Sequence-based typing (SBT) has been the standard molecular typing method for understanding Legionella pneumophila genetic relationships. However, genome-scale typing approaches, namely core-genome (cg) or whole-genome (wg) multilocus sequence typing (MLST), provide higher discriminatory power. To advance these capabilities, the Legionella International Typing (LIT) workgroup was established to develop, evaluate, and disseminate a novel cgMLST schema with enhanced wgMLST resolution for L. pneumophila investigations. We created and populated the LIT cg/wgMLST schema with chewBBACA software using more than 9000 genome assemblies representative of the species diversity. We applied a multi-step refinement workflow, considering loci prevalence, diversity and presence/absence profile across the species tree, to select the final cg/wgMLST loci, and compared the performance of the LIT cgMLST schema with the previously used 1521-loci schema and assessed its congruence with SBT. The LIT schema includes 2009 loci present in 98% of the dataset, forming the static cgMLST schema for routine genomic surveillance, plus 2698 accessory loci for an in-depth wgMLST analysis of clusters of interest. The LIT cgMLST schema maintains moderate agreement with SBT and presents high clustering congruence with the 1521-loci schema, while providing increased resolution. Analysis of epidemiologically related isolates using the LIT cgMLST schema for initial cluster delineation, followed by cluster-specific dynamic wgMLST analysis extending the cgMLST with accessory loci shared among isolates within each cluster, demonstrated increased confidence for outbreak investigation and source identification. The LIT schema is expected to contribute to harmonizing genomic surveillance of Legionnaires’ disease at both local and global levels. The schema and associated resources for local implementation are available on Zenodo ( https://doi.org/10.5281/zenodo.17871973 ).

Following an extended period of declining prevalence of the epidemic Clostridioides difficile ribotype 027 (RT027) in Portugal, a genetically distinct, multidrug-resistant (MDR) RT027 strain with reduced susceptibility to vancomycin has emerged, causing a 15-month outbreak. This investigation provides epidemiological and genomic evidence for renewed circulation and evolutionary adaptation of this high-risk lineage. A comprehensive outbreak investigation was conducted in a tertiary-care hospital in northern Portugal between 2023 and 2025. Epidemiological and clinical data, antimicrobial exposures, and infection-control measures were analysed. Whole-genome sequencing (WGS) was performed to characterize the outbreak clone. Sixty-six RT027 C. difficile infection (CDI) cases were confirmed, with incidence peaking at 5.46 cases per 10,000 patient-bed days in April 2024. WGS revealed an unusual accumulation of AMR determinants conferring a broad MDR phenotype. Notably, all isolates harboured the VanR T115A substitution, a rare mutation previously linked to reduced vancomycin susceptibility, raising concerns regarding evolving antimicrobial tolerance within RT027. The close relatedness to 2016-2018 USA isolates suggests a recent emergence of this clone. Transmission was facilitated by structural constraints, limited isolation capacity and shared sanitary facilities. This prolonged outbreak documents the re-emergence and genomic evolution of a hypervirulent RT027 lineage, characterized by a concerning expansion of antimicrobial resistance and decreased vancomycin susceptibility and a high recurrence rate (25%). These findings highlight the ongoing adaptive potential of C. difficile under antimicrobial pressure and underscore the need for strengthened surveillance, genomic monitoring, and infection-prevention strategies to mitigate re-establishment of epidemic RT027 strains in Europe and beyond.

