Journal of Medical Microbiology Papers in Press. Published May 22, 2015 as doi:10.1099/jmm.0.000094
Journal of Medical Microbiology Incidence of Burkholderia contaminans at a cystic fibrosis center with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance --Manuscript Draft-Manuscript Number:
JMM-D-14-00046R6
Full Title:
Incidence of Burkholderia contaminans at a cystic fibrosis center with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance
Short Title:
Burkholderia contaminans at a cystic fibrosis center
Article Type:
Standard
Section/Category:
Epidemiology
Corresponding Author:
Isabel Sá-Correia 1Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av Rovisco Pais, P-1049001 Lisbon, Portugal Lisboa, PORTUGAL
First Author:
Carla P. Coutinho, PhD
Order of Authors:
Carla P. Coutinho, PhD Celeste Barreto Luísa Pereira Luís Lito José Melo Cristino Isabel Sá-Correia
Abstract:
The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here re-classified as Burkholderia contaminans, including the former B. cepacia IST408, which was on the focus of previous studies regarding the biosynthesis of the exopolysaccharide "cepacian". The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.
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1
Incidence of Burkholderia contaminans at a cystic fibrosis
2
center with an unusually high representation of
3
Burkholderia cepacia during 15 years of epidemiological
4
surveillance
5 6
Carla P. Coutinho1, Celeste Barreto2, Luísa Pereira², Luís Lito3, José Melo
7
Cristino3 and Isabel Sá-Correia1*
8 9
1
iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering,
10
Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001
11
Lisbon, Portugal
12
2
13
Hospitalar Lisboa Norte, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
14
3
15
Norte, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
Cystic Fibrosis Center , Department of Paediatrics, Hospital de Santa Maria, Centro
Laboratório de Patologia Clínica, Hospital de Santa Maria, Centro Hospitalar Lisboa
16 17 18 19 20 21 22
* Corresponding author: Professor Isabel Sá-Correia, Biological Sciences,
23
Department of Bioengineering, Instituto Superior Técnico, Torre Sul, Universidade de
24
Lisboa,
25
[email protected]; Telephone: +351-218417682; Fax: +351-218419062
Av.
Rovisco
Pais,
1049-001,
Lisbon,
Portugal.
E-mail:
26 27
Running title: Burkholderia contaminans at a cystic fibrosis center
28
Content Category: Epidemiology
29 30
Abbreviations: Bcc, Burkholderia cepacia complex; CF, cystic fibrosis; FEV1,
31
forced expiratory volume in 1 second; HSM, Hospital de Santa Maria; MLST,
32
multilocus sequence typing; ST, sequence type.
33
ABSTRACT
34
The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria
35
comprising around 20 related species. These bacteria are important opportunistic
36
pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse
37
prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates
38
retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon,
39
from 1995 to 2006, was re-examined in the present work. These isolates, formerly
40
classified as Burkholderia cepacia (taxon K), are here re-classified as Burkholderia
41
contaminans, including the former B. cepacia IST408, which was on the focus of
42
previous studies regarding the biosynthesis of the exopolysaccharide “cepacian”. The
43
CF population examined has been previously described as having an exceptionally
44
high representation of B. cepacia, presumably due to a contamination arising from
45
saline solutions for nasal application. Twenty-one additional isolates, obtained from a
46
chronically infected patient, from 2006 to 2010, were also identified as B.
47
contaminans. This study also provides insight into the potential clinical impact of B.
48
contaminans, a species that is rarely associated with CF infections. Isolates belonging
49
to this species were shown to be involved in chronic and transient respiratory
50
infections, and were associated with severe lung function deterioration and with a
51
case of death with cepacia syndrome. However, since the patients were co-infected
52
with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played
53
by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to
54
prevail over B. cenocepacia isolates during co-infection of at least one chronically
55
infected patient.
56 57 58 59 60 61 62 63 64
Keywords: Burkholderia contaminans; Burkholderia cepacia, species identification,
65
genotyping;
66
surveillance.
cystic
fibrosis;
chronic
respiratory
infection;
epidemiological
67
INTRODUCTION
68
The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that
69
can cause serious respiratory infections in immunocompromised individuals. Patients
70
with cystic fibrosis (CF) are particularly at risk, where Bcc infections are associated
71
with a worse prognosis and early death (Lipuma, 2010; Peeters et al., 2013). The
72
large majority of CF patients infected with Bcc develop a chronic infection that can
73
last for years, leading to progressive loss of lung function. Also, a number of Bcc-
74
infected patients may develop a rapid and fatal necrotizing pneumonia known as
75
“cepacia syndrome” (Mahenthiralingam et al., 2002). Most of the 20 described Bcc
76
species have been isolated from CF pulmonary infections, but there is a
77
disproportionate distribution of Bcc species, and the prevalence described for each
78
species may vary depending on the country and CF center under consideration
79
(LiPuma et al., 2001; Govan et al., 2007; Drevinek and Mahenthiralingam, 2010).
80
Nevertheless, Burkholderia cenocepacia and Burkholderia multivorans account for
81
most (80%) of the Bcc infections in CF patients worldwide (Drevinek and
82
Mahenthiralingam, 2010; Lipuma, 2010; Peeters et al., 2013), with B. cenocepacia
83
being considered the most serious Bcc pathogen in CF (Mahenthiralingam and
84
Vandamme, 2005; Drevinek and Mahenthiralingam, 2010). Consequently, most of the
85
research dedicated to Bcc pathogenicity in CF has been focused on this species, and
86
information regarding the impact of other Bcc species on clinical outcome is still
87
scarce. However, epidemiological studies have shown that other Bcc species are also
88
associated with poor prognosis, patient-to-patient transmission, chronic or transient
89
infections, and cepacia syndrome (Biddick et al., 2003; Cunha et al., 2003; Kalish et
90
al., 2006; Cunha et al., 2007; Drevinek and Mahenthiralingam, 2010; Lipuma, 2010;
91
Moreira et al., 2014).
