scholarly journals Segregation but Not Replication of the Pseudomonas aeruginosa Chromosome Terminates at Dif

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Bijit K. Bhowmik ◽  
April L. Clevenger ◽  
Hang Zhao ◽  
Valentin V. Rybenkov
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Chromosome Segregation ◽  
Large Scale ◽  
Critical Role ◽  
Genetic Material ◽  
Chromosome Replication ◽  
Content Type ◽  
Chromosome Partitioning ◽  
Bacterial Chromosomes ◽  
Large Domains

ABSTRACT Coordination between chromosome replication and segregation is essential for equal partitioning of genetic material between daughter cells. In bacteria, this is achieved through the proximity of the origin of replication, oriC, and the chromosome partitioning site, parS. We report here that in Pseudomonas aeruginosa, segregation but not replication is also controlled at the terminus region of the chromosome. Using the fluorescent repressor operator system (FROS), we investigated chromosome segregation in P. aeruginosa strain PAO1-UW, wherein the chromosome dimer resolution site, dif, is asymmetrically positioned relative to oriC. In these cells, segregation proceeded sequentially along the two chromosomal arms and terminated at dif. In contrast, chromosome replication terminated elsewhere, opposite from oriC. We further found two large domains on the longer arm of the chromosome, wherein DNA segregated simultaneously. Notably, GC-skew, which reflects a bias in nucleotide usage between the leading and lagging strands of the chromosome, switches polarity at the dif locus but not necessarily at the terminus of replication. These data demonstrate that termination of chromosome replication and segregation can be physically separated without adverse effects on bacterial fitness. They also reveal the critical role of the dif region in defining the global layout of the chromosome and the progression of chromosome segregation and suggest that chromosome packing adapts to its subcellular layout. IMPORTANCE Segregation of genetic information is a central event in cellular life. In bacteria, chromosome segregation occurs concurrently with replication, sequentially along the two arms from oriC to dif. How the two processes are coordinated is unknown. We explored here chromosome segregation in an opportunistic human pathogen, Pseudomonas aeruginosa, using its strain with markedly unequal chromosomal arms. We found that replication and segregation diverge in this strain and terminate at very different locations, whereas the longer chromosomal arm folds into large domains to align itself with the shorter arm. The significance of this research is in establishing that segregation and replication of bacterial chromosomes are largely uncoupled from each other and that the large-scale structure of the chromosome adapts to its subcellular layout.

scholarly journals Chromosomal Rearrangements in Salmonella enterica Serovar Typhi Strains Isolated from Asymptomatic Human Carriers

mBio ◽  
2011 ◽  
Vol 2 (3) ◽  
Author(s):  
T. David Matthews ◽  
Wolfgang Rabsch ◽  
Stanley Maloy
Keyword(s):  
Salmonella Enterica ◽  
Large Scale ◽  
Chromosomal Rearrangements ◽  
Growth Conditions ◽  
Genetic Changes ◽  
Content Type ◽  
Long Term Storage ◽  
Bacterial Chromosomes ◽  
Over Time

ABSTRACTHost-specific serovars ofSalmonella entericaoften have large-scale chromosomal rearrangements that occur by recombination betweenrrnoperons. Two hypotheses have been proposed to explain these rearrangements: (i) replichore imbalance from horizontal gene transfer drives the rearrangements to restore balance, or (ii) the rearrangements are a consequence of the host-specific lifestyle. Although recent evidence has refuted the replichore balance hypothesis, there has been no direct evidence for the lifestyle hypothesis. To test this hypothesis, we determined therrnarrangement type for 20Salmonella entericaserovar Typhi strains obtained from human carriers at periodic intervals over multiple years. These strains were also phage typed and analyzed for rearrangements that occurred over long-term storage versus routine culturing. Strains isolated from the same carrier at different time points often exhibited different arrangement types. Furthermore, colonies isolated directly from the Dorset egg slants used to store the strains also had different arrangement types. In contrast, colonies that were repeatedly cultured always had the same arrangement type. Estimated replichore balance of isolated strains did not improve over time, and some of the rearrangements resulted in decreased replicore balance. Our results support the hypothesis that the restricted lifestyle of host-specificSalmonellais responsible for the frequent chromosomal rearrangements in these serovars.IMPORTANCEAlthough it was previously thought that bacterial chromosomes were stable, comparative genomics has demonstrated that bacterial chromosomes are dynamic, undergoing rearrangements that change the order and expression of genes. While mostSalmonellastrains have a conserved chromosomal arrangement type, rearrangements are very common in host-specificSalmonellastrains. This study suggests that chromosome rearrangements in the host-specificSalmonella entericaserovar Typhi, the causal agent of typhoid fever, occur within the human host over time. The results also indicate that rearrangements can occur during long-term maintenance on laboratory medium. Although these genetic changes do not limit survival under slow-growth conditions, they may limit the survival ofSalmonellaTyphi in other environments, as predicted for the role of pseudogenes and genome reduction in niche-restricted bacteria.