Background Orf virus (ORFV) is a neglected zoonotic virus. In Portugal, the few human cases have historically been identified through clinical suspicion rather than molecular confirmation, resulting in significant gaps in the regional molecular epidemiology and genomic diversity. Methods ORFV was detected via real-time PCR targeting the B2L gene in a biopsy taken from a 23-year-old woman presenting with a painful nodule on her finger following a goat bite. A near-complete genome was obtained directly from the clinical biopsy using the Twist Comprehensive Viral Research Panel (hybrid capture) and Illumina MiSeq sequencing. Results Molecular testing resolved a histopathological misdiagnosis of chondroma, confirming ORFV infection. Sequencing yielded a 129.5 kb genome (ORFV/PT001/INSA2025) with a mean coverage of 1000x. Of the 130 OV-SA-00 reference strain genes, 117 were detected, with 110 showing > 95% similarity. Phylogenetic analysis placed the isolate within a goat-associated cluster, which is distinct from the sheep-associated IHUMI-1and B029 genomes, the only ones of human origin reported to date. Conclusions This report presents the first laboratory-confirmed human ORFV case in Portugal and the first near-complete goat-associated human-derived ORFV genome worldwide. The results demonstrate that hybrid capture enables high-depth genomic characterization directly from clinical samples, thereby bridging the diagnostic-genomic gap and emphasizing the necessity of a One Health approach to zoonotic poxviruses.

West Nile virus (WNV) is an emerging mosquito-borne pathogen in Europe, responsible for recurrent outbreaks affecting humans, horses, and wildlife. Although WNV circulation has been documented in Portugal, genomic data from mosquito populations remain scarce, limiting our understanding of viral diversity and transmission dynamics in the western Iberian Peninsula. Here, we investigated the presence and genetic diversity of WNV in mosquitoes collected from wetland ecosystems in southern Portugal in 2025. Mosquitoes were collected and grouped into pools by collection date, location, and species. Pools were screened for WNV RNA by RT-qPCR, and positive samples were subjected to whole-genome sequencing using a hybrid-capture target enrichment method. Phylogenetic analyses were conducted to determine the evolutionary relationships of the detected viruses with previously reported WNV strains circulating worldwide. WNV RNA was detected in several mosquito pools, predominantly in Culex univittatus , from which seven near-complete viral genomes were recovered. All sequences belonged to WNV lineage 1A but segregated into two distinct genetic clusters. One cluster grouped with strains previously reported from the Iberian Peninsula, whereas the second formed a divergent variant previously undetected. These findings indicate the co-circulation of genetically distinct WNV strains in Portuguese wetlands. Our results provide the phylogenetic characterization of WNV from mosquito populations in Portugal and reveal previously undocumented viral diversity in the region. This study highlights the importance of integrating entomological surveillance with genomic approaches to better understand the mechanisms underlying WNV introduction, maintenance, and spread in southern Europe.

In 2024, an outbreak of Salmonella typhimurium affected two regions in Portugal. To identify the vehicle, we conducted a case-case study using a 'same disease, different time period' design. We compared S. typhimurium cases linked by whole-genome sequencing (WGS) (cluster cases) with salmonellosis cases notified in 2023 (historical cases) and calculated odds ratios (OR) for food exposures in surveillance data using logistic regression. We performed WGS on 58 isolates from the outbreak period (11/03/2024-2118/06/2024), and all belonged to a single cgMLST cluster (HierCC HC5_410,410). Compared with the 552 historical cases, cluster cases more frequently reported fresh cheese consumption (OR 18; 95% CI: 8.5-38). We visited the implicated cheese production site, identified food safety non-conformities, and enforced hygiene measures. Environmental and product specimens collected at the visit tested negative for Salmonella spp. Taken together, the most plausible vehicle in this outbreak was fresh cheese. The case-case design enabled a rapid, low-cost analysis to support targeted investigation using surveillance data. Using WGS cluster cases as the case definition, rather than all S. typhimurium cases during the outbreak period, yielded a higher OR in the case-case study, increasing confidence in the findings. We recommend this combined approach as part of the toolkit for foodborne outbreak investigations in Portugal in similar contexts.

