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Funding Information: This work is financed by national funds from FCT?Funda??o para a Ci?ncia e a Tecnologia, I.P., in the scope of the project UIDP/04378/2020 of the Research Unit on Applied Molecular Biosciences?UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy?i4HB. FCT/MCTES through project grants PTDC/QUIQUI/119116/2010, PTDC/GEO-FIQ/5162/2014 and PTDC/BIA-MIC/31645/2017. Thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020), through national funds. This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) programme. APA is thankful for the Ant?nio Coutinho Prize, awarded supported by Instituto Gulbenkian de Ci?ncia (IGC) under the reference 11/BI-PD/20. LL thanks ERASMUS Mundus for the funding. JRS acknowledges FCT/MCTES for fellowship SFRH/BD/148671/2019 and the STSM grant funded by COST Action CA18238 (Ocean4Biotech). CFR is funded by national funds (OE), through FCT?Funda??o para a Ci?ncia e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July. Funding Information: Funding: This work is financed by national funds from FCT—Fundação para a Ciência e a Tecnolo-gia, I.P., in the scope of the project UIDP/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB. FCT/MCTES through project grants PTDC/QUIQUI/119116/2010, PTDC/GEO-FIQ/5162/2014 and PTDC/BIA-MIC/31645/2017. Thanks are due to FCT/MCTES for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020), through national funds. This publication is based upon work from COST Action CA18238 (Ocean4Biotech), supported by COST (European Cooperation in Science and Technology) programme. APA is thankful for the António Coutinho Prize, awarded supported by Instituto Gulbenkian de Ciência (IGC) under the reference 11/BI-PD/20. LL thanks ERASMUS Mundus for the funding. JRS acknowledges FCT/MCTES for fellowship SFRH/BD/148671/2019 and the STSM grant funded by COST Action CA18238 (Ocean4Biotech). CFR is funded by national funds (OE), through FCT—Fundação para a Ciência e a Tecnologia, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of 29 August, changed by Law 57/2017, of 19 July. Publisher Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. Made available in DSpace on 2022-02-02T23:15:49Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-01 publishersversion published

The search for new and effective strategies to reduce bacterial biofilm formation is of utmost importance as bacterial resistance to antibiotics continues to emerge. The use of anti-biofilm agents that can disrupt recalcitrant bacterial communities can be an advantageous alternative to antimicrobials, as their use does not lead to the development of resistance mechanisms. Six MAR4 Streptomyces strains isolated from the Madeira Archipelago, at the unexplored Macaronesia Atlantic ecoregion, were used to study the chemical diversity of produced hybrid isoprenoids. These marine actinomycetes were investigated by analysing their crude extracts using LC-MS/MS and their metabolomic profiles were compared using multivariate statistical analysis (principal component analysis), showing a separation trend closely related to their phylogeny. Molecular networking unveiled the presence of a class of metabolites not previously described from MAR4 strains and new chemical derivatives belonging to the napyradiomycin and marinone classes. Furthermore, these MAR4 strains produce metabolites that inhibit biofilm formation of Staphylococcus aureus and Marinobacter hydrocarbonoclasticus. The anti-biofilm activity of napyradiomycin SF2415B3 (1) against S. aureus was confirmed.

The undesired attachment of micro and macroorganisms on water-immersed surfaces, known as marine biofouling, results in severe prevention and maintenance costs (billions €/year) for aquaculture, shipping and other industries that rely on coastal and off-shore infrastructures. To date, there are no sustainable, cost-effective and environmentally safe solutions to address this challenging phenomenon. Therefore, we investigated the antifouling activity of napyradiomycin derivatives that were isolated from actinomycetes from ocean sediments collected off the Madeira Archipelago. Our results revealed that napyradiomycins inhibited ≥80% of the marine biofilm-forming bacteria assayed, as well as the settlement of Mytilus galloprovincialis larvae (EC50 &lt, 5 µg/ml and LC50/EC50 &gt, 15), without viability impairment. In silico prediction of toxicity end points are of the same order of magnitude of standard approved drugs and biocides. Altogether, napyradiomycins disclosed bioactivity against marine micro and macrofouling organisms, and non-toxic effects towards the studied species, displaying potential to be used in the development of antifouling products.

