RESUMO
Abstract Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplificaron and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and car-bapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates. © 2019 Asociación Argentina de Microbiología. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (https://creativecommons.org/licenses/by-nc-nd/4.0/).
Resumen Se emplearon diversas técnicas fenotípicas y moleculares para describir la distribución de diferentes especies del género Achromobacter en pacientes con fibrosis quística (FQ) en Argentina, y se evaluó el perfil de resistencia a los antibióticos. Se realizó la identificación fenotípica por pruebas bioquímicas convencionales, galerías comerciales y MALDI-TOF MS. El enfoque genético incluyó la detección del marcador especie-específico de A. xylosoxidans bla[PRESERVECIRC]tu, la amplificación y la secuenciación de los genes ARNr 16S, nrdA y secuencia completa de blaOXA, y el análisis por MLST. Los enfoques fenotípicos, incluso la técnica de MALDI-TOF, proporcionaron resultados no concluyentes o erróneos. Por el contrario, se obtuvieron resultados concordantes entre la secuenciación del gen nrdA o el análisis de secuenciotipos (ST) y la secuenciación completa de blaOXA, lo que permitió una discriminación confiable de las diferentes especies de Achromobacter. A. xylosoxidans representó el 63% de las infecciones por Achromobacter y A. ruhlandii representó el 17%. Las especies restantes correspondieron a A. insuavis, A. dolens, A. marplatensis y A. pulmonis. Se determinó la sensibilidad a antimicrobianos por el método de dilución en agar de acuerdo al CLSI. Los antibióticos más activos fueron piperacilina, piperacilina/tazobactam y carbapenemes. Sin embargo, se detectó la emergencia de aislamientos resistentes a carbapenemes. En conclusión, resultaron necesarias herramientas de identificación rápida y precisas para determinar las diferentes especies del género Achro-mobacter capaces de colonizar/infectar las vías respiratorias de los pacientes con FQ. Asimismo, la terapia antimicrobiana debería llevarse a cabo en función del perfil de sensibilidad de los aislamientos individuales de Achromobacter spp. © 2019 Asociacion Argentina de Microbiología. Publicado por Elsevier Espana, S.L.U. Este es un artículo Open Access bajo la licencia CC BY-NC-ND (https://creativecommons.org/licenses/by-nc-nd/4.0/).
Assuntos
Humanos , Fibrose Cística/microbiologia , Achromobacter/isolamento & purificação , Fenótipo , Argentina , Farmacorresistência Bacteriana , Achromobacter/classificação , Achromobacter/efeitos dos fármacos , Achromobacter/genética , Antibacterianos/farmacologiaRESUMO
Different phenotype-based techniques and molecular tools were used to describe the distribution of different Achromobacter species in patients with cystic fibrosis (CF) in Argentina, and to evaluate their antibiotic resistance profile. Phenotypic identification was performed by conventional biochemical tests, commercial galleries and MALDI-TOF MS. Genetic approaches included the detection of A. xylosoxidans specific marker blaoxa-114, the amplification and sequencing of the 16S rRNA gene, nrdA and blaOXA complete sequence, and MLST analysis. Phenotypic approaches, even MALDI-TOF, rendered inconclusive or misleading results. On the contrary, concordant results were achieved with the nrdA sequencing or sequence type (ST) analysis, and the complete blaOXA sequencing, allowing a reliable discrimination of different Achromobacter species. A. xylosoxidans accounted for 63% of Achromobacter infections and A. ruhlandii accounted for 17%. The remaining species corresponded to A. insuavis, A. dolens, A. marplatensis and A. pulmonis. Antimicrobial susceptibilities were determined by the agar dilution method according to CLSI guidelines. Piperacillin, piperacillin/tazobactam and carbapenems were the most active antibiotics. However, the emergence of carbapenem-resistant isolates was detected. In conclusion, prompt and accurate identification tools were necessary to determine that different Achromobacter species may colonize/infect the airways of patients with CF. Moreover, antimicrobial therapy should be administered based on the susceptibility profile of individual Achromobacter sp. isolates.
Assuntos
Achromobacter/isolamento & purificação , Fibrose Cística/microbiologia , Achromobacter/classificação , Achromobacter/efeitos dos fármacos , Achromobacter/genética , Antibacterianos/farmacologia , Argentina , Farmacorresistência Bacteriana , Humanos , FenótipoRESUMO
Achromobacter spp. are opportunistic pathogens increasingly recovered from adult patients with cystic fibrosis (CF). We report the characterization of 122 Achromobacter spp. isolates recovered from 39 CF patients by multilocus sequence typing, virulence traits, and susceptibility to antimicrobials. Two species, A. xylosoxidans (77%) and A. ruhlandii (23%) were identified. All isolates showed a similar biofilm formation ability, and a positive swimming phenotype. By contrast, 4·3% and 44·4% of A. xylosoxidans and A. ruhlandii, respectively, exhibited a negative swarming phenotype, making the swimming and swarming abilities of A. xylosoxidans significantly higher than those of A. ruhlandii. A. xylosoxidans isolates from an outbreak clone also exhibited significantly higher motility. Both species were generally susceptible to ceftazidime, ciprofloxacin, imipenem and trimethoprim/sulphamethoxazole and there was no significant difference in susceptibility between isolates from chronic or sporadic infection. However, A. xylosoxidans isolates from chronic and sporadic cases were significantly more resistant to imipenem and ceftazidime than isolates of the outbreak clone.