scholarly journals A Screen for Antibiotic Resistance Determinants Reveals a Fitness Cost of the Flagellum in Pseudomonas aeruginosa

2019 ◽  
Vol 202 (6) ◽  
Author(s):  
E. A. Rundell ◽  
N. Commodore ◽  
A. L. Goodman ◽  
B. I. Kazmierczak
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Outer Membrane ◽  
Critical Role ◽  
Fitness Cost ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Fitness Advantage ◽  
Membrane Barrier ◽  
Flagellar Assembly ◽  
Cell Envelopes

ABSTRACT The intrinsic resistance of Pseudomonas aeruginosa to many antibiotics limits treatment options for pseudomonal infections. P. aeruginosa’s outer membrane is highly impermeable and decreases antibiotic entry into the cell. We used an unbiased high-throughput approach to examine mechanisms underlying outer membrane-mediated antibiotic exclusion. Insertion sequencing (INSeq) identified genes that altered fitness in the presence of linezolid, rifampin, and vancomycin, antibiotics to which P. aeruginosa is intrinsically resistant. We reasoned that resistance to at least one of these antibiotics would depend on outer membrane barrier function, as previously demonstrated in Escherichia coli and Vibrio cholerae. This approach demonstrated a critical role of the outer membrane barrier in vancomycin fitness, while efflux pumps were primary contributors to fitness in the presence of linezolid and rifampin. Disruption of flagellar assembly or function was sufficient to confer a fitness advantage to bacteria exposed to vancomycin. These findings clearly show that loss of flagellar function alone can confer a fitness advantage in the presence of an antibiotic. IMPORTANCE The cell envelopes of Gram-negative bacteria render them intrinsically resistant to many classes of antibiotics. We used insertion sequencing to identify genes whose disruption altered the fitness of a highly antibiotic-resistant pathogen, Pseudomonas aeruginosa, in the presence of antibiotics usually excluded by the cell envelope. This screen identified gene products involved in outer membrane biogenesis and homeostasis, respiration, and efflux as important contributors to fitness. An unanticipated fitness cost of flagellar assembly and function in the presence of the glycopeptide antibiotic vancomycin was further characterized. These findings have clinical relevance for individuals with cystic fibrosis who are infected with P. aeruginosa and undergo treatment with vancomycin for a concurrent Staphylococcus aureus infection.

scholarly journals Chromosome Dynamics in Bacteria: Triggering Replication at the Opposite Location and Segregation in the Opposite Direction

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Ady B. Meléndez ◽  
Inoka P. Menikpurage ◽  
Paola E. Mera
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Opposite Direction ◽  
Caulobacter Crescentus ◽  
Chromosome Replication ◽  
Opposite Orientation ◽  
Circular Chromosome ◽  
Content Type ◽  
Cell Pole ◽  
Protein Gradients

