Search

In 2023, a second wave of the global mpox epidemic, which is mainly affecting men who have sex with men (MSM), was observed in some countries. Herein, we benefited from a large viral sequence sampling (76/121; 63%) and vast epidemiological data to characterise the re-emergence and circulation of the Monkeypox virus (MPXV) in Portugal during 2023. We also modelled transmission and forecasted public health scenarios through a compartmental susceptible-exposed-infectious-recovered (SEIR) model. Our results suggest that the 2023 mpox wave in Portugal resulted from limited introduction(s) of MPXV belonging to C.1.1 sublineage, hypothetically from Asia, followed by sustained viral transmission and potential exportation to other countries. We estimated that the contribution of the MSM high sexual activity group to mpox transmission was 120 (95% CrI: 30–3553) times higher than that of the low sexual activity group. However, among the high sexual activity group, vaccinated individuals likely contributed approximately eight times less [0.123 (95% CrI: 0.068–0.208)] than the unvaccinated ones. Vaccination was also linked to potential reduced disease severity, with a Mpox Severity Score of 6.0 in the vaccinated group compared to 7.0 in unvaccinated individuals. Scenario analysis indicated that transmission is highly sensitive to sexual behaviour, projecting that a slight increase in the MSM sub-population with high sexual activity can trigger new mpox waves. This study strongly supports that continued vaccination, targeted awareness among risk groups and routine genomic epidemiology is needed to anticipate and respond to novel MPXV threats (e.g. global dissemination of clade I viruses).

IntroductionFrancisella tularensis is a highly infectious bacterium that causes the zoonotic disease tularemia. The development of genotyping methods, especially those based on whole-genome sequencing (WGS), has recently increased the knowledge on the epidemiology of this disease. However, due to the difficulties associated with the growth and isolation of this fastidious pathogen in culture, the availability of strains and subsequently WGS data is still limited.MethodsTo surpass these constraints, we aimed to implement a culture-free approach to capture and sequence F. tularensis genomes directly from complex samples. Biological samples obtained from 50 common voles and 13 Iberian hares collected in Spain were confirmed as positive for F. tularensis subsp. holarctica and subjected to a WGS target capture and enrichment protocol, using RNA oligonucleotide baits designed to cover F. tularensis genomic diversity.ResultsWe obtained full genome sequences of F. tularensis from 13 animals (20.6%), two of which had mixed infections with distinct genotypes, and achieved a higher success rate when compared with culture-dependent WGS (only successful for two animals). The new genomes belonged to different clades commonly identified in Europe (B.49, B.51 and B.262) and subclades. Despite being phylogenetically closely related to other genomes from Spain, the detected clusters were often found in other countries. A comprehensive phylogenetic analysis, integrating 599 F. tularensis subsp. holarctica genomes, showed that most (sub)clades are found in both humans and animals and that closely related strains are found in different, and often geographically distant, countries.DiscussionOverall, we show that the implemented culture-free WGS methodology yields timely, complete and high-quality genomic data of F. tularensis, being a highly valuable approach to promote and potentiate the genomic surveillance of F. tularensis and ultimately increase the knowledge on the genomics, ecology and epidemiology of this highly infectious pathogen.

We estimated comparative primary and booster vaccine effectiveness (VE) of SARS-CoV-2 Omicron BA.5 and BA.2 lineages against infection and disease progression. During April–June 2022, we implemented a case–case and cohort study and classified lineages using whole-genome sequencing or spike gene target failure. For the case–case study, we estimated the adjusted odds ratios (aORs) of vaccination using a logistic regression. For the cohort study, we estimated VE against disease progression using a penalized logistic regression. We observed no reduced VE for primary (aOR 1.07 [95% CI 0.93–1.23]) or booster (aOR 0.96 [95% CI 0.84–1.09]) vaccination against BA.5 infection. Among BA.5 case-patients, booster VE against progression to hospitalization was lower than that among BA.2 case-patients (VE 77% [95% CI 49%–90%] vs. VE 93% [95% CI 86%–97%]). Although booster vaccination is less effective against BA.5 than against BA.2, it offers substantial protection against progression from BA.5 infection to severe disease.

