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28 The Family Burkholderiaceae
Tom Coeny e
Laboratory of Pharmaceutical Microbiology, Ghent University, Ghent, Belgium
Taxonomy: Historical and Current . . ..................... 759
Short Description of the Family . . . . . . . . . .. . . . . . . . . . . . . . . 759
Taxonomic History and Phylogenetic Structure of
the Family and Its Genera . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 759
Molecular Analyses . . . . . . .................................. 760
DNA-DNA Hybridization Studies . . . .. . . . . . . . . . . . . . . . . . . 760
Phylogenetic Analysis Based on Housekeeping Genes . . . 761
Multilocus Sequence Analysis .. . . . . . . . . . . . . . . . . . . . . . . . . . 761
MALDI-TOF MS .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 761
PCR-Based Assays for Identification of Species
Belonging to the Family Burkholderiaceae ..............763
Ribotyping and Riboprinting . . . . . . . . . . . . . . . . . . . . . .. . . . . 763
REP-PCR . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 763
Genome Comparison . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . 764
Phenotypic Analyses .. . . . .................................. 765
Burkholderia Yabuuchi et al. 1993 Emend.
Gillis et al. 1995 ..........................................765
Chitinimonas Chang et al. 2004 Emend.
Kim et al. 2006 ...........................................766
Cupriavidus Makkar and Casida 1987 Emend.
Vandamme and Coenye 2004 ...........................766
Lautropia Gerner-Smidt et al. 1995 . . . . . . . . . . . . . . . . . . . . . 766
Limnobacter Spring et al. 2001 Emend. Lu et al. 2011 . . . 767
Pandoraea Coenye et al. 2000 ............................767
Paucimonas Jendrossek 2001 ............................768
Polynucleobacter Heckmann and Schmidt 1987
Emend. Hahn et al. 2009 ................................768
Ralstonia Yabuuchi et al. 1996 . . . . . . . . . . . . . . . . . . .. . . . . . . . 768
Thermothrix Caldwell et al. 1981 . . . . . . . . . . . . . . . . . . . . . . . . 768
Wautersia Vaneechoutte et al. 2004 .....................769
Isolation, Enrichment, and Maintenance Procedures . . . . . 769
Ecology . . . . . . ............................................... 770
Pathogenicity: Clinical Relevance . . . . ..................... 770
Burkholderia pseudomallei and Burkholderia mallei .....770
Respiratory Tract Infections in Cystic
Fibrosis Patients .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
Application . ............................................... 770
Burkholderia cepacia Complex Bacteria as Biocontrol,
Bioremediation, and Plant-Growth-Promoting
Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . 770
Heavy-Metal Resistance in the Genus Cupriavidus .....771
Abstract
The family Burkholderiaceae belongs to the order Burkholderiales
within the class Betaproteobacteria. It contains the genera
Burkholderia (the type genus), Chitinimonas, Cupriavidus,
Lautropia,Limnobacter,Pandoraea,Paucimonas,
Polynucleobacter,Ralstonia,Thermothrix, and Wautersia. The
family is characterized by the presence of ecologically extremely
diverse organisms and contains truly environmental saprophytic
organisms, phytopathogens, opportunistic pathogens, as well as
primary pathogens for humans and animals.
Taxonomy: Historical and Current
Short Description of the Family
Burk.hol.de.ri.a’ce.ae. M.L. fem. n. Burkholderia type genus of
the family, -aceae ending to denote family; M.L. pl. n.
Burkholderiaceae, the Burkholderia family.
The description is an emended version of the description
given in Bergey’s Manual, 2nd edition (Garrity et al. 2005).
Family is phenotypically, metabolically, and ecologically
extremely diverse and includes both strict aerobic and faculta-
tively anaerobic chemoorganotrophs, as well as obligate and
facultative chemolithotrophs. Based on phylogenetic analysis
of 16S rRNA gene sequences, the family Burkholderiaceae
contains the genera Burkholderia (the type genus), Cupriavidus,
Lautropia,Limnobacter,Pandoraea,Paucimonas,
Polynucleobacter,Ralstonia, and Thermothrix. In addition to
these genera, the family also contains the more recently
described genera Chitinimonas and Wautersia. Together with
the families Alcaligenaceae, Comamonadaceae, and Oxalobac-
teraceae, the family Burkholderiaceae belongs to the order
Burkholderiales within the class Betaproteobacteria.
Taxonomic History and Phylogenetic Structure
of the Family and Its Genera
Historically, the taxonomy of the family Burkholderiaceae has its
basis in studies on the genus Pseudomonas (reviewed by Kersters
et al. 1996). Due to its broad and vague phenotypic definition,
a lot of incompletely characterized, polarly flagellated, rod-
shaped, aerobic, Gram-negative bacteria were placed in the
genus Pseudomonas, but following results obtained by rRNA-
DNA hybridizations, the genus was divided into five so-called
rRNA groups by Palleroni et al. (1973). Later De Vos et al.
(1985) demonstrated that these five groups were only distantly
E. Rosenberg et al. (eds.), The Prokaryotes – Alphaproteobacteria and Betaproteobacteria, DOI 10.1007/978-3-642-30197-1_239,
#Springer-Verlag Berlin Heidelberg 2014
related to each other and that the genuine genus Pseudomonas
had to be restricted to the group containing the type species
Pseudomonas aeruginosa (rRNA group I or the Pseudomonas
fluorescens rRNA branch). The seven species ([Pseudomonas]
solanacearum,[Pseudomonas]pickettii,[Pseudomonas]cepacia,
[Pseudomonas]gladioli,[Pseudomonas]mallei,[Pseudomonas]
pseudomallei, and [Pseudomonas]caryophylli belonging to rRNA
group II (the so-called solanacearum rRNA branch)) were trans-
ferred to the new genus Burkholderia in 1992 by Yabuuchi et al.
[Burkholderia]pickettii and [Burkholderia]solanacearum
were subsequently transferred to the novel genus Ralstonia
(Yabuuchi et al. 1995). The genus Burkholderia belongs to
rRNA superfamily III sensu De Ley (1992) or subgroup beta-3
of the Betaproteobacteria sensu Woese (1987). In the last decades,
the genus Burkholderia has been expanded considerably, now
containing well over 60 species (see http://www.bacterio.cict.fr/
b/burkholderia.html for an up-to-date overview). The
closest neighbor of the genus Burkholderia is the genus
Pandoraea, described by Coenye et al. in 2000. Besides the
previously described species [Burkholderia]norimbergensis, the
genus initially contained four other species (Pandoraea apista,
Pandoraea pnomenusa, Pandoraea pulmonicola, and Pandoraea
sputorum) and was more recently expanded with the species
Pandoraea thiooxydans (Anandham et al. 2010) and Pandoraea
oxalativorans,Pandoraea faecigallinarum, and Pandoraea
vervacti (Sahin et al. 2011). Besides these named species, the
genus Pandoraea also contains four unnamed genomospecies
(Coenye et al. 2000; Daneshvar et al. 2001).
A second major phylogenetic cluster within the family
contains the genera Cupriavidus, Ralstonia, and Wautersia. As
already mentioned above, the genus Ralstonia was created to
accommodate two misclassified Pseudomonas species (Ralstonia
pickettii and Ralstonia solanacearum) as well as a misclassified
Alcaligenes species (Ralstonia eutropha) (Yabuuchi et al. 1995).
Over the next few years, a number of other Ralstonia species
were described (either novel species or previously misclassified
taxa), including Ralstonia basilensis (Steinle et al. 1998;Goris
et al. 2001), Ralstonia campinensis (Goris et al. 2001), Ralstonia
gilardii (Coenye et al. 1999), Ralstonia insidiosa (Coenye et al.
2003), Ralstonia mannitolilytica (De Baere et al. 2001), Ralstonia
metallidurans (Goris et al. 2001), Ralstonia oxalatica (Sahin et al.
