Isabel Maida

Sponge-associated microbial Antarctic communities exhibiting antimicrobial activity against Burkholderia cepacia complex bacteria.

The aerobic heterotrophic bacterial communities isolated from three different Antarctic sponge species were analyzed for their ability to produce antimicrobial compounds active toward Cystic Fibrosis opportunistic pathogens belonging to the Burkholderia cepacia complex (Bcc). The phylogenetic analysis performed on the 16S rRNA genes affiliated the 140 bacterial strains analyzed to 15 genera. Just three of them (Psychrobacter, Pseudoalteromonas and Arthrobacter) were shared by the three sponges. The further Random Amplified Polymorphic DNA analysis allowed to demonstrate that microbial communities are highly sponge-specific and a very low degree of genus/species/strain sharing was detected. Data obtained revealed that most of these sponge-associated Antarctic bacteria and belonging to different genera were able to completely inhibit the growth of bacteria belonging to the Bcc. On the other hand, the same Antarctic strains did not have any effect on the growth of other pathogenic bacteria, strongly suggesting that the inhibition is specific for Bcc bacteria. Moreover, the antimicrobial compounds synthesized by the most active Antarctic bacteria are very likely Volatile Organic Compounds (VOCs), a finding that was confirmed by the SPME-GC-MS technique, which revealed the production of a large set of VOCs by a representative set of Antarctic bacteria. The synthesis of these VOCs appeared to be related neither to the presence of pks genes nor the presence of plasmid molecules. The whole body of data obtained in this work indicates that sponge-associated bacteria represent an untapped source for the identification of new antimicrobial compounds and are paving the way for the discovery of new drugs that can be efficiently and successfully used for the treatment of CF infections.

Molecular and phenotypic characterization of Acinetobacter strains able to degrade diesel fuel

Characterization of bacterial communities in oil-contaminated soils and evaluation of their degradation capacities may serve as a guide for improving remediation of such environments. Using physiological and molecular methods, the aim of this work was to characterize 17 Acinetobacter strains (13 species) able to use diesel fuel oil as sole carbon and energy source. The strains were first tested for their ability to grow on different alkanes on minimal medium containing high NaCl concentrations. The envelope hydrophobicity of each strain was assessed by microbial adhesion to the hydrocarbon test (MATH) when grown in LB medium or minimal medium containing succinate or diesel fuel. Most strains were hydrophobic both in LB and minimal medium, except for strain Acinetobacter venetianus VE-C3 that was hydrophobic only in minimal medium. Furthermore, two A. venetianus strains, RAG-1(T) and LUH 7437, and strain ATCC 17905 (genomic species 13BJ) displayed biosurfactant activity. The alkM gene encoding alkane hydroxylase was detected in the chromosome of the 15 strains by PCR amplification, sequencing and Southern blot analysis. Phenotype microarray analysis performed on the five A. venetianus strains revealed that they differentially used purines as N-source and confirmed that they are unable to use carbohydrates.

Exploring the HME and HAE1 efflux systems in the genus Burkholderia

BackgroundThe genus Burkholderia includes a variety of species with opportunistic human pathogenic strains, whose increasing global resistance to antibiotics has become a public health problem. In this context a major role could be played by multidrug efflux pumps belonging to Resistance Nodulation Cell-Division (RND) family, which allow bacterial cells to extrude a wide range of different substrates, including antibiotics. This study aims to i) identify rnd genes in the 21 available completely sequenced Burkholderia genomes, ii) analyze their phylogenetic distribution, iii) define the putative function(s) that RND proteins perform within the Burkholderia genus and iv) try tracing the evolutionary history of some of these genes in Burkholderia.ResultsBLAST analysis of the 21 Burkholderia sequenced genomes, using experimentally characterized ceoB sequence (one of the RND family counterpart in the genus Burkholderia) as probe, allowed the assembly of a dataset comprising 254 putative RND proteins. An extensive phylogenetic analysis revealed the occurrence of several independent events of gene loss and duplication across the different lineages of the genus Burkholderia, leading to notable differences in the number of paralogs between different genomes. A putative substrate [antibiotics (HAE1 proteins)/heavy-metal (HME proteins)] was also assigned to the majority of these proteins. No correlation was found between the ecological niche and the lifestyle of Burkholderia strains and the number/type of efflux pumps they possessed, while a relation can be found with genome size and taxonomy. Remarkably, we observed that only HAE1 proteins are mainly responsible for the different number of proteins observed in strains of the same species. Data concerning both the distribution and the phylogenetic analysis of the HAE1 and HME in the Burkholderia genus allowed depicting a likely evolutionary model accounting for the evolution and spreading of HME and HAE1 systems in the Burkholderia genus.ConclusionA complete knowledge of the presence and distribution of RND proteins in Burkholderia species was obtained and an evolutionary model was depicted. Data presented in this work may serve as a basis for future experimental tests, focused especially on HAE1 proteins, aimed at the identification of novel targets in antimicrobial therapy against Burkholderia species.

