Decoding Pseudomonas aeruginosa: Genomic insights into adaptation, antibiotic resistance, and the enigmatic role of T6SS in interbacterial dynamics.

Pseudomonas aeruginosa demonstrates a remarkable capacity for adaptation and survival in diverse environments. Furthermore, its clinical importance is underscored by its intrinsic and acquired resistance to a wide range of antimicrobial agents, posing a substantial challenge in healthcare settings. Amidst this complex landscape of resistance, the Type VI Secretion System (T6SS) in P. aeruginosa adds yet another layer of intricacy and allows bacteria to engage in interbacterial competition, potentially influencing their resilience and pathogenicity. Whole genome sequencing (WGS) was conducted on the five isolates under investigation, enabling the identification of antibiotic resistance genes (ARGs) and mutations associated with resistance. All isolates exhibit class C and D ß-lactamases, displaying variant differences. The Resistance-nodulation-division (RND) antibiotic efflux pumps, crucial for multidrug resistance, have been encoded chromosomally. When exploring the role of the T6SS in urinary tract infections involving other bacteria, it was noted that P. aeruginosa isolates exhibited reduced counts when co-cultivated with other bacteria. The downregulation of the tssJ gene, associated with the T6SS under bacterial stress, and the exclusion of several cluster genes in this study suggest the hypothesis of a basal state rather than an attack/defence mechanism in the initial contact.

Red Grape By-Products from the Demarcated Douro Region: Chemical Analysis, Antioxidant Potential and Antimicrobial Activity against Food-Borne Pathogens.

Wine production is one of the most important agricultural activities. The winemaking process generates a considerable volume of different residues characterized as by-products, such as pomace, seeds, stems, and skins. By-products are rich in polyphenols with antioxidant and antibacterial properties and may act as bacteriostatic or bactericidal agents against food-borne pathogens, improving food safety by enhancing antibiotic efficacy and reducing bacterial resistance. The aim of this study was to evaluate the phenolic composition and antioxidant activity of grape components (skins, seeds, and stems) from three red grape varieties (Periquita, Gamay, and Donzelinho Tinto) and determine their antibacterial activity against antibiotic-resistant bacteria, including Escherichia coli in food-producing animals and Listeria monocytogenes from food products and food-related environments. Ten phenolic compounds were quantified in these red grape varieties, with specific compounds found in different parts of the grape, including phenolic acids and flavonoids. Flavonoids are abundant in seeds and stems, malvidin-3-O-glucoside being the main anthocyanin in skins. The ethanolic extract from the seeds showed in vitro concentration-dependent activity against reactive species like •NO and O2•-. Gamay extract was the most effective, followed by Donzelinho Tinto and Periquita. Extracts showed varying antibacterial activity against Gram-positive and Gram-negative bacteria, with stronger effects on Gram-positive bacteria. L. monocytogenes was more susceptible, while E. coli was limited to three strains. Seeds exhibited the strongest antibacterial activity, followed by stems. The results of our study provide evidence of the potential of grape by-products, particularly seeds, as sources of bioactive compounds with antioxidant and antibacterial properties, offering promising avenues for enhancing food safety and combating antibiotic resistance in food production and related environments.

A novel gluten knowledge base of potential biomedical and health-related interactions extracted from the literature: Using machine learning and graph analysis methodologies to reconstruct the bibliome.

