Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas aeruginosa small colony variant.

1 Transcriptional and mutational profiling of an aminoglycoside resistant Pseudomonas 1 aeruginosa small colony variant 2 3 Monika Schniederjans,a,b Michal Koska,b Susanne Häussler,a,b # 4 Department of Molecular Bacteriology, Helmholtz Centre for Infection Research, Braunschweig, Germany a 5 Institute for Molecular Bacteriology, TWINCORE GmbH, Centre for Clinical and Experimental Infection 6 Research, a joint venture of the Hannover Medical School and the Helmholtz Centre for Infection Research, 7 Hannover, Germanyb 8 9 10 11 Running head: Antibiotic resistance profiling 12 13 # Address correspondence to Susanne Häussler, [email protected]. 14 15 AAC Accepted Manuscript Posted Online 5 September 2017 Antimicrob. Agents Chemother. doi:10.1128/AAC.01178-17 Copyright © 2017 American Society for Microbiology. All Rights Reserved. on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 2 Abstract 16 Pseudomonas aeruginosa is a major causative agent of both acute and chronic infections. 17 Although aminoglycoside antibiotics are very potent drugs to fight such infections, antibiotic 18 failure is steadily increasing mainly due to increasing resistance of the bacteria. Many 19 molecular mechanisms that determine resistance such as acquisition of genes encoding for 20 aminoglycoside-inactivating enzymes or overexpression of efflux pumps have been 21 elucidated. However, there are additional, less-well described mechanisms of 22 aminoglycoside resistance. In this study we have profiled a clinical tobramycin resistant P. 23 aeruginosa strain that exhibited a small colony variant (SCV) phenotype. Both, the resistance 24 and the colony morphology phenotypes were lost upon passaging the isolate under rich 25 medium conditions. Transcriptional and mutational profiling revealed that the SCV harbored 26 activating mutations in the two two-component systems AmgRS and PmrAB. Introduction of 27 these mutations singularly into the type strain PA14 conferred tobramycin and colistin 28 resistance, respectively. However, their combined introduction had an additive effect on the 29 tobramycin resistance phenotype. Activation of the AmgRS system slightly reduced the 30 colony size of the PA14 wild-type, whereas the simultaneous overexpression of gacA, the 31 response regulator of the GacSA two component system, further reduced colony size. In 32 conclusion, we uncovered combinatorial influences of two-component systems on clinically 33 relevant phenotypes, such as resistance and the expression of the SCV phenotype. Our 34 results clearly demonstrate that combined activation of P. aeruginosa two-component 35 systems exhibit pleiotropic effects with unforeseen consequences. 36 37 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 3 Introduction 38 Pseudomonas aeruginosa is one of the most important opportunistic pathogens and a 39 serious concern for public health. Its high potential for intrinsic and acquired antimicrobial 40 resistance is a major anticipated problem in hospitals (1-3). Aminoglycosides are highly 41 potent, broad-spectrum antibiotics. They are particularly used to treat P. aeruginosa 42 pulmonary infections in cystic fibrosis patients, where the drugs are often inhaled to reach 43 high local concentration in the bronchial sections (4). Intravenously administered 44 combination therapies mainly with β-lactam antibiotics are also common (5). However, as 45 for most antimicrobial classes, higher rates of inappropriate empiric antimicrobial treatment 46 are associated with the increasing resistance to the antimicrobials in use. 47 Diverse mechanisms have been described to confer resistance towards aminoglycosides in P. 48 aeruginosa. The most prominent ones include drug inactivation through the activity of 49 aminoglycoside-modifying enzymes and decreased drug accumulation inside the bacterial 50 cell via active efflux or diminished cell wall permeability. Active efflux of aminoglycosides in 51 P. aeruginosa can be achieved via the upregulation of the MexXY efflux system (6) mainly 52 caused by mutations in regulatory genes (7). Furthermore, an adaptive expression of the 53 MexXY efflux pump, e.g. in the presence of ribosome-targeting antibiotics has been 54 described (8). In cystic fibrosis isolates, active MexXY efflux is very common (9-11). 