ABSTRACT Maintaining the integrity of the genome is essential to cell survival. In the bacterium Caulobacter crescentus, the single circular chromosome exhibits a specific orientation in the cell, with the replication origin (ori) residing at the pole of the cell bearing a stalk. Upon initiation of replication, the duplicated centromere-like region parS and ori move rapidly to the opposite pole where parS is captured by a microdomain hosting a unique set of proteins that contribute to the identity of progeny cells. Many questions remain as to how this organization is maintained. In this study, we constructed strains of Caulobacter in which ori and the parS centromere can be induced to move to the opposite cell pole in the absence of chromosome replication, allowing us to ask whether once these chromosomal foci were positioned at the wrong pole, replication initiation and chromosome segregation can proceed in the opposite orientation. Our data reveal that DnaA can initiate replication and ParA can orchestrate segregation from either cell pole. The cell reconstructs the organization of its ParA gradient in the opposite orientation to segregate one replicated centromere from the new pole toward the stalked pole (i.e., opposite direction), while displaying no detectable viability defects. Thus, the unique polar microdomains exhibit remarkable flexibility in serving as a platform for directional chromosome segregation along the long axis of the cell. IMPORTANCE Bacteria can accomplish surprising levels of organization in the absence of membrane organelles by constructing subcellular asymmetric protein gradients. These gradients are composed of regulators that can either trigger or inhibit cell cycle events from distinct cell poles. In Caulobacter crescentus, the onset of chromosome replication and segregation from the stalked pole are regulated by asymmetric protein gradients. We show that the activators of chromosome replication and segregation are not restricted to the stalked pole and that their organization and directionality can be flipped in orientation. Our results also indicate that the subcellular location of key chromosomal loci play important roles in the establishment of the asymmetric organization of cell cycle regulators.

scholarly journals Metapopulation Structure of CRISPR-Cas Immunity inPseudomonas aeruginosaand Its Viruses

mSystems ◽  
2018 ◽  
Vol 3 (5) ◽  
Author(s):  
Whitney E. England ◽  
Ted Kim ◽  
Rachel J. Whitaker
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Microbial Populations ◽  
Bacterial Strains ◽  
Viral Control ◽  
Data Set ◽  
Content Type ◽  
Global Population

ABSTRACTViruses that infect the widespread opportunistic pathogenPseudomonas aeruginosahave been shown to influence physiology and critical clinical outcomes in cystic fibrosis (CF) patients. To understand how CRISPR-Cas immune interactions may contribute to the distribution and coevolution ofP. aeruginosaand its viruses, we reconstructed CRISPR arrays from a highly sampled longitudinal data set from CF patients attending the Copenhagen Cystic Fibrosis Clinic in Copenhagen, Denmark (R. L. Marvig, L. M. Sommer, S. Molin, and H. K. Johansen, Nat Genet 47:57–64, 2015,https://doi.org/10.1038/ng.3148). We show that new spacers are not added to or deleted from CRISPR arrays over time within a single patient but do vary among patients in this data set. We compared assembled CRISPR arrays from this data set to CRISPR arrays extracted from 726 additional publicly availableP. aeruginosasequences to show that local diversity in this population encompasses global diversity and that there is no evidence for population structure associated with location or environment sampled. We compare over 3,000 spacers from our global data set to 98 lytic and temperate viruses and proviruses and find a subset of related temperate virus clusters frequently targeted by CRISPR spacers. Highly targeted viruses are matched by different spacers in different arrays, resulting in a pattern of distributed immunity within the global population. Understanding the multiple immune contexts thatP. aeruginosaviruses face can be applied to study ofP. aeruginosagene transfer, the spread of epidemic strains in cystic fibrosis patients, and viral control ofP. aeruginosainfection.IMPORTANCEPseudomonas aeruginosais a widespread opportunistic pathogen and a major cause of morbidity and mortality in cystic fibrosis patients. Microbe-virus interactions play a critical role in shaping microbial populations, as viral infections can kill microbial populations or contribute to gene flow among microbes. Investigating howP. aeruginosauses its CRISPR immune system to evade viral infection aids our understanding of how this organism spreads and evolves alongside its viruses in humans and the environment. Here, we identify patterns of CRISPR targeting and immunity that indicateP. aeruginosaand its viruses evolve in both a broad global population and in isolated human “islands.” These data set the stage for exploring metapopulation dynamics occurring within and between isolated “island” populations associated with CF patients, an essential step to inform future work predicting the specificity and efficacy of virus therapy and the spread of invasive viral elements and pathogenic epidemic bacterial strains.

