The increase in Klebsiella spp. multidrug-resistant strains represent a serious threat to public health. Thus, currently human, and veterinary medicine is threatened worldwide by a growing resistance to antibiotics, particularly present in opportunistic Enterobacterial pathogens (e.g., Klebsiella spp.). However, comprehensive, and comparable data on its occurrence in surface waters are scarce, as well as phenotypic and genotypic characteristics for several countries including Portugal. Given this scenario, we aimed to investigate the prevalence of Klebsiella spp. in surface waters, as well as characterize the isolates phenotypically and genotypically. Sixty-five samples were collected from different surface waters in the North of Portugal, of which 31 (47,7%) were positive for Klebsiella spp. The antimicrobial susceptibility was performed by the Kirby-Bauer disc diffusion method against 11 antibiotics. High rates of antibiotic resistance were observed among these isolates for cefotaxime (41,9%), on the other hand, low rates of antibiotic resistance were observed among these isolates for meropenem (3,2%). The presence of different carbapenem genes (blaOXA, blaIMP, and blaKPC) was analyzed by PCR/sequencing in Klebsiella spp. isolates, as well as the presence of other resistance genes (e.g. blaCTX). The one with the highest prevalence was blaCTX (22,6%). However, in the carbapenem genes, amplification was not verified. Our study provides information on the prevalence of Klebsiella spp., which is of clinical importance in surface water in Portugal. These findings indicate their possible dissemination in the environment and the potential risk of colonization and/or infection of humans and livestock associated with exposure to contaminated water sources.

Foodborne diseases are a serious public health problem worldwide. Food‐producing animals are considered a major source of these diseases through contamination of food-products, where pathogenic and drug-resistant Escherichia coli and Salmonella spp. are among the principal bacterial agents. Here, we aimed to assess the role of pig reservoir as potential transmission vehicle to Humans, contributing for the understanding of the epidemiology and population structure of these zoonotic agents in Portugal, since data is still limited.

Fecal samples were collected in different Portuguese slaughterhouses. After bacterial isolation in non- and selective media, serotyping of Salmonella and pathotyping of E. coli isolates were performed. Antimicrobial susceptibility was tested for a sub-set, by the disk diffusion method and interpreted according to EUCAST guidelines.

Overall, 124 stool samples were studied, mostly from Centro and Lisboa e Vale do Tejo Regions. While no Salmonella spp. was detected, E. coli was identified in all pig samples, with Shiga toxin-producing E. coli (STEC) detected in two samples. For a set of 50 E. coli isolates, preliminary antimicrobial resistance results showed that, with exception of one isolate, all were resistant to at least one of the 19 tested antibiotics (mostly erythromycin), where 17 of them displayed a multidrug-resistance profile (MDR). One of these MDR isolates was a STEC.

Although this study is still ongoing, our preliminary findings revealed the presence of MDR E. coli isolates in fecal samples of pigs slaughtered for human consumption, highlighting the role of this animal reservoir as potential source of these bacteria.

Most conventional dressings present a passive action against microorganisms. Here, a multifunctional sandwich-like system was proposed and examined for its potential in fighting CW infections. The scaffolding system was made of three layers: (outer) fibrous mat of polycaprolactone (PCL) working as a barrier for preventing other microorganisms and impurities from reaching the wound bed, and was prepared at 14% w/v in chloroform/dimethylformamide (CHF/DMF) at 90/10% v/v (4 h at 50ºC); (middle) sodium alginate (SA) hydrogel loaded with the antimicrobial agents, polyethylenimine (PEI) and ceftazidime (CEF), examined against Staphylococcus aureus and Pseudomonas aeruginosa using the broth micro-dilution assay (determination of minimum inhibitory concentration or MIC), and used to maintain a moisture environment and absorb exudates. The hydrogel was obtained by solvent casting-phase inversion method using a 2%w/v SA solution and coagulation bath of 2%w/v CaCl₂. Finally, (inner) a second fibrous mat composed of PCL and polyethylene glycol (PEG), also examined against S.aureus e P.aeruginos (MIC of 256 µg/mL), was used to facilitate cell integration and recognition and reduce mat hydrophobicity. Mats were produced via electrospinning (12kV, 0.7mL/h, 17cm, 18G). The sandwich-like system was generated by pouring the hydrogel solution onto a casting mold lined with the inner layer and depositing the outer layer at the top of the viscous solution. After 1h of intimate contact, the casting solution was added and afterwards the scaffolding system was ready and totally detached from the mold. Data reports the effectiveness of this sandwich-like system for potential applications in CW care.

