The past two years have been marked by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. This virus is found in the intestinal tract and reaches the wastewater system, and, consequently, the natural receiving water bodies. As such, inefficiently treated wastewater (WW) can be a means of contamination. The currently used disinfection methods of WW can be expensive, lead to the formation of toxic compounds, or be inefficient. So, new alternative approaches must be considered for this matter, namely photodynamic inactivation (PDI). In this work, the bacteriophage Phi6 was used as a surrogate model for the SARS-CoV-2. First, to understand the virus survival in the environment, phage Phi6 was subjected to different laboratory-simulated environmental conditions of temperature, pH, salinity, and solar and UV-B irradiation, and its persistence over time was monitored. Thereafter, the PDI efficiency towards virus was assessed, both in phosphate-buffered saline (PBS), as control aqueous matrix, and in real secondarily treated WW. Then, the safety of WW PDI-treated was assessed through the determination of the viability of native marine water microorganisms after their contact with the PDI-treated effluent, since generally WW is discharged into the marine environment after treatment. The results revealed that phage Phi6 (SARS-CoV-2 surrogate) remained viable in different environmental conditions for a considerable period. Also, PDI was confirmed to be an effective approach in the virus inactivation, and the PDI-treated effluent showed no toxicity to native marine microorganisms under realistic conditions of treated WW dilutions, supporting PDI as an efficient and safe WW tertiary disinfection method. Though the studies were performed with phage Phi6, considered a suitable model of SARS-CoV-2, further studies using the SARS-CoV-2 are needed, nevertheless, the observed results envisage the potential of the PDI to control SARS-CoV-2 in WW.

Acknowledgments: Thanks are due to the University of Aveiro and FCT/ MEC for the financial support to LAQV-REQUIMTE (UIDB/50006/2020) and CESAM (UID/AMB/50017/2019 and UIDB/50017/ 2020 + UIDP/50017/2020) research units, and to the FCT projects (PREVINE-FCT-PTDC/ASP-PES/29576/2017) through national founds (OE) and, where applicable, co-financed by the FEDER-Operational Thematic Program for Competitiveness and Internationalization−COMPETE 2020, within the PT2020 Partnership Agreement, and to the Portuguese NMR Network. The doctoral grants to M.B. and C.V. are funded by FCT (SFRH/BD/121645/2016, SFRH/BD/150358/2019, respectively).

Burn wounds can lead to numerous severe complications including bacterial infections causing patient morbidity and mortality, mostly in low- and middle-income countries. The considerable increase of the microbial resistance against traditional antibiotics is leading towards alternative strategies to treat bacterial infections. Nisin Z is an antimicrobial peptide which exhibits a significant antibacterial activity against Gram-positive bacteria. Its efficacy against Gram-negative bacteria is limited, nonetheless it can be improved with the addition of surfactants, such as ethylenediaminetetraacetic acid (EDTA). The incorporation of peptide and other biomolecules within a biopolymer matrix provides protection maintaining their antimicrobial potential. Bacterial nanocellulose (BNC) has been widely used as wound dressings. Its impressive water retention capacity (> 99 %) and porosity are beneficial to manage wounds due to its potential to absorb exudates, providing a breathable and humid environment. In this work, the functionalization of BNC with Nisin Z (BNC-NZ) via vacuum filtration is reported. The entrapment of the peptide inside the BNC films was confirmed through morphological characterization using Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectrometry. Typical absorbance peaks of Nisin Z are easily identifiable at 1647 cm^-1 (amide group) and 1520 cm^-1 (bending of primary amines). Thermal Gravimetric Analysis (TGA) suggested that Nisin Z did not interfere with the BNC matrix. The antimicrobial activity of Nisin Z against five of the most common bacteria found in burn wounds was verified by Minimum Bactericidal Concentration (MBC) ranging 8.0-256.0 µg/mL. Agar Diffusion and Shake Flask methods revealed the potential of BNC-NZ for prospective applications in burn wound dressings.

