DECRYPTING THE DIVERSITY OF MICROBIOME IN AQUACULTURE

Aquaculture can play an important role in reducing the overexploitation of natural re- sources and feeding the world’s growing population. However, the use of e.g., antibiotics in aquaculture can favor the development of resistant bacteria and jeopardize the safety of its products. Thus, this Ph.D. thesis aimed to contribute to the deciphering of aquaculture’s mi- crobiome and resistome, as well as to the understanding of the role of mobile genetic elements (MGE) in the dissemination of resistance genes in these environments. Several approaches were used, to obtain the results that most reflect the microbiome and resistome of seabream and bivalve mollusks from aquaculture. All microbiomes studied were very diverse, encompassing commensal and pathogenic bacteria from seabream and bivalve mollusks (e.g., Aeromonas,Kocuria, Pseudomonas and Vibrio genera), as well as bacteria important in human medicine (e.g., Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae). Twenty-one new se- quence types were described in Aeromonas spp., Citrobacter sp., Enterobacter spp., Shewanella spp., Staphylococcus sp. and Vibrio spp. Decreased susceptibilities to phenicols, oxytetracy- cline, β-lactams (namely carbapenems), quinolones, glycopeptides, mupirocin, erythromycin, and colistin were found. The resistome also revealed a great diversity of genes in all samples studied associated with antibiotics (e.g., blaTEM-1B, mecA, sul2, mcr-9.1), disinfectants (e.g.,formA-type), and heavy metals (e.g., sil) resistance. Twenty-five different genes related with increased virulence were also detected. Thirteen new β-lactams resistance genes were identi- fied (e.g., blaCTX-M-246, blaFOX-18, and blaOXA-958) and 35 other resistance genes, namely for antibi- otics (e.g., mcr-9 and qnrD2), heavy metals (e.g., emrA and mdtE) and disinfectants ( sitABCD- type), and virulence factors (e.g., astA and hlyF) were here described for the first time associated with aquaculture. Our results suggest that some of these resistance genes (e.g., erm(T)-type,qnrB19, catA1-type, tet(A), dfrA-type, aph(6)-Id, qacE∆ 1 and merA) are being disseminated by MGE such as plasmids, class 1 integrons, and Tn As1. These findings not only expand our knowledge about aquaculture’s microbiome and resistome, but also provide the necessary xiv information to implement the most suitable measures to control antibiotic resistance in aqua- culture environments.A aquacultura pode desempenhar um papel importante na redução da sobre-explora- ção dos recursos naturais e no fornecimento de alimentos para a crescente população mundial. No entanto, o uso de, por exemplo, antibióticos em aquacultura pode favorecer o desenvolvi- mento de bactérias resistentes e comprometer a segurança dos seus produtos. Assim, esta tese de Doutoramento teve como objetivo contribuir para a decifração do microbioma e resistoma em aquacultura, bem como compreender o papel dos elementos genéticos móveis na disse- minação de genes de resistência nestes ambientes. Foram usadas diversas abordagens com o intuito de obter os resultados que melhor refletem o microbioma e o resistoma das douradas e moluscos bivalves de aquacultura. Os microbiomas estudados apresentaram uma grande diversidade, englobando bactérias comensais e patogénicas das douradas e moluscos bivalves (ex.: os géneros Aeromonas, Kocuria, Pseudomonas e Vibrio), assim como bactérias importan- tes em medicina humana (ex.: Staphylococcus aureus, Escherichia coli e Klebsiella pneumo- niae). Foram descritos 21 novos sequence type em Aeromonas spp., Citrobacter sp., Entero- bacter spp., Shewanella spp., Staphylococcus sp. e Vibrio spp. Foram encontradas suscetibili- dades diminuídas aos fenicois, oxitetraciclina, β-lactâmicos (como carbapenemes), quinolonas, glicopéptidos, mupirocina, eritromicina e colistina. Também os resistomas revelaram uma grande diversidade de genes associados a resistências aos antibióticos (ex.: blaTEM-1B, mecA,sul2, mcr-9.1), desinfetantes (ex.: formA-type) e metais pesados (ex.: sil). Foram também dete- tados 25 genes diferentes, associados a uma maior virulência. Encontram-se aqui descritos 13 novos genes de resistência aos β-lactâmicos (ex.: blaCTX-M-246, blaFOX-18, blaOXA-958) e outros 35 genes de resistência, nomeadamente aos antibióticos (ex.: mcr-9 e qnrD2), metais pesados (ex.:emrA e mdtE) e desinfetantes ( sitABCD-type), e fatores de virulência (ex.: astA e hlyF) são aqui identificados pela primeira vez associados à aquacultura. Os nossos resultados sugerem que alguns destes genes de resistência (como erm(T)-type, qnrB19, catA1-type, tet(A), dfrA-type, aph(6)-Id, qacE∆ 1 e merA) estarão a ser disseminados por elementos genéticos móveis, como plasmídeos, integrões de classe 1 e Tn As1. Estes estudos não só ampliam o nosso conheci- mento sobre o microbioma e o resistoma em aquacultura, mas também providenciam a infor- mação necessária para a implementação das medidas mais adequadas ao controlo da resis- tência aos antibióticos em aquacultura