2000), Ralstonia paucula (Vandamme et al. 1999), Ralstonia
respiraculi (Coenye et al. 2003b), Ralstonia syzygii (formerly
known as [Pseudomonas syzygii]) (Vaneechoutte et al. 2004),
and Ralstonia taiwanensis (Chen et al. 2001). In 2004
Vaneechoutte et al. reported that two sublineages (>4 % dis-
similarity in the 16S rRNA gene sequence) could be distin-
guished within the genus Ralstonia and proposed to reclassify
the species belonging to the so-called Ralstonia eutropha lineage
(R. basilensis,R. campinensis,R. eutropha,R. gilardii,
R. metallidurans,R. oxalatica,R. paucula,R. respiraculi, and
R. taiwanensis) in the novel genus Wautersia, with Wautersia
eutropha as the type species. However, in 2004, Vandamme and
Coenye demonstrated that W. eutropha (the type species of the
genus Wautersia) is a later heterotypic synonym of Cupriavidus
necator (the type species of the genus Cupriavidus, described by
Makkar and Casida in 1987). According to Rule 37a of the
Bacteriological Code, this meant that the name Wautersia had
to be replaced by Cupriavidus, and all Wautersia species were
renamed as Cupriavidus species. Only a single species remains in
the genus Wautersia, Wautersia numazuensis (described in 2005
by Kageyama et al.), but its close phylogenetic relationship with
Cupriavidus species strongly suggests it should be reclassified in
this genus. Since its creation, three novel species were added to
the genus Cupriavidus, namely, Cupriavidus pinatubonensis and
Cupriavidus laharis (Sato et al. 2006) and Cupriavidus pampae
(Cuadrado et al. 2010). Estrada-de Los Santos et al. (2012)
recently proposed the name Cupriavidus alkaliphilus for plant-
associated isolates. At the time of writing, this name has not been
validated yet.
Besides the Burkholderia/Pandoraea group and the
Cupriavidus/Ralstonia/Wautersia group, several other genera
belong to the family Burkholderiaceae, namely, the genera
Chitinimonas,Lautropia,Limnobacter,Paucimonas,Poly-
nucleobacter, and Thermothrix.
The genus Chitinimonas (Chang et al. 2004), containing the
species Chitinimonas taiwanensis (Chang et al. 2004) and
Chitinimonas koreensis (Kim et al. 2006), forms a separate branch
in the 16S rRNA-based phylogenetic tree and is only distantly
related to other members of this family. Actually, this genus appears
to be more closely related to members of the Neisseriaceae and its
exact phylogenetic affiliation probably will need to be revised.
Limnobacter and Lautropia species (Limnobacter thiooxidans,
Limnobacter litoralis, and Lautropia mirabilis) (Gerner-Smidt
et al. 1994; Spring et al. 2001; Lu et al. 2011) occupy a position
between both major phylogenetic groups but are more closely
associated with the Burkholderia/Pandoraea group.
The genus Paucimonas (containing a single species,
Paucimonas lemoignei) (Jendrossek 2001) appears to occupy
a position somewhat between the Burkholderiaceae and the
Oxalobacteraceae.
The genus Polynucleobacter contains five species
(Polynucleobacter acidophilus, Polynucleobacter cosmopolitanus,
Polynucleobacter difficilis, Polynucleobacter necessarius [with sub-
species necessarius and asymbioticus], and Polynucleobacter
rarus) (Heckmann and Schmidt 1987; Hahn et al. 2009,2010,
2011a,b,2012). Although it occupies a distinct position within
the tree, its closest relatives are members of the Cupriavidus/
Ralstonia/Wautersia group.
Finally, the genus Thermothrix, containing the species
Thermothrix azorensis and Thermothrix thiopara (Odintsova
et al. 1996; Caldwell et al. 1976), appears to be unrelated to any
other member of this family and its deep-branching position
suggests it should be reclassified.
Molecular Analyses
DNA-DNA Hybridization Studies
The vast majority of descriptions of species of the genera
belonging to the family Burkholderiaceae include results of
760 28 The Family Burkholderiaceae
DNA-DNA hybridization experiments. Overall species can easily
be distinguished from each other as DNA-DNA
binding values between representatives of different species are
below 30 %. However, within the Burkholderia cepacia complex,
representatives of different species generally have DNA-DNA
hybridization values between 30 % and 60 %, while values
obtained from strains belonging to the same species are generally
higher than 70 %. DNA-DNA binding values obtained with
other Burkholderia species are generally below 30 % (Coenye
et al. 2001). The same appears to be true to some extent for
Pandoraea species (with values up to 45 % between
strains from different species) (Coenye et al. 2000) and certain
Ralstonia species, e.g., hybridization values up to 58 %
between Ralstonia pickettii and Ralstonia mannitolilytica
(De Baere et al. 2001).
Phylogenetic Analysis Based on
Housekeeping Genes
Ribosomal RNA genes evolve slowly, and due the associated
limited taxonomic resolution, analysis of the 16S rRNA gene
often does not allow to reliably distinguish between closely
related species (Fox et al. 1992; Palys et al. 2000). This problem
is most obvious in the genera Burkholderia (especially
in the Burkholderia cepacia complex) and in the genus Pandoraea
(>Fig. 28.1). The sequencing of several ‘‘housekeeping’’
genes (i.e., protein-coding genes with an essential function)
(e.g., gyrB,rpoB,infB, and recA) has been proposed to
complement the phylogenetic information obtained
from sequencing the 16S rRNA gene (Eisen 1995; Yamamoto
and Harayama 1995,1998; Venkateswaran et al. 1998;
Hedegaard et al. 1999; Mare
´chal et al. 2000). The housekeeping
genes most often used in phylogenetic analyses of taxa of
the family Burkholderiaceae are recA (encoding recombinase A)
and gyrB (encoding gyrase B).
Phylogenetic analyses based on the recA gene have played
an important role in taxonomic studies of the genus
Burkholderia and in the development of diagnostic PCR assays
(see below), especially for the Burkholderia cepacia complex
(Mahenthiralingam et al. 2000; Payne et al. 2005). In phyloge-
netic trees based on (nearly) complete recA sequences, most
Burkholderia cepacia complex species will form discrete clusters
that can easily be discerned from other species, unlike in trees
based on 16S rRNA gene sequences. A notable exception
is Burkholderia cenocepacia, in which four different recA
lineages (designated IIIA through IIID) can be found
(Vandamme et al. 2003; Vanlaere et al. 2009). Other protein-
coding housekeeping genes that have been used for
deducing relationships between Burkholderia species include
hisA (encoding a 1-(5-phosphoribosyl)-5-[(5-phosphoribo-
sylamino)methylideneamino]imidazole-4-carboxamide isom-
erase involved in histidine biosynthesis) (Papaleo et al. 2010)
and parB and repA, two genes involved in the partitioning
and replication, respectively, of the multireplicon Burkholderia
cepacia complex genomes (Drevinek et al. 2008).
Sequence analysis of the gene encoding the gyrase B protein
(gyrB) was shown to be very useful to deduce phylogenetic
relationships between Pandoraea species. Sequence analysis of
gyrB confirmed the separate status of Pandoraea genomospecies
1, 3, and 4 and suggested a close relationship between Pandoraea
genomospecies 2 and Pandoraea sputorum (Coenye and LiPuma
2002; Sahin et al. 2011).