Methylation microarray and bisulphite sequencing are often used to study 5’-methylcytosine (5’-mC) modification of CpG dinucleotides in the human genome. Although both technologies produce trustworthy results, the evaluation of the methylation status of CpG sites suffers from the potential side effects of DNA modification by bisulphite and/or the ambiguity of mapping short reads in repetitive and highly homologous genomic regions, respectively. Nanopore sequencing is an attractive alternative for the study of 5’-mC since it allows sequencing of native DNA molecules, whereas the long reads produced by this technology help to increase the resolution of those genomic regions. In this work, we show that nanopore sequencing with 10X coverage depth, using DNA from a human cell line, produces 5’-mC methylation frequencies consistent with those obtained by 450k microarray, digital restriction enzyme analysis of methylation, and reduced representation bisulphite sequencing. High correlation between methylation frequencies obtained by nanopore sequencing and the other methodologies was also noticeable in either low or high GC content regions, including CpG islands and transcription start sites. We also showed that a minimum of five reads per CpG yields strong correlations (>0.89) in replicate nanopore sequencing runs and an almost uniform linearity of the methylation frequency variation between zero and one. Furthermore, nanopore sequencing was able to correctly display methylation frequency patterns based on genomic annotations of CpG regions. These results demonstrate that nanopore sequencing is a fast, robust, and reliable approach to the study of 5’-mC in the human genome with low coverage depth.

The 2022 multicountry mpox outbreak concurrent with the ongoing Coronavirus Disease 2019 (COVID-19) pandemic further highlighted the need for genomic surveillance and rapid pathogen whole-genome sequencing. While metagenomic sequencing approaches have been used to sequence many of the early mpox infections, these methods are resource intensive and require samples with high viral DNA concentrations. Given the atypical clinical presentation of cases associated with the outbreak and uncertainty regarding viral load across both the course of infection and anatomical body sites, there was an urgent need for a more sensitive and broadly applicable sequencing approach. Highly multiplexed amplicon-based sequencing (PrimalSeq) was initially developed for sequencing of Zika virus, and later adapted as the main sequencing approach for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Here, we used PrimalScheme to develop a primer scheme for human monkeypox virus that can be used with many sequencing and bioinformatics pipelines implemented in public health laboratories during the COVID-19 pandemic. We sequenced clinical specimens that tested presumptively positive for human monkeypox virus with amplicon-based and metagenomic sequencing approaches. We found notably higher genome coverage across the virus genome, with minimal amplicon drop-outs, in using the amplicon-based sequencing approach, particularly in higher PCR cycle threshold (Ct) (lower DNA titer) samples. Further testing demonstrated that Ct value correlated with the number of sequencing reads and influenced the percent genome coverage. To maximize genome coverage when resources are limited, we recommend selecting samples with a PCR Ct below 31 Ct and generating 1 million sequencing reads per sample. To support national and international public health genomic surveillance efforts, we sent out primer pool aliquots to 10 laboratories across the United States, United Kingdom, Brazil, and Portugal. These public health laboratories successfully implemented the human monkeypox virus primer scheme in various amplicon sequencing workflows and with different sample types across a range of Ct values. Thus, we show that amplicon-based sequencing can provide a rapidly deployable, cost-effective, and flexible approach to pathogen whole-genome sequencing in response to newly emerging pathogens. Importantly, through the implementation of our primer scheme into existing SARS-CoV-2 workflows and across a range of sample types and sequencing platforms, we further demonstrate the potential of this approach for rapid outbreak response.

Este artigo faz uma breve análise, em primeiro lugar, da actual conjuntura internacional, designadamente no que diz respeito aos EUA, à República Popular da China e à Federação Russa. Em segundo lugar, o papel que Portugal desempenha no âmbito das Relações Internacionais, não esquecendo, como é evidente, a relevância da Região Autónoma dos Açores e em particular a base das Lajes no âmbito da Política Externa portuguesa.

