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Advances in Burkholderia cepacia complex
PAEDIATRIC RESPIRATORY REVIEWS (2002) 3, 230±235 doi: 10.1016/S1526±0542(02)00185-9, available online at http://www.idealibrary.com on
SERIES: NEW BIOLOGY OF THE AIRWAYS
Advances in Burkholderia cepacia complex David Paul Speert Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia and British Columbia's Children's and Women's Health Centre and the Canadian Bacterial Diseases Network, Room 377, Research Centre, 950 West 28th Avenue, Vancouver, BC, V5Z 4H4, Canada KEYWORDS Burkholderia cepacia, Burkholderia cepacia complex, cystic ®brosis, chronic granulomatous disease, epidemiology, antibiotic resistance, quorum sensing, bio®lms, multiple combination antimicrobial testing
Summary Burkholderia cepacia is an important opportunistic pathogen in certain compromised hosts, particularly those with either cystic ®brosis (CF) or chronic granulomatous disease. The ``family'' of bacteria known as B. cepacia is highly heterogeneous and is composed of at least nine discrete species or genomovars, constituting the B. cepacia complex. Bacteria from the B. cepacia complex are particularly virulent in susceptible hosts, often causing necrotising invasive infection and death. Whereas the microbial determinants of virulence in B. cepacia complex are currently not de®ned, the bacteria appear to have features facilitating survival within host cells. Burkholderia cepacia is highly resistant to antibiotics and to neutrophil-mediated non-oxidative killing; infection should be treated with combination antimicrobial therapy. Burkholderia cepacia can spread from one CF patient to another. Transmission appears to be facilitated by close personal contact and by certain bacterial factors. Published by Elsevier Science Ltd.
INTRODUCTION Burkholderia (formerly Pseudomonas) cepacia comprises a complex of bacteria of evolving importance in certain patients with compromised immunity. What had previously been considered to be a single species, is now recognised to be a highly heterogeneous group of bacteria that is referred to as the B. cepacia complex. This complex comprises at least nine discrete groups, each of which is suf®ciently different to constitute a unique species. Species designation has been assigned to those that are physically (phenotypically) unique. Those groups of bacteria within the B. cepacia complex that are unique genetically but dif®cult to differentiate phenotypically are de®ned as genomovars. Burkholderia cepacia complex was initially described as a pathogen of plants, causing sour skin in onions.1 More recently, it has evolved as a particularly virulent pathogen in certain patients with cystic ®brosis (CF)2 and in chronic granulomatous disease (CGD).3,4 Furthermore, it has the capacity to spread readily among patients with CF.5 The purpose of this article is to examine the changing face of this evolving human opportunistic pathogen. I will brie¯y describe the human infections caused by B. cepacia complex, review the evolving taxonomy, summarise the 1526±0542/02/$ ± see front matter
epidemiology (particularly in patients with CF) and summarise current concepts of its virulence mechanisms. Finally, I will suggest therapeutic strategies for infections with this complex of micro-organisms with high intrinsic antibiotic resistance and raise some questions that seem most pressing for investigative solution.
SPECTRUM OF DISEASES CAUSED BY B. CEPACIA COMPLEX Burkholderia cepacia complex is an opportunistic pathogen ± it causes disease only in hosts whose capacity to resist infection is impaired. It is also an opportunistic phytopathogen, preying on damaged onions in particular.6 The two human diseases in which B. cepacia infection is predominantly seen are CF7 and CGD.3,4 Whereas these two diseases are quite dissimilar, commonalities between them may shed some light on the pathogenetic mechanisms of B. cepacia. CGD is an inborn error of leukocyte function in which phagocytic cells are unable to generate bactericidal reactive oxygen radicals.8 Bacteria and other potential pathogens can be ingested normally but they cannot be killed by oxidative means.9 Patients with CGD are susceptible to Published by Elsevier Science Ltd.