Self-reported Diabetes in Portugal and its association with health-related quality of life and medical resources consumption using a nationwide prospective cohort

Trabalho de projeto de mestrado, Bioestatística, Universidade de Lisboa, Faculdade de Ciências, 2022Diabetes is a chronic metabolic disease characterised by high blood glucose levels. It is a major burden on the national healthcare system as it brings serious problems associated with it over time, such as heart, eye, kidney, nerve and blood vessel problems, and can increase the consumption of healthcare resources. Diabetes greatly disrupts the lives of individuals being associated with poorer quality of life and increased early mortality. Therefore, it is important to characterise the Portuguese diabetic population as well as the comorbidities that most often accompany diabetes. (Organization, 2000). This study is part of a project proposed by EpiDoC Unit at Nova Medical School, where a prospective cohort study entitled Epidemiology in chronic diseases (EpiDoC) was developed in 2011. To this end, this study was designed to be representative of the Portuguese adult population. EpiDoC is a prospective closed cohort study, including a nationally representative sample of adults (older than 18 years) who were not institutionalised and lived in private homes in Mainland Portugal and Islands (Ac¸ores and Madeira). The main objective of this project was to characterise the diabetic population in Portuguese adults using the EpiDoC cohort. Data collected at the three follow-up waves were analysed to assess the impact of diabetes on the loss of quality of life and on the consumption of medical resources, namely the occurrence of hospitalisations and the number of medical appointments. The specific objectives were as follows: • To characterise the population regarding self-reported diabetes among Portuguese adults, taking into account the geographical distribution, socio-demographic characteristics and comorbidities associated with the disease. To this end, a broad exploratory analysis was conducted. • To assess the impact of diabetes on quality of life - measured by the EuroQol Five Dimensional Questionnaire score - at each data collection wave, as well as in a longitudinal perspective, in order to assess the evolution of this measure over time. To answer this question, the Tobit regression model was used. • To assess the impact of diabetes on the occurrence of hospital admissions and the number of medical appointments through longitudinal generalized linear models for binary data and counts, respectively. By the analysis of the prevalences, and analysing the responses of individuals in the first wave, it is concluded that there are more diabetic women than men, yet this difference is not significant. Furthermore, there is a higher prevalence of diabetics of Caucasian ethnicity, aged 66 to 75 years and married. Regarding NUTS II, there is a higher proportion of diabetics in the Norte region. Regarding the level of education, there is a higher proportion of diabetics whose schooling varies between 1 and 4 years. However, age may be considered a confounding factor, since there is a higher probability of low levels of education being associated with individuals with a more advanced age. The fact that most diabetics were retired corroborates the idea of age being a confounding factor. With regard to lifestyle habits, we concluded that most diabetics drink alcohol, do not smoke and do exercise. As expected, most diabetics have a Body Mass Index (BMI) corresponding to the obesity category. Finally, high blood pressure proved to be the most prevalent disease in diabetics. Regarding the quality of life models, it decreases with the presence of diabetes. As regards the occurrence of hospital admissions, a first analysis showed that only 14.5% of diabetic individuals had been hospitalized. Through the analysis of the model, concluded that the chance of an individual being hospitalized increases in diabetic patients. With regard to the number of consultations, it was found that diabetes causes the expected number of medical appointments to increase