Borges et al. study the dynamics of SARS-CoV-2 in Portugal during 2020. The authors use phylodynamic modelling to infer patterns of SARS-CoV-2 introduction into Portugal and its subsequent spread, providing a comprehensive genomic epidemiology study of the early SARS-CoV-2 epidemic in Portugal.

Introduction: The prevalence of malnutrition in hospitalized children is high and is associated with negative health impact. The aim of this study was to characterize the nutritional status of hospitalized children as well as comparing nutritional risk stratification using the STRONGkids® tool and anthropometric assessment. Material and Methods: A prospective study was conducted between March and June 2019 in a paediatric unit of a district hospital. Children with ages between one month and 17 years were included. Nutritional screening (STRONGkids®) was performed, and demographic and anthropometric variables were assessed by trained doctors and nurses (z-scores for height-for-age, weight-for-age, weight-for-height and body mass index were compared to the World Health Organization reference values) and related to the underlying condition (cause of hospitalization; hospital stay; the presence of chronic disease). Results: A total of 209 children were evaluated, 188 of whom were included. Median age was 4.6 years and median hospital length of stay was four days. Fifty-four per cent were classified with “moderate risk” and 2% with “high risk” of developing malnutrition; 25% were effectively malnourished. Of the 105 children for which it was possible to calculate the z-scores, 6% presented acute malnutrition and nearly 14% presented chronic malnutrition. The STRONGkids® score correlated positively with nutritional status on admission, disease type on admission, and presence of previous underlying disease (p < 0.05). Conclusion: STRONGkids® is a simple, quick nutritional screening tool for hospitalized children that is related to nutritional status on admission. Given that a considerably high percentage of children were identified as being at risk for malnutrition, it is essential to identify this early and provide nutritional intervention during hospitalization.

DNA methylation is an epigenetic mechanism that involves the addition of a methyl group to a cytosine residue in CpG dinucleotides, which are particularly abundant in gene promoter regions. Several studies have highlighted the role that modifications of DNA methylation may have on the adverse health effects caused by exposure to environmental toxicants. One group of xenobiotics that is increasingly present in our daily lives are nanomaterials, whose unique physicochemical properties make them interesting for a large number of industrial and biomedical applications. Their widespread use has raised concerns about human exposure, and several toxicological studies have been performed, although the studies focusing on nanomaterials’ effect on DNA methylation are still limited. The aim of this review is to investigate the possible impact of nanomaterials on DNA methylation. From the 70 studies found eligible for data analysis, the majority were in vitro, with about half using cell models related to the lungs. Among the in vivo studies, several animal models were used, but most were mice models. Only two studies were performed on human exposed populations. Global DNA methylation analyses was the most frequently applied approach. Although no trend towards hypo- or hyper-methylation could be observed, the importance of this epigenetic mechanism in the molecular response to nanomaterials is evident. Furthermore, methylation analysis of target genes and, particularly, the application of comprehensive DNA methylation analysis techniques, such as genome-wide sequencing, allowed identifying differentially methylated genes after nanomaterial exposure and affected molecular pathways, contributing to the understanding of their possible adverse health effects.

Re-positivity of SARS-CoV-2 tests is widely reported, raising discussion about guidance for patient discharge and ending isolation. The unsuccessful recovery of replication-competent virus and/or absence of secondary cases has suggested that re-positive patients are not contagious. This study reports SARS-CoV-2 re-positivity in a healthcare professional 16 days after three negative tests, with viral genome sequencing supporting contagiousness leading to secondary cases.