scholarly journals The Pseudomonas aeruginosa PrrF Small RNAs Regulate Iron Homeostasis during Acute Murine Lung Infection

2017 ◽  
Vol 85 (5) ◽  
Author(s):  
Alexandria A. Reinhart ◽  
Angela T. Nguyen ◽  
Luke K. Brewer ◽  
Justin Bevere ◽  
Jace W. Jones ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Small Rnas ◽  
Iron Homeostasis ◽  
Critical Role ◽  
Opportunistic Pathogen ◽  
Lung Infection ◽  
Content Type ◽  
The Individual

ABSTRACT Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that requires iron for virulence. Iron homeostasis is maintained in part by the PrrF1 and PrrF2 small RNAs (sRNAs), which block the expression of iron-containing proteins under iron-depleted conditions. The PrrF sRNAs also promote the production of the Pseudomonas quinolone signal (PQS), a quorum sensing molecule that activates the expression of several virulence genes. The tandem arrangement of the prrF genes allows for expression of a third sRNA, PrrH, which is predicted to regulate gene expression through its unique sequence derived from the prrF1-prrF2 intergenic (IG) sequence (the PrrHIG sequence). Previous studies showed that the prrF locus is required for acute lung infection. However, the individual functions of the PrrF and PrrH sRNAs were not determined. Here, we describe a system for differentiating PrrF and PrrH functions by deleting the PrrHIG sequence [prrF(ΔHIG)]. Our analyses of this construct indicate that the PrrF sRNAs, but not PrrH, are required for acute lung infection by P. aeruginosa. Moreover, we show that the virulence defect of the ΔprrF1-prrF2 mutant is due to decreased bacterial burden during acute lung infection. In vivo analysis of gene expression in lung homogenates shows that PrrF-mediated regulation of genes for iron-containing proteins is disrupted in the ΔprrF1-prrF2 mutant during infection, while the expression of genes that mediate PrrF-regulated PQS production are not affected by prrF deletion in vivo. Combined, these studies demonstrate that regulation of iron utilization plays a critical role in P. aeruginosa's ability to survive during infection.

scholarly journals Regulation of Rab5 Function during Phagocytosis of Live Pseudomonas aeruginosa in Macrophages

2013 ◽  
Vol 81 (7) ◽  
pp. 2426-2436 ◽  
Author(s):  
Sushmita Mustafi ◽  
Nathalie Rivero ◽  
Joan C. Olson ◽  
Philip D. Stahl ◽  
M. Alejandro Barbieri
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Trafficking ◽  
Rho Gtpase ◽  
Critical Role ◽  
Rab Gtpases ◽  
Content Type ◽  
Host Cell Proteins ◽  
Opportunistic Human Pathogen ◽  
Hospital Acquired ◽  
Adp Ribosyltransferase

ABSTRACTPseudomonas aeruginosa, a Gram-negative opportunistic human pathogen, is a frequent cause of severe hospital-acquired infections. Effectors produced by the type III secretion system disrupt mammalian cell membrane trafficking and signaling and are integral to the establishment ofP. aeruginosainfection. One of these effectors, ExoS, ADP-ribosylates several host cell proteins, including Ras and Rab GTPases. In this study, we demonstrated that Rab5 plays a critical role during early stages ofP. aeruginosainvasion of J774-Eclone macrophages. We showed that live, but not heat-inactivated,P. aeruginosainhibited phagocytosis and that this occurred in conjunction with downregulation of Rab5 activity. Inactivation of Rab5 was dependent on ExoS ADP-ribosyltransferase activity, and in J744-Eclone cells, ExoS ADP-ribosyltransferase activity caused a more severe inhibition of phagocytosis than ExoS Rho GTPase activity. Furthermore, we found that expression of Rin1, a Rab5 guanine exchange factor, but not Rabex5 and Rap6, partially reversed the inactivation of Rab5 during invasion of liveP. aeruginosa. These studies provide evidence that liveP. aeruginosacells are able to influence their rate of phagocytosis in macrophages by directly regulating activation of Rab5.

