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Sortase A confers protection against Streptococcus pneumoniae in 1
mice. 2
3
Claudia Gianfaldoni, Silvia Maccari, Laura Pancotto, Giacomo Rossi 1, Markus 4
Hilleringmann, Werner Pansegrau, Antonia Sinisi, Monica Moschioni, Vega 5
Masignani, Rino Rappuoli, Giuseppe Del Giudice, Paolo Ruggiero *. 6
Novartis Vaccines and Diagnostics s.r.l., Research Center, Siena, Italy 7
1 University of Camerino, Dept. of Veterinary Sciences, Matelica, Italy 8
9
10
* corresponding author: Paolo Ruggiero, Novartis Vaccines and Diagnostics s.r.l., 11
Research Center, I-53100 Siena, Italy. Tel +39.0577.243234. Fax +39.0577.243564. 12
E-mail [email protected] 13
14
15
16
17
Running title: Streptococcus pneumoniae Sortase A is protective in mice 18
19
Keywords: Streptococcus pneumoniae, pneumococcus, vaccine, Sortase A20
Copyright © 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01516-08 IAI Accepts, published online ahead of print on 11 May 2009
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ABSTRACT 21
Streptococcus pneumoniae Sortase A (SrtA) is a transpeptidase highly conserved 22
among pneumococcal strains, which involvement in adhesion/colonization has been 23
reported. We found that intraperitoneal immunization with recombinant SrtA 24
conferred protection to mice against S. pneumoniae intraperitoneal challenge, and that 25
passive transfer of immune serum before intraperitoneal challenge was also 26
protective. Moreover, in the intranasal challenge model, we observed a significant 27
reduction of bacteremia when mice were intraperitoneally immunized with SrtA, 28
while a moderate decrease of lung infection was achieved by intranasal 29
immunization, even though no influence on nasopharynx colonization was seen. 30
Taken together, our results suggest that SrtA is a good candidate for inclusion in a 31
multi-component, protein-based, pneumococcal vaccine. 32
33
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INTRODUCTION 34
Streptococcus pneumoniae colonizes the nasopharynx of humans and represents a 35
leading cause of severe diseases such as otitis media, pneumonia, and meningitis. S. 36
pneumoniae is one of the major causes of bacterial pneumonia in developing 37
countries (19). It is estimated that each year nearly 1 million children worldwide die 38
because of pneumococcal diseases (10). Besides children, groups at high risk of 39
pneumococcal infection are immunocompromised subjects and elderly, in which high 40
case fatality rate is also observed. The last decades have seen the investigation on 41
protein antigens growing up and several protein candidates to be proposed for a 42
vaccine against S. pneumoniae (2), to overcome the problems inherent to the currently 43
available polysaccharide-based vaccines. In fact, the 23-valent polysaccharide 44
pneumococcal vaccine (PPV) is not effective in children under 2 years of age, whose 45
immune system is unable to mount the T-independent response to polysaccharides. 46
On the other hand, the 7-valent polysaccharide conjugate vaccine (PCV), although 47
efficacious, induces serotype replacement (5, 20). Moreover, while more than 90 S. 48
pneumoniae serotypes are presently known, both PPV and PCV are effective only 49
against the serotypes included in the vaccine. Efforts in identifying new S. 50
pneumoniae factors that play a role in colonization and pathogenesis may contribute 51
to indicate possible targets of either new therapeutic agents or vaccines. 52
Sortase A (SrtA) is a membrane-anchored transpeptidase expressed by Gram-positive 53
bacteria (12). The role of SrtA in processing of sorting signals at the LPXTG motif to 54
anchor surface proteins to the cell wall envelope was firstly described in 55
Staphylococcus aureus (21), in which isogenic SrtA mutation resulted in strongly 56
reduced ability to infect animals (13, 23). SrtA has been shown to participate in 57
colonization and/or pathogenesis in several Streptococcus species (1, 6, 8, 22, 24). 58
