Antimicrobial resistance and plasmid replicons in Yersinia enterocolitica strains isolated in Brazil in 30 years

Abstract Some studies evaluated the resistance profile of the Y. enterocolitica strains isolated in diverse countries. However, in Brazil the isolation and the study of Y. enterocolitica are not common and therefore information about the antimicrobial resistance profile of this species in this country is scarce. Therefore, the aim of this study was to evaluate the antimicrobial resistance of Y. enterocolitica of biotypes 1A, 2 and 4 isolated from clinical and non-clinical sources between 1979 and 2012, in Brazil. This study showed that some Yersinia enterocolitica of different biotypes remain susceptible to antimicrobials used for gastroenteritis treatment. Moreover, neither acquired resistance genes nor diversity of plasmids replicons were found; however, variation in the in vitro intrinsic resistant pattern was detected, except the non-resistance to cefoxitin in all strains. Notwithstanding, due to epidemiological link between Y. enterocolitica and the pork production chain, monitoring plasmid acquired resistance in Y. enterocolitica could also be considered for antimicrobial resistance control purposes and food safety measures.

Draft genome sequence of Bacillus thuringiensis 147, a Brazilian strain with high insecticidal activity

Bacillus thuringiensisis a ubiquitous Gram-positive and sporulating bacterium. Its crystals and secreted toxins are useful tools against larvae of diverse insect orders and, as a consequence, an alternative to recalcitrant chemical insecticides. We report here the draft genome sequence ofB. thuringiensis147, a strain isolated from Brazil and with high insecticidal activity. The assembled genome contained 6,167,994 bp and was distributed in seven replicons (a chromosome and 6 plasmids). We identified 12 coding regions, located in two plasmids, which encode insecticidal proteins.

Estratégias moleculares para utilização do vírus da febre amarela como vetor vacinal / Molecular strategies to use the yellow fever virus as a vaccine vector

A vacina da febre amarela 17D (YFV-17D) é bastante segura e uma dose única confere imunidade potente e duradoura. Por essas e outras características, diferentes tecnologias têm sido propostas para a utilização da cepa 17D como vetor vacinal. Estratégias promissoras para o desenvolvimento de novas vacinas têm se baseado na construção de quimeras YFV-17D com inserção de seqüências heterólogas e produção em larga escala de replicons empacotados em partículas pseudo-infecciosas (PPIs), no entanto, ainda não existe um consenso da melhor estratégia a ser utilizada para esses fins. O presente estudo teve por objetivo avaliar diferentes estratégias de construção para a utilização do YFV-17D como vetor vacinal. Para isso foram construídos duas quimeras do YFV-17D com inserção de um gene repórter YFP (Yellow Fluorescent Protein) na junção E/NS1 e dois replicons subgenômicos do YFV-17D expressando o gene repórter luciferase. Para a produção de PPIs foi desenvolvida a linhagem HEK-YFV-prM/E-opt. O YFV-YFPSSE revelou instabilidade genética com perda do gene YFP e correlação negativa entre expressão de proteínas virais e do gene repórter. O YFV-YFP-DENV1linker mostrou-se estável geneticamente com expressão eficiente de YFP e proteínas virais, e mostrou-se ser o mais adequado para ser utilizado como vetor viral. Os replicons do YFV-17D mostraram-se funcionais e capazes de expressar eficientemente o gene heterólogo. E, embora a linhagem HEK-YFV-prM/E-opt tenha expressado as proteínas estruturais prM e E eficientemente, poucas partículas pseudo-infecciosas foram produzidas. Diante do exposto, as diferentes estratégias de manipulação genética do YFV avaliadas neste trabalho constituem ferramentas viáveis e aplicáveis ao processo de desenvolvimento de vacinas, havendo, porém, necessidade de otimização dessas estratégias para assegurar maiores segurança e eficácia.