92
In the last few years, the taxonomy of the Bcc has evolved significantly, with the
93
description of novel species (Yabuuchi et al., 2000; Coenye et al., 2001; Vandamme
94
et al., 2002; Vermis et al., 2002; Vermis et al., 2004; Lipuma, 2005; Vanlaere et al.,
95
2008; Vanlaere et al., 2009; Peeters et al., 2013; Smet et al., 2015). Until 2009,
96
multicenter studies revealed the existence of several clusters of isolates that do not
97
group with the Bcc species described at the time. One divergent group was classified
98
as belonging to Burkholderia cepacia complex recA RFLP type K (Vermis et al.,
99
2002; Baldwin et al., 2005; Dalmastri et al., 2005; Payne et al., 2005;
100
Mahenthiralingam et al., 2006; Dalmastri et al., 2007). After that date, Vanlaere and
101
co-workers re-examined the taxonomic position and structure of taxon K (also known
102
as group K), and proposed two novel species, Burkholderia contaminans and
103
Burkholderia lata (Vanlaere et al., 2009). The group of previously unclassified Bcc
104
isolates of taxon K (Vermis et al., 2002; Baldwin et al., 2005; Dalmastri et al., 2005;
105
Payne et al., 2005; Mahenthiralingam et al., 2006; Dalmastri et al., 2007) includes
106
bacteria isolated worldwide from human and environmental sources, namely from
107
sputum cultures of CF patients in the UK, Italy, Portugal, USA, Canada, China,
108
Brazil, Argentina, and Australia (Cunha et al., 2003; Campana et al., 2005; Assaad et
109
al., 2006; Mahenthiralingam et al., 2006; Cunha et al., 2007; Jorda-Vargas et al.,
110
2008; Fang et al., 2010; Martina et al., 2013; Ramsay et al., 2013), as well as from
111
river water, human-built water reservoirs, soil, roots, animals, pharmaceutical
112
products, such as contaminated nasal spray, personal care products, and domestic
113
products (Souza et al., 2004; Mahenthiralingam et al., 2006; Mahenthiralingam et al.,
114
2008; Martin et al., 2011). B. contaminans has a low prevalence in CF patients
115
worldwide, with remarkable exceptions in Argentina and in Spain (Martina et al.,
116
2013). Several reports of B. contaminans outbreaks amongst non-CF patients in
117
Argentina and Brazil were also published, suggesting the existence of regional
118
spreading of B. contaminans and other Bcc species between bordering countries
119
(Mahenthiralingam et al., 2000; Agodi et al., 2002; Magalhaes et al., 2003; Shehabi et
120
al., 2004; Souza et al., 2004; Woods et al., 2004). In the present work we have re-
121
examined the taxonomic position, at the species level, of several isolates from our Bcc
122
collection, recovered from CF patients receiving care at the major Portuguese CF
123
Treatment Center, at Hospital de Santa Maria (HSM), in Lisbon. These isolates were
124
obtained over 15 years of epidemiological surveillance of respiratory infections
125
involving Bcc, and the study is focused on isolates previously classified as B. cepacia
126
complex recA RFLP type K. This study also extends to isolates more recently
127
obtained from a chronically infected patient known to harbour B. cepacia complex
128
recA RFLP type K isolates, and provides insights into the potential clinical impact of a
129
species that is rarely associated with CF infections.
130
METHODS
131
Bacterial isolates and culture conditions. The Bcc isolates used in this work (Table
132
1) were obtained from the HSM CF Center, Lisbon, Portugal, over the period of 1995
133
to 2010, from CF patients’ respiratory secretions. According to the hospital’s routine,
134
sputum samples are obtained from CF patients every two to three months, during
135
periodic consultations to monitor their clinical status, or more often for patients
136
showing clinical deterioration. The seven patients indicated in Table 1 were found to
137
be infected with Burkholderia cepacia complex recA RFLP type K, and the isolates
138
were gathered from the date of their detection until the date of eradication or until
139
patient death. Some of these isolates were previously classified as B. cepacia (Cunha
140
et al., 2003; Cunha et al., 2007), while others were molecularly examined for the first
141
time in the present work and were obtained from a CF patient who continued to be
142
chronically colonized with B. cepacia recA RFLP profile K after the date of
143
previously published studies. Bacterial cultures are stored at -80°C in 1:1 (vol/vol)
144
glycerol. When in use, bacteria are cultivated on Pseudomonas Isolation Agar (PIA;
145
Difco, Sparks, MD) plates or on Luria-Bertani agar (LB agar; Difco, Sparks, MD)
146
plates.
147
Species identification and genotyping. Total genomic DNAs from each isolate,
148
previously cultured overnight in LB medium with orbital agitation at 37°C, was
149
extracted using a cell and tissue kit (Gentra Systems, Qiagen, Germany). The
150
concentration of genomic DNA solutions was estimated using an ND-1000
151
spectrophotometer (NanoDrop).
152
The recA PCR RFLP analysis was performed by amplification of the entire
153
recA gene (1,040 bp) using specific primers for bacteria belonging to the Bcc (BCR1-
154
5’
155
previously described by Mahenthiralingam et al. (Mahenthiralingam et al., 2000). Bcc
156
recA amplicons were digested with HaeIII (Amersham Biosciences) restriction
157
endonuclease and the restriction fragments were separated by electrophoresis in 2%
158
(w/v) agarose gels. The RFLP patterns obtained for the Bcc isolates examined were
159
compared with those reported in the literature (Mahenthiralingam et al., 2000; Vermis
160
et al., 2002; Leite et al., 2011).
TGACCGCCGAGAAGAGCAA3’; BCR2-5’CTCTTCTTCGTCCATCGCCTC3’) as
161
The identification of the clinical isolates at the species level was performed
162
using two different methods: multilocus sequence typing (MLST) analysis and hisA
163
gene sequence analysis. MLST analysis was performed as previously described
164
(Coutinho et al., 2011). Amplification was carried out using a Gene Amp® PCR
165
System 2700 (Applied Biosystems, USA). The amplification products were confirmed
166
following separation of PCR products by 0.9% (w/v) horizontal agarose gel
167
electrophoresis, and the bands were excised, purified using a gel extraction kit
168
(JETquick spin column technique; Genomed, Germany) and sequenced. The sequence
169
data for each isolate was compared with known sequences from the Bcc MLST
170
database
171
(www.mbio.ncsu.edu/bioedit/bioedit.html). The alleles for each of the seven loci were
172
assigned and the allelic profile (string of seven integers) was used to define the
173
sequence type (ST), using the same database. The allelic profile of the isolates under
174
study was submitted and added to the Bcc MLST database.
(http://pubmlst.org/bcc),
using
BioEdit
software
175
The hisA gene sequence analysis was performed by PCR amplification of the
176
hisA gene region (89 to 530bp) of 442bp as previously described by Papaleo et al.
177
(Papaleo et al., 2010). Briefly, in a 50 µl reaction mixture containing 50 ng of DNA
178
template, 2 U Taq DNA polymerase (Biotaq Taq polymerase; Bioline), 250 mM each
179
dNTP, 0.6 mM each primer (forward – AGGACCCGGCGGCGAT; reverse –
180
TGCAGCATCCCGTCGCG), 1.5 mM MgCl2 and 1PCR buffer (10; supplied by
181
the polymerase manufacturer) were added. The amplification reaction was carried out
182
using an initial step of denaturation for 2 min at 95°C, followed by 30 cycles of 30 s
183
at 95°C for denaturation, 45 s at 67°C for primer annealing, and 1 min at 72°C for
184
polymerization, followed by a final extension step of 72°C for 10 min, using a
185
GeneAmp PCR System 2700 (Applied Biosystems). Amplification was confirmed by
186
electrophoresis in 0.9% (w/v) agarose gels. The PCR products were purified using the
187
PCR product purification spin kit (JETquick spin column technique; Genomed),
188
according to the manufacturer’s instructions, and sequenced. The sequences were
189
compared with the eleven-letter code for the Bcc hisA gene, which allows species
190
identification as detailed by Papaleo et al. (Papaleo et al., 2010).
191
Patients’ clinical course. The clinical course of patient AF, before detection of Bcc
192
infection and throughout the period of chronic respiratory infection, was assessed
193
based on values of forced expiratory volume in 1 s (FEV1) during regular lung
194
function tests performed at the hospital to monitor the patient’s respiratory function.
195
There are no available records of FEV1 values for the other patients.
196
Ethics. Studies involving clinical Bcc isolates obtained as part of the hospital routine
197
were approved by the hospital ethics committee and the patients’ anonymity is
198
preserved. Consent was obtained from the patients’ legal guardian for the use of these
199
isolates in research.
