Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections James, C, Davies, E, Fothergill, J, Walshaw, M, Beale, C, Brockhurst, M and Winstanley, C http://dx.doi.org/10.1038/ismej.2014.223 Title Lytic activity by temperate phages of Pseudomonas aeruginosa in long-term cystic fibrosis chronic lung infections Authors James, C, Davies, E, Fothergill, J, Walshaw, M, Beale, C, Brockhurst, M and Winstanley, C Type Article URL This version is available at: http://usir.salford.ac.uk/id/eprint/33271/ Published Date 2014 USIR is a digital collection of the research output of the University of Salford. Where copyright permits, full text material held in the repository is made freely available online and can be read, downloaded and copied for non- commercial private study or research purposes. Please check the manuscript for any further copyright restrictions. For more information, including our policy and submission procedure, please contact the Repository Team at: [email protected].
23
Embed
Lytic activity by temperate phages of Pseudomonas ...usir.salford.ac.uk/33271/1/6615_1_merged_1410254325.pdf5 89 knowledge this represents the first longitudinal study of a bacterial
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Lytic a c tivity by t e m p e r a t e p h a g e s of Ps e u do mo n a s
a e r u ginos a in long-t e r m cys tic fib rosis c h ro nic lun g infec tions
Ja m e s, C, Davies, E, Foth e r gill, J, Walsh aw, M, Be ale, C, Brock h u r s t , M a n d Wins t a nley, C
h t t p://dx.doi.o rg/1 0.10 3 8/is m ej.20 1 4.2 2 3
Tit l e Lytic a c tivity by t e m p e r a t e p h a g e s of Ps e u do mo n a s a e r u ginos a in long-t e r m cys tic fib rosis c h ro nic lun g infec tions
Aut h or s Jam e s, C, Davies, E, Foth e r gill, J, Walsh aw, M, Be ale, C, Brockh u r s t , M a n d Wins t a nley, C
Typ e Article
U RL This ve r sion is available a t : h t t p://usir.s alfor d. ac.uk/id/e p rin t/33 2 7 1/
P u bl i s h e d D a t e 2 0 1 4
U SIR is a digi t al collec tion of t h e r e s e a r c h ou t p u t of t h e U nive r si ty of S alford. Whe r e copyrigh t p e r mi t s, full t ex t m a t e ri al h eld in t h e r e posi to ry is m a d e fre ely availabl e online a n d c a n b e r e a d , dow nloa d e d a n d copied for no n-co m m e rcial p riva t e s t u dy o r r e s e a r c h p u r pos e s . Ple a s e c h e ck t h e m a n u sc rip t for a ny fu r t h e r copyrig h t r e s t ric tions.
For m o r e info r m a tion, including ou r policy a n d s u b mission p roc e d u r e , ple a s econ t ac t t h e Re posi to ry Tea m a t : u si r@s alford. ac.uk .
1, p < 0.001), consistent with on-going lytic activity by the temperate phages, but no effect of 184
time or exacerbations on total free-phage densities. Further, we observed a negative linear 185
relationship between the phage-to-bacterium ratio and bacterial density (Figure 2b & Table S2c 186
9
bacterial coefficient -3.206+- 0.484, LRT = 108.4, d.f. = 1, p < 0.001), suggesting a role for 187
phage lysis in regulation of bacterial densities. Time and exacerbations had no significant effect 188
on phage-to-bacterium ratios. It is perhaps surprising that exacerbations were not associated with 189
either a change in phage densities or a change in the phage-to-bacterium ratio (Figure 3), given 190
that these episodes are associated with the administration of high-dose intravenous antibiotics. 191
However, it should be noted that these patients were all subject to variable cocktails of 192
antibiotics over several years irrespective of exacerbations (Table 1). Moreover, clinical data on 193
antibiotic use in these patients was too incomplete to be used in analyses, and therefore effects 194
of particular antibiotics on phage dynamics may have been missed. 195
196
Abundance heirarchy among individual phages within lungs 197
Next, we considered each phage individually, observing a general hierarchy of free-phage 198
densities, though the precise patterns were clearly influenced by the fact that the LES 199
populations for each patient did not all share the same prophage complement (Table 1). Figure 4 200
illustrates the free-phage densities of individual LES phages for each of the patients. In the 201
majority of patients (CF2-CF9) similar free-phage dynamics were observed in that the density of 202
free LESφ2 was consistently higher than that of the other LES phages, closely followed by 203
LES4 (Figure 4). A positive correlation was observed between LESφ2 and LESφ4 densities in 204