Identification of species of the Burkholderia cepacia complex by sequence analysis of the hisA gene.

Bacteria of the Burkholderia cepacia complex (Bcc) are opportunistic human pathogens that can cause serious infections in the lungs of cystic fibrosis patients. The Bcc is a complex taxonomic group and comprises 17 closely related species of both biotechnological and clinical importance that have been discriminated by a polyphasic taxonomic approach. In this study we focused on the hisA gene, which encodes a 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide isomerase involved in histidine biosynthesis, as a new target gene to discriminate among the Bcc species. PCR primers were designed to amplify a hisA DNA fragment of 442 bp from 78 strains representative of all the 17 Bcc species known at the time of writing. The nucleotide sequences of the amplicons were determined and aligned with the 54 Bcc sequences available in databases. Then a phylogenetic tree was constructed on the basis of this alignment and this revealed that this hisA region allows discrimination of all Bcc species, suggesting that this gene fragment can be used for the identification of Bcc strains. In addition, an 11 nucleotide letter code for the rapid discrimination of Bcc species was identified.

Genome‐scale metabolic reconstruction and constraint‐based modelling of the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125

The Antarctic strain Pseudoalteromonas haloplanktis TAC125 is one of the model organisms of cold-adapted bacteria and is currently exploited as a new alternative expression host for numerous biotechnological applications. Here, we investigated several metabolic features of this strain through in silico modelling and functional integration of -omics data. A genome-scale metabolic model of P. haloplanktis TAC125 was reconstructed, encompassing information on 721 genes, 1133 metabolites and 1322 reactions. The predictive potential of this model was validated against a set of experimentally determined growth rates and a large dataset of growth phenotypic data. Furthermore, evidence synthesis from proteomics, phenomics, physiology and metabolic modelling data revealed possible drawbacks of cold-dependent changes in gene expression on the overall metabolic network of P. haloplanktis TAC125. These included, for example, variations in its central metabolism, amino acid degradation and fatty acid biosynthesis. The genome-scale metabolic model described here is the first one reconstructed so far for an Antarctic microbial strain. It allowed a system-level investigation of variations in cellular metabolic fluxes following a temperature downshift. It represents a valuable platform for further investigations on P. haloplanktis TAC125 cellular functional states and for the design of more focused strategies for its possible biotechnological exploitation.

Genomic analysis of three sponge-associated Arthrobacter Antarctic strains, inhibiting the growth of Burkholderia cepacia complex bacteria by synthesizing volatile organic compounds.

In this work we analyzed the ability of three Arthrobacter strains (namely TB23, TB26 and CAL618), which were isolated from the Antarctic sponges Haliclonissa verrucosa and Lyssodendrix nobilis, to specifically inhibit the growth of a panel of 40 Burkholderia cepacia complex strains, representing a major cause of infections in patients that are affected by Cystic Fibrosis. The inhibitory activity was due to the synthesis of antimicrobial compounds, very likely volatile organic compounds (VOCs), and was partially dependent on the growth media that were used for Antarctic strains growth. The phylogenetic analysis revealed that two of them (i.e. CAL 618 and TB23) were very close and very likely belonged to the same Arthrobacter species, whereas the strain TB26 was placed in a distant branch. The genome of the strains TB26 and CAL618 was also sequenced and compared with that of the strain TB23. The analysis revealed that TB23 and CAL618 shared more genomic properties (GC content, genome size, number of genes) than with TB26. Since the three strains exhibited very similar inhibition pattern vs Bcc strains, it is quite possible that genes involved in the biosynthesis of antimicrobial compounds very likely belong to the core genome.

The genome sequence of the hydrocarbon-degrading Acinetobacter venetianus VE-C3

Here we report the genome sequence of Acinetobacter venetianus VE-C3, a strain isolated from the Venice Lagoon and known to be able to degrade n-alkanes. Post sequencing analyses revealed that this strain is relatively distantly related to the other Acinetobacter strains completely sequenced so far as shown by phylogenetic analysis and pangenome analysis (1285 genes shared with all the other Acinetobacter genomes sequenced so far). A. venetianus VE-C3 possesses a wide range of determinants whose molecular functions are probably related to the survival in a strongly impacted ecological niche. Among them, genes probably involved in the metabolism of long-chain n-alkanes and in the resistance to toxic metals (e.g. arsenic, cadmium, cobalt and zinc) were found. Genes belonging to these processes were found both on the chromosome and on plasmids. Also, our analysis documented one of the possible genetic bases underlying the strategy adopted by A. venetianus VE-C3 for the adhesion to oil fuel droplets, which could account for the differences existing in this process with other A. venetianus strains. Finally, the presence of a number of DNA mobilization-related genes (i.e. transposases, integrases, resolvases) strongly suggests an important role played by horizontal gene transfer in shaping the genome of A. venetianus VE-C3 and in its adaptation to its special ecological niche.