BACKGROUND: In return for their nutritional properties and broad availability, cereal crops have been associated with different alimentary disorders and symptoms, with the majority of the responsibility being attributed to gluten. Therefore, the research of gluten-related literature data continues to be produced at ever-growing rates, driven in part by the recent exploratory studies that link gluten to non-traditional diseases and the popularity of gluten-free diets, making it increasingly difficult to access and analyse practical and structured information. In this sense, the accelerated discovery of novel advances in diagnosis and treatment, as well as exploratory studies, produce a favourable scenario for disinformation and misinformation. OBJECTIVES: Aligned with, the European Union strategy "Delivering on EU Food Safety and Nutrition in 2050″ which emphasizes the inextricable links between imbalanced diets, the increased exposure to unreliable sources of information and misleading information, and the increased dependency on reliable sources of information; this paper presents GlutKNOIS, a public and interactive literature-based database that reconstructs and represents the experimental biomedical knowledge extracted from the gluten-related literature. The developed platform includes different external database knowledge, bibliometrics statistics and social media discussion to propose a novel and enhanced way to search, visualise and analyse potential biomedical and health-related interactions in relation to the gluten domain. METHODS: For this purpose, the presented study applies a semi-supervised curation workflow that combines natural language processing techniques, machine learning algorithms, ontology-based normalization and integration approaches, named entity recognition methods, and graph knowledge reconstruction methodologies to process, classify, represent and analyse the experimental findings contained in the literature, which is also complemented by data from the social discussion. RESULTS AND CONCLUSIONS: In this sense, 5814 documents were manually annotated and 7424 were fully automatically processed to reconstruct the first online gluten-related knowledge database of evidenced health-related interactions that produce health or metabolic changes based on the literature. In addition, the automatic processing of the literature combined with the knowledge representation methodologies proposed has the potential to assist in the revision and analysis of years of gluten research. The reconstructed knowledge base is public and accessible at https://sing-group.org/glutknois/.

Genomic evolution of the human and animal coronavirus diseases.

Different coronaviruses have emerged due to their ability to infect, mutate and recombine multiple species and cell types, suggesting that these viruses will carry on to evolve and origin both veterinary and human diseases. So far, more than fifteen coronavirus-related diseases have been described in animals and seven in humans. Of which recently, a novel human betacoronavirus designated by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), an emerging zoonotic coronavirus is the causative agent of the coronavirus disease 2019. This virus emerged in China and spread rapidly worldwide. At the end of January 2020, the WHO declared the pandemic as a public health emergency of international concern. In this pandemic, the SARS-CoV-2 virus has infected more than 198 million people, with 4.2 million deaths worldwide (as of 2 August 2021). In the past two decades, this is the third betacoronavirus that has crossed the interspecies barrier from animals to infect humans and other animal species. The diseases caused mainly severe respiratory infections. The aim of this review is to summarize and provide an overview of the coronaviruses that can affect animals and humans and the diseases that ensue, as well as, its genomic relationship.

Genomic and Metabolic Characteristics of the Pathogenicity in Pseudomonas aeruginosa.

In recent years, the effectiveness of antimicrobials in the treatment of Pseudomonas aeruginosa infections has gradually decreased. This pathogen can be observed in several clinical cases, such as pneumonia, urinary tract infections, sepsis, in immunocompromised hosts, such as neutropenic cancer, burns, and AIDS patients. Furthermore, Pseudomonas aeruginosa causes diseases in both livestock and pets. The highly flexible and versatile genome of P. aeruginosa allows it to have a high rate of pathogenicity. The numerous secreted virulence factors, resulting from its numerous secretion systems, the multi-resistance to different classes of antibiotics, and the ability to produce biofilms are pathogenicity factors that cause numerous problems in the fight against P. aeruginosa infections and that must be better understood for an effective treatment. Infections by P. aeruginosa represent, therefore, a major health problem and, as resistance genes can be disseminated between the microbiotas associated with humans, animals, and the environment, this issue needs be addressed on the basis of an One Health approach. This review intends to bring together and describe in detail the molecular and metabolic pathways in P. aeruginosa's pathogenesis, to contribute for the development of a more targeted therapy against this pathogen.

Valorization of Winemaking By-Products as a Novel Source of Antibacterial Properties: New Strategies to Fight Antibiotic Resistance.

The emergence of antibiotic-resistance in bacteria has limited the ability to treat bacterial infections, besides increasing their morbidity and mortality at the global scale. The need for alternative solutions to deal with this problem is urgent and has brought about a renewed interest in natural products as sources of potential antimicrobials. The wine industry is responsible for the production of vast amounts of waste and by-products, with associated environmental problems. These residues are rich in bioactive secondary metabolites, especially phenolic compounds. Some phenolics are bacteriostatic/bactericidal against several pathogenic bacteria and may have a synergistic action towards antibiotics, mitigating or reverting bacterial resistance to these drugs. Complex phenolic mixtures, such as those present in winemaking residues (pomace, skins, stalks, leaves, and especially seeds), are even more effective as antimicrobials and could be used in combined therapy, thereby contributing to management of the antibiotic resistance crisis. This review focuses on the potentialities of winemaking by-products, their extracts, and constituents as chemotherapeutic antibacterial agents.