ADVANCES IN BURKHOLDERIA CEPACIA COMPLEX infection with a relatively narrow range of micro-organisms. Burkholderia cepacia complex has evolved as the most virulent Gram-negative bacterial pathogen.3 Phagocytic cells are armed to kill their ingested prey by either oxidative or non-oxidative means. The former is disabled in CGD and the latter depends upon the direct action of cationic peptides (``nature's own antibiotics''), which reside in lysosomes but attack ingested microbes. Since oxidative killing is disabled in CGD, the cells must rely on their non-oxidative defences. Burkholderia cepacia complex bacteria are remarkably resistant to non-oxidative killing,10 explaining why they are such successful pathogens in CGD. Burkholderia cepacia infections in patients with CGD are remarkably aggressive and many fatalities have been reported. Respiratory infection is the most common manifestation of disease and bacteraemic spread may occur. Bacteraemia in CGD is relatively uncommon with other organisms, suggesting that B. cepacia has virulence determinants that set it apart from other CGD pathogens. CF is another inborn defect in cell function but it is quite different from CGD. Epithelial cells from patients with CF have defective chloride channel function due to mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR).11 These patients are highly susceptible to infection with Pseudomonas aeruginosa.12 However, B. cepacia complex has evolved as a particularly virulent pathogen in these patients over the past two decades.7 A clear explanation for the tropism of B. cepacia for the CF lung has not yet been found but clues may come from the observations in patients with CGD. It is possible that there exists an oxidant/antioxidant imbalance in the CF lung protecting the bacteria from oxidative killing. The mucoid exopolysaccharide of P. aeruginosa is able to quench oxidative radicals;13 this may provide a privileged and protected niche for B. cepacia to reside, replicate and cause disease after a chronic infection with mucoid P. aeruginosa has been established. The prevalence of B. cepacia infection in patients with CF varies markedly among treatment centres, ranging from zero to about 40%. Furthermore, the severity of disease is highly variable, even in patients infected with the same strain of B. cepacia. A minority of patients with CF who become infected with B. cepacia experience a rapid deterioration in clinical status with systemic toxicity, bacteraemia and death;2 this is known as the ``cepacia syndrome'' and is distinctly different from the disease associated with P. aeruginosa infection in CF. Patients with CF who become infected with B. cepacia experience deterioration of lung function more rapidly than those infected with P. aeruginosa alone or with neither of these two pathogens.14 Patients with CF undergoing lung transplantation do substantially worse if they are infected with B. cepacia prior to transplantation15 and certain strains of B. cepacia are associated with a particularly poor prognosis.16
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TAXONOMY The classi®cation of members of the B. cepacia complex has evolved dramatically over the past decade. Changes have derived from the fact that bacterial strains can now be compared to one another on the basis of their genetic (genotypic) rather than only their physical (phenotypic) properties. Genus and species designation are therefore determined by comparing the genetic endowment of groups of bacteria, which may appear to be very similar phenotypically but prove to be quite different genotypically. New information, derived from such genetic analysis, allows the reclassi®cation of bacteria and their assignment to novel genera and species. This process of re-classi®cation is clearly evident in the taxonomic history of B. cepacia complex. This diverse group of bacteria phenotypically resembles P. aeruginosa and was originally called Pseudomonas cepacia. The species was re-analysed by Yabuuchi et al.17 and was found to be quite distinct genetically from other species in the Pseudomonas genus; it was therefore re-classi®ed to the novel genus Burkholderia (along with several other species), in honour of William Burkholder who had originally described the cause of sour skin in onions.1 Subsequently, Vandamme and colleagues18 have determined that the ``species'' B. cepacia is actually a complex of multiple genetically distinct groups of bacteria differing from one another suf®ciently to warrant individual unique species designation. Those groups of bacteria within the complex that have a unique phenotype (allowing them to be distinguished from others on the basis of physical traits) have been given species names (e.g. Burkholderia multivorans; formerly genomovar II). Those groups that cannot yet be distinguished phenotypically, but are unique from the other species genotypically are called ``genomovars'' (e.g. genomovar III). To date nine genomovars/species have been described within the B. cepacia complex (see Table 1). Genomovar III and Burkholderia multivorans are the predominant species recovered from patients with CF.19 A complete analysis of the taxonomy of B. cepacia is beyond the scope of this review but has been described recently.20
IDENTIFICATION OF B. CEPACIA Members of the B. cepacia complex can be very dif®cult to identify in the clinical diagnostic laboratory. Prior to the advent of enhanced culture and identi®cation techniques in the last decade, B. cepacia was often misidenti®ed, even in laboratories that handled a large volume of CF sputum samples. Identi®cation is now facilitated by the use of specialised selective agar.21 Other bacterial species with which B. cepacia has been confused include Stenotrophomonas maltophilia and many other Gram-negative organisms.22,23 When a CF patient is ®rst infected with a putative member of the B. cepacia complex, it is critically important that the organism's identity be con®rmed. Specialised
232 Table 1
D. P. SPEERT Genomovars/species of the Burkholderia cepacia complex.
Species designation Transmissibility in patients with CF Frequency of recovery from patients with CF Phytopathogenicity Recovery from patients with CGD
Genomovar I
Genomovar II
Burkholderia cepacia
Genomovar III
Genomovar IV
Genomovar V
Genomovar VI
Genomovar VII
Genomovar VIII
Genomovar IX
Burkholderia multivorans
Burkholderia stabilis
Burkholderia vietnamiensis
Burkholderia ambifaria
Burkholderia anthina
Burkholderia pyrrocinia
?
? Yes
? Yes
? ?
? Yes
? ?
? ?
? ?
? ?
CF, cystic fibrosis; CGD, chronic granulomatous disease.
research and referral laboratories exist in the USA, Canada and the UK to which isolates can be sent for con®rmation of identity, for genomovar/species identi®cation and for genetic typing. Location of the Canadian and American laboratories can be found on the International Burkholderia cepacia Working Group website (http://allserv.rug.ac.be/ ppvandam/cepacia/). Subsequent isolates from the same patient that appear to be the same phenotypically as the initial isolate may be assumed to be the same strain and treated as such.
EPIDEMIOLOGY An interest in the epidemiology of B. cepacia has been heightened by the observations from several groups that B. cepacia can be spread from one CF patient to another.24 Although the mechanism of spread had not been determined, the adverse consequences of acquisition can be profound. It appears that certain strains and genomovars/species of the B. cepacia complex are more readily transmitted than others.19,25 The factors underlying patient-to-patient spread are currently under investigation.
