Gene expression data from Pseudomonas aeruginosa PAO1 and mutator (Δ mutS) evolved for 940 generations in LB with and without sub-inhibitory concentrations of ciprofloxacin (0.05µg/ml)
ABSTRACT: Gene expression of P. aerruginosa changes after short-term exposure to ciprofloxacin at sub-inhibitory concentrations but the effect of long-term exposure which select for the most fitted subpopulations is not known. We used microarrays to investigate the changes in gene expression of P.aeruginosa PAO1 and mutator (Δ mutS) after long-term evolution (94 daily passages) in LB in the presence and absence of ciprofloxacin Three different colonies from the ancestral populations of PAO1 and mutator (Δ mutS) as well as from the evolved populations (day 94) of each of the three lineages (A;B;C) in the presence or absence of ciprofloxacin at a concentration of 0.05 µg/ml were used for overnight cultures in LB and total RNA was extracted at OD600nm=1 and hybridized on P. aeruginosa Affymetrix chip.
Project description:We compared the dynamics and mechanisms of resistance development to ceftazidime, meropenem, ciprofloxacin, and ceftolozane-tazobactam in wild-type (PAO1) and mutator (PAOMS, ∆mutS) P. aeruginosa. The strains were incubated for 24 h with 0.5 to 64× MICs of each antibiotic in triplicate experiments. The tubes from the highest antibiotic concentration showing growth were reinoculated in fresh medium containing concentrations up to 64× MIC for 7 consecutive days. The susceptibility profiles and resistance mechanisms were assessed in two isolated colonies from each step, antibiotic, and strain. Ceftolozane-tazobactam-resistant mutants were further characterized by whole-genome analysis through RNA sequencing (RNA-seq). The development of high-level resistance was fastest for ceftazidime, followed by meropenem and ciprofloxacin. None of the mutants selected with these antibiotics showed cross-resistance to ceftolozane-tazobactam. On the other hand, ceftolozane-tazobactam resistance development was much slower, and high-level resistance was observed for the mutator strain only. PAO1 derivatives that were moderately resistant (MICs, 4 to 8 ug/ml) to ceftolozane-tazobactam showed only 2 to 4 mutations, which determined global pleiotropic effects associated with a severe fitness cost. High-level-resistant (MICs, 32 to 128 ug/ml) PAOMS derivatives showed 45 to 53 mutations. Major changes in the global gene expression profiles were detected in all mutants, but only PAOMS mutants showed ampC overexpression, which was caused by dacB or ampR mutations. Moreover, all PAOMS mutants contained 1 to 4 mutations in the conserved residues of AmpC (F147L, Q157R, G183D, E247K, or V356I). Complementation studies revealed that these mutations greatly increased ceftolozane-tazobactam and ceftazidime MICs but reduced those of piperacillin-tazobactam and imipenem, compared to those in wild-type ampC. Therefore, the development of high-level resistance to ceftolozane-tazobactam appears to occur efficiently only in a P. aeruginosa mutator background, in which multiple mutations lead to overexpression and structural modifications of AmpC. Mutants of Pseudomonas aeroginosa PAO1 and PAO1 ∆mutS against Ceftolozane-tazobactam were generated and analysed using RNA-Seq
Project description:Pseudomonas aeruginosa PAO1 persister and normal cells were treated with and without Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) to understand the effect of GM-CSF on gene expression of PAO1. We used DNA microarrays to identify the down-regulated and up-regulated genes after GM-CSF treatment. Pseudonmonas aeruginosa PAO1 normal cells were obtained from stationary phase culture and persister cells were isolated by lysing normal cells with ciprofloxacin. The normal cells and isolated persister cells were treated with GM-CSF and selected for RNA extraction and hybridization on Affymetrix microarrays. The experiment was performed in two biological replicates. The samples for the biological replciates were: Control C1, Treatment T1, Control C2, and Treatment T2.
