Burkholderia cepacia complex species: health hazards and biotechnological potential

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Burkholderia cepacia complex species: health hazards and biotechnological potential Luigi Chiarini1, Annamaria Bevivino1, Claudia Dalmastri1, Silvia Tabacchioni1 and Paolo Visca2,3 1

Department of Biotechnology, Protection of Health and Ecosystems, C.R. Casaccia, ENEA, 00060 Rome, Italy Department of Biology, University RomaTre, 00146 Rome, Italy 3 Molecular Microbiology Unit, National Institute for Infectious Diseases “L. Spallanzani”, 00149 Rome, Italy 2

The Burkholderia cepacia complex is a group of nine closely related bacterial species that have useful properties in the natural environment as plant pest antagonists, plant growth promoters and degradative agents of toxic substances. Because these species are human opportunistic pathogens, especially in cystic fibrosis patients, biotechnological applications that involve environmental releases have been severely restricted. Recent progress in understanding the taxonomy, epidemiology and ecology of the B. cepacia complex species has unravelled considerable variability in their pathogenicity and ecological properties, which has set the basis for a reassessment of the risk posed by individual species to human health.

Burkholderia cepacia complex: an overview The Burkholderia cepacia complex (Bcc) is a group of genetically distinct but phenotypically similar bacteria that are divided into at least nine species (Table 1). The Bcc species were isolated from a variety of natural habitats such as plant rhizosphere (see Glossary), soil and river water and from several urban environments such as playgrounds and athletic fields [1–3]. They have useful properties as plant pest antagonists, plant-growthpromoting (PGP) rhizobacteria and degradative agents of toxic substances (Table 2) [4–8]. In contrast to these beneficial characteristics, Bcc species can also colonize and/or infect cystic fibrosis (CF) patients and, occasionally, immunocompromised individuals, and they are collectively regarded as human opportunistic pathogens [9]. The wide environmental spread of Bcc has raised concern about the existence of natural reservoirs of pathogenic strains. Indeed, genotypically identical Bcc strains have been isolated from both CF patients and environmental sources, which suggests that the acquisition of pathogenic strains can occur directly from the natural environment [10,11]. As a result of the clinical relevance of Bcc species and their close interspecies relatedness, the biotechnological applications of all Bcc species have been severely Corresponding author: Chiarini, L. ([email protected]).

restricted by the U.S. Environmental Protection Agency (EPA) [12]. However, recent advances in the taxonomy of Bcc and the implementation of DNA-based identification methods have set the basis for a more accurate evaluation of the ecology, clinical importance and biotechnological potential of the individual species within the complex (Box 1). Excellent reviews covering different aspects of Bcc biology have been published in the past five years [13–18]. This article presents highlights from recent studies on the taxonomy, ecology, epidemiology, pathogenicity and biotechnological impact of the distinct Bcc species with the aim of discussing whether or not the restrictions on the biotechnological use of Bcc strains deserve reconsideration based on existing interspecies diversity.

The evolving taxonomy of Bcc Burkholderia is the genus that was proposed by Yabuuchi et al. [19] to accommodate a few rRNA group II Pseudomonas species. According to current taxonomy, the Burkholderia genus contains 34 validly described species (Figure 1) [18]. The genus name was assigned in recognition of the pioneering work of W. Burkholder, who first described the Gram-negative Pseudomonas cepacia (now Burkholderia Glossary Biocontrol: the use of biological means (e.g. bacteria) to control a pest. Bioremediation: the use of living organisms (e.g. bacteria) to remove pollutants from soil, water and wastewater. ‘Cepacia syndrome’: the rapid decline of lung function observed in CF subpopulations infected with some Bcc species, accompanied by marked pyrexia, multi-organ failure and positive blood cultures. Culture-independent methods: these methods involve direct extraction of nucleic acids (DNA or RNA) from samples and their subsequent amplification by PCR. The amplified products can either be analyzed directly or cloned and sequenced to assess the molecular diversity in the sample. Cystic fibrosis: a genetic disease that affects w1 in 2000 individuals, caused by mutations in the CFTR gene. CFTR encodes a protein called the cystic fibrosis transmembrane conductance regulator (CFTR) that functions as a chloride-ion transporter. Mutations in CFTR result in pancreatic insufficiency and the build up of sticky mucus in the lungs, which predisposes to infection and compromises breathing. Plant-growth-promoting rhizobacteria: bacteria that grow in association with a host plant and stimulate the growth of their host. Rhizosphere: the soil zone that immediately surrounds plant roots, which is modified by the increased number of microorganisms that live there in association with plant roots.

