Microbiology Papers in Press. Published April 30, 2014 as doi:10.1099/mic.0.074203-0
1
Inhibition of co-colonising cystic fibrosis-associated pathogens by
2
Pseudomonas aeruginosa and Burkholderia multivorans
3 4 5
Running Title: Inhibition among co-colonising CF pathogens
6 7
Microbial Pathogenesis
8 9
Anne Costello1, F. Jerry Reen2, Fergal O’Gara2, 3, Máire Callaghan1, and Siobhán McClean1*
10 11
1, Centre of Microbial Host Interactions, Centre of Applied Science for Health, Institute of
12
Technology Tallaght, Old Blessington Road, Tallaght, Dublin 24, Ireland
13
2, BIOMERIT Research Centre, Department of Microbiology, University College Cork,
14
Ireland; 3, Curtin University, School of Biomedical Sciences, Perth WA 6845, Australia.
15 16 17
*Corresponding author : Siobhán McClean
18
[email protected]
19 20
# words summary: 249
21
#words in the main text 5,932
22
1
23
Summary
24
Cystic fibrosis (CF) is a recessive genetic disease characterised by chronic respiratory
25
infections and inflammation causing permanent lung damage.
26
caused by Gram negative antibiotic resistant bacterial pathogens such as Pseudomonas
27
aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen, genus
28
Pandoraea.
29
investigated. Pandoraea and Bcc both elicit potent pro-inflammatory responses which were
30
significantly greater than Ps. aeruginosa. The original aim was to examine whether
31
combinations of pro-inflammatory pathogens would further exacerbate inflammation.
32
contrast, when these pathogens were colonised in the presence of Ps. aeruginosa the pro-
33
inflammatory response was significantly decreased.
34
bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the
35
growth of B. multivorans, B. cenocepacia, P. pulmonicola and P. apista, which may be a
36
factor in its dominance as a coloniser of CF patients. Ps. aeruginosa cell free supernatant
37
also suppressed growth of these pathogens, indicating that inhibition was innate rather than a
38
response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library
39
highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of
40
B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia
41
strain, J2315.
42
aeruginosa growth. B. multivorans also inhibited B. cenocepacia and P. apista.
43
conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and
44
virulence of co-colonising CF pathogens.
Recurrent infections are
In this study, the interactions between co-colonising CF pathogens were
In
Real time PCR quantification of
B. multivorans was the only species that could significantly inhibit Ps. In
45 46
Keywords: Cystic fibrosis pathogens; co-colonisation; Burkholderia cepacia complex; genus
47
Pandoraea; Pseudomonas aeruginosa; pyoverdine.
48 2
49
INTRODUCTION
50
Cystic fibrosis (CF) is an autosomal recessive genetic disorder which is characterised by
51
chronic lung infection resulting in permanent lung damage caused by excessive
52
inflammation. Many CF pathogens are highly antibiotic resistant and these chronic infections
53
are difficult to manage in patients. A hierarchy of pathogens colonise the CF lung throughout
54
a patient’s life, beginning with Staphylococcus aureus and Haemophilus influenzae, followed
55
by Ps. aeruginosa later. This Gram negative bacterium is the most common among CF
56
patients, colonising up to 80% of adults (Lipuma, 2010). Other Gram negative pathogenic
57
bacteria, including Burkholderia cepacia complex (Bcc) and an emerging pathogen, genus
58
Pandoraea also cause opportunistic infections in CF patients, usually in later life. Bcc is a
59
diverse group of eighteen species, which impacts on the morbidity and mortality of CF
60
patients (Govan et al., 2007).
61
cenocepacia and B. multivorans comprising 85 to 90% of all Bcc infection among CF
62
patients (Drevinek & Mahenthiralingam, 2010). B. cenocepacia is widely accepted as the
63
most virulent pathogen among CF patients, resulting in the most rapid decline relative to
64
other species (Courtney et al., 2004; De Soyza et al., 2010). In the past ten years, B.
