it from B. mallei, which is in contrast, exquisitely sensitive to a large number of antibiotics. For environmental specimens only, differentiation
from the non-pathogenic B. thailandensis
using an arabinose
test is necessary (B. thailandensis is never isolated from clinical specimens). The laboratory identification of B. pseudomallei has been described in the literature.
The classic textbook description of B. pseudomallei in clinical samples is of a intracellular bipolar-staining Gram-negative rod, but this is of little value in identifying the organism from clinical samples. It has been suggested by some that the Wayson stain
is useful for this purpose, but this has been shown not to be the case.
Laboratory identification of B. pseudomallei can be difficult, especially in Western countries where B. pseudomallei is rarely seen. The large wrinkled colonies look like environmental contaminants and are therefore often discarded as being of no clinical significance. The organism grows more slowly than other bacteria that may be present in clinical specimens, and in specimens from non-sterile sites, is easily overgrown. Non-sterile specimens should therefore be cultured in selective media (e.g., Ashdown's or B. cepacia medium. Even when the isolate is recognised to be significant, commonly used identification systems may misidentify the organism as Chromobacterium violaceum
or other non-fermenting gram-negative bacilli such as Burkholderia cepacia or Pseudomonas aeruginosa
. Again, because the disease is rarely seen in western countries, identification of the bacterium B. pseudomallei in cultures may not actually trigger alarm bells in physicians unfamiliar with the disease. Routine biochemical methods for identification of bacteria vary widely in their identification of this organism: the API 20NE system accurately identifies B. pseudomallei in 99% of cases, as does the automated Vitek 1 system, but the automated Vitek 2 system only identifies 19% of isolates.
The pattern of resistance to antimicrobials is distinctive, and helps to differentiate the organism from P. aeruginosa. The majority of B. pseudomallei isolates are intrinsically resistant to all aminoglycosides (via an efflux pump mechanism), but sensitive to co-amoxiclav: this pattern of resistance almost never occurs in P. aeruginosa and is helpful in identification.
Molecular methods (PCR
) of diagnosis are possible, but not routinely available for clinical diagnosis. Fluorescence in situ hybridisation has also been described, but has not been clinical validated and it not commercially available . In Thailand, a latex agglutination assay is widely used. A rapid immunofluorescence technique is also available in a small number of centres in Thailand.
, iodine
, mercuric chloride, potassium permanganate
, 1% sodium hypochlorite, 70% ethanol
, 2% glutaraldehyde
and to a lesser extent, phenolic preparations. B. pseudomallei is effectively killed by the commercial disinfectants, Perasafe® and Virkon®. The microorganism can also be destroyed by heating to above 74°C for 10 min or by UV irradiation. B. pseudomallei is not reliably disinfected by chlorine.
. The mortality of melioidosis is 20 to 50% even with treatment.
. While various antibiotic
s are active in vitro (e.g., chloramphenicol
, doxycycline
, co-trimoxazole
), they have been proven to be inferior in vivo for the treatment of acute melioidosis. Disc diffusion tests are unreliable when looking for co-trimoxazole resistance in B. pseudomallei (they greatly over-estimate resistance) and Etests or agar dilution tests should be used in preference. The actions of co-trimoxazole and doxycycline are antagonistic, which suggests that these two drugs ought not to be used together.
The organism is intrinsically resistant to gentamicin
and to colistin
, and this fact is helpful in the identification of the organism. Kanamycin
is used to kill B. pseudomallei in the laboratory, but the concentrations used are much higher than those achievable in humans.
Strains which cause disease in humans differ from those causing disease in other animals by possessing certain genomic islands. It may have the ability to cause disease in humans because of DNA it has acquired from other microorganisms. The mutation rate is also high, and the organism continues to evolve even after infecting the host.
B. pseudomallei is able to invade cells (it is an intracellular pathogen). It is able to polymerise actin
and to spread from cell to cell, causing cell fusion and the formation of multinucleate giant cells. The bacterium also expresses a toxin called lethal factor 1. B. pseudomallei is one of the first proteobacteria to be identified as containing an active Type 6 secretion system. it is also the only organism identified that contains up to six different type 6 secretion systemsz.
