Combining Functional Genomics and Whole-Genome Sequencing to Detect Antibiotic Resistance Genes in Bacterial Strains Co-Occurring Simultaneously in a Brazilian Hospital.

(1) Background: The rise of multi-antibiotic resistant bacteria represents an emergent threat to human health. Here, we investigate antibiotic resistance mechanisms in bacteria of several species isolated from an intensive care unit in Brazil. (2) Methods: We used whole-genome analysis to identify antibiotic resistance genes (ARGs) and plasmids in 34 strains of Gram-negative and Gram-positive bacteria, providing the first genomic description of Morganella morganii and Ralstonia mannitolilytica clinical isolates from South America. (3) Results: We identified a high abundance of beta-lactamase genes in resistant organisms, including seven extended-spectrum beta-lactamases (OXA-1, OXA-10, CTX-M-1, KPC, TEM, HYDRO, BLP) shared between organisms from different species. Additionally, we identified several ARG-carrying plasmids indicating the potential for a fast transmission of resistance mechanism between bacterial strains. Furthermore, we uncovered two pairs of (near) identical plasmids exhibiting multi-drug resistance. Finally, since many highly resistant strains carry several different ARGs, we used functional genomics to investigate which of them were indeed functional. In this sense, for three bacterial strains (Escherichia coli, Klebsiella pneumoniae, and M. morganii), we identified six beta-lactamase genes out of 15 predicted in silico as those mainly responsible for the resistance mechanisms observed, corroborating the existence of redundant resistance mechanisms in these organisms. (4) Conclusions: Systematic studies similar to the one presented here should help to prevent outbreaks of novel multidrug-resistant bacteria in healthcare facilities.

Microbial Community Profiling in Intensive Care Units Expose Limitations in Current Sanitary Standards.

Hospital-associated infections (HAIs) are a leading cause of morbidity and mortality in intensive care units (ICUs) and neonatal intensive care units (NICUs). Organisms causing these infections are often present on surfaces around the patient. Given that microbiota may vary across different ICUs, the HAI-related microbial signatures within these units remain underexplored. In this study, we use deep-sequencing analyses to explore and compare the structure of bacterial communities at inanimate surfaces of the ICU and NICU wards of The Medical School Clinics Hospital (Brazil). The data revealed that NICU presents higher biodiversity than ICU and surfaces closest to the patient showed a peculiar microbiota, distinguishing one unit from the other. Several facultative anaerobes or obligate anaerobes HAI-related genera were classified as biomarkers for the NICU, whereas Pseudomonas was the main biomarker for ICU. Correlation analyses revealed a distinct pattern of microbe-microbe interactions for each unit, including bacteria able to form multi-genera biofilms. Furthermore, we evaluated the effect of concurrent cleaning over the ICU bacterial community. The results showed that, although some bacterial populations decreased after cleaning, various HAI-related genera were quite stable following sanitization, suggesting being well-adapted to the ICU environment. Overall, these results enabled identification of discrete ICU and NICU reservoirs of potentially pathogenic bacteria and provided evidence for the presence of a set of biomarkers genera that distinguish these units. Moreover, the study exposed the inconsistencies of the routine cleaning to minimize HAI-related genera contamination.

Genetically Engineered Proteins to Improve Biomass Conversion: New Advances and Challenges for Tailoring Biocatalysts.

Protein engineering emerged as a powerful approach to generate more robust and efficient biocatalysts for bio-based economy applications, an alternative to ecologically toxic chemistries that rely on petroleum. On the quest for environmentally friendly technologies, sustainable and low-cost resources such as lignocellulosic plant-derived biomass are being used for the production of biofuels and fine chemicals. Since most of the enzymes used in the biorefinery industry act in suboptimal conditions, modification of their catalytic properties through protein rational design and in vitro evolution techniques allows the improvement of enzymatic parameters such as specificity, activity, efficiency, secretability, and stability, leading to better yields in the production lines. This review focuses on the current application of protein engineering techniques for improving the catalytic performance of enzymes used to break down lignocellulosic polymers. We discuss the use of both classical and modern methods reported in the literature in the last five years that allowed the boosting of biocatalysts for biomass degradation.

Engineering the affinity of a family 11 carbohydrate binding module to improve binding of branched over unbranched polysaccharides.

Carbohydrate binding modules (CBMs) are non-catalytic domains within larger multidomain polypeptides. The CelH from Ruminoclostridium (Clostridium) thermocellum contains a family 11 CBM (RtCBM11) with high binding affinity for the linear polysaccharide ß-glucan, and low affinity for the branched xyloglucan. Screening a random RtCBM11 mutant phage library created by error prone PCR for xyloglucan binding identified RtCBM11 mutants with enhanced xyloglucan affinity. Subsequent recombination of the selected variants by site-directed mutagenesis generated the H102L/Y152F and Y46N/G52D/H102L/Y152F mutants. Fusion of the quadruple RtCBM11 mutant with the xyloglucanase from Aspergillus niveus increased the catalytic efficiency of the enzyme by 38%. Isothermal titration calorimetry demonstrated increased xyloglucan affinity for both mutants and reduced affinity for ß-glucan in the H102L/Y152F mutant. Molecular dynamics simulations indicated that the increased xyloglucan specificity results both from formation of a xylosyl binding pocket in the carbohydrate binding cleft, and via modulation of a hydrogen bond network between the oligosaccharide ligand and the protein. These results explain the improved xyloglucan binding in the RtCBM11 H102L/Y152F mutant and advance the understanding of the structural determinants of CBMs binding that discriminate between branched and unbranched polysaccharides.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

A xylose-stimulated xylanase-xylose binding protein chimera created by random nonhomologous recombination.