65
multivorans has been the most frequently isolated Bcc species among newly colonised CF
66
patients (Lipuma, 2010). More recently, genus Pandoraea has been implicated as the cause
67
of declining lung function and even death in some chronically colonised patients and has
68
recently been reported as a virulent pathogen with multiple mechanisms of pathogenicity
69
(Costello et al., 2011; Jorgensen et al., 2003; Stryjewski et al., 2003). Pandoraea
70
colonisation has been associated with CF and non CF patients and often occurs when the
71
patient has already succumbed to chronic colonisation with another pathogen (Schneider et
72
al., 2006). Nine species have been identified within the genus to date (Coenye et al., 2000;
73
Daneshvar et al., 2001; Sahin et al., 2011). P. apista has been associated with epidemic
The two most clinically relevant Bcc species are B.
3
74
spread and rapid patient decline (Atkinson et al., 2006; Jorgensen et al., 2003), while P.
75
pulmonicola is the most commonly identified Pandoraea species among Irish CF patients
76
(Costello et al., 2011).
77 78
It has recently been established that the CF microbiome is a complex, polymicrobial
79
environment (Sibley et al., 2011). CF patients are rarely colonised by one single pathogen but
80
by multiple pathogens with a range of possible virulence determinants which are regulated in
81
response to changes in the bacterial environment (Duan et al., 2009). The investigation of the
82
virulence of a particular bacterial species is usually carried out on single pathogens in
83
isolation; co-colonising bacteria are generally not taken into account. Competition between
84
pathogens may result in the inhibition of one co-colonising species by another in an
85
environment where the bacterial species must compete for limited nutrients. It was recently
86
shown that Ps. aeruginosa can inhibit planktonic B. cenocepacia; however, no other CF-
87
associated pathogens were examined (Bragonzi et al., 2012). Alternatively, interactions
88
between pathogens may have implications on the virulence associated with bacteria including
89
repression or complementation of the virulence of another pathogen (Lavigne et al., 2008;
90
Sibley et al., 2008). Co-colonising pathogens can interact via their quorum sensing systems,
91
for example, B. cenocepacia can utilise, and is regulated by acyl-homoserine lactones
92
(AHLs) from Ps. aeruginosa and vice versa (Lewenza et al., 2002; Riedel et al., 2001).
93
Furthermore, in the case of CF pathogens, the pro-inflammatory host responses elicited by
94
many of these contribute to hyperinflammation in the CF airways leading to irreversible
95
tissue damage (Bonfield et al., 1995; Machen, 2006).
96
cenocepacia and members of genus Pandoraea were all capable of eliciting potent pro-
97
inflammatory responses (Bamford et al., 2007; Caraher et al., 2008), we wanted to
Given that B. multivorans, B
4
98
investigate the pro-inflammatory response in the presence of more than one of these
99
pathogens.
100
In this study the interactions between Ps. aeruginosa, Burkholderia cepacia complex (Bcc)
101
and the emerging CF pathogen Pandoraea were examined. Our starting hypothesis was that
102
these pathogens would result in a further elevation in inflammation when these pathogens are
103
co-colonising.
104
induced by Ps. aeruginosa, Bcc and Pandoraea species either singly or in combination were
105
examined. The lack of an enhanced cytokine response when two pathogens colonised lung
106
cells in combination led us to examine the competition between co-colonising pathogens and
107
the factors that may contribute to the ability of Ps. aeruginosa to inhibit co-colonising
108
pathogens.
To examine this, levels of IL-6 and IL-8 secreted from lung epithelial cells
5
109
METHODS
110
Bacterial Strains. The strains used in this study are listed in Table 1. Bcc and Pandoraea
111
strains were purchased from BCCM/LMG, University of Ghent, Belgium, while Ps.
112
aeruginosa ATCC27853 was obtained from the American Type Culture Collection. PA14
113
mutant library (Liberati et al., 2006) and Pandoraea isolates were routinely grown on Tryptic
114
Soy Agar (TSA) agar or in TS broth at 37oC. Bcc and Ps. aeruginosa strains were routinely
115
grown on Luria–Bertani (LB) agar or in LB broth at 37oC.
116 117
Cytokine secretion from CF lung epithelial cells. CF bronchial epithelial (CFBE41o-) cell
118
line, is homozygous for the most common CF mutation (F508 mutation). These cells were
119
routinely cultured on coated flasks as recently described (Bevivino et al., 2012). IL-6 and IL-
120
8 secretion from CFBE41o- cells were measured using ELISA as previously described
121
(Caraher et al., 2008). CFBE41o- cells were seeded at 4 x 105 cells per well and incubated
122
for 24 hours prior to infection with bacterial strains individually for 24 hours (MOI 1:50)
123
prior to analysis of cytokine levels. For double infections, the cells were exposed to the first
124
strain (0.6 OD600; MOI 1:25) 24 hours after seeding and then the second strain (0.6 OD600;
125
MOI 25:1) a further 24 hours later, in order to model an incoming additional infection to an
126
already colonised epithelium. The infected cell cultures were then incubated an additional 24
127
hours before pro-inflammatory cytokine analysis of supernatants.