these patients (Table S4). The dynamics observed in samples from patient CF10 (Figure 3) 205
exhibited a change in the hierarchy of free phage, with considerably higher free LES4 densities 206
observed. Despite consistent carriage of LES prophage 3, very little free LESφ3 was detected in 207
patients CF2 - CF10. However, higher levels of free LESφ3 (3.29 x 107 l-1) were observed in all 208
sputa from patient CF1 (Figure 4), whose P. aeruginosa were the only populations not to carry 209
prophage 2 (Table 1). We showed previously that LES populations exhibit genotypic diversity, 210
10
including variation in carriage of LES prophages. In particular, the carriage of LES prophage 5 211
was not consistent in all individuals of a given LES population (Fothergill, et al., 2010). In this 212
study, prophage 5 was intermittently detectable in the sputum from patients 7 (up to 105 copies 213
µl-1) and 10 (102 – 104 copies µl-1). This explains the low density of free LESφ5 in these patients. 214
Free copies of LESφ6 were not detected in the majority of sputum samples. Where free copies 215
were detected, the density was lower than the host bacterial load (6.7 x 103 – 1 x 107 copies µl-1). 216
217
Discussion 218
The levels of free LES phages detected in all patients throughout this study suggest an active 219
lytic cycle that may be promoted by the presence of H2O2 or DNA damaging antibiotics in the 220
CF lung (Fothergill et al 2011, McGrath et al 1999). Surprisingly, we observed no effect of 221
patient exacerbation on total free-phage density, although this is consistent with previous studies 222
showing that neither fluctuations in P. aeruginosa populations (Mowat et al 2011a), nor in the 223
wider bacterial population (Fodor et al 2012), show any relationship with the exacerbation period 224
in chronically infected patients, despite the use of high level intravenous antibiotic therapy. It is 225
known that particular antibiotics can induce phage lysis, and it is possible that different 226
antibiotics regimes may have influenced differential induction of phages between patients. 227
Indeed, we have shown previously that LES induction varies in response to different antibiotics 228
(Fothergill et al 2011). Unfortunately, because records of antibiotic treatments for these patients 229
were very incomplete, we were unable to explicitly test for effects of particular antibiotics in this 230
study. This would in any case be difficult because of the extensive and varied use of antibiotics in 231
this group of patients (Table 1), which was not restricted to periods of exacerbation. 232
Our data do however suggest that on-going phage lysis may play a role in regulating 233
bacterial density in the CF lung. Treatments which induced the lytic cycle of temperate phages 234
11
could therefore offer a promising alternative or addition to standard antibiotic therapies which in 235
themselves often do not successfully reduce P. aeruginosa densities in long-term chronically 236
infected patients (Foweraker 2009, Mowat et al 2011b). Several studies have demonstrated 237
effective phage-antibiotic synergism in the reduction of bacterial numbers in vitro and in vivo 238
(Comeau et al 2007, Hagens et al 2006, Knezevic et al 2013). However, this strategy would need 239
to be considered with caution. Antibiotic therapies that induce stx phages of Shiga-toxigenic E. 240
coli have been shown to increase expression of shiga toxin genes that are encoded in the late 241
region of the phage genome and thus increase cytotoxic damage and exacerbate symptoms 242
(Matsushiro et al 1999). Although we have not identified any obvious virulence factors encoded 243
in the late gene region of the LES phages (Winstanley, et al., 2009, James, et al., 2012), we cannot 244
ignore the possibility that the lytic cycle might induce upregulation of virulence genes. 245
We demonstrate here that LES2 was the most abundant free phage in 9-out-of-10 LES-246
infected patients. The hierarchy of free LES phage in patient sputa was also observed in our 247
previous studies of LES phage induction in in vitro bacterial cultures (James et al 2012). This 248
suggests therefore that LES2 is generally more readily induced or exhibits a more efficient lytic 249
cycle than the other phages both in vitro and in vivo. In the sputa of patient CF1, who was infected 250
by a LES that lacked prophage 2, LES3 reached far higher abundances than observed in other 251
