DDI - Apresentações orais em encontros internacionais
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- Pathogenic Fungi: an unacknowledged risk at coastal resorts? New insights on microbiological sand quality in PortugalPublication . Brandão, JoãoPathogenic Fungi: an unacknowledged risk at coastal resorts? New insights on microbiological sand quality in Portugal Raquel Sabino, Cristina Veríssimo, Célia Alves, Filipa C Ferreira, Maria Ana Cunha, Bela Wergikoski, Raquel Rodrigues, Helena Parada, Leonor Falcão, Laura Rosado, Catarina Pinheiro, Eleonora Paixão, João Brandão Affiliation: Reference Unit for Systemic Infections and Zoonosis - Department of Infectious Diseases, Instituto Nacional de Saúde/National Institute of Health Dr. Ricardo Jorge, Av. Padre Cruz 1649-016 Lisboa, Portugal (www.insa.pt) Contacts: E-mail: joao.brandao@insa.min-saude.pt, Tel: (+351) 217.519.247, Fax: (+351) 217.526.400, www.insa.pt Whilst the potential impact on beach users from microorganisms in water has received considerable attention, there has been relatively little investigation into microbial contaminants in sand. Between 2000 and 2002, a project involving several national institutions took place, aiming to define beach sand quality parameters and associated methods: “Microbiologic Quality of Coastal Beach Sands”. During this project we split Portugal into 5 regions and from each region 3 beaches were selected: One blue flag awarded (thus with documented good maintenance and water quality), one wild (with the least possible human intervention), and one with documented poor water quality. Samples were collected every 2 months for 13 months. Biological, biochemical and chemical parameters were studied. In 2006, Portugal initiated a voluntary sand quality microbiological monitoring programme through the Blue Flag association which lasted through 2010. With this activity data was collected which allowed to revise/update the initial project’s results: For this analysis, 33 beaches across Portugal were analyzed during a five year period (2006–2010) to determine the presence of yeasts, pathogenic fungi, dermatophytes, total coliforms, Escherichia coli and intestinal enterococci in sand. Our results showed that 60.4% of the samples were positive for fungi and that 25.2% were positive for the bacterial parameters. The most frequent fungal species found were Candida sp. and Aspergillus sp., whereas intestinal enterococci were the most frequently isolated bacteria. Positive associations were detected among analyzed parameters and country-regions but none among those parameters and sampling period. Regarding threshold values, we propose 15 cfu/g for yeasts, 17 cfu/g for potential pathogenic fungi, 8 cfu/g for dermatophytes. Twenty-five cfu/g for E. coli, and 10 cfu/g for intestinal enterococci.
- Molecular biology versus conventional methods – Complementary methodologies to understand occupational exposure to fungiPublication . Viegas, C.; Malta-Vacas, J.; Sabino, R.
- Accessing the molecular basis of transferable quinolone resistance in Escherichia coli and Salmonella spp from food-producing animals and productsPublication . Caniça, Manuela; Jones-Dias, Daniela; Francisco, Ana Patrícia; Manageiro, Vera; Ferreira, EugéniaBackground: Salmonella and Escherichia coli resistant to quinolones frequently arise in animals, being easily transferred to humans through the food chain, which can ultimately lead to the development of untreatable infectious diseases. The aim of the present study was to investigate the presence of PMQR determinants among Salmonella spp and E. coli from food-producing animals and derivative food products. Methods: Salmonella spp (n=183) and E. coli (n=182) isolates were collected from food-producing animals (n=274) and derivative food products (n=91). Antimicrobial susceptibility testing was performed by standard disk diffusion method, according to the CA-SFM veterinary guidelines. PCR and sequencing were used to detect PMQR- (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6’)-Ib-cr, and qepA) and β-lactamase-encoding genes (blaTEM, blaSHV, blaOXA and ampC) and to examine the QRDR of gyrA, gyrB, parC and parE genes in PMQR positive isolates. Plasmid characterization was accessed by conjugation followed by replicon-typing. Genetic relatedness of PMQR positive E. coli was examined by MLST and Salmonella isolates were serotyped according to the Kauffmann-White scheme. Mobile genetic elements were also investigated through PCR mapping assays. Results: Overall, 4.7% (17/365) harbored Qnr-encoding genes from qrnB and qnrS families, specifically qnrB2 (n=3), qrnB19 (n=3), and qnrS1 (n=11). All but one isolate presented at least one mutation in QRDR region of genes gyrA, parC or parE genes. 35.3% of Qnr-producing isolates presented resistance to β-lactam antibiotics that were justified by the presence of β-lactamases from TEM (TEM-1, n=10; and TEM-135, n=1) and SHV (SHV-108, n=1) families in QnrB19- and QnrS1-harbouring isolates. All but one Qnr-producing isolates were positively typed by replicon-typing, varying among IncN (n=2), IncFIB (n=11), IncFIC (n=3), IncI1 (n=2), IncHI2 (n=5), IncY (n=1) and IncL/M (n=3) and were, mostly, genetic unrelated. Qnr genes were detected nearby several mobile elements like ISEcl2, IS26 and ISCR1. Conclusions: This study illustrated the existence of Qnr-producing E. coli and Salmonella from food-producing animals, associated to specific mobile elements that can mediate their transference between species and among distinct settings. Epidemiology of PMQR mechanisms and the dissemination of plasmids carrying Qnr-encoding genes in veterinary isolates can compromise the efficacy of fluroquinolone treatments in both animals and humans.