128
6
129
Determination of growth inhibition by agar diffusion assay. Agar well diffusion plates
130
were prepared with 60mls of TSA containing 2% (v/v) Tween 80 and 5% v/v of stationary
131
phase indicator cultures (Ps. aeruginosa ATCC27853, P. pulmonicola LMG18108, P. apista
132
LMG16407, B. multivorans LMG13010 or B. cenocepacia J2315). Wells (18 x 6 mm) were
133
inoculated with challenge culture (100l) or cell free supernatant (CFS, 100l) and incubated
134
for 24 hours at 37°C. After incubation, zones of inhibition and overgrowth were measured
135
using digital callipers. Three independent experiments were performed for each pair of
136
strains examined.
137
deviation.
The results are presented as the mean zone of clearance +/- standard
138 139
Quantification of bacterial growth by real time PCR. Quantification of individual strains
140
in co-cultures was determined by real time PCR. Species-specific primers were designed for
141
B. cenocepacia J2315 and B. multivorans LMG13010 against the 16S ribosomal GidB
142
methyl-transferase gene and partial 16S sequences were used to design primers for P. apista
143
LMG16407, P. pulmonicola LMG18108 and Ps. aeruginosa ATCC27853 to enable
144
quantification of a single strain in a mixed culture. Primer3 software was used for primer
145
generation and Blast software for specificity. All primers (Table 2) were 18 to 20 bp long
146
with a melting temperature of 58 to 60°C and a GC content of 40 – 60% generating products
147
of 100 to 120 bp in length. The specificity of each set of primer pairs was confirmed using
148
real time PCR. Primer efficiency for each pair was calculated using standard curves ranging
149
from 2 x 107 to 2 x 103 CFU. In order to ascertain whether one pathogen had an effect on the
150
growth or survival of the other, each culture was grown singly in 1.5 ml as control and mixed
151
in 3 ml volumes.
7
152
Log phase cultures (2 x 107 log phase CFU) of each strain were inoculated into separate and
153
mixed growth tubes. DNA extraction, from standardised cultures (2 x 107 CFU), was carried
154
out using Qiagen DNeasy blood and tissue DNA extraction kit. Real time PCR analysis was
155
carried out on 24 hour co-cultured bacterial samples and single controls. Standard curves
156
were run for each strain 2 x 107 – 2 x 103 CFU to ascertain bacterial number in single and co-
157
cultured samples. Each analysis was run in triplicate on three separate occasions.
158 159
Inhibition of bacterial growth by cell free supernatants. The effect of secreted factors on
160
pathogen growth was examined using single or co-culture CFS. Log phase cultures (2 x 107
161
CFU) were inoculated individually into 5 ml broth. Co-cultures were prepared by inoculating
162
2 x 107 CFU of each pathogen into 10 ml of LB, for 24 h, at 37°C. Bacterial cultures were
163
centrifuged and the supernatants were removed and filter sterilised (0.22 m filters). CFS
164
from either a single culture or a co-culture was added to LB at a ratio of 1:1, in a total of 3
165
ml, into which a single bacterial culture was inoculated (2 x 107 CFU). The growth of an
166
individual pathogen in its own CFS and co-culture CFS was examined. A pathogen grown in
167
CFS from a previous culture of the same species (with 1:1 broth) was taken as the control.
168
Sample tubes were incubated on shaker at 37°C for 24 hours. CFU for each sample was then
169
calculated by serial dilution and plating (in duplicate). Each test was carried out at least 3
170
times.
171
A range of dilutions of Ps. aeruginosa or B. multivorans CFS were tested against B.
172
cenocepacia J2315 growth to determine the potency of each pathogen CFS. Serial dilutions
173
of CFS (10 to 100%) were prepared in sterile water and an aliquot of 3 ml added to 3 ml of
174
fresh media. B. cenocepacia J2315 (2 x 107 CFU) was inoculated into each sample and
175
incubated with shaking at 37°C for 24 hour. B. cenocepacia J2315 CFU was determined as
176
described.
