Browsing by Author "Almeida, Joana"
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- Carbapenemase-producing multidrug resistant Enterobacteriaceae in Portugal: class A and BPublication . Caniça, Manuela; Manageiro, Vera; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Ferreira, EugéniaIntroduction: Carbapenems are often the antimicrobials of last resort to treat infections due to extended-spectrum β-lactamase (ESBL)- or plasmid-mediated AmpC (PMAβ)-producing Enterobacteriaceae isolates. Unfortunately, carbapenem non-susceptible Enterobacteriaceae have been reported worldwide mainly, because of the acquisition of carbapenemase-encoding genes. A large variety of carbapenemases has increasingly been identified in each of the four Ambler molecular classes. Thus, the main aim of this study was to search and characterize carbapenemase-producing Enterobacteriaceae (CPE) isolates recovered from Portuguese health care institutions. Methods: This study included 165 clinical isolates of Enterobacteriaceae nonsusceptible to ertapenem, identified among 2105 isolates, all recovered from different Portuguese healthcare institutions, between 2006 and 2013 that were sent to the NIH for antibiotic susceptibility confirmation in the context of its reference function. Screening of antimicrobial susceptibility was performed by disc diffusion method (www.sfm-microbiologie.org/). Clinical isolates with resistance or with decreased susceptibility to ertapenem were considered putative carbapenemase producers. For all CPE isolates and respective transconjugants, MICs of selected antibiotics were determined by the microdilution broth method (http://www.eucast.org/clinical_breakpoints/). PCR and sequencing were applied to detect and identify the main CPE-encoding genes from class A, B and/or D. The presence of blaESBL, blaPMAβ, and PMQR-encoding genes, were also investigated. Transferability of the carbapenemase genes was attended by broth mating-out assays. The plasmids obtained from transconjugants were characterized by PCR-based replicon typing. Genetic diversity of K. pneumoniae isolates was investigated by multilocus sequence typing (MLST), using the protocol developed by the Institute Pasteur (www.pasteur.fr/mlst/Kpneumoniae.html). Results: Thirty-five (21.2%) of the 165 positive isolates were confirmed to be CPE, of which the majority were collected from the urine (54.3%) of elderly (≥65 years old) male patients (54.3%), and admitted at the emergency room/ambulatory (22.9%) or internal medicine (17.1%) wards. All were multidrug-resistant with nonsusceptibility to at least one carbapenem; colistin was the only antibiotic effective against all CPE isolates. Tigecycline and ciprofloxacin were effective against 57.1% and 31.4%, respectively, of carbapenemase-producing isolates. We identify 30 blaKPC-3, 4 blaGES-5 and one blaVIM-2, alone or in combination with other bla and/or PMQR-encoding genes. MLST of the 30 K. pneumoniae isolates showed an important diversity, belonging to distinct STs. Conclusion: This study provides new data regarding the widespread of CPE in Portugal, which encoding genes are carried by similar plasmids. Overall, our results emphasize the need of a concerted action to manage carbapenem use.
- CNV Characterization, Inheritance and Phenotypic Correlations in Families With AutismPublication . C. Conceição, Inês; Correia, Catarina; Oliveira, Bárbara; M. Rama, Maria; Café, Cátia; Almeida, Joana; Mouga, Susana; Duque, Frederico; Oliveira, Guiomar; M. Vicente, AstridAutism Spectrum Disorders (ASD) have a strong genetic component, with an estimated heritability of over 90%1. Recent studies carried out by the Autism Genome Project (AGP) consortium suggest that rare Copy Number Variants (CNVs), characterized by submicroscopic chromosomal deletions and duplications, are more frequent in ASD compared to controls, and may play an important role in susceptibility to this disorder2. However, to adequately assess pathogenicity, a detailed characterization of patients CNVs and phenotype is required. The goal of this study was to establish the clinical and etiological relevance for ASD of potentially pathogenic CNVs identified in a Portuguese population sample by whole genome CNV analysis, through the detailed characterization of CNVs and correlation with clinical phenotypes. Analysis of the AGP genome-wide CNV results using 1M SNP microarray2 identified a total of 14218 CNVs in 342 Portuguese probands. We selected 291 CNVs, present in 191 individuals (19 females and 172 males), using the following criteria: 1) CNVs that contained implicated/candidate genes for ASD; 2) CNVs in genomic regions known to be implicated/candidate for ASD; 3) CNVs in regions associated with syndromes with ASD symptoms; and 4) high confidence CNVs that did not overlap more than 20% with controls in available databases. We explored recurrence rates, genic content, regulatory elements, inheritance patterns and phenotypic correlations.