The yellow fever vaccine 17D (YFV-17D) is safe and a single dose confers lastingand powerful immunity. For these, different technologies have been proposed for the use of YF-17D strain as a vaccine vector. Promising strategies for the development of new vaccines has been based on chimeric YFV-17D with insertion of heterologous genes and subgenomic replicons packaged into pseudo-infectious particles (PIPs).However, there is still no consensus in the best strategy to be used for suchpurposes. This study aimed to evaluate different strategies for building for the use of YFV-17D as a vector vaccine. For that were constructed two chimeric YFV-17D with insertion of a reporter gene YFP (yellow fluorescent protein) at E/NS1 junction and two subgenomic replicons of YFV-17D expressing the luciferase reporter gene. For the production of PIPs a recombinant cell line were developed (HEK-YFV-prM/E-opt). The YFV-YFP-SSE showed genetic instability with loss of the YFP gene and negative correlation between expression of viral proteins and reporter gene. The YFV-YFPDENV1linker proved to be genetically stable with efficient expression of YFP and viral proteins, and proved to be the most suitable for use as a viral vector. The replicons ofYFV-17D were shown to be functional and able to efficiently express heterologous gene. And although the cell line HEK-YFV-prM/E-opt has expressed efficiently structural viral proteins prM and E, a few of pseudoinfectious particles were produced. In this light, the different strategies of genetic manipulation of YFV measured in this study are feasible and applicable tools to the process of vaccine development, however, the optimization of these strategies is important to ensure greater safety and efficacy.

Construction of a cassette for cloning and analysis of replicons / Construccion de un cassette para el clonamiento y analisis de replicones

The aim of this work was the construction of a cassette, i.e., a non-replicative molecule formed by linkage of an antibiotic resistance gene and a multiple cloning site. This cassette would allow the cloning and analysis of a wide range of replicons. The aac(6')-lc amikacin gene was isolated and ligated to the multiple clining site of the pUC18 vector. This construction was HindIII digested and cloned in the HindIII site of the vector. The resulting pHJ13 clone conferred to the recipient cells the ability to grow in presence of amikacin (cassette marker) and ampicillin (vector gene). By restriction analysis, the cassette orientation was established. Cassette versatility is provided by the presence of the unaltered multiple cloning site segment, and also because it allows sequencing of any replication origin inserted. Cassette funcionality was demonstrated by ligation to a replicative region of H plasmid pHH1457. Presence of the ori region from pHH1457 and the aac(6')-lc gene was confirmed in E. coli transformed clones. The incompatibility properties of the pHH1457 and its capability to replicate in a Poll defective strain were preserved in the pHJII14 construct. Currently, the amikacin cassette is being used in the characterization of H Complex plasmids.

El objetivo de este trabajo es la construcción de un cassette ­ molécula no replicativa ­ formada por un gen de resistencia a un antibiótico y una región de múltiple sitios de clonamiento. Este cassette permitirá el clonamiento y análisis de una amplia variedad de replicones. El gen que determina resistencia a amikacina (aac (6')-Ic) fue aislado y ligado a la región de múltiple sitios de clonamiento del vector pUC18. La construcción resultante fue digerida con Hind III y clonada en el sitio Hind III del vector. El clon pHJ13 resultante confirió a las células receptoras la capacidad de crecer en presencia de amikacina (marcador del cassette) y ampicilina (marcador del vector). Mediante análisis con enzimas de restricción se determinó la orientación del cassette. La versatilidad del cassette se sustenta en la presencia, sin modificaciones, de la región de múltiple sitios de clonamiento, que permitirá obtener la secuencia de nucleótidos de cualquier origen de replicación insertado. La funcionalidad del cassette fue demostrada mediante el ligamiento a una región de replicación del plásmido pHH1457 (Complejo H). La presencia de la región ori de pHH1457 y del gen aac (6')-Ic fue confirmada en clones de E. coli. Las propiedades de incompatibilidad del plásmido H y su capacidad para replicarse en una cepa defectiva en PolI se conservaron en el plásmido pHJII14 construido. El cassette de amikacina está siendo utilizado en la caracterización de plásmidos del Complejo H. P