200
201
RESULTS AND DISCUSSION
202
Identification and molecular typing of B. contaminans clinical isolates
203
The 56 Bcc isolates examined in this study were obtained from seven paediatric CF
204
patients from 1995 to 2010. They include those formerly classified as Burkholderia
205
cepacia recA profile K and those more recently isolated from one chronically infected
206
patient found to be infected with profile K isolates in previously published
207
epidemiological surveys (Cunha et al., 2003; Cunha et al., 2007). The profile K was
208
confirmed for all the isolates based on recA gene amplification and restriction with
209
HaeIII of the amplified products followed by RFLP analysis. However, since recA
210
sequence-based-identification of the so-called taxon K bacteria is considered
211
problematic and can only solve part of the species complexity in this lineage
212
(Vanlaere et al., 2009), a number of isolates, retrieved from chronically infected
213
patients at different colonization times, were examined by MLST analysis by
214
assessing seven housekeeping genes (atpD, gltB, gyrB, recA, lepA, phaC, and trpB)
215
(Baldwin et al., 2005). All the taxon K isolates examined were considered to be
216
isogenic given their identical allelic profile (all of them gave rise to the same
217
sequence type (ST), ST-96) (Table 1). Since MLST analysis is a costly and time-
218
consuming procedure, it is limited to the analysis of a small number of isolates.
219
Therefore, our option was to molecularly characterize the remaining isolates using the
220
hisA gene sequence analysis, since this method was also validated for Bcc species
221
determination (Papaleo et al., 2010). Altogether, the molecular analyses performed
222
revealed that the isolates previously classified as belonging to B. cepacia recA profile
223
K, as well as the isolates more recently retrieved from one patient that had been
224
infected with recA profile K isolates from 2004 to 2006 and continued infected with
225
the same taxon K bacteria until 2010, are B. contaminans. Moreover, this was the
226
only species of taxon K found among the Bcc isolates examined during the referred
227
surveillance period.
228 229
Clinical impact of B. contaminans infections in CF
230
Two of the paediatric patients with Bcc (G and AF) were considered chronically
231
infected with B. contaminans, i.e. with 50% or more of positive Bcc cultures within a
232
12-month period (Lee et al., 2003). Both patients died, one with cepacia syndrome
233
(patient G) and the other following long-term infection (six years) and clinical
234
deterioration (patient AF). However, patient AF was also found to be co-infected with
235
Burkholderia cenocepacia IIIA and IIIB and with bacteria from other genera
236
(Pseudomonas aeruginosa, Staphylococcus aureus and Haemophilus influenza).
237
Patient G was co-infected with P. aeruginosa and with Bcc bacteria for eight months
238
and, during this time, three Bcc isolates were obtained during periodic visits to
239
monitor the patient’s clinical status: a B. cenocepacia IIIB isolate was collected first,
240
followed by a B. contaminans isolate, and the last isolate, obtained before the
241
patient´s death with cepacia syndrome, belonged to B. cenocepacia IIIB (Table 2).
242
Since patient G was co-infected with B. cenocepacia, his death with cepacia
243
syndrome cannot simply be attributed to B. contaminans, especially since the last
244
isolate retrieved close to the patient’s death was classified as B. cenocepacia IIIB.
245
Patient AF was infected with Bcc during almost six years but most of the isolates
246
retrieved (29) belong to the B. contaminans species. However, isolates belonging to
247
B. cepacia (two), Burkholderia dolosa (one), B. cenocepacia IIIB (one) and B.
248
cenocepacia IIIA species (eight) were also obtained (Table 2).
249
Remarkably, although transient co-infection with B. cenocepacia IIIA and IIIB
250
occurred, B. contaminans prevailed until the patient’s death as recently reported for B.
251
dolosa (Moreira et al., 2014), contrasting with the generalized idea that B.
252
cenocepacia bacteria replace other Bcc species present in the lungs (Bernhardt et al.,
253
2003). The registered co-colonization of the patients with B. cenocepacia and other
254
non-Burkholderia bacteria raises the question whether B. contaminans was the main
255
cause of death. However, in the case of patient AF, during the last year of the life,
256
only B. contaminans isolates were retrieved suggesting that B. contaminans
257
contributed to the clinical outcome deterioration of the infected patient (Table 1 and
258
2). Following the first B. contaminans bacterium isolation, patient AF was
259
hospitalized several times (one during the first year and three during the second year
260
following isolation) and the FEV1 values, which reached 60% one year before the
261
isolation of the first B. contaminans bacterium, decreased to 44% during the first year
262
following isolation of Bcc bacteria that consisted mostly of B. contaminans. In
263
summary, the inexorable decline in the FEV1 value appears to have been related with
264
B. contaminans infection, although it is impossible to be sure about the clinical impact
265
of this species in a co-infected patient.
266
The other five patients (A, E, F, W and X) infected with B. contaminans cannot be
267
considered as chronically infected. Patients A, E and F were infected with Bcc
268
bacteria for less than one month. Two isolates belonging to B. contaminans and B.
269
cenocepacia IIIB were isolated from patient A, but spontaneous clearance occurred.
270
For the other two patients, only one isolate of B. contaminans was apparently
271
retrieved because, following the initial B. contaminans isolation, the patients died.
272
Both patients were also co-infected with P. aeruginosa (E patient) or S. aureus, H.
273
influenza and P. aeruginosa (patient F). Patients W and X, who were also co-infected
274
with S. aureus, H. influenza and P. aeruginosa, were able to spontaneously eradicate
275
B. contaminans after five or three months of the first isolation, respectively. In
276
summary, the species B. contaminans was associated either with chronic infection in
277
CF patients (the case of patients G and AF), or with a transient infection that, in
278
certain cases, led to spontaneous clearance (the case of patients A, W and X).
279 280
The cepacian producing strain IST408 belongs to B. contaminans and not to B.
281
cepacia
282
Among the isolates whose taxonomic position was re-examined at the species level in
283
the present work is IST408, an isolate that was previously classified as B. cepacia
284
(Richau et al., 2000; Cunha et al., 2003; Moreira et al., 2003; Cunha et al., 2004;
285
Ferreira et al., 2011). B. contaminans IST408 has been on the focus of our studies
286
regarding exopolysaccharide (EPS) biosynthesis in Bcc (Cescutti et al., 2000; Richau
287
et al., 2000; Sist et al., 2003; Ferreira et al., 2011), since it was consistently the
288
highest producing strain tested among the clinical isolates available, at that time, in
289
our culture collection. The designation of the EPS, which was chemically and
290
structurally characterized, as “cepacian”, was based on the idea that the producing
291
isolate belonged to the species B. cepacia (Cescutti et al., 2000; Richau et al., 2000;
292
Sist et al., 2003). Remarkably, all of the previously characterized B. contaminans
293
isolates (formerly considered B. cepacia) were found to be, in general, high EPS
294
producers, compared with isolates from the other species examined (B. cenocepacia,
295
B. cepacia, B. multivorans and B. stabilis) (Cunha et al., 2004)). Although the number
296
of Bcc isolates tested concerning the level of EPS production was small and includes
297
a large number of isogenic variants, this observation concerning the high EPS
298
producing capacity of B. contaminans isolates certainly deserves attention in future
299
studies.
300 301
B. contaminans infections in the Portuguese CF population examined
302
Based on the epidemiological survey carried out for the past two decades in the HSM
303
CF Treatment Center, we found B. contaminans isolates in 17% of the CF population
304
infected with Bcc (i.e. in seven patients - three girls and four boys - of a total of 41
305
patients). The prevalence of B. contaminans in CF patients worldwide is low, with
306
remarkable exceptions registered in Argentina (Martina et al., 2013) and Spain
307
(Medina-Pascual et al., 2015), presumably resulting from patient to patient
308
transmission or other sources of infection, such as environmental or pharmaceutical
309
products.
