Burkholderia cenocepacia phenotypic clonal variation during a 3.5-year colonization in the lungs of a cystic fibrosis patient.

Abstract

Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF) patients. Bacteria of the Burkholderia cepacia complex are the most threatening pathogens in CF, and a better understanding of how these bacteria adapt to the CF airway environment and resist the host defense mechanisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies adopted by Burkholderia cenocepacia during long-term colonization, we carried out a phenotypic assessment of 11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient during 3.5 years of colonization of the lungs, until the patient's death with cepacia syndrome. Phenotypic characterization included susceptibility assays against different classes of antimicrobials and characterization of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition, growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed. The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However, the values for antimicrobial susceptibility and swarming motility for the first B. cenocepacia isolate, thought to have initiated the infection, were consistently above those for the clonal variants obtained during the course of infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization, described here for the first time, involved the alteration of membrane fatty acid composition, in particular a reduction of the degree of fatty acid saturation, in the B. cenocepacia variants retrieved, along with the deterioration of pulmonary function and severe oxygen limitation.

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INFECTION AND IMMUNITY, July 2011, p. 2950–2960
0019-9567/11/$12.00doi:10.1128/IAI.01366-10
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 7
Burkholderia cenocepacia Phenotypic Clonal Variation during a
3.5-Year Colonization in the Lungs of a Cystic Fibrosis Patient?
Carla P. Coutinho,1,2Carla C. C. R. de Carvalho,1,2Andreia Madeira,1,2
Ana Pinto-de-Oliveira,1and Isabel Sa ´-Correia1,2*
Institute for Biotechnology and Bioengineering, Centre for Biological and Chemical Engineering, Instituto Superior Te ´cnico,
Av. Rovisco Pais, 1049-001 Lisbon, Portugal,1and Department of Bioengineering, Instituto Superior Te ´cnico,
Av. Rovisco Pais, 1049-001 Lisbon, Portugal2
Received 8 December 2010/Returned for modification 4 February 2011/Accepted 16 April 2011
Chronic lung infection is the major cause of morbidity and premature mortality in cystic fibrosis (CF)
patients. Bacteria of the Burkholderia cepacia complex are the most threatening pathogens in CF, and a better
understanding of how these bacteria adapt to the CF airway environment and resist the host defense mech-
anisms and therapeutically administered antibiotics is crucial. To provide clues to the adaptive strategies
adopted by Burkholderia cenocepacia during long-term colonization, we carried out a phenotypic assessment of
11 clonal variants obtained at the major Portuguese CF Center in Lisbon from sputa of the same CF patient
during 3.5 years of colonization of the lungs, until the patient’s death with cepacia syndrome. Phenotypic
characterization included susceptibility assays against different classes of antimicrobials and characterization
of cell motility, cell hydrophobicity and zeta potential, colony and cell morphology, fatty acid composition,
growth under iron limitation/load conditions, exopolysaccharide production, and size of the biofilms formed.
The results suggest the occurrence of clonal expansion during long-term colonization. For a number of the
characteristics tested, no isolation time-dependent consistent alteration pattern could be identified. However,
the values for antimicrobial susceptibility and swarming motility for the first B. cenocepacia isolate, thought to
have initiated the infection, were consistently above those for the clonal variants obtained during the course of
infection, and the opposite was found for the zeta potential. The adaptive strategy for long-term colonization,
described here for the first time, involved the alteration of membrane fatty acid composition, in particular a
reduction of the degree of fatty acid saturation, in the B. cenocepacia variants retrieved, along with the
deterioration of pulmonary function and severe oxygen limitation.
Morbidity and mortality in cystic fibrosis (CF) relate to
chronic airway infection with a variety of bacterial species,
which contributes significantly to tissue destruction and con-
tinuous deterioration of lung function (39, 40). In particular,
when Pseudomonas aeruginosa and Burkholderia cepacia com-
plex (BCC) bacteria become established, these bacteria are
difficult to eradicate from CF lungs due to their intrinsic resis-
tance to multiple antibiotics and to antimicrobial peptides of
the innate immunity and also to a rapid development of mul-
tidrug resistance. The BCC is a heterogeneous group that
comprises at least 17 closely related species that are ubiquitous
in the environment (28). A few recent studies have gained
insights into the complexity of the strategies developed by P.
aeruginosa cells in order to adapt to the stressing conditions to
which they are exposed in CF airways (16, 39, 40). However,
equivalent studies on BCC bacteria are still lacking, although
infections involving these bacteria, especially Burkholderia
cenocepacia, are particularly feared by CF patients because, in
contrast to the case for P. aeruginosa, a subset of patients
infected with these bacteria succumb to “cepacia syndrome,”
which is characterized as a fatal necrotizing pneumonia with
bacteremia (8, 28). Moreover, CF patients infected with B.
cenocepacia have a substantially worse prognosis than those
infected with P. aeruginosa only, and many centers refuse to
perform lung transplantation on CF patients colonized with
BCC bacteria (29, 41).
In many human infections, hosts and pathogens may coexist
for years. During chronic colonization of a CF patient’s air-
ways, bacteria of the BCC experience changing selection pres-
sures, in particular those resulting from challenges of the im-
mune defenses, antimicrobial therapy, and oxygen limitation
(21). These stressing conditions were found to lead to the
adaptive evolution of P. aeruginosa, a major respiratory patho-
gen in CF (40). Over time, during long-term colonization,
multiple phenotypic variants of the underlying clonal popula-
tion emerge and become established in the patient’s airways as
the result of genetic adaptations (28). Clonal variants of BCC
bacteria recovered from several chronically colonized CF pa-
tients at the major Portuguese CF treatment center, at Santa
Maria Hospital (HSM), were found to exhibit various antimi-
crobial susceptibility levels (25, 27). In general, the more re-
sistant variants were isolated following pulmonary exacerba-
tion and aggressive antibiotic therapy (25). In order to obtain
clues to the adaptive strategies developed by B. cenocepacia
during long-term colonization of CF lungs, in the present study
we carried out a phenotypic assessment of a number of rele-
vant characteristics of 11 sequential isolates of B. cenocepacia
(recA lineage III-A) obtained at HSM from the same CF pa-
tient during molecular epidemiological studies carried out by
* Corresponding author. Mailing address: Institute for Biotechnol-
ogy and Bioengineering, Centre for Biological and Chemical Engineer-
ing, Department of Bioengineering, Instituto Superior Te ´cnico, Av.
Rovisco Pais, 1049-001 Lisbon, Portugal. Phone: 351-218417682. Fax:
351-218419199. E-mail: isacorreia@ist.utl.pt.
?Published ahead of print on 2 May 2011.
2950