scholarly journals Pseudomonas aeruginosa ExsA Regulates a Metalloprotease, ImpA, That Inhibits Phagocytosis of Macrophages

2019 ◽  
Vol 87 (12) ◽  
Author(s):  
Zhenyang Tian ◽  
Sen Cheng ◽  
Bin Xia ◽  
Yongxin Jin ◽  
Fang Bai ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Critical Role ◽  
Surface Protein ◽  
Host Cells ◽  
Bacterial Cells ◽  
Mobility Shift ◽  
Content Type ◽  
Direct Binding ◽  
Mobility Shift Assay ◽  
Opportunistic Pathogenic

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogenic bacterium whose type III secretion system (T3SS) plays a critical role in acute infections. Translocation of the T3SS effectors into host cells induces cytotoxicity. In addition, the T3SS promotes the intracellular growth of P. aeruginosa during host infections. The T3SS regulon genes are regulated by an AraC-type regulator, ExsA. In this study, we found that an extracellular metalloprotease encoded by impA (PA0572) is under the regulation of ExsA. An ExsA consensus binding sequence was identified upstream of the impA gene, and direct binding of the site by ExsA was demonstrated via an electrophoretic mobility shift assay. We further demonstrate that secreted ImpA cleaves the macrophage surface protein CD44, which inhibits the phagocytosis of the bacterial cells by macrophages. Combined, our results reveal a novel ExsA-regulated virulence factor that cooperatively inhibits the functions of macrophages with the T3SS.

scholarly journals Choreography of the Mycobacterium Replication Machinery during the Cell Cycle

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Damian Trojanowski ◽  
Katarzyna Ginda ◽  
Monika Pióro ◽  
Joanna Hołówka ◽  
Partycja Skut ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Mycobacterium Smegmatis ◽  
Chromosome Replication ◽  
Specific Cell ◽  
Replication Machinery ◽  
Content Type ◽  
Chromosome Dynamics ◽  
Mycobacterial Cell ◽  
Slow Growing

ABSTRACT It has recently been demonstrated that bacterial chromosomes are highly organized, with specific positioning of the replication initiation region. Moreover, the positioning of the replication machinery (replisome) has been shown to be variable and dependent on species-specific cell cycle features. Here, we analyzed replisome positions in Mycobacterium smegmatis, a slow-growing bacterium that exhibits characteristic asymmetric polar cell extension. Time-lapse fluorescence microscopy analyses revealed that the replisome is slightly off-center in mycobacterial cells, a feature that is likely correlated with the asymmetric growth of Mycobacterium cell poles. Estimates of the timing of chromosome replication in relation to the cell cycle, as well as cell division and chromosome segregation events, revealed that chromosomal origin-of-replication (oriC) regions segregate soon after the start of replication. Moreover, our data demonstrate that organization of the chromosome by ParB determines the replisome choreography. IMPORTANCE Despite significant progress in elucidating the basic processes of bacterial chromosome replication and segregation, understanding of chromosome dynamics during the mycobacterial cell cycle remains incomplete. Here, we provide in vivo experimental evidence that replisomes in Mycobacterium smegmatis are highly dynamic, frequently splitting into two distinct replication forks. However, unlike in Escherichia coli, the forks do not segregate toward opposite cell poles but remain in relatively close proximity. In addition, we show that replication cycles do not overlap. Finally, our data suggest that ParB participates in the positioning of newly born replisomes in M. smegmatis cells. The present results broaden our understanding of chromosome segregation in slow-growing bacteria. In view of the complexity of the mycobacterial cell cycle, especially for pathogenic representatives of the genus, understanding the mechanisms and factors that affect chromosome dynamics will facilitate the identification of novel antimicrobial factors.