310
Some of the isolates that were re-classified as B. contaminans in this study had been
311
previously classified as B. cepacia. Remarkably, the CF population under surveillance
312
at the CF Treatment Center of HSM is well known for having an exceptionally high
313
representation of B. cepacia (Fig. 1) (Cunha et al., 2007). This abnormal prevalence
314
was associated with an outbreak in 2003-2005 presumably caused by contaminated
315
non-sterile saline solutions for nasal application, detected with our help during routine
316
market surveillance by the Portuguese Medicines and Health Products Authority
317
(INFARMED). B. cepacia strains with ribopatterns 17, 18, 19 and 20 were identified
318
in several batches of the saline solutions examined and in infected CF patients.
319
Notably, the isolates that were now re-classified as B. contaminans and that were
320
retrieved during this critical period have ribopatterns 2 and 5 (Cunha et al., 2007).
321
Therefore, a direct causal link between the contaminated saline solutions and B.
322
contaminans infections cannot be established at this time. Nevertheless, this
323
hypothesis can neither be excluded. Indeed, several B. contaminans isolates were
324
collected from patients during the period of maximal contamination with B. cepacia,
325
which coincided with the identification of the contaminated saline solutions, and even
326
if ribopatterns 2 and 5 were not detected in those solutions, the coincidence in time
327
raises the possibility of a common source of infection. Also, a B. cepacia isolate with
328
ribopattern 17 was collected from patient AF (IST4221), who was found to be
329
simultaneously infected with B. contaminans since 2004 (Table 1).
330 331
CONCLUDING REMARKS
332
This work describes the identification of 41 B. contaminans isolates retrieved from
333
seven paediatric cystic fibrosis (CF) patients, two of them chronically infected over
334
the course of 15 years of epidemiological surveillance of Bcc respiratory infections at
335
the major Portuguese CF center. The same CF population examined had been
336
previously reported as having an exceptionally high representation of B. cepacia
337
isolates (Cunha et al., 2007). This fact was considered to be at least partially
338
associated with a contamination of saline solutions for nasal application. However,
339
20 of those B. cepacia isolates, formerly classified as recA RFLP type K, were
340
reclassified in this work as B. contaminans. Twenty-one additional B. contaminans
341
isolates were obtained later from a chronically infected patient. This study reinforces
342
the observation that this Portuguese CF population is infected with an abnormally
343
high percentage of Bcc species that are rarely found in CF respiratory infections,
344
namely B. cepacia and B. contaminans, and provides insight into the potential clinical
345
impact of B. contaminans species. Isolates belonging to this species were retrieved
346
from chronic and transient respiratory infections and were associated with lung
347
function deterioration and in the patient deceased from the cepacia syndrome.
348
However, since these patients were co-infected with B. cenocepacia and other non-
349
Burkholderia bacteria, the role played by B. contaminans is unclear. During co-
350
infection of at least one chronically infected patient, B. contaminans isolates were
351
found to prevail over B. cenocepacia isolates.
352 353 354 355
ACKNOWLEDGMENTS
356
This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT),
357
Portugal
358
SFRH/BPD/81220/2011- to C. P. C.).
(contract
UID/BIO/04565/2013
and
a
post-doctoral
grant-
359 360
DECLARATION OF INTEREST
361
All authors report no potential conflicts of interest. Only the authors are responsible
362
for the content and writing of the paper.
363 364 365 366 367 368 369 370 371 372 373 374
375
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376 377 378 379 380 381
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560 561 562 563
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568 569 570 571
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576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601
602
Table 1. Isolates used in this study Patient
Isolate
Isolation Date
A
IST401 IST404 IST406 IST408 IST405 IST410 IST411 IST480 IST487 IST488 IST492 IST493 IST4101 IST4120 IST4126 IST4193 IST4194 IST4186 IST4188 IST4192 IST4206 IST4207 IST4221 IST4238 IST4237 IST4240 a)† IST4240 b) † IST4240 c) † IST4251 IST4253 a) † IST4253 b) † IST4258 a) † IST4258 b) † IST4272 IST4276 IST4303 IST4319 IST4322 IST4391 IST4402 a) † IST4402 b) † IST4408 IST4412 IST4420 a) † IST4420 d) † IST4435 IST4497 IST4456 IST4474 IST4496 IST4485 IST4490 IST4550 IST4513 IST4517 IST4566
28/08/95 05/09/95 15/12/95 31/01/95 07/04/95 01/06/95 09/12/95 21/02/01 06/04/01 21/04/01 07/05/01 10/05/01 04/07/01 24/01/02 18/04/02 09/12/04 10/02/05 14/04/05 04/05/05 30/06/05 08/10/05 08/10/05 23/11/05 14/03/06 20/03/06 10/05/06 10/05/06 10/05/06 14/06/06 07/07/06 07/07/06 02/08/06 02/08/06 11/12/06 22/11/06 28/03/07 23/04/07 23/04/07 19/03/08 09/07/08 09/07/08 13/08/08 16/09/08 17/10/08 17/10/08 23/11/09 11/01/10 29/03/10 18/05/10 22/02/10 10/05/10 16/06/10 19/07/10 09/08/10 10/10/10 29/10/10
E F G
W
X AF
603 604 605
(*)
Burkholderia species this work
B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cenocepacia IIIB B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cepacia -
B. contaminans B. cenocepacia IIIB B. contaminans B. contaminans B. cenocepacia IIIB B. contaminans B. cenocepacia IIIB B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. cenocepacia IIIB B. cepacia B. contaminans B. contaminans B. cenocepacia IIIA B. contaminans B. cepacia B. cenocepacia IIIA B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. dolosa B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans
Notes: all isolates were identified by hisA gene sequence analysis; * as identified by Cunha et al. J. Clin. Microbiol. 45:1628-1633,2007; # Information in http://pubmlst.org/bcc/, partially provided by our laboratory, (-) not done; † a) b) c) and d) refer to different isolates from the species B. cenocepacia IIIA, B. contaminans, B. cepacia and B. dolosa, respectively, obtained at the same isolation date.
ST# 96 96 96 96 96 96 96 96 96 96 96 -
Table 2. Multi-Bcc-species’ infection in the seven CF patients from whom B. contaminans isolates were retrieved during 15 years of epidemiological surveillance
Patient
Gender*
Age at CF diagnosis / 1st Bcc isolation¶
A
F
3m/4m
E
M
3m/3m
F
F
10m/2y
G
F
4m/10y
W
M
5m/5m
Duration of Bcc infection¶
Co-infecting bactéria#
clinical outcome
Journal of Medical Microbiology Incidence of Burkholderia contaminans at a cystic fibrosis center with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance --Manuscript Draft-Manuscript Number:
JMM-D-14-00046R6
Full Title:
Incidence of Burkholderia contaminans at a cystic fibrosis center with an unusually high representation of Burkholderia cepacia during 15 years of epidemiological surveillance
Short Title:
Burkholderia contaminans at a cystic fibrosis center
Article Type:
Standard
Section/Category:
Epidemiology
Corresponding Author:
Isabel Sá-Correia 1Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av Rovisco Pais, P-1049001 Lisbon, Portugal Lisboa, PORTUGAL
First Author:
Carla P. Coutinho, PhD
Order of Authors:
Carla P. Coutinho, PhD Celeste Barreto Luísa Pereira Luís Lito José Melo Cristino Isabel Sá-Correia
Abstract:
The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria comprising around 20 related species. These bacteria are important opportunistic pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon, from 1995 to 2006, was re-examined in the present work. These isolates, formerly classified as Burkholderia cepacia (taxon K), are here re-classified as Burkholderia contaminans, including the former B. cepacia IST408, which was on the focus of previous studies regarding the biosynthesis of the exopolysaccharide "cepacian". The CF population examined has been previously described as having an exceptionally high representation of B. cepacia, presumably due to a contamination arising from saline solutions for nasal application. Twenty-one additional isolates, obtained from a chronically infected patient, from 2006 to 2010, were also identified as B. contaminans. This study also provides insight into the potential clinical impact of B. contaminans, a species that is rarely associated with CF infections. Isolates belonging to this species were shown to be involved in chronic and transient respiratory infections, and were associated with severe lung function deterioration and with a case of death with cepacia syndrome. However, since the patients were co-infected with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to prevail over B. cenocepacia isolates during co-infection of at least one chronically infected patient.