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our research group (9, 10). This patient (patient J) was chron-
ically colonized with the same B. cenocepacia strain for 3.5
years, from January 1999 to July 2002, until the patient’s death
with cepacia syndrome following progressive deterioration of
pulmonary function (8, 9). The clonal nature of the B. cenoce-
pacia isolates under study, as shown by the common ribopat-
tern with EcoRI produced by these variants (9), was confirmed
in the present work by their multilocus sequence typing
(MLST) profiles (2). A preliminary phenotypic assessment of
some of these clonal variants had been performed before by
comparing antimicrobial susceptibility profiles (25). In the
present study, this work was extended to all of the isolates
retrieved from the patient and to a number of other relevant
phenotypes in the context of persistent respiratory infections in
CF patients.
MATERIALS AND METHODS
Bacterial isolates and culture conditions. Eleven B. cenocepacia recA lineage
III-A isolates (9), obtained at the HSM Cystic Fibrosis Center in Lisbon, Por-
tugal, were used in this work (Table 1). Isolates were obtained from January 1998
to July 2002, as part of the hospital routine, from respiratory secretions of the
same chronically infected CF patient during prolonged colonization. According
to this routine, sputum samples are obtained from CF patients every 2 to 3
months, during periodic consultations to monitor their clinical status, or more
often for patients showing clinical deterioration. Isolates IST4116A and
IST4116B, with different colony morphologies, were obtained in the same iso-
lation procedure. These isolates belong to the same clonal complex, as do all
other isolates tested in this study (9). Bacterial cultures were stored at ?80°C in
1:1 (vol/vol) glycerol. When in use, bacteria were cultivated on Luria-Bertani
agar (LB agar; Difco, Sparks, MD) plates.
MLST analysis. Total genomic DNAs were extracted from B. cenocepacia
isolates harvested from overnight growth in LB medium with orbital agitation at
37°C, using a cell and tissue kit (Gentra Systems, Qiagen, Germany). The con-
centrations of genomic DNA solutions were estimated using an ND-1000 spec-
trophotometer (NanoDrop). MLST analysis was performed using the primers
and conditions described by Baldwin et al. (2) for seven conserved housekeeping
genes (atpD, gltB, gyrB, recA, lepA, phaC, and trpB). Amplification reactions for
all primers were carried out using an initial step of denaturation for 2 min at
94°C, followed by 30 cycles of 1 min at 94°C for denaturation, 1 min at 58°C for
primer annealing, and 2 min at 72°C for polymerization, followed by a final
extension step of 72°C for 7 min. The amplification mixtures contained the
following, in a total volume of 50 ?l: 75 ng of DNA template, 1 ?l of 0.8 mM
(each) deoxynucleoside triphosphates, 1.5 ?l of 50 mM MgCl2, 1 ?l of 20 pmol
(each) forward and reverse primers, 1.25 U of Taq polymerase (Biotaq Taq
polymerase; Bioline), and 5 ?l of reaction buffer (10?; supplied by the polymer-
ase manufacturer). The amplification products were confirmed following the
separation of the PCR products by 0.7% (wt/vol) horizontal agarose gel electro-
phoresis, and the bands were excised, purified using a gel extraction kit
(JETquick spin column technique; Genomed, Germany), and sequenced. The
sequence data for each isolate were added to a group of known sequences,
available in the Burkholderia cepacia complex MLST database (http://pubmlst
.org/bcc), to simultaneously be aligned and edited to the correct sequence length
using ClustalW software (www.clustal.org). Alleles for each of the seven loci
were assigned, and the allelic profile (string of seven integers) was used to define
the sequence type (ST), using the same database. A new ST number was assigned
to a novel allelic combination that was submitted and added to the BCC MLST
database under the designation ST-614.
Antimicrobial susceptibility assays. The susceptibilities of the isolates under
study to several antimicrobials of different classes were compared using the Etest
and broth microdilution methods. Due to the high antibiotic resistance of the
isolates tested, the antibiotic concentrations used in the Etest strips were not
high enough to differentiate the MIC values for a number of isolates and anti-
microbials (e.g., gentamicin and ceftazidime). Given this, the MIC values were
also assessed using the broth microdilution method.
(i) Etest method. Isolated colonies from LB agar plates were grown in LB
broth (Difco) at 37°C until mid-exponential phase and then diluted to an optical
density at 640 nm (OD640) of 0.2 in 0.9% NaCl (wt/vol), and 100 ?l of this cell
suspension was plated onto Mueller-Hinton (MH) agar (Difco) plates. Etest
strips (AB Biodisk, Solna, Sweden) with the antimicrobial agents to be tested
(ceftazidime, 0.016 to 256 ?g ml?1; imipenem, 0.002 to 32 ?g ml?1; meropenem,
0.002 to 32 ?g ml?1; tobramycin, 0.064 to 1,024 ?g ml?1; gentamicin, 0.064 to
1,024 ?g ml?1; ciprofloxacin, 0.002 to 32 ?g ml?1; and trimethoprim-sulfa-
methoxazole, 0.002 to 32 ?g ml?1) were used, and results were interpreted
according to CLSI guidelines (7). MIC values are mean values for at least three
independent experiments.
(ii) Broth microdilution susceptibility test method. Liquid cultures grown in
LB medium at 37°C until the mid-exponential phase were harvested by centrif-
ugation and then suspended in MH (Difco) broth and diluted to a standardized
TABLE 1. Clonal isolates of B. cenocepacia obtained from the same persistently colonized CF patienta
IsolateIsolation date (day-mo-yr)
Allelic profileb
STID
gyrBlepAatpDphaCtrpBrecAgltB
IST439
IST4103
IST4110
IST4112
IST4113
IST4116Ac
IST4116Bc
IST4131
IST4129
IST4130
IST4134
30-01-1999
03-07-2001
01-10-2001
11-10-2001
06-11-2001
11-02-2002
11-02-2002
26-02-2002
26-03-2002
14-05-2002
02-07-2002
186
186
11
11
11
11
11
11
11
11
11
11
11
132
132
132
132
132
132
132
132
132
132
132
6
6
6
6
6
6
6
6
6
6
6
79
79
79
79
79
79
79
79
79
79
79
14
14
14
14
14
14
14
14
14
14
14
157
157
157
157
157
157
157
157
157
157
157
218
218
614
614
614
614
218
218
218
218
218
963
964
965
966
967
968
969
972
970
971
973
1
1
1
1
186
186
186
186
186
aAll isolates had the same ribopattern (ribopattern 11), as determined by Cunha et al. (9). The species and ribopattern of IST4134 were determined in the present
work.
bAllelic profiles were determined as proposed by Baldwin et al. (2).
cIsolates IST4116A and IST4116B were obtained during the same isolation procedure.
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culture OD640of 0.21. Of these cell suspensions, 190 ?l was used to inoculate the
wells of a 96-well polystyrene microtiter plate (Greiner Bio-One, Frickenhausen,
Germany) containing 10 ?l of the antibiotic solution to be tested. This test was
performed in accordance with the recommendations of NCCLS (31), in at least
three independent experiments. Ceftazidime, gentamicin, and tobramycin were
obtained from Sigma-Aldrich (St. Louis, MO), and ciprofloxacin was obtained
from ICN Biomedicals Inc. (OH); all antimicrobials were in powder form. The
range of concentrations used varied depending on the antimicrobial tested, with
ranges of 128 to 1,700 ?g ml?1for gentamicin, 1.75 to 10 ?g ml?1for cipro-
floxacin, 128 to 512 ?g ml?1for tobramycin, 60 to 1,150 ?g ml?1for ceftazidime,
and 12 to ?32 ?g ml?1for meropenem. The microplates were incubated, and the
OD640values of the cultures in the wells were measured in a VERSAmax tunable
microplate reader (Molecular Devices Corporation, Sunnyvale, CA) after 24 h of
incubation at 37°C. The microplate reader was connected to a computer running
SoftMax Pro 4.8 spectrophotometric software (Molecular Devices Corporation).
Positive (without antibiotic) and negative (not inoculated) controls were in-
cluded.
Colony morphotypes. The comparison of colony morphologies was performed
as described by Bernier et al. (3). The isolates were grown in LB (Difco) broth
at 37°C for approximately 24 h with shaking (250 rpm), and after serial dilution
into fresh LB broth, they were plated onto LB agar plates and incubated at 37°C
for 48 h, followed by an additional period of 24 h at room temperature to more
clearly distinguish the three different colony morphologies, identified as
“smooth,” “rough,” and “semirough.” A Canon digital camera using a Stemi
2000-C stereomicroscope at a magnification of ?50 was used to capture pictures
of the colonies.
Cell morphology. Cell size and morphological parameters, such as equivalent
diameter, circularity, and elongation, were determined by fluorescence micros-
copy. Cells were harvested and stained with a Live/Dead BacLight bacterial
viability kit from Molecular Probes (Invitrogen Co., Spain). The samples were
prepared according to the protocol provided by the manufacturer and were
observed using an Olympus CX40 microscope with an Olympus U-RFL-T burner
and a U-MWB mirror cube unit (BP450-480 excitation filter and BA515 barrier
filter). Images were captured by an Evolution MP5.1 charge-coupled device
(CCD) color camera using Image-Pro Plus software, both from Media Cyber-
netics, Inc. Image analysis was carried out using Visilog 5 software from Noesis
(13). At least 10 images were taken for each sample. The error associated with
image analysis was ?6.8%, based on the standard deviation and sample mean for
10 repeated images of the same sample, quoted for a confidence interval of
99.5%.
Zeta potential assay. The zeta potential of cells collected in the mid-exponen-
tial phase, washed twice, and suspended in 10 mM KNO3was calculated from the
measured electrophoretic mobility by considering the Smoluchowski approxima-
tion by use of Zetasizer v6.12 software from Malvern Instruments, Ltd. (United
Kingdom). The electrophoretic mobility was measured using a Zetasizer Nano
ZS Doppler electrophoretic light scattering analyzer from Malvern Instru-
ments, Ltd.
Determination of bacterial hydrophobicity and cell migration velocity. The
cell surface hydrophobicity of B. cenocepacia isolates under study was assessed by
measuring the adhesion of cells to n-hexadecane, based on the method proposed
by Rosenberg (38). Cells from cultures in the mid-exponential growth phase were
washed twice with 50 mM phosphate-buffered saline (PBS; pH 7.0) and resus-
pended in the same buffer. Bacterial suspensions (1.2 ml) were overlaid with 0.2
ml of n-hexadecane (Sigma-Aldrich) in test tubes. These were agitated at full
speed for 45 s on a vortex machine and allowed to stand still for 15 s. The
migration of the cells was assessed by measuring the OD600of the aqueous phase
at 1-min intervals after removing the organic phase.
EPS production. Exopolysaccharide (EPS) production was assessed by con-
fluent growth of the different isolates on LB agar plates. Plates were inoculated
with 100 ?l of a suspension of cells harvested during the exponential phase of
growth and resuspended to obtain a standardized OD640of 0.2 ? 0.02. These
plates were incubated for 5 days at 37°C and then scraped, and the material
obtained was resuspended in 0.9% NaCl (wt/vol). The bacterial cells present in
these suspensions were removed by centrifugation at 20,000 ? g for 15 min. The
EPS was precipitated from the cell-free supernatant by the addition of 2.5
volumes of cold ethanol, air dried, and redissolved in distilled water. The total
sugar content was assessed by the phenol-sulfuric acid method (15), using the
EPS produced by isolate B. cepacia IST408 as a standard. For this purpose, the
EPS solution was further dialyzed against distilled water at 4°C for 24 h and then
recovered by freeze-drying. The cell pellets obtained from each plate were
washed once with 0.9% NaCl, and the protein content was quantified by the
biuret method, using bovine serum albumin (BSA; Sigma-Aldrich) as a standard
(22). EPS production was expressed in grams of total sugars per protein (g ? g
protein?1). The results are means for at least three independent cultivations and
three determinations of the total sugar and protein contents for each sample.
Biofilm assays. The biofilm formation assay was based on the methodology
described by O’Toole and Kolter. Liquid cultures grown in LB medium at 37°C
until the mid-exponential phase were diluted to a standardized culture OD640of
0.5, and 20 ?l of cell suspension was used to inoculate the wells of a 96-well
polystyrene microtiter plate (Greiner Bio-One, Frickenhausen, Germany) con-
taining 180 ?l of LB medium. Wells containing sterile growth medium were used
as negative controls. These microtiter plates were incubated at 37°C for 24 h
without shaking. Quantification of the amount of biomass attached to the
microtiter dish was carried out as described before, using crystal violet stain-
ing (33, 38).
Effect of iron availability on bacterial growth. The effect of iron availability on
the growth of the different B. cenocepacia isolates examined was assessed using
an iron-poor chemically defined medium (CDM) (6), containing 48 mM glucose,
7.4 mM KCl, 6 mM NaCl, 48 mM (NH4)2SO4, 0.5 mM MgSO4? 7H2O, 60
mM MOPS (morpholinepropanesulfonic acid), 3.8 mM K2HPO4, and 0.1%
Casamino Acids (Bacto; Becton Dickinson and Company). Iron-loaded CDM
was obtained by CDM supplementation with 100 ?M FeCl3. Cells of the different
clonal isolates in the mid-exponential phase of growth were inoculated, at an
initial OD640of 0.05, into CDM either supplemented or not supplemented with
iron. Growth was followed by measuring the culture OD640and, in certain cases,
also the number of viable cells, determined based on the number of CFU. The
values for CFU/ml shown are the mean values for three independent growth
experiments.
Lipid extraction and determination of fatty acid composition. Bacterial cells
used to assess fatty acid composition were grown under both aerophilic and
microaerophilic conditions in LB agar plates incubated for 24 h at 37°C. The
microaerophilic atmosphere, containing 5 to 8% oxygen and 12 to 15% carbon
dioxide, was generated in sealed jars by use of a GENbox microaerator (bio-
Me ´rieux, Marcy L’Etoile, France). The extraction of cellular lipids was carried
out as described by Findlay et al. (17). The fatty acids were derivatized into fatty
acid methyl esters (FAME) and analyzed by gas chromatography on an Agilent
6890N gas chromatograph with a flame ionization detector (FID) and a 7683 B
series injector equipped with a 30-m HP-5 capillary column from J&W Scientific,
as described before (12). Peak identification was achieved using qualitative
standard mixtures of bacterial FAME and polyunsaturated fatty acids, both from
Supelco (Sigma-Aldrich), and a mixture of methyl cis-11-octadecenoate from
Sigma-Aldrich. The average error associated with the GC quantification of each
FAME, calculated based on seven independently prepared standard solutions,
was ?5.1%, quoted for a confidence interval of 99.5%.
Motility assays. (i) Swimming assay. Dry swim agar plates containing 30 ml
of 1% (wt/vol) tryptone (Difco), 0.5% (wt/vol) NaCl, and 0.3% (wt/vol) agar
(Oxoid, Cambridge, United Kingdom) were point inoculated by use of a sterile
toothpick with bacterial cultures from isolated colonies grown on Pseudomonas
isolation agar (PIA; Difco) for 24 h at 37°C. These swim agar plates were
incubated at 37°C for 24 h, and the circular turbid zone formed by the bacterial
cells migrating away from the point of inoculation was measured. Values are the
means for at least three independent growth experiments.
(ii) Swarming assay. Dry swarm agar plates containing 30 ml of 0.8% (wt/vol)
nutrient broth (Difco), 0.5% (wt/vol) glucose, and 0.5% (wt/vol) agar (Oxoid)
were point inoculated and incubated as described for the swimming assay. The
diameter of the zone of growth around the point of inoculation was measured.
Values are means for at least three independent growth experiments.
RESULTS
MLST analysis. MLST of the 11 B. cenocepacia sequential
isolates under study was performed through assessment of the
allelic variation of seven housekeeping locus sequences pro-
posed by Baldwin et al. (2). These genes were amplified and
sequenced, and the allelic types of the seven loci were assigned
(Table 1) and submitted to the Burkholderia cepacia complex
MLST database. They are available publicly at http://pubmlst
.org/bcc/. Six of the tested isolates (IST4103, IST4116B,
IST4131, IST4129, IST4130, and IST4134) had the same allelic
profile as the first isolate obtained from CF patient J (IST439)
and were assigned to ST-218. This ST was already present in
the BCC MLST database before this work, since isolate
IST439 had been analyzed by Baldwin et al. (2). The above
2952COUTINHO ET AL.INFECT. IMMUN.

