Luigi Michaud

Biodiversity of cultivable psychrotrophic marine bacteria isolated from Terra Nova Bay (Ross Sea, Antarctica)

A set of 146 Antarctic marine isolates from the Ross Sea was characterized by a combination of molecular techniques in order to determine the degree of inter- and intraspecific variability. Isolates were analyzed by amplified rDNA restriction analysis (ARDRA) using the tetrameric enzyme AluI, resulting in 52 different groups, corresponding to at least 52 different bacterial species, indicating a high degree of interspecific variability. The phylogenetic position of bacteria belonging to some ARDRA groups was obtained by sequencing of 16S rDNA. Random amplified polymorphic DNA (RAPD) analysis, carried out on the largest ARDRA groups, revealed a high intraspecific genetic variability, too. The analysis of plasmid content revealed the existence of horizontal gene transfer between strains belonging to the same and to different species. A comparison of the whole body of morphological, physiological and biochemical data was finally carried out.

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.

Phylogenetic characterization of the heterotrophic bacterial communities inhabiting a marine recirculating aquaculture system.

Aims:  The aim of the present work was to characterize the heterotrophic bacterial community of a marine recirculating aquaculture system (RAS).

Marine Bacterioplankton Diversity and Community Composition in an Antarctic Coastal Environment

The bacterial community inhabiting the water column at Terra Nova Bay (Ross Sea, Antarctica) was examined by the fluorescent in situ hybridization (FISH) technique and the genotypic and phenotypic characterization of 606 bacterial isolates. Overall, the FISH analysis revealed a bacterioplankton composition that was typical of Antarctic marine environments with the Cytophaga/Flavobacter (CF) group of Bacteroidetes that was equally dominant with the Actinobacteria and Gammaproteobacteria. As sampling was performed during the decay of sea-ice, it is plausible to assume the origin of Bacteroidetes from the sea-ice compartment where they probably thrive in high concentration of DOM which is efficiently remineralized to inorganic nutrients. This finding was supported by the isolation of Gelidibacter, Polaribacter, and Psychroflexus members (generally well represented in Antarctic sea-ice) which showed the ability to hydrolyze macromolecules, probably through the production of extracellular enzymes. A consistently pronounced abundance of the Gammaproteobacteria (67.8%) was also detected within the cultivable fraction. Altogether, the genera Psychromonas and Pseudoalteromonas accounted for 65.4% of total isolates and were ubiquitous, thus suggesting that they may play a key role within the analyzed bacterioplankton community. In particular, Pseudoalteromonas isolates possessed nitrate reductase and were able to hydrolyze substrates for protease, esterase, and β-galactosidase, thus indicating their involvement in the carbon and nitrogen cycling. Finally, the obtained results highlight the ability of the Actinobacteria to survive and proliferate in the Terra Nova Bay seawater as they generally showed a wide range of salt tolerance and appeared to be particularly competitive with strictly marine bacteria by better utilizing supplied carbon sources.

Characterization of the volatile profile of Antarctic bacteria by using solid‐phase microextraction‐gas chromatography‐mass spectrometry

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are significant pathogens in Cystic Fibrosis (CF) patients and are resistant to a plethora of antibiotics. In this context, microorganisms from Antarctica are interesting because they produce antimicrobial compounds inhibiting the growth of other bacteria. This is particularly true for bacteria isolated from Antarctic sponges. The aim of this work was to characterize a set of Antarctic bacteria for their ability to produce new natural drugs that could be exploited in the control of infections in CF patients by Bcc bacteria. Hence, 11 bacterial strains allocated to different genera (e.g., Pseudoalteromonas, Arthrobacter and Psychrobacter) were tested for their ability to inhibit the growth of 21 Bcc strains and some other human pathogens. All these bacteria completely inhibited the growth of most, if not all, Bcc strains, suggesting a highly specific activity toward Bcc strains. Experimental evidences showed that the antimicrobial compounds are small volatile organic compounds, and are constitutively produced via an unknown pathway. The microbial volatile profile was obtained by SPME-GC-MS within the m/z interval of 40-450. Solid phase micro extraction technique affords the possibility to extract the volatile compounds in head space with a minimal sample perturbation. Principal component analysis and successive cluster discriminant analysis was applied to evaluate the relationships among the volatile organic compounds with the aim of classifying the microorganisms by their volatile profile. These data highlight the potentiality of Antarctic bacteria as novel sources of antibacterial substances to face Bcc infections in CF patients.

Lipolytic activity of Antarctic cold-adapted marine bacteria (Terra Nova Bay, Ross Sea)

Aims:  The aim of this study was to investigate the lipolytic activity of cold‐adapted Antarctic marine bacteria and, furthermore, the combined effect of some environmental factors on this enzymatic process.

