Selection of Pseudomonas aeruginosa reference genes for RT-qPCR analysis from sputum of cystic fibrosis patients

Costaglioli, Patricia; Barthe, Christophe; Fayon, Michaël; Christoflour, N.; Bui, Stéphanie; Derlich, Laurence; Domblides, Philippe; Crouzet, Marc; Vilain, Sébastien; Garbay, Bertrand

doi:10.1016/j.mcp.2013.09.003

Cited by 10 publications

(10 citation statements)

References 14 publications

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“…Samples (2 ml) were immediately treated with sputolysin after collection and RNA was extracted as previously described, using the Qiagen RNeasy mini kit (QIAGEN, Hilden, Germany) protocol including an on‐column DNA digestion (Costaglioli et al . ). Bacteriological analysis was performed on each sputum sample.…”

Section: Methodsmentioning

confidence: 97%

“…qPCR experiments, relative quantification and data analysis were performed as previously reported (Costaglioli et al . ). Pseudomonas aeruginosa sequences encoding PA0996 ( pqsA ), PA2513 ( antB ), PA3540 ( algD ), PA3724 ( lasB ) and PA5261 ( algR ) were retrieved from GenBank.…”

Section: Methodsmentioning

confidence: 97%

“…clpX and oprL genes) (Costaglioli et al . ). For each primer pair, the PCR efficiency was within the range of 1·8 and 2 using standard curves in triplicate serial dilutions of PAO1 cDNA.…”

Section: Methodsmentioning

confidence: 97%

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Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients

Barthe

Nandakumar

Derlich

et al. 2015

Lett Appl Microbiol

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Pseudomonas aeruginosa infection leads to high morbidity and mortality in cystic fibrosis patients. The identification of Ps. aeruginosa-assigned factors is important to eradicate the colonization. We started a pilot study to evaluate the gene expression of Ps. aeruginosa directly from the sputum of infected patients. Preliminary results suggest that beyond the heterogeneity of the Ps. aeruginosa phenotype in sputum, we observe a correlation between the expression of antB and pqsA and a low level of lasB transcripts. This approach could shed some light on the behaviour of Ps. aeruginosa during pulmonary infection and may reveal some important elements for optimizing therapy.

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Section: Methodsmentioning

confidence: 97%

Section: Methodsmentioning

confidence: 97%

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Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients

Barthe

Nandakumar

Derlich

et al. 2015

Lett Appl Microbiol

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“…The qRT-PCR primers are listed in Table S3. Relative gene expression was calculated using the 2 −ΔΔCT method with DNA gyrase subunit gene gyrA as a normalization control for S. sanguinis (73, 74) and PA2875 as the reference gene for P. aeruginosa (75).…”

Section: Methodsmentioning

confidence: 99%

Availability of Zinc Impacts Interactions BetweenStreptococcus sanguinisandPseudomonas aeruginosain Co-culture

Gifford

Hampton

et al. 2019

Preprint

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23Airway infections associated with cystic fibrosis (CF) are polymicrobial. We reported 24 previously that clinical isolates of P. aeruginosa promote the growth of a variety of 25 streptococcal species. To explore the mechanistic basis of this interaction, we performed a 26 genetic screen to identify mutants of Streptococcus sanginuis SK36 whose growth was no 27 longer enhanced by P. aeruginosa PAO1. Mutations in zinc uptake systems of S. sanginuis 28 SK36 reduced growth of these strains by 1-3 log compared to wild-type S. sanginuis SK36 29 when grown in coculture with P. aeruginosa PA01, while exogenous zinc (0.1-10 μm) rescued 30 the coculture defect of zinc uptake mutants of S. sanginuis SK36. Zinc uptake mutants of S. 31 sanginuis SK36 had no obvious growth defect in monoculture. Consistent with a competition 32 for zinc driving coculture dynamics, S. sanginuis SK36 grown in coculture with P. aeruginosa 33 showed increased expression of zinc uptake genes compared to S. sanginuis grown alone. 34 Strains of P. aeruginosa PAO1 defective in zinc transport also supported more robust growth 35 by S. sanginuis compared to coculture with wild-type P. aeruginosa PAO1. An analysis of 118 36 CF sputum samples revealed that total zinc levels varied from ~5-145 μM. At relatively low 37 zinc levels, Pseudomonas and Streptococcus were found in approximately equal abundance; 38 at higher zinc levels, we observed an increasing relative abundance of Pseudomonas and 39 decline of Streptococcus, perhaps as a result of increasing zinc toxicity. Together, our data 40 indicate that the relative abundance of these microbes in the CF airway may be impacted by 41 zinc levels. 42 IMPORTANCE. Polymicrobial infections in CF likely impact patient health, but the 43 mechanism(s) underlying such interactions are poorly understood. Here we show that 44 interactions between Pseudomonas and Streptococcus are modulated by zinc availability 45 using an in vitro model system, and clinical data are consistent with this model. Together with 46 previous studies, our work supports a role for metal homeostasis as a key factor driving 47 49Cystic fibrosis (CF) is a monogenic autosomal recessive disorder caused by mutations in the 50 cystic fibrosis transmembrane conductance regulator (CFTR) gene (1). It is estimated that 51 ~70,000 individuals in the world are affected by CF and the most common mutation, caused 52 by a deletion of phenylalanine at the 508th amino acid within the CFTR protein (ΔF508), is 53 found in approximately 70% of this population (2, 3). CFTR dysfunction affects several body 54 systems, and progressive lung disease due to chronic and recurrent microbial infections is 55 the leading cause of morbidity and mortality in individuals with CF (4, 5). It has been shown 56 that CF airway infections are polymicrobial (6, 7), and the composition and interspecies 57 interactions within the polymicrobial communities can have profound and diverse 58 consequences, including on bacterial growth (8-10), as well as disease progression and 59 thera...