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S. pneumoniae SrtA has been described to play a role in adhesion to human 59
pharyngeal cells in vitro (7), in nasopharyngeal colonization in chinchilla (3), and in 60
pneumonia, bacteremia and nasopharyngeal colonization in murine models (15). 61
Although SrtA seems to be dispensable in pilus biogenesis, its possible role has been 62
very recently proposed in repressing pilus islet expression (9). SrtA has been found to 63
be widely expressed among S. pneumoniae isolates and highly conserved, with DNA 64
identity of 99-100% (15). Although all of these findings suggest that pneumococcal 65
SrtA might be useful as a protein vaccine, to the best of our knowledge no data have 66
been provided so far on protective efficacy afforded by SrtA immunization in animal 67
models. Thus, we investigated the protective role of SrtA in murine models of S. 68
pneumoniae infection. 69
MATERIALS AND METHODS 70
Protein expression and purification. 71
The gene portion corresponding to the amino acid sequence 30-247 of the SrtA of the 72
pneumococcal strain D39 (218 amino acids, calculated molecular mass 24.81 kDa), 73
was cloned into the pET151/D-TOPO vector (Invitrogen). Recombinant SrtA was 74
then expressed in Escherichia coli with 6-Histidine tag and purified from bacterial 75
lysate by affinity chromatography on His-Trap high-performance columns (GE 76
Healthcare), equilibrated and eluted following the manufacturer’s instructions. 77
Finally, the SrtA, obtained in soluble form, was dialyzed against saline. The protein 78
purity was higher than 90%, as evaluated by SDS-PAGE gel scanning densitometry. 79
Bacterial strains and culture. 80
The following S. pneumoniae strains were used: TIGR4 (serotype 4), D39 (serotype 81
2), and 35B-SME15 (serotype 35B). Bacteria were grown 24 hrs at 37°C under 5% 82
CO2 atmosphere on Tryptic Soy Agar (TSA, Difco) plates containing colistine 10 83
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mg/l, oxolinic acid 5 mg/l, and 5% defibrinated sheep blood. Bacteria were then 84
harvested and used to inoculate liquid cultures in Tryptic Soy Broth (TSB, Difco). 85
Liquid cultures were carried out statically at 37°C under 5% CO2 atmosphere until 86
reaching A600 = 0.25. Bacteria were then harvested by centrifugation at 3,500 rcf for 87
20 min at 4°C, and either used for challenge or frozen at -80°C in TSB containing 88
20% glycerol and 20% Fetal Bovine Serum. The value of CFU/ml for each bacterial 89
preparation was determined by plating culture aliquots at serial dilutions and counting 90
CFU after 24 hrs of culture carried out as above. 91
Animal treatments and evaluation of infection and mortality. 92
Animal experiments were done in compliance with the current Italian law and 93
approved by the internal Animal Ethics Committee. 94
Female, 6-week-old, specific pathogen-free BALB/c mice (Charles River) received 95
three intraperitoneal administrations, two weeks apart, of 20 µg of SrtA along with 96
Freund’s adjuvant. Controls received the same course of saline plus adjuvant. In other 97
experiments, mice were immunized intranasally with the same schedule. For 98
intranasal immunization, animals were anesthetized by intraperitoneal injection of 99
xylazine and ketamine (0.1 and 0.01 mg/g of body weight, respectively), then 100
received into the nostrils 10 µl of saline containing 20 µg of SrtA along with 2 µg of 101
LTK63, the non-toxic mutant of heat-labile E. coli enterotoxin, as mucosal adjuvant 102
(16). Two weeks after the completion of the immunization cycle, samples of sera 103
were obtained for evaluation of antibody response. 104
Three weeks after the last immunization, the animals were challenged 105
intraperitoneally with one of the following S. pneumoniae strains: TIGR4, 1.4 x 102 106
CFU/mouse; D39, 103 CFU/mouse; 35B-SME15, 7 x 10
3 CFU/mouse. For 107
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intraperitoneal challenge frozen bacteria were thawed and brought to the desired 108
concentration in saline. 109