www.sciencedirect.com 0966-842X/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.04.006

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Table 1. Characteristics of Bcc speciesa Species

Source

Burkholderia ambifaria

Rhizosphere, soil, humans (CF and non-CF) Rhizosphere, soil, water, hospital environment, humans (CF and non-CF) Rhizosphere, soil, water, hospital environment, humans (CF and non-CF) Rotten onion, rhizosphere, soil, water, humans (CF and non-CF) Rhizosphere, humans (CF) Rhizosphere, soil, water, humans (CF and non-CF) Rhizosphere, soil, water, humans (CF) Hospital environment, humans (CF and non-CF) Rhizosphere, soil, water, humans (CF and non-CF)

Burkholderia anthina

Burkholderia cenocepacia

Burkholderia cepacia

Burkholderia dolosa Burkholderia multivorans Burkholderia pyrrocinia Burkholderia stabilis Burkholderia vietnamiensis a

PGP activity and/or biocontrol C

Bioremediation

ND

Frequency of recovery from CF patients (%)b !1

ND

ND

C

‘Cepacia syndrome’

Transmissibility among CF patients

Refs

NR

Decreased survival in CF patients NR

NR

[4,5,23,36, 57,58]

!1

NR

NR

NR

[3,36,69,70]

ND

45.6

C

C

C

[3,32,33,36, 46,58,69]

C

C

3.1

NR

NR

C

[3,20,36,46, 58,69,71]

ND

ND

3.8

C

C

C

[42,46,58]

ND

ND

38.7

C

C

C

[3,33,36,46, 58,69]

ND

ND

!1

NR

NR

C

[36,37,58, 69]

ND

ND

!1

NR

NR

NR

[36,58,72]

C

C

5.9

NR

NR

NR

[6,7,36]

Abbreviations: ND, not determined; NR, not reported; plus symbol (C), activity or property reported. Data from Ref. [36].

b

cepacia) as the phytopathogen responsible for the bacterial rot of onions [20]. Bacteria that were formerly identified as B. cepacia actually represent a complex of closely related species, collectively referred to as the Bcc. These species share low levels (30–60%) of DNA–DNA hybridization but have a remarkably high degree (98–100%) of 16S rDNA sequence similarity [13]. To define the interspecies diversity within the Bcc, Vandamme et al. [21] pioneered a polyphasic taxonomic approach in 1997 (Box 1), which resulted in the description of five species: B. cepacia, Burkholderia multivorans, Burkholderia cenocepacia, Burkholderia stabilis and Burkholderia vietnamiensis [13]. In the following five years, four new Bcc species were identified: Burkholderia dolosa, Burkholderia ambifaria, Burkholderia anthina and Burkholderia pyrrocinia [15], and an identification scheme based on recA gene polymorphism was developed (Figure 1; Box 1 and reference therein). However, the taxonomy of the Bcc has not yet been entirely resolved. Indeed, the taxonomic position of a putative tenth species, Burkholderia ubonensis, has not been conclusively established [18]. Recently, Baldwin et al. [22] published the first multilocus sequence typing (MLST) scheme for Bcc, which demonstrated the existence of four novel potential species within the complex (Box 1). Finally, the presence of discrete genetic lineages within individual species such as B. cepacia and B. cenocepacia adds an additional level of taxonomic complexity [11,22]. www.sciencedirect.com

The dual personality of Bcc Several Bcc species are considered to be beneficial in the natural environment. Strains that belong to the species B. cepacia, B. cenocepacia, B. ambifaria and B. pyrrocinia protect commercially valuable crop plants against fungal diseases such as root rot, which is caused by Aphanomyces euteiches, and damping-off, a disease caused by Pythium species and Rhizoctonia solani that affects seedlings (Table 2). Both PGP and biocontrol activities have been documented most extensively for a variety of strains definitely classified as B. ambifaria [4,5,23]. In particular, B. ambifaria strain MCI7 promotes maize growth and protects against the phytopathogen Fusarium verticillioides (Figure 2) [4]. The B. ambifaria strain BC-F suppresses cucumber and soybean diseases caused by a variety of soil-borne fungal pathogens [24] and antagonizes the plant parasitic nematode Meloidogyne incognita both in vitro and on pepper roots [25]. Indigenous rhizosphere populations of B. cenocepacia provide effective defence for crop plants against the attack of fungal phytopathogens [26]. The nitrogen-fixing B. vietnamiensis strain TVV75 is highly effective in enhancing rice crop yields in South East Asia [6]. The effectiveness of Bcc isolates as biocontrol and PGP agents is based on a wide array of beneficial properties including the production of indoleacetic acid, the ability to fix atmospheric nitrogen and the production of a wide array