- Infeção por H. pylori em endoscopia digestiva: evolução da prevalência e perfil clínicoPublication . Almeida, Mónica; Rodrigues, Teresa; Palha, Ana; Oleastro, Mónica; Lopes, Ana IsabelIntrodução: A diminuição da prevalência da infecção por H. pylori (Hp) tem sido reportada mundialmente, paralelamente à melhoria das condições socio‑económicas. Tanto quanto é do nosso conhecimento, não existem em Portugal estudos de prevalência em crianças sintomáticas submetidas a endoscopia digestiva. Objectivos: Constituíram objectivos deste estudo: avaliar a prevalência da infecção por Hp num período de 10 anos (3 anos representativos) numa amostra da população pediátrica Portuguesa sintomática submetida a endoscopia digestiva; descrever aspectos clínicos associados à infecção na mesma amostra. Métodos: Estudo descritivo e analítico retrospectivo. Efectuada revisão de 359 endoscopias diagnósticas realizadas em 2002, 2006 e 2011 num centro terciário de Gastroenterologia Pediátrica (Lisboa), idade ≤18 anos, com realização de biopsia gástrica, para avaliação da prevalência global de infecção e associação do status Hp com variáveis clínicas, indicações para realização de endoscopia, achados endoscópicos e histológicos. Considerou‑se status Hp(+) se histologia e/ou cultura positivas; status Hp(‑) se histologia e cultura simultaneamente negativas. Estatística: teste Qui‑quadrado, teste Exacto de Fisher; α=0,05. Resultados: A indicação mais frequente para endoscopia foi dor abdominal/ epigastralgias (53.8%); 175 (48,7%) crianças/jovens evidenciaram status Hp(+): 11,4% ≤5anos, 48% 5≤11anos, 40.6% 11≤18 anos. A prevalência média da infecção foi de 57.1% em 2002, 55.5% em 2006 e 41,3% em 2011, sendo a diminuição significativa no último período (p=0,02). Diminuição verificada nos três subgrupos etários, significativa apenas no subgrupo 5≤11 anos (p=0,042). Achados endoscópicos associados ao status Hp(+) VS Hp(‑): esófago normal (p=0,032), nodularidade antral/corpo (p<0,001) e úlcera duodenal (p=0,013); achados histológicos associados ao status Hp(+) VS Hp(‑): inflamação moderada (p<0,001), actividade ligeira e moderada (p<0,001) e presença de folículos/ agregados linfóides (p<0,005). Conclusões: Contrariamente a estudos efectuados em idêntico contexto noutras populações, constatou‑se uma prevalência ainda elevada da infecção por Hp, sugerindo embora uma tendência recente para a sua diminuição, principalmente em crianças em idade escolar. Alguns achados endoscópicos e histológicos associaram‑se de forma significativa à presença de infecção por Hp. Estes resultados enfatizam a relevância da infecção por Hp na população pediátrica Portuguesa sintomática e a necessidade de adopção de estratégias de abordagem com adequada relação custo‑efectividade
- The European Diphtheria Surveillance Network (ESDN) a strong model to combat a rare disease and use resources efficiently, share knowledge openly, and give support effectivelyPublication . Neal, Shona; Efstratiou, Androulla; The European Diphtheria Surveillance Network, EDSNBACKGROUND: As diphtheria is uncommon within the EU, complacency and minimal awareness is constantly observed. However, diphtheria is still seen sporadically in many EU countries and recent outbreaks have occurred worldwide. From the success of the EU-funded Diphtheria Network (DIPNET), the European Diphtheria Surveillance Network (EDSN) was established in 2010 under the auspices of ECDC, and continues to integrate epidemiologists and microbiologists. Microbiological activities have been tendered to the Health Protection Agency and managed as work packages; coordination; diagnostic external quality assurance (EQA) scheme; serology EQA; training workshops and molecular typing. The main objective is to strengthen laboratory diagnosis to ensure accurate and comparative diphtheria surveillance across Europe. METHODS: Thirty laboratory experts were nominated from the EU and EEA countries. Since February 2010, there have been two network meetings, two diagnostic EQAs, one serology EQA and two training workshops. RESULTS: The first diagnostic EQA generated unacceptable identification and toxigenicity reports of 14% and 10%, respectively from 26 countries. The two workshops provided the opportunity for non-DIPNET countries and those that performed poorly in the EQA to improve in diphtheria diagnostic methods; good feedback was received from all participants. Preliminary results of the second EQA revealed slight improvements in results where only 8% gave an unacceptable identification and 10% unacceptable toxigenicity reports (24 countries). CONCLUSIONS: All network members agree that EDSN should continue under ECDC, as this continues to provide the infrastructure essential for harmonised and improved public health management for diphtheria across the EU. However, EQA results emphasised that further training and EQA exercises should continue so as to maintain expertise, assess capabilities and aid standardisation for diphtheria diagnostics and ultimately surveillance.
