RESUMO
Abstract In the last decade Achromobacter spp. has been associated with chronic colonizationin patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequentspecies recovered within this genus, other species such as A. ruhlandii have also been reportedin these patients. Descriptions of mobile elements are scarce in Achromobacter and none ofthem have been originated in A. ruhlandii. The aim of this study was to report the full char-acterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates.Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patientwith CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequencedby Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plas-mid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotidesubstitution translated into one amino acid change among them. These plasmids have a class 1integron containing the aac-(6)-Ib gene, a mercury resistance operon region and the relE/stbEtoxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 fromPseudomonas aeruginosa. This is the first full description and characterization of a plasmid fromA. ruhlandii which was maintained over time.
Resumen Durante la última década, Achromobacter spp. han sido asociadas con la colonización crónica en pacientes con fibrosis quística. Si bien Achromobacter xylosoxidans es la especie más frecuentemente recuperada, otras especies como Achromobacter ruhlandii también fueron reportadas en nuestra región. Sin embargo, pocos reportes se han centrado en la descripción de elementos móviles, y ninguno de ellos los documenta en A. ruhlandii. El objetivo de este estudio fue reportar la caracterización completa de un plásmido conservado en 4 aislamientos clonalmente relacionados de A. ruhlandii. Se recuperaron 9 aislamientos de A. ruhlandii entre 2013 y 2015 de un único paciente con fibrosis quística proveniente de un hospital pediátrico de Buenos Aires, Argentina. Se realizó la secuenciación completa del genoma de los 4 aislamientos seleccionados según el perfil de resistencia antibiótica en un equipo Illumina MiSeq. Estos fueron anotados mediante RAST y curados manualmente. Se detectó la presencia de un solo plásmido de 34.096 pb y 50CDS en los 4 aislamientos, observándose únicamente un cambio nucleotídico traducido en un cambio aminoacídico en un aislamiento. Los plásmidos ensamblados se caracterizaron por presentar un integrón de clase 1 que contenía el gen aac-(6')-Ib, un operón de resistencia a mercurio y el sistema de toxina-antitoxina relE/stbE. Cabe destacar que estos plásmidos poseen un 79% de similitud y un 99% de identidad con el plásmido pmatvim-7 de Pseudomonas aeruginosa. Esta es la primera descripción y caracterización completa de un plásmido proveniente de A. ruhlandii.
RESUMO
In the last decade Achromobacter spp. has been associated with chronic colonization in patients with cystic fibrosis (CF). Although Achromobacter xylosoxidans is the most frequent species recovered within this genus, other species such as A. ruhlandii have also been reported in these patients. Descriptions of mobile elements are scarce in Achromobacter and none of them have been originated in A. ruhlandii. The aim of this study was to report the full characterization of a plasmid which was maintained in four clonally related A. ruhlandii isolates. Between 2013 and 2015, nine A. ruhlandii isolates were recovered from a pediatric patient with CF at a hospital in Buenos Aires. Four selected clonally related isolates were sequenced by Illumina MiSeq, annotated using RAST and manually curated. The presence of a unique plasmid of 34096-bp and 50 CDS was observed in the four isolates, displaying only 1 nucleotide substitution translated into one amino acid change among them. These plasmids have a class 1 integron containing the aac-(6')-Ib gene, a mercury resistance operon region and the relE/stbE toxin/antitoxin system. Plasmids showed 79% similarity and 99% identity with pmatvim-7 from Pseudomonas aeruginosa. This is the first full description and characterization of a plasmid from A. ruhlandii which was maintained over time.
Assuntos
Achromobacter , Fibrose Cística , Infecções por Bactérias Gram-Negativas , Criança , Fibrose Cística/complicações , Humanos , Plasmídeos/genéticaRESUMO
OBJECTIVES: The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii. METHODS: Kinetic parameters for purified OXA-258a and OXA-258b were determined measuring the rate of hydrolysis of a representative group of antimicrobial agents. Whole-genome shotgun sequencing was performed on A. ruhlandii 38 (producing OXA-258a) and A. ruhlandii 319 (producing OXA-258b), and in silico analysis of antimicrobial resistance determinants was conducted. Substrates of the AxyABM efflux pump were investigated by inhibition assays using phenylalanine-arginine ß-naphthylamide (PAßN). Outer membrane protein profiles were resolved by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: Kinetic measurements of purified OXA-258 variants displayed an overall weak catalytic efficiency toward ß-lactams. A detectable hydrolysis of imipenem was observed. In silico genomic analysis confirmed the presence of 32 and 35 putative efflux pump-encoding genes in A. ruhlandii strains 38 and 319, respectively. Complete sequences for AxyABM and AxyXY efflux pumps, previously described in Achromobacter xylosoxidans, were detected. Decreases in the MICs for chloramphenicol, nalidixic acid and trimethoprim/sulfamethoxazole were observed in the presence of the inhibitor PAßN, suggesting that these antibiotics are substrates of AxyABM. AxyXY-encoding genes of A. ruhlandii 38 and A. ruhlandii 319 displayed 99% identity. No differences were observed in the outer membrane protein profiles. CONCLUSIONS: The contribution of OXA-258 enzymes to the final ß-lactam resistance profile may be secondary. Further studies on other putative resistance markers identified in the whole-genome analysis should be conducted to understand the carbapenem resistance observed in A. ruhlandii.
Assuntos
Achromobacter/enzimologia , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Sequenciamento Completo do Genoma/métodos , Resistência beta-Lactâmica , beta-Lactamases/genética , Achromobacter/genética , Antibacterianos/química , Proteínas de Bactérias/genética , Cloranfenicol/química , Cloranfenicol/farmacologia , Simulação por Computador , Variação Genética , Hidrólise , Imipenem/química , Imipenem/farmacologia , Testes de Sensibilidade Microbiana , Ácido Nalidíxico/química , Ácido Nalidíxico/farmacologia , Combinação Trimetoprima e Sulfametoxazol/química , Combinação Trimetoprima e Sulfametoxazol/farmacologiaRESUMO
Achromobacter species are being increasingly isolated from the respiratory tract of cystic fibrosis patients. Recent reports indicate that Achromobacter ruhlandii is a potential human pathogen in cystic fibrosis-related infections. Here we report the draft genome of four A. ruhlandii strains isolated from cystic fibrosis patients in Brazil. This report describes A. ruhlandii as a potential opportunistic pathogen in cystic fibrosis and provides a framework to for additional enquires into potential virulence factors and resistance mechanisms within this species.