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Table 2. Bcc strains with useful biotechnological propertiesa,b Species Bioremediation Bcc Burkholderia cepacia Burkholderia vietnamiensis

Strain

Origin

Degraded substrates or activity

Other relevant information

Refs

CRE-7 ATCC17759 (LMG2161) G4 (ATCC53617; R1808)

Soil Forest soil (Trinidad) Wastewater (USA)

Polycyclic aromatic hydrocarbons Benzoate, 4-fluorobenzoate, 4-hydroxybenzoate Benzene, meta-cresol, ortho-cresol, para-cresol, phenol, toluene, trichloroethylene

None None

[73] [71]

U.S. patents 4925802 and 55443317 (derivative strain ENV435)

[7,8]

RAL-3

Unknown

Application by Agrium Inc. (1998), indoor use only; withdrawn in 2000 by PMRA and EPA

[74]

B. ambifaria

AMMDR1T (LMG19182)

None

[5]

B. ambifaria

BC-F

Pea rhizosphere (USA) Maize rhizosphere (USA)

Antifungal activity in field assays

[24,25]

B. ambifaria

M54 (R-5142)

Maize rhizosphere (USA)

PGP activity and biocontrol of fungi (Pythium, Fusarium, Cylindrocarpon, Botritis and Rhizoctonia species) on seed and/or seedlings of conifers and deciduous trees Biocontrol of fungi (Pythium aphanidermatum and Aphanomyces euteiches) in pea plants Biocontrol of fungi (Rhizoctonia solani, Pythium ultimum, Fusarium oxysporum, Sclerotium rolfsii) in cucumber and soybean and of the nematode Meloidogyne incognita in pepper Biocontrol of fungi

[14]

B. ambifaria

J82 (R-5140)

Maize rhizosphere (USA)

Biocontrol of nematodes and fungi (Sclerotinia sclerotiorum) in sunflowers

B. ambifaria

PHP7

PGP activity and biocontrol of fungi in maize plants

B. ambifaria

MCI7

None

[4]

Burkholderia cenocepacia

M36

Maize rhizosphere (France) Maize rhizosphere (Italy) Maize rhizosphere (USA)

Registered biopesticide 006465 (1996); commercial use as DENYTM, Stine Microbial Products, USA; tolerance for residues in commercial products revoked by EPA in 2004 Registered biopesticide 006464 (1996); commercial use as DENYTM, Stine Microbial Products, USA; tolerance revoked by EPA in 2004 None

[14]

B. cenocepacia

BC-1

Biocontrol of fungi (Pythium) on maize seeds

B. cenocepacia

BC-2

Biocontrol of fungi on maize seeds

None

[76]

B. cepacia Burkholderia pyrrocinia B. pyrrocinia

ATCC49709 BC11

Maize rhizosphere (USA) Maize rhizosphere (USA) Unknown Soil

Registered biopesticide (1996); commercial use as Blue Circle, Stine Microbial Products, USA; withdrawn from commercial use in 2003 None

Biocontrol of fungi on grass seeds Biocontrol of fungi in peanuts

None None

[77] [78]

Biocontrol against fungi

None

[79]

B. vietnamiensis

LMG10925 T (TVV75)

Maize field soil Rice rhizosphere (Vietnam)

PGP activity on rice

None

[6]

Biocontrol Burkholderia ambifaria

a

ATCC39277

PGP activity and biocontrol of fungi (F. oxysporum, Fusarium verticillioides) in maize plants Biocontrol of fungi

[14,75]

[23]

[76]

Abbreviation: PMRA, Pest Management Regulatory Agency, Canada. Adapted, with permission, from Ref. [11].