B. pseudomallei is intrinsically resistant to a large number of antimicrobial agents. One important mechanism is that it is able to pump drugs out of the cell, and this mediates resistance to aminoglycosides (AmrAB-OprA), tetracyclines, fluoroquinolones and macrolides (BpeAB-OprB).
, methionine
, and threonine
in minimal medium. The Δasd bacterium (bacterium with the asd gene removed) protects against inhalational melioidosis in mice.
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
, bipolar, aerobic
Aerobic organism
An aerobic organism or aerobe is an organism that can survive and grow in an oxygenated environment.Faculitative anaerobes grow and survive in an oxygenated environment and so do aerotolerant anaerobes.-Glucose:...
, motile rod-shaped bacterium. It infects humans and animals and causes the disease melioidosis
Melioidosis
Melioidosis is an infectious disease caused by a Gram-negative bacterium, Burkholderia pseudomallei, found in soil and water. It is of public health importance in endemic areas, particularly in Thailand and northern Australia. It exists in acute and chronic forms. Symptoms may include pain in...
. It is also capable of infecting plants.
B. pseudomallei measures 2-5 μm in length and 0.4-0.8 μm in diameter and are capable of self-propulsion using flagellae. The bacteria can grow in a number of artificial nutrient environments, especially betaine- and arginine
Arginine
Arginine is an α-amino acid. The L-form is one of the 20 most common natural amino acids. At the level of molecular genetics, in the structure of the messenger ribonucleic acid mRNA, CGU, CGC, CGA, CGG, AGA, and AGG, are the triplets of nucleotide bases or codons that codify for arginine during...
-containing.
in vitro
In vitro
In vitro refers to studies in experimental biology that are conducted using components of an organism that have been isolated from their usual biological context in order to permit a more detailed or more convenient analysis than can be done with whole organisms. Colloquially, these experiments...
, optimal proliferation temperature is reported around 40°C in pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
-neutral or slightly acidic environments (pH 6.8–7.0). The majority of strains are capable of fermentation
Fermentation (biochemistry)
Fermentation is the process of extracting energy from the oxidation of organic compounds, such as carbohydrates, using an endogenous electron acceptor, which is usually an organic compound. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen,...
of sugar
Sugar
Sugar is a class of edible crystalline carbohydrates, mainly sucrose, lactose, and fructose, characterized by a sweet flavor.Sucrose in its refined form primarily comes from sugar cane and sugar beet...
s without gas formation (most importantly, glucose
Glucose
Glucose is a simple sugar and an important carbohydrate in biology. Cells use it as the primary source of energy and a metabolic intermediate...
and galactose
Galactose
Galactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....
, older cultures are reported to also metabolize maltose
Maltose
Maltose , or malt sugar, is a disaccharide formed from two units of glucose joined with an αbond, formed from a condensation reaction. The isomer "isomaltose" has two glucose molecules linked through an α bond. Maltose is the second member of an important biochemical series of glucose chains....
and starch
Starch
Starch or amylum is a carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. This polysaccharide is produced by all green plants as an energy store...
). Bacteria produce both exo-
Exotoxin
An exotoxin is a toxin excreted by a microorganism, like bacteria, fungi, algae, and protozoa. An exotoxin can cause damage to the host by destroying cells or disrupting normal cellular metabolism. They are highly potent and can cause major damage to the host...
and endo-
Endotoxin
Endotoxins are toxins associated with some Gram-negative bacteria. An "endotoxin" is a toxin that is a structural molecule of the bacteria that is recognized by the immune system.-Gram negative:...
toxins. The role of the toxins identified in the process of melioidosis symptom development has not been fully elucidated.
Identification
B. pseudomallei is not fastidious and will grow on a large variety of culture media (blood agar, MacConkey agarMacConkey agar
MacConkey agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation.-Contents:It contains bile salts MacConkey agar is a culture medium designed to grow Gram-negative bacteria and stain them for lactose fermentation.-Contents:It contains bile salts...
, EMB
Eosin methylene blue
Eosin methylene blue is a slightly selective stain for Gram-negative bacteria. It is a blend of two stains, eosin and methylene blue in the ratio of 6:1...