Advances in quantification and analysis of the celiac-related immunogenic potential of gluten.

Gluten-free products have emerged in response to the increasing prevalence of gluten-related disorders, namely celiac disease. Therefore, the quantification of gluten in products intended for consumption by individuals who may suffer from these pathologies must be accurate and reproducible, in a way that allows their proper labeling and protects the health of consumers. Immunochemical methods have been the methods of choice for quantifying gluten, and several kits are commercially available. Nevertheless, they still face problems such as the initial extraction of gluten in complex matrices or the use of a standardized reference material to validate the results. Lately, other methodologies relying mostly on mass spectrometry-based techniques have been explored, and that may allow, in addition to quantitative analysis, the characterizationof gluten proteins. On the other hand, although the level of 20 mg/kg of gluten detected by these methods is sufficient for a product to be considered gluten-free, its immunogenic potential for celiac patients has not been clinically validated. In this sense, in vitro and in vivo models, such as the organoid technology applied in gut-on-chip devices and the transgenic humanized mouse models, respectively, are being developed for investigating both the gluten-induced pathogenesis and the treatment of celiac disease. Due to the ubiquitous nature of gluten in the food industry, as well as the increased prevalence of gluten-related disorders, here we intend to summarize the available methods for gluten quantification in food matrices and for the evaluation of its immunogenic potential concerning the development of novel therapies for celiac disease to highlight active research and discuss knowledge gaps and current challenges in this field.

Implications of antibiotics use during the COVID-19 pandemic: present and future.

COVID-19 is caused by the SARS-CoV-2 virus, which has infected more than 4 million people with 278 892 deaths worldwide as of 11 May 2020. This disease, which can manifest as a severe respiratory infection, has been declared as a public health emergency of international concern and is being treated with a variety of antivirals, antibiotics and antifungals. This article highlights the administration of antimicrobials in COVID-19 patients worldwide, during the 2019-20 pandemic. It is imperative to be aware of the unreported amounts of antibiotics that have been administered worldwide in just a few months and a marked increase in antimicrobial resistance should therefore be expected. Due to the lack of data about antimicrobial use during this pandemic, the global impact on the emergence of new antimicrobial resistance is as yet unknown. This issue must be at the forefront of public health policymaking and planning in order that we are prepared for the potentially severe consequences for human and animal health and the environment.

Efficacy of dalbavancin against MRSA biofilms in a rat model of orthopaedic implant-associated infection.

OBJECTIVES: The aim of this study was to evaluate the efficacy of dalbavancin against MRSA biofilm-related infection in orthopaedic implants in vivo. METHODS: One MRSA strain isolated from human osteomyelitis was used to promote biofilm formation on the surface of screws. The implants were inserted in the proximal tibia under general anaesthesia. Thirty-nine Wistar rats were divided into three groups [control group (no treatment), Group 1 (7 days of treatment) and Group 2 (14 days of treatment)]; both treatment groups were administered dalbavancin intraperitoneally and euthanized after treatment. cfu of bacteria present in both the tibia and the implant were quantified. The infection severity was assessed by histopathology and scored from 0 (no infection) to 4 (severe infection). RESULTS: The high number of cfu/g and cfu/mL present in the control group indicated a well-established infection. There was a significant reduction in cfu in rats treated with dalbavancin both in the tibia (2.8 × 105 cfu/g) and the implant (1.1 × 106 cfu/mL) in Group 1 (1.8 × 103 cfu/g and 2.4 × 105 cfu/mL, respectively) and in Group 2 (8.2 cfu/g and 8.2 × 103 cfu/mL, respectively). Most animals from the control group presented an infection scored as 3 (severe). At the end of the experiment, most rats from Groups 1 and 2 presented an infection scored as 2 (moderate) and 0 (no infection), respectively. CONCLUSIONS: Although there was a marked decrease in cfu number, signs of biofilm-induced infection prevailed after 14 days of treatment. Further studies should be carried out to evaluate the potential of dalbavancin in the treatment of bone and orthopaedic implant-associated MRSA infections.

