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.

Fast and accurate identification of pathogens is an essential task in healthcare settings. Second-generation sequencing platforms such as Illumina have greatly expanded the capacity with which different organisms can be detected in hospital samples, and third-generation nanopore-driven sequencing devices such as Oxford Nanopore's minION have recently emerged as ideal sequencing platforms for routine healthcare surveillance due to their long-read capacity and high portability. Despite its great potential, protocols and analysis pipelines for nanopore sequencing are still being extensively validated. In this work, we assess the ability of nanopore sequencing to provide reliable community profiles based on 16S rRNA sequencing in comparison to traditional Illumina platforms using samples collected from Intensive Care Units of a hospital in Brazil. While our results demonstrate that lower throughputs may be a shortcoming of the method in more complex samples, we show that the use of single-use Flongle flowcells in nanopore sequencing runs can provide insightful information on the community composition in healthcare settings.

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel β-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel β-glucosidase – named Lfa2 – displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing β-glucose, β-galactose, β-xylose, β-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, β-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that β-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

26As the field of synthetic biology moves towards the utilization of novel bacterial chassis, 27 there is a growing need for biological parts with enhanced performance in a wide number of 28 hosts. Is not unusual that biological parts (such as promoters and terminators), initially 29 characterized in the model bacteria Escherichia coli, do not perform well when implemented 30 in alternative hosts, such as Pseudomonas, therefore limiting the construction of synthetic 31 circuits in industrially relevant bacteria. In order to address this limitation, we present here the 32 mining of transcriptional terminators through functional metagenomics to identify novel parts 33 with broad host-range activity. Using a GFP-based terminator trap strategy and a broad host-34 range plasmid, we identified 20 clones with potential terminator activity in Pseudomonas 35 putida. Further characterization allowed the identification of 4 unique sequences between 58 36 bp and 181 bp long that efficiently terminates transcription in P. putida, E. coli, Burkholderia 37 phymatum and two Pseudomonas strains isolated from Antarctica. Therefore, this work 38 presents a new set of biological parts useful for the engineering of synthetic circuits in 39 Proteobacteria. 40 41 45 efficient set of tools that can be used in organisms with relevant features related to the 46 application intended. For example, E. coli is a classical host in terms of protein production 47 and metabolic engineering 3 , while gram-positive bacteria such as Streptomyces hold the 48potential for the production of small bioactive molecules 4 . By the same token, the natural 49 features of Salmonella make them a suitable host for tumor targeting applications 5 , while the 50 3 metabolic robustness and versatility of Pseudomonas make these bacteria promising chassis 51 for harsh and intense industrial conditions 6 . Yet, most of genetic tools have been initially 52 constructed for E. coli and it is not unusual that biological parts required for synthetic circuit 53 assemble are not fully functional in alternative hosts 2 . In these cases, the initial failure of 54 synthetic circuits constructed in alternative hosts leads to the need of intense re-adaptation of 55 these parts to the new hosts 7,8 . Therefore, as synthetic biologists turn towards non-classical 56 bacterial chassis, there is a growing demand for orthogonal systems that could efficiently 57 operate in a broad number of hosts 9,10 . Attempts in this direction have been made towards the 58 construction of novel expression systems 11 , transformation/DNA delivery strategies 12,13 and 59 plasmid vectors 14 , among others. 61Recently, several reports have described the construction of novel expression systems, based 62 on inducible as well as constitutive promoters, both with narrow or wide host-range 7,10,15,16,2 . 63 Yet, while much is known about transcription initiation, considerably less information is 64 available about transcriptional termination 17,18 . In Bacteria, transcription termination occurs 65 mainly...

Adoption of microorganisms as platforms for sustainable biobased production requires host cells to be able to withstand harsh industrial conditions, which are usually far from the ones where these organisms are naturally adapted to thrive. However, novel survival mechanisms unearthed by the study of microbiomes from extreme habitats may be exploited to enhance microbial robustness under the strict conditions needed for different applications. In this work, synthetic biology approaches were used to engineer enhanced acidic tolerance in Escherichia coli under extreme conditions through the characterization of a library of twenty-seven unique operons composed of combinatorial assemblies of three novel genes from an extreme environment and three synthetic ribosome binding sites. The results here presented illustrate the efficacy of combining different metagenomic genes for tolerance in truly synthetic genetic operons, as expression of these gene clusters increased hundred-fold the survival percentage of cells exposed to an acidic shock in minimal media at pH 1.9 under aerobic conditions.