Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Identification of Marker Compounds and In Vitro Toxicity Evaluation of Two Portuguese Asphodelus Leaf Extracts.

The leaves of Asphodelus bento-rainhae subsp. bento-rainhae, an endemic Portuguese species, and Asphodelus macrocarpus subsp. macrocarpus have been used as food, and traditionally as medicine, for treating ulcers, urinary tract, and inflammatory disorders. The present study aims to establish the phytochemical profile of the main secondary metabolites, together with the antimicrobial, antioxidant and toxicity assessments of both Asphodelus leaf 70% ethanol extracts. Phytochemical screenings were conducted by the TLC and LC-UV/DAD-ESI/MS chromatographic technique, and quantification of the leading chemical classes was performed by spectrophotometric methods. Liquid-liquid partitions of crude extracts were obtained using ethyl ether, ethyl acetate, and water. For in vitro evaluations of antimicrobial activity, the broth microdilution method, and for the antioxidant activity, the FRAP and DPPH methods were used. Genotoxicity and cytotoxicity were assessed by Ames and MTT tests, respectively. Twelve known compounds including neochlorogenic acid, chlorogenic acid, caffeic acid, isoorientin, p-coumaric acid, isovitexin, ferulic acid, luteolin, aloe-emodin, diosmetin, chrysophanol, and ß-sitosterol were identified as the main marker compounds, and terpenoids and condensed tannins were found to be the major class of secondary metabolites of both medicinal plants. The ethyl ether fractions demonstrated the highest antibacterial activity against all the Gram-positive microorganisms, (MIC value of 62 to 1000 µg/mL), with aloe-emodin as one of the main marker compounds highly active against Staphylococcus epidermidis (MIC value of 0.8 to 1.6 µg/mL). Ethyl acetate fractions exhibited the highest antioxidant activity (IC50 of 800 to 1200 µg/mL, respectively). No cytotoxicity (up to 1000 µg/mL) or genotoxicity/mutagenicity (up to 5 mg/plate, with/without metabolic activation) were detected. The obtained results contribute to the knowledge of the value and safety of the studied species as herbal medicines.

Investigation of 29 Antimicrobial Compounds in Soil Using Newly Developed UHPLC-MS/MS Method.

While the prudent and reasonable use of veterinary antimicrobial agents in food-producing animals is necessary, researchers over the decades have shown that these antimicrobial agents can spread into the environment through livestock manure and wastewater. The analysis of the occurrence of antimicrobial compounds in soil samples is of a great importance to determine potential impacts on human and animal health and the environment. In this study, an affordable, rugged and simple analytical method has been developed for the determination of twenty-nine antimicrobial compounds from five different classes (tetracyclines, fluoro(quinolones), macrolides, sulfonamides and diaminopirimidines). Liquid-liquid extraction (LLE) with extract filtration combined with ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was the best strategy for the simultaneous determination of all analytes. The developed method was validated according to the Commission Implementing Regulation (EU) 2021/808. The limit of detections (LODs) ranged from 0.5 to 2.0 µg/kg, while the limit of quantitation (LOQ) was established at 1.0 to 20.0 µg/kg. The developed method was successfully applied for the determination of antimicrobial residues in one hundred and eighteen soil samples obtained from four European countries (Austria, Czech Republic, Estonia and Portugal). Doxycycline in the concentration levels of 9.07 µg/kg-20.6 µg/kg was detected in eight of the analysed samples. Samples were collected from areas where natural fertilizers (swine or cow manure) were applied. Our method can be efficiently used to monitor anti-microbial compounds in soil samples.

Characterizing Antimicrobial Resistance in Clinically Relevant Bacteria Isolated at the Human/Animal/Environment Interface Using Whole-Genome Sequencing in Austria.

