OCORRÊNCIA DE blaKPC E blaVIM EM ISOLADOS DE Pseudomonas aeruginosa MULTIDROGA RESISTENTES PROVENIENTES DE INFECÇÃO EM PACIENTES DE UM HOSPITAL PÚBLICO EM RECIFEPE.

2021 ◽  
Author(s):  
Érica Maria de Oliveira ◽  
Nathaly Bruna de Oliveira Silva ◽  
Ana Catarina de Souza Lopes
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genomic Dna ◽  
Dna Purification

Introdução: Pseudomona aeruginosa é uma bactéria gram negativa oportunista considerada uma séria ameaça à saúde pública e são responsáveis por uma variedade de Infecções Relacionadas à Assistência à Saúde (IRAS), como pneumonias, infecções em tecidos moles, no trato urinário, e bacteremias(1).A emergência de P. aeruginosa resistentes aos carbapenêmicos, produtora de carbapenemases, dificulta ainda mais o tratamento e são associadas a altas taxas de mortalidade(2). Objetivos: O objetivo desse estudo foi analisar o perfil de susceptibilidade bem como detectar a presença dos genes de resistência para carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP e blaGES) em dois isolados de P. aeruginosa (Ps1-A2 e Ps2-A2) provenientes de secreção traqueal e ponta de cateter respectivamente, de dois pacientes de um hospital público de Recife-PE no ano de 2021. Métodos: A identificação bioquímica e o perfil de susceptibilidade dos isolados foi realizada através do sistema automatizado BD PhoenixTM. O DNA total foi extraído utilizando o kit comercial Wizard genomic DNA Purification e a pesquisa dos genes de resistência foram realizadas por Reação em Cadeia da Polimerase (PCR). Resultados: O isolado Ps1-A2 foi resistente a todos os antibióticos testados, com exceção da polimixina B. Já o isolado Ps1-A2 apresentou sensibilidade a ampicilina, gentamicina e polimixina B. Os genes blaNDM, blaIMP e blaGES não foram detectados. O gene blaKPC foi detectado no isolado Ps2-A2, e o isolado Ps1-A2 apresentou simultaneamente os genes blaKPC e blaVIM. A concomitância desses genes merece destaque pelo acúmulo desses mecanismos genéticos de resistência em uma mesma espécie bacteriana. Conclusão: Os genes para carbapenemases detectados nos isolados de P. aeruginosa, revelam o grande potencial dessa espécie em adquirir e consequentemente disseminar esses mecanismos de resistência no ambiente hospitalar. Precisamos considerar isso como um grande problema de saúde pública, levando em conta as limitações terapêuticas para tratar as infecções causadas por essa espécie. Sendo assim, devemos ressaltar a importância da detecção dessas enzimas nos laboratórios de microbiologia para que sejam adotadas medidas de controle e prevenção da disseminação desses microrganismos multirresistentes(3).

scholarly journals Deteksi Transgen (Glu-1Dx5) pada Populasi Padi (Oryza sativa L.) Putative Transgenik Kultivar Fatmawati

Agrikultura ◽  
2010 ◽  
Vol 21 (1) ◽  
Author(s):  
Nono Carsono ◽  
Sri Nurlianti ◽  
Inez Nur Indrayani ◽  
Ade Ismail ◽  
Tri Joko Santoso ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Oryza Sativa ◽  
Dna Polymerase ◽  
Genomic Dna ◽  
Oryza Sativa L ◽  
Dna Purification ◽  
Coding Region ◽  
Chain Reaction ◽  
Taq Dna Polymerase ◽  
Polymerase Chain