Project description:To provide a more detailed survey of adaptive changes in the physiology of P. aeruginosa (PA) during long-term infection of the cystic fibrosis (CF) lung, we performed a comparative proteome and transcriptome analysis of a set of isogenic sequential non-mutator and mutator isolates from three selected CF patients. Recently, we showed that during CF lung persistence PA mutators converge to a virulence-attenuated phenotype. In this study, we demonstrate that besides virulence-associated traits (VATs) the adaptation process of PA predominantly comprises metabolic pathways. In end-stage mutator strains, transcripts of genes encoding VATs, chemotaxis, degradation of aromatic compounds and several two-component regulatory systems were decreased. In contrast, several transcripts of genes or proteins involved in metabolism of fatty acids, nucleotides, amino acids and the generation of energy were increased. Of particular interest is the increased expression level of genes involved in (i) the anaerobic arginine-deiminase pathway, (ii) the anaerobic respiration such as nitrate-uptake protein OprF, redox-active azurin and cytchrome c551 peroxidase, (iii) the micro-aerobic respiration such as high oxygen-affinity cytochrome oxidase cbb3 (iv) the tricarboxylic acid cycle (TCA), glyoxylate shunt and anaplerotic carboxylation reactions to oxaloacetate. Strikingly, an increased transcription of the anaerobic regulator gene anr correlates with the up-regulation of ANR-dependent genes. In conclusion, these changes in transcriptome and proteome indicate an adaptive shift towards constitutive expression of genes of metabolic pathways obviously required for growth under micro-aerobic and nutritional conditions of suppurative CF lung tissue. Finally, these results provide us with new targets for antimicrobial agents and biomarkers reflecting adaptation of PA towards progressive CF lung disease. Experiment Overall Design: P. aeruginosa isolates recovered from different time points of chronic cystic fibrosis lung disease were cultered in vitro, harvested for RNA extraction and hybridization on Affymetrix microarrays. We compared the transcriptome (triplicate microarrays) of early non-mutator P. aeruginosa isolates with late mutator isolates with high mutation frequency probably the driving force of an efficient adaptation to changing environements to conclude from differences in gene expression to the requirements of CF lung environment. Experiment Overall Design: Second publication of array data to be added later
Project description:PAO1 was grown in sub-inhibitory ciprofloxacin (0.1x-, 0.3x-, 1x-MIC) until log phase. Microarrays were done on total RNA isolated from these cultures. Loop design and dye swapping were used.
Project description:Evolution of antibiotic resistance in microbes is frequently achieved by acquisition of spontaneous mutations during antimicrobial therapy. Here we demonstrate that inactivation of a central regulator of iron homeostasis (fur) facilitates laboratory evolution of ciprofloxacin resistance in Escherichia coli. To decipher the underlying molecular mechanisms, we first performed a global transcriptome analysis and demonstrated a substantial reorganization of the Fur regulon in response to antibiotic treatment. We hypothesized that the impact of Fur on evolvability under antibiotic pressure is due to the elevated intracellular concentration of free iron and the consequent enhancement of oxidative damage-induced mutagenesis. In agreement with expectations, over-expression of iron storage proteins, inhibition of iron transport, or anaerobic conditions drastically suppressed the evolution of resistance, while inhibition of the SOS response-mediated mutagenesis had no such effect in fur deficient population. In sum, our work revealed the central role of iron metabolism in de novo evolution of antibiotic resistance, a pattern that could influence the development of novel antimicrobial strategies. We used microarrays to identify genotype specific transcriptional changes under severe DNA damaging conditions (antibiotic ciprofloxacin). We treated Escherichia coli cells with a highly toxic level of ciprofloxacin (gyrase inhibitor) for RNA extraction and hybridization on Affymetrix microarrays. We planned to find genotype specific transcriptional responses using WT control (BW25113) and fur-knockout mutant (selected from the KEIO collection) strains during antibiotic treatments. For each treatment type we used two biological replicates.
