Endangered mussels as antimicrobial-resistant E. coli and other species (Enterobacteriaceae family) reservoirs

Freshwater unionoids are one of the most threatened animal groups worldwide, suffering dramatic regressions globally. The freshwater pearl mussel Margaritifera margaritifera, currently listed as critically endangered in Europe, and Potomida littoralis, listed as endangered, are both present in the River Tua basin (Portugal). The inappropriate use of antibiotics has led to the emergence and spread of resistant bacteria, recognized today as a serious public health problem. Based in the concept of “One health”, the objective of this study was to examine the antibacterial resistance rates in Escherichia coli and other Enterobacteriaceae isolates obtained from both bivalves collected in the River Tua Basin during the summer of 2018. Activity of 22 antibiotics was studied using disc diffusion (Kirby–Bauer) method. Multidrug resistant in E. coli and Enterobacteriaceae isolates was observed only from P. littoralis. Overall, results show that M. margaritifera inhabits more pristine sectors of river not subject to the development of multidrug resistance, unlike P. littoralis which, although does not tolerate high levels of contamination, is naturally found in lower stretches is subject to greater pressure and, as such, a greater probability of the appearance of multi-resistant organisms. The presence of E. coli in freshwater bivalves can represents serious public health problems once bacteria are one the most important biological contaminants in foodborne diseases. Given the conservation status of both studied species their protection against this silent/invisible enemy should be a priority, although work done with UnionidMussels fromNorthern Portugal showed that they had a great antimicrobial and antibiofilm activity.info:eu-repo/semantics/publishedVersio

Multiresistant bacteria: invisible enemies of freshwater mussels

Freshwater mussels are among the most endangered groups of fauna anywhere in world. The indiscriminate use of antibiotics has led to the emergence of resistant strains. These antibiotic-resistant bacteria play a key role in increasing the risk allied with the use of surface water and in spread of resistance genes. Two endangered freshwater mussel species, Margaritifera margaritifera and Potomida littoralis, were sampled at 4 sampling sites along a 50 km stretch of River Tua. Water samples were taken at same sites. Of the total of 135 isolates, 64.44% (39.26% from water and 25.19% from mussels) were coliform bacteria. Site T1, with the lowest concentration of coliform bacteria, and site T2 were the only ones where M. margaritifera was found. No E. coli isolates were found in this species and the pattern between water and mussels was similar. P. littoralis, which was present at T3/T4 sites, is the one that faces the highest concentration of bacterial toxins, which are found in treated wastewater effluents and around population centers. Sites T3/T4 have the isolates (water and mussels) with the highest resistance pattern, mainly to β-lactams. Water and P. littoralis isolates (T3/T4) showed resistance to penicillins and their combination with clavulanic acid, and to cephalosporins, precisely to a fourth generation of cephalosporin antibiotics. The analysis provides important information on the risk to water systems, as well as the need to investigate possible management measures. It is suggested that future studies on the health status of freshwater bivalves should incorporate measures to indicate bacteriological water quality.This work was supported by the Portuguese Foundation for Science and Technology (FCT) and the Operational Competitiveness Programme (COMPETE), under projects UIDB/04033/2020 and UIDP/04033/2020 (CITAB-Inov4Agro-UTAD). This research was also funded by the Conselleira de Educación, Universidade e Formación Profesional, Xunta de Galicia, Spain, under project R815 131H 64502 (Xana Álvarez). Funding for open access charge was from the Universidade de Vigo/CISUG.info:eu-repo/semantics/publishedVersio

Antibiotic resistance profile among Shiga toxin-producing Escherichia coli isolated from dairy cattle