b

of compounds endowed with antimicrobial activity, including cepacin, cepaciamide, cepacidines, altericidins, pyrrolnitrin, quinolones, phenazine, siderophores and a lipopeptide (for a review, see Ref. [14]). In the early 1990s, these useful properties led to the registration of four Bcc strains for use as biopesticides by the EPA, three of which were later classified as B. ambifaria and one as B. cenocepacia (Table 2). Bcc isolates have also been used in bioremediation by virtue of their ability to degrade several man-made toxic www.sciencedirect.com

agents, in particular chlorinated aromatic compounds (Table 2). B. vietnamiensis G4 can degrade aromatic pollutants such as toluene and chlorinated solvents such as trichloroethylene [7]. Field trials with B. vietnamiensis ENV 435, derived from strain G4, showed reduction of the chlorinated solvent content in a sand aquifer by O 70% [27]. While Bcc isolates were increasingly exploited for biotechnological applications, a growing body of clinical evidence raised the awareness of the infectious risk due to

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Box 1. Identification and typing of Bcc species† To unravel the taxonomic structure of the Bcc, Vandamme et al. [21] developed a ‘polyphasic’ approach based on a number of genotypic and phenotypic methods. These include whole-cell protein profile and fatty acid analysis, DNA–DNA hybridization, 16S rRNA and recA gene sequencing and conventional biochemical tests [13]. These methods made it possible to divide the Bcc into at least nine species.

species because of high identity (98–100%) between sequences. The recA gene shows a sequence variability (interspecies identity, 94–95%) that enables the Bcc to be differentiated from closely related bacteria and sorted into species [81]. The recA-based identification tests comprise restriction fragment length polymorphism analysis with the enzyme HaeIII and species-specific PCR reactions in addition to sequencing of the entire recA gene.

Phenotypic methods for Bcc identification

Molecular typing methods

Identification of Bcc isolates by commercial methods such as API 20 NE and Vitek GNI (bioMe´rieux), Crystal E/NF (Becton Dickinson), Microscan GNP (Dade International), RapID NF Plus (Innovative Diagnostics Systems), Remel NF System (Remel) and Sherlock GLC (MIDI) lacks sufficient accuracy and results must be confirmed by the following phenotypic tests: growth on Burkholderia cepacia selective agar medium (Hardy Diagnostics), biochemical tests for lysine and ornithine decarboxylase, oxidase activity, sucrose and adonitol oxidation, hemolysis, pigment production and growth at 428C. With this information, appropriate molecular tests can be chosen for subclassification within the Bcc.

Several genotypic methods are being used to establish relationships between Bcc isolates, including macrorestriction digestion of chromosomal DNA followed by pulsed-field gel electrophoresis (PFGE) and various PCR-based fingerprinting techniques with short random primers (RAPD) or primers directed against repetitive sequences in the bacterial genome (BOX-PCR) [82]. PFGE and RAPD techniques are better suited to small-scale studies such as the investigation of hospital outbreaks in which a limited number of samples is collected within a narrow time frame. BOX-PCR fingerprinting is preferred for global epidemiological questions. Recently, the use of genotyping methods in global epidemiology studies and population structure analyses and the need for suitable inter-laboratory data exchange led to the development of a MLST scheme, in which allelic variation at seven housekeeping genes is indexed directly by nucleotide sequencing of internal fragments of w450 base pairs and the resulting data are stored on a central database held on the internet [22]. As a less expensive and less time-consuming alternative to MLST, a multilocus restriction typing method has been proposed in which variation at several loci is indexed by restriction analysis of PCR-amplified genes [83].

Taxonomy

Molecular methods for Bcc species identification Sequence variation in the 16S rRNA gene was originally used to identify Bcc bacteria before their recognition as a complex [80]. 16S rDNA sequence variation is no longer applied to discriminate the Bcc † Text adapted, with permission, from Ref. [15]. q (2005) Macmillan Magazines Ltd. (http://www.nature.com/reviews).