- Characterization of Arcobacter butzleri isolates from poultry and slaughterhouse EnvironmentPublication . Ferreira, Susana; Fraqueza, Maria João; Queiroz, João; Domingues, Fernanda; Oleastro, MónicaThe genus Arcobacter is an emerging pathogen associated with several clinical symptoms, namely diarrhoea, abdominal pain or bacteraemia. This genus is widely distributed and has been isolated from environmental, animal, food and human samples, being poultry considered the major reservoir. In this study, forty three Arcobacter butzleri strains were isolated from poultry of three flocks from different farms and environment samples at a Portuguese slaughterhouse, also three reference strains were included. All isolates were confirmed at species level by multiplex PCR and genomic DNA fingerprints of all isolates were determined using Pulsed Field Gel Electrophoresis (PFGE) after enzymatic digestion with SmaI and the isolates with undistinguishable SmaI patterns were further analyzed with a second restriction enzyme, SacII. Phenotypic resistance profiles to nine antibiotics were assessed by broth microdilution method. Biofilm formation assays were performed in the 36 out of the 43 A. butzleri isolates, either presenting different pulsetypes or similar PFGE patterns but different origin or even different resistance patterns. PFGE patterns obtained using restriction enzymes SmaI and SacII revealed genetic diversity, with 32 distinct PFGE patterns. A high percentage of A. butzleri isolates was found to be resistant, recording four different resistance profiles. Twenty four of the 43 isolates presented a phenotypic resistance to ciprofloxacin, in contrast to the great susceptibility against gentamicin and chloramphenicol. Among the 36 selected A. butzleri isolates, 13.9% were categorized as moderately adherent, while 58.3% were defined as weakly adherent. Overall, the results showed a high degree of genetic heterogeneity among A. butzleri isolates, along with high levels of resistance to several antibiotics. Biofilm formation ability of A. butzleri can possibly favour dispersion and cross-contamination along the slaughterhouse processing line. These findings may represent a contribution to get insight the survival and even persistence mechanisms of this organism in the environment and on its relevance as a potential hazard for foodborne infections.
- Assessment of Francisella Tularensis in PortugalPublication . Lopes de Carvalho, I.; Carvalho, C.L.; Kingry, L.R.; Zé-Zé, Líbia; Petersen, J.M.; Núncio, M.S.
- Routine screening of harmful microorgansims in beach sands should be implemented: implications to public healthPublication . Brandão, JoãoBeaches worldwide provide recreational opportunities and economic industry to hundreds of millions of people. Beach water is often monitored for microbiological quality to detect the presence of indicators of human sewage contamination so as to prevent public health outbreaks associated with water contact. However, growing evidence suggests that beach sand can harbor microbes harmful to human health, often in concentrations greater than the beach water. Currently, there are no standards for monitoring, sampling, analyzing, or managing beach sand quality. In addition to indicator microbes, growing evidence has identified pathogenic bacteria, viruses, and fungi in a variety of beach sands worldwide. During a workshop of experts that convened in Lisbon, Portugal, it was determined that 1) beach sand should be screened for a variety of pathogens harmful to human health, and monitoring should then be initiated alongside water monitoring; 2) sampling and analysis protocols should be standardized to allow comparisons among beach locations; and 3) further studies are needed to estimate human health risk with exposure.
- 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.