Chlamydia trachomatis causes common sexually transmitted infections. The use of whole-genome sequencing for its classification and surveillance is limited due to technical challenges and the lack of standardized typing frameworks. In particular, core-genome multilocus sequence typing (cgMLST), a scalable and portable approach widely applied to other bacterial pathogens, remains little explored for C. trachomatis. Here, we compiled and curated a diverse C. trachomatis genome dataset (1230 samples from 26 countries), including publicly available and newly generated assemblies, to develop a cgMLST system (n = 846 loci) optimized for standardized local deployment. Benchmarking assays demonstrated its high typeability and clustering congruence with core-SNP approaches and the existing online cgMLST schema (PubMLST). Our cgMLST framework recapitulated the four major evolutionary lineages of C. trachomatis and enabled in-depth exploration of global phylogenomic diversity and evolution, including intra-lineage diversity for detection and tracking of contemporary strains. By enabling a direct link between loci/alleles and specific phylogenomic/phenotypic traits, this cgMLST approach also elucidated the C. trachomatis genome-wide recombination landscape and identified lineage-specific alleles (and disrupted loci) with potential diagnostic and/or functional relevance. Our cgMLST schema is publicly available for local implementation accompanied by a hierarchical cgMLST-based nomenclature, promoting harmonized genogroup tracking across laboratories and countries.

Tuberculosis (TB) remains a global health challenge, exacerbated by the emergence of drug-resistant Mycobacterium tuberculosis strains. Most methods for drug susceptibility testing (DST) are culture-dependent and time consuming, possibly delaying optimal TB-treatment. This study aimed to develop an extensive targeted next-generation sequencing (tNGS) approach for rapid genotypic DST directly from clinical samples. We designed a tNGS panel comprising 30 amplicons targeting 19 genomic regions associated with resistance to 20 antibiotics. This method was applied to 71 smear-positive (0-3+) pulmonary TB clinical samples collected at the Portuguese National Reference Laboratory. DNA was extracted and amplified using multiplex PCRs, followed by sequencing on Oxford Nanopore Technologies MinION platform. Sequencing data were using TB-Profiler and the tNGS results compared to phenotypic DST and whole genome sequencing (WGS) data from corresponding isolates. The tNGS demonstrated high concordance with both phenotypic and WGS-based DST across different sample types and smear positivity levels. For first-line drugs, tNGS showed 88% categorical agreement (CA) with pDST, increasing to 97% when excluding undetermined results. Compared to WGS across all analysed antibiotics, tNGS achieved 92% CA, increasing to >99% when excluding undetermined results. Validation of the tNGS panel showed 90% (1,895/2,076) of amplicons reaching >10x coverage at all analysed positions and 43 (61%) samples with all complete amplicons above this threshold. Non-specific amplification of contaminant bacterial DNA was minimal, with most mapped off-target reads being of human origin. This method enables comprehensive resistance prediction directly from clinical samples and signifies an important development in TB diagnostics and resistance monitoring.

Environmental pollution is a growing concern. Here, we assessed the occurrence of two groups of persistent organic pollutants (POPs—polycyclic aromatic hydrocarbons (PAHs) and microplastics (MPs)) and bacterial populations in the topsoil of three tourist spots located at the Alqueva’s edges during 1 year, once per season. Soil chemical analysis revealed low content of total organic carbon, pH close to neutrality, and nitrogen and phosphorus levels consistent with acquisition of these nutrients only by atmospheric deposition. PAH’s concentrations were in the range of ng/kg, being significantly below the “reference values” for contaminated soils. Nevertheless, potentially carcinogenic PAHs, detected at all locations, raise ecotoxicological concerns. Polyamide, polyester, polystyrene, and styrene acrylonitrile resin MPs were found. Six bacterial phyla constitute the core microbiome in the three locations and include genera of bacteria reported as plastic degraders, such as Bacillus, Exiguobacterium, Paenibacillus, and Pseudomonas. The presence of POPs, even at low levels, in the soil at the edges of a water reservoir should be monitored. The identification of bacteria reported as plastic degraders in the soil, and previously in the water, is promising, and their ability to spontaneously ensure the detoxification of the ecosystem should be further investigated.