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause serious diseases in humans, including bloody diarrhoea and kidney failure. Ruminants, such as cattle, are considered the main reservoirs and source of STEC. Human infection can occur through contaminated food and water, or direct contact with infected animals. STEC antimicrobialresistance (AMR) is increasingly frequent in patients with serious disease. It is necessary to understand the epidemiology, the emergence, and the prevalence of AMR in STEC isolated from cattle to investigate how resistance spreads from ruminants to humans. Thus, susceptibility tests were performed on 55 STEC strains belonging to 29 serogroups. The strains were isolated from healthy dairy cattle faeces (cows and heifers) in the North of Portugal. Antibiotic susceptibility testing (AST) was performed by disc diffusion method following European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2020) and Clinical and Laboratory Standards Institute (CLSI, 2020). The antibiotics used included penicillins (ampicillin; amoxicillin-clavulanic acid), cephalosporins (ceftazidime; cefotaxime; cefoxitin; cephalothin), carbapenems (imipenem; meropenem), aminoglycosides (kanamycin), phenicol (chloramphenicol), sulphonamides/ trimethoprim (trimethoprim-sulfamethoxazole), fluoroquinolones (moxifloxacin; levofloxacin) and tetracyclines (tetracycline; tigecycline). Results reveal low level of resistance among the isolates tested. However, five (9%) STEC isolates were resistant to one antibiotic, and three (5,5%) to three or more antibiotic classes (multidrug resistance-MDR). The MDR strains were resistant to trimethoprim-sulfamethoxazole, tetracycline and other antibiotics commonly used to treat gastroenteritis. Two strains MDR belonged to O91 serogroup and were founded in heifers in the same farm. O91 is an important serogroup to public health surveillance, as it is commonly associated with contamination of products from animal origin, and it has been isolated from patients with severe gastrointestinal disease. Overall, the AMR did not seem to be widely spread in STEC isolates from cattle; but serotype O91 might be of special concern as two O91-multidrug resistance profiles have been identified.This study was financially supported by: i) strategic project UIDB/AGR/04033/2020 by National Funds thought FCT; ii) project PhageSTEC (POCI-01-0145-FEDER-029628) funded by FEDER through COMPETE2020 (Programa Operacional Competitividade e Internacionalização) and by National Funds thought FCT (Fundação para a Ciência e a Tecnologia); iii) strategic project UIDB/04469/2020 unit and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by FCT under the scope of the European Regional Development Fund (Norte2020 – Programa Operacional Regional do Norte); iv) Base Funding - UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy – LEPABE - funded by national funds through the FCT/MCTES (PIDDAC).info:eu-repo/semantics/publishedVersio

Antibiotic Resistance Profile among Shiga Toxin-Producing Escherichia coli Isolated from dairy cattle.

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens that can cause serious diseases in humans, including bloody diarrhoea and kidney failure. Ruminants, such as cattle, are considered the main reservoirs and source of STEC. Human infection can occur through contaminated food and water, or direct contact with infected animals.N/

Biofilms in diabetic foot ulcers: impact, risk factors and control strategies

Diabetic foot ulcers (DFUs) are a serious complication from diabetes mellitus, with a huge economic, social and psychological impact on the patients life. One of the main reasons why DFUs are so difficult to heal is related to the presence of biofilms. Biofilms promote wound inflammation and a remarkable lack of response to host defences/treatment options, which can lead to disease progression and chronicity. In fact, appropriate treatment for the elimination of these microbial communities can prevent the disease evolution and, in some cases, even avoid more serious outcomes, such as amputation or death. However, the detection of biofilm-associated DFUs is difficult due to the lack of methods for diagnostics in clinical settings. In this review, the current knowledge on the involvement of biofilms in DFUs is discussed, as well as how the surrounding environment influences biofilm formation and regulation, along with its clinical implications. A special focus is also given to biofilm-associated DFU diagnosis and therapeutic strategies. An overview on promising alternative therapeutics is provided and an algorithm considering biofilm detection and treatment is proposed.This work was supported by: Base Funding—UIDB/00511/2020 of the Laboratory for Process Engineering, Environment, Biotechnology and Energy—LEPABE—funded by national funds through the FCT/MCTES (PIDDAC); Project Biocide_for_Biofilm-PTDC/BII-BTI/30219/2017- POCI-01-0145-FEDER-030219, ABFISH–PTDC/ASP-PES/28397/2017-POCI-01-0145-FEDER- 028397 and Germirrad-POCI-01-0247-FEDER-072237, funded by FEDER funds through COMPETE2020— Programa Operacional Competitividade e Internacionalização (POCI) and by national funds (PID DAC) through FCT/MCTES. The authors also thank the CITAB (Centre for the Research and Technol ogy of Agro-Environmental and Biological Sciences) under the scope of the FCT funds with reference UIDB/AGR/04033/2020. Ana Afonso (2020.04773.BD) and Diana Oliveira (SFRH/BD/138217/2018) acknowledge the FCT grants. Anabela Borges thanks the FCT for the financial support of their work contract through the Scientific Employment Stimulus—Individual Call—[CEECIND/01261/2017].info:eu-repo/semantics/publishedVersio

Antimicrobial, Antibiofilm, and Antioxidant Properties of Boletus edulis and Neoboletus luridiformis Against Multidrug-Resistant ESKAPE Pathogens