Bacteremia was evaluated in blood samples taken 24 hrs post-challenge and plated on 110
blood-agar plates at serial dilutions made in saline, starting from 1:5 dilution. After 111
24 hrs of culture, carried out as above, CFU were counted and the CFU/ml of blood 112
calculated. Mortality was observed for 10 days post-challenge, twice per day for the 113
first 4 days, and then daily. For the passive protection experiment, each mouse 114
received intraperitoneally 0.3 ml of anti-SrtA rabbit serum 15 min before TIGR4 115
intraperitoneal challenge. Controls received 0.3 ml of normal rabbit serum. 116
For intranasal challenge, animals, anesthetized by xylazine and ketamine as above, 117
received into the nostrils 50 µl of bacterial suspension containing 107 CFU of freshly 118
harvested TIGR4. Two days after intranasal challenge, animals were sacrificed and 119
samples of blood, nasal wash and lung wash were taken. Nasal washes were 120
obtained by flushing 0.5 ml saline through the nostrils. Lung washes were obtained in 121
a final volume of 1 ml by two cycles of injection/extraction of 0.5 ml saline into the 122
lungs. Samples were then plated at serial dilutions, starting from 1:5 dilution (blood) 123
or undiluted (nasal and lung washes), for CFU count as above, and for cytological 124
analysis (nasal and lung washes) as detailed below. 125
The limit of detection of bacteria in blood was 125 CFU/ml, while in nasal and lung 126
washes was 25 CFU/ml. 127
Cytological analysis. 128
Aliquots of nasal and lung washes (undiluted and diluted 1:5 in saline, respectively), 129
taken, as above described, two days after TIGR4 challenge from mice previously 130
immunized intranasally, were subjected to cytological analysis. Samples were 131
cytocentrifuged onto a microscopic slide by a Shandon Cytospin 4 (Thermo Electron 132
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Corp.), and then subjected to May Grünwald - Giemsa staining. Macrophages and 133
polymorphonucleates (PMN) were counted for each mouse as the mean of three 134
different microscope fields at 400x magnification, and the mean for each group 135
calculated. 136
Antibody titer evaluation. 137
Quantification of Immunoglobulin G (IgG) or A (IgA) was made by enzyme-linked 138
immunosorbent assay (ELISA) in mouse sera. Single sera were analyzed for 139
immunized mice, while control sera were analyzed in pool. 140
Serial dilutions of sera were dispensed in Maxisorp 96-well plates (Nalge Nunc Int.) 141
coated with recombinant SrtA at 0.2 µg/well. Antibody binding was detected by 142
alkaline phosphatase-conjugated anti-mouse IgG or IgA (Southern Biotechnology 143
Ass.), followed by the substrate p-nitrophenyl-phosphate (Sigma). Absorbance was 144
measured at 405 nm. Antibody titer was expressed as the reciprocal of serum dilution 145
giving A405 = 1, and reported as mean Log 10 titer ± Standard Deviation. 146
Statistical analysis. 147
Data of antibody titers, bacteremia, and mortality course were analyzed by one-tailed 148
Mann-Whitney U test. Data of cytological analysis were analyzed by two-tailed 149
Mann-Whitney U test. Values of P ≤ 0.05 were considered significant. 150
RESULTS AND DISCUSSION 151
SrtA is immunogenic in mice. 152
Specific IgG response was quantified in sera of mice immunized intraperitoneally 153
with SrtA (N = 46). Good IgG response was detected in all immunized animals, with 154
a mean Log10 titer of 4.87 ± 0.38, corresponding to a serum dilution of about 155
1:75,000. The IgG titer quantified in immunized mice was significantly higher (P < 156
0.0001) than the background value of 2.06 ± 0.42, detected in control sera (N = 40). 157
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Specific IgG response was also measured in the serum from mice immunized 158
intranasally with SrtA (N = 8). The IgG mean Log10 titer resulted 3.23 ± 0.81, more 159
than 40 times lower than that obtained by intraperitoneal immunization, and with 160
higher variability, including two non responders. In the control sera (N = 8), an IgG 161