Transformasi gen Glu-1Dx5, pengendali utama karakter elastisitas dan daya mengembang adonan dari gandum, telah berhasil ditransfer ke dalam genom tanaman padi kultivar Fatmawati dengan menggunakan penembakan partikel, dengan tujuan untuk memperbaiki kualitas adonan tepung beras. Galur-galur harapan telah diperoleh, tetapi karena telah mengalami penyerbukan sendiri selama 1-2 generasi yang menyebabkan transgen mengalami segregasi, maka diperlukan upaya pendeteksian transgen pada populasi putative transgenik ini. Upaya ini dapat dilakukan, antara lain dengan menggunakan teknik Polymerase Chain Reaction (PCR) yang memungkinkan perbanyakan fragmen DNA yang spesifik (gen) secara cepat dalam jumlah banyak.  Percobaan ini bertujuan untuk mendapatkan tanaman padi transgenik yang memiliki gen Glu-1Dx5 pada dua generasi yang sedang bersegregasi. DNA genom dari 149 tanaman padi (generasi T1 sebanyak 14 tanaman, generasi T2 sebanyak 134 tanaman, dan satu tanaman non-transgenik) telah diekstraksi menggunakan Genomic DNA Purification Kit dari Fermentas. Plasmid pK+Dx5 digunakan sebagai positif kontrol, selain itu digunakan juga enzim Taq DNA polymerase dari Go Green Taq® Master Mix (Promega) dan 2 primer spesifik yang mengamplifikasi coding region dari Glu-1Dx5 (2,5 kb). Hasil percobaan menunjukkan, tanaman padi yang memiliki gen Glu-1Dx5 pada generasi T2-7 sebanyak 26 tanaman, T2-11 : 12 tanaman, T2-12 : 3 tanaman, T2-40 : 3 tanaman dan T2-45 : 5 tanaman. Seluruh tanaman generasi T1 tidak memiliki insert. Hasil ini menunjukkan bahwa gen Glu-1Dx5 sudah terintegrasi ke dalam genom tanaman padi kultivar Fatmawati dan diwariskan dari satu generasi ke generasi berikutnya.

scholarly journals Pseudomonas aeruginosa Requires the DNA-Specific Endonuclease EndA To Degrade Extracellular Genomic DNA To Disperse from the Biofilm

2019 ◽  
Vol 201 (18) ◽  
Author(s):  
Kathryn E. Cherny ◽  
Karin Sauer
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Genomic Dna ◽  
Early Stage ◽  
Content Type ◽  
Biofilm Dispersion ◽  
Biofilm Structure ◽  
First Time ◽  
Biofilm Matrix ◽  
Dispersed Cells

ABSTRACT The dispersion of biofilms is an active process resulting in the release of planktonic cells from the biofilm structure. While much is known about the process of dispersion cue perception and the subsequent modulation of the c-di-GMP pool, little is known about subsequent events resulting in the release of cells from the biofilm. Given that dispersion coincides with void formation and an overall erosion of the biofilm structure, we asked whether dispersion involves degradation of the biofilm matrix. Here, we focused on extracellular genomic DNA (eDNA) due to its almost universal presence in the matrix of biofilm-forming species. We identified two probable nucleases, endA and eddB, and eddA encoding a phosphatase that were significantly increased in transcript abundance in dispersed cells. However, only inactivation of endA but not eddA or eddB impaired dispersion by Pseudomonas aeruginosa biofilms in response to glutamate and nitric oxide (NO). Heterologously produced EndA was found to be secreted and active in degrading genomic DNA. While endA inactivation had little effect on biofilm formation and the presence of eDNA in biofilms, eDNA degradation upon induction of dispersion was impaired. In contrast, induction of endA expression coincided with eDNA degradation and resulted in biofilm dispersion. Thus, released cells demonstrated a hyperattaching phenotype but remained as resistant to tobramycin as biofilm cells from which they egress, indicating EndA-dispersed cells adopted some but not all of the phenotypes associated with dispersed cells. Our findings indicate for the first time a role of DNase EndA in dispersion and suggest weakening of the biofilm matrix is a requisite for biofilm dispersion. IMPORTANCE The finding that exposure to DNase I impairs biofilm formation or leads to the dispersal of early stage biofilms has led to the realization of extracellular genomic DNA (eDNA) as a structural component of the biofilm matrix. However, little is known about the contribution of intrinsic DNases to the weakening of the biofilm matrix and dispersion of established biofilms. Here, we demonstrate for the first time that nucleases are induced in dispersed Pseudomonas aeruginosa cells and are essential to the dispersion response and that degradation of matrix eDNA by endogenously produced/secreted EndA is required for P. aeruginosa biofilm dispersion. Our findings suggest that dispersing cells mediate their active release from the biofilm matrix via the induction of nucleases.