Multilocus Sequence Analysis
Initially developed as a typing tool, the sequence analysis
of multiple (protein-coding) housekeeping genes (multilocus
sequence analysis, MLSA) has emerged as a powerful
taxonomic tool (Gevers et al. 2005; Coenye et al. 2005). Within
the genus Burkholderia, MLSA schemes have been developed
for the Burkholderia pseudomallei/Burkholderia mallei/
Burkholderia thailandensis group (Godoy et al. 2003) and the
Burkholderia cepacia complex (Baldwin et al. 2005). In order to
keep up with the increasing taxonomic complexity of the
Burkholderia cepacia complex and to be able to include other
Burkholderia species, an ‘‘expanded’’ MLSA scheme was devel-
oped and published in 2009 (Spilker et al. 2009). The use of
degenerate primers allowed expansion of the Burkholderia
cepacia complex MLSA scheme to include other clinically
relevant (e.g., Burkholderia gladioli) or important environmen-
tal (e.g., Burkholderia glumae) species. In the Burkholderia
cepacia complex MLSA scheme, the following genes are used:
atpD (encoding ATP synthase bchain), gltB (large subunit
of glutamate synthase), gyrB (gyrase B), recA (recombinase A),
lepA (GTP-binding protein), phaC (acetoacetyl-CoA reductase),
and trpB (subunit B of tryptophan synthase). In the
Burkholderia pseudomallei MLSA scheme, the following genes
are used: aceA (encoding acetyl coenzyme A reductase), gltB
(large subunit of glutamate synthase), gmhD (ADP
glycerol-mannoheptose epimerase), lepA (GTP-binding elonga-
tion factor), lipA (lipoic acid synthetase), narK (nitrite extrusion
protein), and ndh (NADH dehydrogenase).
MALDI-TOF MS
Matrix-assisted laser desorption/ionization time-of-flight mass
spectrometry (MALDI-TOF MS) of intact cells has recently
emerged as a powerful tool for the characterization and identifi-
cation of microorganisms. Most studies so far have focussed on
the Burkholderia cepacia complex. In their pioneering study,
Vanlaere et al. (2008) demonstrated that MALDI-TOF MS is
a powerful approach for the rapid identification of Burkholderia
cepacia complex bacteria. Seventy-five strains belonging to the
nine at that time established Burkholderia cepacia complex spe-
cies as well as some species commonly misidentified as belong-
ing to the Burkholderia cepacia complex were included. All
Burkholderia cepacia complex strains clustered together and
could be separated from non-Burkholderia cepacia complex
strains. Within the Burkholderia cepacia complex, most strains
The Family Burkholderiaceae 28 761
.Fig. 28.1
Phylogenetic reconstruction of the family Burkholderiaceae based on 16S rRNA and created using the neighbor-joining algorithm with
the Jukes-Cantor correction. The sequence datasets and alignments were used according to the All-Species Living Tree Project (LTP)
database (Yarza et al., 2010; http://www.arb-silva.de/projects/living-tree). The tree topology was stabilized with the use of a
representative set of nearly 750 high quality type strain sequences proportionally distributed among the different bacterial and archaeal
phyla. In addition, a 40% maximum frequency filter was applied in order to remove hypervariable positions and potentially misplaced
bases from the alignment. Scale bar indicates estimated sequence divergence
762 28 The Family Burkholderiaceae
grouped in species-specific clusters, except for Burkholderia
anthina and Burkholderia pyrrocinia strains which constituted
a single cluster. Subsequent studies in the context of cystic
fibrosis microbiology confirmed that MALDI-TOF MS is
a useful addition to the toolbox to identify Burkholderia cepacia
complex species and related taxa (including Ralstonia and
Cupriavidus species) (Degand et al. 2008; Minan et al. 2009;
Ferna
´ndez-Olmos et al. 2012; Desai et al. 2012). Two recent
studies also demonstrated that this technique is appropriate
for the confirmation of Burkholderia pseudomallei (Inglis et al.
2012; Lau et al. 2012).
PCR-Based Assays for Identification of Species
Belonging to the Family Burkholderiaceae
Several candidate PCR assays have been developed for the iden-
tification of the Burkholderia cepacia complex species, but most
of these assays were developed before the recognition that
several species constitute the Burkholderia cepacia complex,
and most relied on published DNA sequence data derived
from analyses of culture collection strains that, in retrospect,
are poorly representative of the total diversity within this group.
However, in 1999, LiPuma et al. and Bauernfeind et al. (1999)
presented rRNA-gene-based PCR assays which allowed the iden-
tification of Burkholderia multivorans and Burkholderia
vietnamiensis and Burkholderia cepacia, Burkholderia
cenocepacia, and Burkholderia stabilis as a group (but the latter
three species could not be differentiated from each other). They
also developed a PCR assay that, in retrospect, showed a positive
reaction for the majority of Burkholderia, Ralstonia,
Cupriavidus, and Pandoraea isolates investigated (unpublished
data). Around the same time, Whitby et al. (2000) described an
rRNA-gene-based PCR assay for the identification of
Burkholderia gladioli. Vermis et al. (2004) developed a 16S
rRNA-based assay for the identification of Burkholderia dolosa.
16S rRNA-gene-based PCR assays with excellent sensitivity and
specificity were also developed for the genus Pandoraea as
a whole and a number of individual Pandoraea species
(Pandoraea sputorum, Pandoraea norimbergensis, Pandoraea
pnomenusa, and Pandoraea apista/Pandoraea pulmonicola)
(Coenye et al. 2001a). Similar assays were developed for
Ralstonia pickettii and Ralstonia mannitolilytica (Coenye et al.
2002) and Ralstonia insidiosa (Coenye et al. 2003a).
Due to the inherent limitations imposed by the use of the
16S rRNA gene, the possibility to use alternative targets for PCR-
based identification tools has been explored. The recA gene was
identified as such a target that turned out to be extremely useful
for the differentiation of Burkholderia species, including mem-
bers of the Burkholderia cepacia complex. Analysis of recA
sequences from a large number of isolates enabled the design
of a Burkholderia genus-specific recA PCR as well as the design of
aBurkholderia cepacia complex-specific recA PCR
(Mahenthiralingam et al. 2000; Payne et al. 2005). In addition,
PCR assays were developed for a number of individual
Burkholderia cepacia complex species, including Burkholderia
cepacia, Burkholderia multivorans, Burkholderia cenocepacia,
Burkholderia stabilis and Burkholderia vietnamiensis
(Mahenthiralingam et al. 2000), Burkholderia ambifaria (Coenye
et al. 2001), and Burkholderia dolosa (Vermis et al. 2004).
A thorough evaluation by Vermis et al. (2002) (508 Burkholderia
cepacia complex isolates representing nine species) revealed that
the recA-based assays for the identification of Burkholderia
multivorans, Burkholderia cenocepacia, and Burkholderia
ambifaria were most efficient but that the assay directed against
Burkholderia cepacia lacked sensitivity and cross-reacted with all
Burkholderia pyrrocinia isolates examined. In an attempt to
develop a single PCR assay to distinguish all Burkholderia
cenocepacia subgroups from other Burkholderia cepacia complex
species, Drevinek et al. (2008) explored the sequence diversity in
the parB and repA genes. The Burkholderia cenocepacia repA
sequences were distinct from other species, which enabled the
design of a species-specific multiplex PCR that allowed to iden-
tify the former species with 100 % sensitivity and 93 %
specificity.
Ribotyping and Riboprinting
In ribotyping, chromosomal restriction fragment length poly-
morphisms (RFLP) are detected by probing restriction-digested
genomic DNA with rRNA (Stull et al. 1988; Bingen et al. 1994)
and RFLPs result mainly from nucleotide sequence variation in
regions flanking rRNA genes. A modification of ribotyping,
referred to as PCR ribotyping, employs PCR to amplify the
16S–23S intergenic spacer region of the bacterial rRNA operon
to detect sequence length polymorphisms therein (Kostman
et al. 1995). Both ribotyping and PCR ribotyping were used in
early investigations of Burkholderia cepacia complex epidemiol-
ogy (LiPuma et al. 1988,1990,1994; Dasen et al. 1994).
Moissenet et al. (2001) evaluated PCR ribotyping as a tool for
species and strain differentiation within the genera Ralstonia and
Cupriavidus but concluded that the resolution was insufficient.
The generation of RiboPrint patterns for Burkholderiaceae using
the RiboPrinter microbial characterization system has been
evaluated in a limited number of studies. Brisse et al. (2000)
showed that this method was potentially useful for species-level
identification within the Burkholderia cepacia complex. The
method was also evaluated for typing of Burkholderia
pseudomallei (Inglis et al. 2002) and Burkholderia mallei (Grif
et al. 2003). Riboprinting has occasionally been used for other
taxa within this family, including Cupriavidus pauculus
(Clermont et al. 2001).