8
177
To investigate if inhibition was proteinaceous in nature, proteinase K (50 g/ml) was
178
incubated with individual CFS from each of the strains for 24 hours followed by protease
179
inhibitor prior to testing. Alternatively, CFS was heated for 15 minutes at 100°C. Growth
180
inhibition testing was then carried out as previously described.
181 182
Functional screening of the non redundant PA14 transposon mutant library. The genetic
183
factors underpinning Ps. aeruginosa antagonism of CF associated pathogens were
184
investigated by both global and targeted approaches. The complete PA14 mutant library
185
(Liberati et al., 2006) was transferred to bioassay agar plates and grown overnight at 37oC.
186
The following day, an overlay of B. cenocepacia J2315 in 0.3% (w/v) soft agar was added to
187
the plates and incubated overnight at 37oC. Zones of growth surrounding colonies were
188
subsequently marked and the mutants involved were identified in the mutant library database.
189
In tandem with this approach, mutants were selected for singular analysis on the basis of their
190
role in virulence and pathogenesis in Ps. aeruginosa. Singular analysis of all candidate
191
mutants was performed using CFS, which was extracted from overnight cultures by pelleting
192
bacteria for 10 minutes before sterile filtering with 0.2 µm filters. B. cenocepacia J2315
193
overnight culture was diluted to an OD600nm of 0.1 with sterile broth and combined 1:1 with
194
mutant CFS in a 100 well Bioscreen plate, to a total volume of 200 µl. The OD600nm of each
195
plate was read every 15 minutes for 42 hours on a Bioscreen analyser (Oy Growth Curves Ab
196
Ltd, Helsinki). To investigate whether the antagonistic role of PQS/HHQ was direct or
197
indirect, the growth of B. cenocepacia J2315 was also monitored in the presence of synthetic
198
HHQ and PQS (10 µM). Each analysis was performed in duplicate at least three times.
9
199
Effect of pyoverdine on growth of CF pathogens.
200
pyoverdine on growth of the other species was examined by inoculating log phase cultures (2
201
x 107 CFU) into wells of a 96 well plate, and incubating with a range of concentrations of
202
desferripyoverdine (0.39 - 25 g/ml, EMC Microcollections GmBH) in duplicate and
203
measurement of OD600 after 24 hour. The effect of proteinase K or heat treatment on the
204
inhibitory capacity of pyoverdine was assessed by incubation of 40 µM pyoverdine with
205
50ug/ml proteinase K for 24 hours followed by addition of protease inhibitor or incubation at
206
100°C for 15 minutes. Inhibition studies were carried out as above for untreated pyoverdine
207
(MIC) at a final pyoverdine concentration of 20 µM. Each analysis was performed in
208
duplicate at least three times. Pyoverdine was quantified from Ps. aeruginosa ATCC27853
209
as previously described (Baysse et al., 2000) by spectrophotometric analysis of replicate CFS
210
to obtain the max and then the absorption measured at 380nm. Pyoverdine was quantified
211
against a pyoverdine standard curve.
The role of Ps. aeruginosa
212 213
Statistical analysis. Statistical analysis of variance was carried out using Minitab software
214
with 95% confidence interval.
215
10
216
RESULTS
217
Analysis of the pro-inflammatory cytokine response of CFBE41o- cells elicited by Ps.
218
aeruginosa, Bcc and Pandoraea during single and co-infection.
219
CF pathogens and in particular, Bcc and Pandoraea strains have been shown to elicit
220
considerable pro-inflammatory responses in lung epithelial cells (Caraher et al., 2008; Kaza
221
et al., 2010). Combinations of pro-inflammatory co-colonising pathogens were examined to
222
investigate whether dual infections exacerbated the pro-inflammatory effects. As previously
223
described, secretion of inflammatory cytokines IL-8 and IL-6 (Fig. 1) from CFBE41o- cells
224
indicated a potent inflammatory response was elicited following infection with either
225
Pandoraea or Bcc strains which was greater than that of Ps. aeruginosa, (P
1
Inhibition of co-colonising cystic fibrosis-associated pathogens by
2
Pseudomonas aeruginosa and Burkholderia multivorans
3 4 5
Running Title: Inhibition among co-colonising CF pathogens
6 7
Microbial Pathogenesis
8 9
Anne Costello1, F. Jerry Reen2, Fergal O’Gara2, 3, Máire Callaghan1, and Siobhán McClean1*
10 11
1, Centre of Microbial Host Interactions, Centre of Applied Science for Health, Institute of
12
Technology Tallaght, Old Blessington Road, Tallaght, Dublin 24, Ireland
13
2, BIOMERIT Research Centre, Department of Microbiology, University College Cork,
14
Ireland; 3, Curtin University, School of Biomedical Sciences, Perth WA 6845, Australia.
