Maysa M. Clementino

Environmental Shaping of Sponge Associated Archaeal Communities

Background Archaea are ubiquitous symbionts of marine sponges but their ecological roles and the influence of environmental factors on these associations are still poorly understood. Methodology/Principal Findings We compared the diversity and composition of archaea associated with seawater and with the sponges Hymeniacidon heliophila, Paraleucilla magna and Petromica citrina in two distinct environments: Guanabara Bay, a highly impacted estuary in Rio de Janeiro, Brazil, and the nearby Cagarras Archipelago. For this we used metagenomic analyses of 16S rRNA and ammonia monooxygenase (amoA) gene libraries. Hymeniacidon heliophila was more abundant inside the bay, while P. magna was more abundant outside and P. citrina was only recorded at the Cagarras Archipelago. Principal Component Analysis plots (PCA) generated using pairwise unweighted UniFrac distances showed that the archaeal community structure of inner bay seawater and sponges was different from that of coastal Cagarras Archipelago. Rarefaction analyses showed that inner bay archaeaoplankton were more diverse than those from the Cagarras Archipelago. Only members of Crenarchaeota were found in sponge libraries, while in seawater both Crenarchaeota and Euryarchaeota were observed. Although most amoA archaeal genes detected in this study seem to be novel, some clones were affiliated to known ammonia oxidizers such as Nitrosopumilus maritimus and Cenarchaeum symbiosum. Conclusion/Significance The composition and diversity of archaeal communities associated with pollution-tolerant sponge species can change in a range of few kilometers, probably influenced by eutrophication. The presence of archaeal amoA genes in Porifera suggests that Archaea are involved in the nitrogen cycle within the sponge holobiont, possibly increasing its resistance to anthropogenic impacts. The higher diversity of Crenarchaeota in the polluted area suggests that some marine sponges are able to change the composition of their associated archaeal communities, thereby improving their fitness in impacted environments.

Archaeal communities in a tropical estuarine ecosystem: Guanabara Bay, Brazil.

Guanabara Bay is an eutrophic estuarine system located in a humid tropical region surrounded by the second largest metropolitan area of Brazil. This study explores the contrasting environmental chemistry and microbiological parameters that influence the archaeaplankton diversity in a pollution gradient in Guanabara Bay ecosystem. The environments sampled ranged from completely anoxic waters in a polluted inner channel to the adjacent, relatively pristine, coastal Atlantic Ocean. Partial archaeal 16S rDNA sequences in water samples were retrieved by polymerase chain reaction (PCR) and analyzed using denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Sequences were subjected to phylogenetic and diversity analyses. Community structure of the free-living archaeal assemblages was different from that of the particle-attached archaea according to DGGE. Gene libraries revealed that phylotype identification was consistent with environmental setting. Archaeal phylotypes found in polluted anoxic waters and in more pristine waters were closely related to organisms that have previously been found in these environments. However, inner bay archaea were related to organisms found in oil, industrial wastes, and sewage, implying that water pollution controls archaea communities in this system. The detection of a substantial number of uncultured phylotypes suggests that Guanabara Bay harbors a pool of novel archaeaplankton taxa.

Archaeal diversity in naturally occurring and impacted environments from a tropical region

Aims:  To evaluate archaeal diversity in natural and impacted habitats from Rio de Janeiro state, Brazil, a tropical region of South America.

PCR analyses of tRNA intergenic spacer, 16S-23S internal transcribed spacer, and randomly amplified polymorphic DNA reveal inter- and intraspecific relationships of Enterobacter cloacae strains.

ABSTRACT PCR analysis of tRNA intergenic spacer (tDNA-PCR) and of the 16S-23S internal transcribed spacer (ITS-PCR) and random amplified polymorphic DNA (RAPD) analysis were evaluated for their usefulness in characterization of Enterobacter cloacae strains isolated from both clinical origins and vaccine microbial contamination. tDNA-PCR presented specific and reproducible patterns for Enterobacter sakazakii ATCC 29004,Enterobacter aerogenes ATCC 13048, andEnterobacter cloacae ATCC 13047 and 23355 that presented the same profile for all 16 E. cloacae isolates, offering an alternative tool for species-level identification. ITS-PCR and RAPD analysis yielded completely different banding patterns for the 20 strains studied, except for E. cloacae strains isolated from different batches of vaccine that exhibited a unique pattern, suggesting contamination by the same strain. The combined use of tDNA-PCR and ITS-PCR in a one-step protocol allows accurate identification and typing of E. cloacae strains a few hours after the colony has been isolated.