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1
Incidence of Burkholderia contaminans at a cystic fibrosis
2
center with an unusually high representation of
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Burkholderia cepacia during 15 years of epidemiological
4
surveillance
5 6
Carla P. Coutinho1, Celeste Barreto2, Luísa Pereira², Luís Lito3, José Melo
7
Cristino3 and Isabel Sá-Correia1*
8 9
1
iBB-Institute for Bioengineering and Biosciences, Department of Bioengineering,
10
Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001
11
Lisbon, Portugal
12
2
13
Hospitalar Lisboa Norte, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
14
3
15
Norte, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal
Cystic Fibrosis Center , Department of Paediatrics, Hospital de Santa Maria, Centro
Laboratório de Patologia Clínica, Hospital de Santa Maria, Centro Hospitalar Lisboa
16 17 18 19 20 21 22
* Corresponding author: Professor Isabel Sá-Correia, Biological Sciences,
23
Department of Bioengineering, Instituto Superior Técnico, Torre Sul, Universidade de
24
Lisboa,
25
[email protected]; Telephone: +351-218417682; Fax: +351-218419062
Av.
Rovisco
Pais,
1049-001,
Lisbon,
Portugal.
E-mail:
26 27
Running title: Burkholderia contaminans at a cystic fibrosis center
28
Content Category: Epidemiology
29 30
Abbreviations: Bcc, Burkholderia cepacia complex; CF, cystic fibrosis; FEV1,
31
forced expiratory volume in 1 second; HSM, Hospital de Santa Maria; MLST,
32
multilocus sequence typing; ST, sequence type.
33
ABSTRACT
34
The Burkholderia cepacia complex (Bcc) is a heterogeneous group of bacteria
35
comprising around 20 related species. These bacteria are important opportunistic
36
pathogens, especially in cystic fibrosis (CF) patients, and are associated with a worse
37
prognosis and decreased life expectancy. The taxonomic position of 20 Bcc isolates
38
retrieved from CF patients receiving care at Hospital Santa Maria (HSM), in Lisbon,
39
from 1995 to 2006, was re-examined in the present work. These isolates, formerly
40
classified as Burkholderia cepacia (taxon K), are here re-classified as Burkholderia
41
contaminans, including the former B. cepacia IST408, which was on the focus of
42
previous studies regarding the biosynthesis of the exopolysaccharide “cepacian”. The
43
CF population examined has been previously described as having an exceptionally
44
high representation of B. cepacia, presumably due to a contamination arising from
45
saline solutions for nasal application. Twenty-one additional isolates, obtained from a
46
chronically infected patient, from 2006 to 2010, were also identified as B.
47
contaminans. This study also provides insight into the potential clinical impact of B.
48
contaminans, a species that is rarely associated with CF infections. Isolates belonging
49
to this species were shown to be involved in chronic and transient respiratory
50
infections, and were associated with severe lung function deterioration and with a
51
case of death with cepacia syndrome. However, since the patients were co-infected
52
with Burkholderia cenocepacia and other non-Burkholderia bacteria, the role played
53
by B. contaminans is unclear. Nevertheless, B. contaminans isolates were found to
54
prevail over B. cenocepacia isolates during co-infection of at least one chronically
55
infected patient.
56 57 58 59 60 61 62 63 64
Keywords: Burkholderia contaminans; Burkholderia cepacia, species identification,
65
genotyping;
66
surveillance.
cystic
fibrosis;
chronic
respiratory
infection;
epidemiological
67
INTRODUCTION
68
The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacteria that
69
can cause serious respiratory infections in immunocompromised individuals. Patients
70
with cystic fibrosis (CF) are particularly at risk, where Bcc infections are associated
71
with a worse prognosis and early death (Lipuma, 2010; Peeters et al., 2013). The
72
large majority of CF patients infected with Bcc develop a chronic infection that can
73
last for years, leading to progressive loss of lung function. Also, a number of Bcc-
74
infected patients may develop a rapid and fatal necrotizing pneumonia known as
75
“cepacia syndrome” (Mahenthiralingam et al., 2002). Most of the 20 described Bcc
76
species have been isolated from CF pulmonary infections, but there is a
77
disproportionate distribution of Bcc species, and the prevalence described for each
78
species may vary depending on the country and CF center under consideration
79
(LiPuma et al., 2001; Govan et al., 2007; Drevinek and Mahenthiralingam, 2010).
80
Nevertheless, Burkholderia cenocepacia and Burkholderia multivorans account for
81
most (80%) of the Bcc infections in CF patients worldwide (Drevinek and
82
Mahenthiralingam, 2010; Lipuma, 2010; Peeters et al., 2013), with B. cenocepacia
83
being considered the most serious Bcc pathogen in CF (Mahenthiralingam and
84
Vandamme, 2005; Drevinek and Mahenthiralingam, 2010). Consequently, most of the
85
research dedicated to Bcc pathogenicity in CF has been focused on this species, and
86
information regarding the impact of other Bcc species on clinical outcome is still
87
scarce. However, epidemiological studies have shown that other Bcc species are also
88
associated with poor prognosis, patient-to-patient transmission, chronic or transient
89
infections, and cepacia syndrome (Biddick et al., 2003; Cunha et al., 2003; Kalish et
90
al., 2006; Cunha et al., 2007; Drevinek and Mahenthiralingam, 2010; Lipuma, 2010;
91
Moreira et al., 2014).
