Transcription profiling by array of compare two pairs of Burkholderia cenocepacia ST32 clinical isolates (from cystic fibrosis patients) subjected to cultivation in two different conditions
ABSTRACT: Burkholderia cenocepacia sequence type 32 (ST32) represents one of the most globally distributed strains from Bukrholderia cepacia complex (Bcc), which infected 30% of Czech cystic fibrosis (CF) patients. The aim of this study was to compare gene expression in two pairs of ST32 clinical isolates that were subjected to cultivation in two different conditions, characteristic for chronic B. cenocepacia infection in CF patients. ST32 strain is known to be a problematic epidemic strain, which caused a serious outbreak at the Prague CF centre.
Cepacia syndrome (CS) is a fatal septic condition that develops in approximately 20% of cystic fibrosis (CF) patients chronically infected with the Burkholderia cepacia complex (Bcc). The most common causative agent is Burkholderia cenocepacia, a clinically dominant Bcc species that contains the globally distributed epidemic strain sequence type 32 (ST32). Using microarrays, we compared the transcriptomes of ST32 isolates from the bloodstream at the time of CS with their sputum counterparts reco ...[more]
Project description:B. cenocepacia J2315 was grown to mid-log phase in different media: LB broth, iso-sensitest broth, basal salt medium with glucose<br>at pH 7 and pH 6, basal salt medium at pH 7 with a reduced iron content, and basal salt medium with glycerol
Project description:B. cenocepacia J2315 was grown to mid-stationary phase in basal salt medium with two different substrates: 20 mM glucose or 40 mM glycerol. Cells were harvested after 30 hours incubation at 37 degrees centigrade. <br>The expression profile was compared to cells grown on the same medium and harvested in mid-log phase.
Project description:RNA-Seq data encompass transcriptomes from two sequential isolates of B. contaminans ST872. Each isolate was cultivated in three conditions (serum, sputum and BSM medium) in three biological replicates.
Project description:The response of antibiotic adapted resistant mutants of B. cenocepacia J2315 to antibiotic stress was investigated using expression profiling of three biological replicates and comparing the profiles to the J2315 parent control grown without antibiotics.<br>A reference design was used with Cy3 labeled genomic DNA of B. cenocepacia J2315 as common reference. Three test conditions with three biological replicates each were compared to three replicates of the control condition.<br>Test conditions: J2315-A grown in the presence of 250 ug per ml amikacin, J2315-M grown in the presence of 8 ug per ml meropenem and J2315-T grown in the presence of 60 ug per ml trimethoprim and 300 ug per ml sulfamethoxazole.<br>Control condition: J2315 parent strain grown without antibiotics.
Project description: Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum  Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment Overall design:  J2315 vs. HI2424 cells in the same condition.  Two-condition experiment. Biological replicates: 4 replicates.
Project description: Transcription profiling of one Burkholderia cenocepacia clinical isolate, J2315, versus a soil isolate, HI2424, in conditions mimicking CF sputum  Transcription profiling of Burkholderia cenocepacia isolates J2315 and HI2424 in media mimicking CF sputum or the soil environment  J2315 vs. HI2424 cells in the same condition.  Two-condition experiment. Biological replicates: 4 replicates.
Project description:One of the hallmarks of Pseudomonas aeruginosa cystic fibrosis (CF) infection is very high-cell-density (HCD) replication in the lung, allowing this bacterium to induce virulence controlled by HCD quorum-sensing systems. However, the nutrient sources sustaining HCD replication in this chronic infection is largely unknown. Hence, understanding the nutrient factors contributing to HCD in the CF lung will yield new insights into the 'metabolic pathogenicity' and potential treatment of CF infections caused by P. aeruginosa. Herein, we performed microarray studies of P. aeruginosa directly isolated from the CF lung to demonstrate its metabolic capability and virulence in vivo. Our in vivo microarray data, confirmed by real-time reverse-transcription-PCR, indicated P. aeruginosa expressed several genes for virulence, drug-resistance, and utilization of multiple nutrient sources (lung surfactant lipids and amino acids) contributing to HCD replication. The data also indicates deregulation of several pathways, suggesting in vivo evolution by deregulation of a large portion of the transcriptome during chronic CF infection. To our knowledge, this is the first in vivo transcriptome of P. aeruginosa in a natural CF infection, and it indicates several important aspects of pathogenesis, drug-resistance, and nutrient-utilization never before observed in vivo. Experiment Overall Design: The purpose of the experiment was to observe which genes are upregulated in P. aeruginosa during chronic CF lung infection as compared to PAO1. All in vitro studies were grown in 1x M9 minimal media supplemented with 20 mM citrate and grown to mid-log phase prior to RNA isolation. The in vivo RNA was isolated directly from CF sputum samples after TRIzol treatment. Each in vitro sample (both for PAO1 and the CF sputum pool isolate) were processed individually and in triplicate. Two in vivo isolations from sputum were conducted from the same patient but two different sputum samples. After isolation of total RNA, samples were processed for microarrays (i.e. cDNA synthesis, fragmentation, labeling, etc) as recommended by Affymetrix, and processed on the GeneChip as recommended by Affymetrix.
Project description:Chronic airway infection with P. aeruginosa (PA) is a hallmark of cystic fibrosis (CF) disease. The mechanisms producing PA persistence in CF therapies remain poorly understood. To gain insight on PA physiology in patient airways and better understand how in vivo bacterial functioning differs from in vitro conditions, we investigated the in vivo proteomes of PA in 35 sputum samples from 11 CF patients. We developed a novel bacterial-enrichment method enabling improved identification of PA proteome with CF sputum samples. The in vivo PA proteomes were compared with the proteomes of ex vivo-grown PA populations from the same patient sample. We detected 1528 PA proteins (encoded by 1458 core genes and 70 accessory genes) that were expressed in CF airways, of which 1178 proteins were commonly identified with the ex vivo-grown PA populations. Label-free quantitation and proteome comparison revealed the in vivo up-regulation of siderophore TonB-dependent receptors, remodeling in central carbon metabolism including glyoxylate cycle and lactate utilization, and alginate overproduction. Knowledge of these in vivo proteome differences or others derived using the presented methodology could lead to future treatment strategies aimed at altering PA physiology in vivo to compromise infectivity or improve antibiotic efficacy.