The modern diet emphasizes the consumption of ready-to-eat (RTE) or minimally processed fruits and vegetables essential for a healthy lifestyle. Ensuring the microbiological safety of these produce items is crucial given the highly susceptible Young, Old, Pregnant, and Immuno-compromised (YOPI) citizens. Contamination of fresh produce can occur through various activities from farm to fork, such as using contaminated water for overhead irrigation. This study investigated the suitability of the uMsunduzi River water over the different seasons for overhead irrigation of RTE fresh produce by local smallholder farmers. The microbial burden of river water samples was analyzed, and selected hygiene indicators were determined, including aerobic plate counts, total coliforms, fecal coliforms, Escherichia coli, Enterococcus spp., and the common foodborne pathogen Salmonella spp. The results were concerning, as the river water frequently exceeded the safe irrigation limit of 1000 MPN/100ml E. coli stipulated by the World Health Organization. The river water samples analyzed over the four seasons were generally of unsatisfactory quality, with aerobic plate counts reaching up to 6.18 log₁₀ cfu/ml. In addition, total and fecal coliform and E. coli levels of up to 5.81 log₁₀, 5.66 log_10, and 5.23 log₁₀ MPN/100ml were recorded, respectively. Furthermore, high counts of fecal enterococci were regularly present, while presumptive Salmonella spp. were often detected. Based on these findings, it is evident that the uMsunduzi river water is unsuitable for overhead irrigation of produce that is typically consumed raw, highlighting potential risks to food safety when such water is used for overhead irrigation.

Antimicrobial resistance (AMR) of microorganisms is defined as the ability to withstand or resist the action of one or more antimicrobial agents. AMR is widespread and the efficacy in treating certain life-threatening infections is already compromised. Thus, fermented products are considered notable reservoirs of antimicrobial resistant bacteria. Therefore, this work aimed to analyse the prevalence of antimicrobial-resistant lactic acid bacteria (LAB) throughout the production of a raw milk cheese (Idiazabal PDO).

Four artisanal dairies were selected and raw milk, whey, fresh and ripened cheese samples were collected. LAB isolates were obtained in MRS agar and identified by sequencing of the V1–V3 regions of the 16S rRNA. The minimum inhibitory concentration was evaluated by broth microdilution for the most commonly used antibiotics (amoxicillin, dihydrostreptomycin benzylpenicillin and polymyxin B). The resistance was interpreted according to the microbiological cut-off values proposed by the European Food Safety Authority and the European Committee on Antimicrobial Susceptibility Testing.

More than 170 LAB isolates were obtained throughout the cheese-making process. Enterococcus faecalis (36.4%) and Bacillus thuringiensis (13.6%) predominated in raw milk, while Bacillus cereus (15.8%) and Bacillus sp. (13.2%) did it in whey and Bacillus sp. (22.0%), Enterococcus faecalis (14.6%) or Enterococcus hirae (14.6%) in fresh cheeses. In ripened cheeses, instead, Lactobacillus sp. (20.8%) and Lacticaseibacillus paracasei (16.7%) predominated. Bacillus and Enterococcus species showed intermediate-high resistance to all antimicrobials (average 64.0% and 75.0%, respectively), while Lactobacillus and Lacticaseibacillus were more sensible (34.0% and 36.0%), indicating the shaping effect of the cheese-making process to minimize AMRs.

Marine environments harbour diverse and rich microbial communities presenting unique adaptations to this stressful habitat. As a result, marine bacteria developed several stress resistance mechanisms, including the ability to synthesize a wide range of bioactive molecules of biotechnological interest.

In this work, the phenotypic and genotypic characterization of sixteen marine bacteria isolated from seawater collected in diverse locations across the Sado estuary in Portugal is presented. In addition, the isolated bacteria were tested for their ability to promote the growth and the accumulation of valuable compounds in two microalgae, Phaeodactylum tricornutum and Tetraselmis striata.

The obtained results revealed that several bacterial isolates were capable of producing diverse functional and stable extracellular lytic enzymes; showed the ability to synthesize phytohormones such as auxins (indole-3-acetic acid) and produced ammonia, PHAs, as well as carotenoids. Comprehensive genomic analysis revealed the presence of gene clusters involved in the biosynthesis of a wide range of secondary metabolites such as extracellular enzymes, carotenoids, polyunsaturated fatty acids, and polyhydroxy acids in the diverse bacterial isolates.

When co-cultivated with Phaeodactylum tricornutum and Tetraselmis striata, four isolates induced a significant increase in microalgae cell count, cell size, auto-fluorescence, and fucoxanthin content, indicating their microalgae growth-promoting effects.

Ultimately, these findings bring new insights into the untapped potential of marine bacteria and their use in a vast array of biotechnological applications.

Bacteriophages can prevent the spread of foodborne pathogens. Salmonella is a foodborne pathogen posing a global public health risk, evidenced by frequent outbreaks due to ready-to-eat (RTE) produce consumption. Using tomato as a model crop, this study aimed to isolate and characterize a lytic bacteriophage against Salmonella Enteritidis (ATCC13076) (SE), one of the major serovars linked to salmonellosis in Sub-Saharan Africa.

Salmonella phage SCF was isolated from chicken feces using Salmonella Enteritidis as a host. Based on its icosahedral capsid and long, thin tail detected by transmission electron microscopy, the phage was assigned to the class Caudoviricetes. Its genome has 108.56 kb, a G+C% of 39.02, and is closely related to Salmonella phage SE11 (Genbank: NC048786). Virulence or lysogenic genes were not detected. It lysed Salmonella Typhimurium (ATCC14028) but not other γ-Proteobacteria (e.g., E. coli, S. marcescens, E. cloacae, P. aeruginosa, K. pneumoniae). The one-step growth curve revealed a burst size of around 72 virus particles/host cell and a latency time of ~20 minutes. The phage SCF was stable between 8˚C-50˚C and pH 4-8. Phage SCF prevented biofilm formation and significantly decreased existing 72 h biofilms generated by SE. It was used successfully to control SE on the tomato surface. A 2-hour treatment of SE-contaminated tomatoes with phage SCF at ambient temperature reduced SE counts by up to 5.3 log₁₀ units.