The indiscriminate use of antibiotics generates several problems for human health, the main one being bacterial resistance, responsible for making drugs ineffective, making it difficult to treat diseases and representing a major obstacle for researchers and health professionals. Natural alternatives have been widely studied, such as essential oils and their phytoconstituents, in order to verify their antibacterial action. In order to identify scientific evidence of the antibacterial activity of alpha-pinene, an integrative review was carried out whose bibliographic survey used the LILACS and MEDLINE research bases, through the Virtual Health Library, PubMed and Web of Science, using the MeSH terms : anti-bacterial, alpha-pinene and anti-microbial agents associated with the Boolean operators “AND” and “OR”. Studies with full text available, published in the last 5 years, in Portuguese, English and Spanish and that addressed the antibacterial effect on different bacterial strains of the alpha-pinene compound were included, while literature reviews, books, editorials, dissertations and theses were included. were excluded. At the end of reading the articles in full, 10 works were selected to compose this review. The action of alpha-pinene was evaluated in the form of its racemic mixture and its positive and negative enantiomers, as well as its action alone or in association with antibiotics on several bacterial species, including Staphylococcus aureus, Escherichia coli, Mycobacterium tuberculosis, Salmonella Enterica and Campylobacter. fasting. The results indicate that alpha-pinene has a wide potential in antimicrobial therapy, acting to inhibit the development of bacteria; however, when used against certain bacterial strains, it has bactericidal and bacteriostatic limitations. Therefore, it is concluded that the development of scientific research is relevant to analyze the effectiveness of this compound to the most diverse microorganisms that affect human health.

Diabetic foot ulcers (DFUs) are a complex secondary complication that affect the lower extremities caused by diabetes mellitus. More than half of the DFUs are colonized with Staphylococcus aureus and infection progresses due to the immunosuppression triggered by the disease^1,2. Typically, the treatment includes the use of systemic antibiotics, however, that may result in antibiotic resistance and the risk of drug-related toxic effects³. To minimize these negative impacts the use of topical antibiotics is proposed, especially in combination with natural adjuvants. In this study, 2 different plant-based secondary metabolites (phytochemicals) namely, a phenolic compound (chalcone) and a sesquiterpenoid constituent of essential oils (farnesol) were selected to be tested as antibiotic potentiators. After the determination of the minimum inhibitory and bactericidal concentrations (MIC and MBC) of each molecule against a clinical S. aureus isolate from diabetic foot wound (MJMC109), it was evaluated their potentiation effect on the antibiotics mupirocin and gentamicin through the disc diffusion method. Then, the combined effect of both phytochemicals and antibiotics were evaluated on the potential to eradicate a pre-formed S. aureus biofilm. The results showed a significant biomass removal and culturability reduction with the combination of chalcone with mupirocin and farnesol with gentamicin when compared with its constituents (an increase of at least 20% on biomass removal and 1-log CFU/mL reduction). Farnesol combined with mupirocin was able to significantly reduce the cell culturability (4-log CFU/mL). With respect to the metabolic activity, only chalcone combined with mupirocin was able to increase in 20% the effect when compared to its counterparts. Overall, this study reveals the great potential for the topical application of different phytochemicals as adjuvants to mupirocin to combat multi-drug resistant wound infections.

References:

¹ Lipsky, B. A., et al. Clinical Infectious Diseases 39.7 (2004): 885–910.

² Kadam, S., et al. Biomedicines 7.2 (2019): 35.

³ Afonso, A., et al. International journal of molecular sciences 22.15 (2021): 8278.

Acknowledgements:

This research was funded by: LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE) funded by national funds through the FCT/MCTES (PIDDAC; Lisbon, Portugal). Projects PTDC/BIIBTI/30219/2017—POCI-01-0145-FEDER-030219, POCI-01-145-FEDER-006939, POCI-01-0247-FEDER035234, POCI-01-0247-FEDER-072237, funded by FEDER funds through COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PIDDAC) through FCT/MCTES; national funds from Fundação para a Ciência e a Tecnologia (grant numbers: UIDP/ 04378/2020, UIDB/04378/2020, and 2020.01423.CEECIND/CP1596/CT0003); Project “HealthyWaters— Identification, Elimination, Social Awareness and Education of Water Chemical and Biological Micropollutants with Health and Environmental Implications”, with reference NORTE-01-0145-FEDER000069, supported by Norte Portugal Regional Operational Programme (NORTE 2020) under the PORTUGAL 2020 Partnership Agreement through the European Regional Development Fund (ERDF). Grant attributed by Portuguese Foundation for Science and Technology (FCT) to Diana Oliveira (SFRH/BD/138217/2018). Anabela Borges thanks the FCT for the financial support of her work contract through the Scientific Employment Stimulus—Individual Call—[CEECIND/01261/2017].