Occurrence of ESBL-producing Escherichia coli in soils subjected to livestock grazing in Azores archipelago: an environment-health pollution issue?

Antibiotics are successful drugs used in human and animal therapy; however, they must be considered as environmental pollutants. This study aims to isolate and characterize the extended-spectrum ß-lactamase (ESBL) producing Escherichia coli soil from Azores Archipelago subjected to livestock agricultural practices. Twenty-four soil samples were collected from three different pasture systems with different number of cattle heads, and from a control site. Antibiotic susceptibility method was performed by Kirby-Bauer disk diffusion method against 16 antibiotics, and the presence of genes encoding lactamases, antimicrobial resistance genes, virulence factors, and phylogenetic groups was determined by polymerase chain reaction (PCR). Nine ESBLs were recovered from the three grazing sites, and all isolates presented the beta-lactamase genes blaCTX-M-3 and blaSHV. E. coli isolates were resistance to tetracycline and streptomycin and harbored the tetB, strA, and strB genes. One isolate also showed resistance to sulfonamides, and the genes sul1 and sul2 were detected. The isolates were grouped into the following phylogenic groups: B1 (n = 6), D (n = 2), and A (n = 1). The presence of antibiotics and resistance genes in soils may be the source to the development of antimicrobial resistance, which may have negative consequences in human and animal health.

Emergence of community-acquired methicillin-resistant Staphylococcus aureus EMRSA-15 clone as the predominant cause of diabetic foot ulcer infections in Portugal.

Methicillin-resistant Staphylococcus aureus (MRSA) are often found in infected diabetic foot ulcers, in which the prevalence may reach 40%. These complications are one of the main causes of morbidity in diabetic patients. The objectives of this study were to investigate the prevalence and antimicrobial resistance of MRSA strains in infected diabetic foot ulcers and to characterize their genetic lineages. Samples collected from 42 type 2 diabetic patients, presenting infected foot ulcers, were seeded onto ORSAB plates with 2 mg/L of oxacillin for MRSA isolation. Susceptibility to 14 antimicrobial agents was tested by the Kirby-Bauer disk diffusion method. The presence of resistance genes, virulence factors, and the immune evasion cluster system was studied by PCR. All isolates were characterized by MLST, accessory gene regulator (agr), spa, and staphylococcal chromosomal cassette mec (SCCmec) typing. Twenty-five MRSA strains were isolated. All isolates showed resistance to penicillin and cefoxitin. Sixteen isolates showed phenotypic resistance to erythromycin being 7 co-resistant to clindamycin. Resistance to trimethoprim-sulfamethoxazole was found in 2 isolates harboring the dfrA and dfrG genes. The IEC genes were detected in 80% of isolates, 16 of which were ascribed to IEC-type B. Isolates were assigned to 12 different spa types. The MLST analysis grouped the isolates into 7 sequence types being the majority (68%) ascribed to SCCmec type IV. In this study, there was a high prevalence of the EMRSA-15 clone presenting multiple resistances in diabetic foot ulcers making these infections complicated to treat leading to a higher morbidity and mortality in diabetic patients.

High Efficacy of Ozonated Oils on the Removal of Biofilms Produced by Methicillin-Resistant Staphylococcus aureus (MRSA) from Infected Diabetic Foot Ulcers.

Ozone has a high wound healing capacity and antibacterial properties and can be used as a complementary treatment in infections. Methicillin-resistant S. aureus (MRSA) is the most common pathogen found in infected diabetic foot ulcers. Most of MRSA are resistant to several classes of antibiotics and, therefore, there is a need for new, effective, and well-tolerated agents. Thus, we aimed evaluate the antimicrobial and antibiofilm potentials of ozonated vegetable oils against MRSA strains isolated from diabetic foot ulcers. Six ozonated oils were produced with concentrations of ozone ranging from 0.53 to 17 mg of ozone/g of oil. The peroxide values were determined for each oil. Ozonated oils content on fatty acid was determined by gas chromatography equipped with a flame ionization detector. The antimicrobial susceptibility testing was performed by the Kirby-Bauer disk diffusion method and the effect of ozonated oils on biofilm formation ability and on established biofilms was investigated. In general, the content in identified unsaturated fatty acid in oils decreased with the increase of ozonation time and, consequently, the peroxide value increased. Most bacterial strains were inhibited by ozonated oil at a concentration of 4.24 mg/g. Ozonated oils had moderate to high ability to remove adhered cells and showed a high capacity to eradicate 24 h old biofilms. Our results show promising use of ozonated oils on the treatment of infections, in particular those caused by multidrug-resistant MRSA strains.