ABSTRACTGenomic surveillance of SARS-CoV-2 was rapidly implemented in Portugal by the National Institute of Health in collaboration with a nationwide consortium of >50 hospitals/laboratories. Here, we track the geotemporal spread of a SARS-CoV-2 variant with a mutation (D839Y) in a potential host-interacting region involving the Spike fusion peptide, which is a target motif of anti-viral drugs that plays a key role in SARS-CoV-2 infectivity. The Spike Y839 variant was most likely imported from Italy in mid-late February and massively disseminated in Portugal during the early epidemic, becoming prevalent in the Northern and Central regions of Portugal where it represented 22% and 59% of the sampled genomes, respectively, by 30 April. Based on our high sequencing sampling during the early epidemics [15.5% (1275/8251) and 6.0% (1500/24987) of all confirmed cases until the end of March and April, respectively], we estimate that, between 14 March and 9 April (covering the epidemic exponential phase) the relative frequency of the Spike Y839 variant increased at a rate of 12.1% (6.1%–18.2%, CI 95%) every three days, being potentially associated with 24.8% (20.8–29.7%, CI 95%; 3177–4542 cases, CI 95%) of all COVID-19 cases in Portugal during this period. Our data supports population/epidemiological (founder) effects contributing to the Y839 variant superspread. The potential existence of selective advantage is also discussed, although experimental validation is required. Despite huge differences in genome sampling worldwide, SARS-CoV-2 Spike D839Y has been detected in 13 countries in four continents, supporting the need for close surveillance and functional assays of Spike variants.

Tracking the within-patient evolution of SARS-CoV-2 is key to understanding how this pandemic virus shapes its genome toward immune evasion and survival. In the present study, by monitoring a long-term COVID-19 immunocompromised patient, we observed the concurrent emergence of mutations potentially associated with immune evasion and/or enhanced transmission, mostly targeting the SARS-CoV-2 key host-interacting protein and antigen.

Ectopic decidual reaction (or deciduosis) can be rarely seen in the peritoneum and most of the cases in the literature are associated with pregnancy. It is more commonly found in the ovaries, uterus and cervix. Although its pathophysiology is not totally understood, it is accepted that peritoneal deciduosis develops as a result of the progesterone induced metaplasia of subserosal stromal cells during pregnancy. It is important to distinguish this entity from oncologic conditions, namely metastatic carcinoma and mesothelioma. We report an unusual case of a 40 year-old non pregnant patient that presented with imaging findings suggestive of peritoneal carcinomatosis.

Necrotizing fasciitis is a severe soft tissue infection with a high mortality rate and therefore requires emergent surgical treatment. Several microorganisms can cause this infection, Photobacterium damselae being one of them, with only eight cases previously published in the literature. We report the first ever case of necrotizing fasciitis, caused by this microorganism, in Portugal. In this case report the patient survived after several debridement procedures and reconstruction of the upper limb with acellular dermal matrix and skin graft. A brief review of the Photobacterium damselae soft tissue infection reports as well as the clinical presentation, diagnosis, pathophysiology and treatment of necrotizing fasciitis can also be found in this paper.

Background:. Free flap reconstruction in the head and neck region is a complex field in which patient comorbidities, radiation therapy, tumor recurrence, and variability of clinical scenarios make some cases particularly challenging and prone to devastating complications. Despite low free flap failure rates, the impact of flap failure has enormous consequences for the patients. Methods:. Acknowledging and predicting high risk intra- and postoperative situations and having planned strategies on how to deal with them can decrease their rate and improve the patient’s reconstructive journey. Results:. Herein, the authors present 4 examples of significant complications in complex microvascular head and neck cancer reconstruction, encountered for the last 10 years: compression and kinking of the vascular pedicle, lack of planning of external skin coverage in osteoradionecrosis, management of the vessel-depleted neck, and vascular donor site morbidity after fibula harvest. Conclusion:. The authors reflect on the causes and propose preventative strategies in each peri-operative stage.

Case. A 61-year-old male presented with chronic shoulder pain resistant to conservative treatment. Imaging identified a nodular lesion in the deltoid muscle, which histology after resection identified as a glomus tumor. After surgery, the patient became asymptomatic and at 4-year follow-up has not shown signs of recurrence. Conclusion. Glomus tumors around the shoulder should be considered when investigating chronic shoulder pain, as they are more common than thought. Despite being elusive, when diagnosed, excellent outcomes may be expected, with surgery resection being curative.

