Browsing by Author "Salgueiro, Vanessa"
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- Antibiotic-resistant bacteria from air samples collected in nursing homesPublication . Salgueiro, Vanessa; Manageiro, Vera; Jones-Dias, Daniela; Cano, Manuela; Ferreira, Eugénia; Caniça, ManuelaIntroduction: Antibiotic resistance is currently a worldwide public health problem that can include diverse mechanisms and bacterial species. The understanding and monitoring of such phenomena is essential to avoid further dissemination. The main aim of this study was the characterization of antibiotic resistance mechanisms in bacterial strains collected in air samples from nursing homes and compare them with clinical samples isolated from the same geographic area. Materials and methods: Air samples were collected from bedrooms, living rooms and outdoor of 4 nursing homes located in Lisbon, Portugal. Screening of antimicrobial susceptibility of 18 Gram-negative (5 Acinetobacter spp., 1 Klebsiella oxytoca, 4 Pantoea spp., 7 Pseudomonas spp., 1 Sphingomonas paucimobilis) and 12 Gram-positive (3 Micrococcus luteus, 3 Staphylococcus spp.) isolates was performed by disk diffusion method. Different antibiotic resistant genes were searched by PCR. PFGE was used to evaluate clonality between K. oxytoca isolated from the air environment and other strains from clinical origin. Results and discussion: The majority of strains were susceptible to all antibiotics tested. Among beta-lactam antibiotics, reduced susceptibility to cefoxitin was detected in Staphylococcus capitis, through expression of the mecA gene, and to ampicillin, piperacillin and piperacillin-tazobactam in 1 K. oxytoca expressing a blaOXY-5-type beta-lactamase. Nonsusceptibility to meropenem and piperacillin-tazobactam was observed in 2 Pseudomonas putida however no antibiotic resistance gene was detected. Regarding quinolones, non-susceptibility was found in 1 S S. capitis, 2 Staphylococcus haemolyticus, and 1 K. oxytoca. The molecular characterization of the mecA-positive S. capitis from the nursing homes and from a hospital within the same region suggests a potential dissemination of strains between these two environments. The genetic relatedness of K. oxytoca from nursing homes (n=1) and clinical isolates (n=9) recovered within the same region, allowed to conclude that they were not genetically related. Conclusions: Globally, nursing homes 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.
- Bacterial Diversity and Antibiotic Susceptibility of Sparus aurata from AquaculturePublication . Salgueiro, Vanessa; Manageiro, Vera; Bandarra, Narcisa M.; Reis, Lígia; Ferreira, Eugénia; Caniça, ManuelaIn a world where the population continues to increase and the volume of fishing catches stagnates or even falls, the aquaculture sector has great growth potential. This study aimed to contribute to the depth of knowledge of the diversity of bacterial species found in Sparus aurata collected from a fish farm and to understand which profiles of diminished susceptibility to antibiotics would be found in these bacteria that might be disseminated in the environment. One hundred thirty-six bacterial strains were recovered from the S. aurata samples. These strains belonged to Bacillaceae, Bacillales Family XII. Incertae Sedis, Comamonadaceae, Enterobacteriaceae, Enterococcaceae, Erwiniaceae, Micrococcaceae, Pseudomonadaceae and Staphylococcaceae families. Enterobacter sp. was more frequently found in gills, intestine and skin groups than in muscle groups (p ≤ 0.01). Antibiotic susceptibility tests found that non-susceptibility to phenicols was significantly higher in gills, intestine and skin samples (45%) than in muscle samples (24%) (p ≤ 0.01) and was the most frequently found non-susceptibility in both groups of samples. The group of Enterobacteriaceae from muscles presented less decreased susceptibility to florfenicol (44%) than in the group of gills, intestine and skin samples (76%). We found decreased susceptibilities to β-lactams and glycopeptides in the Bacillaceae family, to quinolones and mupirocin in the Staphylococcaceae family, and mostly to β-lactams, phenicols and quinolones in the Enterobacteriaceae and Pseudomonadaceae families. Seven Enterobacter spp. and five Pseudomonas spp. strains showed non-susceptibility to ertapenem and meropenem, respectively, which is of concern because they are antibiotics used as a last resort in serious clinical infections. To our knowledge, this is the first description of species Exiguobacterium acetylicum, Klebsiella michiganensis, Lelliottia sp. and Pantoea vagans associated with S. aurata (excluding cases where these bacteria are used as probiotics) and of plasmid-mediated quinolone resistance qnrB19-producing Leclercia adecarboxylata strain. The non-synonymous G385T and C402A mutations at parC gene (within quinolone resistance-determining regions) were also identified in a Klebsiella pneumoniae, revealing decreased susceptibility to ciprofloxacin. In this study, we found not only bacteria from the natural microbiota of fish but also pathogenic bacteria associated with fish and humans. Several antibiotics for which decreased susceptibility was found here are integrated into the World Health Organization list of "critically important antimicrobials" and "highly important antimicrobials" for human medicine.
- Bacterial resistancesPublication . Manageiro, Vera; Salgueiro, Vanessa; Ferreira, Eugénia; Caniça, ManuelaHere we review several factors involved in the emergence of antibiotic resistance. They are numerous, and the constant adaptation of microorganisms to the selective pressure exerted by antibiotics is extraordinary. The monitoring systems to assess antibiotic resistance levels and the extent of dissemination were highlighted. In addition, the success of spread of certain bacterial lineages and resistant mechanisms remains sometimes difficult to determine. The need to enlarge research in the area of antibiotic resistance was also stated, not only to better understand the dynamics of dissemination of resistance between different bacteria and different ecosystems, but also to enlarge the pharmaceutical pipeline of antibacterials against multidrug-resistant pathogens. It is manifest the severe consequences of antibiotic resistances to humans, animals and environment, constituting a global public health priority. In consequence, it should be tackled on all fronts in view to the essential concept of “One World-One Medicine-One Health”.