92
In the last few years, the taxonomy of the Bcc has evolved significantly, with the
93
description of novel species (Yabuuchi et al., 2000; Coenye et al., 2001; Vandamme
94
et al., 2002; Vermis et al., 2002; Vermis et al., 2004; Lipuma, 2005; Vanlaere et al.,
95
2008; Vanlaere et al., 2009; Peeters et al., 2013; Smet et al., 2015). Until 2009,
96
multicenter studies revealed the existence of several clusters of isolates that do not
97
group with the Bcc species described at the time. One divergent group was classified
98
as belonging to Burkholderia cepacia complex recA RFLP type K (Vermis et al.,
99
2002; Baldwin et al., 2005; Dalmastri et al., 2005; Payne et al., 2005;
100
Mahenthiralingam et al., 2006; Dalmastri et al., 2007). After that date, Vanlaere and
101
co-workers re-examined the taxonomic position and structure of taxon K (also known
102
as group K), and proposed two novel species, Burkholderia contaminans and
103
Burkholderia lata (Vanlaere et al., 2009). The group of previously unclassified Bcc
104
isolates of taxon K (Vermis et al., 2002; Baldwin et al., 2005; Dalmastri et al., 2005;
105
Payne et al., 2005; Mahenthiralingam et al., 2006; Dalmastri et al., 2007) includes
106
bacteria isolated worldwide from human and environmental sources, namely from
107
sputum cultures of CF patients in the UK, Italy, Portugal, USA, Canada, China,
108
Brazil, Argentina, and Australia (Cunha et al., 2003; Campana et al., 2005; Assaad et
109
al., 2006; Mahenthiralingam et al., 2006; Cunha et al., 2007; Jorda-Vargas et al.,
110
2008; Fang et al., 2010; Martina et al., 2013; Ramsay et al., 2013), as well as from
111
river water, human-built water reservoirs, soil, roots, animals, pharmaceutical
112
products, such as contaminated nasal spray, personal care products, and domestic
113
products (Souza et al., 2004; Mahenthiralingam et al., 2006; Mahenthiralingam et al.,
114
2008; Martin et al., 2011). B. contaminans has a low prevalence in CF patients
115
worldwide, with remarkable exceptions in Argentina and in Spain (Martina et al.,
116
2013). Several reports of B. contaminans outbreaks amongst non-CF patients in
117
Argentina and Brazil were also published, suggesting the existence of regional
118
spreading of B. contaminans and other Bcc species between bordering countries
119
(Mahenthiralingam et al., 2000; Agodi et al., 2002; Magalhaes et al., 2003; Shehabi et
120
al., 2004; Souza et al., 2004; Woods et al., 2004). In the present work we have re-
121
examined the taxonomic position, at the species level, of several isolates from our Bcc
122
collection, recovered from CF patients receiving care at the major Portuguese CF
123
Treatment Center, at Hospital de Santa Maria (HSM), in Lisbon. These isolates were
124
obtained over 15 years of epidemiological surveillance of respiratory infections
125
involving Bcc, and the study is focused on isolates previously classified as B. cepacia
126
complex recA RFLP type K. This study also extends to isolates more recently
127
obtained from a chronically infected patient known to harbour B. cepacia complex
128
recA RFLP type K isolates, and provides insights into the potential clinical impact of a
129
species that is rarely associated with CF infections.
130
METHODS
131
Bacterial isolates and culture conditions. The Bcc isolates used in this work (Table
132
1) were obtained from the HSM CF Center, Lisbon, Portugal, over the period of 1995
133
to 2010, from CF patients’ respiratory secretions. According to the hospital’s routine,
134
sputum samples are obtained from CF patients every two to three months, during
135
periodic consultations to monitor their clinical status, or more often for patients
136
showing clinical deterioration. The seven patients indicated in Table 1 were found to
137
be infected with Burkholderia cepacia complex recA RFLP type K, and the isolates
138
were gathered from the date of their detection until the date of eradication or until
139
patient death. Some of these isolates were previously classified as B. cepacia (Cunha
140
et al., 2003; Cunha et al., 2007), while others were molecularly examined for the first
141
time in the present work and were obtained from a CF patient who continued to be
142
chronically colonized with B. cepacia recA RFLP profile K after the date of
143
previously published studies. Bacterial cultures are stored at -80°C in 1:1 (vol/vol)
144
glycerol. When in use, bacteria are cultivated on Pseudomonas Isolation Agar (PIA;
145
Difco, Sparks, MD) plates or on Luria-Bertani agar (LB agar; Difco, Sparks, MD)
146
plates.
147
Species identification and genotyping. Total genomic DNAs from each isolate,
148
previously cultured overnight in LB medium with orbital agitation at 37°C, was
149
extracted using a cell and tissue kit (Gentra Systems, Qiagen, Germany). The
150
concentration of genomic DNA solutions was estimated using an ND-1000
151
spectrophotometer (NanoDrop).
152
The recA PCR RFLP analysis was performed by amplification of the entire
153
recA gene (1,040 bp) using specific primers for bacteria belonging to the Bcc (BCR1-
154
5’
155
previously described by Mahenthiralingam et al. (Mahenthiralingam et al., 2000). Bcc
156
recA amplicons were digested with HaeIII (Amersham Biosciences) restriction
157
endonuclease and the restriction fragments were separated by electrophoresis in 2%
158
(w/v) agarose gels. The RFLP patterns obtained for the Bcc isolates examined were
159
compared with those reported in the literature (Mahenthiralingam et al., 2000; Vermis
160
et al., 2002; Leite et al., 2011).
TGACCGCCGAGAAGAGCAA3’; BCR2-5’CTCTTCTTCGTCCATCGCCTC3’) as
161
The identification of the clinical isolates at the species level was performed
162
using two different methods: multilocus sequence typing (MLST) analysis and hisA
163
gene sequence analysis. MLST analysis was performed as previously described
164
(Coutinho et al., 2011). Amplification was carried out using a Gene Amp® PCR
165
System 2700 (Applied Biosystems, USA). The amplification products were confirmed
166
following separation of PCR products by 0.9% (w/v) horizontal agarose gel
167
electrophoresis, and the bands were excised, purified using a gel extraction kit
168
(JETquick spin column technique; Genomed, Germany) and sequenced. The sequence
169
data for each isolate was compared with known sequences from the Bcc MLST
170
database
171
(www.mbio.ncsu.edu/bioedit/bioedit.html). The alleles for each of the seven loci were
172
assigned and the allelic profile (string of seven integers) was used to define the
173
sequence type (ST), using the same database. The allelic profile of the isolates under
174
study was submitted and added to the Bcc MLST database.
(http://pubmlst.org/bcc),
using
BioEdit
software
175
The hisA gene sequence analysis was performed by PCR amplification of the
176
hisA gene region (89 to 530bp) of 442bp as previously described by Papaleo et al.
177
(Papaleo et al., 2010). Briefly, in a 50 µl reaction mixture containing 50 ng of DNA
178
template, 2 U Taq DNA polymerase (Biotaq Taq polymerase; Bioline), 250 mM each
179
dNTP, 0.6 mM each primer (forward – AGGACCCGGCGGCGAT; reverse –
180
TGCAGCATCCCGTCGCG), 1.5 mM MgCl2 and 1PCR buffer (10; supplied by
181
the polymerase manufacturer) were added. The amplification reaction was carried out
182
using an initial step of denaturation for 2 min at 95°C, followed by 30 cycles of 30 s
183
at 95°C for denaturation, 45 s at 67°C for primer annealing, and 1 min at 72°C for
184
polymerization, followed by a final extension step of 72°C for 10 min, using a
185
GeneAmp PCR System 2700 (Applied Biosystems). Amplification was confirmed by
186
electrophoresis in 0.9% (w/v) agarose gels. The PCR products were purified using the
187
PCR product purification spin kit (JETquick spin column technique; Genomed),
188
according to the manufacturer’s instructions, and sequenced. The sequences were
189
compared with the eleven-letter code for the Bcc hisA gene, which allows species
190
identification as detailed by Papaleo et al. (Papaleo et al., 2010).
191
Patients’ clinical course. The clinical course of patient AF, before detection of Bcc
192
infection and throughout the period of chronic respiratory infection, was assessed
193
based on values of forced expiratory volume in 1 s (FEV1) during regular lung
194
function tests performed at the hospital to monitor the patient’s respiratory function.
