Avaliação do perfil de resistência aos antimicrobianos em cepas de Escherichia coli e Staphylococcus spp. no canal do Marambaia em Balneário Camboriú, Santa Catarina / Evaluation of the antimicrobial resistance profile of isolated strains of Escherichia coli and Staphylococcus spp. in the Marambaia river in Balneário Camboriú, Santa Catarina

Introdução: Durante as últimas décadas, diversos micropoluentes orgânicos foram liberados no meio ambiente como resultado de atividades antropogênicas. Entre eles, se destacam os antibióticos, substâncias pouco biodegradáveis e muito persistentes que, ao se  acumularem nos sistemas aquáticos, proporcionam um ambiente favorável à seleção e proliferação de microrganismos resistentes a antimicrobianos. Objetivo: Avaliar o perfil de resistência aos antimicrobianos em cepas de Escherichia coli e Staphylococcus spp., isoladas no rio Marambaia e na praia adjacente, em Balneário Camboriú, Santa Catarina. Método: A amostragem foi dividida em seis coletas, totalizando 36 amostras e abrangendo todas as estações do ano. Foram realizadas análises microbiológicas fenotípicas, incluindo morfologia e testes de susceptibilidade aos antimicrobianos. A análise de dados foi realizada com o auxílio dos softwares Excel e Past, versão 4.03, através de análise explorativa e descritiva. Resultados: Foi verificada a presença de coliformes totais e termotolerantes, em todos os pontos amostrais, indicando a influência da desembocadura do rio na praia, além disso, a concentração de E. coli encontrada nos pontos amostrais está em desacordo com a Resolução Conama n° 357/2005, exceto para os pontos Marambaia 1 e Praia 3. De forma geral, cinco cepas apresentaram resistência a pelo menos três antibióticos, sendo a mais expressiva a ampicilina, com 14 cepas resistentes. O índice MAR para Staphylococcus spp. mostrou altos riscos para multirresistência, não apresentando diferenças para os fatores momento e local de coleta, sendo que 96 cepas apresentaram resistência para pelo menos um antibiótico. Foi encontrado um  número maior de cepas resistentes aos antimicrobianos eritromicina e clindamicina. Conclusões: A presença de E. coli é preocupante por se tratar de um coliforme termotolerante, indicativo de contaminação fecal recente. A existência de mecanismos de resistência nessas bactérias pode modificar a microbiota local, colocando em risco a saúde da população. Quanto aos Staphylococcus spp., através da avaliação dos dados do índice MAR, verificou-se riscos altos de multirresistência para a maioria dos locais amostrados e em qualquer momento das coletas. Além disso, poucas cepas foram sensíveis para todos os antibióticos testados. A análise do antibiograma revelou que 96 cepas apresentaram resistência para pelo menos uma classe de antimicrobiano. Os mecanismos de resistência que ocorrem em Gram-positivos, apesar de divergirem daqueles ocorridos em Gram-negativos, são igualmente preocupantes, seja do ponto de vista  icrobiológico ou em questões de saúde pública.

Introduction: During the last few decades, various organic micropollutants, including antibiotics, have been released into the environment because of anthropogenic activities. These drugs are poorly biodegradable and very persistent, such that when they accumulate in aquatic systems, they provide a favorable environment for the selection and proliferation of resistant microorganisms. Objective: Evaluate the antimicrobial resistance profile of isolated strains of Escherichia coli and Staphylococcus spp taken from the Marambaia river and its adjacent beach, in Balneário Camboriú, in the Brazilian state of Santa Catarina. Method: The sampling was divided into six collections, with a total of thirty-six samples, across all seasons of the year.  Subsequently, microbiological analyses and antibiotic sensitivity tests were performed. Data analysis was performed using the software programs Excel and Past, version 4.03, through exploratory and descriptive analyses. Results: The results showed the presence of total and thermotolerant coliforms at all sampling points, indicating the influence of the river outflow on the beach. Moreover, the concentrations of E. coli found in the sampling points are not compliant with Conama Resolution 357/2005,  except for the points Marambaia 1 and Praia 3. The MAR index for Staphylococcus showed high risks for multidrug resistance, with no differences for the time and place of collection. Ninety-six strains were resistant to at least one antibiotic. The isolated strains showed greater resistance to antibiotics erythromycin and clindamycin. Conclusions: The presence of E. coli is worrisome because it is a thermotolerant coliform, indicative of recent fecal contamination. The presence of resistance mechanisms in these bacteria can modify the local microbiota, putting the health of the population at risk. As for Staphylococcus spp., through the evaluation of the MAR index, high risks of multidrug resistance were found for most of the sampled locations and at any time of collection. In addition, few strains were sensitive to all antibiotics tested. The resistance mechanisms that occur in Gram-positives, despite differing from those that occur in Gram-negatives, are equally worrisome, whether from a microbiological point of view or in terms of public health.