Varicella is a common viral infection in children and most of them recover without sequelae, but serious complications can follow this infection and 6% have been reported to be musculoskeletal. A previously healthy 3-year-old Caucasian male presented with odynophagia, anorexy, fever, refusal to bear weight, and vesicular exanthema. Varicella was diagnosed, but he sustained fever around 39°C and local tenderness on the proximal lateral portion of the right leg maintaining an antalgic position. Ultrasonography and plain radiography were performed, but the magnetic resonance imaging (MRI) was performed due to the clinically worsening diagnosed pyomyositis. He was subjected to surgical debridement, and we started intravenous antibiotherapy. Streptococcus pyogenes grew in the microbiologic culture. At a 6-month follow-up, the boy did not suffer from any sequelae. The regular course of varicella is benign; however, it can occasionally develop into a more serious illness. The initial presentation of pyomyositis is often subacute, and the first symptoms may be vague. The awareness of musculoskeletal complications is imperative, and the combination of varicella’s exanthema and fever followed by some limb complaint should lead to an alert attitude.

We describe imipenem-resistant and imipenem-susceptible clinical isolates of Clostridium difficile ribotype 017 in Portugal. All ribotype 017 isolates carried an extra penicillin-binding protein gene, pbp5, and the imipenem-resistant isolates had additional substitutions near the transpeptidase active sites of pbp1 and pbp3. These clones could disseminate and contribute to imipenem resistance.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli strain C999 was isolated of a Spanish patient with urinary tract infection. Previous genotyping indicated that this strain presented a multidrug-resistance phenotype and carried beta-lactamase genes encoding CTX-M-15, TEM-1, and OXA-1 enzymes. The whole-cell proteome, and the membrane, cytoplasmic, periplasmic and extracellular sub-proteomes of C999 were obtained in this work by two-dimensional gel electrophoresis (2DE) followed by fingerprint sequencing through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). A total of 602 proteins were identified in the different cell fractions, several of which are related to stress response systems, cellular responses, and antibiotic and drug responses, consistent with the multidrug-resistance phenotype. In parallel, whole genome sequencing (WGS) and RNA sequencing (RNA-Seq) was done to identify and quantify the genes present and expressing. The in silico prediction following WGS confirmed our strain as being serotype O25:H4 and sequence type ST131. The presence of proteins related to antibiotic resistance and virulence in an O25:H4-ST131 E. coli clone are serious indicators of the continued threat of antibiotic resistance spread amongst healthcare institutions. On a positive note, a multiomics approach can facilitate surveillance and more detailed characterization of virulent bacterial clones from hospital environments.

We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.

Gastrointestinal infections caused by nontyphoidal Salmonella (NTS) remain one of the main causes of foodborne illness worldwide. Within the multiple existing Salmonella enterica serovars, the serovar Rissen is rarely reported, particularly as a cause of human salmonellosis. Between 2015 and 2017, the Portuguese National Reference Laboratory of Gastrointestinal Infections observed an increase in the number of clinical cases caused by multidrug-resistant (MDR) S. enterica serovar Rissen, particularly from the Azores archipelago. In the present study, we analyzed by whole genome sequencing (WGS) all clinical, animal, food, and environmental isolates received up to 2017 in the Portuguese Reference Laboratories. As such, through a wgMLST-based gene-by-gene analysis, we aimed to identify potential epidemiological clusters linking clinical and samples from multiple sources, while gaining insight into the genetic diversity of S. enterica serovar Rissen. We also investigated the genetic basis driving the observed multidrug resistance. By integrating 60 novel genomes with all publicly available serovar Rissen genomes, we observed a low degree of genetic diversity within this serovar. Nevertheless, the majority of Portuguese isolates showed high degree of genetic relatedness and a potential link to pork production. An in-depth analysis of these isolates revealed the existence of two major clusters from the Azores archipelago composed of MDR isolates, most of which were resistant to at least five antimicrobials. Considering the well-known spread of MDR between gastrointestinal bacteria, the identification of MDR circulating clones should constitute an alert to public health authorities. Finally, this study constitutes the starting point for the implementation of the “One Health” approach for Salmonella surveillance in Portugal.