mean Log10 titer of 2.18 ± 0.08 was detected (P = 0.032; P = 0.0003 excluding the 162
two non responders). In the same sera specific IgA mean Log10 titer was 1.41 ± 0.34, 163
while control group gave an IgA mean Log10 titer of 0.93 ± 0.12 (P = 0.002; P = 164
0.001 excluding the two non responders). Indeed, mucosal immunization is expected 165
to elicit measurable amount of IgA isotype, even though it can be very low as in this 166
case. 167
Immunization with SrtA protects mice against intraperitoneal challenge. 168
Results of protection afforded by SrtA against intraperitoneal challenge are shown in 169
Figure 1 and 2. 170
After challenge with the TIGR4 strain, 15 out of 16 control mice were bacteremic, 171
and only 6 survived at 10 days. In contrast, bacteremia was detectable only in 4 out of 172
16 mice immunized with SrtA, with a geometric mean about 2 Logs lower than that 173
of the control group (Figure 1A, 3.3 x 102 vs. 4.1 x 10
4 CFU/ml, P = 0.00005). 174
Survival of SrtA-immunized mice was significantly increased (P = 0.023), with 11 175
out of 16 mice alive at 10 days (Figure 1B). 176
When challenge was carried out with the strain D39, all control mice (N = 8) were 177
bacteremic and died within 3 days post-challenge, while in SrtA-immunized group (N 178
= 7) a 40-fold reduction of bacteremia was achieved (Figure 1C, 2.7 x 103 vs. 1.1 x 179
105 CFU/ml, P = 0.047) and survival significantly increased (Figure 1D, P = 0.005). 180
Using the strain 35B-SME15 for the challenge, about 15-fold reduction of bacteremia 181
was achieved in immunized mice (N = 16) as compared with controls (N = 24) 182
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(Figure 1E, 2.5 x 104 vs. 3.6 x 10
5 CFU/ml, P = 0.010), and a good trend of survival 183
increasing was observed, although not significant (Figure 1F, P = 0.121). It must be 184
pointed out that, in our experimental model of infection, 35B-SME15 strain 185
efficiently infects mice, as judged on the basis of bacteremia quantified 24 hrs post-186
challenge; on the other hand, it requires relatively high doses to cause mortality, with 187
high variability, as observed in the experiments done to define the challenging dose 188
with this strain (data not shown). 189
To investigate the involvement of antibodies in the protection conferred by SrtA, an 190
experiment of passive protection against strain TIGR4 was carried out. Significant 191
reduction of bacteremia was achieved, the group receiving the anti-SrtA serum (N = 192
24) having over 20-fold reduced CFU/ml as compared with control group (N = 24) 193
that had received the same amount of normal rabbit serum (Figure 2A, 2.1 x 102 vs. 194
4.6 x 103 CFU/ml, P = 0.012), and a corresponding survival increasing was observed, 195
(Figure 2B, P = 0. 016). 196
Immunization with SrtA protects mice against intranasal challenge at either 197
systemic or mucosal level depending on the immunization route. 198
Intraperitoneal or intranasal immunization route conferred protection against 199
intranasal challenge at different levels. Intraperitoneal immunization resulted in about 200
50-fold decrease of bacteremia (Figure 3A , 2.2 x 103 vs. 1.3 x 10
5 CFU/ml, P = 201
0.014; N = 8 for both immunized and ctrl group), but did not influence 202
nasopharyngeal carriage or lung infection (Figure 3B and C, 1.4 x 105 vs. 1.3 x 10
5 203
and 2.7 x 103 vs. 4.3 x 10
3 CFU/ml, respectively). Conversely, intranasal 204
immunization did not affect bacteremia or nasopharyngeal carriage (Figure 3D and E, 205
2.3 x 103 vs. 2.8 x 10
3 and 1.0 x 10
5 vs. 1.4 x 10
5 CFU/ml, respectively; N = 8 for 206
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both immunized and ctrl group), while was able to decrease the lung infection of 207
about one Log (Figure 3F, 1.4 x 104 vs. 1.5 x 10
5 CFU/ml, P = 0.032). 208
The results of the cytological analysis of nasal and lung washes samples, taken after 209
challenge from mice immunizad intranasally, are summarized in Table 1. In the nasal 210
