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Complete sequencing of plasmids carrying antibiotic resistance genes in bacteria of interest and genome analysis of Pseudomonas aeruginosa-producing SPM-1 and KPC-2 lines by large-scale sequencing

Grant number: 15/11728-0
Support type:Scholarships in Brazil - Post-Doctorate
Effective date (Start): August 01, 2015
Effective date (End): April 06, 2019
Field of knowledge:Biological Sciences - Microbiology - Applied Microbiology
Principal Investigator:Ana Lúcia da Costa Darini
Grantee:Renata Galetti
Home Institution: Faculdade de Ciências Farmacêuticas de Ribeirão Preto (FCFRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil
Associated research grant:14/14494-8 - Molecular epidemiology of gram-negative bacteria and genetic of antibiotic resistance, AP.TEM
Associated scholarship(s):17/26311-3 - High-throughput genome sequencing of Enterobacteriaceae resistance plasmids isolated from poultry, BE.EP.PD

Abstract

Nowadays, the main mechanism of resistance to antibiotics in gram-negative bacteria is the production of enzymes that degrade antibiotics, highlighting the beta-lactamase. Most beta-lactamase genes encoding are inserted in mobile genetic elements (MGE- Mobile Genetic Elements -Integrons, transposons, and insertion sequences) carried or not by plasmids. Sequencing of complete genome and bacterial plasmid allows knowledge of their entire gene content, such as MGE, resistance genes and virulence genes carried by epidemic strains. New generation sequencing are available and provide accurate sequencing of these genomes at a relatively low cost. Objectives: Sequence and annotation of draft genome of two strains of P. aeruginosa, one carrying blaSPM-1 gene and other carrying blaKPC-2 gene, identify the possible presence of MGE, virulence and pathogenicity islands in each of these genomes and identify probable presence of plasmids inserted in the chromosome of these P. aeruginosa, and/or analyze the presence of "scar" that indicate how and how often transposable elements were inserted and/or excised from the bacterial chromosome; Complete sequencing of plasmid not typeable by conventional techniques or major epidemiological concern that carrying resistance genes in gram-negative bacteria of clinical and/or veterinarian interest; To compare the sequenced plasmids of this project with the plasmids deposited in public databases. Materials and Methods: Selection of genomes and plasmids: Two draft genomes of P. aeruginosa strains previously characterized in our laboratory, P. aeruginosa HC84 carrying blaSPM-1 gene from Hospital das Clinicas-Ribeirao Preto in 2007 and P. aeruginosa BH6 carrying the blaKPC-2 gene from hospital-Belo Horizonte in 2012. Approximately 4-6 plasmids non-typable will be selected for complete sequencing from enterobacteria and/or gram-negative non-fermenting. Those plasmids carrying more than one resistance gene and those in epidemiologically important bacteria may also be selected. Sequencing of genome and plasmids: plasmideal DNA will be extracted using the PureLinkTM HiPure Plasmid Filter Midiprep (Invitrogen) and experiments of electroporation and/or conjugation will be realized in selected isolates, when necessary, using techniques already described. The sequencing will be carried out using Illumina platform MiSeq (Illumina, Inc. Assembly and annotation of genomes and plasmids: The quality of the sequencing will be checked using the FastQC program (Babraham Bioinformatics) and the assembly 'denovo' will be performed using the Velvet program (European Bioinformatics Institute (EMBL-EBI). The union of contigs will be confirmed by PCR followed by sequencing by the Sanger method. Then, the data will be analyzed using Prokka tool and CLC Genomics Workbench (CLCbio, Qiagen), the softwares Artemis Version 8 (Sanger Institute) and Sequin. The confirmation and validation of ORFs will be held using BLASTN and BLASTP. Project Execution: The performance of sequencing analysis will be carried out at the Laboratório Especial de Bacteriologia e Epidemiologia Molecular-LEBEM by the selected postdoctoral researcher. For sequencing analysis support, Dr. Alessandra Carattoli, researcher at the Istituto Superiore di Sanita, Rome, IT, and Dr. Alessandro de Mello Varani, Researcher III of the Department of Technology Faculty of Agriculture and Veterinary Sciences - UNESP will be consulted. Expected results: It is expected, with the conclusion of these projects, unravels the genome of two strains of P. aeruginosa and completely sequenced plasmids carrying carbapenemases genes. (AU)

Scientific publications (4)
(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)
BALLABEN, ANELISE STELLA; GALETTI, RENATA; ANDRADE, LEONARDO NEVES; FERREIRA, JOSEANE CRISTINA; GARCIA, DOROTI DE OLIVEIRA; DOI, YOHEI; COSTA DARINI, ANA LUCIA. Extensively drug-resistant IMP-16-producing Pseudomonas monteilii isolated from cerebrospinal fluid. INFECTION GENETICS AND EVOLUTION, v. 87, JAN 2021. Web of Science Citations: 0.
GALETTI, RENATA; ANDRADE, LEONARDO NEVES; VARANI, ALESSANDRO M.; COSTA DARINI, ANA LUCIA. A Phage-Like Plasmid Carrying bla(KPC-2) Gene in Carbapenem-Resistant Pseudomonas aeruginosa. FRONTIERS IN MICROBIOLOGY, v. 10, MAR 20 2019. Web of Science Citations: 0.
GALETTI, RENATA; CASARIN PENHA FILHO, RAFAEL ANTONIO; FERREIRA, JOSEANE CRISTINA; VARANI, ALESSANDRO M.; COSTA DARINI, ANA LIICIA. Antibiotic resistance and heavy metal tolerance plasmids: the antimicrobial bulletproof properties of Escherichia fergusonii isolated from poultry. INFECTION AND DRUG RESISTANCE, v. 12, p. 1029-1033, 2019. Web of Science Citations: 1.
CACADOR, NATALIA CANDIDO; DA COSTA CAPIZZANI, CAROLINA PAULINO; MONTEIRO MARIN TORRES, LFDIA ALICE GOMES; GALETTI, RENATA; CIOFU, OANA; DA COSTA DARINI, ANA LTICIA; FLEFIBY, NIELS. Adaptation of Pseudomonas aeruginosa to the chronic phenotype by mutations in the algTmucABD operon in isolates from Brazilian cystic fibrosis patients. PLoS One, v. 13, n. 11 NOV 29 2018. Web of Science Citations: 3.

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