Typing methods Epidemiological investigation of any bacterium is dependent upon the capacity to determine if two isolates are from the same strain or not. If they are from the same strain (i.e. are genetically almost identical) one can conclude that they have been derived from the same source or have been spread from one patient to another. Historically, the typing of bacterial strains has been based upon the degree of phenotypic (physical) relatedness. Such methods have proven to be unreliable in CF because genetically diverse groups may appear to be identical physically and phenotypically different isolates may be genetically homologous.26 With the advent of molecular microbiology as a discipline, the capacity to compare bacteria in terms of genetic relatedness has become possible. Various methods have been employed to type B. cepacia genotypically. The method reported initially for typing B. cepacia was ribotyp-
ing and enabled LiPuma and co-workers5 to determine that B. cepacia could be spread from one CF patient to another. Subsequent investigations, based upon whole genome analysis, have con®rmed the ®rst observations. The most widely used typing methods are now pulsed ®eld gel electrophoresis and random ampli®ed polymorphic DNA analysis.25
Transmissibility factors The observation that B. cepacia could be transmitted among patients and that some strains (but not others) are clustered in certain CF centres, suggested that these ``epidemic'' strains encode factors that enhance transmissibility. The ®rst such putative factor was identi®ed in the strain of B. cepacia that infects a large number of patients in the United Kingdom and Canada. Spread of this strain has been associated with contact among patients both within and outside the hospital and it is now designated ET12. Isolates from this strain elaborate a unique cable pilus27 that appears to enhance adhesion to epithelial cells.28 A role for the cable pilus in patient-to-patient spread had been suggested but not yet formally proven. The gene for the cable pilus is found predominantly, but not exclusively, in strain ET12. A second putative transmissibility factor is the B. cepacia epidemic strain marker (BCESM);29 this gene appears to be a negative transcriptional regulator and is encoded by many, but not all, ``epidemic'' strains that cluster in individual CF treatment centres. The role of BECSM in transmission or virulence of B. cepacia has not been determined.
Evidence for patient-to-patient spread The literature on B. cepacia epidemiology in CF is now replete with descriptions of patient-to-patient spread. Clustering of strains in patients by geographical location has been described in the USA,30 Canada,19 the United Kingdom,31 Australia32 and Italy.33 It appears that direct personal contact is required for patient-to-patient spread, suggesting that droplet rather than aerosol transmission is responsible. Speci®c activities, which enhance the
ADVANCES IN BURKHOLDERIA CEPACIA COMPLEX likelihood that B. cepacia will be spread from one CF patient to another, have not yet been determined. Until more is learned about the risk factors for patient-to-patient spread, it is prudent to restrict interaction between patients infected with B. cepacia and those who are culturenegative. Furthermore, there is evidence that certain strains of B. cepacia can replace others when patients with different strains are cohorted together, and such replacement can be associated with a poor clinical outcome.25 It is therefore inadvisable to cohort B. cepacia culture-positive CF patients and they should be discouraged from having any direct contact with one another.
VIRULENCE There is a wide variation in disease severity among CF patients infected with B. cepacia. Certain strains seem to be associated with a more severe illness than others.16,25 These observations have raised the possibility that only some strains are endowed with speci®c features that suit them better for human disease. The virulence determinants (disease-producing capacity of bacterial factors) from B. cepacia have been sought by several groups of investigators and reviewed by Nelson et al.34 The investigative efforts are ongoing and updated regularly by the International Burkholderia cepacia Working Group on their website (http:// allserv.rug.ac.be/ppvandam/cepacia/). A wide array of putative virulence determinants have been described but none has yet ``won its spurs'' in a fashion analagous to the capsular polysaccharide of Haemophilus in¯uenzae type b or the toxin of Clostridium tetani. Among the candidate virulence determinants are pili (particularly the cable pilus), lipopolysaccharide (endotoxin), protease, haemolysin, phospholipase C, siderophores, melanin pigment and exopolysaccharide. Since none of these individual factors has been clearly shown to enhance disease severity in humans, features of the bacteria as a whole have been investigated as described, below.
Intracellular parasitism Burkholderia cepacia complex is closely related to a bone ®de intracellular pathogen, B. pseudomallei,35 which causes melioidosis, a disease that is very similar clinically to tuberculosis. This raises the possibility (which has been suggested by a number of investigators) that B. cepacia owes its virulence, in part, to its capacity for intracellular parasitism. Burkholderia cepacia can invade epithelial cells36 and can be found within host cells of infected mice.37 A recent report correlated the capacity of strains to penetrate epithelial cells with mouse infectivity.38 Burkholderia cepacia can also survive within macrophages39 and in amoebae.40 The latter cell is particularly interesting, since it could provide insights into the reservoir within which B. cepacia survives in the environment. If B. cepacia does indeed have the capacity for intracellular parasitism, this raises important issues with
233 regard to therapy; the optimal drug would be one that is able to penetrate eukaryotic cells.
Resistance to non-oxidative killing Infections with B. cepacia complex bacteria are particularly frustrating to treat because of their high level intrinsic resistance to many antibiotics, including the cationic antibiotic polymixin B.41 Resistance to cationic antimicrobial peptides is one feature shared by all members of the B. cepacia complex and this renders them resistant to nonoxidative killing by phagocytic cells10 (see above). In addition to facilitating their capacity to cause infection in patients with CGD, this enhances their infectivity under conditions in which the generation of reactive oxygen radicals is compromised. Macrophages are far less competent than polymorphonuclear leukocytes in elaborating toxic oxygen radicals; this may explain why B. cepacia are able to persist inside macrophages, perhaps enhancing their capacity for intracellular parasitism. Under other conditions in which reactive oxygen intermediates are quenched, B. cepacia would appear to be better suited to cause infection than those bacterial species that are susceptible to non-oxidative killing.