patients, suggesting potential suppression of LES3 lysis by LES2 in vivo. In accordance with 252
our previous in vitro observations of co-induction of lysis by prophages, we observed a degree of 253
synchronisation of free-phage dynamics in vivo, suggesting that the phages may be responding to 254
shared signals, which could include a wide variety of human host, bacterial and environmental 255
triggers(Little 2005). It is exceptionally difficult to disentangle to drivers of microbial dynamics in 256
vivo due to the complexity of host microenvironments; future studies using laboratory models of 257
the infection environment alowing the constituent drivers to be decomposed will be necessary to 258
elucidate this (Fothergill et al 2014, Wright et al 2013). 259
12
The long-term maintenance of intact, active temperate phages in the LES genome 260
despite substantial cell lysis suggests some selective or competitive advantage in vivo, consistent 261
with previous work highlighting a loss of competitiveness observed following the introduction of 262
mutations to some LES prophage regions (Winstanley et al 2009). One possibility is that free-263
phage particles produced by a subpopulation of LES could kill competing bacteria (Brown et al 264
2006). Indeed, LESφ2, LESφ3 and LESφ4 are capable of infecting and lysing other clinical P. 265
aeruginosa isolates (James et al 2012). Thus frequent induction of the lytic cycle may enhance the 266
competitive ability of LES by promoting superinfection, which has been observed clinically 267
(McCallum et al 2001), and preventing invasion of the lung by other strains of P. aeruginosa. 268
Alternatively the prophages may contain accessory genes that contribute directly to LES fitness 269
in the CF lung, which are only expressed during the lytic cycle, as observed for other pathogens 270
(Wagner et al 2001). 271
Little is known about the consequences for the human host of the presence of large 272
numbers of phage in the lung. However, high titre phage preparations have recently been found 273
to interact with the immune system in vivo (Letkiewicz et al 2010). It has also been suggested that, 274
following adherence to mucous, some phages may act as a form of innate host immunity 275
enhancing host defences against bacterial pathogens (Barr et al 2013). Our findings of high free-276
phage abundances in CF lungs highlight the urgent need for research into the interaction of 277
phages with host immunity, particularly in CF where dysfunctional immune responses contribute 278
to pathological processes. 279
280
Supplementary information is available on The ISME journal website 281
282
Acknowledgements 283
13
This work was supported by the Wellcome Trust (089215/Z/09/Z) to CW and MAB. 284
We declare that there are no competing commercial interests in relation to this submitted work 285
286
References 287
Al-Aloul M, Crawley J, Winstanley C, Hart CA, Ledson MJ, Walshaw MJ (2004). Increased morbidity 288 associated with chronic infection by an epidemic Pseudomonas aeruginosa strain in CF patients. 289 Thorax 59: 334-336. 290
291 Ashish A, Shaw M, Winstanley C, Ledson MJ, Walshaw MJ (2012). Increasing resistance of the 292 Liverpool Epidemic Strain (LES) of Pseudomonas aeruginosa (Psa) to antibiotics in cystic fibrosis (CF)--293 a cause for concern? J Cyst Fibros 11: 173-179. 294
295 Barr JJ, Auro R, Furlan M, Whiteson KL, Erb ML, Pogliano J et al (2013). Bacteriophage adhering to 296 mucus provide a non-host-derived immunity. Proc Natl Acad Sci U S A 110: 10771-10776. 297
298 Bobay LM, Touchon M, Rocha EP (2014). Pervasive domestication of defective prophages by 299 bacteria. Proc Natl Acad Sci U S A 111: 12127-12132. 300
301 Breitbart M, Hewson I, Felts B, Mahaffy JM, Nulton J, Salamon P et al (2003). Metagenomic analyses 302 of an uncultured viral community from human feces. J Bacteriol 185: 6220-6223. 303
304 Brown SP, Le Chat L, De Paepe M, Taddei F (2006). Ecology of microbial invasions: amplification 305 allows virus carriers to invade more rapidly when rare. Curr Biol 16: 2048-2052. 306
307 Brussow H, Canchaya C, Hardt WD (2004). Phages and the evolution of bacterial pathogens: from 308 genomic rearrangements to lysogenic conversion. Microbiol Mol Biol Rev 68: 560-602. 309
313 Desiere F, McShan WM, van Sinderen D, Ferretti JJ, Brussow H (2001). Comparative genomics reveals 314 close genetic relationships between phages from dairy bacteria and pathogenic Streptococci: 315 evolutionary implications for prophage-host interactions. Virology 288: 325-341. 316