REP-PCR
The genomes of most bacteria contain specific conserved repeti-
tive sequences. Versalovic et al. (1991) showed that these repeats
can be used to amplify the intervening DNA sequences.
Three main sets of repeats have been used for typing:
(i) repetitive extragenic palindromic (REP) elements
The Family Burkholderiaceae 28 763
(35–40 bp), (ii) enterobacterial repetitive intergenic consensus
(ERIC) sequences (124–127 bp), and (iii) BOX elements
(154 bp) (Olive and Bean 1999). Depending on which sequences
are targeted, these assays are referred to as REP-, ERIC-, or
BOX-PCR, respectively. Collectively, these methods are referred
to as rep-PCR. Targets most commonly used for the
typing of Burkholderia cepacia complex organisms are
the ERIC (Liu et al. 1995; Cimolai and Trombley 1996;
Seo and Tsuchiya 2005) and BOX elements (Chen et al. 2001;
LiPuma et al. 2002; Biddick et al. 2003; Coenye et al. 2004; Seo
and Tsuchiya 2005). This approach has also been used to inves-
tigate Burkholderia pseudomallei (Currie et al. 2007) (BOX),
Ralstonia solanacearum (Smith et al. 1995; Norman et al. 2009;
Stevens and van Elsas 2010; Xue et al. 2011) (ERIC, REP, and
BOX), Ralstonia pickettii and Ralstonia insidiosa
(Ryan et al. 2011) (BOX), and Pandoraea apista (Atkinson
et al. 2006) (ERIC, REP, and BOX). Finally, BOX-PCR was also
used to compare root-nodule isolates with reference
isolates of Burkholderia phymatum,Burkholderia tuberum,
Burkholderia mimosarum,Burkholderia nodosa,Burkholderia
sabiae,Burkholderia caribensis, and Cupriavidus taiwanensis
(Liu et al. 2011).
Genome Comparison
Genome sequences are currently available for many
species of this family, but clinically relevant species like
B. cenocepacia, B. mallei, and B. pseudomallei,
.Table 28.1
Selection of strains of the family Burkholderiaceae for which a complete genome sequence is available. Data obtained from the Genome
Atlas Database (http://www.cbs.dtu.dk/services/GenomeAtlas/)
Species and strain designation
No. of
replicons Total size (bp) No. of genes
No. of 16S
rRNA genes
No. of tRNA
genes %GC
Burkholderia ambifaria AMMD 4 7,528,567 6,617 6 69 66.8
Burkholderia ambifaria MC40-6 4 7,642,536 6,697 6 68 66.4
Burkholderia cenocepacia AU 1054 3 7,279,116 6,477 6 67 66.9
Burkholderia cenocepacia HI2424 4 7,702,840 6,919 6 67 66.8
Burkholderia cenocepacia J2315 4 8,055,782 6,485 6 73 66.9
Burkholderia cenocepacia MC0-3 3 7,971,389 7,008 6 67 66.4
Burkholderia glumae BGR1 6 7,284,683 5,773 5 66 67.9
Burkholderia mallei ATCC 23344 2 5,835,527 5,025 4 56 68.5
Burkholderia mallei NCTC 10229 2 5,742,303 5,510 4 56 68.5
Burkholderia mallei NCTC 10247 2 5,848,380 5,852 4 55 68.5
Burkholderia mallei SAVP1 2 5,232,401 5,189 5 55 68.4
Burkholderia multivorans ATCC 17616 4 7,008,622 6,259 5 65 66.7
Burkholderia phymatum STM815 4 8,676,562 7,496 6 62 62.3
Burkholderia phytofirmans PsJN 3 8,214,658 7,241 6 63 62.3
Burkholderia pseudomallei 1106a 2 7,089,249 7,183 4 59 68.3
Burkholderia pseudomallei 1710b 2 7,308,054 6,347 4 60 68.0
Burkholderia pseudomallei 668 2 7,040,403 7,230 4 59 68.3
Burkholderia pseudomallei K96243 2 7,247,547 5,855 4 60 68.1
Burkholderia thailandensis E264 2 6,723,972 5,634 4 58 67.6
Burkholderia vietnamiensis G4 8 8,391,070 7,617 6 67 65.7
Burkholderia xenovorans LB400 3 9,731,138 8,702 6 64 62.6
Cupriavidus metallidurans CH34 4 6,913,352 6,319 4 62 63.5
Cupriavidus necator H16 3 7,416,678 6,626 5 61 66.3
Cupriavidus taiwanensis 3 6,476,522 1,611 5 63 67.0
Polynucleobacter necessarius subsp.
asymbioticus QLW-P1DMWA-1
1 2,159,490 2,077 1 38 44.8
Polynucleobacter necessarius subsp.
necessarius STIR1
1 1,560,469 1,508 1 37 45.6
Ralstonia pickettii 12D 5 5,685,358 5,361 3 54 63.3
Ralstonia pickettii 12 J 3 5,325,729 4,952 3 55 63.6
Ralstonia solanacearum GMI1000 2 5,810,922 5,120 4 57 67.0
764 28 The Family Burkholderiaceae
phytopathogens like R. solanacearum, and strains from species
with potential for specific applications like C. necator are
overrepresented. A selection of genomes of representatives of
the Burkholderiaceae and their key properties are shown in
>Table 28.1. More information can be found in reviews by
Mahenthiralingam and Drevinek (2007) and Vandamme and
Dawyndt (2011).
Many members of the Burkholderiaceae (most notably
species from the genera Burkholderia, Cupriavidus, and
Ralstonia) have large and unusual multireplicon genomes that
lie at the basis of the remarkable phenotypic diversity demon-
strated by these bacteria (Mahenthiralingam and Drevinek
2007). Early research carried out by Cheng and Lessie
(1994) showed that B. multivorans ATCC 17616 contained
three large circular replicons of 3.4, 2.5, and 0.9 Mb. Each of
these replicons contained rRNA genes, so they could be consid-
ered as real chromosomes. Rodley et al. (1995) showed that
the type strain of B. cepacia and strains belonging to B. glumae
and B. glathei also contained multiple replicons, as did strains
belonging to R. pickettii,R. solanacearum, and R. eutropha.
Further research by Lessie et al. (1996) showed that representa-
tives of all members of the B. cepacia complex contain two or
three chromosomes, with the total genome size ranging from
4.7 to 8.1 Mb. Research from others also demonstrated the
multiple replicon structure in B. gladioli (Wigley and Burton
2000). Interestingly, large genomes have been described as dis-
proportionately enriched in regulation and secondary
metabolism genes and depleted in protein duplication, DNA
replication, cell division, and nucleotide metabolism genes
in comparison to small-sized genomes (Konstantinidis and
Tiedje 2004). This may explain why species with large genomes
dominate in environments where resources are scarce but diverse,
such as in soils (Konstantinidis and Tiedje 2004). Recent work
demonstrated that the smallest chromosome in the B. cepacia
complex (chromosome 3) is not essential, but should be con-
sidered as a large plasmid that encodes virulence,
secondary metabolism, and other accessory functions
in B. cepacia complex bacteria (Agnoli et al. 2012). Based on
a bioinformatics approach, Juhas et al. (2012) recently
claimed that the core genome of the order Burkholderiales
consists of 649 genes. All but two of these identified genes
were located on the largest chromosome (chromosome 1)
of B. cenocepacia, including the known essential genes
infB, gyrB,ubiB, and valS, as well as the so far uncharacterized
genes BCAL1882, BCAL2769, BCAL3142, and BCAL3369.