Draft genome sequence of Psychrobacter nivimaris LAMA 639 and its biotechnological potential.

Bacteria of the genus Psychrobacter are known for their psychrophilic characteristics, being extremophilic organisms capable of surviving and reproducing in hostile environments of low temperature and high pressure. Among many of the genus characteristics, there is the ability to produce enzymes and molecules of industrial biotechnology importance, such as pigments and proteins related to heavy metal bioremediation. The bacterium strain Psychrobacter nivimaris LAMA 639 was isolated from sediments from the Walvis Ridge ocean crest at a depth of 4.400 m (33.40 S 2.35 E). It is a nonmotile, halotolerant, cream-colored gram-negative aerobic bacterium. Its cultivation was performed in marine agar plates and inoculated into test tubes with NaCl at an optimal temperature of 30 °C and with shaking at 100 rpm. Genome extraction was performed with the DNeasy Blood & Tissue Kit (QIAGEN®). Sequencing was performed by Macrogen using the NovaSeq® 6000 platform (Illumina) applying the whole genome shotgun (WGS) method. Thereafter, 14.712.526 reads of 151 bp were generated, totaling 2.2 G bp with a GC content of 42.9%. Assembly and mapping were performed with a CLC Genomics Workbench. The best assembly considered was the one with the lowest number of contigs and the highest base length pair. The assemblies were evaluated using QUAST, and the best resulting variant was selected for annotation. Genome annotation was performed with RAST and PATRIC; the antiSMASH tool was used for secondary metabolites; NaPDoS was used for domains; and three-dimensional structural prediction of relevant proteins was performed using Phyre2. Annotation with ClassicRAST generated 2,891 coding sequences (CDSs) distributed in 402 subsystems. Annotation with PATRIC generated 2,896 coding sequences, among them 776 hypothetical proteins. The antiSMASH tool visualized a beta-lactone cluster in contig 06. In the search for natural products with NaPDoS, two ketosynthase domains were identified. The search for relevant proteins was performed using the AMFEP list as a criterion. From these data, 34 possible enzymes with biotechnological potential were found. Finally, the organism is presented as a new reference regarding the potential of deep-sea marine bacteria, demonstrating that, from the annotated and cured genome, it is possible to find in its genetic repertory products of interest for biotechnological applications.

Evidence of selective pressure in whale fall microbiome proteins and its potential application to industry.

The present study addresses the microbiome of the first whale fall (YOKO 16) that has been described in the deep sea in the southern Atlantic Ocean (São Paulo Plateau; 4204 m depth), in terms of its metabolic uniqueness. Sets of ten thousand protein sequences from YOKO 16 and 29 public domain metagenomes (SRA and GenBank databases) that represent various marine, terrestrial and gut-associated microbial communities were analyzed. The determination of protein functionality, based on the KAAS server, indicated that the YOKO 16 microbiome has industrially-relevant proteins, such as proteases and lipases, that have low similarity (~50%) with previously-described enzymes. The amino acid usage in the YOKO 16 protein sequences (based on blastp and Clustal analysis) revealed a pattern of preference similar to that of extremophiles, with an increased usage of polar, charged and acidic amino acids and a decreased usage of nonpolar residues. We concluded that the targeted microbiome is of potential biotechnological use, which justifies the allocation of resources for the discovery of enzymes in deep-sea whale fall communities.

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria.

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)-a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Prospecting for Marine Bacteria for Polyhydroxyalkanoate Production on Low-Cost Substrates.

Polyhydroxyalkanoates (PHAs) are a class of biopolymers with numerous applications, but the high cost of production has prevented their use. To reduce this cost, there is a prospect for strains with a high PHA production and the ability to grow in low-cost by-products. In this context, the objective of this work was to evaluate marine bacteria capable of producing PHA. Using Nile red, 30 organisms among 155 were identified as PHA producers in the medium containing starch, and 27, 33, 22 and 10 strains were found to be positive in media supplemented with carboxymethyl cellulose, glycerol, glucose and Tween 80, respectively. Among the organisms studied, two isolates, LAMA 677 and LAMA 685, showed strong potential to produce PHA with the use of glycerol as the carbon source, and were selected for further studies. In the experiment used to characterize the growth kinetics, LAMA 677 presented a higher maximum specific growth rate (µmax = 0.087 h-1) than LAMA 685 (µmax = 0.049 h-1). LAMA 677 also reached a D-3-hydroxybutyrate (P(3HB)) content of 78.63% (dry biomass), which was 3.5 times higher than that of LAMA 685. In the assay of the production of P(3HB) from low-cost substrates (seawater and biodiesel waste glycerol), LAMA 677 reached a polymer content of 31.7%, while LAMA 685 reached 53.6%. Therefore, it is possible to conclude that the selected marine strains have the potential to produce PHA, and seawater and waste glycerol may be alternative substrates for the production of this polymer.