Campylobacter jejuni is a leading cause of bacterial gastroenteritis, and fluoroquinolone-resistant strains represent a major public health concern. Still, C. jejuni can also cause invasive disease, particularly in immunocompromised individuals, highlighting the need to elucidate the adaptive mechanisms behind bloodstream invasion and persistence. This study aimed to characterize the within-patient genomic evolution of an invasive C. jejuni strain. Whole-genome sequencing was performed on same-patient isolates from stool (n = 1) and blood (n = 2), followed by in-depth genomic comparisons and antimicrobial susceptibility testing. Here we show that rapid within-patient microevolution is driven by amino acid substitutions and small inactivating indels. Most mutated loci have predicted or reported functions related to motility (including flagella- and energy taxis/chemotaxis-associated proteins), adherence, cell shape/envelope organization and host interactions (e.g., immune evasion and resistance to blood environment). All isolates are resistant to ciprofloxacin (MIC = 16 mg/L) due to the canonical GyrA Thr86Ile substitution. After about one month of infection (including a 21-day ciprofloxacin treatment), ciprofloxacin MIC increased to 128 mg/L and a newly acquired resistance to moxifloxacin (MIC > 32 mg/L) was observed. This expanded resistance profile correlates with the emergence of the GyrA Asp90Gly substitution (previously unreported in C. jejuni) and a 1-bp deletion in the cmeABC promoter region (previously demonstrated to increase efflux pump expression). Dynamic phase variation of several loci was also observed during bloodstream persistence, including ON-phase switching of the cell invasion protein A (CipA). These genomic findings provide insight into within-host population shifts and the emergence of antibiotic-resistant clones, contributing to better understanding the dynamics associated with C. jejuni bloodstream invasion and persistence. Campylobacter jejuni (C. jejuni) is a species of bacteria that commonly causes foodborne illness. In rare cases, especially in people with weakened immune systems, it can spread from the gut into the bloodstream causing severe infection. We wanted to understand how this bacterium changes inside the body during this type of infection and how it becomes more resistant to antibiotics. We compared the complete DNA of bacteria collected from the stool and blood of the same patient over about one month, together with laboratory tests of antibiotic susceptibility. We found that the bacterium rapidly accumulated genetic changes potentially linked to survival in the bloodstream and developed increased resistance to important antibiotics during treatment. These findings improve our understanding of how C. jejuni adapts during invasive infection and may help improve diagnosis, treatment, and future surveillance of antibiotic-resistant strains. Borges et al. investigate the within-host evolution of an invasive Campylobacter jejuni strain using stool and blood isolates collected from a patient over one month. Observed genetic changes may be associated with bloodstream persistence and the emergence of increased antimicrobial resistance, suggesting their possible role in adaptation and invasive disease.

Since 2010, dengue virus (DENV) has caused sporadic outbreaks across Europe, namely in Croatia, Spain, France, Italy and the Portuguese island of Madeira. Aedes aegypti mosquito is established in the Autonomous Region of Madeira, and along the eastern Black Sea coast of Cyprus. In Madeira Island, an outbreak of DENV serotype 1 occurred between 2012 and 2013, resulting in 1080 confirmed cases. Despite ongoing entomological surveillance, no further local transmission was detected in the following decade. In January 2025, following two suspected dengue cases on Madeira Island, increased entomological surveillance efforts were implemented to confirm a local event transmission of DENV. A network of mosquito traps was complemented by targeted surveillance using 17 BG-PRO traps positioned in the vicinity of suspected human cases. Daily collections of adult A. aegypti, collected from 10 January to 31 March 2025, were screened by reverse transcription polymerase chain reaction (RT-PCR) for Aedes-borne viruses in the reference laboratory. Viral sequencing was performed using target enrichment and bioinformatics with INSaFLU-TELEVIR. The climate-driven suitability for dengue transmission by A. aegypti was also investigated. Serological and molecular tests were conducted on samples from suspected human cases. Out of 80 analysed A. aegypti pools (N = 393 mosquitoes), 1 pool, with 9 mosquitoes collected near the home of suspected human cases, tested positive for DENV. The dengue whole genome sequence from this sample was determined and classified as DENV-2 lineage 2II_F.1.1.3. The same virus was retrospectively confirmed in one of the clinical cases. Analysis of mosquito abundance and climate data confirmed the occurrence of this local transmission event during a period of low mosquito abundance and low climatic suitability. Here, we report an in-depth analysis of a local dengue transmission event that occurred in Funchal, the capital of Madeira Island, in January 2025, with whole-genome evidence of DENV-2II_F.1.1.3 in field-caught A. aegypti mosquitoes. Retrospective analysis confirmed the presence of the same virus in one of the two clinical cases, establishing a direct link between human and mosquito infections, and highlighting the risk of off-season arboviral introductions.