, etc.). Ashdown's medium
Ashdown's medium
Ashdown's medium is a selective culture medium for the isolation and characterisation of Burkholderia pseudomallei .Ashdown's medium was first described by LR Ashdown in 1979....
(or Burkholderia cepacia medium) may be used for selective isolation.
Cultures typically become positive in 24 to 48 hours (this rapid growth rate differentiates the organism from B. mallei
Burkholderia mallei
Burkholderia mallei is a gram-negative bipolar aerobic bacterium, a Burkholderia-genus human and animal pathogen causing Glanders; the Latin name of this disease gave name to the causative agent species...
, which typically takes a minimum of 72 hours to grow). Colonies are wrinkled, have a metallic appearance, and possess an earthy odour. On Gram staining
Gram staining
Gram staining is a method of differentiating bacterial species into two large groups ....
, the organism is a Gram-negative
Gram-negative
Gram-negative bacteria are bacteria that do not retain crystal violet dye in the Gram staining protocol. In a Gram stain test, a counterstain is added after the crystal violet, coloring all Gram-negative bacteria with a red or pink color...
rod with a characteristic "safety pin" appearance (bipolar staining). On sensitivity testing, the organism appears highly resistant (it is innately resistant to a large number of antibiotics including colistin
Colistin
Colistin is a polymyxin antibiotic produced by certain strains of Bacillus polymyxa var. colistinus. Colistin is a mixture of cyclic polypeptides colistin A and B. Colistin is effective against most Gram-negative bacilli and is used as a lingerdoodle. It is one of the last-resort antibiotics for...
and gentamicin
Gentamicin
Gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms. However, gentamicin is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophila...
) and that again differentiates it from B. mallei, which is in contrast, exquisitely sensitive to a large number of antibiotics. For environmental specimens only, differentiation
from the non-pathogenic B. thailandensis
Burkholderia thailandensis
Burkholderia thailandensis is a non-fermenting motile Gram-negative bacillus that occurs naturally in soil. It is closely related to Burkholderia pseudomallei, but unlike B. pseudomallei, it only rarely causes disease in humans or animals. The lethal inoculum is approximately 1000 times higher...
using an arabinose
Arabinose
Arabinose is an aldopentose – a monosaccharide containing five carbon atoms, and including an aldehyde functional group.For biosynthetic reasons, most saccharides are almost always more abundant in nature as the "D"-form, or structurally analogous to D-glyceraldehyde.For sugars, the D/L...
test is necessary (B. thailandensis is never isolated from clinical specimens). The laboratory identification of B. pseudomallei has been described in the literature.
The classic textbook description of B. pseudomallei in clinical samples is of a intracellular bipolar-staining Gram-negative rod, but this is of little value in identifying the organism from clinical samples. It has been suggested by some that the Wayson stain
Wayson stain
The Wayson stain is a basic fuchsin-methylene blue, ethyl alcohol-phenol microscopic staining procedure. It was originally a modified methylene blue stain used for diagnosing bubonic plague...
is useful for this purpose, but this has been shown not to be the case.
Laboratory identification of B. pseudomallei can be difficult, especially in Western countries where B. pseudomallei is rarely seen. The large wrinkled colonies look like environmental contaminants and are therefore often discarded as being of no clinical significance. The organism grows more slowly than other bacteria that may be present in clinical specimens, and in specimens from non-sterile sites, is easily overgrown. Non-sterile specimens should therefore be cultured in selective media (e.g., Ashdown's or B. cepacia medium. Even when the isolate is recognised to be significant, commonly used identification systems may misidentify the organism as Chromobacterium violaceum
Chromobacterium violaceum
Chromobacterium violaceum is a Gram-negative, facultative anaerobic, non-sporing coccobacillus. It is part of the normal flora of water and soil of tropical and sub-tropical regions of the world. It produces a natural antibiotic called violacein, which may be useful for the treatment of colon and...
or other non-fermenting gram-negative bacilli such as Burkholderia cepacia or Pseudomonas aeruginosa
Pseudomonas aeruginosa
Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres, but also in hypoxic atmospheres, and has, thus, colonized many...