Quorum sensing and biofilm formation Many bacterial infections evolve in the form of communities of bacteria (bio®lms) in which there is a type of communication among the individual bacteria when they sense a ``quorum''.42 This phenomenon of quorum sensing occurs when the bacteria achieve a certain density. It is associated with enhanced resistance to antimicrobial agents43 and with the elaboration of quorum sensing molecules (known as autoinducers).44 These autoinducers, which are acylated homoserine lactones (AHL), have substantial pleiotropic effects on the expression of various genes. Several of the genes in P. aeruginosa, the expression of which is controlled in part by autoinducers, are considered to be virulence determinants. Thus bacterial density (as occurs when bio®lms develop) is probably very important in dictating the level of toxin production as well as antibiotic resistance during chronic P. aeruginosa infection, as occurs in CF. Bacteria from all of the B. cepacia complex genomovars encode the gene(s) for elaboration of AHLs.45 We have recently demonstrated that all of these bacteria also elaborate AHLs but to greatly different degrees (B. Conway & D.P. Speert, unpublished results). Many of the strains also form bio®lms but there is no correlation between the extent of bio®lm formation and the extent of AHL production. Another study has shown that in one strain there was a direct correlation between AHL production and bio®lm formation.46 Since B. cepacia causes chronic infection in some patients with CF and since bio®lms are such an important feature of chronic infections, the role of quorum sensing in B. cepacia infection deserves further investigation.
234
THERAPY Therapy of infections with B. cepacia complex is fraught with problems that result from the organism's very high level of intrinsic resistance to many antibiotics and its capacity to develop further resistance during therapy. This problem of induced resistance during therapy is particularly problematic in CF patients, in whom chronic infection is a hallmark of the disease. The cell envelope of B. cepacia is unusual among Gramnegative bacteria by virtue of its unique lipopolysaccharide, a molecule that is a major constituent of the outer membrane. The unusual chemical composition of the lipopolysaccharide is thought to render the bacteria intrinsically resistant to aminoglycosides (such as gentamicin and tobramycin) and to polymixins.47 In fact, one of the differential features used to aid in the identi®cation of B. cepacia in diagnostic laboratories is this high level intrinsic resistance. Many strains of B. cepacia are resistant to a multitude of antibiotics, limiting the choice of therapeutic options. It is not uncommon for patients with CF who have been infected with B. cepacia for a prolonged period and who have received multiple courses of antibiotics, to be infected with a strain of B. cepacia that is resistant to all major classes of antibiotics. Antimicrobial agents to which B. cepacia bacteria are often susceptible include semisynthetic penicillins (such as ticarcillin), carbapenems (such as meropenem), cephalosporins (particularly ceftazidime), quinolones (such as cipro¯oxacin) and trimethoprim/sulfamethoxazole. It is not possible to predict a priori which antimicrobial agents will be effective against any particular isolate of B. cepacia, so each should be subjected to susceptibility testing. Furthermore, since resistance to antibiotics may develop during the course of therapy, any additional isolates from persistently infected patients should also undergo susceptibility testing. Therapy for B. cepacia in chronically infected CF patients must often be initiated before the results of antimicrobial susceptibility tests are available. Therapy should be based upon the most recent antimicrobial susceptibility results. Combination therapy should be used for serious infections and should ideally include drugs from two different classes to enhance the likelihood of achieving synergistic activity. Even though all strains of B. cepacia are resistant in vitro to aminglycosides, that class of antimicrobials appears to be bene®cial in treating B. cepacia infections when used in combination with other agents.41 Dual b-lactam therapy should be avoided whenever possible as it appears to induce expression of b-lactamases, resulting in apparent antagonism. Whereas combination therapy is advisable for serious B. cepacia infections, the effects of each drug on the activity of the other cannot be predicted by evaluating each in isolation of the others. Some combinations are synergistic, some merely additive and some antagonistic.41 In an effort to optimise antimicrobial therapy, some research laboratories are evaluating the use of multiple combination antimicrobial testing (MCT).41 These ``checkerboard'' evaluations permit
D. P. SPEERT one to determine the antimicrobial activity when more than one antibiotic is evaluated simultaneously. Either two or three drugs are mixed and their capacity to inhibit or kill the bacteria is assessed. From large in vitro studies of multiresistant isolates of B. cepacia, the following antibiotic combinations were found to be the best: meropenem±minocycline, meropenem±amikacin and meropenem±cefazidime.41
ACKNOWLEDGEMENTS Work in my laboratory is supported with funds from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes for Health Research, the Canadian Bacterial Diseases Network and the British Columbia Lung Association. I thank Deborah Henry for critically reviewing the manuscript.
RESEARCH DIRECTIONS Despite considerable growth in research on B. cepacia over the past decade, much more remains to be learned about this evolving opportunistic pathogen. Among the most pressing questions are the following: Why has B. cepacia, among all the environmental saprophytic bacteria, evolved as such an important pathogen in patients with cystic fibrosis (CF)? What microbial and host factors favour the spread of B. cepacia from one individual to another and how can the spread be prevented? How can acquisition of B. cepacia be prevented by compromised hosts, such as those with CF or chronic granulomatous disease (CGD)? What is the optimal therapy for patients infected with B. cepacia? What are the most important host factors that defend against infection with B. cepacia? What are the most important microbial factors that enhance the infectivity of B. cepacia?
