Single-Nucleotide-Polymorphism Mapping of the
            <i>Pseudomonas aeruginosa</i>
            Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

Ajayi, Temitayo; Allmond, Leonard R.; Sawa, Teiji; Wiener-Kronish, Jeanine P.

doi:10.1128/jcm.41.8.3526-3531.2003

Cited by 72 publications

(44 citation statements)

References 40 publications

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“…The variable loci were associated with type III secretion systems and effectors, type VI secretion systems and effectors, and pyoverdine siderophore biosynthesis. These structures vary among strains (37)(38)(39)(40). This finding suggests that Nanostring analysis of some transcripts across different genotypes requires the inclusion of probes that target multiple alleles, as demonstrated here for fliC, and/or DNA sequence analysis to confirm the target sequence will hybridize to the probe.…”

Section: Resultsmentioning

confidence: 91%

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

et al. 2016

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The discovery of therapies that modulate Pseudomonas aeruginosa virulence or that can eradicate chronic P. aeruginosa lung infections associated with cystic fibrosis (CF) will be advanced by an improved understanding of P. aeruginosa behavior in vivo. We demonstrate the use of multiplexed Nanostring technology to monitor relative abundances of P. aeruginosa transcripts across clinical isolates, in serial samples, and for the purposes of comparing microbial physiology in vitro and in vivo. The expression of 75 transcripts encoded by genes implicated in CF lung disease was measured in a variety of P. aeruginosa strains as well as RNA serial sputum samples from four P. aeruginosa-colonized subjects with CF collected over 6 months. We present data on reproducibility, the results from different methods of normalization, and demonstrate high concordance between transcript relative abundance data obtained by Nanostring or transcriptome sequencing (RNA-Seq) analysis. Furthermore, we address considerations regarding sequence variation between strains during probe design. Analysis of P. aeruginosa grown in vitro identified transcripts that correlated with the different phenotypes commonly observed in CF clinical isolates. P. aeruginosa transcript profiles in RNA from CF sputum indicated alginate production in vivo, and transcripts involved in quorum-sensing regulation were less abundant in sputum than strains grown in the laboratory. P. aeruginosa gene expression patterns from sputum clustered closely together relative to patterns for laboratory-grown cultures; in contrast, laboratory-grown P. aeruginosa showed much greater transcriptional variation with only loose clustering of strains with different phenotypes. The clustering within and between subjects was surprising in light of differences in inhaled antibiotic and respiratory symptoms, suggesting that the pathways represented by these 75 transcripts are stable in chronic CF P. aeruginosa lung infections. Several factors complicate the comparison of in vitro studies on Pseudomonas aeruginosa with infections in the cystic fibrosis (CF) lung. First, many different P. aeruginosa strains cause CF lung infections; thus, reliance on commonly used laboratory strains might limit our understanding of P. aeruginosa in CF (1-3). Second, laboratory or cell culture studies rarely incorporate coinfecting species and lack components of the lung environment, such as immune response factors, that shape P. aeruginosa phenotypes (4, 5). We know little about how these environmental stimuli influence P. aeruginosa (6-8). To complicate matters further, a single CF sputum sample contains mixtures of P. aeruginosa genotypes and phenotypes. A recent study has suggested that a mixture of strains influenced traits such as drug response in ways that were difficult to predict from the study of single strains (9). Clinical isolates of P. aeruginosa, even from the same respiratory sample, can have striking differences in phenotypes, including colony morphology, quorum-sensing regulation, and motili...

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Section: Resultsmentioning

confidence: 91%

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

et al. 2016

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“…The type III secretion toxins-encoding genes were amplified by multiplex PCR 16 with specific primers (Table 1). PCR was carried out with 2 ul DNA template (100-200 ng), 1 ul equivalent to 100 pM each primer ( total 8 ul PCR primers), 2.5 U Taq DNA polymerase, 200 uM of each dNTPs in a total volume 25 ul using PureTaq Ready-To-Go PCR beads (Amersham Biosciences, UK).…”