Enterococcus spp. are opportunistic bacteria capable of acquiring antimicrobial resistance and virulence traits, facilitating their adaptation to multiple ecological niches. Sternal bursitis, a condition affecting poultry welfare and carcass quality, remains poorly characterized from a microbiological perspective. This study provides the first genomic and phenotypic characterization of Enterococcus isolates from bursitis lesions in broilers, aiming to assess their antimicrobial resistance profiles, virulence determinants, and genetic diversity within a One Health framework. A total of 44 Enterococcus isolates were recovered from 48 sternal bursitis lesions, all identified as E. faecalis. Resistance was common for tetracycline (70.5%) and erythromycin (27.3%), while all isolates remained susceptible to critically important antimicrobials, including vancomycin and linezolid. Whole-genome sequencing revealed a genetically diverse population, comprising multiple sequence types, plasmid replicons, and virulence gene profiles, including determinants for adhesion, biofilm formation, capsule synthesis, and extracellular proteases. Ionophore resistance genes (narA, narB) were also detected in several lineages. The coexistence of antimicrobial resistance and virulence determinants, often linked to mobile genetic elements, highlights the potential of lesion-associated with E. faecalis to act as reservoirs of relevant genes with zoonotic implications. Overall, this work highlights the diverse and ecological role of Enterococcus in extraintestinal poultry infections, reinforcing the need for continued genomic surveillance to promote animal health, food safety, and antimicrobial stewardship.

Genome sequencing has become a crucial tool in the management and understanding of infectious disease outbreaks. By decoding the entire genetic material of pathogens, this technology allows scientists to track the spread and evolution of infectious agents with unprecedented precision. During an outbreak, one of the key applications of genome sequencing is in tracing the transmission pathways of the disease. By comparing the genetic sequences of pathogens from different patients and sources, epidemiologists can determine the source of the outbreak and how the disease is spreading through populations. In addition, genome sequencing has the potential to identify mutations that may affect transmissibility, virulence, or resistance to treatment, providing critical insights for public health responses, for instance enabling targeted interventions, such as specific treatments or vaccines. During the last years, this was particularly evident for multiple internationally occurring severe outbreaks caused by either bacteria or virus, with special emphasis during the COVID-19 pandemic, where genome sequencing played a vital role in monitoring the emergence of new variants and informing vaccine strategies. As sequencing technologies continue to advance and associated costs decline, their integration into public health strategies will be essential for more effective control and prevention of infectious disease outbreaks in the future.

Multidrug resistance in Pseudomonas aeruginosa, particularly resistance to carbapenem, represents a major challenge for public health. This study investigated resistance mechanisms in three P. aeruginosa isolates: HU63 (blaGES-6 carbapenemase-positive), HU141 (carbapenem-resistant without carbapenemase), and PAO1 (control). Genomic analysis revealed distinct sequence types (ST235 for HU63, ST253 for HU141) and chromosomal integration of resistance genes. HU63 harbored diverse resistance mechanisms, including β-lactamases (blaGES-6, blaPDC-35, blaOXA-488) and efflux pumps. Minimum inhibitory concentration assays demonstrated HU63's resistance to all β-lactams tested (meropenem, imipenem-cilastatin, ceftazidime, piperacillin-tazobactam), while HU141 remained susceptible except to cefoxitin and cloxacillin. Time-kill assays revealed tolerance phenotypes, with HU63 showing regrowth after 8-24 h despite initial reductions in bacterial density. Gene expression varied significantlydepending on the antibiotic and the isolate. The HU63 isolate (GES-6 positive) stands out for its marked induction of blaGES-6 in all the antibiotics tested, contributing to its resistance to carbapenems and broad-spectrum cephalosporins. These expression profiles corroborate the classic molecular mechanisms of resistance: regulation of entry pores (oprD), activation of efflux pumps (mexA) and production of β-lactamases (blaGES-6, ampC) adapted to each situation. These findings underscore the multifactorial nature of resistance in Carbapenem-resistant Pseudomonas aeruginosa (CRPA), combining enzymatic inactivation, efflux, and genetic adaptability. The study emphasizes the urgent need for genomic surveillance to track high-risk clones and develop therapies targeting tolerance mechanisms alongside traditional resistance.