Bcc organisms, especially in CF or otherwise critically ill patients [28,29]. In fact, Bcc disease in CF provides a paradigmatic example of bacterial opportunism because infection occurs only in predisposed individuals who have become heavily colonized by bacteria with multifactorial pathogenic potential, which induces an inflammatory response in the host. Indeed, the hallmark of Bcc infection in CF is the inflammation that results from the unusual chemistry and biological properties of lipopolysaccharide, which induces local release of pro-inflammatory factors and consequent immunopathological disorder [30]. In the early 1990s, the prevalence of Bcc among CF patients in different countries reached 20–35% because of extensive person-to-person spread. Nowadays, the implementation of stringent infection control measures has, in general terms, reduced prevalence to 5–8% and new infections are mostly acquired from the environment [31,32]. Bcc infection in CF is often secondary to Pseudomonas aeruginosa infection but the proportional hazard for severe clinical decline and death is substantially increased following infection with Bcc [9]. The mean survival of CF patients decreases by w10 years in the Bcc-culture-positive population compared with the P. aeruginosa-positive population*. Three major outcomes of Bcc infection have been described so far: ‘cepacia syndrome’, which leads to an acute clinical decline and is frequently fatal [33], chronic infection and apparently asymptomatic carriage. Although all Bcc species have been isolated from lung secretions of CF patients, B. cenocepacia and B. multivorans are by far the most frequently isolated species [31,34]. A dramatic drawback of Bcc infection is that it limits the selection of CF * S.C. FitzSimmons, minutes of the International Burkholderia cepacia Working Group Meeting, Victoria BC, Canada, May 1997. www.sciencedirect.com

patients for lung transplantation because of the high risk of post-operative sepsis and death [35]. This worrying scenario has severely delayed further research on biotechnological applications of Bcc and prompted the EPA to reconsider the use of commercial products containing Bcc bacteria. In response to petitions from the U.S. Cystic Fibrosis Foundation and on the basis of the scientific evidence on Bcc infection available at that time, the EPA cancelled the registration of all pesticide products containing Bcc isolates in 2003 and a stringent rule was issued to limit new uses of Bcc to bioremediation processes [12].

Are all Bcc species equally pathogenic in CF? Distribution of Bcc species among CF patients The distribution of Bcc species among isolates from CF patients is clearly disproportionate (Table 1). In the most comprehensive study published so far [36], B. cenocepacia and B. multivorans account for the vast majority of Bcc infection in CF patients from the USA (45.6% and 38.7% of patients, respectively) whereas a minority of patients harbour B. vietnamiensis, B. dolosa and B. cepacia (5.9%, 3.8% and 3.1%, respectively). Remarkably, carriage of B. stabilis, B. ambifaria, B. anthina or B. pyrrocinia was low (!1% of patients). Also, in Canada and Europe the prevalent species are B. cenocepacia and B. multivorans, with marked variation in local isolation rates [34,37,38]. This unequal distribution poses the question of whether distinct Bcc species are endowed with different capacities for pulmonary colonization and/or interpatient spread or whether it simply reflects a disproportionate distribution of Bcc species in their natural reservoirs.

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Burkholderia spp. LMG21262 Burkholderia spp. R15273 88 Burkholderia fungorum LMG16225T 99 Burkholderia spp. LMG19510 Burkholderia spp. R20943 Burkholderia xenovorans LB400 98 Burkholderia spp. LMG20580 Burkholderia caledonica LMG19076T Burkholderia spp. R13392 73 Burkholderia graminis LMG18924T 100 Burkholderia spp. R8349 Burkholderia terricola LMG20594T Burkholderia tuberum LMG21444T Burkholderia caribensis LMG18531T 88 97 Burkholderia phymatum LMG21445T Burkholderia phenazinium LMG2247T Burkholderia spp. R701 Burkholderia kururiensis LMG19447T 91 Burkholderia sacchari LMG19450T Burkholderia caryophylli LMG2155T Burkholderia glathei LMG14190T Burkholderia plantarii LMG9035T 100 71 Burkholderia glumae LMG2196T Burkholderia gladioli LMG2216T 100 Burkholderia mallei ATCC23344 99 Burkholderia pseudomallei K96243T Burkholderia thailandensis LMG20219T 61 Burkholderia multivorans ATCC17616T Burkholderia vietnamiensis LMG10929T 60 Burkholderia dolosa LMG18943T Burkholderia ambifaria ATCC53266T Burkholderia cenocepacia J2315T Burkholderia cepacia Burkholderia anthina C1765 complex Burkholderia stabilis LMG7000 Burkholderia cenocepacia PC184T Burkholderia pyrrocinia LMG14191T 84 Burkholderia cepacia ATCC25416T Pandoraea apista Patient W 100 Pandoraea pnomenusa “K” 100 Pandoraea pulmonicola #40 Ralstonia eutropha LMG1197 Burkholderia spp. R15821 Bordetella avium 35086 Bordetella hinzii 51730 Bordetella pertussis Bordetella parapertussis 15311 Xanthomonas axonopodis Neisseria meningitidis MC58 Pseudomonas aeruginosa PAO1 100

89

96

99

88

92

71

79 64 100

0.1

Figure 1. Phylogeny of the Burkholderia genus using recA gene sequences. Species name and strain designation are indicated (T denotes type strain). The tree was constructed using 760 nucleotides of the recA gene sequence and the neighbour-joining algorithm. Bootstrap values and genetic distance scale are shown. Bcc species (red text) are clustered in the shaded region of the tree. Figure adapted, with permission, from Ref. [11].

