Taxonomía y epidemiología del género aeromonas

Durante la presente tesis doctoral se ha establecido la presencia de variabilidad interoperónica en el gen ARNr 16S de Aeromonas y observado que ésta afectaba a la taxonomía del género, limitando la identificación de A. caviae, A. media y A. veronii. La secuenciación del gen rpoD permitió identificar las cepas con variabilidad interoperónica a nivel de especie y reconocer 5 nuevas especies del género: A. fluvialis, A. taiwanensis, A. sanarelii, A. piscicola y A. rivuli. En colaboración con diversos hospitales españoles se han descrito los primeros aislados de A. aquariorum de origen extraintestinal, el segundo caso en adultos de síndrome urémico hemolítico (SUH) asociado a Aeromonas y se ha podido demostrar la presencia en este género del gen stx2 asociado al SUH. Se han revisado las características clínicas y microbiológicas de las infecciones de herida quirúrgicas relacionadas con Aeromonas. Finalmente, se ha descrito el primer caso de inducción in vivo de resistencia al imipenem en una cepa de A. veronii. Los resultados de esta tesis han dado lugar o contribuido a la publicación de 13 artículos científicos en revistas internacionales.During this thesis has established the presence of inter-operon variability in 16S rRNA gene of Aeromonas and observed that it affected the taxonomy of the genus, limiting the identification of A. caviae, A. media and A. veronii. The rpoD gene sequences identified strains with variability at the species level and recognize 5 new Aeromonas species: A. fluvialis, A. taiwanensis, A. sanarelii, A. piscicola and A. rivuli. In collaboration with various Spanish hospitals have described the first isolation of A. aquariorum from extraintestinal origin, the second adult case of hemolytic uremic syndrome (HUS) associated with Aeromonas and demonstrated the presence of stx2 gene associated with HUS in Aeromonas. We reviewed the clinical and microbiological characteristics of surgical wound infections associated with Aeromonas. Finally, we described the first case of in vivo induction of resistance to imipenem in a strain of A. veronii. The results of this thesis have led or contributed to the publication of 13 scientific papers in international journals

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells

Trabajo presentado al: "Workshops Current Trends in Biomedicine" organizado por la Universidad Internacional de Andalucía y celebrado en Baeza (España) del 24 al 26 de octubre de 2011.Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ΔvirB mutant strain was transfer deficient, while a ΔtrwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs.This work was supported by grants BIO2008-00133 and BIO2007-63656 from the Spanish Ministry of Science and Innovation to M.L. and F.J.S., respectively, and by grant 31003A-109925 from the Swiss National Science Foundation to C.D.Peer reviewe

Analysis of 16S rRNA gene mutations in a subset of Aeromonas strains and their impact in species delineation

Characterization of 999 Aeromonas strains using a published 16S rDNA RFLP identification method showed that 8.1% of the strains produced unexpected (hereafter called "atypical") restriction patterns, making their identification uncertain. Atypical patterns were due to the presence of nucleotide polymorphisms among the rrn operons of the 16S rRNA gene (so-called microheterogeneities). Double sequencing signals at certain positions revealed the nucleotide composition was responsible for the microheterogeneities. Although the number of microheterogeneities was relatively low (0.06-0.66%), trees inferred from the 16S rRNA gene led either to a misidentification or to an inconclusive result for the majority of these strains. Strains with atypical patterns were, however, correctly identified using the rpoD gene sequences, as belonging to Aeromonas caviae, A. veronii, and A. media. All of them, but particularly the two former species, are associated with human disease. Microheterogeneities in 16S rRNA gene sequence were significantly (P 0.01) more prevalent in clinical than in environmental strains. This work also analyzed the effects of these microheterogeneities on the taxonomic position of the investigated strains. The results suggest the need for recording microheterogeneities in the 16S rRNA gene

DNA transfer and genomic integration by conjugative relaxases. Using Type IV Secretion System as a genomic modification tool

Resumen del trabajo presentado en el 41 Congreso de la Sociedad Española de Bioquímica y Biología Molecular SEBBM, celebrado en Santander (España) del 10 al 13 de septiembre de 2018

Role of conjugative relaxases in DNA transfer and integration. Development of biotechnological tools

Trabajo presentado en la XII Reunión del Grupo de Microbiología Molecular de la SEM, celebrada en Zaragoza (España) del 05 al 07 de diciembre de 2018

Transfer of R388 derivatives by a pathogenesis-associated type IV secretion system into both bacteria and human cells

Bacterial type IV secretion systems (T4SSs) are involved in processes such as bacterial conjugation and protein translocation to animal cells. In this work, we have switched the substrates of T4SSs involved in pathogenicity for DNA transfer. Plasmids containing part of the conjugative machinery of plasmid R388 were transferred by the T4SS of human facultative intracellular pathogen Bartonella henselae to both recipient bacteria and human vascular endothelial cells. About 2% of the human cells expressed a green fluorescent protein (GFP) gene from the plasmid. Plasmids of different sizes were transferred with similar efficiencies. B. henselae codes for two T4SSs: VirB/VirD4 and Trw. A ?virB mutant strain was transfer deficient, while a ?trwE mutant was only slightly impaired in DNA transfer. DNA transfer was in all cases dependent on protein TrwC of R388, the conjugative relaxase, implying that it occurs by a conjugation-like mechanism. A DNA helicase-deficient mutant of TrwC could not promote DNA transfer. In the absence of TrwB, the coupling protein of R388, DNA transfer efficiency dropped 1 log. The same low efficiency was obtained with a TrwB point mutation in the region involved in interaction with the T4SS. TrwB interacted with VirB10 in a bacterial two-hybrid assay, suggesting that it may act as the recruiter of the R388 substrate for the VirB/VirD4 T4SS. A TrwB ATPase mutant behaved as dominant negative, dropping DNA transfer efficiency to almost null levels. B. henselae bacteria recovered from infected human cells could transfer the mobilizable plasmid into recipient Escherichia coli under certain conditions, underscoring the versatility of T4SSs

Clinical Relevance of the Recently Described Species Aeromonas aquariorum▿

Twenty-two human extraintestinal isolates (11 from blood) and three isolates recovered from patients with diarrhea were genetically characterized as Aeromonas aquariorum, a novel species known only from ornamental fish. The isolates proved to bear a considerable number of virulence genes, and all were resistant to amoxicillin (amoxicilline), cephalothin (cefalotin), and cefoxitin. Biochemical differentiation from the most relevant clinical species is provided