In 2024, unprecedented outbreaks of dengue and Oropouche were reported in the Americas. We describe a documented co-infection with dengue and Oropouche viruses in a 35-year-old traveller from Cuba detected in Portugal. RT-PCR and next-generation sequencing confirmed both viruses. Our findings highlight the need for multiplex arboviral diagnostics in travellers from regions with concurrent outbreaks.

OBJECTIVES:Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990-2021) in Portugal. METHODS:The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. RESULTS:ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. CONCLUSIONS:This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.

Objectives To survey genetic markers of potential antimicrobial resistance (AMR) to macrolides and fluoroquinolones among Chlamydia trachomatis-positive samples from the collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections (STIs), and explore a multiplex PCR approach coupled with NGS to provide complementary information regarding a strain's genomic backbone.Methods A total of 502 C. trachomatis-positive samples, mostly anorectal exudates, were subjected to PCR and sequencing of five targets, including loci potentially driving AMR (23S rRNA, gyrA and parC) and loci potentially informative about a strain's genomic backbone with emphasis on differentiation of lymphogranuloma venereum (LGV)/non-LGV and L2/L2b (a 9 bp insertion in pmpH, a 74 bp insertion upstream from CT105 and the polymorphic CT442).Results No samples evidenced 23S rRNA mutations recognizably linked to macrolide resistance. Three samples harboured the Ser83Ile mutation in GyrA putatively driving fluoroquinolone resistance: two recombinant L2-L2b/D-Da (0.4%) and one L2 (0.2%). The screened regions in pmpH, upstream CT105 and CT442 were fully concordant with LGV/non-LGV differentiation. As expected, the pmpH L2b-specific genetic trait locus was detected in all L2b and recombinant L2-L2b/D-Da ompA genotypes, but also in 96.0% of L2 specimens, which also likely possess an L2b genomic backbone. The insertion upstream from CT105 exhibited full LGV specificity, constituting a promising target for the development of rapid LGV diagnostic assays.Conclusions This study contributes to enhancing the knowledge of C. trachomatis molecular epidemiology, suggesting that the known genetic determinants of AMR are not disseminated in clinical C. trachomatis strains, and presents an exploratory approach that can be suitable for LGV/non-LGV and L2/L2b genomic background differentiation.

BACKGROUND:Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by Chlamydia trachomatis ompA-genotypes L1-L3, with increasing numbers of detected cases across Europe. Here, we analysed diversity and temporal distribution of the LGV ompA-subvariants detected in Portugal between 2007 and 2023, in order to better understand the dissemination and diversification landscape of LGV strains. METHODS:The collection of the Portuguese National Reference Laboratory includes 1188 LGV ompA-genotyped samples between 2007 and 2023. In-depth analysis of the diversity of LGV ompA-subvariants circulating in Portugal across the years was performed, identifying newly described subvariants and integrating this data in a comprehensive compilation with all representative LGV ompA-subvariants described globally. RESULTS:L2 ompA-variant (L2/434/Bu) was consistently the most frequently detected in our collection, with annual proportions ranging from 34.0% to 82.9%, between 2016 and 2023. L2bV5 was the second most frequent followed by L2b, ranging from 5.0% to 27.9% and 2.6% to 23.7% across the years, respectively, from 2017 to 2023. We highlighted the emergence and considerable increase in circulation of L1-like ompA-subvariants in recent years, representing 13.7% of LGV sequences in 2023. We also identified 13 novel LGV ompA-subvariants that had not been described before, differing by up to three mutations from the respective genotype reference sequences. CONCLUSIONS:This study contributes to the worldwide picture of the LGV molecular epidemiology, highlighting the importance of long-term molecular surveillance to monitor the circulation and geographical spread of LGV and to timely identify and track new strains, such as the recently emerging L1-like ompA-subvariants.