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isolates are considered clonal because they are indistinguish-
able at the seven loci. Isolates IST4110, IST4112, IST4113, and
IST4116A produced a new ST that was designated ST-614 and
added to the BCC MLST database (Table 1). Although the
isolates that exhibit sequence types ST-218 and ST-614 are not
identical clones, they are considered part of the same clonal
complex because ST-614 is a single-locus variant (SLV) of
ST-218 (Table 1). This variation is registered at the gyrB locus
(encoding subunit B of DNA gyrase) and involves 14 different
nucleotides between gyrB1 and gyrB186 (Fig. 1). The differ-
ences registered in gyrB suggest that other mutations might
have occurred in other loci during prolonged colonization in
the airways of the CF patient and during aggressive antibiotic
therapy. The MLST profiles of the isolates analyzed in this
study confirmed that they all belong to the B. cenocepacia
species and are consistent with the clonal nature of these
isolates proposed before, based on other molecular methods
(9). The species and ribopattern of the last isolate obtained
from the CF patient (IST4134) were also determined in this
study because this isolate was missing in the cohort of isolates
examined previously (9).
Antimicrobial susceptibility patterns of B. cenocepacia
clonal isolates. The susceptibility patterns of the sequential B.
cenocepacia clonal isolates under study were determined by
the Etest method, using seven antimicrobials of five distinct
classes: cephalosporins (ceftazidime) and carbapenems (imi-
penem and meropenem), targeting cell wall biosynthesis; ami-
noglycosides, targeting protein synthesis by binding to the 30S
ribosomal unit (gentamicin and tobramycin); fluoroquinolones,
targeting DNA replication (ciprofloxacin); and the folate path-
way inhibitor trimethoprim-sulfamethoxazole. The 11 clonal
isolates under analysis showed different resistance profiles
against all antibiotics tested (Fig. 2), consistent with previous
results reported for the first 10 isolates retrieved from patient
J for piperacillin-tazobactam, ceftazidime, and imipenem (25).
The MIC values obtained based on the Etest method varied,
with ranges of 192 to ?1,024 ?g ml?1(gentamicin), 1 to 12 ?g
ml?1(ciprofloxacin), 48 to 512 ?g ml?1(tobramycin), 0.5 to
3.5 ?g ml?1(trimethoprim-sulfamethoxazole), 12 to ?32 ?g
ml?1(imipenem), 2 to ?32 ?g ml?1(meropenem), and 8 to
?256 ?g ml?1(ceftazidime). The MIC values were also ob-
tained based on the broth microdilution method for the 11
isolates, especially for those antimicrobials whose MIC values
for the different isolates could not be differentiated by the
Etest method due to these being the maximal concentration
that could be tested. Values varied within wide ranges of con-
centrations: 128 to 1,700 ?g ml?1for gentamicin, 1.75 to 10 ?g
ml?1for ciprofloxacin, 128 to 512 ?g ml?1for tobramycin, 12
to 22 ?g ml?1for imipenem, and 60 to 1,150 ?g ml?1for
ceftazidime. The first isolate was found to be consistently more
susceptible to all antimicrobials tested than all of the sequen-
tial clonal variants retrieved from the patient.
Colony morphotype, cell size and morphology, zeta poten-
tial, and hydrophobicity. The B. cenocepacia clonal variants
under study exhibited three different colony morphotypes (Fig.
3A). The colony morphotype of the initial isolate (IST439) was
considered smooth, as well as the morphotype exhibited by
isolates IST4103 and IST4129. The isolates IST4112, IST4113,
and IST4116B were considered rough, while the remaining
isolates, IST4110, IST4116A, IST4130, IST4131, and IST4134,
which exhibited an intermediary morphotype, were considered
semirough.
The cell size of the B. cenocepacia clonal variants also varied,
with isolates IST4112, IST4113, and IST4116B exhibiting cell
sizes 2.4-, 2.9-, and 1.6-fold higher, respectively, than the av-
erage size of the remaining isolates (Fig. 3B). Curiously, these
FIG. 1. Polymorphic sites within the gyrB locus of the BCC MLST scheme corresponding to the new sequence type, ST-614, described in this
work, compared to ST-218. Nucleotide sites are numbered from the first nucleotide position in the gyrB gene.
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Page 5