Antimicrobial resistance (AMR) is a public health issue attributed to the misuse of antibiotics in human and veterinary medicine. Since AMR surveillance requires a One Health approach, we sampled nine interconnected compartments at a hydrological open-air lab (HOAL) in Austria to obtain six bacterial species included in the WHO priority list of antibiotic-resistant bacteria (ARB). Whole genome sequencing-based typing included core genome multilocus sequence typing (cgMLST). Genetic and phenotypic characterization of AMR was performed for all isolates. Eighty-nine clinically-relevant bacteria were obtained from eight compartments including 49 E. coli, 27 E. faecalis, 7 K. pneumoniae and 6 E. faecium. Clusters of isolates from the same species obtained in different sample collection dates were detected. Of the isolates, 29.2% were resistant to at least one antimicrobial. E. coli and E. faecalis isolates from different compartments had acquired antimicrobial resistance genes (ARGs) associated with veterinary drugs such as aminoglycosides and tetracyclines, some of which were carried in conjugative and mobilizable plasmids. Three multidrug resistant (MDR) E. coli isolates were found in samples from field drainage and wastewater. Early detection of ARGs and ARB in natural and farm-related environments can identify hotspots of AMR and help prevent its emergence and dissemination along the food/feed chain.

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.

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.

Influence of agricultural practice on mobile bla genes: IncI1-bearing CTX-M, SHV, CMY and TEM in Escherichia coli from intensive farming soils.

Many calls have been made to address antibiotic resistance in an environmental perspective. With this study, we showed the widespread presence of high-level antibiotic resistant isolates on a collection of non-susceptible Gram-negative bacteria (n = 232) recovered from soils. Bacteria were selected using amoxicillin, cefotaxime and imipenem, from sites representing different agricultural practices (extensive, intensive and organic). Striking levels of non-susceptibility were noticed in intensive soils for norfloxacin (74%), streptomycin (50.7%) and tetracycline (46.6%); indeed, the exposure to intensive agricultural practices constituted a risk factor for non-susceptibility to many antibiotics, multidrug resistance and production of extended-spectrum ß-lactamases (ESBL). Analyses of non-susceptibility highlighted that environmental and clinical bacteria from the same species might not share the same intrinsic resistance patterns, raising concerns for therapy choices in environment-borne infections. The multiple sequence-type IncI1-driven spread of penicillinases (blaTEM-1, blaTEM-135), ESBL (blaSHV-12 and blaCTX-M-1) and plasmid-mediated AmpC ß-lactamases (blaCMY-2), produced by isolates that share their molecular features with isolates from humans and animals, suggests contamination of agricultural soils. This is also the first appearance of IncI1/ST28-harbouring blaCTX-M-1, which should be monitored to prevent their establishment as successfully dispersed plasmids. This research may help disclose paths of contamination by mobile antibiotic resistance determinants and the risks for their dissemination.

New Class 2 Integron In2-4 Among IncI1-Positive Escherichia coli Isolates Carrying ESBL and PMAß Genes from Food Animals in Portugal.

The impact of extended-spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC ß-lactamases (PMAßs) of animal origin constitutes a public health concern. In this study, 179 Escherichia coli from food animals and products were analyzed, among which, 15 cephalosporin-resistant isolates harboring ESBL (CTX-M-1 [n = 8], CTX-M-14 [n = 1], SHV-12 [n = 2]) or PMAß [CMY-2, n = 5]) were identified in poultry and swine, from different farms of distinct regions of Portugal. The multiple sequence-type IncI1-driven spread of ESBLs and PMAßs, flanked by widely disseminated mobile elements, was guaranteed by ST26/IncI1-harboring blaSHV-12, ST12/IncI1-harboring blaCMY-2, ST3 and ST38/IncI1-harboring blaCTX-M-1, and ST1/IncI1-harboring blaCTX-M-14. An IS10-disrupted In2-4, presenting a new attI2 recombination site, was also detected in a SHV-12/CTX-M-1-harboring isolate. This study highlights the fact that animals may act as persistent sources of ESBL- and PMAß-harboring plasmids genes that might be transferred to humans through direct contact or via the food chain.

Predominance of KPC-3 in a survey for carbapenemase-producing Enterobacteriaceae in Portugal.

Among the 2,105 Enterobacteriaceae tested in a survey done in Portugal, 165 were nonsusceptible to carbapenems, from which 35 (26 Klebsiella pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes, and 3 Enterobacter cloacae isolates and 1 Klebsiella oxytoca isolate) were confirmed to be carbapenemase producers by the presence of 30 Tn4401d-blaKPC-3, 4 intI3-blaGES-5, and one intI1-blaVIM-2 gene, alone or in combination with other bla genes. The dissemination of blaKPC-3 gene carried by an IncF plasmid suggests lateral gene transfer as a major mechanism of dissemination.

Two novel CMY-2-type ß-lactamases encountered in clinical Escherichia coli isolates.