Clinical impact Several studies on the clinical follow-up of CF patients infected by Bcc species provided an epidemiological link between B. cenocepacia and, to a lesser extent, B. multivorans infection and poor clinical outcome [31,32,34,39,40] (Table 1). In the case of B. cenocepacia, this is ascribed to the emergence of epidemic strains endowed with an increased virulence [15,41]. Recently, www.sciencedirect.com

chronic CF infection with a single strain of B. dolosa, a species formerly indistinguishable from B. multivorans, has been associated with accelerated loss of lung function and decreased survival [42]. No epidemiological data are available for the other Bcc species, probably because the assembly of comprehensive clinical information has been hampered by their low incidence in the CF population. Moreover, broad epidemiological studies determine

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The infrequent isolation from CF secretions of some Bcc species that are encountered more often in natural habitats than in clinical settings – such as B. ambifaria, B. anthina and B. pyrrocinia (see later) – is difficult to interpret in terms of clinical significance. For example, no strong causal association has so far been established between the presence of such species in pulmonary secretions and the health decline in CF patients. Hence, it is questionable if some Bcc species such as B. ambifaria, B. pyrrocinia and B. anthina are genuine opportunistic pathogens like B. cenocepacia, B. multivorans and B. dolosa.

B. ambifaria MCI7 F. verticillioides

+

–

+

–

+

+

–

–

Figure 2. Effect of Zea mays seed bacterization with Burkholderia ambifaria MCI7 on biocontrol of the pathogenic fungus Fusarium verticillioides and PGP activity. From left to right: Z. mays plant inoculated with B. ambifaria MCI7 and grown in soil artificially infested with F. verticillioides ITEM-504; non-inoculated plant grown in artificially infested soil; inoculated plant grown in uninfested soil; non-inoculated plant grown in uninfested soil. PGP activity and biocontrol assays were carried out under greenhouse conditions, as described in Ref. [4]. Plants were collected after 20 days of growth.

overall prevalence data but do not accurately reflect outcomes of individual patients with different Bcc species infections. B. cenocepacia and B. multivorans are associated with most cases of chronic infection whereas B. ambifaria and B. stabilis account for a small number of cases [43]. Once acquired, B. cenocepacia is difficult to eradicate by antibiotic therapy [44]. In a CF patient co-infected with both B. cenocepacia and B. vietnamiensis, the latter was eradicated by antibiotic therapy while the former persisted [45]. Higher post-transplantation mortality rates were observed in CF patients infected preoperatively with B. cenocepacia than in those infected with other species [35]. In addition, the capacity for inter-patient spread has been observed primarily in B. cenocepacia and less frequently in B. multivorans [46]. Single outbreaks caused by B. cepacia, B. dolosa and B. pyrrocinia have been described whereas transmission among CF patients has not been conclusively demonstrated for the remaining Bcc species [37,46]. A frequently fatal outcome of Bcc infection is ‘cepacia syndrome’, which occurs in almost 20% of Bcc-infected patients [47]. The syndrome has been observed in patients infected with strains of B. cenocepacia, B. multivorans, B. vietnamiensis and B. dolosa [33,40,42,48]. Bloodstream infection, the main pathognomonic feature of ‘cepacia syndrome’, has also been reported in non-CF patients infected with B. stabilis [49]. The severity of infection is related to the ability of Bcc bacteria to invade respiratory epithelial cells and cause sepsis. This ability resides primarily in B. cenocepacia and B. multivorans [50], both of which show a common invasion pattern in vitro involving biofilm formation, endocytosis and epithelial necrosis [51]. By contrast, B. stabilis, which has not been associated with a worsening of CF pulmonary disease, penetrates airway epithelia by paracytosis and does not form biofilms or cause necrosis [51]. www.sciencedirect.com

