Genomic characterization of a diazotrophic microbiota associated with maize aerial root mucilage.

A geographically isolated maize landrace cultivated on nitrogen-depleted fields without synthetic fertilizer in the Sierra Mixe region of Oaxaca, Mexico utilizes nitrogen derived from the atmosphere and develops an extensive network of mucilage-secreting aerial roots that harbors a diazotrophic (N2-fixing) microbiota. Targeting these diazotrophs, we selected nearly 600 microbes of a collection obtained from mucilage and confirmed their ability to incorporate heavy nitrogen (15N2) metabolites in vitro. Sequencing their genomes and conducting comparative bioinformatic analyses showed that these genomes had substantial phylogenetic diversity. We examined each diazotroph genome for the presence of nif genes essential to nitrogen fixation (nifHDKENB) and carbohydrate utilization genes relevant to the mucilage polysaccharide digestion. These analyses identified diazotrophs that possessed the canonical nif gene operons, as well as many other operon configurations with concomitant fixation and release of >700 different 15N labeled metabolites. We further demonstrated that many diazotrophs possessed alternative nif gene operons and confirmed their genomic potential to derive chemical energy from mucilage polysaccharide to fuel nitrogen fixation. These results confirm that some diazotrophic bacteria associated with Sierra Mixe maize were capable of incorporating atmospheric nitrogen into their small molecule extracellular metabolites through multiple nif gene configurations while others were able to fix nitrogen without the canonical (nifHDKENB) genes.

In vivo development of fluconazole resistance in serial Cryptococcus gattii isolates from a cat.

Elevated fluconazole minimum inhibitory concentrations (MICs) are more frequently observed in Cryptococcus gattii compared to C. neoformans isolates; however, the development of in vivo resistance and the molecular mechanisms responsible have not been reported for this species. We report a case of Cryptococcus gattii (molecular type VGIII) that developed reduced susceptibility to fluconazole during therapy and delineate the molecular mechanisms responsible. Multilocus sequence typing and quantitative DNA analysis of the pre- and post-treatment isolates was performed using well-characterized methods. Pre- and post-treatment clinical isolates were confirmed isogenic, and no differences in ERG11 or PDR11 sequences were found. qPCR found an overexpression of ERG11 and the efflux pump PDR11 in the resistant isolate compared to the isolate collected prior to initiation of antifungal therapy. Reversion to wild-type susceptibility was observed when maintained in antifungal-free media confirming the in vivo development of heteroresistance. The in vivo development of heteroresistance to fluconazole in our patient with C. gattii is secondary to overexpression of the efflux pump PDR11 and the drug target ERG11. Additional work in other clinical isolates with elevated fluconazole MICs is warranted to evaluate the frequency of heteroresistance versus point mutations as a cause of resistance.

Fluconazole Susceptibility in Cryptococcus gattii Is Dependent on the ABC Transporter Pdr11.

Cryptococcus gattii isolates from the Pacific Northwest have exhibited higher fluconazole MICs than isolates from other sites. The mechanism of fluconazole resistance in C. gattii is unknown. We sought to determine the role of the efflux pumps Mdr1 and Pdr11 in fluconazole susceptibility. Using biolistic transformation of the parent isolate, we created a strain lacking Mdr1 (mdr1Δ) and another strain lacking Pdr11 (pdr11Δ). Phenotypic virulence factors were assessed by standard methods (capsule size, melanin production, growth at 30 and 37 °C). Survival was assessed in an intranasal murine model of cryptococcosis. Antifungal MICs were determined by the M27-A3 methodology. No differences in key virulence phenotypic components were identified. Fluconazole susceptibility was unchanged in the Mdr1 knockout or reconstituted isolates. However, fluconazole MICs decreased from 32 µg/ml for the wild-type isolate to <0.03 µg/ml for the pdr11Δ strain and reverted to 32 µg/ml for the reconstituted strain. In murine models, no difference in virulence was observed between wild-type, knockout, or reconstituted isolates. We conclude that Pdr11 plays an essential role in fluconazole susceptibility in C. gattii. Genomic and expression differences between resistant and susceptible C. gattii clinical isolates should be assessed further in order to identify other potential mechanisms of resistance.