Ostracodos da passagem Jurássico médio-superior da Bacia Lusitânica, praia de Pedrogão (Leiria)

Tese de mestrado em Geologia, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, em 2018O principal objetivo deste trabalho foi aumentar o conhecimento taxonómico e paleoecológico sobre a associação de ostracodos da transição Caloviano-Oxfordiano da Praia de Pedrógão, Leiria. Foi estudado o corte da Praia de Pedrógão que compreende uma sucessão de margas e calcários, exposta ao longo da linha de costa. Foram estudadas 13 amostras do Caloviano e 22 do Oxfordiano; a associação de ostracodos encontrada apresenta baixa a moderada diversidade e baixa afinidade específica com associações do Caloviano e do Oxfordiano do NW da Europa. No total foram triados 19 823 ostracodos e reconhecidas 34 espécies, das quais 17 marinhas, 13 de água doce/salobra e 4 como incertae sedis. Foram também identificados outros microfósseis: foraminíferos aglutinados e algas calcárias, como carófitas e dasicladáceas; identificaram-se no campo exemplares de macrofósseis: moluscos (gastrópodes, bivalves), amonites e equinodermes. A análise feita às descrições iniciais das espécies e subespécies atribuídas por Helmdach (1972) ao género Theriosynoecum da Praia de Pedrógão permitiu rever e considerar, nesta dissertação, 3 espécies como válidas: Theriosynoecum fluxans, Theriosynoecum levis e Theriosynoecum sp. 1. Em relação ao género Macrodentina, procedeu-se à revisão de 37 descrições iniciais de espécies atribuídas aos subgéneros Macrodentina (Macrodentina) Malz, Macrodentina (Mediodentina) Bate, e Macrodentina (Polydentina) Malz. Esta mesma análise, permitiu concluir que algumas atribuições anteriores a este género necessitam de uma revisão cuidada. Considerando as diferentes espécies que ocorrem, sua quantidade, diversidade e composição das associações definiram-se 7 intervalos paleoecológicos ao longo do corte da Praia de Pedrógão: 3 no Caloviano, 4 no Oxfordiano. Os 3 intervalos inseridos no Caloviano representam um paleoambiente com características marinhas de rampa carbonatada, com domínio das espécies Praeschuleridea lisensis Pais, Cabral & Lord e Neurocythere multiforma Pais, Lord & Cabral e presença de outras formas marinhas Praebythoceratina parascrobiculata Lord, Cabral & Pais, Virgulacytheridea posteroacuminata Cabral, Lord e Pais, Polycope cf. P. sububiquita Whatley, Cytherella cf. C. fullonica Jones & Sherborn e Cytherella cf. C. index Oertli. Para topo da sucessão do Caloviano surgem géneros não marinhos, Septacandona e Theriosynoecum, que refletem a transição para condições de água doce/salobra. Esta passagem reflete uma regressão e formação de uma desconformidade. A sucessão superior está inserida no Oxfordiano devido à presença da dasicladácea Barattoloporellopsis lusitanica e os 4 intervalos identificados representam um paleoambiente margino-marinho de água doce/salobra, de baixa profundidade e com episódios de exposição subaérea. Surgem espécies típicas de água doce/salobra como Theriosynoecum fluxans Helmdach, Theriosynoecum helmdachi Sohn, Theriosynoecum sp. 1, Alicenula cf. A. incurva (Bate), Alicenula sp. 1. e a espécie eurihalina Sinuocythere pedrogaensis Cabral & Colin. Para topo verificam-se alguns episódios de influência marinha, registados pelo aparecimento de Macrodentina (Macrodentina) sp. 1 e Schuleridea sp. 1. As variações de salinidade observadas estão de acordo com trabalhos anteriores e com as variações relativas do nível do mar descritas para o Caloviano-Oxfordiano da Bacia Lusitânica.This dissertation's main goal is to improve the taxonomic and palaeoecological knowledge of the ostracod association of the Callovian-Oxfordian’s transition from Praia de Pedrógão, Leiria. A study of the Praia de Pedrógão’s section was made, which contains a succession of marls and limestones, exposed along the coastline. 