BACKGROUND: Chromosomally encoded AmpC ß-lactamases may be acquired by transmissible plasmids which consequently can disseminate into bacteria lacking or poorly expressing a chromosomal bla AmpC gene. Nowadays, these plasmid-mediated AmpC ß-lactamases are found in different bacterial species, namely Enterobacteriaceae, which typically do not express these types of ß-lactamase such as Klebsiella spp. or Escherichia coli. This study was performed to characterize two E. coli isolates collected in two different Portuguese hospitals, both carrying a novel CMY-2-type ß-lactamase-encoding gene. FINDINGS: Both isolates, INSRA1169 and INSRA3413, and their respective transformants, were non-susceptible to amoxicillin, amoxicillin plus clavulanic acid, cephalothin, cefoxitin, ceftazidime and cefotaxime, but susceptible to cefepime and imipenem, and presented evidence of synergy between cloxacilin and cefoxitin and/or ceftazidime. The genetic characterization of both isolates revealed the presence of bla CMY-46 and bla CMY-50 genes, respectively, and the following three resistance-encoding regions: a Citrobacter freundii chromosome-type structure encompassing a blc-sugE-bla CMY-2-type -ampR platform; a sul1-type class 1 integron with two antibiotic resistance gene cassettes (dfrA1 and aadA1); and a truncated mercury resistance operon. CONCLUSIONS: This study describes two new bla CMY-2-type genes in E. coli isolates, located within a C. freundii-derived fragment, which may suggest their mobilization through mobile genetic elements. The presence of the three different resistance regions in these isolates, with diverse genetic determinants of resistance and mobile elements, may further contribute to the emergence and spread of these genes, both at a chromosomal or/and plasmid level.

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.

Vitex Genus as a Source of Antimicrobial Agents.

Vitex L. is the largest genus of the Lamiaceae family, and most of its species are used in the traditional medicinal systems of different countries. A systematic review was conducted, according to the PRISMA methodology, to determine the potential of Vitex plants as sources of antimicrobial agents, resulting in 2610 scientific publications from which 141 articles were selected. Data analysis confirmed that Vitex species are used in traditional medicine for symptoms of possible infectious diseases. Conducted studies showed that these medicinal plants exhibited in vitro antimicrobial activity against Bacillus subtilis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. Vitex agnus-castus L. and Vitex negundo L. have been the most studied species, not only against bacterial strains but also against fungi such as Aspergillus niger and Candida albicans, viruses such as HIV-1, and parasites such as Plasmodium falciparum. Natural products like agnucastoside, negundol, negundoside, and vitegnoside have been identified in Vitex extracts and their antimicrobial activity against a wide range of microbial strains has been determined. Negundoside showed significant antimicrobial activity against Staphylococcus aureus (MIC 12.5 µg/mL). Our results show that Vitex species are potential sources of new natural antimicrobial agents. However, further experimental studies need to be conducted.

Genetic Complexity of CC5 Staphylococcus aureus Isolates Associated with Sternal Bursitis in Chickens: Antimicrobial Resistance, Virulence, Plasmids, and Biofilm Formation.

Sternal bursitis, a common inflammatory condition in poultry, poses significant challenges to both animal welfare and public health. This study aimed to investigate the prevalence, antimicrobial resistance, and genetic characteristics of Staphylococcus aureus isolates associated with sternal bursitis in chickens. Ninety-eight samples were collected from affected chickens, and 24 S. aureus isolates were identified. Antimicrobial susceptibility testing revealed resistance to multiple agents, with a notable prevalence of aminoglycoside resistance genes. Whole genome sequencing elucidated the genetic diversity and virulence profiles of the isolates, highlighting the predominance of clonal complex 5 (CC5) strains. Additionally, biofilm formation assays demonstrated moderate biofilm production capacity among the isolates. These findings underscore the importance of vigilant monitoring and targeted interventions to mitigate the impact of sternal bursitis in poultry production systems.