were the same isolates that formed rough colonies (Fig. 3A).
The cells of these isolates had the shape of almost perfect
circles (circularity of ca. 1 and elongation of ca. 1.1) (data not
shown), while the other isolates exhibited more elongated
forms (circularity and elongation, ca. 1.2 to 1.3). The value of
the zeta potential (an indirect measure of the cell surface
charge [13]) of the first isolate, IST439, was significantly below
the values of all other clonal isolates (?9.5 mV, compared with
values of around ?30 mV) (Fig. 3C). The bacterial cell surface
hydrophobicity was determined by the microbial adhesion to
hydrocarbons (MATH) method (13, 18), using n-hexadecane
as a solvent. This test has commonly been used to assess the
hydrophobicity of bacterial cells, although cell migration to-
ward the n-hexadecane layer may be the result of both hydro-
phobic and electrostatic interactions (18). The velocity of cell
migration toward the n-hexadecane layer in the MATH test
showed no significant differences between the tested isolates
(data not shown). However, the fraction of cells at the inter-
face suggested that isolate IST4113 was the most hydrophobic
compared with the first isolate, IST439, while isolates IST4110,
IST4116A, and IST4131 had the lowest surface hydrophobicity
values (Fig. 3D).
Exopolysaccharide production and size of the biofilms
formed. The amount of EPS produced (Fig. 3E) and the size of
the biofilm formed (Fig. 3F) in vitro by the different clonal
variants were also found to vary along the colonization period.
Isolates IST4112, IST4113, and IST4116B formed the largest
biofilms, while the biofilms produced by IST4110, IST4116A,
IST4131, IST4130, and IST4134 were much smaller, with the
first isolate, IST439, exhibiting an intermediary value (Fig. 3F).
In general, a correlation could be established between the
amounts of EPS produced and the sizes of the biofilms formed
by the different B. cenocepacia clonal isolates, with the excep-
tion of IST4103 and IST4116A. This observation is consistent
with the concept that the EPS produced contributes to the
development of thicker and more stable biofilms in bacteria of
the B. cepacia complex (11).
Growth efficiency under iron limitation. The growth curves
of the isolates under study were compared using an iron-lim-
ited minimal CDM and the same medium supplemented with
100 ?M Fe3?(iron-loaded medium). Examples of the growth
curves obtained for isolates IST439, IST4113, IST4116A, and
IST4134 in both media are shown in Fig. 4A. As expected, the
biomass attained (associated with the culture OD640) after the
same incubation time (when the culture entered the stationary
phase of growth after 24 h of incubation) under iron limitation
was significantly below the levels attained under iron satura-
tion conditions (Fig. 4A and B). The results also show that the
first isolate, IST439, exhibited less efficient growth under iron
limitation than the other isolates, while IST4113’s growth per-
FIG. 2. MIC values, determined using the Etest method (A) and the microdilution method (B), for the activities of the indicated antimicrobial
agents of different classes against B. cenocepacia clonal isolates obtained during long-term colonization of CF patient J. Vertical lines indicate when
the CF patient received intravenous antibiotic therapy with ceftazidime and gentamicin.
2954 COUTINHO ET AL.INFECT. IMMUN.