Assuntos
Achromobacter/isolamento & purificação , Fibrose Cística/complicações , Infecções por Bactérias Gram-Negativas/microbiologia , Fatores de Virulência/análise , Achromobacter/classificação , Achromobacter/efeitos dos fármacos , Achromobacter/fisiologia , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Humanos , Locomoção , Testes de Sensibilidade Microbiana , Tipagem de Sequências MultilocusRESUMO
Achromobacter xylosoxidans is increasingly being documented in cystic fibrosis patients. The bla(OXA-114) gene has been recognized as a naturally occurring chromosomal gene, exhibiting different allelic variants. In the population under study, the bla(OXA-114)-like gene was found in 19/19 non-epidemiological-related clinical isolates of A. xylosoxidans with ten different alleles including 1 novel OXA-114 variant.
Assuntos
Achromobacter/genética , Alelos , Cromossomos Bacterianos , Variação Genética , beta-Lactamases/genética , Achromobacter/classificação , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Alinhamento de Sequência , beta-Lactamases/químicaRESUMO
A new blaOXA-258 gene is described as a species-specific taxonomic marker for Achromobacter ruhlandii isolates (all recovered from cystic fibrosis patients). Even though OXA-258 differs from OXA-114 variants, isolates could be misidentified as A. xiloxosidans by the amplification of an inner fragment from the OXA-coding gene. A robust identification of A. ruhlandii can be achieved by sequencing this single OXA gene, as well as by a more laborious recently proposed multilocus sequence-typing (MLST) scheme.
Assuntos
Achromobacter/classificação , Achromobacter/enzimologia , Infecções por Bactérias Gram-Negativas/diagnóstico , beta-Lactamases/genética , Achromobacter/genética , Sequência de Aminoácidos , Sequência de Bases , Genes Bacterianos , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Tipagem de Sequências Multilocus , RNA Ribossômico 16S/genética , beta-Lactamases/químicaRESUMO
A multilocus sequence analysis (MLSA) scheme was developed for characterization of strains and species from the genus Achromobacter, which are increasingly recovered from patients with cystic fibrosis (CF). Five conserved housekeeping genes were selected for the MLSA, which was applied to a diverse collection of 77 strains originating from Europe, Asia, and South America and including type strains of the seven recognized Achromobacter species, six environmental strains, eight non-CF clinical strains, and 56 CF clinical strains. The discriminatory power of MLSA, based on 2,098 nucleotides (nt), was much superior to a 16S rRNA gene comparison based on 1,309 nt. Congruence was observed between single-gene trees and a concatenated gene tree. MLSA differentiated all seven current Achromobacter species and also demonstrated the presence of at least four novel potential species within the genus. CF isolates were predominantly Achromobacter xylosoxidans (64%), an undescribed Achromobacter species (18%), and Achromobacter ruhlandii (7%). A clone of Achromobacter, which has spread among patients from Danish CF centers in Aarhus and Copenhagen, was identified as Achromobacter ruhlandii. MLSA facilitates the specific identification of isolates of Achromobacter necessary for describing their role in clinical infections.
Assuntos
Achromobacter/classificação , Achromobacter/genética , Fibrose Cística/complicações , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Tipagem de Sequências Multilocus , Achromobacter/isolamento & purificação , Ásia , Análise por Conglomerados , Microbiologia Ambiental , Europa (Continente) , Genótipo , Humanos , Dados de Sequência Molecular , América do SulRESUMO
This study was designed to isolate and characterize endophytic bacteria from sunflower (Helianthus annuus) grown under irrigation and water stress (drought) conditions, to analyze growth of isolated bacteria under drought condition, and to evaluate the ability of bacteria isolated from plants cultivated under drought to produce jasmonates (JAs) and abscisic acid (ABA). Bacteria were isolated from soil samples collected when sunflower plants were at the end of the vegetative stage. A total of 29 endophytic strains were isolated from plants grown under irrigation or drought condition. Eight strains (termed SF1 through SF8) were selected based on nitrogen-fixing ability. All eight strains showed positive catalase and oxidase activities; five strains (SF2, SF3, SF4, SF5, SF7) solubilized phosphates; none of the strains produced siderophores. Strains SF2, SF3, SF4, and SF5, the ones with the highest phosphate solubilization ability, strongly inhibited growth of the pathogenic fungi Verticillum orense and Sclerotinia sclerotiorum but had less inhibitory effect on Alternaria sp. Among the eight strains, SF2 showed 99.9% sequence homology with Achromobacter xiloxidans or Alcaligenes sp., while the other seven showed 99.9% homology with Bacillus pumilus. Strains SF2, SF3, and SF4 grown in control medium produced jasmonic acid (JA), 12-oxo-phytodienoic acid (OPDA), and ABA. These three strains did not differ in amount of JA or OPDA produced. ABA content was higher than that of JA, and production of both ABA and JA increased under drought condition. The characteristics of these isolated bacterial strains have technological implications for inoculant formulation and improved growth of sunflower crops.