Extraction of genomic DNA from Pseudomonas aeruginosa: a comparison of three methods

2003 ◽  
Vol 60 (1) ◽  
pp. 34-35 ◽  
Author(s):  
Lena Clarke ◽  
B Cherie Millar ◽  
John E. Moore
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Genomic Dna

scholarly journals Outbreak of Multidrug-resistant Pseudomonas Aeruginosa Endophthalmitis Due to Contaminated Trypan Blue Solution

2019 ◽  
Author(s):  
Pritam Bawankar ◽  
Harsha Bhattacharjee ◽  
Manabjyoti Barman ◽  
Ronel Soibam ◽  
Hemalata Deka ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Cataract Surgery ◽  
Genomic Dna ◽  
Trypan Blue ◽  
Dna Typing ◽  
Multidrug Resistant ◽  
Diffusion Method ◽  
Postoperative Endophthalmitis ◽  
Retrospective Case ◽  
Multiple Drugs

Purpose: To report the investigation of an outbreak of multidrug-resistant (MDR) Pseudomonas aeruginosa endophthalmitis in 13 patients after cataract surgery and to emphasize on the importance of clinical profile, risk factors, and treatment outcomes. Methods: This was a hospital-based, retrospective case study with 13 consecutive patients who had manual small-incision cataract surgery with intraocular lens (IOL) implantation and developed acute postoperative Pseudomonas aeruginosa endophthalmitis. The anterior chamber taps, vitreous aspirates, and environmental surveillance specimens were inoculated for culturing. Antibiotic susceptibility testing was performed using the agar diffusion method. Pulsed-field gel electrophoresis (PFGE) was used to determine the relationship between bacterial isolates recovered from study patients and contaminated surveillance samples. Results: Pseudomonas aeruginosa was isolated from all 13 eyes with acute postoperative endophthalmitis and the trypan blue solutions used during surgery. Sensitivity tests revealed that all isolates had an identical resistance to multiple drugs and were only susceptible to imipenem. Genomic DNA typing of Pseudomonas aeruginosa isolates recovered from patients and trypan blue solutions showed an identical banding pattern on the PFGE. Despite the prompt use of intravitreal antibiotics and early vitrectomy with IOL explantation in some patients, the outcome was poor in about 50% of patients. Conclusion: Positive microbiology and genomic DNA typing results proved that the contaminated trypan blue solutions were the source of infection in this outbreak. Postoperative endophthalmitis caused by Pseudomonas aeruginosa is often associated with a poor visual prognosis despite prompt treatment with intravitreal antibiotics.

scholarly journals Optimasi Isolasi Genom untuk Analisis Keragaman Mikrob pada Fermentasi Singkong "Peyem" dengan Teknik Terminal Restriction Fragment Length Polymorphism (T-RFLP)

2013 ◽  
Vol 18 (1) ◽  
Author(s):  
Tati Barus
Keyword(s):  
Restriction Fragment Length Polymorphism ◽  
16S Rdna ◽  
Restriction Fragment ◽  
Length Polymorphism ◽  
Fragment Length ◽  
Genomic Dna ◽  
Fragment Length Polymorphism ◽  
Dna Purification ◽  
Restriction Fragment Length