. Again, because the disease is rarely seen in western countries, identification of the bacterium B. pseudomallei in cultures may not actually trigger alarm bells in physicians unfamiliar with the disease. Routine biochemical methods for identification of bacteria vary widely in their identification of this organism: the API 20NE system accurately identifies B. pseudomallei in 99% of cases, as does the automated Vitek 1 system, but the automated Vitek 2 system only identifies 19% of isolates.
The pattern of resistance to antimicrobials is distinctive, and helps to differentiate the organism from P. aeruginosa. The majority of B. pseudomallei isolates are intrinsically resistant to all aminoglycosides (via an efflux pump mechanism), but sensitive to co-amoxiclav: this pattern of resistance almost never occurs in P. aeruginosa and is helpful in identification.
Molecular methods (PCR
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
) of diagnosis are possible, but not routinely available for clinical diagnosis. Fluorescence in situ hybridisation has also been described, but has not been clinical validated and it not commercially available . In Thailand, a latex agglutination assay is widely used. A rapid immunofluorescence technique is also available in a small number of centres in Thailand.
Disinfection
B. pseudomallei is susceptible to numerous disinfectants including benzalkonium chlorideBenzalkonium chloride
Benzalkonium chloride, also known as alkyldimethylbenzylammonium chloride and ADBAC, is a mixture of alkylbenzyldimethylammonium chlorides of various even-numbered alkyl chain lengths. This product is a nitrogenous cationic surface-acting agent belonging to the quaternary ammonium group...
, iodine
Iodine
Iodine is a chemical element with the symbol I and atomic number 53. The name is pronounced , , or . The name is from the , meaning violet or purple, due to the color of elemental iodine vapor....
, mercuric chloride, potassium permanganate
Potassium permanganate
Potassium permanganate is an inorganic chemical compound with the formula KMnO4. It is a salt consisting of K+ and MnO4− ions. Formerly known as permanganate of potash or Condy's crystals, it is a strong oxidizing agent. It dissolves in water to give intensely purple solutions, the...
, 1% sodium hypochlorite, 70% ethanol
Ethanol
Ethanol, also called ethyl alcohol, pure alcohol, grain alcohol, or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug and one of the oldest recreational drugs. Best known as the type of alcohol found in alcoholic beverages, it is also used in thermometers, as a...
, 2% glutaraldehyde
Glutaraldehyde
Glutaraldehyde is an organic compound with the formula CH22. A pungent colorless oily liquid, glutaraldehyde is used to disinfect medical and dental equipment...
and to a lesser extent, phenolic preparations. B. pseudomallei is effectively killed by the commercial disinfectants, Perasafe® and Virkon®. The microorganism can also be destroyed by heating to above 74°C for 10 min or by UV irradiation. B. pseudomallei is not reliably disinfected by chlorine.
Medical importance
B. pseudomallei infection in humans is called melioidosisMelioidosis
Melioidosis is an infectious disease caused by a Gram-negative bacterium, Burkholderia pseudomallei, found in soil and water. It is of public health importance in endemic areas, particularly in Thailand and northern Australia. It exists in acute and chronic forms. Symptoms may include pain in...
. The mortality of melioidosis is 20 to 50% even with treatment.
Antibiotic treatment and sensitivity testing
The antibiotic of choice is ceftazidimeCeftazidime
Ceftazidime is a third-generation cephalosporin antibiotic. Like other third-generation cephalosporins, it has broad spectrum activity against Gram-positive and Gram-negative bacteria. Unlike most third-generation agents, it is active against Pseudomonas aeruginosa, however it has weaker activity...
. While various antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
s are active in vitro (e.g., chloramphenicol
Chloramphenicol
Chloramphenicol is a bacteriostatic antimicrobial that became available in 1949. It is considered a prototypical broad-spectrum antibiotic, alongside the tetracyclines, and as it is both cheap and easy to manufacture it is frequently found as a drug of choice in the third world.Chloramphenicol is...
, doxycycline
Doxycycline
Doxycycline INN is a member of the tetracycline antibiotics group, and is commonly used to treat a variety of infections. Doxycycline is a semisynthetic tetracycline invented and clinically developed in the early 1960s by Pfizer Inc. and marketed under the brand name Vibramycin. Vibramycin...