55 Besides these well-described strategies to resist aminoglycoside therapy, growth within 56 biofilms (12), conversion to mucoidy (13) or the emergence of persister cells (14) can 57 contribute to survival in the presence of the antibiotic. Furthermore slow-growing small 58 colony variant (SCVs) populations have been associated with persistent infections and 59 increased resistance towards antibiotics, including aminoglycosides (15). It has also been 60 shown that aminoglycoside exposure can induce the formation of SCVs in vivo and in vitro 61 (16, 17). While in Staphylococcus aureus the morphotypic switch to SCVs is linked to 62 electron-transport deficiency in strains that are auxotroph for menadione or haemin or to 63 thymidine auxotrophy (18), the underlying mechanisms of P. aeruginosa SCV generation 64 seem to be more heterogeneous (19-22). In P. aeruginosa the SCV phenotype is often, but 65 not exclusively, associated with elevated intracellular levels of the second messenger cyclic66 di-GMP (12, 19, 23-25). Although the antibiotic susceptibility phenotype may vary a lot (26, 67 27), many clinical SCV isolates exhibit resistance towards several antibiotics and express 68 further phenotypic features like hyperpiliation or increased biofilm formation capabilities 69 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 4 (25, 26). Also overproduction of the exopolysaccharides Pel and Psl (28, 29) and more 70 recently, the involvement of a prophage genomic region (22) has been linked to SCV 71 formation. 72 In this study we describe the underlying genetic determinants of clinical P. aeruginosa 73 isolates that produce not only small colonies on agar plates but exhibit also an 74 aminoglycoside resistant phenotype. Three clonally related SCVs produced revertants 75 following passaging under rich medium conditions, which exhibited larger colony 76 morphologies and aminoglycoside susceptibility By transcriptional and mutational profiling 77 we uncovered a combinatorial impact of the three two-component systems PmrAB, AmgRS 78 and GacSA on aminoglycoside resistance and the SCV morphotype. 79 80 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 5 Results 81 Aminoglycoside resistance is linked to a SCV phenotype. We have previously identified 82 three clonally related tobramycin resistant clinical P. aeruginosa isolates (30). The resistance 83 phenotype of those isolates could not be explained by the presence of a gene encoding for 84 an aminoglycoside-modifying enzyme, nor did they express the mexXY multidrug efflux 85 pump genes at highly elevated levels. (All three SCVs exhibited a below 2-fold increased 86 mexX and mexY expression as compared to the PA14 reference strain). All three clinical 87 isolates however were small colony variants (SCVs) (Fig. 1). The formation of SCVs has been 88 associated with persistent infections and increased resistance towards aminoglycoside 89 antibiotics (15). We therefore hypothesized that there might be a link between the 90 phenotypic variation and aminoglycoside resistance in the three P. aeruginosa SCVs 91 MHH8607, MHH9536 and MHH9604. As shown in Fig.1, even among the closely related 92 SCVs, differences in colony morphologies were observed. MHH8607 seemed to exhibit an 93 even smaller colony morphology than the other two SCV isolates. Via passaging the SCVs in 94 rich medium, we generated stable revertants with larger surface colonies. Morphological 95 differences were also observed for the revertants. REV_MHH8607 appeared as shiny circular 96 colonies, while the other two exhibited rather flat, dull colonies with a rough texture. Of 97 note, all three revertants exhibited an increased susceptibility towards aminoglycosides 98 (Table 1). However, only the revertant of MHH8607 (REV_MHH8607) reached a minimal 99 inhibitory concentration (MIC) value for tobramycin that was categorized as susceptible. 