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SERIES: NEW BIOLOGY OF THE AIRWAYS
Advances in Burkholderia cepacia complex David Paul Speert Division of Infectious and Immunological Diseases, Department of Pediatrics, University of British Columbia and British Columbia's Children's and Women's Health Centre and the Canadian Bacterial Diseases Network, Room 377, Research Centre, 950 West 28th Avenue, Vancouver, BC, V5Z 4H4, Canada KEYWORDS Burkholderia cepacia, Burkholderia cepacia complex, cystic ®brosis, chronic granulomatous disease, epidemiology, antibiotic resistance, quorum sensing, bio®lms, multiple combination antimicrobial testing
Summary Burkholderia cepacia is an important opportunistic pathogen in certain compromised hosts, particularly those with either cystic ®brosis (CF) or chronic granulomatous disease. The ``family'' of bacteria known as B. cepacia is highly heterogeneous and is composed of at least nine discrete species or genomovars, constituting the B. cepacia complex. Bacteria from the B. cepacia complex are particularly virulent in susceptible hosts, often causing necrotising invasive infection and death. Whereas the microbial determinants of virulence in B. cepacia complex are currently not de®ned, the bacteria appear to have features facilitating survival within host cells. Burkholderia cepacia is highly resistant to antibiotics and to neutrophil-mediated non-oxidative killing; infection should be treated with combination antimicrobial therapy. Burkholderia cepacia can spread from one CF patient to another. Transmission appears to be facilitated by close personal contact and by certain bacterial factors. Published by Elsevier Science Ltd.
INTRODUCTION Burkholderia (formerly Pseudomonas) cepacia comprises a complex of bacteria of evolving importance in certain patients with compromised immunity. What had previously been considered to be a single species, is now recognised to be a highly heterogeneous group of bacteria that is referred to as the B. cepacia complex. This complex comprises at least nine discrete groups, each of which is suf®ciently different to constitute a unique species. Species designation has been assigned to those that are physically (phenotypically) unique. Those groups of bacteria within the B. cepacia complex that are unique genetically but dif®cult to differentiate phenotypically are de®ned as genomovars. Burkholderia cepacia complex was initially described as a pathogen of plants, causing sour skin in onions.1 More recently, it has evolved as a particularly virulent pathogen in certain patients with cystic ®brosis (CF)2 and in chronic granulomatous disease (CGD).3,4 Furthermore, it has the capacity to spread readily among patients with CF.5 The purpose of this article is to examine the changing face of this evolving human opportunistic pathogen. I will brie¯y describe the human infections caused by B. cepacia complex, review the evolving taxonomy, summarise the 1526±0542/02/$ ± see front matter
epidemiology (particularly in patients with CF) and summarise current concepts of its virulence mechanisms. Finally, I will suggest therapeutic strategies for infections with this complex of micro-organisms with high intrinsic antibiotic resistance and raise some questions that seem most pressing for investigative solution.
SPECTRUM OF DISEASES CAUSED BY B. CEPACIA COMPLEX Burkholderia cepacia complex is an opportunistic pathogen ± it causes disease only in hosts whose capacity to resist infection is impaired. It is also an opportunistic phytopathogen, preying on damaged onions in particular.6 The two human diseases in which B. cepacia infection is predominantly seen are CF7 and CGD.3,4 Whereas these two diseases are quite dissimilar, commonalities between them may shed some light on the pathogenetic mechanisms of B. cepacia. CGD is an inborn error of leukocyte function in which phagocytic cells are unable to generate bactericidal reactive oxygen radicals.8 Bacteria and other potential pathogens can be ingested normally but they cannot be killed by oxidative means.9 Patients with CGD are susceptible to Published by Elsevier Science Ltd.
ADVANCES IN BURKHOLDERIA CEPACIA COMPLEX infection with a relatively narrow range of micro-organisms. Burkholderia cepacia complex has evolved as the most virulent Gram-negative bacterial pathogen.3 Phagocytic cells are armed to kill their ingested prey by either oxidative or non-oxidative means. The former is disabled in CGD and the latter depends upon the direct action of cationic peptides (``nature's own antibiotics''), which reside in lysosomes but attack ingested microbes. Since oxidative killing is disabled in CGD, the cells must rely on their non-oxidative defences. Burkholderia cepacia complex bacteria are remarkably resistant to non-oxidative killing,10 explaining why they are such successful pathogens in CGD. Burkholderia cepacia infections in patients with CGD are remarkably aggressive and many fatalities have been reported. Respiratory infection is the most common manifestation of disease and bacteraemic spread may occur. Bacteraemia in CGD is relatively uncommon with other organisms, suggesting that B. cepacia has virulence determinants that set it apart from other CGD pathogens. CF is another inborn defect in cell function but it is quite different from CGD. Epithelial cells from patients with CF have defective chloride channel function due to mutations in the gene that encodes the CF transmembrane conductance regulator (CFTR).11 These patients are highly susceptible to infection with Pseudomonas aeruginosa.12 However, B. cepacia complex has evolved as a particularly virulent pathogen in these patients over the past two decades.7 A clear explanation for the tropism of B. cepacia for the CF lung has not yet been found but clues may come from the observations in patients with CGD. It is possible that there exists an oxidant/antioxidant