Fundacao para a Ciencia e a Tecnologia (FCT) through grants PTDC/FIS-NAN/ 0117/2014 and PTDC/BIA-MIC/31645/2017. project UID/Multi/04378/2019 (Unidade de Ciencias Biomoleculares Aplicadas-UCIBIO), funded by FCT/MCTES; project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular), funded by FEDER through COMPETE2020-Programa Operacional Competitividade e Internacionalizacao (POCI); national funds through FCT; by project ONEIDA (LISBOA-01-0145-FEDER-016417), cofunded by FEEI (Fundos Europeus Estruturais e de Investimento) from the Programa Operacional Regional Lisboa 2020; and by national funds from FCT. Funding was also provided by European Society of Clinical Microbiology and Infectious Diseases research grant 2015, awarded to R.G.S. B.V.G., T.A.F., and I.R.G. were supported by fellowships SFRH/BD/131623/2017 respectively. J.S.D. acknowledges the National NMR Network (PTNMR) and Infrastructure Project ROTEIRO/0031/2013-PINFRA/22161/2016 (cofinanced by FEDER through COMPETE 2020, POCI, PORL, and FCT through PIDDAC). Glutamate amidation, a secondary modification of the peptidoglycan, was first identified in Staphylococcus aureus. It is catalyzed by the protein products of the murT and gatD genes, which are conserved and colocalized in the genomes of most sequenced Gram-positive bacterial species. The MurT-GatD complex is required for cell viability, full resistance to β-lactam antibiotics, and resistance to human lysozyme and is recognized as an attractive target for new antimicrobials. Great effort has been invested in the study of this step, culminating recently in three independent reports addressing the structural elucidation of the MurT-GatD complex. In this work, we demonstrate through the use of nonstructural approaches the critical and multiple roles of the C-terminal domain of MurT, annotated as DUF1727, in the MurT-GatD enzymatic complex. This domain provides the physical link between the two enzymatic activities and is essential for the amidation reaction. Copurification of recombinant MurT and GatD proteins and bacterial two-hybrid assays support the observation that the MurT-GatD interaction occurs through this domain. Most importantly, we provide in vivo evidence of the effect of substitutions at specific residues in DUF1727 on cell wall peptidoglycan amidation and on the phenotypes of oxacillin resistance and bacterial growth. publishersversion published

This work was supported by Medical Research Council partnership grants (G1001787/1 and MR/N002660/1) held between the Department of Veterinary Medicine and School of Clinical Medicine at the University of Cambridge, the Moredun Research Institute and the Wellcome Sanger Institute. This publication presents independent research supported by the Health Innovation Challenge Fund (WT098600 and HICF-T5-342)—a parallel funding partnership between the Department of Health and Wellcome Trust. E.M.H. is supported by a UK Research and Innovation Fellowship (MR/S00291X/1). F.C. is supported by the Wellcome Trust (201344/Z/16/Z). X.B. is supported by a UK–China AMR Partnership Grant (MR/P007201/1). Antibiotic resistance in bacterial pathogens threatens the future of modern medicine. One such resistant pathogen is methicillin-resistant Staphylococcus aureus (MRSA), which is resistant to nearly all β-lactam antibiotics, limiting treatment options. Here, we show that a significant proportion of MRSA isolates from different lineages, including the epidemic USA300 lineage, are susceptible to penicillins when used in combination with β-lactamase inhibitors such as clavulanic acid. Susceptibility is mediated by a combination of two different mutations in the mecA promoter region that lowers mecA-encoded penicillin-binding protein 2a (PBP2a) expression, and in the majority of isolates by either one of two substitutions in PBP2a (E246G or M122I) that increase the affinity of PBP2a for penicillin in the presence of clavulanic acid. Treatment of S. aureus infections in wax moth and mouse models shows that penicillin/β-lactamase inhibitor susceptibility can be exploited as an effective therapeutic choice for ‘susceptible’ MRSA infection. Finally, we show that isolates with the PBP2a E246G substitution have a growth advantage in the presence of penicillin but the absence of clavulanic acid, which suggests that penicillin/β-lactamase susceptibility is an example of collateral sensitivity (resistance to one antibiotic increases sensitivity to another). Our findings suggest that widely available and currently disregarded antibiotics could be effective in a significant proportion of MRSA infections. Postprint

Introduction: Actinomycetes Streptomyces genus is recognized for their ability to produce two of the most valuable families of natural products: non-ribosomal peptides (NRPs) and poliketides (PKs) [1]. In particular, Streptomyces aculeolatus (Figure 1), belonging to the MAR4 group, is known to produce a specific group of bioactive secondary metabolites named hybrid isoprenoids (HI) [2–4]. These compounds have great bioactive potential as antibacterial, antifungal, and anticancer agents making them potentially attractive to the pharmaceutical industry [3–5]. Four S. aculeolatus stains isolated from marine sediments collected off the Madeira Archipelago (coded PTM-29, PTM-129, PTM-346 and PTM-398) revealed the production of secondary metabolites with distinct bioactivities. To date, small organic compounds are the most widely explored but now, our main goal is the screening of extracellular water soluble protein species with biological activities secreted by S. aculeolatus. Materials and methods: The antimicrobial activity of S. aculeolatus extracellular extracts (aqueous phase) was evaluated by screening the biological activity against two Methicillin Resistant strains of the human pathogen Staphyloccocus aureus, namely COL and MW2. Minimal inhibitory concentrations (MIC) were determined by growth inhibition halos and microdilution analysis. Results: Preliminary results showed antimicrobial activity against MRSA from extracellular extracts of strains PTM-29, PTM-129, PTM-346 and PTM-398, with MIC values ranging from 0.010-0.020 µg/µl. Discussion and conclusions: All the aqueous extracellular extracts exhibited inhibitory growth effect against COL and MW2 MRSA strains. In addition, the extract from PTM-346 showed the highest inhibitory activity whereas PTM-29 showed the lowest level of activity. Interestingly, PTM-129, whose organic crude extract did not show biological activity, revealed antimicrobial activity in the aqueous extracellular extract obtained in this study. In the future, the bioactive peptide species will be isolated from the extracellular extracts, purified and characterized by biochemical techniques. Ultimately, this work will deliver novel bioactive peptides that might be conducted to clinical trials, in the pursuing of lead-like agents.