Section: Pcr Amplification Of Type III Secretion Toxinsencoding Genesmentioning

confidence: 99%

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Gawish¹

2013

Jmid

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Objective: Pseudomonas aeruginosa uses type 3 secretion system (T3SS) to directly inject four toxins into host cells. The aim of the study was to determine the prevalence of the toxins-encoding genes among P. aeruginosa isolates collected from nosocomial infections and environmental sources.Methods: Clonal relatedness of the studied P. aeruginosa isolates (n=85) was studied by RAPD typing. Multiplex PCR was performed on the 85 P. aeruginosa isolates (68 nosocomial isolates obtained from hospitalized patients present in different wards and departments, and 17 environmental isolates including 2 isolates from health-care worker hands from ICU) to detect the secretion toxins-encoding genes.Results: RAPD fingerprinting demonstrated that most strains were of distinct genotypes and determined the presence of 54 RAPD patterns. The prevalence of the genes among all isolates was as follows; exoT-100%, exoS-40%, exoY-83.5% and exoU-62.4%. No significant differences in the prevalence of these genes were observed between nosocomial and environmental isolates or between isolates cultured from different sites of infection. The part of P. aeruginosa strains harboring either exoS (37.6%) or exoU (60%) gene was significantly higher (P<0.001) than that contained both genes (2.4%). Conclusion:ExoT was present in all isolates and exoS, exoY and exoU are variable traits with exoU gene was the third in prevalence after exoT and exoY. No significant differences in exoS, exoY and exoU prevalence were observed between nosocomial and environmental isolates or between isolates from different sites of infection. ExoS and exoU genes were mutually exclusive that almost all isolates contain either exoS without exoU or exoU without exoS. Yöntemler: Çalışılan P. aeruginosa izolatlarının klonal yakınlığı RAPD tiplendirme metodu ile bakılmıştır. Sekretuar toksin kodlayan genleri saptamak için 85 P. aeruginosa izolatında (farklı klinik ve bölümlerde yatan hastalardan izole edilen 68 hastane kaynaklı izolatı ve ikisi yoğun bakım ünitesindeki sağlık çalışanının ellerinden izole edilen 17 çevresel izolat) multiplex PCR çalışıldı.Bulgular: RAPD fingerprint analizi ile varlığı tespit edilen 54 RAPD paterninin varlığı suşların çoğunun uzak genotiplere sahip olduğunu gösterdi. Tüm izolatlarda saptanan genlerin prevalansı sırasıyla exoT (% 100), exoS (% 40), exoY (% 83,5) ve exoU (% 62,4) şeklindeydi. Farklı enfeksiyon bölgelerinden üretilen izolatlar arasında yada hastane ve çevresel kaynaklardan elde edilen izolatlar arasında bu genlerin prevalansı açısından anlamlı bir fark yoktu. P. aeruginosa suşla-rından exoS (% 37,6) veya exoU (% 60) genlerinden birini taşıyanlar her iki geni taşıyanlardan (% 2,4) anlamlı olarak daha fazlaydı (p<0.001). Sonuç:ExoT tüm izolatlarda bulunurken exoS, exoY ve exoU genlerinin varlığı değişkenlik gösteriyordu. ExoU geni exoT ve exoY geninden sonra üçüncü sıklıkta görülmekteydi. exoS, ExoY ve exoU görülme prevalansı hastane kaynaklı ve çev-re kaynaklı suşlar arasında veya değişik bölge veya enfeksiyonlar arasında an...

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“…Types of bla VIM genes were identified by sequencing analysis among selected bla-VIM -positive strains after comparison with a data bank (http: //blast.ncbi.nlm.nih.gov/Blast.cgi). In all isolates, TTSS genes (exoS, exoT, exoU, exoY) were investigated by PCR (10).…”

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confidence: 99%

Spread of Multidrug-Resistant Pseudomonas aeruginosa Clones in a University Hospital

et al. 2013

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An outbreak of multidrug-resistant Pseudomonas aeruginosa (MDRPA) infections in a university hospital is described. Phenotypic and genotypic analysis of 240 isolates revealed that 152 patients, mainly in the intensive care unit (ICU), were colonized or infected with MDRPA, the majority with O11. All metallo-␤-lactamase (MBL)-positive isolates carried the bla VIM-2 or bla VIM-1 gene. One or more type III secretion system toxin genes were detected in most isolates. Five dominant pulsed-field gel electrophoresis (PFGE) types were characterized, associated with ST235, ST111, ST253, ST309, and ST639.

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Single-Nucleotide-Polymorphism Mapping of the Pseudomonas aeruginosa Type III Secretion Toxins for Development of a Diagnostic Multiplex PCR System

Cited by 72 publications

References 40 publications

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

Use of a Multiplex Transcript Method for Analysis of Pseudomonas aeruginosa Gene Expression Profiles in the Cystic Fibrosis Lung

An investigation of type 3 secretion toxins encoding-genes of Pseudomonas aeruginosa isolates in a University Hospital in Egypt

Spread of Multidrug-Resistant Pseudomonas aeruginosa Clones in a University Hospital

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