100 101 Transcriptional profiling reveals a down-regulation of the PmrAB two-component system 102 in the revertants. Transcriptional profiles of the three SCV isolates have been recorded 103 before (31, 32) and were complemented in this study by RNA-sequencing of the respective 104 revertant strains. Gene expression analysis revealed a range of 64 to 84 genes that were 105 differentially expressed between the SCVs and their respective revertants (Fig. 2, 106 Supplementary Table 1). Among those genes, we found an overlap of 14 genes differentially 107 regulated in all three SCV / revertant pairs (Table 2). Most of them (10 genes) belonged to 108 the PmrAB two-component regulatory pathway. 109 110 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 6 The sensor kinase encoding gene pmrB and the response regulator encoding gene pmrA 111 were most strongly down-regulated in all revertants. Furthermore, many genes of the 112 downstream regulated gene operon arnBCADTEF (33) and the spermidine synthesis gene 113 cluster speD (PA14_63110) and speE (PA14_63120) (34) were expressed at lower levels in 114 the revertants. Expression of the arnBCADTEF operon is known to limit the interaction and 115 self-promoted uptake of polycationic antibiotics (35), whereas spermidine is a polyamine 116 and has been described before to protect cells from antibiotic treatment and oxidative stress 117 (34, 36). 118 Next to those commonly regulated genes, we found in each revertant an individual set of 119 differentially expressed genes (Supplementary Table 1). A closer inspection of the isolate 120 MHH8607 (where the REV_MHH8607 reached aminoglycoside MIC levels in the susceptible 121 range) revealed the downregulation of a further two-component system sensor kinase 122 encoding gene, amgS (PA14_68680, annotated as envZ in the PA14 reference genome) (log2 123 fold change of 3.67). The corresponding response regulator amgR (PA14_68700, annotated 124 as ompR in the PA14 reference genome) also appeared to be downregulated by a log2 fold 125 change of 2.31 (although p-value significance was not reached). The AmgRS system was 126 described before to be involved in aminoglycoside resistance, as it has been identified in a 127 screen of a transposon mutant library for genes whose inactivation increased tobramycin 128 sensitivity (37). The AmgRS system is a membrane-stress responsive two-component system 129 involved in the transcriptional regulation of membrane proteases and other membrane 130 proteins (38). In line with this, we found a reduced expression of the protease HtpX 131 (encoded by PA14_27480, log2 fold change of 3.99) and a membrane protein of unknown 132 function (encoded by PA14_72930, log2 fold change of 3.97) in the REV_MHH8607 strain. 133 In conclusion, it seems that the down-regulation of the PmrAB system in the revertants is 134 involved in the regained aminoglycoside susceptibility, whereas an additional down135 regulation of the AmgRS in the REV_MHH8607 might account for the regained full 136 susceptibility against tobramycin of this particular strain. 137 138 The aminoglycoside resistant SCVs exhibit increased pmrAB and amgRS expression levels. 139 We next explored whether the PmrAB and AmgRS two component systems exhibited an 140 overall increased expression in the SCVs and thus might explain the aminoglycoside 141 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 7 resistance phenotype. We therefore compared the expression levels of the respective genes 142 with those of a P. aeruginosa type strain cultivated under identical conditions. Indeed all 143 three SCV isolates exhibited a high gene expression level of both systems (up to 43-fold 144 increased expression for pmrA and up to 5-fold for amgS; data not shown) as compared to 145 the PA14 reference strain. Also in comparison to the median expression level in 151 clinical 146 isolates that have been sequenced before (32), we found higher expression of both two147 component systems (data not shown). 