Prokaryotic diversity in one of the largest hypersaline coastal lagoons in the world

Araruama Lagoon is an environment characterized by high salt concentrations. The low raining and high evaporation rates in this region favored the development of many salty ponds around the lagoon. In order to reveal the microbial composition of this system, we performed a 16S rRNA gene survey. Among archaea, most clones were related to uncultured environmental Euryarchaeota. In lagoon water, we found some clones related to Methanomicrobia and Methanothermococcus groups, while in the saline pond water members related to the genus Haloarcula were detected. Bacterial community was dominated by clones related to Gamma-proteobacteria, Actinobacteria, and Synechococcus in lagoon water, while Salinibacter ruber relatives dominated in saline pond. We also detected the presence of Alpha-proteobacteria, Pseudomonas-like bacteria and Verrucomicrobia. Only representatives of the genus Ralstonia were cosmopolitan, being observed in both systems. The detection of a substantial number of clones related to uncultured archaea and bacteria suggest that the hypersaline waters of Araruama harbor a pool of novel prokaryotic phylotypes, distinct from those observed in other similar systems. We also observed clones related to halophilic genera of cyanobacteria that are specific for each habitat studied. Additionally, two bacterioplankton molecular markers with ecological relevance were analyzed, one is linked to nitrogen fixation (nifH) and the other is linked to carbon fixation by bacterial photosynthesis, the protochlorophyllide genes, revealing a specific genetic distribution in this ecosystem. This is the first study of the biogeography and community structure of microbial assemblages in Brazilian tropical hypersaline environments. This work is directed towards a better understanding of the free-living prokaryotic diversity adapted to life in hypersaline waters.

Gut Bacterial Communities in the Giant Land Snail Achatina fulica and Their Modification by Sugarcane-Based Diet

The invasive land snail Achatina fulica is one of the most damaging agricultural pests worldwide representing a potentially serious threat to natural ecosystems and human health. This species is known to carry parasites and harbors a dense and metabolically active microbial community; however, little is known about its diversity and composition. Here, we assessed for the first time the complexity of bacterial communities occurring in the digestive tracts of field-collected snails (FC) by using culture-independent molecular analysis. Crop and intestinal bacteria in FC were then compared to those from groups of snails that were reared in the laboratory (RL) on a sugarcane-based diet. Most of the sequences recovered were novel and related to those reported for herbivorous gut. Changes in the relative abundance of Bacteroidetes and Firmicutes were observed when the snails were fed a high-sugar diet, suggesting that the snail gut microbiota can influence the energy balance equation. Furthermore, this study represents a first step in gaining a better understanding of land snail gut microbiota and shows that this is a complex holobiont system containing diverse, abundant and active microbial communities.

Coastal bacterioplankton community diversity along a latitudinal gradient in Latin America by means of V6 tag pyrosequencing

The bacterioplankton diversity of coastal waters along a latitudinal gradient between Puerto Rico and Argentina was analyzed using a total of 134,197 high-quality sequences from the V6 hypervariable region of the small-subunit ribosomal RNA gene (16S rRNA) (mean length of 60 nt). Most of the OTUs were identified into Proteobacteria, Bacteriodetes, Cyanobacteria, and Actinobacteria, corresponding to approx. 80% of the total number of sequences. The number of OTUs corresponding to species varied between 937 and 1946 in the seven locations. Proteobacteria appeared at high frequency in the seven locations. An enrichment of Cyanobacteria was observed in Puerto Rico, whereas an enrichment of Bacteroidetes was detected in the Argentinian shelf and Uruguayan coastal lagoons. The highest number of sequences of Actinobacteria and Acidobacteria were obtained in the Amazon estuary mouth. The rarefaction curves and Good coverage estimator for species diversity suggested a significant coverage, with values ranging between 92 and 97% for Good coverage. Conserved taxa corresponded to aprox. 52% of all sequences. This study suggests that human-contaminated environments may influence bacterioplankton diversity.