Endophytic and rhizospheric bacterial communities isolated from the medicinal plants Echinacea purpurea and Echinacea angustifolia

The Iberian Pyrite Belt, located in Southwestern Spain, represents one of the worlds largest accumulations of mine wastes and acid mine drainages. This study reports the comparative microbial ecology of the water column of Nuestra Señora del Carmen acid pit lake with the extreme acidic Río Tinto basin. The canonical correspondence analysis identified members of the Leptospirillum, Acidiphilium, Metallibacterium, Acidithiobacillus, Ferrimicrobium and Acidisphaera genera as the most representative microorganisms of both ecosystems. The presence of archaeal members is scarce in both systems. Only sequences clustering with the Thermoplasmata have been retrieved in the bottom layer of Nuestra Señora del Carmen and one station of Río Tinto. Although the photosynthetically active radiation values measured in this lake upper layer were low, they were sufficient to activate photosynthesis in acidophilic microorganisms. All identified photosynthetic microorganisms in Nuestra Señora del Carmen (members of the Chlamydomonas, Zygnemopsis and Klebsormidium genera) are major members of the photosynthetic eukaryotic community characterized in Río Tinto basin. This study demonstrates a close relationship between the microbial diversity of Nuestra Señora del Carmen pit lake and the diversity detected in the Río Tinto basin, which underlain the influence of the shared mineral substrates in the microbial ecology of these ecosystems.In this work we analyzed the composition and structure of cultivable bacterial communities isolated from the stem/leaf and root compartments of two medicinal plants, Echinacea purpurea (L.) Moench and Echinacea angustifolia (DC.) Hell, grown in the same soil, as well as the bacterial community from their rhizospheric soils. Molecular PCR-based techniques were applied to cultivable bacteria isolated from the three compartments of the two plants. The results showed that the two plants and their respective compartments were characterized by different communities, indicating a low degree of strain sharing and a strong selective pressure within plant tissues. Pseudomonas was the most highly represented genus, together with Actinobacteria and Bacillus spp. The presence of distinct bacterial communities in different plant species and among compartments of the same plant species could account for the differences in the medicinal properties of the two plants.

Phenotypic and genomic characterization of the Antarctic bacterium Gillisia sp. CAL575, a producer of antimicrobial compounds

Microorganisms from Antarctica have evolved particular strategies to cope with cold. Moreover, they have been recently reported as producers of antimicrobial compounds, which inhibit the growth of other bacteria. In this work we characterized from different viewpoints the Gillisia sp. CAL575 strain, a psychrotrophic bacterium that produces microbial volatile organic compounds involved in the growth inhibition of Burkholderia cepacia complex members. Sequencing and analysis of the whole genome of Gillisia sp. CAL575 revealed that it includes genes that are involved in secondary metabolite production, adaptation to cold conditions, and different metabolic pathways for the production of energy. All these features make Gillisia sp. CAL575 a possible tool for biotechnology.

Exploring the Anti-Burkholderia cepacia Complex Activity of Essential Oils: A Preliminary Analysis

In this work we have checked the ability of the essential oils extracted from six different medicinal plants (Eugenia caryophyllata, Origanum vulgare, Rosmarinus officinalis, Lavandula officinalis, Melaleuca alternifolia, and Thymus vulgaris) to inhibit the growth of 18 bacterial type strains belonging to the 18 known species of the Burkholderia cepacia complex (Bcc). These bacteria are opportunistic human pathogens that can cause severe infection in immunocompromised patients, especially those affected by cystic fibrosis (CF), and are often resistant to multiple antibiotics. The analysis of the aromatograms produced by the six oils revealed that, in spite of their different chemical composition, all of them were able to contrast the growth of Bcc members. However, three of them (i.e., Eugenia caryophyllata, Origanum vulgare, and Thymus vulgaris) were particularly active versus the Bcc strains, including those exhibiting a high degree or resistance to ciprofloxacin, one of the most used antibiotics to treat Bcc infections. These three oils are also active toward both environmental and clinical strains (isolated from CF patients), suggesting that they might be used in the future to fight B. cepacia complex infections.