washes no difference in macrophage or PMN count was observed between 211
immunized and control group, while in the lung washes the immunized group showed 212
a trend of reduction of macrophage number (P = 0.094) and a very marked decrease 213
of PMN (P = 0.0003). 214
The finding that inflammatory cells were decreased in the lungs of immunized mice 215
reinforces the observation that in the same site the number of detectable bacteria were 216
decreased, suggesting that mice immunized intranasally with SrtA cleared bacteria 217
from the lower respiratory tract with faster kinetics than controls. These results 218
suggest that at least partial protection against pneumonia can be afforded by mucosal 219
immunization with SrtA. Conversely, immunization with SrtA did not afford 220
protection against nasopharynx colonization. The relatively low antibody titers 221
measured after intranasal immunization might indicate that an optimal immune 222
response was not achieved. Therefore, additional work to enhance protection, 223
including optimization of adjuvants and of the immunization schedule and routes, 224
could lead to an improved efficacy. On the other hand, our results could apparently 225
suggest a scarce relevance of SrtA to colonization in the model used. A role of SrtA 226
in colonization has been already described (15): however, in that study different 227
bacterial and mouse strains were used, and, more importantly, the results were based 228
on the evaluation of colonization ability of pneumococcal SrtA deletion mutants. Our 229
results do not exclude the possible relevance of SrtA to colonization, while suggest 230
that immunity against SrtA might be scarcely or not effective at this stage. In other 231
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words, in the model we used, SrtA might be scarcely or not accessible to the immune 232
system effectors during colonization, while it might become more accessible during 233
lung infection and invasion. 234
CONCLUSIONS. 235
SrtA has been reported to be highly conserved among S. pneumoniae strains (15), and 236
to be involved in adhesion/colonization (3, 7, 15). Results reported here clearly show 237
that SrtA is immunogenic in mice and is able to confer protection in the mouse model 238
of intraperitoneal challenge against three different S. pneumoniae strains (TIGR4, 239
D39, 35B-SME15) by active immunization with the recombinant protein. Moreover, 240
passive transfer of immune serum conferred protection against intraperitoneal 241
challenge with strain TIGR4, indicating a role of antibodies in the protective 242
mechanism. Finally, we observed at least partial protection against intranasal 243
challenge with strain TIGR4, either at blood or lung level, depending on the route of 244
immunization, intraperitoneal or intranasal, respectively. 245
The body of evidence presently available supports the notion that it is unlikely that a 246
single protein antigen can afford protection against all S. pneumoniae serotypes, even 247
though it can confer either partial protection against a broad range of pneumococcal 248
strains or high protection against a subset of strains. Conceivably, an effective 249
vaccine should be multi-component (2, 4, 14), as it is the case for some vaccines on 250
the market (e.g. acellular pertussis vaccines) (17) and others under development (e.g. 251
protein-based vaccines against group B meningococci or against group B 252
streptococci) (11, 18). 253
Our results indicate that SrtA is a promising pneumococcal antigen that could be 254
considered for inclusion in a multi-component protein vaccine against S. pneumoniae. 255
ACKNOWLEDGEMENTS 256
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TIGR4, D39, and 35B-SME15 strains were kindly provided by Gianni Pozzi, Stanley 257
Falkow, and Birgitta Henriques Normark, respectively. Giacomo Matteucci and 258
Roberto Sabato managed the Animal Resources. Animal treatments were carried out 259
by Marco Tortoli, Stefania Torricelli, Riccardo Bastone and Elena Amantini. All 260