148 149 The aminoglycoside resistant SCVs exhibit gain-of-function mutations in the two150 component systems. As mutational activation of both the PmrAB and the AmgRS systems 151 have been described previously (39-41), the gene alleles of the SCV isolates were 152 consequently screened for sequence alterations, which could be gain-of-function mutations. 153 Indeed, in all three clonal SCV isolates, non-synonymous sequence variations in comparison 154 to the PA14 reference strain were discovered in both genes of the pmrAB operon as well as 155 in the sensor kinase amgS. The pmrA sequence variation caused an amino acid substitution 156 in the corresponding protein (Leu71Arg) that is located within the signal receiver domain of 157 the response regulator, which also contains the phosphorylation site of the protein. Within 158 pmrB, two sequence variations were detected; one caused an amino acid exchange in the 159 secretion signal of the protein (Thr4Ala) while the second is located in close proximity to the 160 histidine kinase A and the ATP binding domain (Leu323His) (40, 42). To explore whether 161 these sequence variations act as activating mutations, we introduced them into the PA14 162 reference strain isolated or in various combinations and analyzed the resulting phenotype 163 (Table 3). 164 Aminoglycoside-susceptibility was slightly affected by the mutation in amgS, which led to a 165 2-fold increase of the tobramycin MIC (Table 3). The activating mutations within the PmrAB 166 system did not influence the tobramycin MIC. However, the combined activation of AmgS 167 and the PmrAB system led to a 4-fold increase in the tobramycin MIC. PmrAB is known to be 168 involved in polymyxin resistance by activating the arnBCADTEF operon that in turns modifies 169 the LPS structure. We therefore also tested the generated mutant strains for changes in 170 colistin susceptibility. Single mutations in pmrA or pmrB led to none or only a slight decrease 171 in colistin susceptibility, while both pmrB mutations in combination resulted in high-level 172 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 8 resistance (MIC 64 µg/ml). The aminoglycoside resistance inducing mutation in amgS did not 173 further enhance polymyxin susceptibility. 174 175 Intriguingly, the three investigated clinical clonal SCV isolates did not exhibit colistin 176 resistance, although carrying the gain-of-function mutations in pmrAB and exhibiting 177 overexpression of the system and the downstream genes. There was also no difference in 178 colistin susceptibility between the SCVs and their respective revertants even though changes 179 in arnBCADTEF expression occurred. We did not identify any insertions or deletions within 180 the SCV arn genes. Nevertheless, we found overall 19 sequence variations (Supplementary 181 Table 2) within the arn genes of the SCVs as compared to the PA14 WT. It cannot be 182 excluded that among them there is an inactivating mutation that might explain colistin 183 susceptibility despite anr gene overexpression. 184 Susceptibility towards Colistin in the SCVs could also not be explained by the expression 185 levels of the two-component systems PhoPQ-OprH operon or ParRS. (We found an up to 2.3186 fold decreased expression for phoQ, phoP and oprH as compared to the PA14 wild-type, and 187 an up to 2.8-fold increased expression for parS, the expression for parR was not significantly 188 changed; data not shown). 189 190 191 Mutations that led to the switch to the revertant phenotype. The PmrAB pathway has been 192 previously described to contribute to an antibiotic resistance phenotype. As this pathway 193 was down regulated in all three revertants as compared to their respective SCVs, we further 194 explored whether this was due to a mutational inactivation of the system in the revertants. 195 We therefore used our RNA-sequencing dataset to identify mutations that have been 196 acquired by the revertants during the course of their phenotypic switch. A range of 197 polymorphic genes or regulatory pathways that were affected in more than one revertant 198 strain were detected (Table 4). This included the PmrAB two-component system encoding 199 genes, in which all revertants had acquired a mutation in either the sensor kinase or the 200 response regulator. Additionally and in line with the transcriptional data, the two201 component system AmgRS harbored a nonsense mutation in amgS in REV_MHH8607. 