imbalance in the CF lung protecting the bacteria from oxidative killing. The mucoid exopolysaccharide of P. aeruginosa is able to quench oxidative radicals;13 this may provide a privileged and protected niche for B. cepacia to reside, replicate and cause disease after a chronic infection with mucoid P. aeruginosa has been established. The prevalence of B. cepacia infection in patients with CF varies markedly among treatment centres, ranging from zero to about 40%. Furthermore, the severity of disease is highly variable, even in patients infected with the same strain of B. cepacia. A minority of patients with CF who become infected with B. cepacia experience a rapid deterioration in clinical status with systemic toxicity, bacteraemia and death;2 this is known as the ``cepacia syndrome'' and is distinctly different from the disease associated with P. aeruginosa infection in CF. Patients with CF who become infected with B. cepacia experience deterioration of lung function more rapidly than those infected with P. aeruginosa alone or with neither of these two pathogens.14 Patients with CF undergoing lung transplantation do substantially worse if they are infected with B. cepacia prior to transplantation15 and certain strains of B. cepacia are associated with a particularly poor prognosis.16
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TAXONOMY The classi®cation of members of the B. cepacia complex has evolved dramatically over the past decade. Changes have derived from the fact that bacterial strains can now be compared to one another on the basis of their genetic (genotypic) rather than only their physical (phenotypic) properties. Genus and species designation are therefore determined by comparing the genetic endowment of groups of bacteria, which may appear to be very similar phenotypically but prove to be quite different genotypically. New information, derived from such genetic analysis, allows the reclassi®cation of bacteria and their assignment to novel genera and species. This process of re-classi®cation is clearly evident in the taxonomic history of B. cepacia complex. This diverse group of bacteria phenotypically resembles P. aeruginosa and was originally called Pseudomonas cepacia. The species was re-analysed by Yabuuchi et al.17 and was found to be quite distinct genetically from other species in the Pseudomonas genus; it was therefore re-classi®ed to the novel genus Burkholderia (along with several other species), in honour of William Burkholder who had originally described the cause of sour skin in onions.1 Subsequently, Vandamme and colleagues18 have determined that the ``species'' B. cepacia is actually a complex of multiple genetically distinct groups of bacteria differing from one another suf®ciently to warrant individual unique species designation. Those groups of bacteria within the complex that have a unique phenotype (allowing them to be distinguished from others on the basis of physical traits) have been given species names (e.g. Burkholderia multivorans; formerly genomovar II). Those groups that cannot yet be distinguished phenotypically, but are unique from the other species genotypically are called ``genomovars'' (e.g. genomovar III). To date nine genomovars/species have been described within the B. cepacia complex (see Table 1). Genomovar III and Burkholderia multivorans are the predominant species recovered from patients with CF.19 A complete analysis of the taxonomy of B. cepacia is beyond the scope of this review but has been described recently.20
IDENTIFICATION OF B. CEPACIA Members of the B. cepacia complex can be very dif®cult to identify in the clinical diagnostic laboratory. Prior to the advent of enhanced culture and identi®cation techniques in the last decade, B. cepacia was often misidenti®ed, even in laboratories that handled a large volume of CF sputum samples. Identi®cation is now facilitated by the use of specialised selective agar.21 Other bacterial species with which B. cepacia has been confused include Stenotrophomonas maltophilia and many other Gram-negative organisms.22,23 When a CF patient is ®rst infected with a putative member of the B. cepacia complex, it is critically important that the organism's identity be con®rmed. Specialised
232 Table 1
D. P. SPEERT Genomovars/species of the Burkholderia cepacia complex.
Species designation Transmissibility in patients with CF Frequency of recovery from patients with CF Phytopathogenicity Recovery from patients with CGD
Genomovar I
Genomovar II
Burkholderia cepacia
Genomovar III
Genomovar IV
Genomovar V
Genomovar VI
Genomovar VII
Genomovar VIII
Genomovar IX
Burkholderia multivorans
Burkholderia stabilis
Burkholderia vietnamiensis
Burkholderia ambifaria
Burkholderia anthina
Burkholderia pyrrocinia
?
? Yes
? Yes
? ?
? Yes
? ?
? ?
? ?
? ?
CF, cystic fibrosis; CGD, chronic granulomatous disease.
research and referral laboratories exist in the USA, Canada and the UK to which isolates can be sent for con®rmation of identity, for genomovar/species identi®cation and for genetic typing. Location of the Canadian and American laboratories can be found on the International Burkholderia cepacia Working Group website (http://allserv.rug.ac.be/ ppvandam/cepacia/). Subsequent isolates from the same patient that appear to be the same phenotypically as the initial isolate may be assumed to be the same strain and treated as such.
EPIDEMIOLOGY An interest in the epidemiology of B. cepacia has been heightened by the observations from several groups that B. cepacia can be spread from one CF patient to another.24 Although the mechanism of spread had not been determined, the adverse consequences of acquisition can be profound. It appears that certain strains and genomovars/species of the B. cepacia complex are more readily transmitted than others.19,25 The factors underlying patient-to-patient spread are currently under investigation.