Strategic responses to institutional pressures for sustainability

2017 ◽  
Vol 30 (8) ◽  
pp. 1677-1710 ◽  
Author(s):  
Chaminda Wijethilake ◽  
Rahat Munir ◽  
Ranjith Appuhami
Keyword(s):  
Large Scale ◽  
Critical Role ◽  
Organisational Change ◽  
Management Control ◽  
Institutional Pressures ◽  
Strategic Responses ◽  
Apparel Manufacturing ◽  
Content Type ◽  
Sustainability Challenges ◽  
Outsourcing Contracts

Purpose The purpose of this paper is to examine the role of management control systems (MCS) in strategically responding to institutional pressures for sustainability (IPS). Drawing on institutional theory (DiMaggio and Powell, 1983) and strategic responses to institutional pressures framework (Oliver, 1991), the study argues that organisations strategically respond to IPS using MCS. Design/methodology/approach Data were collected by interviewing sustainability managers of a large-scale multinational apparel manufacturing organisation with its headquarters in Sri Lanka. Findings The study finds that organisations actively respond to IPS using acquiescence, compromise, avoidance, defiance, and manipulation strategies. The results not only reveal that formal MCS play a critical role in complying with IPS, but also in more active responses, including compromise, avoidance, defiance, and manipulation. The findings highlight that organisations use MCS as a medium to respond strategically to IPS, and in turn, the use of MCS has important implications for organisational change and improvement. Practical implications The study has implications for Western organisations, finding that suppliers committed to sustainability in Asia strategically respond to IPS as a means of strengthening outsourcing contracts, instead of blindly accepting. Findings indicate that organisational changes and success seem to be a function of strategically responding to IPS rather than operating an organisation by neglecting sustainability challenges. The organisational ability to use MCS in strategically responding to IPS has the potential for long-term value creation. Originality/value This study provides novel insights into the MCS, strategy and sustainability literatures by exploring different uses of MCS tools in strategically responding to IPS. More specifically, it shows how the use of MCS tools varies in supporting strategic responses, and with respective IPS. In doing so, it enhances our understanding of the importance of the use of MCS in dynamics of institutional change and practical variances in strategically responding to IPS.

scholarly journals A Large-Scale Whole-Genome Comparison Shows that Experimental Evolution in Response to Antibiotics Predicts Changes in Naturally Evolved Clinical Pseudomonas aeruginosa

2019 ◽  
Vol 63 (12) ◽  
Author(s):  
Samuel J. T. Wardell ◽  
Attika Rehman ◽  
Lois W. Martin ◽  
Craig Winstanley ◽  
Wayne M. Patrick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Experimental Evolution ◽  
Large Scale ◽  
Opportunistic Pathogen ◽  
Clinical Isolates ◽  
Genome Comparison ◽  
Chronic Infections ◽  
Content Type ◽  
Large Deletions ◽  
Wide Range

ABSTRACT Pseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have mutations that make them antibiotic resistant, making treatment difficult. To identify resistance-associated mutations, we experimentally evolved the antibiotic-sensitive strain P. aeruginosa PAO1 to become resistant to three widely used antipseudomonal antibiotics, namely, ciprofloxacin, meropenem, and tobramycin. Mutants could tolerate up to 2,048-fold higher concentrations of antibiotics than strain PAO1. Genome sequences were determined for 13 mutants for each antibiotic. Each mutant had between 2 and 8 mutations. For each antibiotic, at least 8 genes were mutated in multiple mutants, demonstrating the genetic complexity of resistance. For all three antibiotics, mutations arose in genes known to be associated with resistance but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in this study, we analyzed the corresponding genes in 558 isolates of P. aeruginosa from patients with chronic lung disease and in 172 isolates from the general environment. Many genes identified through experimental evolution had predicted function-altering changes in clinical isolates but not in environmental isolates, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. Additionally, large deletions of up to 479 kb arose in experimentally evolved meropenem-resistant mutants, and large deletions were present in 87 of the clinical isolates. These findings significantly advance understanding of antibiotic resistance in P. aeruginosa and demonstrate the validity of experimental evolution in identifying clinically relevant resistance-associated mutations.

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