There is a growing interest in using information derived from
whole-genome sequencing projects for taxonomic purposes
(Coenye and Vandamme 2003b; Coenye et al. 2005; Gevers
et al. 2005). Several of the approaches were evaluated
by Vanlaere et al. (2009; on 13 genomes) and by Vandamme
and Dawyndt (2011; on 44 genomes) to determine their ability
to contribute to our taxonomic understanding of the
Burkholderiaceae (with a strong focus on the genus
Burkholderia). Parameters that have been included in these
studies are average nucleotide identity (ANI), average amino
acid identity (AAI), and core gene identity (CGI)
(Konstantinidis and Tiedje 2005a,b,2007; Goris et al. 2007).
ANI and CGI analysis of 44 genomes (43 Burkholderia genomes
+C. metallidurans CH34) resulted in highly correlated similarity
matrices for both methods (r
2
= 0.95) (Vandamme and Dawyndt
2011). Trees based on these similarity matrices showed
a much better resolved picture of the phylogenetic relationships
of more distantly related species. For example, the B. cepacia
complex and the B. mallei/B. pseudomallei group were extremely
well resolved with high bootstrap values and both formed dis-
tinct lines of descent (Vandamme and Dawyndt 2011).
Phenotypic Analyses
Burkholderia Yabuuchi et al. 1993 Emend.
Gillis et al. 1995
Burk.hold.er’i.a. M.L. dim. ending -ia; M.L. fem. n.
Burkholderia, named after W. H. Burkholder, the American
bacteriologist who first discovered the etiological agent of sour
skin of onions.
The following genus description was presented in 1995 by
Gillis et al. and was based on the 11 species described at that
time. Burkholderia cells are Gram-negative, nonfermentative,
straight rods that have a single polar flagellum or a tuft of
polar flagella. A single species, B. mallei, is atrichous and
nonmotile. Catalase is produced, and oxidase activity varies
between species. The cellular fatty acids are characterized by
the presence of 3-hydroxy C16:0. The type strains of several
species are characterized by the presence of two types of orni-
thine lipids. Most species grow at 40 C. All species can grow
with the following substrates as sole carbon sources: glucose,
glycerol, inositol, galactose, sorbitol, and mannitol. Some spe-
cies are pathogenic for humans, animals, or plants. They
are isolated from plant material, soil, or clinical samples and
can be recognized on the basis of 16S rRNA characteristics.
Most strains accumulate polyhydroxybutyrate as carbon reserve
material and are capable of ortho cleavage of protocatechuate.
The G + C content is 59.0–69.5 mol%. The type species is
Burkholderia cepacia.
.Table 28.2
Phenotypic characteristics that allow the differentiation of
Burkholderia mallei,Burkholderia pseudomallei, and Burkholderia
thailandensis (Based on data compiled by LiPuma et al. (2011))
B. mallei B. pseudomallei B. thailandensis
Growth at 42 C++
Gas from nitrate ++
Acid from maltose ++
Acid from mannitol ++
Acid from arabinose ND +
Motile No Yes Yes
+, >90 % of isolates are positive; ,<10 % of isolates are positive; ND, not
determined
The Family Burkholderiaceae 28 765
Phenotypic characteristics distinguishing the closely related
species Burkholderia mallei,Burkholderia pseudomallei, and
Burkholderia thailandensis are shown in >Table 28.2.Pheno-
typic characteristics distinguishing the members of the
Burkholderia cepacia complex and Burkholderia gladioli from
each other are shown in >Table 28.3.
Chitinimonas Chang et al. 2004 Emend.
Kim et al. 2006
Chi.ti.ni.mo’nas N.L. n. chitinum chitin; Gr. n. monas
unit, monad; N.L. fem. n. Chitinimonas, a chitin-utilizing
monad.
Cells are Gram-negative rods and motile by means of single
polar flagella. Poly-b-hydroxybutyrate granules are stored as
reserve material. Endospores are not formed. Grows well by
using chitin as the exclusive carbon, nitrogen and energy source
both under aerobic and anaerobic conditions. Growth occurs
at 4–39 C, pH 4 to 10, and 0–1 % NaCl salinity. Catalase
and oxidase activity is present. Nitrate is reduced, no indole
production or glucose fermentation. Esculin, gelatin, and urea
may or may not be hydrolyzed. The major fatty acid components
are 16:0, 18:1 o7c, and summed feature 3 (16:1 o7cor 15 iso
2-OH or both). The type species is Chitinimonas taiwanensis.
Cupriavidus Makkar and Casida 1987 Emend.
Vandamme and Coenye 2004
Cup.ri.a.vi’dus. L. n. cuprum copper; L. adj. avidus eager for,
loving; M.L. neut. N. Cupriavidus lover of copper.
Cells are Gram-negative, peritrichously flagellated rods, and
chemoheterotrophic or chemolithotrophic. The metabolism is
oxidative. Several amino acids are used as sole carbon and
nitrogen sources. Catalase and oxidase activity is produced.
Resistance to various metals is widespread. The respiratory
quinone Q8 has been reported in [Ralstonia]eutropha
(Yabuuchi et al. 1995). The DNA G + C content
is between 63 and 69 mol%. Species occur in soil and human-
clinical specimens, particularly in samples from
debilitated patients. The type species is Cupriavidus
necator. Characteristics to differentiate Cupriavidus and
Ralstonia species are shown in >Table 28.4.
Lautropia Gerner-Smidt et al. 1995
Lau.tro’pia, in honor of the late Hans Lautrop, Danish
bacteriologist.
Gram-negative cocci occur in at least three forms: small
encapsulated cocci (1–2 mm in diameter), often occurring in
clusters of 10 to more than 100 cells; small unencapsulated cocci
(1–2 mm in diameter), motile by the action of a tuft of three to
nine flagella; and large (>5mm in diameter) spheroplast-like
cells. They are facultative aerobes but grow best under aerobic
conditions with no requirement for CO
2
. They are mesophilic,
growing at temperatures between 30 C and 44 C;
nonpigmented; grow on most enriched media, especially on
chocolate and Levinthal, TGY, and Tween 80 agar; and grow
slower with no hemolysis on horse-blood agar. Three colony
morphologies can be observed, flat, dry, and circular colonies
predominating in young cultures, becoming larger, wrinkled,
crisp, and crateriform on prolonged incubation and smooth,
.Table 28.3
Phenotypic characteristics that allow the differentiation of members of the Burkholderia cepacia complex and Burkholderia gladioli
(Based on data compiled by LiPuma et al. (2011))
Bam Ban Bar Bcen Bce Bco Bdi Bdo Blaa Blae Bme Bmu Bpy Bse Bst Bubo Bvi Bgl
Oxidase + + + + + v + + v + + + v + + + + v
Growth on MacConkey + + + v v + + + + + + + + + + + v +
Acid from
Maltose + + + v v v + + + + + + + + + + +
Lactose + + v + + + + + + + + + + + v + +
D-Xylose + + + + + + + + + + + + + + v + v +
Sucrose + v v + v + v v+vvv++
Adonitol + v + v v + v + v + + + + + + +
Nitrate reduction v v v v v++v +vvvv
Lys decarboxylase + v v + + + + +++v +v++
Orn decarboxylase +v v v vv+
Esculin hydrolase v vvv v+v v
Gelatinase + +v v + v vv+v+++ v
+, >90 % of isolates are positive; ,<10 % of isolates are positive; v, between 10 % and 90 % of strains are positive; ND, not determined
Bam B. ambifaria,Ban B. anthina,Bar B. arboris,Bcen B. cenocepacia,Bce B. cepacia,Bco B. contaminans,Bdi B. diffusa,Bdo B. dolosa,Blaa B. lata,Blae B. latens,Bme B.
metallica,Bmu B. multivorans,Bpy B. pyrrocinia,Bse B. seminalis,Bst,B. stabilis,Bub B. ubonensis,Bvi B. vietnamiensis,Bgl B. gladioli
766 28 The Family Burkholderiaceae
glistering, raised, round, mucoid colonies. The colony diameter
varies between pinpoint size and more than 5 mm; all colony
types may occur in all sizes, largest in older cultures; and colo-
nies are usually adherent to the substrate. Growth in broth is
granular with a coarse sediment and granules adherent to the
side of the tube. Biochemically, strains are oxidase, catalase, and
urease positive; reduce nitrate and nitrites; produce polysaccha-
ride on sucrose agar; ferment glucose, fructose, maltose, sucrose,
and mannitol; and do not ferment lactose, trehalose, raffinose,
inulin, salicin, adonitol, dulcitol, sorbitol, inositol, xylose,
rhamnose or arabinose, or hydrolyse starch. Most strains
hydrolyse esculin. They may produce b-xylosidase, but not
b-galactosidase or b-glucuronidase, do not decarboxylate lysine
or ornithine, and do not produce arginine decarboxylase/
dihydrolase or phenylalanine deaminase. They are VP,
gelatinase, and H
2
S negative, do not hydrolyze hippurate or
Tween 80, and are sensitive to penicillin G, ampicillin,
piperacillin, cefuroxime, gentamicin, and erythromycin. Mean
mol% (G + C) is 65 (range 64.6–65.4). The habitat is the human
oral cavity. The type species is Lautropia mirabilis.