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formance under iron limitation was the best, reaching both the
highest biomass and viable cell concentrations (Fig. 4A to D).
However, in the iron-supplemented medium, the growth of the
first isolate, IST439, was faster, reaching the highest biomass
concentration (Fig. 4A to D). Differences in the growth per-
formance of isolates IST439 and IST4113 in iron-limited or
iron-loaded medium were confirmed based on the viable cell
concentrations of the two cultures (Fig. 4B) and are more
evident using a linear scale (Fig. 4C). The comparison of cul-
ture optical densities reached by all isolates after 24 h of
growth in iron-limiting and iron-loaded CDM (Fig. 4D) indi-
cates that the first isolate was consistently the best suited to
grow under iron-loaded conditions but, in general, exhibited
the worst performance under iron limitation conditions. Ex-
ceptions were registered for isolates that exhibited a poor
growth performance even under iron-loaded conditions.
Fatty acid composition. The most abundant fatty acids in the
clonal variants examined were tetradecanoic (myristic) acid
(C14:0), cis-9-hexadecenoic (palmitoleic) acid (C16:1?7), methyl
3-hydroxytetradecanoicacid
(palmitic) acid (C16:0), cis-9,1-methyleneoctadecanoic acid
(cyC17:0), methyl 2-hydroxyhexadecanoic acid (C16:0 2-OH), cis-
11-octadecenoic (vaccenic) acid (C18:1?7cis), methyl trans-9-
octadecenoate (elaidic) acid (C18:1?9trans), and cis-9,10-methyl-
enehexadecanoic acid (cyC19:0). These fatty acids, which have
previously been identified in Burkholderia strains (5, 42), rep-
resented over 86% of the total amount of fatty acids deter-
mined for the isolates under study. In general, under both
microaerophilic and aerophilic conditions, the content of sat-
urated fatty acids decreased in the isolates retrieved along the
colonization period in the CF patient’s lungs (Fig. 5A and B),
(C14:0 3-OH),hexadecanoic
in particular the content of saturated cyclopropyl branched
fatty acids in those isolates obtained during the last months of
life. The decrease in the content of saturated fatty acids was
followed by an increase in the percentage of unsaturated fatty
acids registered from the first to the last isolates under both
growth conditions, although the fatty acid contents were sig-
nificantly different in cells grown under aerophilic and mi-
croaerophilic conditions.
In general, the degree of saturation of the membrane fatty
acids, defined as the ratio between the saturated fatty acids
C16:0and octadecanoic acid (C18:0) (Fig. 5, a1and b1) and the
unsaturated fatty acids methyl cis-9-hexadecenoate (palmito-
leic) acid (C16:1cis9), C18:1?7cis, and C18:1?9trans(Fig. 5, a2and
b2), was found to be significantly lower in cells grown under
microaerophilic conditions than under aerophilic conditions,
decreasing in cells of the sequential clonal variants as the
colonization time increased, approaching the date of the pa-
tient’s death (Fig. 5C). The decrease of the saturation degree
was accompanied by an increase of the percentage of unsatu-
rated fatty acids (Fig. 5, a3and b3). The cells grown under
microaerophilic conditions exhibited a higher percentage of
unsaturated fatty acids than the cells grown under aerophilic
conditions. In the last isolates retrieved from the patient, the
decrease of the saturation degree was related mainly to a
decrease in the amount of C18:0and C16:0and to an increase in
the amount of cis-11-octadecenoic acid (C18:1?7cis). Since the
presence of cis-11-octadecenoic acid in cell membranes is re-
lated to anaerophilic growth (20), the higher concentration of
this fatty acid (Fig. 5, a3and b3) registered in bacterial isolates
obtained during the last stages of CF infection, when the level
of oxygen in the airways was very limited, is also consistent with
FIG. 3. (A) Colony morphology. I, smooth; II, semirough; III, rough. (B) Cell size. (C) Zeta potential. (D) Fraction of cells at the interface
of water and n-hexadecane. (E) Exopolysaccharide produced. (F) Sizes of biofilms formed by the sequential clonal isolates of B. cenocepacia
retrieved from the same persistently colonized patient J.
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Page 7