202 Furthermore, the GacSA system harbored mutations in two of the three revertants (in 203 REV_MHH8606 in gacS and in REV_MHH9536 44 bp upstream of gacA). Another mutational 204 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 9 hotspot was the alternative sigma factor algU, where SNPs occurred in close proximity in 205 two revertant strains. This latter mutation is likely to be the cause of the observed 206 morphological differences among the three revertants: Only REV_MHH8607 exhibited a 207 shiny and more mucoid colony surface, while the ones with an acquired algU mutation and 208 thus presumably lower alginate production appeared as dull, non-mucoid colonies, as 209 described before (43). 210 211 212 The combined activation of the AmgRS and GacSA system in the type strain PA14 produces 213 an SCV phenotype. The GacSA/RsmAZY signaling system is known to be involved in the 214 phenotypic switch between chronic (persistence, SCV formation) and acute infectious 215 lifestyles. Since the revertants of two of the clinical SCVs harbored mutations in the GacSA 216 pathway, we overexpressed the gacA gene in the PA14 wild-type strain as well as the PA14 217 strain harboring the gain of function mutations in pmrB and/or amgS in order to determine 218 whether we can observe an influence on the colony morphology. Overexpression of the 219 gacA gene in PA14 or in the mutants overexpressing PmrB did not lead to alterations in 220 colony morphology or the resistance phenotype (data not shown). However, in the mutant 221 with an activation of the AmgRS two-component system, gacA overexpression led to an SCV 222 phenotype. The resistance phenotype did not change in this strain background due to gacA 223 overexpression (data not shown). 224 Our data thus indicate that the combined activation of the GacSA and the AmgRS system 225 seem to explain the SCV phenotype in our clinical isolates, whereas the combined activation 226 of the PmrAB and the AmgRS system confers to enhanced aminoglycoside resistance. 227 228 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 16 348 FIG 1: Colony morphology of three clonal small-colony variants and their respective revertants. 349 Small-colony variant MHH8607, MHH9536 and MHH9604 (left) and their respective revertants with larger and 350 diverse colony surfaces (right) after 24 h of growth on Columbia blood agar plates. 351 352 353 354 FIG 2: Differentially expressed genes in the SCV isolates in comparison to their respective 355 revertants. 356 A gene was categorized as differentially expressed when the absolute value of log2 fold change was ≥ 2 and the 357 pval was ≤ 0.01. The Venn diagram was created with VENNY (60). 358 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 17 References 359 1. Breidenstein EB, de la Fuente-Nunez C, Hancock RE. 2011. Pseudomonas aeruginosa: 360 all roads lead to resistance. Trends Microbiol 19:419-426. 361 2. Michiels JE, Van den Bergh B, Verstraeten N, Fauvart M, Michiels J. 2016. In Vitro 362 Emergence of High Persistence upon Periodic Aminoglycoside Challenge in the 363 ESKAPE Pathogens. Antimicrob Agents Chemother 60:4630-4637. 364 3. Lister PD, Wolter DJ, Hanson ND. 2009. Antibacterial-resistant Pseudomonas 365 aeruginosa: clinical impact and complex regulation of chromosomally encoded 366 resistance mechanisms. Clin Microbiol Rev 22:582-610. 367 4. Chiappini E, Taccetti G, de Martino M. 2014. Bacterial lung infections in cystic fibrosis 368 patients: an update. Pediatr Infect Dis J 33:653-654. 369 5. Doring G, Conway SP, Heijerman HG, Hodson ME, Hoiby N, Smyth A, Touw DJ. 2000. 370 Antibiotic therapy against Pseudomonas aeruginosa in cystic fibrosis: a European 371 consensus. Eur Respir J 16:749-767. 372 6. Poole K. 2001. Multidrug resistance in Gram-negative bacteria. Curr Opin Microbiol 373 4:500-508. 