15 16 17
*Corresponding author : Siobhán McClean
18
[email protected]
19 20
# words summary: 249
21
#words in the main text 5,932
22
1
23
Summary
24
Cystic fibrosis (CF) is a recessive genetic disease characterised by chronic respiratory
25
infections and inflammation causing permanent lung damage.
26
caused by Gram negative antibiotic resistant bacterial pathogens such as Pseudomonas
27
aeruginosa, Burkholderia cepacia complex (Bcc) and the emerging pathogen, genus
28
Pandoraea.
29
investigated. Pandoraea and Bcc both elicit potent pro-inflammatory responses which were
30
significantly greater than Ps. aeruginosa. The original aim was to examine whether
31
combinations of pro-inflammatory pathogens would further exacerbate inflammation.
32
contrast, when these pathogens were colonised in the presence of Ps. aeruginosa the pro-
33
inflammatory response was significantly decreased.
34
bacterial DNA from mixed cultures indicated that Ps. aeruginosa significantly inhibited the
35
growth of B. multivorans, B. cenocepacia, P. pulmonicola and P. apista, which may be a
36
factor in its dominance as a coloniser of CF patients. Ps. aeruginosa cell free supernatant
37
also suppressed growth of these pathogens, indicating that inhibition was innate rather than a
38
response to the presence of a competitor. Screening of a Ps. aeruginosa mutant library
39
highlighted a role for quorum sensing and pyoverdine biosynthesis genes in the inhibition of
40
B. cenocepacia. Pyoverdine was confirmed to contribute to the inhibition of B. cenocepacia
41
strain, J2315.
42
aeruginosa growth. B. multivorans also inhibited B. cenocepacia and P. apista.
43
conclusion, both Ps. aeruginosa and B. multivorans are capable of suppressing growth and
44
virulence of co-colonising CF pathogens.
Recurrent infections are
In this study, the interactions between co-colonising CF pathogens were
In
Real time PCR quantification of
B. multivorans was the only species that could significantly inhibit Ps. In
45 46
Keywords: Cystic fibrosis pathogens; co-colonisation; Burkholderia cepacia complex; genus
47
Pandoraea; Pseudomonas aeruginosa; pyoverdine.
48 2
49
INTRODUCTION
50
Cystic fibrosis (CF) is an autosomal recessive genetic disorder which is characterised by
51
chronic lung infection resulting in permanent lung damage caused by excessive
52
inflammation. Many CF pathogens are highly antibiotic resistant and these chronic infections
53
are difficult to manage in patients. A hierarchy of pathogens colonise the CF lung throughout
54
a patient’s life, beginning with Staphylococcus aureus and Haemophilus influenzae, followed
55
by Ps. aeruginosa later. This Gram negative bacterium is the most common among CF
56
patients, colonising up to 80% of adults (Lipuma, 2010). Other Gram negative pathogenic
57
bacteria, including Burkholderia cepacia complex (Bcc) and an emerging pathogen, genus
58
Pandoraea also cause opportunistic infections in CF patients, usually in later life. Bcc is a
59
diverse group of eighteen species, which impacts on the morbidity and mortality of CF
60
patients (Govan et al., 2007).
61
cenocepacia and B. multivorans comprising 85 to 90% of all Bcc infection among CF
62
patients (Drevinek & Mahenthiralingam, 2010). B. cenocepacia is widely accepted as the
63
most virulent pathogen among CF patients, resulting in the most rapid decline relative to
64
other species (Courtney et al., 2004; De Soyza et al., 2010). In the past ten years, B.