These findings demonstrate that Salmonella phage SCF could be employed as a biocontrol agent to tackle SE and improve the safety of RTE produce such as tomatoes.

Weaning often leads to gastrointestinal problems, irritation and diarrhea in infants. Weaning influenced by various factors, involves complex interaction axes, in particular the gut-microbiota axis. Better understanding these responses and the underlying pathways may facilitate the development of targeted therapies to attenuate the intensity of dysbiosis and consequently gut inflammation in infants.

The objective of this study is to introduce a new approach to visualize and understand the interactions between differentially expressed genes (DEGs) induced by dysbiosis and intestinal inflammation during the weaning period in infants, using bioinformatics methodologies.

A set of 117 DEGs was collected from different bibliographic sources with a fold change >1 and p-value<0.05 adjusted. The analysis was performed in the Cytoscape platform (Version: 3.9.1 https://cytoscape.org/) through the different packages: GeneMANIA for gene function prediction from functional association networks, CytoHubba for the identification of hub genes, The Molecular Complex Detection (MCODE) for the determination of highly interconnected network region, and ShinyGO (version 0.65; http://bioinformatics.sdstate.edu/go/) to identify enrichments with the Gene Ontology (GO) domains and the curated pathway KEGG database.

The findings unveiled the presence of six pivotal genes, namely CCL19, CCL5, CXCL9, CXCL8, IFNG, and CXCL10, which appear to play a role in the development and progression of intestinal inflammation in weaned infants. The identified biological processes of significant involvement included the inflammatory response, response to external biotic stimulus, response to cytokine, and regulation of inflammatory response. Additionally, the analysis revealed significant associations between these genes and pathways such as cytokine-cytokine receptor interaction, inflammation mediated by chemokine, cytokine signaling pathway, inflammatory bowel disease, and chemokine signaling pathway.

In conclusion, all the results obtained suggest that the balanced intestinal microbiota plays a crucial role in the intestinal homeostasis of infants during weaning. Thus, a diet rich in prebiotics and probiotics can be a very promising therapeutic alternative.

Essential oils constitute a rich source of bioactive compounds and serve as common ingredients in the cosmetic and pharmaceutical industries. Because they are often applied directly to the skin, we aimed at determining the influence of essential oils on Staphylococcus spp. isolated from healthy skin microbiota.

A microbroth dilution method was used to determine the antimicrobial activity of thyme (Thymus vulgaris L.), mellisa (Melissa officinalis L.), sage (Salvia officinalis L.), peppermint (Mentha piperita L.), tea tree (Melaleuca alternifolia) and oregano (Origanum vulgare L.) essential oils against reference staphylococci as well as 21 strains isolated from healthy skin represented by seven species: Staphylococcus saprophyticus, S. haemolyticus, S. lentus, S. warneri, S. aureus, S. epidermidis, and S. hominis.

The analysis showed that oregano and thyme essentials oils exhibited the highest antistaphylococcal activity (MIC ranging from 0.5 to 2 mg/ml), followed by tee tree and mellisa oils (MIC ranging from 2 to 8 mg/ml), and sage and peppermint oils (MIC ranging from 4 to 16 mg/ml). All tested essential oils showed bactericidal activity.

Because of the low MIC values, part of the examined essential oils showed significant antibacterial activity giving them the potential of usage as supportive agents in the treatment of skin infections. Due to high activity against commensal staphylococci, thyme as well as oregano essential oils can disrupt skin microbiota homeostasis, which underlines the necessity of careful skin application.

Bluetongue is a non-contagious arthropod-borne viral infection that is notifiable in several countries. The disease produces significant economic losses and is a main source of concern for ruminant commerce. Cattle are considered the reservoir of the bluetongue virus (BTV), which is responsible for huge economic losses on ruminant farms. In this study, we evaluated the seroprevalence of BTV in the Campania region, southern Italy, among the cattle and buffalo populations. The infection was widely spread, as attested by the high individual (43.6%) and herd prevalence (85.4%). We also evaluated the correlation between BTV seropositivity and various risk factors. The seropositivity rate did not differ between the two species considered or the type of housing. Surprisingly, seropositivity to SBV did not predispose animals to be positive for BTV, even though these infections share the same vector (Culicoides). A total co-infection rate of 21.8% was found. Age, on the other hand, was found to be a risk factor, as higher prevalences were found in adult animals. Among the climatic factors analyzed, a preponderant role is given by the average temperature, which is capable of influencing the probability of being positive for this infection. Our data, associated with those present in the literature, suggest that the spread of BTV is now transversal in the Mediterranean basin, that the rate of co-infection with SBV is not high, and that climate change has facilitated and continues to improve the spread of this virus.