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“…These include (with the most appropriate genes listed in parentheses): the human gut-associated Bifidobacterium adoloescentis exposed to bile ( gyrA and sdhA ) [ 20 ]; P . aeruginosa from cystic fibrosis patients (genes coding for the conserved hypothetical proteins PA2875 and PA3340) [ 21 ]; Clostridium ljungdahlii ( gyrA , rho , and fotl ) across multiple culture conditions [ 22 ]; the fish pathogen Francisella noatunensis (outer membrane protein fopA , cell division protein ftsZ , and DNA polymerase polA ) [ 23 ]; the nitrogen-fixing endophyte Gluconacetobacter diazotrophicus grown under different carbon sources ( rho , 23S rRNA, and rpoD ) [ 24 ]; the group B streptococci species Streptococcus agalactiae ( recA ) [ 25 ]; Lactobacillus casei , a potential probiotic strain (GAPD, gyrB ) [ 26 ]; and the marine bacterium Zobellia galactanivorans (serine/glycine hydroxymethyltransferase A glyA , NADP-dependent isocitrate dehydrogenase icdA , guanylate kinase gmkA ) [ 27 ].…”

Section: Introductionmentioning

confidence: 99%

Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions

et al. 2015

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The phylum Deinococcus-Thermus is a deeply-branching lineage of bacteria widely recognized as one of the most extremophilic. Members of the Thermus genus are of major interest due to both their bioremediation and biotechnology potentials. However, the molecular mechanisms associated with these key metabolic pathways remain unknown. Reverse-transcription quantitative PCR (RT-qPCR) is a high-throughput means of studying the expression of a large suite of genes over time and under different conditions. The selection of a stably-expressed reference gene is critical when using relative quantification methods, as target gene expression is normalized to expression of the reference gene. However, little information exists as to reference gene selection in extremophiles. This study evaluated 11 candidate reference genes for use with the thermophile Thermus scotoductus when grown under different culture conditions. Based on the combined stability values from BestKeeper and NormFinder software packages, the following are the most appropriate reference genes when comparing: (1) aerobic and anaerobic growth: TSC_c19900, polA2, gyrA, gyrB; (2) anaerobic growth with varied electron acceptors: TSC_c19900, infA, pfk, gyrA, gyrB; (3) aerobic growth with different heating methods: gyrA, gap, gyrB; (4) all conditions mentioned above: gap, gyrA, gyrB. The commonly-employed rpoC does not serve as a reliable reference gene in thermophiles, due to its expression instability across all culture conditions tested here. As extremophiles exhibit a tendency for polyploidy, absolute quantification was employed to determine the ratio of transcript to gene copy number in a subset of the genes. A strong negative correlation was found to exist between ratio and threshold cycle (CT) values, demonstrating that CT changes reflect transcript copy number, and not gene copy number, fluctuations. Even with the potential for polyploidy in extremophiles, the results obtained via absolute quantification indicate that relative quantification is appropriate for RT-qPCR studies with this thermophile.

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Selection of Pseudomonas aeruginosa reference genes for RT-qPCR analysis from sputum of cystic fibrosis patients

Cited by 10 publications

References 14 publications

Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients

Exploring the expression of Pseudomonas aeruginosa genes directly from sputa of cystic fibrosis patients

Availability of Zinc Impacts Interactions BetweenStreptococcus sanguinisandPseudomonas aeruginosain Co-culture

Selection and Evaluation of Reference Genes for Reverse Transcription-Quantitative PCR Expression Studies in a Thermophilic Bacterium Grown under Different Culture Conditions

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