Tuberculosis in the 21th century: Current status of diagnostic methods.

Tuberculosis is an infectious bacterial disease with a high mortality rate worldwide constituting a serious public health problem. The diagnostic methods commonly used by health professionals are slow and expensive and the results may take about sixty days which will cause a delay in administrating the most proper treatment to the patient, as well as increase health care costs and infection transmission possibility. Patients infected simultaneously with human immunodeficiency virus and Mycobacterium tuberculosis are a constant and worrying challenge for the scientific community which will research and develop new methods of diagnosis, new drugs and new therapies. Nowadays there are new tuberculosis diagnosis methods and some of which are already in clinical trial phases. These methods have high sensitivity, but do not replace the microbiological examination for isolation and culture of Mycobacterium spp. However, in clinical practice, microbiological, imaging, clinical and epidemiological data integration provide the best diagnosis and treatment possible. Consequently, throughout this paper, the different methods of diagnosis of human tuberculosis with its advantages and disadvantages will be covered, describing new omics and ultra-fast methods to increase knowledge and obtain a rapid diagnosis of tuberculosis.

Subproteomic signature comparison of in vitro selected fluoroquinolone resistance and ciprofloxacin stress in Salmonella Typhimurium DT104B.

BACKGROUND: Fluoroquinolone resistance in nontyphoidal Salmonella is a situation of serious and international concern, particularly in S. Typhimurium DT104B multiresistant strains. Although known to be multifactorial, fluoroquinolone resistance is still far from a complete understanding. METHODS: Subproteome changes between an experimentally selected fluoroquinolone-resistant strain (Se6-M) and its parent strain (Se6), and also in Se6-M under ciprofloxacin (CIP) stress, were evaluated in order to give new insights into the mechanisms involved. Proteomes were compared at the intracellular and membrane levels by a 2-DE~LC-MS/MS and a shotgun LC-MS/MS approach, respectively. RESULTS: In total, 35 differentially abundant proteins were identified when comparing Se6 with Se6-M (25 more abundant in Se6 and 10 more abundant in Se6-M) and 82 were identified between Se6-M and Se6-M+CIP (51 more abundant in Se6-M and 31 more abundant under ciprofloxacin stress). CONCLUSION: Several proteins with known and possible roles in quinolone resistance were identified which provide important information about mechanism-related differential protein expression, supporting the current knowledge and also leading to new testable hypotheses on the mechanism of action of fluoroquinolone drugs.

Study of InDel genetic markers with forensic and ancestry informative interest in PALOP's immigrant populations in Lisboa.

The migratory phenomenon in Portugal has become one of the main factors for the genetic variability. In the last few years, a new class of autosomal insertion/deletion markers-InDel-has attracted interest in forensic genetics. Since there is no data for InDel markers of Portuguese-speaking African countries (PALOP) immigrants living in Lisboa, our aim is the characterization of those groups of individuals by typing them with at least 30 InDel markers and to compare different groups of individuals/populations. We studied 454 bloodstain samples belonging to immigrant individuals from Angola, Guinea-Bissau, and Mozambique. DNA extraction was performed with the Chelex® 100 method. After extraction, all samples were typed with the Investigator® DIPplex method. Through the obtained results, allelic frequencies show that all markers are at Hardy-Weinberg equilibrium, and we can confirm that those populations show significant genetic distances between themselves, between them, and the host Lisboa population. Because of this, they introduce genetic variability in Lisboa population.

Characterization of Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss) feed and larvae: safety, DNA fingerprinting, and bacteriocinogenicity.