374 7. Matsuo Y, Eda S, Gotoh N, Yoshihara E, Nakae T. 2004. MexZ-mediated regulation of 375 mexXY multidrug efflux pump expression in Pseudomonas aeruginosa by binding on 376 the mexZ-mexX intergenic DNA. FEMS Microbiol Lett 238:23-28. 377 8. Morita Y, Sobel ML, Poole K. 2006. Antibiotic inducibility of the MexXY multidrug 378 efflux system of Pseudomonas aeruginosa: involvement of the antibiotic-inducible 379 PA5471 gene product. J Bacteriol 188:1847-1855. 380 9. Sobel ML, McKay GA, Poole K. 2003. Contribution of the MexXY multidrug 381 transporter to aminoglycoside resistance in Pseudomonas aeruginosa clinical isolates. 382 Antimicrob Agents Chemother 47:3202-3207. 383 10. Morita Y, Tomida J, Kawamura Y. 2012. MexXY multidrug efflux system of 384 Pseudomonas aeruginosa. Front Microbiol 3:408. 385 11. Islam S, Jalal S, Wretlind B. 2004. Expression of the MexXY efflux pump in amikacin386 resistant isolates of Pseudomonas aeruginosa. Clin Microbiol Infect 10:877-883. 387 12. Drenkard E, Ausubel FM. 2002. Pseudomonas biofilm formation and antibiotic 388 resistance are linked to phenotypic variation. Nature 416:740-743. 389 13. Hatch RA, Schiller NL. 1998. Alginate lyase promotes diffusion of aminoglycosides 390 through the extracellular polysaccharide of mucoid Pseudomonas aeruginosa. 391 Antimicrob Agents Chemother 42:974-977. 392 14. Moker N, Dean CR, Tao J. 2010. Pseudomonas aeruginosa increases formation of 393 multidrug-tolerant persister cells in response to quorum-sensing signaling molecules. 394 J Bacteriol 192:1946-1955. 395 15. Haussler S. 2004. Biofilm formation by the small colony variant phenotype of 396 Pseudomonas aeruginosa. Environ Microbiol 6:546-551. 397 16. Gerber AU, Vastola AP, Brandel J, Craig WA. 1982. Selection of aminoglycoside398 resistant variants of Pseudomonas aeruginosa in an in vivo model. J Infect Dis 399 146:691-697. 400 17. Langford PR, Anwar H, Gonda I, Brown MR. 1989. Outer membrane proteins of 401 gentamicin induced small colony variants of Pseudomonas aeruginosa. FEMS 402 Microbiol Lett 52:33-36. 403 18. Garcia LG, Lemaire S, Kahl BC, Becker K, Proctor RA, Denis O, Tulkens PM, Van 404 Bambeke F. 2013. Antibiotic activity against small-colony variants of Staphylococcus 405 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 18 aureus: review of in vitro, animal and clinical data. J Antimicrob Chemother 68:1455406 1464. 407 19. Malone JG, Jaeger T, Manfredi P, Dötsch A, Blanka A, Bos R, Cornelis GR, Häussler S, 408 Jenal U. 2012. The YfiBNR signal transduction mechanism reveals novel targets for 409 the evolution of persistent Pseudomonas aeruginosa in cystic fibrosis airways. PLoS 410 Pathog 8:e1002760. 411 20. Blanka A, Duvel J, Dotsch A, Klinkert B, Abraham WR, Kaever V, Ritter C, Narberhaus 412 F, Haussler S. 2015. Constitutive production of c-di-GMP is associated with mutations 413 in a variant of Pseudomonas aeruginosa with altered membrane composition. Sci 414 Signal 8:ra36. 415 21. Wei Q, Tarighi S, Dötsch A, Häussler S, Müsken M, Wright VJ, Camara M, Williams P, 416 Haenen S, Boerjan B, Bogaerts A, Vierstraete E, Verleyen P, Schoofs L, Willaert R, De 417 Groote VN, Michiels J, Vercammen K, Crabbe A, Cornelis P. 2011. Phenotypic and 418 genome-wide analysis of an antibiotic-resistant small colony variant (SCV) of 419 Pseudomonas aeruginosa. PLoS One 6:e29276. 420 22. Lim WS, Phang KK, Tan AH, Li SF, Ow DS. 2016. Small Colony Variants and Single 421 Nucleotide Variations in Pf1 Region of PB1 Phage-Resistant Pseudomonas 422 aeruginosa. Front Microbiol 7:282. 423 23. D'Argenio DA, Calfee MW, Rainey PB, Pesci EC. 2002. Autolysis and autoaggregation 424 in Pseudomonas aeruginosa colony morphology mutants. J Bacteriol 184:6481-6489. 425 24. Hickman JW, Tifrea DF, Harwood CS. 2005. A chemosensory system that regulates 426 biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci 427 U S A 102:14422-14427. 428 25. Häussler S, Tümmler B, Weissbrodt H, Rohde M, Steinmetz I. 1999. Small-colony 429 variants of Pseudomonas aeruginosa in cystic fibrosis. Clin Infect Dis 29:621-625. 430 26. Badenoch PR, Coster DJ. 1989. Selection of gentamicin-resistant variants of 431 Pseudomonas aeruginosa in the rat cornea. J Ocul Pharmacol 5:19-25. 