- In vitro activity of tigecycline and resistance mechanisms due to efflux pump production in Gram negative isolates, in PortugalPublication . Manageiro, Vera; Almeida, Joana; Pires, Sofia; Jones-Dias, Daniela; Ferreira, Eugénia; Caniça, ManuelaBackground: Tigecycline is increasingly used to treat infections caused by Gram negative isolates, because these bacteria express multidrug-resistance (MDR) phenotypes in an escalating frequency. Thus, this study aimed: 1) to correlate mediated β-lactam and fluoroquinolone resistance mechanisms (RMs) among Gram-negative isolates causing untreatable bacterial infections nowadays; 2) to study the in vitro activity of tigecycline to identify the importance of this antibiotic; 3) and to characterize tigecycline resistance mechanisms (RM) in order to understand how to extend its activity. Material/methods: A total of 2560 unduplicated Gram-negative (2309 Enterobacteriaceae and 251 Acinetobacter baumannii) isolates, collected in healthcare facilities in Portugal (2009-2013), were studied. MICs of tigecycline were determined against all isolates, and interpreted according to EUCAST (Enterobacteriaceae) or CLSI (A. baumannii, Ab) guidelines. Antimicrobial susceptibility of other 17 antibiotics was determined to all clinical isolates by disk diffusion method and interpreted by EUCAST. Fluoroquinolone and β-lactam RMs were reached by PCR and sequencing of 623 Enterobacteriaceae and 133 Ab using specific primers targeting PMQR-, Class A- and D β-lactamase-, Class B/MBL- and PMAβ-encoding genes. Tigecycline resistance due to efflux pump production was studied by molecular methods: ramR gene of Klebsiella pneumoniae and marR gene of Escherichia coli isolates. Results: We identified 27.2%/35.8% and 96.4%/96.2% tigecycline non-susceptible/MDR Enterobacteriaceae and Ab isolates, respectively. The molecular analyses of tigecycline RMs revealed deletions/insertions/point mutations in the ramR gene that might contribute to the overexpression of AcrAB efflux pump in 63 out of 108 K. pneumoniae isolates showing reduced susceptibility to tigecycline. Point mutations observed in marR gene from E. coli isolates with or without tigecycline resistance, might contribute to MDR scenarios. A great diversity of β-lactamases was observed in Enterobacteriaceae isolates: penicillinases, ESBLs (CTX-M-1/-14/-15/-32/-G1-type/-G2-type, TEM-4/-10, SHV-12/-55, GES-7), carbapenemases (KPC-3, GES-5/-6, OXA-48, VIM-2/-34) and PMAβ (CMY-2, DHA-1, MIR-type, ACT-type); Enterobacteriaceae were fully susceptible to tigecycline for: 48.3% of β-lactamase producers; 65.6% carrying PMQR determinants; 69.1% presenting both RMs. Concerning Ab all 133 isolates tested (3.8% tigecycline susceptible) expressed an acquired carbapenemase (OXA-23 and/or OXA-24). Conclusions: This study showed that tigecycline remains a substrate of MDR Ab isolates. However, the in vitro susceptibility of Enterobacteriaceae isolates to tigecycline showed its decisive importance when these bacteria presented resistance to other antibiotic classes, specifically through plasmid-mediated β-lactam and fluoroquinolone RMs. Thus, tigecycline contribution against the most important MDR Gram-negative pathogens should be preserved principally to bacterial infections untreatable by other antibiotics (such as carbapenems and colistin).
