IBR   13079
INSTITUTO DE BIOLOGIA MOLECULAR Y CELULAR DE ROSARIO
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
ISAba825 as a modulator of blaOXA-58 expression and carbapenem resistance in Acinetobacter baumannii
Autor/es:
MUSSI M.A.; RAVASI P.; LIMANSKY A.S.; VIALE A.M.
Lugar:
Puerto Madryn
Reunión:
Congreso; XLVI Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB).; 2010
Institución organizadora:
SAIB
Resumen:
ISAba825 modulates genome plasticity and blaOXA-58 expression in Acinetobacter baumannii Alejandro M. Viale, Pablo Ravasi, Adriana S. Limansky, María Alejandra Mussi. Instituto de Biología Molecular y Celular de Rosario (IBR, CONICET), Fac. Cs. Bioq. y Farm., UNR, Argentina. e-mail: viale@ibr.gov.ar We recently reported the loss of the OM channel CarO of A. baumannii as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825. We evaluated here ISAba825 functionality and impact on the plasticity of the A. baumannii genome. For this purpose, we tagged ISAba825 with a kanamycin resistance cassette and cloned the construction in a suicide plasmid. Transposition was evaluated by transformation of this plasmid in proper hosts, selection and sequencing of Kn-resistant clones. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains was evaluated by PCR using primer combinations for ISAba825 and different blaOXA genes followed by sequencing. We found that ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. ISAba825::Kn inserted into AT rich targets, generating 6-9 bp direct repeats. Moreover, ISAba825 was found truncating the tnpA gene of an ISAba3-like element located upstream of a plasmid-borne blaOXA-58 gene in Ab880 strain. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, solely responsible for the generation of high levels of carbapenem resistance. Overall, a role for this IS in the modulation of carbapenem resistance in A. baumannii is proposed. We recently reported the loss of the OM channel CarO of A. baumannii as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825. We evaluated here ISAba825 functionality and impact on the plasticity of the A. baumannii genome. For this purpose, we tagged ISAba825 with a kanamycin resistance cassette and cloned the construction in a suicide plasmid. Transposition was evaluated by transformation of this plasmid in proper hosts, selection and sequencing of Kn-resistant clones. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains was evaluated by PCR using primer combinations for ISAba825 and different blaOXA genes followed by sequencing. We found that ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. ISAba825::Kn inserted into AT rich targets, generating 6-9 bp direct repeats. Moreover, ISAba825 was found truncating the tnpA gene of an ISAba3-like element located upstream of a plasmid-borne blaOXA-58 gene in Ab880 strain. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, solely responsible for the generation of high levels of carbapenem resistance. Overall, a role for this IS in the modulation of carbapenem resistance in A. baumannii is proposed. We recently reported the loss of the OM channel CarO of A. baumannii as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825. We evaluated here ISAba825 functionality and impact on the plasticity of the A. baumannii genome. For this purpose, we tagged ISAba825 with a kanamycin resistance cassette and cloned the construction in a suicide plasmid. Transposition was evaluated by transformation of this plasmid in proper hosts, selection and sequencing of Kn-resistant clones. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains was evaluated by PCR using primer combinations for ISAba825 and different blaOXA genes followed by sequencing. We found that ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. ISAba825::Kn inserted into AT rich targets, generating 6-9 bp direct repeats. Moreover, ISAba825 was found truncating the tnpA gene of an ISAba3-like element located upstream of a plasmid-borne blaOXA-58 gene in Ab880 strain. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, solely responsible for the generation of high levels of carbapenem resistance. Overall, a role for this IS in the modulation of carbapenem resistance in A. baumannii is proposed. We recently reported the loss of the OM channel CarO of A. baumannii as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825. We evaluated here ISAba825 functionality and impact on the plasticity of the A. baumannii genome. For this purpose, we tagged ISAba825 with a kanamycin resistance cassette and cloned the construction in a suicide plasmid. Transposition was evaluated by transformation of this plasmid in proper hosts, selection and sequencing of Kn-resistant clones. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains was evaluated by PCR using primer combinations for ISAba825 and different blaOXA genes followed by sequencing. We found that ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. ISAba825::Kn inserted into AT rich targets, generating 6-9 bp direct repeats. Moreover, ISAba825 was found truncating the tnpA gene of an ISAba3-like element located upstream of a plasmid-borne blaOXA-58 gene in Ab880 strain. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, solely responsible for the generation of high levels of carbapenem resistance. Overall, a role for this IS in the modulation of carbapenem resistance in A. baumannii is proposed. We recently reported the loss of the OM channel CarO of A. baumannii as a result of the insertional inactivation of its coding gene by a novel insertion sequence, ISAba825. We evaluated here ISAba825 functionality and impact on the plasticity of the A. baumannii genome. For this purpose, we tagged ISAba825 with a kanamycin resistance cassette and cloned the construction in a suicide plasmid. Transposition was evaluated by transformation of this plasmid in proper hosts, selection and sequencing of Kn-resistant clones. Whether ISAba825 is located in close proximity to blaOXA genes in carbapenem-resistant A. baumannii clinical strains was evaluated by PCR using primer combinations for ISAba825 and different blaOXA genes followed by sequencing. We found that ISAba825::Kn could transpose only in A. baumannii, indicating an extremely narrow species range. ISAba825::Kn inserted into AT rich targets, generating 6-9 bp direct repeats. Moreover, ISAba825 was found truncating the tnpA gene of an ISAba3-like element located upstream of a plasmid-borne blaOXA-58 gene in Ab880 strain. RACE analysis indicated that ISAba825 insertion generated an alternative hybrid promoter for blaOXA-58 expression, solely responsible for the generation of high levels of carbapenem resistance. Overall, a role for this IS in the modulation of carbapenem resistance in A. baumannii is proposed.