The increasing global rate of antimicrobial resistance have led to a higher incidence of mortal infections that require alternative methods for their control. Antimicrobial photodynamic therapy (aPDT) is gaining attention as an effective approach against resistant microbial strains. The present work aims to evaluate the photodynamic efficiency of a photosensitizer (PS) based on a low-cost formulation constituted by five cationic porphyrins (FORM) and its combined effect with potassium iodide (KI) on a broad spectrum of microorganisms. To this purpose, the aPDT assays were conducted with different concentrations of FORM (0.1 to 5.0 μM) and 100 mM of KI on planktonic and biofilm forms of Gram-positive (methicillin resistant Staphylococcus aureus – MRSA) and Gram-negative (Escherichia coli resistant to chloramphenicol and ampicillin) bacteria and of the yeast Candida albicans. These studies were performed under white light irradiation (25 W.m^-2) and the results obtained indicate that FORM alone, at low concentrations (< 5.0 μM), had an efficient photodynamic action towards the planktonic forms of the Gram-negative and Gram-positive bacteria and also against the yeast C. albicans. Moreover, the application of KI enhanced the photodynamic effect of FORM towards all tested microorganisms, allowing the reduction of PS concentration and treatment time. The results also showed that the combination FORM+KI was highly efficient in the elimination of the already well-established biofilms of E. coli, S. aureus, and C. albicans. This effect was probably associated with the longer-lived iodine reactive species produced during the aPDT treatment.

Acknowledgments: Thanks are due to the University of Aveiro and FCT/ MEC for the financial support to LAQV-REQUIMTE (UIDB/50006/2020) and CESAM (UID/AMB/50017/2019 and UIDB/50017/ 2020 + UIDP/50017/2020) research units, and to the FCT projects (PREVINE-FCT-PTDC/ASP-PES/29576/2017) through national founds (OE) and, where applicable, co-financed by the FEDER-Operational Thematic Pro-gram for Competitiveness and Internationalization−COMPETE 2020, within the PT2020 Partner-ship Agreement, and to the Portuguese NMR Network. Thanks are also due to the SANAQUA project MAR02.05.01-FEAMP-0004 and I9+Proalga project MAR2020-16-01-03-FMP-0011. The doctoral grants to C.V., M.B. and M.Q.M. are funded by FCT (SFRH/BD/150358/2019, SFRH/BD/121645/2016- and SFRH/ BD/112317/2015, respectively).

Increasing rates of artemisinin-resistant plasmodium strains associated with malarial cases are alarming, hampering effective control of malaria. Artocarpin is a major flavonoid constituent of Artocarpus lakoocha, earlier demonstrated in-vitro potency against Malarial strains. However, efforts to elucidate the exact mechanism of interactions are still ongoing. Aiming to elucidate the probable mechanism of its anti-malarial action as Falcipain-2 (FP-2) inhibition, we investigated molecular modeling analysis. Our Molecular docking analysis for a set of 50 lakoocha bioactive compounds was realized that Artocarpin has the highest binding affinity (docking score: -6.4 Kcal/mol) against FP-2 from Plasmodium falciparum. We further accessed in-silico pharmacokinetics and toxicities for several Artocarpus lakoocha flavonoids. Our results provide critical insights into the mechanism of action of Artocarpin and other Artocarpus lakoocha flavonoids as a potential therapeutic agent (FP-2) against Malaria.

The protein family YjgF/YER057c/UK114 (Rid) is widespread in all domains of life. The only member of this superfamily biochemically well-characterized is the archetypal RidA that is involved in detoxification. Besides RidA, seven families named Rid1 to Rid7 are identified in prokaryotes. A conserved arginine residue is shared by all Rid members with the detoxifying activity. Conversely, other members lack the arginine residue, and their role is mysterious.

A step toward understanding the role of these proteins has been achieved by studying a protein, called Rid7C, in Nonomuraea gerenzanensis, a rare actinomycete used to produce the A40926, which is the precursor of dalbavancin, an FDA approved antibiotic effective against Staphylococcus aureus MRSA. This actinomycete is characterized by the presence of duplicated genes encoding β-subunit of RNA polymerase: rpoB(S) (wild-type gene) and rpoB(R) (specialist rpoB gene). RpoB(R) isoform controls the morphological differentiation and the activation of secondary metabolism (including the production of A40926). Translation of the RpoB(R) mRNA is negatively modulated by a self-complementary hairpin loop in its 5’-UTR which hides the Shine & Dalgarno sequence. Rid7C is the endoribonuclease involved in resolving the hairpin loop by removing a ~80 nt segment from the rpoB(R) mRNA end. In addition, Rid7C may be associated with ribonuclease P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro. Computational, in vitro and in vivo evidence suggest that Rid7C endoribonuclease activity is inhibited by A40926 suggesting the existence of a negative feedback loop on A40926 production, and a role of the A40926 in the modulation of differentiation in this microorganism. This new molecular tool, namely the co-overexpression of RpoB (R) and Rid7C, can be used to increase antibiotic production in actinomycetes. Computational modelling suggests that Rid7 proteins may be involved in the molecular mechanisms of infections in pathogens, so these proteins could be a new target for innovative anti-infective agents.