- Investigating antimicrobial resistance in soil culturable Gram negative bacteriaPublication . Jones-Dias, Daniela; Ferreira, Eugénia; Almeida, Joana; Caniça, ManuelaObjectives: Many calls have been made regarding resistome investigation but only a small number of studies were focused on the role of environmental antibiotic resistance. The soil may be a source of mobile antibiotic resistance genes, but this knowledge needs to be assessed extensively, directing this investigation to resistance mechanisms that are relevant in the clinical setting, such as the production of β-lactamases, which constitutes the main antimicrobial resistance mechanism in clinical Gram negative bacteria. This work aimed to assess the soil resistome and determine whether different agricultural practices influenced the diversity of antibiotic resistant isolates and mechanisms. Methods: Nine soil samples were collected from intensive (n=3), extensive (n=3), and organic (n=3) agricultural settings, in July 2012 and February 2013, covering the two main seasons; GPS coordinates, temperature and humidity values were recorded for all sampling sites. Then, soil samples were incubated in Brain Heart Infusion (BHI) enrichment broth followed by selection of resistant Gram negative bacteria in MacConkey agar plates containing 2μg/μl of cefotaxime (CTX), imipenem (IMP) or 100μg/μl amoxicillin (AMX). The isolates were then identified through 16S rDNA universal sequencing or API20E/NE and evaluated in what regards their antimicrobial susceptibility by standard disk diffusion method against 32 antibiotics from different classes, according to the nonspecific French Society of Microbiology guidelines. The molecular screening for the main mobile β-lactam resistance genes (Ambler class A, B, C, D β-lactamase-encoding genes) and genetic elements (class 1, 2 and 3 integrons) was performed by PCR and further sequencing. Results: Overall, we collected 232 Gram negative resistant isolates distributed mainly among Achromobacter, Ochrobactrum, Pseudomonas, Stenotrophomonas, Enterobacteriaceae groups. Among those, 27.5% of the antibiotic resistant Isolates were recovered from organic agriculture, 26.3% from extensive agriculture and 25.4% from intensive agriculture. The great majority of the isolates gathered were multidrug resistant (85.8%) among which β-lactam antibiotics, cloramphenicol, nitrofurantoin and fosfomycin were the main contributors. These isolates were mostly susceptible to aminoglycosides (83.6%), specifically amikacin (95.7%). The presence of synergy between β-lactam antibiotics and β-lactamase inhibitors frequently suggested the production of metallo β-lactamases, AmpC but also extended-spectrum β-lactamases. The molecular screening detected the occurrence of mobile resistance genes and genetic elements such as blaTEM, dfrA, aadA and class 1 integrons in Enterobacteriaceae and Pseudomonadaceae isolates. Conclusions: The results gathered so far showed that there was a high level of multidrug resistance in soil bacteria, independent on the anthropogenic actions directly related to soil use. The report of mobile antibiotic resistance genes highlights the spread of resistance genes by mobile genetic elements and the possibility of shared resistance genes between environmental and clinical bacteria. Indeed, the evaluation of environmental resistant bacteria is an important assignment that continuously contributes to the anticipation of resistant clinically important pathogens.