Typing methods Epidemiological investigation of any bacterium is dependent upon the capacity to determine if two isolates are from the same strain or not. If they are from the same strain (i.e. are genetically almost identical) one can conclude that they have been derived from the same source or have been spread from one patient to another. Historically, the typing of bacterial strains has been based upon the degree of phenotypic (physical) relatedness. Such methods have proven to be unreliable in CF because genetically diverse groups may appear to be identical physically and phenotypically different isolates may be genetically homologous.26 With the advent of molecular microbiology as a discipline, the capacity to compare bacteria in terms of genetic relatedness has become possible. Various methods have been employed to type B. cepacia genotypically. The method reported initially for typing B. cepacia was ribotyp-
ing and enabled LiPuma and co-workers5 to determine that B. cepacia could be spread from one CF patient to another. Subsequent investigations, based upon whole genome analysis, have con®rmed the ®rst observations. The most widely used typing methods are now pulsed ®eld gel electrophoresis and random ampli®ed polymorphic DNA analysis.25
Transmissibility factors The observation that B. cepacia could be transmitted among patients and that some strains (but not others) are clustered in certain CF centres, suggested that these ``epidemic'' strains encode factors that enhance transmissibility. The ®rst such putative factor was identi®ed in the strain of B. cepacia that infects a large number of patients in the United Kingdom and Canada. Spread of this strain has been associated with contact among patients both within and outside the hospital and it is now designated ET12. Isolates from this strain elaborate a unique cable pilus27 that appears to enhance adhesion to epithelial cells.28 A role for the cable pilus in patient-to-patient spread had been suggested but not yet formally proven. The gene for the cable pilus is found predominantly, but not exclusively, in strain ET12. A second putative transmissibility factor is the B. cepacia epidemic strain marker (BCESM);29 this gene appears to be a negative transcriptional regulator and is encoded by many, but not all, ``epidemic'' strains that cluster in individual CF treatment centres. The role of BECSM in transmission or virulence of B. cepacia has not been determined.
Evidence for patient-to-patient spread The literature on B. cepacia epidemiology in CF is now replete with descriptions of patient-to-patient spread. Clustering of strains in patients by geographical location has been described in the USA,30 Canada,19 the United Kingdom,31 Australia32 and Italy.33 It appears that direct personal contact is required for patient-to-patient spread, suggesting that droplet rather than aerosol transmission is responsible. Speci®c activities, which enhance the
ADVANCES IN BURKHOLDERIA CEPACIA COMPLEX likelihood that B. cepacia will be spread from one CF patient to another, have not yet been determined. Until more is learned about the risk factors for patient-to-patient spread, it is prudent to restrict interaction between patients infected with B. cepacia and those who are culturenegative. Furthermore, there is evidence that certain strains of B. cepacia can replace others when patients with different strains are cohorted together, and such replacement can be associated with a poor clinical outcome.25 It is therefore inadvisable to cohort B. cepacia culture-positive CF patients and they should be discouraged from having any direct contact with one another.
VIRULENCE There is a wide variation in disease severity among CF patients infected with B. cepacia. Certain strains seem to be associated with a more severe illness than others.16,25 These observations have raised the possibility that only some strains are endowed with speci®c features that suit them better for human disease. The virulence determinants (disease-producing capacity of bacterial factors) from B. cepacia have been sought by several groups of investigators and reviewed by Nelson et al.34 The investigative efforts are ongoing and updated regularly by the International Burkholderia cepacia Working Group on their website (http:// allserv.rug.ac.be/ppvandam/cepacia/). A wide array of putative virulence determinants have been described but none has yet ``won its spurs'' in a fashion analagous to the capsular polysaccharide of Haemophilus in¯uenzae type b or the toxin of Clostridium tetani. Among the candidate virulence determinants are pili (particularly the cable pilus), lipopolysaccharide (endotoxin), protease, haemolysin, phospholipase C, siderophores, melanin pigment and exopolysaccharide. Since none of these individual factors has been clearly shown to enhance disease severity in humans, features of the bacteria as a whole have been investigated as described, below.
Intracellular parasitism Burkholderia cepacia complex is closely related to a bone ®de intracellular pathogen, B. pseudomallei,35 which causes melioidosis, a disease that is very similar clinically to tuberculosis. This raises the possibility (which has been suggested by a number of investigators) that B. cepacia owes its virulence, in part, to its capacity for intracellular parasitism. Burkholderia cepacia can invade epithelial cells36 and can be found within host cells of infected mice.37 A recent report correlated the capacity of strains to penetrate epithelial cells with mouse infectivity.38 Burkholderia cepacia can also survive within macrophages39 and in amoebae.40 The latter cell is particularly interesting, since it could provide insights into the reservoir within which B. cepacia survives in the environment. If B. cepacia does indeed have the capacity for intracellular parasitism, this raises important issues with
233 regard to therapy; the optimal drug would be one that is able to penetrate eukaryotic cells.
Resistance to non-oxidative killing Infections with B. cepacia complex bacteria are particularly frustrating to treat because of their high level intrinsic resistance to many antibiotics, including the cationic antibiotic polymixin B.41 Resistance to cationic antimicrobial peptides is one feature shared by all members of the B. cepacia complex and this renders them resistant to nonoxidative killing by phagocytic cells10 (see above). In addition to facilitating their capacity to cause infection in patients with CGD, this enhances their infectivity under conditions in which the generation of reactive oxygen radicals is compromised. Macrophages are far less competent than polymorphonuclear leukocytes in elaborating toxic oxygen radicals; this may explain why B. cepacia are able to persist inside macrophages, perhaps enhancing their capacity for intracellular parasitism. Under other conditions in which reactive oxygen intermediates are quenched, B. cepacia would appear to be better suited to cause infection than those bacterial species that are susceptible to non-oxidative killing.