195
There are no available records of FEV1 values for the other patients.
196
Ethics. Studies involving clinical Bcc isolates obtained as part of the hospital routine
197
were approved by the hospital ethics committee and the patients’ anonymity is
198
preserved. Consent was obtained from the patients’ legal guardian for the use of these
199
isolates in research.
200
201
RESULTS AND DISCUSSION
202
Identification and molecular typing of B. contaminans clinical isolates
203
The 56 Bcc isolates examined in this study were obtained from seven paediatric CF
204
patients from 1995 to 2010. They include those formerly classified as Burkholderia
205
cepacia recA profile K and those more recently isolated from one chronically infected
206
patient found to be infected with profile K isolates in previously published
207
epidemiological surveys (Cunha et al., 2003; Cunha et al., 2007). The profile K was
208
confirmed for all the isolates based on recA gene amplification and restriction with
209
HaeIII of the amplified products followed by RFLP analysis. However, since recA
210
sequence-based-identification of the so-called taxon K bacteria is considered
211
problematic and can only solve part of the species complexity in this lineage
212
(Vanlaere et al., 2009), a number of isolates, retrieved from chronically infected
213
patients at different colonization times, were examined by MLST analysis by
214
assessing seven housekeeping genes (atpD, gltB, gyrB, recA, lepA, phaC, and trpB)
215
(Baldwin et al., 2005). All the taxon K isolates examined were considered to be
216
isogenic given their identical allelic profile (all of them gave rise to the same
217
sequence type (ST), ST-96) (Table 1). Since MLST analysis is a costly and time-
218
consuming procedure, it is limited to the analysis of a small number of isolates.
219
Therefore, our option was to molecularly characterize the remaining isolates using the
220
hisA gene sequence analysis, since this method was also validated for Bcc species
221
determination (Papaleo et al., 2010). Altogether, the molecular analyses performed
222
revealed that the isolates previously classified as belonging to B. cepacia recA profile
223
K, as well as the isolates more recently retrieved from one patient that had been
224
infected with recA profile K isolates from 2004 to 2006 and continued infected with
225
the same taxon K bacteria until 2010, are B. contaminans. Moreover, this was the
226
only species of taxon K found among the Bcc isolates examined during the referred
227
surveillance period.
228 229
Clinical impact of B. contaminans infections in CF
230
Two of the paediatric patients with Bcc (G and AF) were considered chronically
231
infected with B. contaminans, i.e. with 50% or more of positive Bcc cultures within a
232
12-month period (Lee et al., 2003). Both patients died, one with cepacia syndrome
233
(patient G) and the other following long-term infection (six years) and clinical
234
deterioration (patient AF). However, patient AF was also found to be co-infected with
235
Burkholderia cenocepacia IIIA and IIIB and with bacteria from other genera
236
(Pseudomonas aeruginosa, Staphylococcus aureus and Haemophilus influenza).
237
Patient G was co-infected with P. aeruginosa and with Bcc bacteria for eight months
238
and, during this time, three Bcc isolates were obtained during periodic visits to
239
monitor the patient’s clinical status: a B. cenocepacia IIIB isolate was collected first,
240
followed by a B. contaminans isolate, and the last isolate, obtained before the
241
patient´s death with cepacia syndrome, belonged to B. cenocepacia IIIB (Table 2).
242
Since patient G was co-infected with B. cenocepacia, his death with cepacia
243
syndrome cannot simply be attributed to B. contaminans, especially since the last
244
isolate retrieved close to the patient’s death was classified as B. cenocepacia IIIB.
245
Patient AF was infected with Bcc during almost six years but most of the isolates
246
retrieved (29) belong to the B. contaminans species. However, isolates belonging to
247
B. cepacia (two), Burkholderia dolosa (one), B. cenocepacia IIIB (one) and B.
248
cenocepacia IIIA species (eight) were also obtained (Table 2).
249
Remarkably, although transient co-infection with B. cenocepacia IIIA and IIIB
250
occurred, B. contaminans prevailed until the patient’s death as recently reported for B.
251
dolosa (Moreira et al., 2014), contrasting with the generalized idea that B.
252
cenocepacia bacteria replace other Bcc species present in the lungs (Bernhardt et al.,
253
2003). The registered co-colonization of the patients with B. cenocepacia and other
254
non-Burkholderia bacteria raises the question whether B. contaminans was the main
255
cause of death. However, in the case of patient AF, during the last year of the life,
256
only B. contaminans isolates were retrieved suggesting that B. contaminans
257
contributed to the clinical outcome deterioration of the infected patient (Table 1 and
258
2). Following the first B. contaminans bacterium isolation, patient AF was
259
hospitalized several times (one during the first year and three during the second year
260
following isolation) and the FEV1 values, which reached 60% one year before the
261
isolation of the first B. contaminans bacterium, decreased to 44% during the first year
262
following isolation of Bcc bacteria that consisted mostly of B. contaminans. In
263
summary, the inexorable decline in the FEV1 value appears to have been related with
264
B. contaminans infection, although it is impossible to be sure about the clinical impact
265
of this species in a co-infected patient.
266
The other five patients (A, E, F, W and X) infected with B. contaminans cannot be
267
considered as chronically infected. Patients A, E and F were infected with Bcc
268
bacteria for less than one month. Two isolates belonging to B. contaminans and B.
269
cenocepacia IIIB were isolated from patient A, but spontaneous clearance occurred.
270
For the other two patients, only one isolate of B. contaminans was apparently
271
retrieved because, following the initial B. contaminans isolation, the patients died.
272
Both patients were also co-infected with P. aeruginosa (E patient) or S. aureus, H.
273
influenza and P. aeruginosa (patient F). Patients W and X, who were also co-infected
274
with S. aureus, H. influenza and P. aeruginosa, were able to spontaneously eradicate
275
B. contaminans after five or three months of the first isolation, respectively. In
276
summary, the species B. contaminans was associated either with chronic infection in
277
CF patients (the case of patients G and AF), or with a transient infection that, in
278
certain cases, led to spontaneous clearance (the case of patients A, W and X).
279 280
The cepacian producing strain IST408 belongs to B. contaminans and not to B.
281
cepacia
282
Among the isolates whose taxonomic position was re-examined at the species level in
283
the present work is IST408, an isolate that was previously classified as B. cepacia
284
(Richau et al., 2000; Cunha et al., 2003; Moreira et al., 2003; Cunha et al., 2004;
285
Ferreira et al., 2011). B. contaminans IST408 has been on the focus of our studies
286
regarding exopolysaccharide (EPS) biosynthesis in Bcc (Cescutti et al., 2000; Richau
287
et al., 2000; Sist et al., 2003; Ferreira et al., 2011), since it was consistently the
288
highest producing strain tested among the clinical isolates available, at that time, in
289
our culture collection. The designation of the EPS, which was chemically and
290
structurally characterized, as “cepacian”, was based on the idea that the producing
291
isolate belonged to the species B. cepacia (Cescutti et al., 2000; Richau et al., 2000;
292
Sist et al., 2003). Remarkably, all of the previously characterized B. contaminans
293
isolates (formerly considered B. cepacia) were found to be, in general, high EPS
294
producers, compared with isolates from the other species examined (B. cenocepacia,
295
B. cepacia, B. multivorans and B. stabilis) (Cunha et al., 2004)). Although the number
296
of Bcc isolates tested concerning the level of EPS production was small and includes
297
a large number of isogenic variants, this observation concerning the high EPS
298
producing capacity of B. contaminans isolates certainly deserves attention in future
299
studies.