Introduction: The spread of carbapenem-resistant Enterobacterales in hospitals constitutes an important epidemiological and therapeutic problem that especially affects vulnerable patients such as perioperative patients. Materials and Methods: We conducted a descriptive, observational, retrospective case-control study of patients infected with carbapenemase-producing carbapenem-resistant Enterobacterales (CP-CRE) and carbapenem-sensitive Enterobacterales during the perioperative period in a single-institution. Results: Metallo-beta-lactamase was detected in all the 124 CRE isolates, with NDM-type carbapenemase being dominant, while three isolates coproduced KPC-type enzyme. Steroid use (OR: 3.22, p＜0.01), prior use of two or more antibiotics (OR: 4.04, p=0.01), prior use of broad-spectrum cephalosporins (OR: 2.40, p=0.04) and prior use of carbapenem (OR: 4.77, p=0.03) were the independent risk factors for progressing to CP-CRE infection. Bloodstream infections and pneumonia associated with CP-CRE had higher mortality risk. However, colistin-based combination therapy was not found to reduce the mortality risk for CP-CRE infection during hospitalization compared to patients treated by tigecycline- or fosfomycin-based regimens. Conclusion: High mortality is associated with nosocomial infections in the perioperative period caused by NDM carbapenemase-producing Enterobacterales, of which the dissemination in health care settings in Cuba is a public health challenge.

The SOS response was found to be directly involved in the onset of antibiotic resistance. Our research project represents a promising strategy to inhibit this signalling pathway blocking LexA protein autoproteolysis.

For this purpose, we initially identified three nanobodies (NbSOS1-3) that can inhibit LexA autoproteolysis. After extensive characterisation, we father optimised these Nbs by means of rational protein engineering.

Furthermore, biparatopic nanobodies (BiNbSOSs) were constructed, fusing with a flexible amino acid linker two Nbs having different recognition sites to the LexA antigen.

We chose a functional characterisation based on an integrative approach, combining mainly in vitro techniques. We performed assays based on Fluorescence Polarisation (FP) to derive the IC50 values of the NbSOSs tested. This enabled us to detect the inhibitory capacity towards the autoproteolysis of LexA. We also determined KD values of the NbSOSs by Surface Plasmon Resonance technique (SPR) in order to evaluate their binding affinity to LexA.

Finally, NbSOSs, expressed in E. coli, were tested to verify over time their stability and expression in presence and in absence of ciprofloxacin (a strong inducer of exogenous stress capable of activating the SOS response in bacteria).

We demonstrated that these Nbs inhibit LexA autoproteolysis with IC₅₀ values in the low micromolar range. Improvements in terms of both KD, IC₅₀ and expression profile were observed for rationally designed mutants as well as BiNbSOSs.

We believe that these results pave the way for novel approaches in the fight against antibiotic resistance, leaving the door open for further research in this direction.

Marine actinobacteria produce secondary metabolites with many biological activities of interest, including antifungals. As fungal infections have increased in the last decade, it is important to search for new compounds. In this work, we aimed to evaluate the antifungal activities of marine actinobacteria extracts against pathogenic fungi. Thirty extracts of actinobacteria isolated from marine macroalgae and deep-sea samples were screened against fungi: yeasts (Candida albicans ATCC 90028, Candida parapsilosis ATCC 22019, Cryptococcus neoformans PYCC 3957T, Cryptococcus laurentii ZY8) and molds (Aspergillus flavus ATCC 204304, Aspergillus fumigatus ATCC 204305, Aspergillus brasiliensis ATCC 16404). We performed the disk diffusion method (DD), following the CLSI guidelines (M44-A, M38-A2 and M61). To determine the minimum inhibitory/fungicide concentration (MIC/MFC) we choose the extracts with inhibition zones ≥ 15mm, the cut-off for amphotericin B. Also, the effect of the best extracts on biofilm and germ tube formation were studied (Candida spp.).

In all organisms and for DD, the susceptibilities varied with species (p< 0.0001) and the extracts (p<0.0001). Cr. neoformans, and C. albicans were the most susceptible species. The highest MICs were obtained for Cryptococcus spp., C. parapsilosis and A. flavus (all MIC >250 µg/mL). For A. brasiliensis, two extracts had the lowest MICs (15.62 µg/mL). The results for C. albicans were in the range of 15.62-125 µg/mL, and for C. parapisilosis MIC was > 250 µg/mL. The MFC ranged from 15.62 to > 250 µg/mL. In the biofilm assay, the percentage of inhibition varied greatly between extracts (0-96%). Also, some extracts significantly delayed the germ tube formation.

The extracts from deep-sea and Laminaria ochroleuca samples exhibited higher efficacy against fungi, mostly against yeasts and among these C. albicans, (33% of extracts), than Chondrus crispus and Codium tomentosum. The dereplication analysis of the extracts explained the antifungal activity of most of them.