"Peyem" merupakan salah satu pangan fermentasi Indonesia. Kualitas pangan fermentasi bergantung pada aktivitas mikrob yang terdapat selama proses fermentasi berlangsung. Salah satu teknik molekuler yang telah banyak digunakan untuk menganalisis komunitas mikrob pada suatu habitat adalah teknik Terminal–Restriction Fragment Lenght Polymorphism (T-RFLP). Metode isolasi genom dan jenis primer yang digunakan pada saat PCR penting pada teknik T- RFLP dalam mengkaji komunitas mikrob. Oleh sebab itu, penelitian ini bertujuan untuk membandingkan empat metode isolasi genom dan membandingkan penggunaan dua set primer dalam mengkaji komunitas bakteri dari "Peyem" dengan teknik T-RFLP. Genom komunitas bakteri diisolasi dengan menggunakan empat metode, yaitu: 1) QIAamp DNA Stool Mini Kit (G1), 2) QIAamp DNA Stool Mini Kit + lisozim (G2), 3) Genomic DNA Purification Kit (G3), dan 4) Genomic DNA Purification Kit + lisozim (G4). Untuk mengamplifikasi 16S rDNA digunakan dua set primer, yaitu: 1) primer 27F-FAM dan 1492R, 2) primer 63F-FAM dan 1387R. Hasil penelitian menunjukkan isolasi genom dengan metode G4 menghasilkan konsentrasi genom tertinggi (330,20 ng/µl) dibandingkan metode G1, G2, dan G3 (163,50 ng/µl; 183,25 ng/µl, dan 260,80 ng/µl). Primer 27F-FAM menghasilkan jumlah peak yang lebih tertinggi (264) dibandingkan dengan primer 63F-FAM (177). Jumlah peak TRF pada teknik TRFLP menggambarkan keragaman komunitas mikrob. Dengan demikian isolasi genom dengan Genomic DNA Purification Kit + lysozyme dan penggunaan pasangan primer 27F-FAM-1492R adalah yang terbaik untuk menganalisis komunitas bakteri dari "Peyem" dengan teknik T-RFLP.Kata kunci: Genom, Primer, T-RFLP, Mikrob, "Peyem"

Genomic DNA: Purification

2018 ◽  
pp. 1-5 ◽  
Author(s):  
Burkhard Tümmler ◽  
Frauke Stanke
Keyword(s):  
Genomic Dna ◽  
Dna Purification

scholarly journals A 3D-Printed Millifluidic Platform Enabling Bacterial Preconcentration and DNA Purification for Molecular Detection of Pathogens in Blood

Micromachines ◽  
2018 ◽  
Vol 9 (9) ◽  
pp. 472 ◽  
Author(s):  
Yonghee Kim ◽  
Jinyeop Lee ◽  
Sungsu Park
Keyword(s):  
Genomic Dna ◽  
Molecular Detection ◽  
Three Dimensional ◽  
Immunomagnetic Separation ◽  
Automated System ◽  
Dna Purification ◽  
Clinical Samples ◽  
Silica Bead ◽  
3D Printed ◽  
Magnetic Silica

Molecular detection of pathogens in clinical samples often requires pretreatment techniques, including immunomagnetic separation and magnetic silica-bead-based DNA purification to obtain the purified DNA of pathogens. These two techniques usually rely on handling small tubes containing a few millilitres of the sample and manual operation, implying that an automated system encompassing both techniques is needed for larger quantities of the samples. Here, we report a three-dimensional (3D)-printed millifluidic platform that enables bacterial preconcentration and genomic DNA (gDNA) purification for improving the molecular detection of target pathogens in blood samples. The device consists of two millichannels and one chamber, which can be used to preconcentrate pathogens bound to antibody-conjugated magnetic nanoparticles (Ab-MNPs) and subsequently extract gDNA using magnetic silica beads (MSBs) in a sequential manner. The platform was able to preconcentrate very low concentrations (1–1000 colony forming units (CFU)) of Escherichia coli O157:H7 and extract their genomic DNA in 10 mL of buffer and 10% blood within 30 min. The performance of the platform was verified by detecting as low as 1 CFU of E. coli O157:H7 in 10% blood using either polymerase chain reaction (PCR) with post gel electrophoresis or quantitative PCR. The results suggest that the 3D-printed millifluidic platform is highly useful for lowering the limitations on molecular detection in blood by preconcentrating the target pathogen and isolating its DNA in a large volume of the sample.

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