Animal and plant models of infection Studies on the infectivity and pathogenicity of Bcc species in different models – namely the alfalfa plant, the rat agar bead, the mouse [52,53] and the nematodes Caenorhabditis elegans and Panagrellus redivivus – provided contradictory results [54,55]. Differences between Bcc species and considerable intraspecific variability were documented in all models. In particular, the alfalfa plant and the rat agar bead models highlighted the high pathogenic potential of B. cepacia and B. cenocepacia strains. Surprisingly, B. multivorans caused major pathological symptoms only in the mouse and the nematode P. redivivus, whereas B. ambifaria and B. pyrrocinia proved to be pathogenic only to C. elegans. These observations are not fully consistent with the clinical profile of patients infected with these species and make it questionable whether existing models of infection do actually reflect the CF lung environment and to what extent they provide a reliable measure of the pathogenic potential of Bcc species in humans. Bcc in the natural environment In general terms, it is assumed that the capacity to use a wide array of compounds as carbon sources enables Bcc bacteria to colonize extremely diverse habitats. Moreover, the multireplicon Bcc genome harbours an extensive array of insertion sequences that can promote genomic plasticity and metabolic adaptability [56]. But can all Bcc species equally thrive in habitats as apparently diverse as the human lung, the plant rhizosphere and natural surface waters? An increasing body of evidence indicates that the various Bcc species are disproportionately distributed in these habitats. Whereas B. cenocepacia and B. multivorans are prevalent in the CF lung, B. ambifaria and B. cenocepacia are the most common Bcc species in the plant rhizosphere [1,57,58]. B. cepacia, B. vietnamiensis and B. pyrrocinia are also present in the rhizosphere, whereas B. multivorans, B. stabilis, B. dolosa and B. anthina are rarely found in this environment [57,58]. Surprisingly, only B. cepacia, B. multivorans, B. cenocepacia, B. vietnamiensis and B. anthina were recovered from several surface natural waters, whereas B. ambifaria remained undetected [3]. The difficulty in recovering B. multivorans, B. stabilis, B. dolosa and B. anthina from the plant rhizosphere has been attributed to the lack of isolation media that enable the simultaneous growth of all Bcc species and the lack of

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efficient methods for recovery of low-density bacterial populations. The presence of B. cepacia, B. cenocepacia, B. vietnamiensis, B. ambifaria and B. pyrrocinia in the rhizosphere of maize and the absence of all other Bcc species was confirmed by culture-independent methods [57]. However, a colony hybridization assay detected not only these five species but also a few isolates of B. multivorans, B. stabilis and B. dolosa [58]. It is conceivable that the presence of the latter species in the maize rhizosphere is occasional and highly variable on a geographical basis. As anticipated, typical soil species such as B. cepacia, B. cenocepacia, B. vietnamiensis, B. ambifaria and B. pyrrocinia have an important role as biocontrol and PGP agents in the rhizosphere of several crop plants, although it is unknown if other Bcc species have similar properties. It seems that B. cenocepacia is the Bcc species that is better adapted to both the human lung and the plant rhizosphere. The features that make this species more suitable than others for these habitats are still a matter of speculation. B. cenocepacia seems to be capable of displacing competing species from both the human lung and the plant rhizosphere [43] (L. Chiarini, unpublished). This competitive ability might be related to the higher metabolic diversity shown by B. cenocepacia and to its capacity to oxidize various carbon compounds at high rates [59]. In addition, it is speculated that this species can colonize both human lung epithelia and plant root cells because both root and lung colonization might involve partially overlapping mechanisms for recognition of and adherence to host cells [60]. The abundance of B. ambifaria in the rhizosphere and the presence in this species of putative virulence-related determinants pose the question of whether the low prevalence of B. ambifaria among CF patients can be attributed to the absence of some crucial virulence traits and/or to the different regulation of existing ones. Biotechnological use of Bcc: should we keep an option open? It is accepted that different species of the Bcc and even strains within the same species differ widely from each other for transmissibility and clinical impact on CF patients. This has opened discussion on whether infection control measures, which are rigidly applied to all Bccinfected CF patients, could be stratified to meet more selective, human and efficient criteria by taking into account the species and/or strain status of the infectious agent [61]. Similarly, it can be questioned if safety issues on the biotechnological use of Bcc should be reassessed by taking into account the ongoing taxonomy of the Bcc and existing differences in epidemiology and pathogenicity