Microbial community compositional shifts in Bleached colonies of the Brazilian Reef-Building Coral "Siderastrea stellata"

The association of metazoan, protist, and microbial communities with Scleractinian corals forms the basis of the coral holobiont. Coral bleaching events have been occurring around the world, introducing changes in the delicate balance of the holobiont symbiotic interactions. In this study, Archaea, bacteria, and eukaryotic phototrophic plastids of bleached colonies of the Brazilian coral Siderastrea stellata were analyzed for the first time, using 16S rRNA gene libraries. Prokaryotic communities were slightly more diverse in healthy than in bleached corals. However, the eukaryotic phototrophic plastids community was more diverse in bleached corals. Archaea phylogenetic analyses revealed a high percentage of Crenarchaeota sequences, mainly related to Nitrosopumilusmaritimus and Cenarchaeum symbiosum. Dramatic changes in bacterial community composition were observed in this bleaching episode. The dominant bacterial group was Alphaproteobacteria followed by Gammaproteobacteria in bleached and Betaproteobacteria in healthy samples. Plastid operational taxonomic units (OTUs) from both coral samples were mainly related to red algae chloroplasts (Florideophycea), but we also observed some OTUs related to green algae chloroplasts (Chlorophyta). There seems to be a strong relationship between the Bacillariophyta phylum and our bleached coral samples as clones related to members of the diatom genera Amphora and Nitzschia were detected. The present study reveals information from a poorly investigated coral species and improves the knowledge of coral microbial community shifts that could occur during bleaching episodes.

Molecular Characterization of Quinolone-Resistant Neisseria gonorrhoeae Isolates from Brazil

ABSTRACT Despite the rapid spread of antibiotic resistance among gonococci worldwide, limited reports are available from Brazilian locations. In the present study, 25 quinolone-resistant Neisseria gonorrhoeae (QRNG) strains isolated in Rio de Janeiro, Brazil, were characterized by phenotypic and molecular methods, including analysis of mutations in the gyrA and parC genes. They represented 16.5% of the N. gonorrhoeae isolates obtained during a survey performed from 2006 to 2010. A trend for increasing resistance to ciprofloxacin was observed in the period investigated. The most prevalent pattern of mutation observed among QRNG isolates, Ser-91 to Phe and Asp-95 to Gly in gyrA and Ser-87 to Arg in parC, was detected in 40% of the isolates exhibiting MICs ranging from 4 to >32 μg/ml. Rare types of mutations were found in the gyrA gene (Gln-102 to His [12%] and Asp-95 to Tyr [4%]) and in the parC gene (Ser-88 to Thr [4%]). The genetic relationship of the QRNG isolates, evaluated by pulsed-field gel electrophoresis, suggested that the increase in the frequencies of the QRNG isolates in Rio de Janeiro, Brazil, may have arisen as a result of simultaneous spread of two clonal groups. The results also indicate that fluoroquinolones may no longer be used as first line antibiotics for the treatment of gonorrhea in Rio de Janeiro, and that programs for antimicrobial susceptibility surveillance of N. gonorrhoeae should also be implemented in other regions of Brazil.

Presence of qacEΔ1 Gene and Susceptibility to a Hospital Biocide in Clinical Isolates of Pseudomonas aeruginosa Resistant to Antibiotics

Biocides play an important role in healthcare-associated infection control by either minimizing or preventing microorganism dissemination. This study evaluated the susceptibility of Pseudomonas aeruginosa clinical isolates to a quaternary ammonium (QAC) disinfectant and antibiotics, and verified the presence of qacEΔ1, a determinant of resistance to QAC. The disinfectant test was the Association of Official Analytical Chemists Use-Dilution Test, and polymerase chain reaction was used to examine for qacEΔ1. The qacEΔ1 gene was detected in 48% of the isolates. Eighty-eight percent of the multiresistant isolates carried qacEΔ1 gene, while 35% of the non-multiresistant isolates was positive to this gene, and multiresistance well correlated with its presence. Among isolates tested for the disinfectant, 46% showed a reduced susceptibility to the disinfectant. qacEΔ1 gene was present in 70% of the susceptible isolates to the biocide, whereas 90% of the less susceptible strains harbored this gene. Reduced susceptibility to the disinfectant was independent of presence of qacEΔ1 suggesting that it does not play an important role in biocide resistance in P. aeruginosa. As far as we know, it is the first report confirming this fact and testing with disinfectant at its in-use concentration. The evidence of less susceptible strains than the reference bacterium used in disinfectant testing, and the high percentage of qacEΔ1 gene detected are of special concern and suggests continued investigation in laboratory and in situ, not only in healthcare settings, but also in all areas of biocide usage, including different micro-organisms and biocides.