13 samples from Callovian and 22 from Oxfordian were picked to study the ostracod association, which shows low to moderate diversity and low affinity at specific level with associations of Callovian and Oxfordian NW Europe. From those samples were picked 19 823 ostracods; and identified 34 species, of which 17 are marine, 13 are fresh/brackish water, and 4 incertae sedis. Other microfossils were also identified: agglutinated foraminifera and calcareous algae, such as carophytes and dasycladaleans (Barattoloporellopsis lusitanica); on the field, macrofossils were identified: mollusks (gastropods, bivalves), ammonites and echinoderms. The analysis made to the first descriptions of the species and subspecies assigned by Helmdach (1972) to the genus Theriosynoecum in Praia de Pedrógão, that allowed to review and consider 3 species as valid: Theriosynoecum fluxans, Theriosynoecum levis and Theriosynoecum sp. 1. An initial review of 37 first descriptions of species assigned to the subgenus (Macrodentina (Macrodentina) Malz, Macrodentina (Mediodentina) Bate, and Macrodentina (Polydentina) Malz was made, which later allowed the conclusion that some of these assignments should be re-analysed. Based on the species found, and the assemblages' abundance and composition, there were defined 7 palaeoecological intervals along the Praia de Pedrógão’s section: 3 on Calovian and 4 on Oxfordian. The 3 intervals on the Callovian represent a palaeoenvironment with marine characteristics of carbonated ramp, with dominance of Praeschuleridea lisensis Pais, Cabral & Lord and Neurocythere multiforma Pais, Lord & Cabral and other marine forms Praebythoceratina parascrobiculata Lord, Cabral & Pais, Virgulacytheridea posteroacuminata Cabral, Lord & Pais, Polycope cf. P. sububiquita Whatley, Cytherella cf. C. fullonica Jones & Sherborn and Cytherella cf. C. index Oertli. At the top of the Callovian section, non-marine genus arises, such as Septacandona and Theriosynoecum, reflecting the transition to fresh/brackish water. This also reflects a regression and formation of a disconformity. The presence of the dasycladacean Barattoloporellopsis lusitanica shows that the superior section is located on the Oxfordian, and the 4 Oxfordian identified intervals represent a marginal-marine palaeoenvironment of fresh/brackish water, with low depth and subaerial exposure periods. Brackish/freshwater species also emerge, like Theriosynoecum fluxans Helmdach, Theriosynoecum helmdachi Sohn, Theriosynoecum sp. 1, Alicenula cf. A. incurva (Bate), Alicenula sp. 1, and also the eurihaline species Sinuocythere pedrogaensis Cabral & Colin. To the top of Pedrógão section marine episodes are attested by the appearance of Macrodentina (Macrodentina) sp. 1 and Schuleridea sp. 1. The salinity’s variation are in agreement with previous work, and with descriptions of sea level variation for the Callovian-Oxfordian of the Lusitanian basin