Dissemination of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in healthcare institutions affects both patients and health-care workers (HCW), as well as the institutional capacity to provide essential health services. Here, we investigated an outbreak of SARS-CoV-2 in a “non-COVID-19” hospital ward unveiled by massive testing, which challenged the reconstruction of transmission chains. The contacts network during the 15-day period before the screening was investigated, and positive SARS-CoV-2 RNA samples were subjected to virus genome sequencing. Of the 245 tested individuals, 48 (21 patients and 27 HCWs) tested positive for SARS-CoV-2. HCWs were mostly asymptomatic, but the mortality among patients reached 57.1% (12/21). Phylogenetic reconstruction revealed that all cases were part of the same transmission chain. By combining contact tracing and genomic data, including analysis of emerging minor variants, we unveiled a scenario of silent SARS-CoV-2 dissemination, mostly driven by the close contact within the HCWs group and between HCWs and patients. This investigation triggered enhanced prevention and control measures, leading to more timely detection and containment of novel outbreaks. This study shows the benefit of combining genomic and epidemiological data for disclosing complex nosocomial outbreaks, and provides valuable data to prevent transmission of COVID-19 in healthcare facilities.

In Portugal, the epidemiological stage for the spread of carbapenemase-producing Enterobacteriaceae (CPE) increased from sporadic isolates or single hospital clones (2010–2013), to hospital outbreaks, later. Here we report data from a 6-month study performed under the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE). During the study period, 67 isolates (61 Klebsiella pneumoniae and 6 Escherichia coli) non-susceptible to carbapenems were identified in participant hospital laboratories. We detected 37 blaKPC–type (including one new variant: blaKPC–21), 1 blaGES–5, and 1 blaGES–6 plus blaKPC–3, alone or in combination with other bla genes. Bioinformatics analysis of the KPC-21-producing E. coli identified the new variant blaKPC–21 in a 12,748 bp length plasmid. The blaKPC–21 gene was harbored on a non-Tn4401 element, presenting upstream a partial ISKpn6 (ΔISKpn6/ΔtraN) with the related left IR (IRL) and downstream a truncated Tn3 transposon. PFGE and MLST analysis showed an important diversity, as isolates belonged to distinct PFGE and STs profiles. In this study, we highlighted the presence of the high-risk clone E. coli sequence-type (ST) 131 clade C/H30. This worldwide disseminated E. coli lineage was already detected in Portugal among other antibiotic resistance reservoirs. This study highlights the intra- and inter-hospital spread and possible intercontinental circulation of CPE isolates.

A new QepA4 variant was detected in an O86:H28 ST156-fimH38 Escherichia coli, showing a multidrug-resistance phenotype. PAβN inhibition of qepA4-harboring transconjugant resulted in increase of nalidixic acid accumulation. The qepA4 and catA1 genes were clustered in a 26.0-kp contig matching an IncF-type plasmid, and containing a Tn21-type transposon with multiple mobile genetic elements. This QepA variant is worrisome because these determinants might facilitate the selection of higher-level resistance mutants, playing a role in the development of resistance, and/or confer higher-level resistance to fluoroquinolones in association with chromosomal mutations.

Morganella morganii is a commensal bacterium and opportunistic pathogen often present in the gut of humans and animals. We report the 4.3Mbp draft genome sequence of a M. morganii isolated in association with an Escherichia coli from broilers in Portugal that showed macroscopic lesions consistent with coliseptisemia. The analysis of the genome matched the multidrug resistance phenotype and enabled the identification of several clinically important and potentially mobile acquired antibiotic resistance genes, including the plasmid-mediated quinolone resistance determinant qnrD1. Mobile genetic elements, prophages and pathogenicity factors were also detected, improving our understanding towards this human and animal opportunistic pathogen.