Quorum sensing and biofilm formation Many bacterial infections evolve in the form of communities of bacteria (bio®lms) in which there is a type of communication among the individual bacteria when they sense a ``quorum''.42 This phenomenon of quorum sensing occurs when the bacteria achieve a certain density. It is associated with enhanced resistance to antimicrobial agents43 and with the elaboration of quorum sensing molecules (known as autoinducers).44 These autoinducers, which are acylated homoserine lactones (AHL), have substantial pleiotropic effects on the expression of various genes. Several of the genes in P. aeruginosa, the expression of which is controlled in part by autoinducers, are considered to be virulence determinants. Thus bacterial density (as occurs when bio®lms develop) is probably very important in dictating the level of toxin production as well as antibiotic resistance during chronic P. aeruginosa infection, as occurs in CF. Bacteria from all of the B. cepacia complex genomovars encode the gene(s) for elaboration of AHLs.45 We have recently demonstrated that all of these bacteria also elaborate AHLs but to greatly different degrees (B. Conway & D.P. Speert, unpublished results). Many of the strains also form bio®lms but there is no correlation between the extent of bio®lm formation and the extent of AHL production. Another study has shown that in one strain there was a direct correlation between AHL production and bio®lm formation.46 Since B. cepacia causes chronic infection in some patients with CF and since bio®lms are such an important feature of chronic infections, the role of quorum sensing in B. cepacia infection deserves further investigation.
234
THERAPY Therapy of infections with B. cepacia complex is fraught with problems that result from the organism's very high level of intrinsic resistance to many antibiotics and its capacity to develop further resistance during therapy. This problem of induced resistance during therapy is particularly problematic in CF patients, in whom chronic infection is a hallmark of the disease. The cell envelope of B. cepacia is unusual among Gramnegative bacteria by virtue of its unique lipopolysaccharide, a molecule that is a major constituent of the outer membrane. The unusual chemical composition of the lipopolysaccharide is thought to render the bacteria intrinsically resistant to aminoglycosides (such as gentamicin and tobramycin) and to polymixins.47 In fact, one of the differential features used to aid in the identi®cation of B. cepacia in diagnostic laboratories is this high level intrinsic resistance. Many strains of B. cepacia are resistant to a multitude of antibiotics, limiting the choice of therapeutic options. It is not uncommon for patients with CF who have been infected with B. cepacia for a prolonged period and who have received multiple courses of antibiotics, to be infected with a strain of B. cepacia that is resistant to all major classes of antibiotics. Antimicrobial agents to which B. cepacia bacteria are often susceptible include semisynthetic penicillins (such as ticarcillin), carbapenems (such as meropenem), cephalosporins (particularly ceftazidime), quinolones (such as cipro¯oxacin) and trimethoprim/sulfamethoxazole. It is not possible to predict a priori which antimicrobial agents will be effective against any particular isolate of B. cepacia, so each should be subjected to susceptibility testing. Furthermore, since resistance to antibiotics may develop during the course of therapy, any additional isolates from persistently infected patients should also undergo susceptibility testing. Therapy for B. cepacia in chronically infected CF patients must often be initiated before the results of antimicrobial susceptibility tests are available. Therapy should be based upon the most recent antimicrobial susceptibility results. Combination therapy should be used for serious infections and should ideally include drugs from two different classes to enhance the likelihood of achieving synergistic activity. Even though all strains of B. cepacia are resistant in vitro to aminglycosides, that class of antimicrobials appears to be bene®cial in treating B. cepacia infections when used in combination with other agents.41 Dual b-lactam therapy should be avoided whenever possible as it appears to induce expression of b-lactamases, resulting in apparent antagonism. Whereas combination therapy is advisable for serious B. cepacia infections, the effects of each drug on the activity of the other cannot be predicted by evaluating each in isolation of the others. Some combinations are synergistic, some merely additive and some antagonistic.41 In an effort to optimise antimicrobial therapy, some research laboratories are evaluating the use of multiple combination antimicrobial testing (MCT).41 These ``checkerboard'' evaluations permit
D. P. SPEERT one to determine the antimicrobial activity when more than one antibiotic is evaluated simultaneously. Either two or three drugs are mixed and their capacity to inhibit or kill the bacteria is assessed. From large in vitro studies of multiresistant isolates of B. cepacia, the following antibiotic combinations were found to be the best: meropenem±minocycline, meropenem±amikacin and meropenem±cefazidime.41
ACKNOWLEDGEMENTS Work in my laboratory is supported with funds from the Canadian Cystic Fibrosis Foundation, the Canadian Institutes for Health Research, the Canadian Bacterial Diseases Network and the British Columbia Lung Association. I thank Deborah Henry for critically reviewing the manuscript.
RESEARCH DIRECTIONS Despite considerable growth in research on B. cepacia over the past decade, much more remains to be learned about this evolving opportunistic pathogen. Among the most pressing questions are the following: Why has B. cepacia, among all the environmental saprophytic bacteria, evolved as such an important pathogen in patients with cystic fibrosis (CF)? What microbial and host factors favour the spread of B. cepacia from one individual to another and how can the spread be prevented? How can acquisition of B. cepacia be prevented by compromised hosts, such as those with CF or chronic granulomatous disease (CGD)? What is the optimal therapy for patients infected with B. cepacia? What are the most important host factors that defend against infection with B. cepacia? What are the most important microbial factors that enhance the infectivity of B. cepacia?
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