Caracterização dos mecanismos de resistência aos antibióticos em estirpes de origem humana, ambientes associados aos cuidados de saúde e veterinária

Tese de mestrado, Biologia Humana e Ambiente, Universidade de Lisboa, Faculdade de Ciências, 2015A resistência aos antibióticos é atualmente um problema de saúde pública a nível mundial. Este fenómeno envolve diversos mecanismos e espécies bacterianas, revelando-se de extrema importância a sua compreensão, através de investigação e vigilância, para evitar possíveis disseminações. Neste âmbito, este trabalho teve como principal objetivo a caracterização de mecanismos de resistência aos antibióticos em 139 estirpes bacterianas com três origens distintas: hospitais (Acinetobacter baumannii, Exiguobacterium acetylicum, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus capitis e Staphylococ-cus epidermidis), ambiente de instituições de cuidados continuados (Acinetobacter haemolyticus, Acinetobacter lwoffii, Acinetobacter junii, Acinetobacter pittii, Klebsiella oxyto-ca, Micrococcus luteus, Pantoea spp., Pseudomonas fluorescens, Pseudomonas oryzihabi-tans, Pseudomonas putida, Sphingomonas paucimobilis, S. capitis, Staphylococcus capi-tis/caprae/warneri, S. epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis e Staphylococcus xylosos) e animais (S. aureus e Staphylococcus simiae). A realização de testes de suscetibilidade aos antibióticos (difusão em disco e E-teste) permitiu não só a identificação de estirpes com suscetibilidade diminuída às várias classes de antibióticos ensaiadas, mas também a orientação da pesquisa de genes de resistência por PCR (Polymerase Chain Reaction). Procurou-se estudar a diversidade genética das es-tirpes de A. baumannii através da deteção de linhagens clonais com importância internacio-nal e MLST (Multilocus Sequence Typing). No caso particular de S. aureus, a tipagem dos genes spa e agr, assim como MLST, permitiram estabelecer relações genéticas entre estas estirpes. Por outro lado, o método de PFGE (Pulsed-Field Gel Electrophoresis), utilizado para K. oxytoca, possibilitou a avaliação da clonalidade de uma estirpe ambiental em relação a estirpes de origem clínica. A maioria das estirpes coletadas em hospitais possuía um fenótipo de multirresistên-cia, ao contrário das estipes com origem nos lares e em animais. Aí se destaca a suscetibili-dade diminuída à ciprofloxacina e cefoxitina, esta última por expressão do gene mecA, em S. aureus e a suscetibilidade diminuída aos carbapenemes em A. baumannii e P. aerugino-sa, diminuindo as opções de tratamento disponíveis para infeções causadas por estas bactérias. Em A. baumannii foram encontrados os genes blaOXA-66 e blaOXA-23 e a sequência de inserção ISAba1. Em P. aeruginosa detetaram-se os genes blaGES-7-tipo, blaVEB-tipo, blaVIM-2 e blaVIM-11. A identificação do mesmo tipo de spa e ST em estirpes de S. aureus com origem humana e animal demonstra a possível disseminação entre estes dois reservatórios. A caracterização molecular permitiu ainda identificar uma eventual transferência de uma estirpe e/ou de determinantes de resistência entre S. capitis de um hospital e de um lar, situados na mesma área geográfica. Os lares e os animais, em paralelo com o Homem, poderão ser reservatórios de genes de resistência. Assim, é essencial um melhor conhecimento dos mecanismos de resistência e das vias de disseminação nestes reservatórios, para intervir e controlar a sua emergência e expansão.Antibiotic resistance is currently a worldwide public health problem that can include diverse mechanisms and bacterial species. The understanding of such phenomena is essen-tial to avoid further dissemination, namely through research studies and monitoring. The main aim of this study was the characterization of antibiotic resistance mecha-nisms in 139 bacterial strains collected in three different origins: humans admitted to he-althcare facilities (Acinetobacter baumannii, Exiguobacterium acetylicum, Pseudomonas ae-ruginosa, Staphylococcus aureus, Staphylococcus capitis and Staphylococcus epidermidis), air samples from long term care facilities (Acinetobacter haemolyticus, Acinetobacter lwoffii, Acinetobacter junii, Acinetobacter pittii, Klebsiella oxytoca, Micrococcus luteus, Pantoea spp., Pseudomonas fluorescens, Pseudomonas oryzihabitans, Pseudomonas putida, Sphin-gomonas paucimobilis, S. capitis, Staphylococcus capitis/caprae/warneri, S. epidermidis, Staphylococcus haemolyticus, Staphylococcus hominis and Staphylococcus xylosos) and animals (S. aureus and Staphylococcus simiae). Antibiotic susceptibility testing by disk diffusion and E-test allowed not only the identification of strains showing reduced susceptibility to several antibiotic classes, but also oriented the search for antibiotic resistance genes by PCR (Polymerase Chain Reaction). The genetic diversity of A. baumannii was studied using PCR detection of the main clonal lineages and MLST (Multilocus Sequence Typing). For S. aureus, besides MLST, spa and agr typing methods were also applied to evaluate genetic relatedness. PFGE (Pulsed-Field Gel Electrophoresis) was used to evaluate clonality between K. oxytoca isolated from the air environment and other strains from clinical origin. The majority of the hospital acquired strains showed multidrug resistance, while strains from the environment of long term care facilities and animals were mainly susceptible. Among strains from human origin, reduced susceptibility to ciprofloxacin and cefoxitin (the last one through expression of the mecA gene) was detected in S. aureus. It was also observed nonsusceptibility to carbapenems supported by the detection of blaOXA-66 and blaOXA-23 together with ISAba1 in A. baumannii, and blaGES-7-type, blaVEB-type, blaVIM-2 and blaVIM-11 genes in P. aeruginosa. These nonsusceptibility patterns constitute a source for concern because carbapenems constitute last resource antibiotics for the treatment of infections caused by these bacteria. The identification of the same ST and spa types in S. aureus from humans and animals suggests a potential dissemination of strains between these two environments. Moreover, the molecular characterization of S. capitis strains from a hospital and a long term care facilitie within the same region indicates a putative phenomena of transference. Globally, long term care facilities and animal-harboring environments may act as complementary reservoirs of antibiotic resistant bacteria and antibiotic resistance genes. Thus, a better understanding of the antibiotic resistance mechanisms and dissemination pathways in other reservoirs than human is essential to control its emergence and spread