Sternal bursitis is an underexplored lesion in poultry, often overlooked in microbiological diagnostics. In this study, we characterized 36 Escherichia coli isolates recovered from sternal bursitis in broiler chickens, combining phenotypic antimicrobial susceptibility testing, PCR-based screening, and whole genome sequencing (WGS). The genetic analysis revealed a diverse population spanning 15 sequence types, including ST155, ST201, and ST58. Resistance to tetracycline and ciprofloxacin was common, and several isolates carried genes encoding β-lactamases, including blaTEM-1B. Chromosomal mutations associated with quinolone and fosfomycin resistance (e.g., gyrA p.S83L, glpT_E448K) were also identified. WGS revealed a high number of virulence-associated genes per isolate (58–96), notably those linked to adhesion (fim, ecp clusters), secretion systems (T6SS), and iron acquisition (ent, fep, fes), suggesting strong pathogenic potential. Many isolates harbored virulence markers typical of ExPEC/APEC, such as iss, ompT, and traT, even in the absence of multidrug resistance. Our findings suggest that E. coli from sternal bursitis may act as reservoirs of resistance and virulence traits relevant to animal and public health. This highlights the need for including such lesions in genomic surveillance programs and reinforces the importance of integrated One Health approaches.

Different laboratories employ different Whole-Genome Sequencing (WGS) pipelines for Food and Waterborne disease (FWD) surveillance, casting doubt on the comparability of their results and hindering optimal communication at intersectoral and international levels. Through a collaborative effort involving eleven European institutes spanning the food, animal, and human health sectors, we aimed to assess the inter-pipeline clustering congruence across all resolution levels and perform an in-depth comparative analysis of cluster composition at outbreak level for four important foodborne pathogens: Listeria monocytogenes, Salmonella enterica, Escherichia coli, and Campylobacter jejuni. We found a general concordance between allele-based pipelines for all species, except for C. jejuni, where the different resolution power of allele-based schemas led to marked discrepancies. Still, we identified non-negligible differences in outbreak detection and demonstrated how a threshold flexibilization favors the detection of similar outbreak signals by different laboratories. These results, together with the observation that different traditional typing groups (e.g., serotypes) exhibit a remarkably different genetic diversity, represent valuable information for future outbreak case-definitions and WGS-based nomenclature design. This study reinforces the need, while demonstrating the feasibility, of conducting continuous pipeline comparability assessments, and opens good perspectives for a smoother international and intersectoral cooperation towards an efficient One Health FWD surveillance.

As mpox continues to spread globally, proactive monitoring and preparedness are crucial to minimize impact and enhance response strategies. Using a mathematical model combining a negative binomial distribution with Richards' logistic curve, we reconstructed the hidden phase of mpox transmission in Portugal, offering insights into the timing and dynamics of the initial outbreak. The analysis of 950 PCR-positive and 986 negative cases suggested that symptom onset occurred between March 24 and April 2, 2022, with March 27 identified as the most probable date. This study delineates the likely period of silent circulation of MPXV in Portugal, providing a clearer understanding of early outbreak dynamics and surveillance performance. Possible imperfections in early diagnostic testing and limited awareness of mpox may have contributed to delayed recognition of the outbreak. By demonstrating how retrospective mathematical modelling can estimate undetected transmission periods, our findings highlight the value of such approaches in epidemic reconstruction and underscore the importance of strengthening early surveillance systems to detect undiagnosed transmission of mpox in non-endemic countries.