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Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates.

ABSTRACT: Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the selected isolates of the endophytic bacterial community of sugarcane belong to the genera of Burkholderia, Pantoea, Pseudomonas, and Microbacterium. Bacterial isolates belonging to the genus Burkholderia were the most predominant among the endophytic bacteria. Many of the Burkholderia isolates produced the antifungal metabolite pyrrolnitrin, and all were able to grow at 37 degrees C. Phylogenetic analyses of the 16S rRNA gene and recA gene sequences indicated that the endophytic Burkholderia isolates from sugarcane are closely related to clinical isolates of the Burkholderia cepacia complex and clustered with B. cenocepacia (gv. III) isolates from cystic fibrosis patients. These results suggest that isolates of the B. cepacia complex are an integral part of the endophytic bacterial community of sugarcane in Brazil and reinforce the hypothesis that plant-associated environments may act as a niche for putative opportunistic human pathogenic bacteria.

SUBMITTER: Mendes R

PROVIDER: S-EPMC2168197 | biostudies-literature | 2007 Nov

REPOSITORIES: biostudies-literature

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Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates.

Mendes Rodrigo R Pizzirani-Kleiner Aline A AA Araujo Welington L WL Raaijmakers Jos M JM

Applied and environmental microbiology 20070928 22

Bacteria were isolated from the rhizosphere and from inside the roots and stems of sugarcane plants grown in the field in Brazil. Endophytic bacteria were found in both the roots and the stems of sugarcane plants, with a significantly higher density in the roots. Many of the cultivated endophytic bacteria were shown to produce the plant growth hormone indoleacetic acid, and this trait was more frequently found among bacteria from the stem. 16S rRNA gene sequence analysis revealed that the select ...[more]

PMID: 17905875

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Project description:Burkholderia pseudomallei, the founding member of the B. pseudomallei complex (Bpc), is a biothreat agent and causes melioidosis, a disease whose treatment mainly relies on ceftazidime and meropenem. The concern is that B. pseudomallei could enhance its drug resistance repertoire by the acquisition of DNA from resistant near-neighbor species. Burkholderia ubonensis, a member of the B. cepacia complex (Bcc), is commonly coisolated from environments where B. pseudomallei is present. Unlike B. pseudomallei, in which significant primary carbapenem resistance is rare, it is not uncommon in B. ubonensis, but the underlying mechanisms are unknown. We established that carbapenem resistance in B. ubonensis is due to an inducible class A PenB ?-lactamase, as has been shown for other Bcc bacteria. Inducibility is not sufficient for high-level resistance but also requires other determinants, such as a PenB that is more robust than that present in susceptible isolates, as well as other resistance factors. Curiously and diagnostic for the two complexes, both Bpc and Bcc bacteria contain distinct annotated PenA class A ?-lactamases. However, the protein from Bcc bacteria is missing its essential active-site serine and, therefore, is not a ?-lactamase. Regulated expression of a transcriptional penB'-lacZ (?-galactosidase) fusion in the B. pseudomallei surrogate B. thailandensis confirms that although Bpc bacteria lack an inducible ?-lactamase, they contain the components required for responding to aberrant peptidoglycan synthesis resulting from ?-lactam challenge. Understanding the diversity of antimicrobial resistance in Burkholderia species is informative about how the challenges arising from potential resistance transfer between them can be met.IMPORTANCE Burkholderia pseudomallei causes melioidosis, a tropical disease that is highly fatal if not properly treated. Our data show that, in contrast to B. pseudomallei, B. ubonensis ?-lactam resistance is fundamentally different because intrinsic resistance is mediated by an inducible class A ?-lactamase. This includes resistance to carbapenems. Our work demonstrates that studies with near-neighbor species are informative about the diversity of antimicrobial resistance in Burkholderia and can also provide clues about the potential of resistance transfer between bacteria inhabiting the same environment. Knowledge about potential adverse challenges resulting from the horizontal transfer of resistance genes between members of the two complexes enables the design of effective countermeasures.

Project description:Members of the Burkholderia cepacia complex (Bcc) are an important cause of opportunistic or nosocomial infections that may be hard to treat due to a high incidence of multidrug resistance. We characterised a collection of 51 clinical isolates from this complex, assigning them to 18 sequence types using multi-locus sequence type analysis. Resistance to eight commonly used antibiotics was assessed using by using agar-dilution assays to calculate MICs and widespread and heterogeneous multidrug resistance was confirmed, with eight strains proving resistant to all antibiotics tested. Disc diffusion screening of antimicrobial activity of a range of plant essential oils against these Bcc isolates identified six oils with significant activity (lavender, lemongrass, marjoram, peppermint, tea tree and rosewood) and broth microdilution assays indicated that of these lemongrass and rosewood oils had the highest activity, with MIC50 values of 0.5% and MIC90 values of 1%. Comparison of MIC and MBC values showed that four of these six oils, including lemongrass and rosewood, were bacteriocidal rather than bacteriostatic in their effects. Qualitative analysis of the four bacteriocidal essential oils via GC/MS indicated the presence of 55 different component compounds, mostly monoterpenes. We assessed selected essential oil components as anti-Bcc agents and demonstrated that terpinen-4-ol and geraniol were effective with MICs of 0.125-0.5% (v/v) and 0.125-1% (v/v), respectively. Time-kill studies indicate that these two alcohols are effective against non-growing cells in an efflux-dependent manner. Analysis of bacterial leakage of potassium ions and 260 nm UV-absorbing material on treatment with terpinen-4-ol and geraniol suggested that the observed anti-Bcc activity was a consequence of membrane disruption. This finding was supported by a gas chromatography analysis of bacterial fatty acid methyl esters, which indicated changes in membrane fatty acid composition caused by terpinen-4-ol and geraniol. These essential oils or oil components may ultimately prove useful as therapeutic drugs, for example to treat Bcc infections in CF patients.

Dataset Information

Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates.

Publications

Diversity of cultivated endophytic bacteria from sugarcane: genetic and biochemical characterization of Burkholderia cepacia complex isolates.

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