Project description:Gene expression data from Pseudomonas aeruginosa PAO1 and mutator (Δ mutS) evolved for 940 generations in LB with and without sub-inhibitory concentrations of ciprofloxacin (0.05µg/ml)
Project description:The pqs operon of the bacteria P. aeruginosa is responsible for the production of at least 60 secreted compounds mainly hidroxy-quinolones (HAQs). Some of them, like PQS and HHQ, were found to be important signal that control the virulence of the bacteria. We have discover that one of the most abandon molecule produced by the pqs operon, 2 aminoacetophenone (2-AA), a volatile molecule serves as a unique signal. In contrast to the previous described PQS and HHQ, 2-AA is downregulating virulence and acute phase proteins and upregulating chronic phase proteins. In addition we have found that 2-AA promotes the formation of persisters cells that antibiotic tolerant. we have compared the genes profile of wild-type PA14 strain and its derivatives topA which lacks Topoisomerase I and pqsA that do not produces HAQs or 2-AA to cultures that were treated with 3mM of 2-AA. Cell were grown with and without 2-AA (3mM) to OD 2.0.
Project description:We used Affymetrix microarrays to determine the cisplatin-induced gene expression changes in E. coli deficient in dam and mismatch repair (dam, dam mutS, and mutS mutant strains). E. coli deficient in dam are hypersensitive to cisplatin. However introducing an additional mutation in mismatch repair (i.e., a mutation in mutS or mutL) abrogates this sensitivity and essentially restores wildtype levels of resistance. Experiment Overall Design: Overnight cultures were diluted 1000-fold and grown in Luria-Bertani (LB) medium until the cells reached exponential growth as determined by OD600. The exponentially growing cells were resuspended in M9 minimal medium at a cell density of 2 X 108 cells/ml in a volume of 15 ml and treated with 150 uM cisplatin for 2 hours at 37°C. Following treatment cultures were resuspended in 15 ml LB and allowed to recover for 90 minutes at 37°C. OD600 readings were taken after the recovery period, when RNA isolation began. Total RNA was isolated from cells by extraction using the MasterPure RNA Purification Kit (Epicentre Technologies) according to the manufacture’s protocol. The isolated total RNA was quantitated by absorption at 260 nm (typical yield from a 15 ml culture was 250-500 µg of total RNA), and the purity was determined by the ratio of absorption values at 260/280nm. RNA quality was determined by formaldehyde agarose gel electrophoresis (1.2% agarose in FA Buffer pH 7.0 (20mM 3-[N-morpholino]propanesulfonic acid, 5mM sodium acetate, 1mM ethylenadiaminetetraacetic acide (EDTA)) or by analysis on an Agilent 2100 Bioanalyzer. All samples visualized by gel electrophoresis or by the bioanalyzer electropherogram showed clear distinct bands correlating to 16S and 23S ribosomal RNA, indicating that no detectable RNA degradation occurred and that RNA integrity was maintained throughout the RNA isolation procedure.
Project description:Pseudomonas aeruginosa evolving in the cystic fibrosis (CF) lung encounters selection via iron-limitation, antibiotics, immune system effectors and other microbes. Standing genetic variation, which depends on rates of horizontal gene transfer (HGT) and mutation supply, controls response to challenges. HGT may increase if new clones successfully invade, while mutator strains increase new mutations. We sought to ascertain genomic signatures of invasion and whether, in the absence of novel invasion, mutator-containing P. aeruginosa populations from chronically infected CF lung are more genetically variable than nonmutator populations. Forty-nine strains from 14 patients treated over three years at Necker Children’s Hospital in Paris were phenotyped for antibiotic resistance, mucoidy and mutator status, and then genotyped by rep-PCR, PFGE and MLST analysis. Overall, strains exhibited greater genetic similarity within patients than among patients, with initial and terminal clones differing markedly between series, indicating unrelated clones independently established infections and resisted invasions that might enlarge genetic variation by sexual recombination. Mutator series were more likely to be multiply antibiotic-resistant, but were no more genetically variable at the single nucleotide level. DNA microarray analyses of bacterial genomes in two longitudinal series of equal duration, one containing and one lacking mutators, revealed both series conspicuously lack genes encoding proteins involved in attachment, motility and amino acid biosynthesis. The mutator series also contained fewer genes hybridizing to canonical PAO1 genome sequences. These data suggest genetic variation arising from mutators may be limited in scope, transient in nature or not easily resolved by fingerprinting, MLST or comparative genomic analyses. Clinical P. aeruginosa hypermutator and nonhypermutator lineages from CF lung. Array-based genome hybridization.