432 27. Kirisits MJ, Prost L, Starkey M, Parsek MR. 2005. Characterization of colony 433 morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ 434 Microbiol 71:4809-4821. 435 28. Malone JG, Jaeger T, Spangler C, Ritz D, Spang A, Arrieumerlou C, Kaever V, 436 Landmann R, Jenal U. 2010. YfiBNR mediates cyclic di-GMP dependent small colony 437 variant formation and persistence in Pseudomonas aeruginosa. PLoS Pathog 438 6:e1000804. 439 29. Starkey M, Hickman JH, Ma L, Zhang N, De Long S, Hinz A, Palacios S, Manoil C, Kirisits 440 MJ, Starner TD, Wozniak DJ, Harwood CS, Parsek MR. 2009. Pseudomonas aeruginosa 441 rugose small-colony variants have adaptations that likely promote persistence in the 442 cystic fibrosis lung. J Bacteriol 191:3492-3503. 443 30. Khaledi A, Schniederjans M, Pohl S, Rainer R, Bodenhofer U, Xia B, Klawonn F, 444 Bruchmann S, Preusse M, Eckweiler D, Dotsch A, Haussler S. 2016. Transcriptome 445 Profiling of Antimicrobial Resistance in Pseudomonas aeruginosa. Antimicrob Agents 446 Chemother 60:4722-4733. 447 31. Dotsch A, Schniederjans M, Khaledi A, Hornischer K, Schulz S, Bielecka A, Eckweiler D, 448 Pohl S, Haussler S. 2015. The Pseudomonas aeruginosa Transcriptional Landscape Is 449 Shaped by Environmental Heterogeneity and Genetic Variation. MBio 6:e00749. 450 32. Pohl S, Klockgether J, Eckweiler D, Khaledi A, Schniederjans M, Chouvarine P, 451 Tummler B, Haussler S. 2014. The extensive set of accessory Pseudomonas 452 aeruginosa genomic components. FEMS Microbiol Lett 356:235-241. 453 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 19 33. McPhee JB, Lewenza S, Hancock RE. 2003. Cationic antimicrobial peptides activate a 454 two-component regulatory system, PmrA-PmrB, that regulates resistance to 455 polymyxin B and cationic antimicrobial peptides in Pseudomonas aeruginosa. Mol 456 Microbiol 50:205-217. 457 34. Johnson L, Mulcahy H, Kanevets U, Shi Y, Lewenza S. 2012. Surface-localized 458 spermidine protects the Pseudomonas aeruginosa outer membrane from antibiotic 459 treatment and oxidative stress. J Bacteriol 194:813-826. 460 35. Fernandez L, Gooderham WJ, Bains M, McPhee JB, Wiegand I, Hancock RE. 2010. 461 Adaptive resistance to the "last hope" antibiotics polymyxin B and colistin in 462 Pseudomonas aeruginosa is mediated by the novel two-component regulatory 463 system ParR-ParS. Antimicrob Agents Chemother 54:3372-3382. 464 36. Kwon DH, Lu CD. 2006. Polyamines induce resistance to cationic peptide, 465 aminoglycoside, and quinolone antibiotics in Pseudomonas aeruginosa PAO1. 466 Antimicrob Agents Chemother 50:1615-1622. 467 37. Lee S, Hinz A, Bauerle E, Angermeyer A, Juhaszova K, Kaneko Y, Singh PK, Manoil C. 468 2009. Targeting a bacterial stress response to enhance antibiotic action. Proc Natl 469 Acad Sci U S A 106:14570-14575. 470 38. Hinz A, Lee S, Jacoby K, Manoil C. 2011. Membrane proteases and aminoglycoside 471 antibiotic resistance. J Bacteriol 193:4790-4797. 472 39. Lau CH, Fraud S, Jones M, Peterson SN, Poole K. 2013. Mutational activation of the 473 AmgRS two-component system in aminoglycoside-resistant Pseudomonas 474 aeruginosa. Antimicrob Agents Chemother 57:2243-2251. 475 40. Moskowitz SM, Brannon MK, Dasgupta N, Pier M, Sgambati N, Miller AK, Selgrade SE, 476 Miller SI, Denton M, Conway SP, Johansen HK, Hoiby N. 2012. PmrB mutations 477 promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin478 treated cystic fibrosis patients. Antimicrob Agents Chemother 56:1019-1030. 479 41. Moskowitz SM, Ernst RK, Miller SI. 2004. PmrAB, a two-component regulatory system 480 of Pseudomonas aeruginosa that modulates resistance to cationic antimicrobial 481 peptides and addition of aminoarabinose to lipid A. J Bacteriol 186:575-579. 482 42. Olaitan AO, Morand S, Rolain JM. 2014. Mechanisms of polymyxin resistance: 483 acquired and intrinsic resistance in bacteria. Front Microbiol 5:643. 484 43. Yu H, Schurr MJ, Deretic V. 1995. Functional equivalence of Escherichia coli sigma E 485 and Pseudomonas aeruginosa AlgU: E. coli rpoE restores mucoidy and reduces 486 sensitivity to reactive oxygen intermediates in algU mutants of P. aeruginosa. J 487 Bacteriol 177:3259-3268. 