65
multivorans has been the most frequently isolated Bcc species among newly colonised CF
66
patients (Lipuma, 2010). More recently, genus Pandoraea has been implicated as the cause
67
of declining lung function and even death in some chronically colonised patients and has
68
recently been reported as a virulent pathogen with multiple mechanisms of pathogenicity
69
(Costello et al., 2011; Jorgensen et al., 2003; Stryjewski et al., 2003). Pandoraea
70
colonisation has been associated with CF and non CF patients and often occurs when the
71
patient has already succumbed to chronic colonisation with another pathogen (Schneider et
72
al., 2006). Nine species have been identified within the genus to date (Coenye et al., 2000;
73
Daneshvar et al., 2001; Sahin et al., 2011). P. apista has been associated with epidemic
The two most clinically relevant Bcc species are B.
3
74
spread and rapid patient decline (Atkinson et al., 2006; Jorgensen et al., 2003), while P.
75
pulmonicola is the most commonly identified Pandoraea species among Irish CF patients
76
(Costello et al., 2011).
77 78
It has recently been established that the CF microbiome is a complex, polymicrobial
79
environment (Sibley et al., 2011). CF patients are rarely colonised by one single pathogen but
80
by multiple pathogens with a range of possible virulence determinants which are regulated in
81
response to changes in the bacterial environment (Duan et al., 2009). The investigation of the
82
virulence of a particular bacterial species is usually carried out on single pathogens in
83
isolation; co-colonising bacteria are generally not taken into account. Competition between
84
pathogens may result in the inhibition of one co-colonising species by another in an
85
environment where the bacterial species must compete for limited nutrients. It was recently
86
shown that Ps. aeruginosa can inhibit planktonic B. cenocepacia; however, no other CF-
87
associated pathogens were examined (Bragonzi et al., 2012). Alternatively, interactions
88
between pathogens may have implications on the virulence associated with bacteria including
89
repression or complementation of the virulence of another pathogen (Lavigne et al., 2008;
90
Sibley et al., 2008). Co-colonising pathogens can interact via their quorum sensing systems,
91
for example, B. cenocepacia can utilise, and is regulated by acyl-homoserine lactones
92
(AHLs) from Ps. aeruginosa and vice versa (Lewenza et al., 2002; Riedel et al., 2001).
93
Furthermore, in the case of CF pathogens, the pro-inflammatory host responses elicited by
94
many of these contribute to hyperinflammation in the CF airways leading to irreversible
95
tissue damage (Bonfield et al., 1995; Machen, 2006).
96
cenocepacia and members of genus Pandoraea were all capable of eliciting potent pro-
97
inflammatory responses (Bamford et al., 2007; Caraher et al., 2008), we wanted to
Given that B. multivorans, B
4
98
investigate the pro-inflammatory response in the presence of more than one of these
99
pathogens.
100
In this study the interactions between Ps. aeruginosa, Burkholderia cepacia complex (Bcc)
101
and the emerging CF pathogen Pandoraea were examined. Our starting hypothesis was that
102
these pathogens would result in a further elevation in inflammation when these pathogens are
103
co-colonising.
104
induced by Ps. aeruginosa, Bcc and Pandoraea species either singly or in combination were
105
examined. The lack of an enhanced cytokine response when two pathogens colonised lung
106
cells in combination led us to examine the competition between co-colonising pathogens and
107
the factors that may contribute to the ability of Ps. aeruginosa to inhibit co-colonising
108
pathogens.
To examine this, levels of IL-6 and IL-8 secreted from lung epithelial cells
5
109
METHODS
110
Bacterial Strains. The strains used in this study are listed in Table 1. Bcc and Pandoraea
111
strains were purchased from BCCM/LMG, University of Ghent, Belgium, while Ps.
112
aeruginosa ATCC27853 was obtained from the American Type Culture Collection. PA14
113
mutant library (Liberati et al., 2006) and Pandoraea isolates were routinely grown on Tryptic
114
Soy Agar (TSA) agar or in TS broth at 37oC. Bcc and Ps. aeruginosa strains were routinely
115
grown on Luria–Bertani (LB) agar or in LB broth at 37oC.