Limnobacter Spring et al. 2001 Emend.
Lu et al. 2011
Lim.no.bac’ter. Gr. n. limnos lake; M.L. n. bacter rod; M.L.
masc. n. Limnobacter lake rod, referring to the isolation of the
type species from lake sediment.
Cells are Gram-negative, slightly curved rods that are motile
by single polar flagella. Polyhydroxybutyrate is stored as
a reserve material. Endospores are not formed. Limnobacter are
strictly aerobic and oxidase and catalase positive. Growth occurs
between 4 C and 44 C. Carboxylic acids and amino acids are
used as energy and carbon sources, but no carbohydrates or
polyols are used. They are not able to grow autotrophically.
Thiosulfate is oxidized to sulfate in the presence of an organic
carbon source. Major fatty acids are 18:1o7c, 16:1o7c, 16:0, and
10:0 3-OH. The major ubiquinone is Q-8. The G + C content is
between 55 % and 59 %. The type species is Limnobacter
thiooxidans.
Pandoraea Coenye et al. 2000
Pan.do.rae’a. N.L. fem. n. Pandoraea, referring to Pandora’s
box in Greek mythology, which was the origin of diseases
of mankind and thus to the surprisingly diverse members of
this genus.
Pandoraea cells are Gram-negative, nonsporulating straight
rods of 0.5–0.7 by 1.5–4.0 mm. They occur single and are motile
by means of a single polar flagellum. Catalase activity is present.
Growth is observed at 30 C and 37 C. Nitrite is not reduced.
There are no denitrification, no b-galactosidase or DNAse activ-
ity, no liquefaction of gelatin, no esculin hydrolysis, no indole
production, and no hydrolysis of Tween 80. Additional charac-
teristics are listed in the Results section above. The following
fatty acid components are present: 12:0, 12:0 2OH, 16:0, 17:0
cyclo, 16:0 2OH, 16:0 3OH, 19:0 cyclo o8c, 18:1 2OH, summed
feature 3, summed feature 4, and summed feature 7. The
G + C content is between 61.2 and 64.3 mol%. Strains of this
.Table 28.4
Phenotypic characteristics that allow the differentiation of Cupriavidus and Ralstonia species. Based on data reported by Gillis et al.
(1995), Goris et al. (2001), Coenye et al. (2003a), Coenye et al. (2003b), Vaneechoutte et al. (2004), Sato et al. (2006), Cuadrado et al.
(2010), and LiPuma et al. (2011)
Rp Rm Ri Rsyz Rsol Cr Cg Cp Ccam Cbas Cmet Cnec Cpina Clah Cpam Ctai Coxa Calk
Nitrate reduction + +v + v+vv v+ ++
Urease activity + + v ND ++ v+ +
Assimilation of
Citrate + + + v + +++ ++ ++ ++
N-acetylglucosamine + + ND +v
Phenylacetate + +v++ + + + + ++
Acid from
L-Arabinose + + ND ND +ND
Glucose + + + ++ ND
Growth at 41 Cv+NDND + v + ++++ +
Motility + + ND +ND+++ +++
+, >90 % of isolates are positive; ,<10 % of isolates are positive; v, between 10 % and 90 % of strains are positive; ND, not determined
Rp Ralstonia pickettii,Rm Ralstonia mannitolilytica,Ri Ralstonia insidiosa,Rsyz Ralstonia syzygii,Rsol Ralstonia solanacearum,Cr Cupriavidus respiraculi,Cg
Cupriavidus gilardii,Cp Cupriavidus pauculus,Ccam Cupriavidus campinensis,Cbas Cupriavidus basilensis,Cmet Cupriavidus metallidurans,Cnec Cupriavidus
necator,Cpina Cupriavidus pinatubonensis,Clah Cupriavidus laharis,Cpam Cupriavidus pampae,Ctai Cupriavidus taiwanensis,Coxa Cupriavidus oxalaticus,Calk
Cupriavidus alkaliphilus
The Family Burkholderiaceae 28 767
genus are isolated from human clinical samples (mostly cystic
fibrosis patients) and the environment and do not cause soft rot
on onions. The clinical data that are presently available indicate
that at least some of these organisms may cause chronic infec-
tion in and have been transmitted among cystic fibrosis patients.
The type species is Pandoraea apista. Phenotypic characteristics
allowing the differentiation of the different Pandoraea species
are shown in >Table 28.5.
Paucimonas Jendrossek 2001
Pau.ci.mo’nas. L., adj. paucus little, few; Gr. fem. monas unit,
cell; Paucimonas bacterium with restricted (few) catabolic
capacities.
Paucimonas cells are Gram-negative with chemoorga-
notrophic and strictly respiratory metabolism and are catalase
and oxidase positive. Preferred carbon sources are organic acids.
Most sugars (e.g., glucose, fructose, xylose), sugar acids (e.g.,
gluconate), polyalcohols (e.g., sorbitol, glycerol), alcohols (e.g.,
ethanol, butanediol, phenol), amino acids, polypeptides (e.g.,
nutrient broth, gelatin, Luria-Bertani broth), or polysaccharides
(e.g., starch, cellulose) do not support growth. PHB and related
homopolyesters and/or copolymers of hydroxyalkanoic acids
can be accumulated intracellularly. Chemolithoautotrophic or
phototrophic growth has not been observed. Type species of the
genus is Paucimonas lemoignei.
Polynucleobacter Heckmann and Schmidt 1987
Emend. Hahn et al. 2009
Po.ly.nuc0le.o.bac.ter. Gr. adj. polys numerous; L. masc. n.
nucleus nut, kernel; N.L. masc. n. bacter the equivalent of the
Gr. neut. n. bactron a rod; N.L. masc. Polynucleobacter rod with
many nucleoids.
It harbors endosymbiotic strains of several Euplotes
species and free-living strains dwelling in the water column
of freshwater lakes, ponds, and streams and is essential
for their host species and nonmotile. Not all strains affiliated
with the genus possess multiple nucleoid-like structures as indi-
cated by the genus name. The type species is Polynucleobacter
necessarius.
Ralstonia Yabuuchi et al. 1996
Ral.sto’n.ia. M.L. dim. -ia ending; M.L. fem. n. Ralstonia named
after Ericka Ralston, the American bacteriologist who first
described Pseudomonas pickettii.
Ralstonia cells are Gram-negative rods; have no formation of
endospores; are nonmotile or motile by means of a single polar
or peritrichous flagella; are aerobic, with a respiratory metabo-
lism with oxygen as terminal electron acceptor; and are able to
grow on ordinary peptone media. The colony color is beige for
most species. They are oxidase and catalase positive and lysine
and ornithine decarboxylase negative. The major ubiquinone is
Q-8. Cellular lipids contain two kinds of phosphatidylethanol-
amine. Major fatty acids are C16:0, a mixture of C18:0 o9t and
C18:1 o7c, and C14:0 3OH. The G + C content is between 64
and 68 mol%. The type species is Ralstonia pickettii. Character-
istics to differentiate Ralstonia and Cupriavidus species are
shown in >Table 28.4.