In this communication, we describe evidence demonstrating the capacity of Atl, the major Staphylococcus aureus autolytic enzyme to bind DNA. Electrophoretic mobility shift assays (EMSA) show that both the Atl protein and the endo-β-N-acetylglucosaminidase (GL) domain were able to bind DNA of nonspecific sequence. The implications of this unexpected observation for the physiology of S. aureus remain to be explored.

Bacteria have to avoid recognition by the host immune system in order to establish a successful infection. Peptidoglycan, the principal constituent of virtually all bacterial surfaces, is a specific molecular signature recognized by dedicated host receptors, present in animals and plants, which trigger an immune response. Here we report that autolysins from Gram-positive pathogenic bacteria, enzymes capable of hydrolyzing peptidoglycan, have a major role in concealing this inflammatory molecule from Drosophila peptidoglycan recognition proteins (PGRPs). We show that autolysins trim the outermost peptidoglycan fragments and that in their absence bacterial virulence is impaired, as PGRPs can directly recognize leftover peptidoglycan extending beyond the external layers of bacterial proteins and polysaccharides. The activity of autolysins is not restricted to the producer cells but can also alter the surface of neighboring bacteria, facilitating the survival of the entire population in the infected host. DOI: http://dx.doi.org/10.7554/eLife.02277.001, eLife digest While most bacteria are harmless, some can cause diseases as varied as food poisoning and meningitis, so our immune system has developed various ways of detecting and eliminating bacteria and other pathogens. Receptor proteins belonging to the immune system detect molecules that give away the presence of the bacteria and trigger an immune response targeted at the invading pathogen. Peptidoglycan is one telltale molecule that betrays the presence of bacteria. Peptidoglycan is found in the bacterial cell wall, and for many years it was assumed that the immune system detected stray fragments of peptidoglycan that were accidentally shed by the bacteria. However, it was later shown that the immune system could, under certain conditions, detect peptidoglycan when it is still part of the cell wall. This raised an interesting question: do bacteria use other methods to stop peptidoglycan being detected by the immune system? Now, Atilano, Pereira et al. have found that enzymes called autolysins can conceal bacteria from the receptor proteins that detect peptidoglycan. These enzymes are needed to break the bonds within the peptidoglycan present in the rigid bacterial cell wall to allow the bacteria to grow and divide. ‘Knocking out’ the genes for autolysins allowed the receptor proteins from the fruit fly, Drosophila, to bind to the bacteria; however, the mutant bacteria were able to evade the immune system after they had been treated with the purified enzymes. Atilano, Pereira et al. suggest that the autolysins trim the exposed ends of the peptidoglycan molecules on the surface of the cell wall, which could otherwise be detected by the host. The experiments also show that bacterial pathogens—including a strain of MRSA—with mutations that knock out autolysin activity trigger a stronger immune response in fruit flies, and are therefore less able to infect this host. Autolysins also help to conceal Streptococcus pneumoniae—a bacterial pathogen that is a common cause of pneumonia and infant deaths in developing countries—from detection by fruit flies. The findings of Atilano, Pereira et al. highlight how bacteria employ a number of ways to evade detection. If similar behavior is observed when bacteria infect humans, autolysins could represent a new drug target for the treatment of bacterial diseases. DOI: http://dx.doi.org/10.7554/eLife.02277.002

The activity of growing living bacteria was investigated using real-time and in situ rheology -- in stationary and oscillatory shear. Two different strains of the human pathogen Staphylococcus aureus -- strain COL and its isogenic cell wall autolysis mutant -- were considered in this work. For low bacteria density, strain COL forms small clusters, while the mutant, presenting deficient cell separation, forms irregular larger aggregates. In the early stages of growth, when subjected to a stationary shear, the viscosity of both strains increases with the population of cells. As the bacteria reach the exponential phase of growth, the viscosity of the two strains follow different and rich behaviours, with no counterpart in the optical density or in the population's colony forming units measurements. While the viscosity of strain COL keeps increasing during the exponential phase and returns close to its initial value for the late phase of growth, where the population stabilizes, the viscosity of the mutant strain decreases steeply, still in the exponential phase, remains constant for some time and increases again, reaching a constant plateau at a maximum value for the late phase of growth. These complex viscoelastic behaviours, which were observed to be shear stress dependent, are a consequence of two coupled effects: the cell density continuous increase and its changing interacting properties. The viscous and elastic moduli of strain COL, obtained with oscillatory shear, exhibit power-law behaviours whose exponent are dependent on the bacteria growth stage. The viscous and elastic moduli of the mutant have complex behaviours, emerging from the different relaxation times that are associated with the large molecules of the medium and the self-organized structures of bacteria. These behaviours reflect nevertheless the bacteria growth stage., Comment: 9 pages, 10 figures