The use of lactic acid bacteria (LAB) as probiotics constitutes an alternative or complementary strategy to chemotherapy and vaccination for disease control in aquaculture. The objectives of this work were (1) the in vitro safety assessment of 8 Pediococcus acidilactici strains isolated from rainbow trout (Oncorhynchus mykiss, Walbaum) feed and larvae; (2) the evaluation of their genetic relatedness; (3) the study of their antimicrobial/bacteriocin activity against fish pathogens; and (4) the biochemical and genetic characterization of the bacteriocin produced by the strain displaying the greatest antimicrobial activity. Concerning the safety assessment, none of the pediococci showed antibiotic resistance nor produced hemolysin or gelatinase, degraded gastric mucin, or deconjugated bile salts. Four strains (50%) produced tyramine or putrescine, but the corresponding genes were not amplified by PCR. Enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR) fingerprinting allowed clustering of the pediococci into 2 well-defined groups (68% similarity). From the 8 pediococci displaying direct antimicrobial activity against at least 3 out of 9 fish pathogens, 6 strains (75%) were identified as bacteriocin producers. The bacteriocin produced by P. acidilactici L-14 was purified, and mass spectrometry and DNA sequencing revealed its identity to pediocin PA-1 (PedPA-1). Altogether, our results allowed the identification of 4 (50%) putatively safe pediococci, including 2 bacteriocinogenic strains. ERIC-PCR fingerprinting was a valuable tool for genetic profiling of P. acidilactici strains. This work reports for the first time the characterization of a PedPA-1-producing P. acidilactici strain isolated from an aquatic environment (rainbow trout larvae), which shows interesting properties related to its potential use as a probiotic in aquaculture.

Antimicrobial resistance and virulence genes in enterococci from wild game meat in Spain.

A total of 55 enterococci (45 Enterococcus faecium, 7 Enterococcus faecalis, and three Enterococcus durans) isolated from the meat of wild game animals (roe deer, boar, rabbit, pheasant, and pigeon) in North-Western Spain were tested for susceptibility to 14 antimicrobials by the disc diffusion method. All strains showed a multi-resistant phenotype (resistance to between three and 10 antimicrobials). The strains exhibited high percentages of resistance to erythromycin (89.1%), tetracycline (67.3%), ciprofloxacin (92.7%), nitrofurantoin (67.3%), and quinupristin-dalfopristin (81.8%). The lowest values (9.1%) were observed for high-level resistance to gentamicin, kanamycin, and streptomycin. The average number of resistances per strain was 5.8 for E. faecium isolates, 7.9 for E. faecalis, and 5.7 for E. durans. Genes encoding antimicrobial resistance and virulence were studied by polymerase chain reaction. A total of 15 (57.7%) of the 26 vancomycin-resistant isolates harboured the vanA gene. Other resistance genes detected included vanB, erm(B) and/or erm(C), tet(L) and/or tet(M), acc(6')-aph(2″), and aph(3')-IIIa in strains resistant to vancomycin, erythromycin, tetracycline, gentamicin, and kanamycin, respectively. Specific genes of the Tn5397 transposon were detected in 54.8% of the tet(M)-positive enterococci. Nine virulence factors (gelE, agg, ace, cpd, frs, esp, hyl, efaAfs and efaAfm) were studied. All virulence genes, with the exception of the frs gene, were found to be present in the enterococcal isolates. At least one virulence gene was detected in 20.0% of E. faecium, 71.4% of E. faecalis and 33.3% of E. durans isolates, with ace and cpd being the most frequently detected genes (6 isolates each). This suggests that wild game meat might play a role in the spreading through the food chain of enterococci with antimicrobial resistance and virulence determinants to humans.

Evaluation of Enterococcus spp. from rainbow trout (Oncorhynchus mykiss, Walbaum), feed, and rearing environment against fish pathogens.