Bacteria associated with sabellids (Polychaeta: Annelida) as a novel source of surface active compounds

A total of 69 bacteria were isolated from crude oil enrichments of the polychaetes Megalomma claparedei, Sabella spallanzanii and Branchiomma luctuosum, and screened for biosurfactant (BS) production by conventional methods. Potential BS-producers (30 isolates) were primarily selected due to the production of both interesting spots on thin layer chromatography (TLC) plates and highly stable emulsions (E₂₄ ≥ 50%). Only few strains grew on cetyltrimethylammonium bromide and blood agar plates, indicating the probable production of anionic surfactants. The 16S rRNA gene sequencing revealed that selected isolates mainly belonged to the CFB group of Bacteroidetes, followed by Gammaproteobacteria and Alphaproteobacteria. A number of BS-producers belonged to genera (i.e., Cellulophaga, Cobetia, Cohaesibacter, Idiomarina, Pseudovibrio and Thalassospira) that have been never reported as able to produce BSs, even if they have been previously detected in hydrocarbon-enriched samples. Our results suggest that filter-feeding Polychaetes could represent a novel and yet unexplored source of biosurfactant-producing bacteria.

Partaking of Archaea to biogeochemical cycling in oxygen-deficient zones of meromictic saline Lake Faro (Messina, Italy).

We used a combination of molecular and microbiological approaches to determine the activity, abundance and diversity of archaeal populations inhabiting meromictic saline Lake Faro (Messina, Italy). Analysis of archaeal 16S rRNA, amoA, accA and hbd genes and transcripts revealed that sub- and anoxic layers of Lake Faro are primarily inhabited by the organisms related to the clusters of Marine Group I.1a of Thaumarchaeota frequently recovered from oxygen-depleted marine ecosystems. These organisms dominated the metabolically active archaea down to the bottom of the lake, indicating their adaptation to recurrent changes in the levels of water column hypoxia. The upper microaerobic layer of Lake Faro redoxcline has the maximal rates of dark primary production much lower than those of other previously studied pelagic redoxclines, but comparable to the values of meso- and bathypelagic areas of Mediterranean Sea. Application of bacterial inhibitors, especially azide, significantly declined the CO2 fixation rates in the low interface and monimolimnion, whereas archaea-specific inhibitor had effect only in upper part of the redoxcline. Based on these findings, we hypothesize that dark bicarbonate fixation in suboxic zone of Lake Faro results mainly from archaeal activity which is affected by the predicted lack in oxygen in lower layers.

GC–MS volatolomic approach to study the antimicrobial activity of the antarctic bacterium Pseudoalteromonas sp. TB41

Many bacteria produce a wide range of volatile info-chemicals compounds (mVOCs) that constitute an important regulatory factor in the interrelationships among different organisms in microbial ecosystems. It has been shown that Antarctic bacteria isolated from three different sponge species, by producing mVOCs, are able to inhibit specifically the growth of Burkholderia cepacia complex (Bcc) strains (i.e. opportunistic pathogens of cystic fibrosis patients) as demonstrated by cross-streaking inhibition assays. This study reports a metabolomics approach to investigate the volatile profile of both the Antarctic sponge-associated Pseudoalteromonas sp. TB41 (P-sp-TB41) and Burkholderia cenocepacia strain LMG16654 (Bc-LMG16654) under aerobic conditions. Solid phase micro extraction (SPME) in head space of biological samples allowed an in vivo sampling of mVOCs with minimal specimen disturbance. The SPME fiber was termically desorbed in the injection port of gas chromatography–mass spectrometer (GC–MS) system setted in EI scan mode. The raw data were processed using both an automated mass spectra deconvolution and identification system and a metabolomic approach, which allowed a selection of 30 compounds presumably responsible for the inhibition of Bc-LMG16654 growth. The results obtained from samples prepared under cross-streaking conditions also suggest that the presence of Bc-LMG16654 cells neither interferes with the production of mVOCs nor induces the synthesis of different mVOCs. The employing of mass spectrometry played a key role in tuning the experimental system and in the evaluation of results. The use of this approach to study the interaction, in aerobic condition, among other Antarctic bacteria and Bcc and the possibility to extend this approach to other pathogen-antagonist relationship, is currently in progress.

Occurrence and characterization of psychrotolerant hydrocarbon-oxidizing bacteria from surface seawater along the Victoria Land coast (Antarctica)

A total of 253 hydrocarbon-oxidizing bacterial isolates were achieved from eight Antarctic surface seawater samples enriched on diesel oil at 4°C. Isolates were screened by amplified ribosomal DNA restriction analysis prior to 16S rRNA gene sequencing. Sequences were compared to those in available databases using the Basic Local Alignment Search Tool network service to determine their approximate phylogenetic affiliations. The majority of the isolates were affiliated to the Actinobacteria (75.9%) and the Gamma-Proteobacteria (22.9%). The Alpha- and Beta-Proteobacteria represented 0.8 and 0.4% of total isolates, respectively. The Actinobacteria were predominantly allocated to the genera Arthrobacter, Cryobacterium and Rhodococcus. The Gamma-Proteobacteria were mainly found to be related to the genus Pseudomonas. Conversely, the Alpha- and Beta-Proteobacterial isolates shared the highest degree of sequence identity with unclassified bacteria. Differences in the distribution of the detected phylotypes were observed among the analyzed samples. Isolates representing each phylotype were selected for further characterization, including phenotypic assays and screening for the growth ability in the presence of individual hydrocarburic substrates as the sole supplied carbon and energy source. Isolates possessed different patterns of substrate utilization. Aliphatic hydrocarbons supported the growth of a higher number of isolates than aromatics. Results confirm the ability of our Antarctic marine bacteria to utilize hydrocarbons at low temperature and therefore suggesting that isolates with different substrate specificities can act in nature as a consortium in the utilization of complex hydrocarburic mixtures.