Macrophage adaptation leads to parallel evolution of genetically diverseEscherichia colismall-colony variants with increased fitness in vivo and antibiotic collateral sensitivity

Small-colony variants (SCVs) are commonly observed in evolution experiments and clinical isolates, being associated with antibiotic resistance and persistent infections. We recently observed the repeated emergence of Escherichia coli SCVs during adaptation to the interaction with macrophages. To identify the genetic targets underlying the emergence of this clinically relevant morphotype, we performed whole-genome sequencing of independently evolved SCV clones. We uncovered novel mutational targets, not previously associated with SCVs (e.g. cydA, pepP) and observed widespread functional parallelism. All SCV clones had mutations in genes related to the electron-transport chain. As SCVs emerged during adaptation to macrophages, and often show increased antibiotic resistance, we measured SCV fitness inside macrophages and measured their antibiotic resistance profiles. SCVs had a fitness advantage inside macrophages and showed increased aminoglycoside resistance in vitro, but had collateral sensitivity to other antibiotics (e.g. tetracycline). Importantly, we observed similar results in vivo. SCVs had a fitness advantage upon colonization of the mouse gut, which could be tuned by antibiotic treatment: kanamycin (aminoglycoside) increased SCV fitness, but tetracycline strongly reduced it. Our results highlight the power of using experimental evolution as the basis for identifying the causes and consequences of adaptation during host-microbe interactions.

Molecular identification and localization of filamentous symbiotic bacteria associated with the hydrothermal vent annelid Alvinella pompejana

Alvinella pompejana is a polychaetous annelid that inhabits high temperature environments associated with active deep-sea hydrothermal vents along the East Pacific Rise. A unique and diverse epibiotic microflora with a prominent filamentous morphotype is found associated with the worm's dorsal integument. A previous study established the taxonomic positions of two epsilon proteobacterial phylotypes, 13B and 5A, which dominated a clone library of 16S rRNA genes amplified by PCR from the epibiotic microbial community of an A. pompejana specimen. In the present study deoxyoligonucleotide PCR primers specific for phylotypes 13B and 5A were used to demonstrate that these phylotypes are regular features of the bacterial community associated with A. pompejana. Assaying of other surfaces around colonies of A. pompejana revealed that phylotypes 13B and 5A are not restricted to A. pompejana. Phylotype 13B occurs on the exterior surfaces of other invertebrate genera and rock surfaces, and phylotype 5A occurs on a congener, Alvinella caudata. The 13B and 5A phylotypes were identified and localized on A. pompejana by in situ hybridization, demonstrating that these two phylotypes are, in fact, the prominent filamentous bacteria on the dorsal integument of A. pompejana. These findings indicate that the filamentous bacterial symbionts of A. pompejana are epsilon Proteobacteria which do not have an obligate requirement for A. pompejana.

Tafonomia e paleoicnologia do neogénico superior do sector Cacela-Huelva (SE da Ibéria)

Tese de doutoramento em Paleontologia. Faculdade de Ciências do Mar e do Ambiente, Univ. do Algarve, 2005

Indirect and expedite assessment of Ilex paraguariensis commercial yield.

A produção de biomassa comercial é uns dos principais indicadores para seleção de progênies e clones de erva-mate. Técnicas tradicionais para se obter tais informações dependem da colheita das árvores e apresentam, dentre outras limitações, elevado custo e reduzida praticidade. Assim, objetivou-se avaliar a eficiência de métodos indiretos, por meio de estimativa de biomassa comercial e escore de produtividade em função de diferentes procedências, sexo e morfotipos. Em um teste de progênies e procedências instalado em 1997, foram avaliadas em agosto de 2015 (dois anos após a última colheita) duas metodologias de análise visual. Para tanto, participaram cinco avaliadores treinados que determinaram, para cada planta, uma estimativa de biomassa comercial (kg), e uma nota, com base em um escore de produtividade (0-10). Para avaliação da produtividade pelas técnicas tradicionais, todas as plantas foram podadas e tiveram sua biomassa comercial (folhas e ramos finos menores de 7 mm de diâmetro) colhida e avaliada por meio de pesagem (Kg.planta-1). As avaliações foram realizadas em experimento instalado em blocos ao acaso, com cinco repetições, sete procedências e 126 progênies, totalizando 5292 plantas avaliadas. Os métodos avaliados foram eficientes na estimativa da biomassa comercial. Os avaliadores apresentaram boa acuidade nas estimativas, expressando de forma eficiente a maior produtividade determinada por comparação de médias entre as procedências, sexo das matrizes e morfotipos. As maiores correlações foram verificadas na análise geral das médias e a estimativa de biomassa comercial é a melhor metodologia para estimar a biomassa comercial aferida em plantas adultas de erva-mate.