the hypothesis that fatty acid alteration is the result of bacterial
adaptation to the selective pressure faced in CF lungs.
Swarming and swimming motility. Compared with the first
isolate, IST439, all of the other sequential clonal variants ob-
tained during the 3.5 years of bacterial persistence in the pa-
tient’s lungs exhibited lower swarming motilities (Fig. 6A).
However, the variation in swimming motility values did not
exhibit a specific pattern (Fig. 6B).
DISCUSSION
Bacteria of the BCC are ubiquitous in the environment, have
very high metabolic versatility, and can cause chronic oppor-
tunistic infections in immunocompromised patients, in partic-
ular in patients with CF (28). Although these bacteria can
colonize the lungs while causing no symptoms and having no
long-term effect, in general they lead to chronic infection and
to a continuous decline in lung function (28). In the worst-case
scenario, they can cause the fatal “cepacia syndrome” (8, 28).
Chronic infections with BCC bacteria are very difficult to treat
due to their intrinsic resistance to a large number of antimi-
crobials and their ability to develop high-level resistance dur-
ing antibiotic treatment and to adapt to and resist other ad-
verse environmental conditions (4, 25). This fact severely limits
the effective treatment of respiratory infections, making BCC
bacteria very difficult (if it is even possible) to eradicate from
FIG. 4. (A) Growth curves on a semilog scale based on the OD640values of isolates IST439, IST4103, IST4110, and IST4113 in iron-limited
CDM (E) and in the same medium supplemented with 100 ?M Fe3?(F). (B) Growth curves based on the concentrations of viable cells (expressed
as numbers of CFU per ml) in the cultures of isolates IST439 (F and E) and IST4113 (f and ?) in the same iron-limited CDM (E and ?) and
in this medium supplemented with 100 ?M Fe3?(F and f). (C) Growth curves of IST439 (F and E) and IST4113 (f and ?), compared on a linear
scale, in CDM (E and ?) and in CDM supplemented with 100 ?M Fe3?(F and f). (D) Culture optical densities after 24 h of growth in CDM
or CDM supplemented with 100 ?M Fe3?for all sequential clonal variants of B. cenocepacia obtained during long-term colonization of patient J.
2956COUTINHO ET AL.INFECT. IMMUN.