Assessing the bacterial community composition of bivalve mollusks collected in aquaculture farms and respective susceptibility to antibiotics

Research Areas: Infectious Diseases ; Pharmacology & PharmacyAquaculture is a growing sector, providing several products for human consumption, and it is therefore important to guarantee its quality and safety. This study aimed to contribute to the knowledge of bacterial composition of Crassostrea gigas, Mytilus spp. and Ruditapes decussatus, and the antibiotic resistances/resistance genes present in aquaculture environments. Two hundred and twenty-two bacterial strains were recovered from all bivalve mollusks samples belonging to the Aeromonadaceae, Bacillaceae, Comamonadaceae, Enterobacteriaceae, Enterococcaceae, Micrococcaceae, Moraxellaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Staphylococcaceae, Streptococcaceae, Vibrionaceae, and Yersiniaceae families. Decreased susceptibility to oxytetracycline prevails in all bivalve species, aquaculture farms and seasons. Decreased susceptibilities to amoxicillin, amoxicillin/clavulanic acid, cefotaxime, cefoxitin, ceftazidime, chloramphenicol, florfenicol, colistin, ciprofloxacin, flumequine, nalidixic acid and trimethoprim/sulfamethoxazole were also found. This study detected six qnrA genes among Shewanella algae, ten qnrB genes among Citrobacter spp. and Escherichia coli, three oqxAB genes from Raoultella ornithinolytica and blaTEM-1 in eight E. coli strains harboring a qnrB19 gene. Our results suggest that the bacteria and antibiotic resistances/resistance genes present in bivalve mollusks depend on several factors, such as host species and respective life stage, bacterial family, farm’s location and season, and that is important to study each aquaculture farm individually to implement the most suitable measures to prevent outbreaks.info:eu-repo/semantics/publishedVersio

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.VS has her Ph.D. fellowship granted by FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 co-financed by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was financial supported with funding from FCT/MCTES (UIDB/00211/2020) through national funds.info:eu-repo/semantics/publishedVersio

Snapshot of resistome, virulome and mobilome in aquaculture

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with β-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.Highlights: - New STs (17) and possible evolutionary relationships with other STs were identified. - Over 140 resistance genes provided a snapshot of the aquaculture resistome. - Many resistance genes found are common to those of clinical isolates (e.g. qnrB19). - Many ARGs detected (e.g. sul) are associated to antibiotics used in aquaculture. - Several (74.2 %) strains studied were classified as pathogenic to human.V.S. has a Ph.D. fellowship granted by the FCT (Fundação para a Ciência e a Tecnologia) with the reference SFRH/BD/133100/2017 cofinanced by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. This work was supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme (WORLDCOM project), and by FCT/MCTES [UIDB/ 00211/2020] through national funds.info:eu-repo/semantics/publishedVersio