The use of lactic acid bacteria of aquatic origin as probiotics constitutes an alternative strategy to the antibiotic treatment for disease control in aquaculture. Enterococci are currently used as probiotics in human and animal health. In this study, we evaluated the safety of 64 enterococci isolated from rainbow trout (Oncorhynchus mykiss, Walbaum), feed and rearing environment, and their antimicrobial activity against 9 fish pathogens. The 64 enterococcal isolates were identified to the species level by polymerase chain reaction (PCR), using specific primers for the different enterococcal species, and confirmed by superoxide dismutase gene sequencing. Enterococcus faecium and E. hirae were the most common species (42.2 and 35.9%, respectively). A total of 48 isolates (75%) showed phenotypic resistance to at least 1 antibiotic determined by a disk-diffusion method, and 25 isolates (39.1%) harbored at least 1 antibiotic resistance gene [erm(B), tet(M), tet(S), tet(K), tet(L), tet(T), vanC2, and aad(E)], detected by PCR. One (1.6%) isolate produced gelatinase and none produced hemolysin, using a plate assay. The virulence genes gelE (46.9%), efaAfs (17.2%), agg (1.6%), and hyl (1.6%) were detected by PCR. A total of 48 isolates (75%) exerted antimicrobial activity against 1 or more of the tested fish pathogens, using a stab-on-agar test. From these isolates, 21 (43.8%) harbored at least 1 bacteriocin-encoding gene (entP, entL50A and entL50B, hirJM79, entSE-K4, entQ and entA), detected by PCR. None of the enterococci showed bile deconjugation and mucin degradation abilities. A total of 17 enterococcal isolates (26.6%) that did not harbor any antibiotic resistance or virulence factor were considered safe for application as probiotics, including 6 isolates (35.3%) that showed antimicrobial activity against at least 1 fish pathogen and harbored at least 1 bacteriocin-encoding gene. Rainbow trout, feed, and rearing environment constitute an appropriate source for the isolation of enterococci as potential probiotic for aquaculture.

Inhibition of fish pathogens by the microbiota from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment.

This work reports the isolation and taxonomic identification of the cultivable total microbiota (TM) and Lactic Acid Bacteria (LAB) from rainbow trout (Oncorhynchus mykiss, Walbaum) and rearing environment from selected stages of the life-cycle, and the evaluation of the LAB antimicrobial activity against the main fish pathogens. TM and LAB isolates were randomly selected and identified by 16S rRNA and/or superoxide dismutase gene sequencing. Although a great diversity in the TM was observed, Enterobacteriaceae and Aeromonadaceae were clearly prevalent, while the genus Lactococcus was the predominant LAB. From a total of 1620 randomly selected LAB, 1159 isolates (71.5%) showed antimicrobial activity. From these, 248 isolates (21.4%) selected for their activity against, at least, four fish pathogens, were taxonomically identified, being Lactococcus lactis the most common species (164 isolates, 66.1%). Interestingly, 88 isolates (35.5%), including 55 L. lactis isolates, exerted activity against four strains of the rainbow trout pathogen Lactococcus garvieae. Our results demonstrate that rainbow trout and rearing environment are potential sources for the isolation of LAB, mainly lactococci, active against L. garvieae and other fish pathogens. Moreover, this is the first study describing the cultivable TM and LAB from rainbow trout intestine and rearing environment along the fish life-cycle. The host-derived LAB active against fish pathogens comprise potential candidates as probiotics in rainbow trout farming as an alternative or complementary strategy to antibiotics and vaccines for disease prevention.

Vancomycin-resistant enterococci among haemodialysis patients in Portugal: prevalence and molecular characterization of resistance, virulence and clonality.

INTRODUCTION: Vancomycin-resistant enterococci (VRE) among haemodialysis patients has increased rapidly and, to date, there is no report of this incidence in Portugal. METHODS: A total of 121 faecal samples were collected from haemodialysis patients, and then tested for VRE. Antimicrobial resistance, virulence and multilocus sequence typing (MLST) were studied. RESULTS: VRE prevalence was 3.3%. Three VRE isolates, Enterococcus faecium, Enterococcus faecalis and Enterococcus raffinosus, were multi-resistant and vanA-positive. E. faecium and E. faecalis belonged to CC17 and CC2, respectively. CONCLUSION: Haemodialysis patients in Portugal are colonized with virulent, multi-resistant enterococci from high-risk clonal complexes, representing a public health concern.