- Molecular survey of carbapenem resistant Enterobacteriaceae isolates from Portuguese Hospitals: co-production of carbapenemase KPC-3 and the efflux pump OqxABPublication . Manageiro, Vera; Ferreira, Eugénia; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Antimicrobial Resistance Surveillance Program in Portugal (ARSIP); Caniça, ManuelaObjectives: Although there are important studies regarding the different carbapenemase (CARB)-producing Gram-negative bacteria, little is known concerning their molecular epidemiology in Portugal. The main aim of this study was to characterize, by phenotype and molecular typing methods, CARB-producing Enterobacteriaceae isolates recovered from Portuguese health care institutions, and evaluate its impact on treatment strategy. Methods: This study included 2105 clinical isolates, collected between April/2006 and February/2013, in different Portuguese healthcare institutions. Screening of antimicrobial susceptibility was performed by disc diffusion method. Clinical isolates with resistance or with decreased susceptibility to ertapenem were considered presumptively CARB-producers; in these isolates, PCR and sequencing were applied to detect and identify CARB-encoding genes, as well as other bla and plasmid-mediated quinolone resistance (PMQRs) genes. MICs of CARB-producing isolates were tested by microdilution (EUCAST breakpoints). The plasmids obtained from clinical isolates were characterized by PCR-based replicon typing (PBRT). Clonal relatedness of K. pneumoniae isolates was investigated by multilocus sequence typing (MLST), using the protocol developed by the Institute Pasteur (www.pasteur.fr/mlst/Kpneumoniae.html). Results: Among the 2105 isolates tested, 165 (7.8%) were putative CARB-producers and were selected for further analysis. Thirty-five (21.2%) of the 165 positive isolates were confirmed to be CARB-producers, of which the majority were collected from the urine (54.3%) of elderly (≥65 years old) male patients (54.3%), and admitted at the emergency room/ambulatory (22.9%) or internal medicine (17.1%) wards. All were multidrug-resistant, with nonsusceptibility to at least one carbapenem, and with consistent susceptibility only to colistin. In those isolates was detected the following beta-lactamases: 30 KPC-3 (22 K. pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes and 3 Enterobacter clocae), 4 GES-5 (K. pneumoniae) and one VIM-2 (Klebsiella oxytoca). CARB-encoding genes were present alone or in combination with other bla genes, such as blaSHV-12, blaSHV-14, blaSHV-26, blaSHV-36, blaCTX-M-15, and the blaSHV-164. PMQR-encoding genes were also detected, namely qnrA, qnrB, aac(6’)-Ib-cr and the recently identified oqxAB. All blaKPC-3 genes were located on a Tn3-based transposon, Tn4401, while blaGES-5 and blaVIM-2 genes were associated with class 3 and 1 integrons, respectively. In our study, the majority of the blaCARB-harbouring plasmids were nonconjugative, having been typed as IncFrepB by PBRT. Clonal relatedness of the 26 K. pneumoniae isolates, obtained by MLST, showed that they were from distinct STs, namely ST14, ST15, ST34, ST59, ST147, ST416, ST698, and from the two novels ST: ST960 and, among all, the predominant ST1138 (corresponding to KPC-3 plus SHV-36 producers). Conclusion: In conclusion, this study provides new data regarding the molecular epidemiology of CARB-producing Enterobacteriaceae, which appears to be widespread in Portugal. Dissemination of blaCARB seems to be due to carriage of similar CARB-harbouring plasmids within genetically diverse clinical strains. Overall, our results emphasize the need of a concerted action to manage carbapenem use.
- Phenotypic categorization of putative pathogenic CNVs in a population of Autism Spectrum Disorder patientsPublication . Conceição, Inês C.; Correia, Catarina; Oliveira, Bárbara; Rama, Maria M.; Café, Cátia; Almeida, Joana; Mouga, Susana; Duque, Frederico; Oliveira, Guiomar; Vicente, A.M.