authors declare potential conflicting interests. 261
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262
263
Table 1. Cytological analysis from mice immunized intranasally and challenged
intranasally with TIGR4
nasal wash lung wash
SrtA (1, 3) ctrl (2, 3) P SrtA (1, 3) ctrl (2, 3) P
Macrophages 5.9 ± 2.5 6.4 ± 1.1 0.463 9.1 ± 2.1 15.3 ± 2.3 0.094
PMN 16.9 ± 5.7 17.0 ± 4.8 0.955 17.6 ± 4.4 129.9 ± 22.8 0.0003
(1) SrtA-immunized mice (n = 7)
(2) control group receiving adjuvant plus saline (n = 8)
(3) mean cell number ± SE
264
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FIGURE LEGENDS 266
267
Figure 1. Protective efficacy of SrtA intraperitoneal immunization against 268
intraperitoneal challenge with TIGR4 (A and B), D39 (C and D), or 35B-SME15 (E 269
and F) S. pneumoniae strain, as indicated at the right side. Mice were either 270
immunized with Sortase A (SrtA) or received adjuvant plus saline (ctrl). In the left 271
panels (A, C, and E) values of bacteremia at 24 hrs are shown: circles represent 272
values of CFU/ml for single animals, horizontal bars represent the geometric mean 273
for each group, and the dashed line indicates the detection limit (i.e. no CFU were 274
detected in samples positioned below dashed line). In the right panels (B, D, and F) 275
the mortality course is shown: diamonds represent survival days for single animals, 276
horizontal bars represent the median survival time for each group and the dashed line 277
indicates the endpoint of observation ( i.e. animals whose survival time is above the 278
dashed line were alive at the 10th
day post-challenge). * and ** indicate values of P ≤ 279
0.05 and ≤ 0.01, respectively, for each immunized group as compared with the 280
control. 281
282
Figure 2. Protective efficacy of passive anti-SrtA serum transfer against subsequent 283
intraperitoneal challenge with TIGR4 S. pneumoniae strain. The figure includes data 284
coming from three experiments carried out under the same conditions. Symbols are 285
described in Figure 1 legend. 286
287
Figure 3. Protective efficacy of SrtA immunization against intranasal challenge with 288
TIGR4 S. pneumoniae strain. Mice were immunized with SrtA either intraperitoneally 289
(A, B, and C) or intranasally (D, E, and F), as indicated at the right side. Results of 290
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CFU quantification in blood (A and D), nasal washes (B and E), and lung washes (C 291
and F) are shown. Symbols are described in Figure 1 legend. 292
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Figure 1
BACTEREMIA MORTALITY
SrtActrl
A
su
rviv
al ti
me (d
ays)
CF
U/m
l
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
102
103
104
105
106
107
108
102
103
104
105
106
107
108
102
103
104
105
106
107
108
SrtActrl
su
rviv
al ti
me (d
ays)
CF
U/m
l
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
102
103
104
105
106
107
108
102
103
104
105
106
107
108
102
103
104
105
106
107
108
B
su
rviv
al ti
me (d
ays
)
CF
U/m
l
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
102
103
104
105
106
107
108
102
103
104
105
106
107
108
102
103
104
105
106
107
108
C
***
***
**
D
E F
TIG
R4
D3
935
B-S
ME
15
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BACTEREMIA MORTALITY
su
rviv
al
tim
e
(da
ys
)
CF
U/m
l
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
0
1
2
3
4
5
6
7
8
9
10
102
103
104
105
106
107
108
102
103
104
105
106
107
108
102
103
104
105
106
107
108
SrtActrl SrtActrl
* B *
Figure 2
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Figure 3
BACTEREMIA
CF
U/m
l
102
103
104
105
106
107
102
103
104
105
106
107*
A B
102
103
104
105
106
107
102
103
104
105
106
107
NASAL COLONIZATION
C
102
103
104
105
106
107
102
103
104
105
106
107
LUNG INFECTION
CF
U/m
l
102
103
104
105
106
107
102
103
104
105
106
107
SrtActrl SrtActrl
D E
102
103
104
105
106
107
102
103
104
105
106
107
SrtActrl
F
102
103
104
105
106
107*
intr
ap
eri
ton
ea
lim
mu
n,
intr
an
as
alim
mu
n.
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