300 301
B. contaminans infections in the Portuguese CF population examined
302
Based on the epidemiological survey carried out for the past two decades in the HSM
303
CF Treatment Center, we found B. contaminans isolates in 17% of the CF population
304
infected with Bcc (i.e. in seven patients - three girls and four boys - of a total of 41
305
patients). The prevalence of B. contaminans in CF patients worldwide is low, with
306
remarkable exceptions registered in Argentina (Martina et al., 2013) and Spain
307
(Medina-Pascual et al., 2015), presumably resulting from patient to patient
308
transmission or other sources of infection, such as environmental or pharmaceutical
309
products.
310
Some of the isolates that were re-classified as B. contaminans in this study had been
311
previously classified as B. cepacia. Remarkably, the CF population under surveillance
312
at the CF Treatment Center of HSM is well known for having an exceptionally high
313
representation of B. cepacia (Fig. 1) (Cunha et al., 2007). This abnormal prevalence
314
was associated with an outbreak in 2003-2005 presumably caused by contaminated
315
non-sterile saline solutions for nasal application, detected with our help during routine
316
market surveillance by the Portuguese Medicines and Health Products Authority
317
(INFARMED). B. cepacia strains with ribopatterns 17, 18, 19 and 20 were identified
318
in several batches of the saline solutions examined and in infected CF patients.
319
Notably, the isolates that were now re-classified as B. contaminans and that were
320
retrieved during this critical period have ribopatterns 2 and 5 (Cunha et al., 2007).
321
Therefore, a direct causal link between the contaminated saline solutions and B.
322
contaminans infections cannot be established at this time. Nevertheless, this
323
hypothesis can neither be excluded. Indeed, several B. contaminans isolates were
324
collected from patients during the period of maximal contamination with B. cepacia,
325
which coincided with the identification of the contaminated saline solutions, and even
326
if ribopatterns 2 and 5 were not detected in those solutions, the coincidence in time
327
raises the possibility of a common source of infection. Also, a B. cepacia isolate with
328
ribopattern 17 was collected from patient AF (IST4221), who was found to be
329
simultaneously infected with B. contaminans since 2004 (Table 1).
330 331
CONCLUDING REMARKS
332
This work describes the identification of 41 B. contaminans isolates retrieved from
333
seven paediatric cystic fibrosis (CF) patients, two of them chronically infected over
334
the course of 15 years of epidemiological surveillance of Bcc respiratory infections at
335
the major Portuguese CF center. The same CF population examined had been
336
previously reported as having an exceptionally high representation of B. cepacia
337
isolates (Cunha et al., 2007). This fact was considered to be at least partially
338
associated with a contamination of saline solutions for nasal application. However,
339
20 of those B. cepacia isolates, formerly classified as recA RFLP type K, were
340
reclassified in this work as B. contaminans. Twenty-one additional B. contaminans
341
isolates were obtained later from a chronically infected patient. This study reinforces
342
the observation that this Portuguese CF population is infected with an abnormally
343
high percentage of Bcc species that are rarely found in CF respiratory infections,
344
namely B. cepacia and B. contaminans, and provides insight into the potential clinical
345
impact of B. contaminans species. Isolates belonging to this species were retrieved
346
from chronic and transient respiratory infections and were associated with lung
347
function deterioration and in the patient deceased from the cepacia syndrome.
348
However, since these patients were co-infected with B. cenocepacia and other non-
349
Burkholderia bacteria, the role played by B. contaminans is unclear. During co-
350
infection of at least one chronically infected patient, B. contaminans isolates were
351
found to prevail over B. cenocepacia isolates.
352 353 354 355
ACKNOWLEDGMENTS
356
This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT),
357
Portugal
358
SFRH/BPD/81220/2011- to C. P. C.).
(contract
UID/BIO/04565/2013
and
a
post-doctoral
grant-
359 360
DECLARATION OF INTEREST
361
All authors report no potential conflicts of interest. Only the authors are responsible
362
for the content and writing of the paper.
363 364 365 366 367 368 369 370 371 372 373 374
375
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576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592 593 594 595 596 597 598 599 600 601
602
Table 1. Isolates used in this study Patient
Isolate
Isolation Date
A
IST401 IST404 IST406 IST408 IST405 IST410 IST411 IST480 IST487 IST488 IST492 IST493 IST4101 IST4120 IST4126 IST4193 IST4194 IST4186 IST4188 IST4192 IST4206 IST4207 IST4221 IST4238 IST4237 IST4240 a)† IST4240 b) † IST4240 c) † IST4251 IST4253 a) † IST4253 b) † IST4258 a) † IST4258 b) † IST4272 IST4276 IST4303 IST4319 IST4322 IST4391 IST4402 a) † IST4402 b) † IST4408 IST4412 IST4420 a) † IST4420 d) † IST4435 IST4497 IST4456 IST4474 IST4496 IST4485 IST4490 IST4550 IST4513 IST4517 IST4566
28/08/95 05/09/95 15/12/95 31/01/95 07/04/95 01/06/95 09/12/95 21/02/01 06/04/01 21/04/01 07/05/01 10/05/01 04/07/01 24/01/02 18/04/02 09/12/04 10/02/05 14/04/05 04/05/05 30/06/05 08/10/05 08/10/05 23/11/05 14/03/06 20/03/06 10/05/06 10/05/06 10/05/06 14/06/06 07/07/06 07/07/06 02/08/06 02/08/06 11/12/06 22/11/06 28/03/07 23/04/07 23/04/07 19/03/08 09/07/08 09/07/08 13/08/08 16/09/08 17/10/08 17/10/08 23/11/09 11/01/10 29/03/10 18/05/10 22/02/10 10/05/10 16/06/10 19/07/10 09/08/10 10/10/10 29/10/10
E F G
W
X AF
603 604 605
(*)
Burkholderia species this work
B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cenocepacia IIIB B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cepacia B. cenocepacia IIIB B. cepacia B. cepacia B. cepacia -
B. contaminans B. cenocepacia IIIB B. contaminans B. contaminans B. cenocepacia IIIB B. contaminans B. cenocepacia IIIB B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. cenocepacia IIIB B. cepacia B. contaminans B. contaminans B. cenocepacia IIIA B. contaminans B. cepacia B. cenocepacia IIIA B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. contaminans B. cenocepacia IIIA B. dolosa B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans B. contaminans
Notes: all isolates were identified by hisA gene sequence analysis; * as identified by Cunha et al. J. Clin. Microbiol. 45:1628-1633,2007; # Information in http://pubmlst.org/bcc/, partially provided by our laboratory, (-) not done; † a) b) c) and d) refer to different isolates from the species B. cenocepacia IIIA, B. contaminans, B. cepacia and B. dolosa, respectively, obtained at the same isolation date.
ST# 96 96 96 96 96 96 96 96 96 96 96 -
Table 2. Multi-Bcc-species’ infection in the seven CF patients from whom B. contaminans isolates were retrieved during 15 years of epidemiological surveillance
Patient
Gender*
Age at CF diagnosis / 1st Bcc isolation¶
A
F
3m/4m
E
M
3m/3m
F
F
10m/2y
G
F
4m/10y
W
M
5m/5m
Duration of Bcc infection¶
Co-infecting bactéria#
clinical outcome