116 117
Cytokine secretion from CF lung epithelial cells. CF bronchial epithelial (CFBE41o-) cell
118
line, is homozygous for the most common CF mutation (F508 mutation). These cells were
119
routinely cultured on coated flasks as recently described (Bevivino et al., 2012). IL-6 and IL-
120
8 secretion from CFBE41o- cells were measured using ELISA as previously described
121
(Caraher et al., 2008). CFBE41o- cells were seeded at 4 x 105 cells per well and incubated
122
for 24 hours prior to infection with bacterial strains individually for 24 hours (MOI 1:50)
123
prior to analysis of cytokine levels. For double infections, the cells were exposed to the first
124
strain (0.6 OD600; MOI 1:25) 24 hours after seeding and then the second strain (0.6 OD600;
125
MOI 25:1) a further 24 hours later, in order to model an incoming additional infection to an
126
already colonised epithelium. The infected cell cultures were then incubated an additional 24
127
hours before pro-inflammatory cytokine analysis of supernatants.
128
6
129
Determination of growth inhibition by agar diffusion assay. Agar well diffusion plates
130
were prepared with 60mls of TSA containing 2% (v/v) Tween 80 and 5% v/v of stationary
131
phase indicator cultures (Ps. aeruginosa ATCC27853, P. pulmonicola LMG18108, P. apista
132
LMG16407, B. multivorans LMG13010 or B. cenocepacia J2315). Wells (18 x 6 mm) were
133
inoculated with challenge culture (100l) or cell free supernatant (CFS, 100l) and incubated
134
for 24 hours at 37°C. After incubation, zones of inhibition and overgrowth were measured
135
using digital callipers. Three independent experiments were performed for each pair of
136
strains examined.
137
deviation.
The results are presented as the mean zone of clearance +/- standard
138 139
Quantification of bacterial growth by real time PCR. Quantification of individual strains
140
in co-cultures was determined by real time PCR. Species-specific primers were designed for
141
B. cenocepacia J2315 and B. multivorans LMG13010 against the 16S ribosomal GidB
142
methyl-transferase gene and partial 16S sequences were used to design primers for P. apista
143
LMG16407, P. pulmonicola LMG18108 and Ps. aeruginosa ATCC27853 to enable
144
quantification of a single strain in a mixed culture. Primer3 software was used for primer
145
generation and Blast software for specificity. All primers (Table 2) were 18 to 20 bp long
146
with a melting temperature of 58 to 60°C and a GC content of 40 – 60% generating products
147
of 100 to 120 bp in length. The specificity of each set of primer pairs was confirmed using
148
real time PCR. Primer efficiency for each pair was calculated using standard curves ranging
149
from 2 x 107 to 2 x 103 CFU. In order to ascertain whether one pathogen had an effect on the
150
growth or survival of the other, each culture was grown singly in 1.5 ml as control and mixed
151
in 3 ml volumes.
7
152
Log phase cultures (2 x 107 log phase CFU) of each strain were inoculated into separate and
153
mixed growth tubes. DNA extraction, from standardised cultures (2 x 107 CFU), was carried
154
out using Qiagen DNeasy blood and tissue DNA extraction kit. Real time PCR analysis was
155
carried out on 24 hour co-cultured bacterial samples and single controls. Standard curves
156
were run for each strain 2 x 107 – 2 x 103 CFU to ascertain bacterial number in single and co-
157
cultured samples. Each analysis was run in triplicate on three separate occasions.
158 159
Inhibition of bacterial growth by cell free supernatants. The effect of secreted factors on
160
pathogen growth was examined using single or co-culture CFS. Log phase cultures (2 x 107
161
CFU) were inoculated individually into 5 ml broth. Co-cultures were prepared by inoculating
162
2 x 107 CFU of each pathogen into 10 ml of LB, for 24 h, at 37°C. Bacterial cultures were
163
centrifuged and the supernatants were removed and filter sterilised (0.22 m filters). CFS
164
from either a single culture or a co-culture was added to LB at a ratio of 1:1, in a total of 3
165
ml, into which a single bacterial culture was inoculated (2 x 107 CFU). The growth of an
166
individual pathogen in its own CFS and co-culture CFS was examined. A pathogen grown in
167
CFS from a previous culture of the same species (with 1:1 broth) was taken as the control.
168
Sample tubes were incubated on shaker at 37°C for 24 hours. CFU for each sample was then
169
calculated by serial dilution and plating (in duplicate). Each test was carried out at least 3
170
times.
171
A range of dilutions of Ps. aeruginosa or B. multivorans CFS were tested against B.
