SNaPaer: A Practical Single Nucleotide Polymorphism Multiplex Assay for Genotyping of Pseudomonas aeruginosa

Eusébio, Nádia; Pinheiro, Tiago; Amorim, A.; Gamboa, Fredy; Gusmão, Leonor; Amorim, António; Araújo, Ricardo

doi:10.1371/journal.pone.0066083

Cited by 12 publications

(12 citation statements)

References 40 publications

(46 reference statements)

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“…Therefore, many methods have been described for diagnosis of Pseudomonas including traditional methods including phenotypic diagnostics, biochemical tests, and direct molecular strategies such as PCR [6], and here the focus will be on these methods. Solanum nigrum is an important plant in traditional medicines belongs to the family of solanaceae.…”

Section: Introductionmentioning

confidence: 99%

Bacteriological Study of Pseudomonas Aeruginosa Isolated from Different Infections and Study Antimicrobial Activities of Plant Extract Solanum Nigrum Against It

Salman

Amer

Abud-Rahman

2017

ijs

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This study was conducted for the period from 1/9/2016 to 30/3/2017 in Baquba city in Iraq. Eighty nine samples were collected from different infections from Baquba General Hospital and Al-Batool Hospital. Fifteen isolates (16.85%) were found to be Pseudomonas aeruginosa by using biochemical test. The bacterial diagnosis of all these isolates (100%) were confirmed by VITEK2, beside its possession of the 16s rDNA gene as determined by molecular detection. The results of virulence factors that had Pseudomonas aeruginosa showed possession of all isolates many virulence factors and a high production of which increases the pathogenicity of it. All isolates were able to produce hemolysin (100%), protease (73.33%), and biofilm formation (52%). The susceptibility test was applied on these isolates against (10) antibiotics. The results revealed that the highest resistances were for Ampicillin and Cefotaxime with 100% for each, while the lowest resistance were for Impineme (0.0%) and Ciprofloxacin (6.66%). The effects of ethanolic extract of fruits (Solanum nigrum) against P.aerugionsa were studied. At a concentration of 6% mg/ml, Solanum nigrum caused a marked increase in zone of inhibition (mm) on this bacteria growth. Inhibition zones sizes were different and increased according to concentration of extract and again the growth was completely inhibited in the highest concentration.

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Section: Introductionmentioning

confidence: 99%

Bacteriological Study of Pseudomonas Aeruginosa Isolated from Different Infections and Study Antimicrobial Activities of Plant Extract Solanum Nigrum Against It

Salman

Amer

Abud-Rahman

2017

ijs

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“…) or targeting a set of SNPs located in the mlst genes (Eusebio et al . ,b; Trembizki et al . ), but all these methods rely on a specific number of polymorphic alleles located in house‐keeping genes or core genome fragments, losing the key information contained in the accessory genome.…”

Section: Discussionmentioning

confidence: 99%

Extending RAD tag analysis to microbial ecology: a comparison between MultiLocus Sequence Typing and 2b‐RAD to investigate Listeria monocytogenes genetic structure

Pauletto

Carraro

Babbucci

et al. 2015

Molecular Ecology Resources

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The advent of next-generation sequencing (NGS) has dramatically changed bacterial typing technologies, increasing our ability to differentiate bacterial isolates. Despite it is now possible to sequence a bacterial genome in a few days and at reasonable costs, most genetic analyses do not require whole-genome sequencing, which also remains impractical for large population samples due to the cost of individual library preparation and bioinformatics. More traditional sequencing approaches, however, such as MultiLocus Sequence Typing (mlst) are quite laborious and time-consuming, especially for large-scale analyses. In this study, a genotyping approach based on restriction site-associated (RAD) tag sequencing, 2b-RAD, was applied to characterize Listeria monocytogenes strains. To verify the feasibility of the method, an in silico analysis was performed on 30 available complete genomes. For the same set of strains, in silico mlst analysis was conducted as well. Subsequently, 2b-RAD and mlst analyses were experimentally carried out on 58 isolates collected from food samples or food-processing sites. The obtained results demonstrate that 2b-RAD predicts mlst types and often provides more detailed information on population structure than mlst. Moreover, the majority of variants differentiating identical sequence type isolates mapped against accessory fragments, thus providing additional information to characterize strains. Although mlst still represents a reliable typing method, large-scale studies on molecular epidemiology and public health, as well as bacterial phylogenetics, population genetics and biosafety could benefit of a low cost and fast turnaround time approach such as the 2b-RAD analysis proposed here.

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“…A SNP based strategy has recently been developed for genotyping of Pseudomonas aeruginosa presenting a discriminatory power of 0.9993 comparing with MLST [19].…”

Section: Introductionmentioning

confidence: 99%

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection, Identification and Genotyping in Clinical Specimens

et al. 2013

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ObjectiveEarly diagnosis of invasive aspergillosis is essential for positive patient outcome. Likewise genotyping of fungal isolates is desirable for outbreak control in clinical setting. We designed a molecular assay that combines detection, identification, and genotyping of Aspergillus fumigatus in a single reaction.MethodsTo this aim we combined 20 markers in a multiplex reaction and the results were seen following mini-sequencing readings. Pure culture extracts were firstly tested. Thereafter, Aspergillus-DNA samples obtained from clinical specimens of patients with possible, probable, or proven aspergillosis according to European Organization for the Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria.ResultsA new set of designed primers allowed multilocus sequence typing (MLST) gene amplification in a single multiplex reaction. The newly proposed SNaPAfu assay had a specificity of 100%, a sensitivity of 89% and detection limit of 1 ITS copy/mL (∼0.5 fg genomic Aspergillus-DNA/mL). The marker A49_F was detected in 89% of clinical samples. The SNaPAfu assay was accurately performed on clinical specimens using only 1% of DNA extract (total volume 50 µL) from 1 mL of used bronchoalveolar lavage.ConclusionsThe first highly sensitive and specific, time- and cost-economic multiplex assay was implemented that allows detection, identification, and genotyping of A. fumigatus strains in a single amplification followed by mini-sequencing reaction. The new test is suitable to clinical routine and will improve patient management.

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SNaPaer: A Practical Single Nucleotide Polymorphism Multiplex Assay for Genotyping of Pseudomonas aeruginosa

Cited by 12 publications

References 40 publications

Bacteriological Study of Pseudomonas Aeruginosa Isolated from Different Infections and Study Antimicrobial Activities of Plant Extract Solanum Nigrum Against It

Bacteriological Study of Pseudomonas Aeruginosa Isolated from Different Infections and Study Antimicrobial Activities of Plant Extract Solanum Nigrum Against It

Extending RAD tag analysis to microbial ecology: a comparison between MultiLocus Sequence Typing and 2b‐RAD to investigate Listeria monocytogenes genetic structure

SNaPAfu: A Novel Single Nucleotide Polymorphism Multiplex Assay for Aspergillus fumigatus Direct Detection, Identification and Genotyping in Clinical Specimens

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