Genomic and epidemiological insight of an outbreak of carbapenemase-producing Enterobacterales in a Portuguese hospital with the emergence of the new KPC-124.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) is an increasing problem in healthcare settings. This study aimed to identify the source of a CPE outbreak that occurred in 2022, in a tertiary hospital in the North of Portugal, to identify exposed patients, and to assess the risk of becoming CPE-positive following hospital admission. METHODS: A multi-disciplinary investigation was conducted including descriptive, analytical, and molecular epidemiology, environmental screening, and assessment of infection control measures. Clinical and environmental isolates were analyzed using whole-genome sequencing and phylogenetic analysis. Additionally, a prospective observational cohort study was conducted to further investigate the risk factors associated with the emergence of new cases in cohorts of CPE-negative admitted patients. RESULTS: We observed the presence of multispecies KPC-, IMP-, and/or NDM-producing isolates. Genetically indistinguishable clinical and environmental isolates were found on the same room/ward. The ST45 KPC-3-producing Klebsiella pneumoniae clone was the responsible for the outbreak. During patients' treatment, we detected the emergence of resistance to ceftazidime-avibactam, associated with mutations in the blaKPC-3 gene (blaKPC-46, blaKPC-66 and blaKPC-124, the last variant never previously reported), suggesting a vertical evolutionary trajectory. Patients aged ≥ 75 years, hygiene/feeding-care dependent, and/or subjected to secretion aspiration were risk factors for CPE colonization after hospital admission. Additionally, cases with previous admission to the emergency department suggest that CPE dissemination may occur not only during hospitalization but also in the emergency department. CONCLUSION: Overall, the study highlights that selection pressure with antibiotics, like ceftazidime-avibactam, is a contributing factor to the emergence of new ß-lactamase variants and antibiotic resistance. It also shows that the hospital environment can be a significant source of CPE transmission, and that routine use of infection control measures and real-time molecular epidemiology investigations are essential to ensure the long-term termination of CPE outbreaks and prevent future resurgences.

Resistome, Virulome, and Clonal Variation in Methicillin-Resistant Staphylococcus aureus (MRSA) in Healthy Swine Populations: A Cross-Sectional Study.

This cross-sectional study investigates the methicillin-resistant Staphylococcus aureus (MRSA): its prevalence, antimicrobial resistance, and molecular characteristics in healthy swine populations in central Portugal. A total of 213 samples were collected from pigs on twelve farms, and MRSA prevalence was assessed using selective agar plates and confirmed via molecular methods. Antimicrobial susceptibility testing and whole genome sequencing (WGS) were performed to characterize resistance profiles and genetic determinants. Among the 107 MRSA-positive samples (83.1% prevalence), fattening pigs and breeding sows exhibited notably high carriage rates. The genome of 20 isolates revealed the predominance of the ST398 clonal complex, with diverse spa types identified. Antimicrobial susceptibility testing demonstrated resistance to multiple antimicrobial agents, including penicillin, cefoxitin, and tetracycline. WGS analysis identified a diverse array of resistance genes, highlighting the genetic basis of antimicrobial resistance. Moreover, virulence gene profiling revealed the presence of genes associated with pathogenicity. These findings underscore the significant prevalence of MRSA in swine populations and emphasize the need for enhanced surveillance and control measures to mitigate zoonotic transmission risks. Implementation of prudent antimicrobial use practices and targeted intervention strategies is essential to reducing MRSA prevalence and safeguarding public health. Continued research efforts are warranted to elucidate transmission dynamics and virulence potential, ultimately ensuring food safety and public health protection.

Alkyl deoxyglycoside-polymyxin combinations against critical priority carbapenem-resistant gram-negative bacteria.

The escalating antimicrobial resistance crisis urges the development of new antibacterial treatments with innovative mechanisms of action, particularly against the critical priority carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA) and Enterobacteriaceae (CRE). Membrane-disrupting dodecyl deoxyglycosides have been reported for their interesting phosphatidylethanolamine-associated bactericidal activity against Gram-positive strains; however, their inability to penetrate the Gram-negative outer membrane (OM) renders them useless against the most challenging pathogens. Aiming to repurpose alkyl deoxyglycosides against Gram-negative bacteria, this study investigates the antimicrobial effects of five reference compounds with different deoxygenation patterns or anomeric configurations in combination with polymyxins as adjuvants for enhanced OM permeability. The generation of the lead 4,6-dideoxy scaffold was optimized through a simultaneous dideoxygenation step and applied to the synthesis of a novel alkyl 4,6-dideoxy C-glycoside 5, herein reported for the first time. When combined with subtherapeutic colistin concentrations, most glycosides demonstrated potent antimicrobial activity against several multidrug-resistant clinical isolates of CRAB, CRE and CRPA exhibiting distinct carbapenem resistance mechanisms, together with acceptable cytotoxicity against human HEK-293T and Caco-2 cells. The novel 4,6-dideoxy C-glycoside 5 emerged as the most promising prototype structure for further development (MIC 3.1 µg/mL when combined with colistin 0.5 µg/mL against CRPA or 0.25 µg/mL against several CRE and CRAB strains), highlighting the potential of C-glycosylation for an improved bioactive profile. This study is the first to show the potential of IM-targeting carbohydrate-based compounds for the treatment of infections caused by MDR Gram-negative pathogens of clinical importance.