- Caracterização dos mecanismos de resistência aos antibióticos em estirpes de origem humana, ambientes associados aos cuidados de saúde e veterináriaPublication . Salgueiro, Vanessa; Caniça, Manuela; Rebelo, Maria TeresaA 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.
- Decrypting the diversity of microbiome in aquaculturePublication . Salgueiro, Vanessa; Caniça, Manuela; Manageiro, Vera; Nogueira, IsabelAquaculture 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.
- First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirsPublication . Salgueiro, Vanessa; Manageiro, Vera; Bandarra, Narcisa M.; Ferreira, Eugénia; Clemente, Lurdes; Caniça, ManuelaStaphylococcus 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.
- Genetic Relatedness and Diversity of Staphylococcus aureus from Different Reservoirs: Humans and Animals of Livestock, Poultry, Zoo, and AquaculturePublication . Salgueiro, Vanessa; Manageiro, Vera; Bandarra, Narcisa M.; Ferreira, Eugénia; Clemente, Lurdes; Caniça, ManuelaThe main aim of this study was the characterization of antibiotic resistance mechanisms in 82 Staphylococcus aureus strains isolated from humans and animals. Antibiotic susceptibility testing was performed on all S. aureus isolates accordingly, and antibiotic-resistant genes were investigated by genotypic methods. The genetic diversity of S. aureus was studied through spa, multilocus sequence typing (MLST), and agr typing methods. The majority of S. aureus from human sources were resistant to cefoxitin (and harbor the mecA gene) and fluoroquinolones, whereas only four strains of S. aureus from animal sources revealed resistance to ciprofloxacin. In the set of S. aureus isolated from humans, the most frequent spa, MLST, and agr group were t032, ST22, and I, respectively. In strains from animal origin the most common spa, MLST, and agr group found were t2383, ST398, and III/not typable, respectively. S. aureus from humans and animals were identified either in clonal complexes CC5, CC30, and CC398, suggesting that they have the same putative founder in their evolution. Considering the three CCs encompassing strains from human and animal reservoirs with different spa-types, we can hypothesize that this might reflect an adaptation to different phylogenetic lineages in those reservoirs (host species) probably associated to genetic diversification of pre-existing strains.
- Genomic Analysis of a mcr-9.1-Harbouring IncHI2-ST1 Plasmid from Enterobacter ludwigii Isolated in Fish FarmingPublication . Manageiro, Vera; Salgueiro, Vanessa; Rosado, Tânia; Bandarra, Narcisa M.; Ferreira, Eugénia; Smith, Terry; Dias, Elsa; Caniça, ManuelaThis 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.
- Genotypic and phenotypic characterization of Staphylococcus aureus isolates from human and animal origin in Portugal.Publication . Salgueiro, Vanessa; Manageiro, Vera; Clemente, Lurdes; Ferreira, Eugénia; Caniça, ManuelaBackground: Different methicillin-susceptible (MSSA) and methicillin-resistant (MRSA) Staphylococcus aureus clones have been encountered in isolates of human and animal origins. The objective of this study was to identify and characterize S. aureus from humans, and to compare their phenotypic and genotypic characteristics with animal isolates. Material/methods: Fifty-eight S. aureus isolates recovered from different specimens of patients admitted at Portuguese hospitals, comprising community-acquired and nosocomial infections, were studied. Seventeen S. aureus strains isolated from animals were also studied. Antibiotic susceptibility testing was performed to all S. aureus isolates by disk diffusion and E-test, according to EUCAST guidelines. Different antibiotic resistant genes were searched by PCR. MLST, spa and agr typing methods were applied to evaluate diversity and genetic relatedness. Results: The majority of the 58 clinical S. aureus was MRSA (70.7%), with reduced susceptibility to cefoxitin due to expression of the mecA gene; no mecC gene was detected. Additionally, 82.8% had decreased susceptibility to ciprofloxacin. Two (3.4%) linezolid-resistant S. aureus strains (CC5-ST105-t1094-agr 2 and CC8-ST239-t1223-agr 1) harboured mutations in the domain V of the 23S rRNA and/or in the rlmN gene, which explained the resistance phenotype. Decreased susceptibility to daptomycin (17.2%) was related with hospital environment clones, such as CC22-ST22-t032 and CC8-ST239-t037. Eleven (19%) S. aureus had a phenotype of multidrug resistance, with one (1.7%) characterized as hGISA (clone ST5/ST105-t002-agr 2). Among clinical strains, the most frequent spa types were t032 (ST22), followed closely by t002 (ST5/ST105); 2 new spa types (t14878 and t14933) were detected. Contrarily, all S. aureus isolated from animals were MSSA, showing susceptibility profiles to almost all antibiotics tested; four strains isolated from rabbits showed decreased susceptibility to ciprofloxacin. Furthermore, among S. aureus from animals we also identified CC398-ST398-t571 and CC130-t84 that have been described both in humans and animal infections, a new spa type (t15307), and three new ST (ST3254, ST3269 and ST3270). Globally, ST5 and ST34 were found in both reservoirs, while ST22/ST105 and ST121 were the most frequently identified among human and animal isolates, respectively. Conclusions: 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. In countries with high MRSA rates like Portugal, it is necessary to implement strategies in hospitals and also in veterinary areas in order to reduce the spread of this bacterium, which is a public health problem today.
- Snapshot of resistome, virulome and mobilome in aquaculturePublication . Salgueiro, Vanessa; Manageiro, Vera; Rosado, Tânia; Bandarra, Narcisa M.; Botelho, Maria João; Dias, Elsa; Caniça, ManuelaAquaculture 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.