317 Fodor AA, Klem ER, Gilpin DF, Elborn JS, Boucher RC, Tunney MM et al (2012). The adult cystic 318 fibrosis airway microbiota is stable over time and infection type, and highly resilient to antibiotic 319 treatment of exacerbations. PLoS One 7: e45001. 320
14
321 Fothergill JL, Mowat E, Ledson MJ, Walshaw MJ, Winstanley C (2010). Fluctuations in phenotypes 322 and genotypes within populations of Pseudomonas aeruginosa in the cystic fibrosis lung during 323 pulmonary exacerbations. J Med Microbiol 59: 472-481. 324
325 Fothergill JL, Mowat E, Walshaw MJ, Ledson MJ, James CE, Winstanley C (2011). Effect of antibiotic 326 treatment on bacteriophage production by a cystic fibrosis epidemic strain of Pseudomonas 327 aeruginosa. Antimicrob Agents Chemother 55: 426-428. 328
329 Fothergill JL, Walshaw MJ, Winstanley C (2012). Transmissible strains of Pseudomonas aeruginosa in 330 Cystic Fibrosis lung infections. Eur Respir J 40: 227-238. 331
332 Fothergill JL, Ledson MJ, Walshaw MJ, McNamara PS, Southern KW, Winstanley C (2013). 333 Comparison of real time diagnostic chemistries to detect Pseudomonas aeruginosa in respiratory 334 samples from cystic fibrosis patients. J Cyst Fibros 12: 675 - 681. 335
336 Fothergill JL, Neill DR, Loman N, Winstanley C, Kadioglu A (2014). Pseudomonas aeruginosa 337 adaptation in the nasopharyngeal reservoir leads to migration and persistence in the lungs. Nature 338 communications 5: 4780. 339
340 Foweraker J (2009). Recent advances in the microbiology of respiratory tract infection in cystic 341 fibrosis. Br Med Bull 89: 93-110. 342
343 Ghosh D, Roy K, Williamson KE, Srinivasiah S, Wommack KE, Radosevich M (2009). Acyl-homoserine 344 lactones can induce virus production in lysogenic bacteria: an alternative paradigm for prophage 345 induction. Appl Environ Microbiol 75: 7142-7152. 346
347 Goss CH, Burns JL (2007). Exacerbations in cystic fibrosis. 1: Epidemiology and pathogenesis. Thorax 348 62: 360-367. 349
350 Hagens S, Habel A, Blasi U (2006). Augmentation of the antimicrobial efficacy of antibiotics by 351 filamentous phage. Microb Drug Resist 12: 164-168. 352
353 James CE, Stanley KN, Allison HE, Flint HJ, Stewart CS, Sharp RJ et al (2001). Lytic and lysogenic 354 infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage. Appl 355 Environ Microbiol 67: 4335-4337. 356
357 James CE, Fothergill JL, Kalwij H, Hall AJ, Cottell J, Brockhurst MA et al (2012). Differential infection 358 properties of three inducible prophages from an epidemic strain of Pseudomonas aeruginosa. BMC 359 Microbiol 12: 216. 360
361
15
Kim MS, Park EJ, Roh SW, Bae JW (2011). Diversity and abundance of single-stranded DNA viruses in 362 human feces. Appl Environ Microbiol 77: 8062-8070. 363
364 Knezevic P, Curcin S, Aleksic V, Petrusic M, Vlaski L (2013). Phage-antibiotic synergism: a possible 365 approach to combatting Pseudomonas aeruginosa. Res Microbiol 164: 55-60. 366
367 Lawrence JG, Hendrix RW, Casjens S (2001). Where are the pseudogenes in bacterial genomes? 368 Trends Microbiol 9: 535-540. 369
370 Letkiewicz S, Miedzybrodzki R, Klak M, Jonczyk E, Weber-Dabrowska B, Gorski A (2010). The 371 perspectives of the application of phage therapy in chronic bacterial prostatitis. FEMS Immunol Med 372 Microbiol 60: 99-112. 373
374 Lim YW, Evangelista JS, 3rd, Schmieder R, Bailey B, Haynes M, Furlan M et al (2014). Clinical insights 375 from metagenomic analysis of sputum samples from patients with cystic fibrosis. J Clin Microbiol 52: 376 425-437. 377
378 Little JW (2005). Lysogeny, prophage induction and lysogenic conversion. In: Waldor MK, Friedman 379 A, Adhya S (eds). Phages. ASM Press: Washington DC. pp 37-54. 380
381 Martin K, Baddal B, Mustafa N, Perry C, Underwood A, Constantidou C et al (2013). Clusters of 382 genetically similar isolates of Pseudomonas aeruginosa from multiple hospitals in the United 383 Kingdom. J Med Microbiol 62: 988 - 1000. 384
385 Matsushiro A, Sato K, Miyamoto H, Yamamura T, Honda T (1999). Induction of prophages of 386 enterohemorrhagic Escherichia coli O157:H7 with norfloxacin. J Bacteriol 181: 2257-2260. 387
388 McCallum SJ, Corkill J, Gallagher M, Ledson MJ, Hart CA, Walshaw MJ (2001). Superinfection with a 389 transmissible strain of Pseudomonas aeruginosa in adults with cystic fibrosis chronically colonised by 390 P aeruginosa. Lancet 358: 558-560. 391