Genomic Analysis of a mcr-9.1-Harbouring IncHI2-ST1 Plasmid from Enterobacter ludwigii Isolated in Fish Farming

This article belongs to the Special Issue Antibiotic Resistance and Virulence Profiles of Gram-Negative Bacteria.This study analyzed the resistome, virulome and mobilome of an MCR-9-producing Enterobacter sp. identified in a muscle sample of seabream (Sparus aurata), collected in a land tank from multitrophic fish farming production. Average Nucleotide Identity analysis identified INSAq77 at the species level as an Enterobacter ludwigii INSAq77 strain that was resistant to chloramphenicol, florfenicol and fosfomycin and was susceptible to all other antibiotics tested. In silico antimicrobial resistance analyses revealed genes conferring in silico resistance to β-lactams (blaACT-88), chloramphenicol (catA4-type), fosfomycin (fosA2-type) and colistin (mcr-9.1), as well as several efflux pumps (e.g., oqxAB-type and mar operon). Further bioinformatics analysis revealed five plasmid replicon types, including the IncHI2/HI2A, which are linked to the worldwide dissemination of the mcr-9 gene in different antibiotic resistance reservoirs. The conserved nickel/copper operon rcnR-rcnA-pcoE-ISSgsp1-pcoS-IS903-mcr-9-wbuC was present, which may play a key role in copper tolerance under anaerobic growth and nickel homeostasis. These results highlight that antibiotic resistance in aquaculture are spreading through food, the environment and humans, which places this research in a One Health context. In fact, colistin is used as a last resort for the treatment of serious infections in clinical settings, thus mcr genes may represent a serious threat to human health.V.S. had her Ph.D. fellowship granted by FCT (Fundação para a Ciência e a Tecnologia) [SFRH/BD/133100/2017], co-financed by European Social Fund and the Operational Program for Human Capital (POCH), Portugal. The work was supported with funding from FCT/MCTES [UIDB/00211/2020] through national funds. This work was partially supported by funding from the European Union’s Horizon 2020 Research and Innovation programme under grant agreement No 773830: One Health European Joint Programme (WORLDCOM project), and partially from FCT/MCTES [UIDB/00211/2020] through national funds.info:eu-repo/semantics/publishedVersio

Application of the deployment of the quality function in the check-in attendance of airport passengers

Generally, when people travel by plane, they feel nervous and anxious, even before the aircraft takes off, this makes them tend to criticize the first service they receive at the airport or the airline of their choice, since they need to Attention, agility and feel safe to travel. Nowadays, airplane travel is present in several economic classes, so, even though the airlines have their target audience, they have adapted their employees to the market, that is, the front line service is directed to the customer, both for executive passengers, who travel frequently and know many safety procedures, as well as those Brazilian or foreign passengers who have never traveled by plane or have traveled, but need the attention and empathy of the airline employee to make their trip pleasant. This paper presents the application of the QFD (Quality Function Deployment) method in an airline, together with the POP (Standard Operational Procedure) tool, to identify and meet the needs of the passengers, the results obtained show that 60% of customers are satisfied with the attendance of employees, but in terms of infrastructure measures must be adopted to improve levels of passenger satisfactio

AtGRP3 is implicated in root size and aluminum response pathways in Arabidopsis

AtGRP3 is a glycine-rich protein (GRP) from Arabidopsis thaliana shown to interact with the receptor-like kinase AtWAK1 in yeast, in vitro and in planta. In this work, phenotypic analyses using transgenic plants were performed in order to better characterize this GRP. Plants of two independent knockout alleles of AtGRP3 develop longer roots suggesting its involvement in root size determination. Confocal microscopy analysis showed an abnormal cell division and elongation in grp3-1 knockout mutants. Moreover, we also show that grp3-1 exhibits an enhanced Aluminum (Al) tolerance, a feature also described in AtWAK1 overexpressing plants. Together, these results implicate AtGRP3 function root size determination during development and in Al stress