Unveiling Antibiotic Resistance, Clonal Diversity, and Biofilm Formation in E. coli Isolated from Healthy Swine in Portugal.

Escherichia coli, a commensal microorganism found in the gastrointestinal tract of human and animal hosts, plays a central role in agriculture and public health. Global demand for animal products has promoted increased pig farming, leading to growing concerns about the prevalence of antibiotic-resistant E. coli strains in swine populations. It should be noted that a significant portion of antibiotics deployed in swine management belong to the critically important antibiotics (CIA) class, which should be reserved for human therapeutic applications. This study aimed to characterize the prevalence of antibiotic resistance, genetic diversity, virulence characteristics, and biofilm formation of E. coli strains in healthy pigs from various farms across central Portugal. Our study revealed high levels of antibiotic resistance, with resistance to tetracycline, ampicillin, tobramycin, and trimethoprim-sulfamethoxazole. Multidrug resistance is widespread, with some strains resistant to seven different antibiotics. The ampC gene, responsible for broad-spectrum resistance to cephalosporins and ampicillin, was widespread, as were genes associated with resistance to sulfonamide and beta-lactam antibiotics. The presence of high-risk clones, such as ST10, ST101, and ST48, are a concern due to their increased virulence and multidrug resistance profiles. Regarding biofilm formation, it was observed that biofilm-forming capacity varied significantly across different compartments within pig farming environments. In conclusion, our study highlights the urgent need for surveillance and implementation of antibiotic management measures in the swine sector. These measures are essential to protect public health, ensure animal welfare, and support the swine industry in the face of the growing global demand for animal products.

Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria.

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Communicating and disseminating One Health: successes of the One Health European Joint Programme.

The application of a One Health approach recognizes that human health, animal health, plant health and ecosystem health are intrinsically connected. Tackling complex challenges associated with foodborne zoonoses, antimicrobial resistance, and emerging threats is imperative. Therefore, the One Health European Joint Programme was established within the European Union research programme Horizon 2020. The One Health European Joint Programme activities were based on the development and harmonization of a One Health science-based framework in the European Union (EU) and involved public health, animal health and food safety institutes from almost all EU Member States, the UK and Norway, thus strengthening the cooperation between public, medical and veterinary organizations in Europe. Activities including 24 joint research projects, 6 joint integrative projects and 17 PhD projects, and a multicountry simulation exercise facilitated harmonization of laboratory methods and surveillance, and improved tools for risk assessment. The provision of sustainable solutions is integral to a One Health approach. To ensure the legacy of the work of the One Health European Joint Programme, focus was on strategic communication and dissemination of the outputs and engagement of stakeholders at the national, European and international levels.

Clonal diversity of ESBL-producing Escherichia coli in pigs at slaughter level in Portugal.

We aimed to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in fecal samples of healthy pigs, and to evaluate their clonality and associated resistance. Forty-nine percent of pigs sampled (n=35/71) in a slaughterhouse in Portugal revealed ESBL-producing E. coli isolates. Most isolates produced CTX-M-1 enzyme (71.4%; n=25/35), followed by CTX-M-9 (11.4%; n=4/35), CTX-M-14 (5.7%; n=2/35), SHV-12 (5.7%; n=2/35), and CTX-M-32 (5.7%; n=2/35). Ninety-four percent of the isolates presented a phenotype of multi-resistance. Most isolates belonged to phylogroups B1 (42.8%; n=15/35) and A (40%; n=14/35). Multilocus sequence typing (MLST) analysis revealed nine sequence types (STs) under six clonal complexes (CCs) and nine singletons, including overrepresentation of CC10 and three new STs (ST2524, ST2525, ST2528). We observed the frequent presence of CTX-M-producing E. coli in pigs at slaughter level, most of them belonging to CC10, commonly recovered from clinical samples.