Page 8

the CF lung (8, 25, 28). In general, B. cenocepacia is the most
common BCC species recovered in CF centers worldwide, and
it is frequently associated with severe infections (26, 36). How-
ever, there are documented exceptions, in particular in the
major Portuguese Cystic Fibrosis Center at HSM, in Lisbon,
where the species B. cepacia dominates (8, 10). The first epi-
demiological survey of BCC bacteria involved in respiratory
infections among the Portuguese CF population under surveil-
lance at this center was reported by our laboratory in 2000 (37).
This study was followed by others, covering isolates of B. ceno-
cepacia, B. cepacia, Burkholderia multivorans, and Burkholderia
stabilis obtained from 1995 to 2006 (9, 10, 11, 25). The present
study was designed to try to obtain clues to the adaptive strat-
egies adopted by B. cenocepacia during chronic infection
through the systematic assessment of a number of relevant
phenotypic characteristics, in the context of CF infection, of 11
FIG. 5. Fatty acid compositions of sequential clonal variants of B. cenocepacia obtained during long-term colonization of patient J, grown on
LB agar plates under aerophilic (A) and microaerophilic (B) conditions. The main fatty acids analyzed were saturated straight-chain fatty acids
C14:0(dashed line) and C16:0(full line) (a1and b1); saturated cyclopropyl branched fatty acids C17:0 cyclo(dashed line) and C19:0 cyclo(full line) (a2
and b2); unsaturated fatty acids C18:1?9trans(full line), C18:1?7cis(dashed line), and C16:1?7(dashed-dotted line) (a3and b3); and hydroxy-substituted
fatty acids C14:0 3-OH(full line) and C16:0 2-OH(dashed line) (a4and b4). (C) Degrees of saturation of fatty acids in the same sequential clonal isolates
of B. cenocepacia grown under aerophilic (f) or microaerophilic (?) conditions. The fatty acid compositions of these isolates are shown in panels
A and B.
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Page 9

serial clonal variants obtained at HSM from a CF patient who
was chronically infected for 3.5 years, until the patient’s death
with cepacia syndrome. When this work was started, it was
known that the recA restriction fragment length polymorphism
(RFLP) and EcoRI ribopattern profiles of these B. cenocepacia
isolates are indistinguishable (9). The MLST performed during
this study revealed that four of the isolates exhibited a novel
allelic profile, the sequence type ST-614, while the other isolates,
including IST439, considered the variant that started the infec-
tion, belonged to the already described group ST-218. Although
they are not identical clones, all of the isolates are part of the
same clonal based upon related sequence types (BURST) group,
since only one of the seven loci (the gyrB locus) tested exhibited
an alteration (in 14 nucleotides). This result is in agreement with
the idea that a CF patient’s lungs can be infected chronically for
years by one or a few lineages of P. aeruginosa or BCC species (9,
10, 14, 19). Together with other results obtained during this study,
this observation also supports the concept of the occurrence of B.
cenocepacia clonal expansion during chronic lung colonization,
presumably as the result of mutations and selective pressures
occurring in the CF lung environment, in particular those exerted
by the immune defenses, antibiotic therapy, and oxygen limita-
tion, as described for P. aeruginosa (32).
Concerning several of the phenotypic characteristics of the
clonal variants under study, no colonization time-dependent
alteration patterns could be identified. This fact could be the
result of the heterogeneity of the colonizing clonal population
and the changes of the selection pressures occurring during
long-term colonization, in particular those related to the ap-
plication of intravenous antibiotic therapy and the continuous
or rapid deterioration of lung function. In fact, with only one
exception, i.e., isolates IST4116A and IST4116B, obtained dur-
ing the same isolation procedure and known to exhibit differ-
ent colony morphologies, only a single isolate picked up at
random per isolation date was tested. Remarkably, the pheno-
typic traits that did not show a clear time-dependent variation
pattern were also found to be divergent in isolates IST4116A
and IST4116B, obtained during the some isolation procedure.
However, in general, the properties of the isolate considered to
have initiated the infection appeared to differ significantly
from those exhibited by the isolates obtained during the course
of the infection during a period ranging from 29 to 41 months
after the isolation of the first B. cenocepacia isolate. Indeed,
the antimicrobial susceptibility of the first isolate was signifi-
cantly higher than the susceptibilities of the other clonal vari-
ants toward all tested antimicrobials. This pattern is absolutely
clear for the aminoglycoside gentamicin, used as intravenous
therapy during specific periods of the patient’s hospitalization
FIG. 6. Swarming (A) and swimming (B) motilities of sequential clonal variants of B. cenocepacia retrieved during long-term colonization of
patient J.
2958COUTINHO ET AL.INFECT. IMMUN.