Multidrug-resistant ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) has become the most recurrent global cause of skin and soft-tissue infections, belonging to the WHO priority pathogens list. Successful therapy remains challenging and entails the assessment of novel and successful antibiotics. In this study, mushrooms are considered a valuable and unique source of natural antimicrobial compounds. Therefore, this study aimed to evaluate the antimicrobial and antibiofilm properties of Boletus edulis (B. edulis) and Neoboletus luridiformis (N. luridiformis) aqueous and methanolic extracts against ESKAPE isolates from clinical wound infections. Disk diffusion and microdilution methods were used to assess the antimicrobial activity. Phytochemical characterization was achieved by analysis of total phenols, orthodiphenols content, and antioxidant activity as well as by high-performance liquid chromatography-diode array detector (HPLC-DAD). Human foreskin fibroblasts-1 (HFF-1) cell viability was performed by the MTT assay. Aqueous and methanolic extracts of B. edulis and N. luridiformis showed antimicrobial and antibiofilm properties against multidrug-resistant bacteria, although with different efficacy rates. The results showed that there is a convincing relation between the content of phenolic compounds, antioxidant activity, and antimicrobial activity suggesting that the presence of phenolic compounds may explain the biological effects. HPLC analysis revealed high levels of protocatechuic acid, homogentisic acid, pyrogallol, gallic acid, p-catechin, and dihydroxybenzoic acid in the aqueous extract of B. edulis, explaining the highest antimicrobial and antibiofilm properties. Importantly, the mushrooms extracts were non-cytotoxic at all the tested concentrations. Overall, the tested mushrooms extracts are good candidates to further explore its use in the prevention of wound infection, particularly by multidrug-resistant pathogensThis study was funded by the project I&T Companies in Co-promotion FungiTech, Norte-01-0247-FEDER-033788; National Funds by FCT—Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (CITAB-Center for the Research and Technology of Agro-Environmental and Biological Sciences). JG wish to acknowledge the project AquaValor—Centro de Valorização e Transferência de Tecnologia da Água (NORTE-01-0246-FEDER-000053), supported by Norte Portugal Regional Operational Programme (NORTE, 2020). FR was thankful for her contract (CEECIND/01886/2020) financed by FCT/MCTES—CEEC Individual 2020 Program Contractinfo:eu-repo/semantics/publishedVersio

The memory of iron stress in strawberry plants

Research ArticleTo provide information towards optimization of strategies to treat Fe deficiency, experiments were conducted to study the responses of Fe-deficient plants to the resupply of Fe. Strawberry (Fragaria ananassa Duch.) was used as model plant. Bare-root transplants of strawberry (cv. ‘Diamante’) were grown for 42 days in Hoagland's nutrient solutions without Fe (Fe0) and containing 10 mM of Fe as Fe-EDDHA (control, Fe10). For plants under Fe0 the total chlorophyll concentration of young leaves decreased progressively on time, showing the typical symptoms of iron chlorosis. After 35 days the Fe concentration was 6% of that observed for plants growing under Fe10. Half of plants growing under Fe0 were then Fe-resupplied by adding 10 mM of Fe to the Fe0 nutrient solution (FeR). Full Chlorophyll recovery of young leaves took place within 12 days. Root ferric chelate-reductase activity (FCR) and succinic and citric acid concentrations increased in FeR plants. Fe partition revealed that FeR plants expressively accumulated this nutrient in the crown and flowers. This observation can be due to a passive deactivation mechanism of the FCR activity, associated with continuous synthesis of succinic and citric acids at root level, and consequent greater uptake of Fe.info:eu-repo/semantics/publishedVersio

Drinking-water isolated Delftia acidovorans selectively coaggregates with partner bacteria and facilitates multispecies biofilm development

Coaggregation plays an important role in the development of multispecies biofilms in different environments, often serving as an active bridge between biofilm members and other organisms that, in their absence, would not integrate the sessile structure. The ability of bacteria to coaggregate has been reported for a limited number of species and strains. In this study, 38 bacterial strains isolated from drinking water (DW) were investigated for their ability to coaggregate, in a total of 115 pairs of combinations. Among these isolates, only Delftia acidovorans (strain 005P) showed coaggregating ability. Coaggregation inhibition studies have shown that the interactions mediating D. acidovorans 005P coaggregation were both polysaccharide-protein and protein-protein, depending on the interacting partner bacteria. Dual-species biofilms of D. acidovorans 005P and other DW bacteria were developed to understand the role of coaggregation on biofilm formation. Biofilm formation by Citrobacter freundii and Pseudomonas putida strains highly benefited from the presence of D. acidovorans 005P, apparently due to the production of extracellular molecules/public goods favouring microbial cooperation. This was the first time that the coaggregation capacity of D. acidovorans was demonstrated, highlighting its role in providing a metabolic opportunity for partner bacteria.This work was financially supported by LA/P/0045/2020 (ALiCE), UIDB/00511/2020 and UIDP/00511/2020 (LEPABE), funded by national funds through FCT/MCTES (PIDDAC); UIDB/04469/2020 (CEB) and by LABBELS – Associate Laboratory in Biotechnology, Bioengineering and Microelectromechnaical Systems, LA/P/0029/2020; UIDB/04033/2020 (CITAB); Project Biocide_for_Biofilm-PTDC/BII-BTI/30219/2017-POCI-01-0145-FEDER-030219, ABFISH–PTDC/ASP-PES/28397/2017-POCI-01-0145-FEDER- 028397 and Germirrad-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; project HealthyWaters (NORTE-01-0145-FEDER-000069)- NORTE 2020/ERDF; and the FCT grant (2020.04773.BD).info:eu-repo/semantics/publishedVersio