- Polygenic transmission disequilibrium confirms that common and rare variation act additively to create risk for autism spectrum disordersPublication . Weiner, Daniel J.; Wigdor, Emilie M.; Ripke, Stephan; Walters, Raymond K.; Kosmicki, Jack A.; Grove, Jakob; Samocha, Kaitlin E.; Goldstein, Jacqueline I.; Okbay, Aysu; Bybjerg-Grauholm, Jonas; Werge, Thomas; Minshew, Nancy; Merikangas, Alison; McMahon, William M.; McGrew, Susan G.; Ladd-Acosta, Christine; Mattheisen, Manuel; Hougaard, David M.; Jacob, Suma; Bishop, Somer; Iliadou, Bozenna; Arking, Dan E.; Hultman, Christina M.; Hertz-Picciotto, Irva; Hendren, Robert; Hansen, Christine S.; Haines, Jonathan L.; Guter, Stephen J.; Grice, Dorothy E.; Green, Jonathan M.; Taylor, Jacob; Mortensen, Preben Bo; Skuse, David; Green, Andrew; Goldberg, Arthur P.; Gillberg, Christopher; Gilbert, John; Gallagher, Louise; Freitag, Christine M.; Fombonne, Eric; Folstein, Susan E.; Fernandez, Bridget; Fallin, M Daniele; Devlin, Bernie; Børglum, Anders D.; Bækvad-Hansen, Marie; Ercan-Sencicek, A Gulhan; Ennis, Sean; Duque, Frederico; Duketis, Eftichia; Delorme, Richard; De Rubeis, Silvia; De Jonge, Maretha V.; Dawson, Geraldine; Anney, Richard; Cuccaro, Michael L.; Smith, George Davey; Correia, Catarina T.; Dumont, Ashley; Conroy, Judith; Conceição, Inês C.; Chiocchetti, Andreas G.; Celestino-Soper, Patrícia B.S.; Casey, Jillian; Cantor, Rita M.; Mattheisen, Manuel; Café, Cátia; Brennan, Sean; Daly, Mark J.; Bourgeron, Thomas; Bolton, Patrick F.; Hansen, Christine; Bölte, Sven; Bolshakova, Nadia; Betancur, Catalina; Bernier, Raphael; Sanders, Stephan J.; Beaudet, Arthur L.; Battaglia, Agatino; Bal, Vanessa H.; Robinson, Elise B.; Baird, Gillian; Bailey, Anthony J.; Bækvad-Hansen, Marie; Hansen, Thomas F.; Bader, Joel S.; Bacchelli, Elena; Nordentoft, Merete; Anagnostou, Evdokia; Amaral, David; Almeida, Joana; Buxbaum, Joseph D.; Moran, Jennifer; Chakravarti, Aravinda; Cook, Edwin H.; Coon, Hilary; Geschwind, Daniel H.; Howrigan, Daniel; Nørgaard-Pedersen, Bent; Gill, Michael; Hakonarson, Hakon; Hallmayer, Joachim; Palotie, Aarno; Santangelo, Susan; Mors, Ole; Sutcliffe, James S.; Lowe, Jennifer K.; Poterba, Timothy; Martsenkovsky, Igor; Poulsen, Jesper; Stevens, Christine; Anttila, Verneri; Holmans, Peter; Huang, Hailiang; Klei, Lambertus; Lee, Phil H; Medland, Sarah E.; Neale, Benjamin; Lord, Catherine; Martin, Donna M.; Weiss, Lauren A.; Zwaigenbaum, Lonnie; Yu, Timothy W.; Wittemeyer, Kerstin; Willsey, A Jeremy; Wijsman, Ellen M; Wassink, Thomas H.; Waltes, Regina; Walsh, Christopher A.; Wallace, Simon; Mane, Shrikant M.; Levitt, Pat; Vorstman, Jacob A.S.; Vieland, Veronica J.; Vicente, Astrid M.; van Engeland, Herman; Tsang, Kathryn; Thompson, Ann P.; Szatmari, Peter; Svantesson, Oscar; Steinberg, Stacy; Magnusson, Pall; Stefansson, Kari; Martin, Christa Lese; Stefansson, Hreinn; State, Matthew W.; Soorya, Latha; Silagadze, Teimuraz; Scherer, Stephen W.; Schellenberg, Gerard D.; Sandin, Sven; Saemundsen, Evald; Magalhaes, Tiago; Rouleau, Guy A.; Rogé, Bernadette; Ledbetter, David H.; Roeder, Kathryn; Roberts, Wendy; Reichert, Jennifer; Reichenberg, Abraham; Rehnström, Karola; Regan, Regina; Poustka, Fritz; Maestrini, Elena; Poultney, Christopher S.; Piven, Joseph; Pinto, Dalila; Leboyer, Marion; Pericak-Vance, Margaret A.; Pejovic-Milovancevic, Milica; Pedersen, Marianne G.; Pedersen, Carsten B.; Paterson, Andrew D.; Parr, Jeremy R.; Kolevzon, Alexander; Pagnamenta, Alistair T.; Oliveira, Guiomar; Nurnberger, John I.; Nordentoft, Merete; Le Couteur, Ann S.; Murtha, Michael T.; Mouga, Susana; Mors, Ole; Morrow, Eric M.; De Luca, Daniel Moreno; Klauck, Sabine M.; Monaco, Anthony P.Autism spectrum disorder (ASD) risk is influenced by common polygenic and de novo variation. We aimed to clarify the influence of polygenic risk for ASD and to identify subgroups of ASD cases, including those with strongly acting de novo variants, in which polygenic risk is relevant. Using a novel approach called the polygenic transmission disequilibrium test and data from 6,454 families with a child with ASD, we show that polygenic risk for ASD, schizophrenia, and greater educational attainment is over-transmitted to children with ASD. These findings hold independent of proband IQ. We find that polygenic variation contributes additively to risk in ASD cases who carry a strongly acting de novo variant. Lastly, we show that elements of polygenic risk are independent and differ in their relationship with phenotype. These results confirm that the genetic influences on ASD are additive and suggest that they create risk through at least partially distinct etiologic pathways.