Page 10

due to pulmonary exacerbation. The ability of B. cenocepacia
to develop high resistance levels during antibiotic treatment is
in agreement with the generalized idea that this adaptive
mechanism is among the important features contributing to
persistent infection. Although bacterial resistance to antibiot-
ics seems to be linked to the growth of bacteria in communities
embedded in a protective polysaccharide matrix or biofilm, no
correlation could be established for the different variants be-
tween antibiotic resistance and the size of the biofilm formed,
indicating that other relevant mechanisms also contribute to
the increased resistance registered toward several antimicrobi-
als of different classes. Indeed, a number of underlying mech-
anisms were recently hypothesized based on a quantitative
proteomic analysis of the first isolate, IST439, compared with
the highly resistant variant IST4113 (27). In general, a corre-
lation was found between the size of the biofilm and the
amount of the exopolysaccharide cepacian synthesized by each
variant, consistent with the proposed role of cepacian in the
formation of larger biofilms in isogenic strains (11).
The swarming motility of the first isolate was found to be
consistently higher than the swarming motility values of all sub-
sequent clonal isolates, while a less negative value for the zeta
potential was registered for cells of isolate IST439 than for the
other clonal variants. These results suggest that the alteration of
these two traits may be among the strategies used by B. cenoce-
pacia to persist during progressive CF lung disease. Swarming
motility involves the coordinated and rapid movement of a bac-
terial population across a semisolid surface, playing a crucial role
in the establishment of a number of infections, in particular those
in a CF patient’s lungs (43). Although at this time there is no
supported explanation for the higher level of swarming motility
exhibited by the first isolate than by the subsequent isolates, we
are tempted to hypothesize that this could be related to differ-
ences at the level of fatty acid metabolism that may affect biosur-
factant production. Indeed, the swarming process involves the
release of biosurfactants which act by reducing the local surface
tension; rhamnolipids are the biosurfactant involved in swarming
motility in P. aeruginosa (24). The less negative value of the zeta
potential of IST439, which is associated with a less negative cell
surface net charge, could also be related to higher values for
swarming motility, since the cell surface charge has been proven
to be important for cell-cell or cell-surface interactions essential
in swarming motility (30).
During this study, we also compared the growth efficiencies
of the clonal variants of B. cenocepacia under iron-limiting
conditions. The main conclusion was that although the con-
centration of biomass produced by the first isolate, IST439, was
higher than the concentration of biomass produced by all other
isolates when cultures entered the stationary phase of growth
after the same time of incubation in iron-loaded minimal me-
dium, this isolate was less suited to growth under iron limita-
tion conditions than most of the subsequent isolates. Patho-
genic bacteria require iron as a cofactor for numerous
metabolic enzymes, including those involved in aerobic respi-
ration. The free iron concentration in living organisms is usu-
ally too low to be sufficient for bacterial growth, although
higher concentrations may be present in the CF lung (34).
Increased iron in the cystic fibrosis airway was proposed to play
an important role in facilitating P. aeruginosa infection and
contributing to anaerobic biofilm growth (35). Moreover, the
importance of iron homeostasis in the CF lung and its role in
determining the success and chronicity of P. aeruginosa infec-
tion are well documented (34, 35). Bacteria have evolved mul-
tiple metabolic pathways for efficient iron acquisition to suc-
cessfully become established in the lungs, such as the synthesis
of siderophores. Based on results from a quantitative pro-
teomic analysis carried out recently in our laboratory, it is
known that the clonal variant that exhibited the highest growth
efficiency under iron limitation conditions (IST4113) has a
higher content of proteins involved in binding and transport of
iron ions than the first isolate, IST439, which showed one of
the lowest growth performances under iron limitation condi-
tions (27). These proteins include the TonB-dependent sidero-
phore receptor, the TonB-dependent copper receptor, and the
FAD-binding 9 siderophore-interacting protein (MxcB) (27).
The results of the present work also strongly suggest that the
alteration of B. cenocepacia’s ability to synthesize membranes
with a different fatty acid composition, in particular at the level
of fatty acid saturation, constitutes an important adaptation
strategy to long-term colonization, especially at the end stage
of CF lung disease. Indeed, the isolates recovered during the
last 10 months of the patient’s life exhibited a very evident
time-dependent decrease of fatty acid saturation. At first, this
adaptive response was considered intriguing because oxidative
stress is among the stresses that bacteria are expected to be
exposed to during colonization and because unsaturated fatty
acids are more susceptible than saturated fatty acids to oxy-
radical-mediated lipid peroxidation (44). Given that a mini-
mum level of saturation was registered for the isolates ob-
tained during the last months of life of patient J, we
hypothesized that this adaptive trait could be due to severe
oxygen depletion in the CF lungs. Consistent with this pro-
posal, the fatty acid saturation degree and the amount of sat-
urated cyclopropyl branched fatty acids in the isolates grown
under microaerophilic conditions were significantly below
those in isolates grown under aerophilic conditions. According
to the hospital records, when isolate IST439 was obtained, the
forced expiratory value in the first second (FEV1) was 22%,
below the value obtained prior to its isolation (27%). No fur-
ther values of FEV1 were registered during the later stages of
the patient’s life due to the highly severe deterioration of
pulmonary function (8). It is known that in the lungs of CF
patients, bacteria grow to high densities in mucopurulent ma-
terial that is limited or depleted in oxygen. Although there is a
generalized idea that growth in these circumstances is depen-
dent on anaerobic nitrate respiration, microaerobic respiration
appears to be the predominant mode of P. aeruginosa growth
in CF lungs (1). Remarkably, during chronic infection of the
CF lungs, oxygen-limiting conditions seem to contribute to
persistent infection with P. aeruginosa (42). Despite the impor-
tance of bacterial metabolism under microaerophilic or an-
aerophilic conditions, little is known about the underlying
mechanisms and how anaerobic metabolism contributes to a
persistent infection, even for P. aeruginosa. However, it ap-
pears that the adaptation process affects predominantly meta-
bolic pathways in this species, in particular those involving fatty
acids, amino acids, and energy generation (23). Although it is
too early to make firm conclusions, the adaptive phenomenon
described here for the first time, involving the reduction of
fatty acid saturation in B. cenocepacia along with the deterio-
VOL. 79, 2011 B. CENOCEPACIA CLONAL VARIATION DURING COLONIZATION2959

Page 11

ration of pulmonary function, appears to constitute an adap-
tive shift to a fatty acid metabolism more suited to severely
oxygen-limited conditions.
ACKNOWLEDGMENTS
This work was supported by FEDER and by the Portuguese Foun-
dation for Science and Technology, FCT (contracts PTDC/SAU-MII/
69591/2006 and ERA-PTG/SAU/0001/2008, in the context of the
ADHRES Signature Project of EraNet Pathogenomics; Ph.D. grants
SFRH/BD/32729/2006 [C.P.C.] and SFRH/BD/37012/2007 [A.M.]; and
a contract under the program Cie ˆncia2007 awarded to C.C.C.R.D.C.).
This study was performed under COST Action BM1003, Microbial
Cell Surface Determinants of Virulence as Targets for New Therapeu-
tics in Cystic Fibrosis.
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Editor: S. M. Payne
2960 COUTINHO ET AL.INFECT. IMMUN.