172
cenocepacia J2315 growth to determine the potency of each pathogen CFS. Serial dilutions
173
of CFS (10 to 100%) were prepared in sterile water and an aliquot of 3 ml added to 3 ml of
174
fresh media. B. cenocepacia J2315 (2 x 107 CFU) was inoculated into each sample and
175
incubated with shaking at 37°C for 24 hour. B. cenocepacia J2315 CFU was determined as
176
described.
8
177
To investigate if inhibition was proteinaceous in nature, proteinase K (50 g/ml) was
178
incubated with individual CFS from each of the strains for 24 hours followed by protease
179
inhibitor prior to testing. Alternatively, CFS was heated for 15 minutes at 100°C. Growth
180
inhibition testing was then carried out as previously described.
181 182
Functional screening of the non redundant PA14 transposon mutant library. The genetic
183
factors underpinning Ps. aeruginosa antagonism of CF associated pathogens were
184
investigated by both global and targeted approaches. The complete PA14 mutant library
185
(Liberati et al., 2006) was transferred to bioassay agar plates and grown overnight at 37oC.
186
The following day, an overlay of B. cenocepacia J2315 in 0.3% (w/v) soft agar was added to
187
the plates and incubated overnight at 37oC. Zones of growth surrounding colonies were
188
subsequently marked and the mutants involved were identified in the mutant library database.
189
In tandem with this approach, mutants were selected for singular analysis on the basis of their
190
role in virulence and pathogenesis in Ps. aeruginosa. Singular analysis of all candidate
191
mutants was performed using CFS, which was extracted from overnight cultures by pelleting
192
bacteria for 10 minutes before sterile filtering with 0.2 µm filters. B. cenocepacia J2315
193
overnight culture was diluted to an OD600nm of 0.1 with sterile broth and combined 1:1 with
194
mutant CFS in a 100 well Bioscreen plate, to a total volume of 200 µl. The OD600nm of each
195
plate was read every 15 minutes for 42 hours on a Bioscreen analyser (Oy Growth Curves Ab
196
Ltd, Helsinki). To investigate whether the antagonistic role of PQS/HHQ was direct or
197
indirect, the growth of B. cenocepacia J2315 was also monitored in the presence of synthetic
198
HHQ and PQS (10 µM). Each analysis was performed in duplicate at least three times.
9
199
Effect of pyoverdine on growth of CF pathogens.
200
pyoverdine on growth of the other species was examined by inoculating log phase cultures (2
201
x 107 CFU) into wells of a 96 well plate, and incubating with a range of concentrations of
202
desferripyoverdine (0.39 - 25 g/ml, EMC Microcollections GmBH) in duplicate and
203
measurement of OD600 after 24 hour. The effect of proteinase K or heat treatment on the
204
inhibitory capacity of pyoverdine was assessed by incubation of 40 µM pyoverdine with
205
50ug/ml proteinase K for 24 hours followed by addition of protease inhibitor or incubation at
206
100°C for 15 minutes. Inhibition studies were carried out as above for untreated pyoverdine
207
(MIC) at a final pyoverdine concentration of 20 µM. Each analysis was performed in
208
duplicate at least three times. Pyoverdine was quantified from Ps. aeruginosa ATCC27853
209
as previously described (Baysse et al., 2000) by spectrophotometric analysis of replicate CFS
210
to obtain the max and then the absorption measured at 380nm. Pyoverdine was quantified
211
against a pyoverdine standard curve.
The role of Ps. aeruginosa
212 213
Statistical analysis. Statistical analysis of variance was carried out using Minitab software
214
with 95% confidence interval.
215
10
216
RESULTS
217
Analysis of the pro-inflammatory cytokine response of CFBE41o- cells elicited by Ps.
218
aeruginosa, Bcc and Pandoraea during single and co-infection.
219
CF pathogens and in particular, Bcc and Pandoraea strains have been shown to elicit
220
considerable pro-inflammatory responses in lung epithelial cells (Caraher et al., 2008; Kaza
221
et al., 2010). Combinations of pro-inflammatory co-colonising pathogens were examined to
222
investigate whether dual infections exacerbated the pro-inflammatory effects. As previously
223
described, secretion of inflammatory cytokines IL-8 and IL-6 (Fig. 1) from CFBE41o- cells
224
indicated a potent inflammatory response was elicited following infection with either
225
Pandoraea or Bcc strains which was greater than that of Ps. aeruginosa, (P
