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Doctoral Thesis
DOI
Document
Author
Full name
Luana Claudino de Melo
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2018
Supervisor
Committee
Huenuman, Nilton Erbet Lincopan (President)
Galhardo, Rodrigo da Silva
Minarini, Luciene Andrade da Rocha
Spinosa, Helenice de Souza
Title in Portuguese
Contexto genético e prevalência da resistência do tipo ESBL/pAmpC em enterobactérias isoladas de cães e gatos no Brasil e na França.
Keywords in Portuguese
E. coli
animais de companhia
bactéria comensal
cromossomo
CTX-M
ESBL
multirresistentes
plasmídeos
Abstract in Portuguese
Animais de companhia têm sido apontados como reservatórios de bactérias gram-negativas resistentes a antibióticos utilizados em medicina humana e veterinária. O objetivo deste estudo foi investigar a prevalência da resistência mediada por plasmídeos em bactérias Gram-negativas isoladas de animais de companhia no Brasil e na França, elucidando o papel potencial desses animais como portadores assintomáticos. Amostras de DNA extraídas de quatro coleções de bactérias Gram-negativas produtoras de ESBL foram analisadas por tipagem e sub-tipagem baseados em PCR, análise do polimorfismo de comprimento de fragmentos de restrição (RFLP), dimensionamento baseado em PFGE de nuclease S1 e hibridação Southern blot. Adicionalmente, isolados de Escherichia coli, Klebsiella pneumoniae e Enterobacter cloacae foram caracterizados por PFGE (Pulsed-field Gel Electrophoresis) e Multilocus Sequence Typing, agrupamento filogenético e tipagem de O25b. A presença de plasmídeos IncH12 (~600-kb) e IncFIB (~210-290-kp) carregando genes blaCTX-M-15 e blaCTX-M-9, foi confirmada entre cepas de E. coli isoladas de animais brasileiros, enquanto uma predominância de plasmídeos IncI1 (~200 kb) pertencentes ao complexo clonal (CC) CC12 contendo o gene blaCMY-2 foi observado entre linhagens de E. coli, em filogrupos de baixa virulência A e B1. A presença de plasmídeos do tipo IncHI2 (~ 600kb) carregando o gene blaCTX-M-15 foi confirmada em cepas de E. cloacae ST927 isoladas de fezes e saliva de cães assintomáticos no Brasil. Entre os animais franceses com infecções, os isolados de E. coli pertencentes ao filogrupo A, B1 e B2 apresentaram tamanho de plasmídeo IncF de ~210-290 kb, carregando principalmente genes blaCTX-M-15, além da presença de plasmídeo IncI1 carregando em sua maioria genes blaCTX-M-1, blaCTX-M-9 e blaCMY-2. Em animais franceses saudáveis, além das associações blaCTX-M-15/IncI1, blaCTX-M-1/IncFIB, blaCTX-M-14/IncF e da presença de blaCMY-2 e blaTEM-52b (não tipáveis), foi identificada uma cepa de E. coli carregando um plasmídeo IncL (~60kb) contendo o gene blaOXA-48, sendo esta a primeira descrição desse gene em animais na França. Além disso, os genes blaCTX-M-15, blaCTX-M-2 e blaCTX-M-9 foram localizados no cromossomo em cepas brasileiras e francesas, como observado por Southern blot e Sequenciamento de Nova Geração (NGS). Em resumo, em ambos os países, a prevalência de cepas positivas para a resistência tipo ESBL é grande. Os animais de companhia podem ter um papel importante na disseminação dos genes AmpC e ESBL mediados por plasmídeos.
Title in English
Genetic context and prevalence of ESBL / pAmpC resistance in enterobacteria isolated from dogs and cats in Brazil and France. 2018.
Keywords in English
E. coli
Commensal bacteria
CTX-M
ESBL
Multidrug-resistant
Plasmid
Abstract in English
Companion animals can be reservoirs of Gram-negative bacteria resistant to antibiotics used in human and veterinary medicine. The aim of this study was to investigate the genetic context of plasmid-mediated resistance in Gram-negative bacteria isolated from companion animals in Brazil and France, elucidating the potential role of these animals as asymptomatic carriers. DNA samples, extracted from a collection of ESBL-producing Gram-negative bacteria, were analyzed by PCR-based typing and sub-typing schemes, restriction fragment length polymorphism analysis, S1 nuclease PFGE-based sizing and Southern blot hybridization. Additionally, Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates were characterized by PFGE (Pulsed-field Gel Electrophoresis), Multilocus Sequence Typing, phylogenetic grouping and O25b typing. Presence of IncH12 (~600-kb) and IncFIB (~210-290-kp) plasmids carrying blaCTX-M-15 and blaCTX-M-9, respectively, was confirmed among E. coli strains isolated from Brazilian pets, whereas predominance of IncI1 plasmids (~200 kb) belonging to the clonal complex (CC) CC12 and carrying blaCMY-2 gene was observed among E. coli strains of low-virulence phylogrups A and B1. The presence of IncHI2-type (~ 600-kb) plasmids carrying blaCTX-M-15 gene was confirmed in E. cloacae strains ST927 isolated from feces and saliva from asymptomatic dogs in Brazil. Among French diseased companion animals, E. coli isolates belonging to phylogroup A, B1 and B2 were found carrying IncF-type plasmid with size of ~210-290-kb, which harbored mainly blaCTX-M-15 genes. In addition, presence of IncI1 plasmids carrying blaCTX-M-1, blaCTX-M-9 and blaCMY-2 genes was identified. In healthy French animals, besides associations blaCTX-M-15/IncI1, blaCTX-M-1/IncFIB, blaCTX-M-14/IncF, and the presence of blaCMY-2 and blaTEM-52b (non-typable), it was observed an E. coli strain carrying an IncL plasmid (~ 60kbp) containing the blaOXA-48 gene, representing the first description of this gene in a French dog. In addition, blaCTX-M-15, blaCTX-M-2 and blaCTX-M-9 genes were located on the chromosome in Brazilian and French strains, as observed by Southern Blot and New Generation Sequencing (NGS) analysis. In summary, in both countries, the prevalence of positive strains for ESBL-type resistance in cats and dogs is high. Companion animals may play an important role in the dissemination of the plasmid mediated AmpC and ESBL genes.
 
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Release Date
2021-01-28
Publishing Date
2019-05-07
 
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