, co-trimoxazole
Co-trimoxazole
Trimethoprim/sulfamethoxazole or co-trimoxazole is a sulfonamide antibiotic combination of trimethoprim and sulfamethoxazole, in the ratio of 1 to 5, used in the treatment of a variety of bacterial infections.The name co-trimoxazole is the British Approved Name, and has been marketed worldwide...
), they have been proven to be inferior in vivo for the treatment of acute melioidosis. Disc diffusion tests are unreliable when looking for co-trimoxazole resistance in B. pseudomallei (they greatly over-estimate resistance) and Etests or agar dilution tests should be used in preference. The actions of co-trimoxazole and doxycycline are antagonistic, which suggests that these two drugs ought not to be used together.
The organism is intrinsically resistant to gentamicin
Gentamicin
Gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms. However, gentamicin is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophila...
and to colistin
Colistin
Colistin is a polymyxin antibiotic produced by certain strains of Bacillus polymyxa var. colistinus. Colistin is a mixture of cyclic polypeptides colistin A and B. Colistin is effective against most Gram-negative bacilli and is used as a lingerdoodle. It is one of the last-resort antibiotics for...
, and this fact is helpful in the identification of the organism. Kanamycin
Kanamycin
Kanamycin sulfate is an aminoglycoside antibiotic, available in oral, intravenous, and intramuscular forms, and used to treat a wide variety of infections. Kanamycin is isolated from Streptomyces kanamyceticus.-Mechanism:...
is used to kill B. pseudomallei in the laboratory, but the concentrations used are much higher than those achievable in humans.
Pathogenicity mechanisms and virulence factors
B. pseudomallei is an "accidental pathogen". It is an environmental organism that has no requirement to pass through an animal host in order to replicate. From the point of view of the bacterium, human infection is an evolutionary "dead end".Strains which cause disease in humans differ from those causing disease in other animals by possessing certain genomic islands. It may have the ability to cause disease in humans because of DNA it has acquired from other microorganisms. The mutation rate is also high, and the organism continues to evolve even after infecting the host.
B. pseudomallei is able to invade cells (it is an intracellular pathogen). It is able to polymerise actin
Actin
Actin is a globular, roughly 42-kDa moonlighting protein found in all eukaryotic cells where it may be present at concentrations of over 100 μM. It is also one of the most highly-conserved proteins, differing by no more than 20% in species as diverse as algae and humans...
and to spread from cell to cell, causing cell fusion and the formation of multinucleate giant cells. The bacterium also expresses a toxin called lethal factor 1. B. pseudomallei is one of the first proteobacteria to be identified as containing an active Type 6 secretion system. it is also the only organism identified that contains up to six different type 6 secretion systemsz.
B. pseudomallei is intrinsically resistant to a large number of antimicrobial agents. One important mechanism is that it is able to pump drugs out of the cell, and this mediates resistance to aminoglycosides (AmrAB-OprA), tetracyclines, fluoroquinolones and macrolides (BpeAB-OprB).
Vaccine candidates
There are no vaccine currently available, but a number of vaccines candidates have been suggested. Aspartate-β-semialdehyde dehydrogenase (asd) gene deletion mutants are auxotrophic for diaminopimelate (DAP) in rich medium and auxotrophic for DAP, lysineLysine
Lysine is an α-amino acid with the chemical formula HO2CCH4NH2. It is an essential amino acid, which means that the human body cannot synthesize it. Its codons are AAA and AAG....
, methionine
Methionine
Methionine is an α-amino acid with the chemical formula HO2CCHCH2CH2SCH3. This essential amino acid is classified as nonpolar. This amino-acid is coded by the codon AUG, also known as the initiation codon, since it indicates mRNA's coding region where translation into protein...
, and threonine
Threonine
Threonine is an α-amino acid with the chemical formula HO2CCHCHCH3. Its codons are ACU, ACA, ACC, and ACG. This essential amino acid is classified as polar...
in minimal medium. The Δasd bacterium (bacterium with the asd gene removed) protects against inhalational melioidosis in mice.
External links
- Burkholderia pseudomallei genomes and related information at PATRIC, a Bioinformatics Resource Center funded by NIAID
- Getting a Grip on the Great Mimicker: Secrets of a Stealth Organism from the Wellcome Trust.
- Pathema Burkholderia resource