BACKGROUND: Saccharification of lignocellulosic material by xylanases and other glycoside hydrolases is generally conducted at high concentrations of the final reaction products, which frequently inhibit the enzymes used in the saccharification process. Using a random nonhomologous recombination strategy, we have fused the GH11 xylanase from Bacillus subtilis (XynA) with the xylose binding protein from Escherichia coli (XBP) to produce an enzyme that is allosterically stimulated by xylose. RESULTS: The pT7T3GFP_XBP plasmid containing the XBP coding sequence was randomly linearized with DNase I, and ligated with the XynA coding sequence to create a random XynA-XBP insertion library, which was used to transform E. coli strain JW3538-1 lacking the XBP gene. Screening for active XBP was based on the expression of GFP from the pT7T3GFP_XBP plasmid under the control of a xylose inducible promoter. In the presence of xylose, cells harboring a functional XBP domain in the fusion protein (XBP+) showed increased GFP fluorescence and were selected using FACS. The XBP+ cells were further screened for xylanase activity by halo formation around xylanase producing colonies (XynA+) on LB-agar-xylan media after staining with Congo red. The xylanase activity ratio with xylose/without xylose in supernatants from the XBP+/XynA+ clones was measured against remazol brilliant blue xylan. A clone showing an activity ratio higher than 1.3 was selected where the XynA was inserted after the asparagine 271 in the XBP, and this chimera was denominated as XynA-XBP271. The XynA-XBP271 was more stable than XynA at 55 °C, and in the presence of xylose the catalytic efficiency was ~3-fold greater than the parental xylanase. Molecular dynamics simulations predicted the formation of an extended protein-protein interface with coupled movements between the XynA and XBP domains. In the XynA-XBP271 with xylose bound to the XBP domain, the mobility of a ß-loop in the XynA domain results in an increased access to the active site, and may explain the observed allosteric activation. CONCLUSIONS: The approach presented here provides an important advance for the engineering enzymes that are stimulated by the final product.

Insertion of a xylanase in xylose binding protein results in a xylose-stimulated xylanase.

BACKGROUND: Product inhibition can reduce catalytic performance of enzymes used for biofuel production. Different mechanisms can cause this inhibition and, in most cases, the use of classical enzymology approach is not sufficient to overcome this problem. Here we have used a semi-rational protein fusion strategy to create a product-stimulated enzyme. RESULTS: A semi-rational protein fusion strategy was used to create a protein fusion library where the Bacillus subtilis GH11 xylanase A (XynA) was inserted at 144 surface positions of the Escherichia coli xylose binding protein (XBP). Two XynA insertions at XBP positions 209 ([209]XBP-Xyn-XBP) and 262 ([262]XBP-Xyn-XBP) showed a 20% increased xylanolytic activity in the presence of xylose, conditions where native XynA is inhibited. Random linkers of 1-4 Gly/Ala residues were inserted at the XynA N- and C-termini in the [209]XBP and [262]XBP, and the chimeras 2091A and 2621B were isolated, showing a twofold increased xylanolytic activity in the presence of xylose and k cat values of 200 and 240 s(-1) in the 2091A and 2621B, respectively, as compared to 70 s(-1) in the native XynA. The xylose affinity of the XBP was unchanged in the chimeras, showing that the ~3- to 3.5-fold stimulation of catalytic efficiency by xylose was the result of allosteric coupling between the XBP and XynA domains. Molecular dynamics simulations of the chimeras suggested conformation alterations in the XynA on xylose binding to the XBP resulted in exposure of the catalytic cavity and increased mobility of catalytic site residues as compared to the native XynA. CONCLUSIONS: These results are the first report of engineered glycosyl hydrolase showing allosteric product stimulation and suggest that the strategy may be more widely employed to overcome enzyme product inhibition and to improve catalytic performance. Graphical abstractProtein fusion of a GH11 xylanase (in red) and a xylose binding protein (XBP, in blue) results in a xylanase-XBP chimera that presents allosteric activation of the xylanase activity by xylose (shown as a space-filled molecule bound to the xylanase-XBP chimera).

Reestruturação do Programa de atividade física da terceira idade para a redução do sedentarismo dos idosos no Programa Saúde da Família

Atualmente o sedentarismo é um problema de saúde pública, tanto em países em desenvolvimento quanto em países de primeiro mundo, sendo a inatividade física um importante fator de risco para as doenças cardiovasculares e outras doenças crônicas. Os benefícios da atividade física na prevenção e tratamento de inúmeras doenças parecem estar bem documentados na literatura, de modo que o incentivo à prática de atividades físicas é uma preocupação da agenda de saúde pública mundial. Os idosos são os que mais são prejudicados pelo sedentarismo. Este presente estudo fez uma análise da incidência das doenças causadas ou agravadas pelo sedentarismo no idoso junto à equipe de saúde da UBS de Carmen Defillipo observando um número significativo de idosos que não realizam atividades físicas e que apresentam limitações físicas e doenças relacionadas ao sedentarismo. O principal fator observado da não realização de atividade física por muitos idosos se deve à distância do local onde são realizadas as atividades físicas na terceira idade acompanhada por profissional de Educação Física, um programa para a terceira idade, em relação a suas residências. Com isso, buscou-se desenvolver um projeto de intervenção que amplie o número de locais onde possa ser realizada atividade física do idoso acompanhado por profissional a fim de aumentar o número de idosos participantes desse programa e a consequente espera pela redução de complicações relacionadas ao sedentarismo nessa faixa etária.