- Predominance of KPC-3 in a Survey for Carbapenemase-Producing Enterobacteriaceae in PortugalPublication . Manageiro, Vera; Ferreira, Eugénia; Almeida, Joana; Barbosa, Stephanie; Simões, Constança; Bonomo, Robert A.; Caniça, ManuelaAmong the 2,105 Enterobacteriaceae tested in a survey done in Portugal, 165 were nonsusceptible to carbapenems, from which 35 (26 Klebsiella pneumoniae, 3 Escherichia coli, 2 Enterobacter aerogenes, and 3 Enterobacter cloacae isolates and 1 Klebsiella oxytoca isolate) were confirmed to be carbapenemase producers by the presence of 30 Tn4401d-blaKPC-3, 4 intI3-blaGES-5, and one intI1-blaVIM-2 gene, alone or in combination with other bla genes. The dissemination of blaKPC-3 gene carried by an IncF plasmid suggests lateral gene transfer as a major mechanism of dissemination.
- Relevance of Common and Rare CNVs for Autism EtiologyPublication . C. Conceição, Inês; Correia, Catarina; Oliveira, Bárbara; Rama, Maria Margarida; Café, Cátia; Almeida, Joana; Mouga, Susana; Duque, Frederico; Oliveira, Guiomar; M. Vicente, AstridRecent reports by the Autism Genome Project (AGP) consortium and other groups show that Copy Number Variants (CNVs), while individually rare, collectively may explain a large fraction of the etiology of Autism Spectrum Disorders (ASD). The goal of this study was to establish the clinical and etiological relevance for ASD of potentially pathogenic CNVs identified in a Portuguese population sample by whole genome CNV analysis, through the detailed characterization of CNVs and correlation with clinical phenotypes. Analysis of the Autism Genome Project genome-wide CNV results using 1M SNP microarray1 identified a total of 14218 CNVs in 342 Portuguese probands. We selected 292 CNVs, present in 191 individuals (19 females and 172 males), using the following criteria: 1) CNVs that contained implicated/candidate genes for ASD; 2) CNVs in genomic regions known to be implicated/candidate for ASD; 3) CNVs containing genes associated with syndromes with ASD symptoms; and 4) high confidence CNVs that did not overlap more than 20% with controls in available databases. We explored recurrence rates, genic content, regulatory elements, inheritance patterns and clinical correlations
- SHANK2 mutations: synapse homeostasis in Autism Spectrum Disorders (ASD)Publication . Oliveira, Bárbara; Correia, Catarina; Conceição, Inês; Almeida, Joana; Café, Cátia; Oliveira, Guiomar; M Vicente, AstridASD are a heterogeneous group of neurodevelopmental disorders presenting a complex inheritance pattern. The genetic causes of ASD are diverse, but the majority of genes previously implicated participate in the development and function of neuronal circuits. Mutations in genes encoding synaptic cell adhesion molecules and scaffolding proteins, such as neuroligins (NLGN), neurexins (NRXN) and the SHANK family, have been recurrently reported in patients with ASD. SHANK2 encodes a scaffolding protein located in the postsynaptic density (PSD) of glutamatergic synapses, and mutations in ProSAP1/SHANK2 have been recently associated with both ASD and intellectual disability.