Comparative antioxidant and antimicrobial properties of Lentinula edodes Donko and Koshin varieties against priority multidrug-resistant pathogens

The problematic increase in multidrug-resistant bacteria translates into the urgent need to discover novel and effective antimicrobial substances. Herein, mushrooms could be a promising alternative of natural source of new antimicrobials. The present work aimed to compare the phytochemical composition and antimicrobial activity of methanol and aqueous crude extracts of Lentinula edodes var. Koshin and Donko. Disk diffusion method was used to screen the antimicrobial activity and to assess the synergistic effect of the mushroom extracts. Microdilution method was used to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). Phytochemical characterization of mushrooms extracts was achieved by analysis of total phenols, ortho-diphenols content and its antioxidant activity. The results noticed a positive relation between phenolic compounds content, antioxidant activity, and antimicrobial capacity of the mushroom's extracts. The L. edodes var. Koshin aqueous extracts, which contained the highest amount of total phenolic compounds, exhibited the highest scavenging capacity of ABTS which, in turns, exhibited the highest antimicrobial efficacy in inhibiting the growth of methicillin-resistant Staphylococcus aureus. Moreover, the combination between mushrooms extracts and commercial antibiotics showed favorable synergistic effects against tested bacteria. These results suggest that L. edodes var. Koshin may represent an important and valuable therapeutic source of compounds to be used against multidrug-resistant bacteria.This work was supported by the project I&T Companies in Co- Promotion FungiTech, Norte-01-0247-FEDER-033788; National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UIDB/04033/2020 (CITAB-Center for the Research and Technology of Agro-Environmental and Biological Sciences), Centro de Química – Vila Real (UIDB/00616/2020) and UIDB/00690/2020 (CIMO-Centro de Investigaç˜ao de Montanha).info:eu-repo/semantics/publishedVersio

The role of aquatic ecosystems (River Tua, Portugal) as reservoirs of multidrug-resistant Aeromonas spp.

The inappropriate use of antibiotics, one of the causes of the high incidence of antimicrobialresistant bacteria isolated from aquatic ecosystems, represents a risk for aquatic organisms and the welfare of humans. This study aimed to determine the antimicrobial resistance rates among riverine Aeromonas spp., taken as representative of the autochthonous microbiota, to evaluate the level of antibacterial resistance in the Tua River (Douro basin). The prevalence and degree of antibiotic resistance was examined using motile aeromonads as a potential indicator of antimicrobial susceptibility for the aquatic environment. Water samples were collected from the middle sector of the river, which is most impacted area by several anthropogenic pressures. Water samples were plated on an Aeromonas-selective agar, with and without antibiotics. The activity of 19 antibiotics was studied against 30 isolates of Aeromonas spp. using the standard agar dilution susceptibility test. Antibiotic resistance rates were fosfomycin (FOS) 83.33%, nalidixic acid (NA) 60%, cefotaxime (CTX) 40%, gentamicin (CN) 26.67%, tobramycin (TOB) 26.67%, cotrimoxazole (SXT) 26.67%, chloramphenicol (C) 16.67%, and tetracycline (TE) 13.33%. Some of the nalidixic acid-resistant strains were susceptible to fluoroquinolones. Multiple resistance was also observed (83.33%). The environmental ubiquity, the natural susceptibility to antimicrobials and the zoonotic potential of Aeromonas spp. make them optimal candidates for studying antimicrobial resistance (AMR) in aquatic ecosystems. Aquatic environments may provide an ideal setting for the acquisition and dissemination of antibiotic resistance because anthropogenic activities frequently impact them. The potential risk of multi- and pan-resistant bacteria transmission between animals and humans should be considered in a “One Health-One World” concept.This research was supported by the Portuguese Foundation for Science and Technology (FCT) and the Operational Competitiveness Programme (COMPETE), under the projectsUIDB/04033/2020 (CITAB-UTAD) and the UIDB/00690/2020 (CIMO-IPB).info:eu-repo/semantics/publishedVersio