The spread of antibiotic resistant bacteria throughout the food chain constitutes a public health concern. To understand the contribution of fresh produce in shaping antibiotic resistance bacteria and integron prevalence in the food chain, 333 antibiotic resistance Gram negative isolates were collected from organic and conventionally produced fruits (pears, apples and strawberries) and vegetables (lettuces, tomatoes and carrots). Although low levels of resistance have been detected, the bacterial genera identified in the assessed fresh produce are often described not only as environmental, but mostly as commensals and opportunistic pathogens. The genomic characterization of integron-harboring isolates revealed a high number of mobile genetic elements and clinically relevant antibiotic resistance genes, of which we highlight the presence of as mcr-1, qnrA1, blaGES-11, mphA and oqxAB. The study of class 1 (n=8), class 2 (n=3) and class 3 (n=1) integrons, harbored by species such as Morganella morganii, Escherichia coli, Klebsiella pneumoniae, led to the identification of different integron promoters (PcW, PcH1, PcS and PcWTNG-10) and cassette arrays (containing drfA, aadA, cmlA, estX, sat and blaGES). In fact, the diverse integron backbones were associated with transposable elements (e.g. Tn402, Tn7, ISCR1, Tn2*, IS26, IS1326 and IS3) that conferred greater mobility. This is also the first appearance of In1258, In1259 and In3-13, which should be monitored to prevent their establishment as successfully dispersed mobile resistance integrons. These results underscore the growing concern about the dissemination of acquired resistance genes by mobile elements in the food chain.

Research on the obligate intracellular bacterium Chlamydia trachomatis demands culture in cell-lines, but the adaptive process behind the in vivo to in vitro transition is not understood. We assessed the genomic and transcriptomic dynamics underlying C. trachomatis in vitro adaptation of strains representing the three disease groups (ocular, epithelial-genital and lymphogranuloma venereum) propagated in epithelial cells over multiple passages. We found genetic features potentially underlying phase variation mechanisms mediating the regulation of a lipid A biosynthesis enzyme (CT533/LpxC), and the functionality of the cytotoxin (CT166) through an ON/OFF mechanism. We detected inactivating mutations in CT713/porB, a scenario suggesting metabolic adaptation to the available carbon source. CT135 was inactivated in a tropism-specific manner, with CT135-negative clones emerging for all epithelial-genital populations (but not for LGV and ocular populations) and rapidly increasing in frequency (~23% mutants per 10 passages). RNA-sequencing analyses revealed that a deletion event involving CT135 impacted the expression of multiple virulence factors, namely effectors known to play a role in the C. trachomatis host-cell invasion or subversion (e.g., CT456/Tarp, CT694, CT875/TepP and CT868/ChlaDub1). This reflects a scenario of attenuation of C. trachomatis virulence in vitro, which may take place independently or in a cumulative fashion with the also observed down-regulation of plasmid-related virulence factors. This issue may be relevant on behalf of the recent advances in Chlamydia mutagenesis and transformation where culture propagation for selecting mutants/transformants is mandatory. Finally, there was an increase in the growth rate for all strains, reflecting gradual fitness enhancement over time. In general, these data shed light on the adaptive process underlying the C. trachomatis in vivo to in vitro transition, and indicates that it would be prudent to restrict culture propagation to minimal passages and check the status of the CT135 genotype in order to avoid the selection of CT135-negative mutants, likely originating less virulent strains.

Burkholderia pseudomallei is a Gram-negative bacillus and the causative agent of melioidosis, a serious infection associated with high mortality rate in humans. It can be naturally found as an environmental saprophyte in soil or stagnant water, and rice paddies that predominate in regions of endemicity such as Northeast Thailand. B. pseudomallei is a Biosafety Level 3 organism due to risks of aerosolization and severe disease and is now included in formal emergency preparedness plans and guidelines issued by various authorities in the United States and Europe. Here, we report the first case of imported melioidosis in Portugal. B. pseudomallei was isolated from the patient's blood as well as from a left gluteal abscess pus. The isolate strain showed the unusual resistance profile to first-line eradication therapy trimethroprim/sulfamethoxazole. Whole genome sequencing revealed its similarity with isolates from Southeast Asia, suggesting the Thai origin of this Portuguese isolate, which is in agreement with a recent patient's travel to Thailand.