392 McGrath LT, Mallon P, Dowey L, Silke B, McClean E, McDonnell M et al (1999). Oxidative stress 393 during acute respiratory exacerbations in cystic fibrosis. Thorax 54: 518-523. 394
395 Mowat E, Paterson S, Fothergill JL, Wright EA, Ledson MJ, Walshaw MJ et al (2011a). Pseudomonas 396 aeruginosa population diversity and turnover in cystic fibrosis chronic infections. Am J Respir Crit 397 Care Med 183: 1674-1679. 398
399 Mowat E, Paterson S, Fothergill JL, Wright EA, Ledson MJ, Walshaw MJ et al (2011b). Pseudomonas 400 aeruginosa Population Diversity and Turnover in Cystic Fibrosis Chronic Infections. Am J Respir Crit 401 Care Med. 402
16
403 Ojeniyi B, Birch-Andersen A, Mansa B, Rosdahl VT, Hoiby N (1991). Morphology of Pseudomonas 404 aeruginosa phages from the sputum of cystic fibrosis patients and from the phage typing set. An 405 electron microscopy study. APMIS 99: 925-930. 406
407 Pinheiro JC, Bates DM (2000). Linear mixed-effects models: basic concepts and examples. Springer 408 New York. 409
410 Reyes A, Semenkovich NP, Whiteson K, Rohwer F, Gordon JI (2012). Going viral: next-generation 411 sequencing applied to phage populations in the human gut. Nat Rev Microbiol 10: 607-617. 412
413 Wagner PL, Acheson DW, Waldor MK (2001). Human neutrophils and their products induce Shiga 414 toxin production by enterohemorrhagic Escherichia coli. Infect Immun 69: 1934-1937. 415
416 Willner D, Furlan M, Haynes M, Schmieder R, Angly FE, Silva J et al (2009). Metagenomic analysis of 417 respiratory tract DNA viral communities in cystic fibrosis and non-cystic fibrosis individuals. PLoS One 418 4: e7370. 419
420 Winstanley C, Langille MG, Fothergill JL, Kukavica-Ibrulj I, Paradis-Bleau C, Sanschagrin F et al (2009). 421 Newly introduced genomic prophage islands are critical determinants of in vivo competitiveness in 422 the Liverpool Epidemic Strain of Pseudomonas aeruginosa. Genome Res 19: 12-23. 423
424 Wright EA, Fothergill JL, Paterson S, Brockhurst MA, Winstanley C (2013). Sub-inhibitory 425 concentrations of some antibiotics can drive diversification of Pseudomonas aeruginosa populations 426 in artificial sputum medium. BMC Microbiol 13: 170. 427
428
429
Figure Legends 430
Figure 1: Longitudinal dynamics of total free-phage density and P. aeruginosa density 431
in ten CF patients 432
Q-PCR assays were used to enumerate free LES phage (dotted line) and P. aeruginosa (solid 433
line) densities from the sputa of 10 LES-infected CF patients (CF1-CF5 left to right top row, 434
and CF6-CF10 left to right bottom row) over a two year period. Samples were obtained from 435
patients both during stable periods (black symbols) and during exacerbation of symptoms 436
17
(red symbols). The dotted line represents the mean values of all free LES phages (2,3,4,5 and 437
6) for each patient. The density of free-phage copies of each LES phage was calculated by 438
subtracting prophage copies from total phage copies in each case. 439
Figure 2: Relationships of phage density and phage-to-bacterium ratio with bacterial 440
density. 441
Datapoints represent sputum samples; patient identity is indicated by colour (see visual key 442
for details); regression lines indicate significant relationships between variables. Panel A 443
(upper) shows the positive relationship between log10 phage density and log10 bacterial 444
density; panel B (lower) shows the negative relationship between phage-to-bacterium ratio 445
and log10 bacterial density. 446
447
Figure 3: Phage density and phage-to-bacterium ratio are not affected by exacerbations 448
Outlier box-plots display phage density (upper panel) or phage-to-bacterium ratio (lower 449
panel) in sputa from patients during stable periods (black) and exacerbations (red). 450
451
Figure 4: Densities of individual LES phage types in patient sputa exhibit hierarchical 452
trends. 453
The free-phage densities, calculated for each individual LES phage in the ten CF patients 454
analysed (CF1-CF5 left to right top row, and CF6-CF10 left to right bottom row). Each line 455
represents one LES phage type; LESφ2 (blue); LESφ3 (cyan); LES φ4 (pink); LES φ5 456
(green); LES φ6 (orange); P. aeruginosa (black circles). All Q-PCR assays were performed 457
in triplicate and mean values are presented. The density of free-phage copies of each LES 458
phage was calculated by subtracting prophage copies from total phage copies in each case. 459