488 44. Adams MD, Nickel GC, Bajaksouzian S, Lavender H, Murthy AR, Jacobs MR, Bonomo 489 RA. 2009. Resistance to colistin in Acinetobacter baumannii associated with 490 mutations in the PmrAB two-component system. Antimicrob Agents Chemother 491 53:3628-3634. 492 45. Yeaman MR, Yount NY. 2003. Mechanisms of antimicrobial peptide action and 493 resistance. Pharmacol Rev 55:27-55. 494 46. Lewenza S. 2013. Extracellular DNA-induced antimicrobial peptide resistance 495 mechanisms in Pseudomonas aeruginosa. Front Microbiol 4:21. 496 47. Lee JY, Ko KS. 2014. Mutations and expression of PmrAB and PhoPQ related with 497 colistin resistance in Pseudomonas aeruginosa clinical isolates. Diagn Microbiol Infect 498 Dis 78:271-276. 499 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from 20 48. Jochumsen N, Marvig RL, Damkiaer S, Jensen RL, Paulander W, Molin S, Jelsbak L, 500 Folkesson A. 2016. The evolution of antimicrobial peptide resistance in Pseudomonas 501 aeruginosa is shaped by strong epistatic interactions. Nat Commun 7:13002. 502 49. Pournaras S, Poulou A, Dafopoulou K, Chabane YN, Kristo I, Makris D, Hardouin J, 503 Cosette P, Tsakris A, De E. 2014. Growth retardation, reduced invasiveness, and 504 impaired colistin-mediated cell death associated with colistin resistance development 505 in Acinetobacter baumannii. Antimicrob Agents Chemother 58:828-832. 506 50. Sun S, Negrea A, Rhen M, Andersson DI. 2009. Genetic analysis of colistin resistance 507 in Salmonella enterica serovar Typhimurium. Antimicrob Agents Chemother 53:2298508 2305. 509 51. Hraiech S, Roch A, Lepidi H, Atieh T, Audoly G, Rolain JM, Raoult D, Brunel JM, 510 Papazian L, Bregeon F. 2013. Impaired virulence and fitness of a colistin-resistant 511 clinical isolate of Acinetobacter baumannii in a rat model of pneumonia. Antimicrob 512 Agents Chemother 57:5120-5121. 513 52. Snitkin ES, Zelazny AM, Gupta J, Program NCS, Palmore TN, Murray PR, Segre JA. 514 2013. Genomic insights into the fate of colistin resistance and Acinetobacter 515 baumannii during patient treatment. Genome Res 23:1155-1162. 516 53. Lopez-Rojas R, Dominguez-Herrera J, McConnell MJ, Docobo-Perez F, Smani Y, 517 Fernandez-Reyes M, Rivas L, Pachon J. 2011. Impaired virulence and in vivo fitness of 518 colistin-resistant Acinetobacter baumannii. J Infect Dis 203:545-548. 519 54. Davies JA, Harrison JJ, Marques LL, Foglia GR, Stremick CA, Storey DG, Turner RJ, 520 Olson ME, Ceri H. 2007. The GacS sensor kinase controls phenotypic reversion of 521 small colony variants isolated from biofilms of Pseudomonas aeruginosa PA14. FEMS 522 Microbiol Ecol 59:32-46. 523 55. Malone JG. 2015. Role of small colony variants in persistence of Pseudomonas 524 aeruginosa infections in cystic fibrosis lungs. Infect Drug Resist 8:237-247. 525 56. Liberati NT, Urbach JM, Miyata S, Lee DG, Drenkard E, Wu G, Villanueva J, Wei T, 526 Ausubel FM. 2006. An ordered, nonredundant library of Pseudomonas aeruginosa 527 strain PA14 transposon insertion mutants. Proc Natl Acad Sci U S A 103:2833-2838. 528 57. West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ. 1994. Construction of 529 improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and 530 sequence of the region required for their replication in Pseudomonas aeruginosa. 531 Gene 148:81-86. 532 58. Wiegand I, Hilpert K, Hancock RE. 2008. Agar and broth dilution methods to 533 determine the minimal inhibitory concentration (MIC) of antimicrobial substances. 534 Nat Protoc 3:163-175. 535 59. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP. 1998. A broad-host536 range Flp-FRT recombination system for site-specific excision of chromosomally537 located DNA sequences: application for isolation of unmarked Pseudomonas 538 aeruginosa mutants. Gene 212:77-86. 539 60. Oliveros JC. 2007. VENNY. An interactive tool for comparing lists with Venn Diagrams. 540 http://bioinfogp.cnb.csic.es/tools/venny/index.html 541 542 on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from on September 22, 2017 by Helmholtz-Zentrum fuer Infektionsforschung - BIBLIOTHEK-http://aac.asm.org/Downloaded from