Thermothrix Caldwell et al. 1981
Ther.mo’thrix. Gr. adj. thermos, hot; Gr. fem. n. thrix, hair; N.L.
fem. n. Thermothrix, hot hair.
Thermothrix cells are Gram-negative rods, mobile with
a single polar flagellum, and thermophiles with growth temper-
atures ranging from 63 Cto86C. They are aerobic and are
.Table 28.5
Phenotypic characteristics that allow the differentiation of Pandoraea species (Based on data reported by Coenye et al. (2000),
Anandham et al. (2010), and Sahin et al. (2011))
Pnor Ppul Ppno Papi Pspu Pthio Poxa Pver Pfae
Growth at 42 C++ +v + ++
Enzymatic activity
Urease v ++v+++
C4-esterase v vvv++++
C8-esterase + +++++
Assimilation of
Caprate v + + v v ++
D-Gluconate + + + + + ND ++
+, all isolates are positive; , none of isolates are positive; v, result is isolate dependent; ND, not determined
Pnor Pandoraea norimbergensis,Ppul Pandoraea pulmonicola,Ppno Pandoraea pnomenusa,Papi Pandoraea apista,Pspu Pandoraea sputorum,Pthio Pandoraea
thiooxydans,Poxa Pandoraea oxalativorans,Pver Pandoraea vervacti,Pfae Pandoraea faecigallinarum
768 28 The Family Burkholderiaceae
facultative or obligate chemolithotrophs. Under unfavorable
conditions (lack of oxygen, pH around 8.5, or temperature
close to maximum growth temperature), cells will form
filaments and are isolated from geothermal sources where
the pH is close to neutral. The type species is Thermothrix
thiopara.
Wautersia Vaneechoutte et al. 2004
Wau.ter0si.a. L. fem. n. Wautersia named in honor of the Belgian
microbiologist Georges Wauters.
Wautersia cells are Gram-negative rods that are motile by
means of peritrichous flagella. They are aerobic, form smooth
colonies that reach 1–2 mm within 48 h at 30C on blood agar,
and are positive for catalase and oxidase. Glucose is neither
acidified nor assimilated. They are susceptible to colistin. Cellu-
lar fatty acids are of the saturated and monounsaturated
straight-chain types, mainly C16:1o9c, C16:0, C18:1o11c, and
C14:0. The type species of the genus is Wautersia eutropha.As
mentioned above, only Wautersia numazuensis still remains in
this genus, with all other species (including the type species
Wautersia eutropha) being reclassified as Cupriavidus species. It
is clear that Wautersia numazuensis should also be reclassified as
aCupriavidus species and that the use of the name Wautersia
should be discontinued.
Isolation, Enrichment, and Maintenance
Procedures
Most members of the family Burkholderiaceae grow on
a wide range of media, including (but not limited to) nutrient
agar, tryptic soy agar, Mueller-Hinton agar, Columbia agar, and
LB agar, with or without blood. However, the use of selective
media can be required to recover relevant species (Burkholderia
pseudomallei,Burkholderia cepacia complex) from clinical
samples. Note that attempts to grow Polynucleobacter necessarius
subsp. necessarius outside its eukaryotic host have so far
been unsuccessful (Hahn et al. 2009). Similarly, several obligate
endosymbionts of Psychotria plants (and other Rubiaceae)
have been described, but since attempts to obtain these in
pure culture were unsuccessful, they have been described
as ‘‘Candidatus Burkholderia kirkii’’ (Van Oevelen et al. 2002),
‘‘Candidatus Burkholderia calva’’ and ‘‘Candidatus
Burkholderia nigropunctata’’ (Van Oevelen et al. 2004),
‘‘Candidatus Burkholderia andongensis’’ and ‘‘Candidatus
Burkholderia petitii’’ (Lemaire et al. 2011), ‘‘Candidatus
Burkholderia harborii’’ and ‘‘Candidatus Burkholderia
schumanniana’’ (Verstraete et al. 2011), and, finally,
‘‘Candidatus Burkholderia hispidae,’’ ‘‘Candidatus Burkholderia
rigidae,’’ and ‘‘Candidatus Burkholderia schumannianae’’
(Lemaire et al. 2012).
Several different media have been developed for the selec-
tive isolation of Burkholderia cepacia complex isolates from
sputum of cystic fibrosis, e.g., Pseudomonas cepacia (PC)
medium (containing 300U polymyxin B per ml and 100 mg
ticarcilline per ml) (Gilligan et al. 1985); oxidation-fermentation
agar supplemented with lactose, 300U polymyxin B per ml, and
0.2U of bacitracin per ml (OFPBL agar) (Welch et al. 1987); and
B. cepacia selective agar (BCSA) (containing 1 % lactose and 1 %
sucrose in an enriched base of casein and yeast extract with 600U
of polymyxin B per ml, 10 mg of gentamicin per ml, and 2.5 mg
vancomycin per ml) (Henry et al. 1997). BCSA was reported
superior compared to OFPBL and PCA for the rapidity and
quality of recovery of B. cepacia complex organisms from cystic
fibrosis respiratory specimens and was more inhibitory towards
other organisms (Henry et al. 1999). A comparison of three
commercially available ‘‘Burkholderia cepacia’’ media (MAST
Diagnostics, Bootle, Merseyside, United Kingdom; LAB
M Ltd., Bury, United Kingdom; and Oxoid Ltd., Basingstoke,
United Kingdom), through the analysis of 142 clinical and
environmental isolates, showed that BCSA and Mast B. cepacia
medium supported the growth of Burkholderia cepacia complex
isolates most efficiently (Vermis et al. 2003b). BCSA and MAST
were also compared in terms of sensitivities and specificities for
the isolation of Burkholderia cepacia complex species from spu-
tum specimens from cystic fibrosis patients; in that study, BCSA
was found to be as sensitive as MAST agar but more selective
(Wright et al. 2001). It should be noted that several Pandoraea,
Ralstonia, and Cupriavidus isolates will also grow on BCSA. The
selectivity of the abovementioned media for the isolation of
environmental Burkholderia cepacia complex isolates may be
much lower (Carson et al. 1988), and therefore the use of
other media, like PCAT medium (containing azelaic acid and
tryptamine) (Burbage and Sasser 1982), may be recommended,
although not all Burkholderia cepacia complex species grow
on the latter medium (Vermis et al. 2003a). An enrichment
medium, based on the ability of Burkholderia cepacia complex,
isolates to use L-threonine and L-arabinose as carbon sources,
and their insensitivity to polymyxin B and 9-chloro-
9-(4-diethylaminophenyl)-10-phenylacridan (C-390), was
developed by Vermis et al. (2003b) and successfully used to
demonstrate the presence of Burkholderia cepacia complex in
water and soil samples (Vermis et al. 2003b; Vanlaere et al. 2005).
However, other bacteria were also recovered (including
isolates belonging to the genera Pandoraea, Chryseobacterium,
Comamonas, Ralstonia, Herbaspirillum, and Pseudomonas)
following this enrichment procedure. Ashdown medium
(containing crystal violet and gentamicin as selective agents)
is typically used for the isolation of Burkholderia pseudomallei
(LiPuma et al. 2011). If enrichment is required, an
enrichment broth consisting of Ashdown medium
supplemented with 50 mg of colistin was found to be
superior to standard enrichment broth such as tryptic soy
broth and increases recovery of Burkholderia pseudomallei
(LiPuma et al. 2011).
For short-term preservation (several weeks up to months),
stab cultures in semisolid medium or cultures on slants
(with storage at 4 C) are appropriate. For long-term storage,
media containing glycerol (20 % v/v) and storage at 80 Corin
liquid nitrogen are recommended.