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among Bcc species, and if existing restrictions should not be applied so rigidly to all Bcc isolates irrespective of their species status. Should the ban be lifted on some strains of distinct Bcc species such as B. ambifaria and B. pyrrocinia but not on some other species such as B. cenocepacia, B. multivorans and B. dolosa? Is there a middle ground? An overall ecological description of these species is not yet possible because the preferential ecological niches and/or natural reservoirs of most Bcc species remain ill-defined. However, existing data on their distribution in natural habitats coupled with epidemiological and pathogenicity data point to the high diversity of this group of taxonomically closely related species, and this could warrant a reconsideration of the regulatory status of the individual Bcc species. At present, several issues remain controversial (Box 2). The close genetic relatedness of these species raises concerns about the possibility of DNA exchange among strains that belong to different species. This would, in principle, enable an apparently harmless Bcc strain to acquire pathogenicity traits from a virulent neighbour. Certainly, systems exist in Bcc to facilitate recombination, with an extensive presence of insertion sequences, phage and genomic islands [62–64]. Although no solid evidence is available for genetic exchange in nature between different Bcc species, in vitro transduction of antibiotic-resistance genes, the existence of Bcc phage with broad hostspecificity and the presence of common insertion sequences in different Bcc species make horizontal gene transfer between Bcc species a realistic possibility [64–66]. The phenomenon is probably of broader significance given that the rhizosphere could function as a hot spot for high-rate transfer of genes between related rhizobacteria (e.g. Burkholderia, Pseudomonas and Stenotrophomonas species) [67]. Nevertheless, integration of foreign genes by homologous recombination could be prevented by knocking out the rec system of biotechnologically useful Bcc strains before their release into the environment but the full exploitation of such mutants would require an accurate assessment of their ecological fitness. Moreover, the increasing genomic information available for Bcc species would provide a basis for the systematic inactivation of virulence genes in biotechnological strains to render them avirulent. Finally, it has not been firmly established whether some Bcc species are more involved than others in transient infection and are more susceptible to eradication by antibiotic therapy. Outcome studies on large CF populations are needed to evaluate the relative virulence of all species and specific strains within the Bcc. At present, it can be argued that some species such as B. ambifaria, B. pyrrocinia and B. anthina are mainly

Box 2. Outstanding questions † What are the natural reservoirs of individual Bcc species? † What are the routes of transmission of Bcc bacteria to patients? † What is the relative risk of infection with individual Bcc species? † Which bacterial factors are responsible for patient-to-patient transmission and virulence in the different Bcc species? www.sciencedirect.com

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† Are there differences between the Bcc species in their capacity to cause transient versus chronic infection? † Are there differences in clinical outcome between patients infected with different strains or species of Bcc? † Is it possible to engineer ‘safe’ Bcc strains by knocking out rec and/or virulence-related genes?

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intermittent colonizers of the CF airway without evidence of an associated clinical decline, as already proposed for Stenotrophomonas maltophilia and the harmless fluorescent pseudomonads [68]. If this view is corroborated by robust evidence, it might open the way for a relaxation of restrictions on the use of selected strains of B. ambifaria and B. pyrrocinia as biopesticides in agriculture, which could possibly lead to a differentiation, in this respect, from pathogenic species like B. cenocepacia, B. multivorans and B. dolosa. Concluding remarks Once thought to be a single species, B. cepacia is now regarded as a complex of at least nine closely related species. This taxonomic ‘revolution’ still represents a big challenge to clinical and environmental microbiologists because the ecology and pathogenicity of Bcc species must be reassessed in a considerably more complex context. The availability of reliable identification methods and the gathering of robust epidemiological and clinical data, along with the unravelling of the ecological properties of Bcc species and the search for their natural reservoirs will shed more light on the underlying diversity between the species. A substantial effect on assessment of the genetic relationships between the Bcc species is expected from the extensive implementation of the MLST scheme and genome sequencing of several Bcc isolates that is currently underway. Challenging questions for environmental and clinical microbiologists are whether the existing differences in the pathogenetic and ecological properties among Bcc species warrant a reconsideration of the possibility of using these organisms in biotechnology. Such a reassessment would also be welcomed by clinical scientists as a means of reducing current draconian infection control measures. Acknowledgements Work in our laboratories has been partially supported by Istituto Nazionale per la Prevenzione e la Sicurezza sul Lavoro (ISPESL; grant B56_DIPIA_00) and Fondazione Italiana per la Ricerca sulla Fibrosi Cistica (FFC; grants FFC#9/2003 and FFC#11/2004). We also acknowledge with respect the work of many researchers that has contributed to knowledge in this field, which could not be directly referenced here for space reasons.

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