The Family Burkholderiaceae 28 769
Ecology
For reviews of the ecology of Burkholderia species, see Coenye
and Vandamme (2003) and Compant et al. (2008). Members of
the genus Burkholderia occupy diverse ecological niches and can
be found in the soil, in water, and in (close) association with
plants, animals, and fungi. Several Burkholderia species (includ-
ing Burkholderia vietnamiensis) are capable of nitrogen fixation
and several Burkholderia species are capable of forming root
nodules on several plants. Others, including Burkholderia glad-
ioli and Burkholderia glumae, are notorious plant pathogens
(Gonzalez et al. 2007), as is Ralstonia solanacearum (Genin and
Boucher 2004; Mansfield et al. 2012). Much less is known about
the ecology of other genera of this family.
Pathogenicity: Clinical Relevance
Burkholderia pseudomallei and Burkholderia
mallei
The family Burkholderiaceae contains two highly pathogenic
organisms, Burkholderia pseudomallei and Burkholderia mallei
(LiPuma et al. 2011). Burkholderia pseudomallei causes
melioidosis, an infection characterized by a wide range of clin-
ical manifestations, ranging from asymptomatic colonization to
fulminant sepsis. The most common presentations of
melioidosis include pneumonia, soft-tissue infection, abscesses
of liver and spleen, and septicemia (Dance 1991; LiPuma et al.
2011). Burkholderia pseudomallei is a saprophytic organism,
broadly distributed in soil and water in Southeast Asia and
northern Australia. The majority of infected people acquire the
organism through percutaneous inoculation on exposure to
contaminated soil or water, although the possibility of inhala-
tion or ingestion as modes of infection requires further investi-
gation (Dance et al. 2000; Haase et al. 1995; Currie et al. 2001).
Sporadic cases have resulted from person-to-person or animal-
to-person spread (Dance 2000). Exposure in endemic areas is
quite frequent due to the organism’s ubiquity, and latent infec-
tions are common. Thus, it is difficult to accurately determine
what sort of environmental exposure poses the greatest risk of
melioidosis. Sporadic cases of human melioidosis occur in
regions outside the endemic area, such China, Korea, the
Philippines, Indonesia, India, and West Africa. Most cases in
Europe and North America are thought to be imported by
immigrants or international travelers (Dorman et al. 1998;
Dance et al. 1999). Several reports describe the occurrence of
B. pseudomallei in European cystic fibrosis patients. In
these cases, the organisms were most likely acquired during
travel, although this could not always be confirmed
(Visca et al. 2001; Schulin and Steinmetz 2001; O’Carroll et al.
2003; Engelthaler et al. 2011).
Burkholderia mallei is primarily a pathogen in horses, in
which it causes glanders, a disease characterized by fever, inflam-
mation of the nasal mucosa, necrosis, and obstruction of the
oropharynx. In humans, infection can be limited to
subcutaneous tissues or can disseminate to cause sepsis (LiPuma
et al. 2011). If inhaled, Burkholderia mallei can cause pneumonia
with necrosis of the tracheobronchial tree (Srinivasan et al.
2001). Burkholderia mallei can be spread via contact with
infected animals or through exposure in research laboratories
(Srinivasan et al. 2012; CDC 2000). Glanders has been
virtually eliminated in the Western world due to stringent infec-
tion control measures, including the immediate slaughter
of affected animals. However, research interest in this species
due to recent concerns about biological warfare may result in an
increasing risk of occupational exposure.
Respiratory Tract Infections in Cystic
Fibrosis Patients
Members of the Burkholderia cepacia complex and phenotypi-
cally similar species (including B. gladioli and Pandoraea,
Cupriavidus, and Ralstonia species) are notorious for causing
respiratory tract infections in cystic fibrosis patients. The
reader is referred to several detailed reviews on this topic for
more information (LiPuma 2005,2010, 2011; Coenye and
LiPuma 2003).
Application
Burkholderia cepacia Complex Bacteria as
Biocontrol, Bioremediation, and Plant-Growth-
Promoting Agents
The biological control of plant diseases, insects, and nematodes
by microorganisms (both bacteria and fungi) has been proposed
as an alternative or a supplement to chemical pesticides, and the
use of introduced biological control could have enormous eco-
logical and economic benefits. The two traditional approaches
used for biological control of soilborne plant pathogens in the
field have been (1) crop rotations, to allow time for resident
antagonists to ‘‘sanitize’’ the soil or for propagules of specialized
pathogens to die, and (2) the addition of organic amendments to
soil, to stimulate resident antagonists. However, the greatest
progress towards biological control of soilborne plant pathogens
has been made with antagonists introduced with the planting
material, i.e., biological control with plant-associated microor-
ganisms. It has often been shown that Burkholderia cepacia
complex strains can be used to control seedling and root diseases
in vitro and in field tests and could replace chemical alternatives
like captan, thiram, PCNB, benlate, and thiabendazole (LiPuma
and Mahenthiralingam 1999). Field tests have shown that
Burkholderia cepacia complex can colonize the rhizosphere
of several crops, including corn, maize, rice, pea, sunflower,
and radish and thereby significantly can increase the crop
yield, even in the absence of pathogens (see, e.g., McLoughlin
et al. 1992; Parke et al. 1991; Bowers and Parke 1993;
Hebbar et al. 1998; see Balandreau and Mavingui (2007)
for a review on this topic).
770 28 The Family Burkholderiaceae
In addition, the exceptional metabolic versatility of this
organism can be used for bioremediation purposes. Constitu-
ents of crude oils (including polycyclic aromatic compounds),
herbicides (including 2,4-dichlorophenoxyacetic acid and 2,4,5-
trichlorophenoxyacetic acid, the principal component of ‘‘Agent
Orange’’) TCE, and ether derivatives used as gasoline additives
can be degraded by several Burkholderia cepacia complex isolates
(Kilbane et al. 1982; Folsom et al. 1990; Krumme et al. 1993;
Bhat et al. 1994). Well-characterized biodegradative
strains belonging to the Burkholderia cepacia complex include
G4 (Nelson et al. 1987; Folsom et al. 1990; Shields et al. 1991;
Leahy et al. 1996; McClay et al. 1996; Massol-Deya et al. 1997)
and CRE-7 (Mueller et al. 1996). Potentially useful
strains have also been identified in other Burkholderia species,
including the species Burkholderia xenovorans (strain
LB400, degradation of biphenyl and polychlorinated biphenyls)
(Haddock et al. 1993; Seeger et al. 1995; Billingsley et al. 1997;
Master and Mohn 1998; Bopp 1985; Goris et al. 2004)
and Burkholderia kururiensis (trichloroethylene) (Zhang et al.
2000). For more information, readers are referred to Denef
(2007) for a review on this topic. Another extensively studied
strain with demonstrated biodegradative capacities is
Cupriavidus necator JMP134 (for a recent review, see Perez-
Pantoja et al. 2008).
However, many strains used or under development for those
purposes are taxonomically poorly characterized and their
potential hazard to people with cystic fibrosis is unclear
(Govan and Vandamme 1998; Holmes et al. 1998; Vidaver
et al. 1999; Govan et al. 2000). Until more is known about the
organisms currently used or under development for agricultural
applications and the potential risks for cystic fibrosis patients,
widespread use of these organisms has been forbidden by several
national regulatory agencies, including the United States
Environmental Protection Agency (EPA).
Heavy-Metal Resistance in the Genus Cupriavidus
Many members of the genus Cupriavidus, including Cupriavidus
metallidurans,Cupriavidus campinensis, and Cupriavidus
basilensis, are known for their metal resistance (Goris et al.
2001). The high resistance of certain Cupriavidus strains to
cadmium, copper, zinc, cobalt, lead, and mercury has attracted
considerable attention and opened the possibilities of develop-
ing bacterial biosensors for contamination and the development
of novel approaches for the bioremediation of contaminated
water and soils. For more information, the reader is directed to
the reviews of Mergeay et al. (2003), Diels et al. (2009), and von
Rozycki and Nies (2009) and the references therein.
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