Browsing by Author "Pessanha, Maria Ana"
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- Antimicrobial Resistance and Biofilms Underlying Catheter-Related Bloodstream Coinfection by Enterobacter cloacae Complex and Candida parapsilosisPublication . Štefánek, Matúš; Wenner, Sigurd; Borges, Vítor; Pinto, Miguel; Gomes, João Paulo; Rodrigues, João; Faria, Isabel; Pessanha, Maria Ana; Martins, Filomena; Sabino, Raquel; Veríssimo, Cristina; Nogueira, Isabel D.; Carvalho, Patrícia Almeida; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections are a public health concern especially in the context of healthcare-associated infections such as catheter-related bloodstream infections (CRBSIs). We evaluated the biofilm formation and antimicrobials resistance (AMR) of Enterobacter cloacae complex and Candida parapsilosis co-isolated from a CRBSI patient. Antimicrobial susceptibility of central venous catheters (CVCs) and hemoculture (HC) isolates was evaluated, including whole genome sequencing (WGS) resistome analysis and evaluation of gene expression to obtain insight into their AMR determinants. Crystal violet assay was used to assess dual biofilm biomass and microscopy was used to elucidate a microorganism’s distribution within biofilms assembled on different materials. Bacteria were multidrug-resistant including resistance to colistin and beta-lactams, likely linked to the mcr-9-like phosphoethanolamine transferase and to an ACT family cephalosporin-hydrolyzing class C beta-lactamase, respectively. The R398I and Y132F mutations in the ERG11 gene and its differential expression might account for C. parapsilosis resistance to fluconazole. The phenotype of dual biofilms assembled on glass, polystyrene and polyurethane depends on the material and how biofilms were initiated by one or both pathogens. Biofilms assembled on polyurethane were denser and richer in the extracellular polymeric matrix, and microorganisms were differently distributed on the inner/outer surface of the CVC.
- Biofilms and catheter related bloodstream infection: a tale of two kigdomsPublication . Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel; Faria, Isabel; Pessanha, Maria Ana; Verissimo, Cristina; Sabino, Raquel; Rodrigues, Joao; Matias, Rui; Martins, Filomena; Carvalho, Patricia; Gomes, Joao Paulo; Jordão, LuísaBackground: Biofilm-associated infections are a public health concern in the context of healthcare-associated infections (HAI) such as catheter-related bloodstream infections (CRBSI). Here, we studied two top ten CRBS etiological agents, Enterobacter cloacae and Candida parapsilosis, isolated from a patient with CRBSI in order to understand the role played by biofilms on this HAI. Materials/methods: E.cloacae and C.parapsilosis were isolated from CVC and peripheral blood by standard procedures. EUCAST guidelines were followed for antimicrobial susceptibility evaluation. Single and/or mixed biofilms were assembled on different materials in Mueller-Hinton broth with 2% glucose. Biofilm assembly was assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification and to assess microorganisms distribution within the biofilm (3D reconstruction). In addition, Focus Ion Beam (FIB)-SEM was used to assess biofilms assembled on inner and outer surfaces of CVCs and construct tomograms. CVC and hemoculture (HC) isolates were subjected to whole-genome sequencing (WGS). Results: All Enterobacter and Candida isolates were antimicrobial resistant. Of note, E. cloacae-CVC revealed an additional resistance (ceftolozame-tazobactam) in comparison to the HC- isolate. Both microorganisms assembled biofilms on glass, polystyrene and polyurethane. Mixed biofilms were denser when both microorganisms were present from the beginning. Biofilm phenotype was not dependent of biofilm initiation by E.cloacae or C.parapsilosis. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Environmental isolates of the same species were also tested, showing that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), we observed denser mixed biofilms richer in EPS. FIB-SEM preliminary results suggest that biofilms assembled on inner and outer catheter surface might differ on microorganisms’ distribution. WGS confirmed the genetic identity of the CVC/HC pairs while corroborating the virulence potential and antimicrobial resistant character of the CRBSI-driving pathogens. Conclusions: The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Biofilms within the human body and its clinical implicationsPublication . Jordão, Luísa; Subtil, João; Lavado, Paula; Rodrigues, João; Reis, Lúcia; Faria, Isabel; Pessanha, Maria AnaBiofilms with medical implications could be find on medical devices or on organs. Here we discuss the results obtained in two studies one associated with an organ (adenoid) and another associated with a medical device (central venous catheters- CVC). In the first study, we evaluate the association between biofilm assembly on adenoids and the incidence of recurrent infections in a paediatric population comparing adenoid samples from adenoidectomy groups with and without infectious indication. Biofilms were present in 27.4% of the adenoid samples. For H. influenzae, S. aureus, S. pyogenes, S. pneumococcus and M. catarrhalis, no association was found between ability to assemble biofilms in vitro and the presence of biofilms on adenoids, and the same was found for antibiotic resistance. The most isolated bacterium was H. influenzae that revealed after further characterization to be non-typeable (NT). No statistical difference was found on biofilm presence between the two groups, infectious versus non-infectious diagnosis. The same was true for biofilm assembling ability of bacteria found on adenoid surface and core. As in other studies, we did not find a correlation between biofilm formation and susceptibility or resistance to antibiotics and this raise the question of the importance of biofilms on the pathogenesis of infectious diseases. In the second study, we explore the relation between the presence of biofilms on central venous catheters and central venous catheter-related bloodstream infection (CRBSI). Our preliminary results (relative to data collected over 10 months) show that Staphylococci, either coagulase negative or positive, are major etiologic agents of this healthcare associated infection.
- Catheter related bloodstream infection caused by E. cloacae and Candida parapsilosis: Are biofilms guilty?Publication . Štefánek, Matúš; Borges, Vítor; Wenner, Sigurd; Nogueira, Isabel D.; Pinto, Miguel; Faria, Isabel; Pessanha, Maria Ana; Veríssimo, Cristina; Sabino, Raquel; Rodrigues, João; Matias, Rui; Carvalho, Patrícia Almeida; Gomes, João Paulo; Bujdáková, Helena; Jordao, LuisaBiofilm-associated infections is a public health concern in the context of healthcare associated infections (HAI) such as catheter related bloodstream infections (CRBSI). Here the dynamics of two top ten etiological agents of CRBSI, Enterobacter cloacae and Candida parapsilosis isolated from a CRBSI’s patient, were studied to get insights on the role played by biofilms on this HAI. Antimicrobial susceptibility of CVC and HC’s isolates was evaluated according to EUCAST guidelines. Single and/or mixed biofilms assembled on different materials in Mueller-Hinton broth with 2% glucose were assessed by crystal violet assay and scanning electron microscopy (SEM). Fluorescence in situ hybridization (FISH) was used for identification purposes and to assess microorganisms distribution within the biofilm (3D reconstruction) complemented with Focus Ion Beam (FIB)-SEM to assess biofilms assembled on the inner/outer CVC’s surfaces (tomograms). Whole-genome sequencing (WGS) was performed for all isolates. All isolates were antimicrobial resistant. Of note E.cloacae resistance to collistin and an additional resistance of the CVC compared to HC-isolate (ceftolozame-tazobactam) probably linked to a mutation in rpoB gene. Candida resistance to fluconazol might be explained by ERG11 gene mutation. Enterobacter and Candida assembled biofilms on glass, polystyrene and polyurethane being mixed biofilms denser when both microorganism were present from the beginning. FISH and SEM analysis showed that biofilm bottom layer was in all cases richer in E.cloacae. Using environmental isolates of the same species we showed that this biofilm phenotype is not a general feature. Using polyurethane catheters (shape/material factor), denser mixed biofilms richer in EPS were observed. A distinct phenotype was present on the patient’s CVC by SEM and FIB/SEM. WGS confirmed the genetic identity of the pair CVC/HC isolates, while corroborating the virulence potential and observed antimicrobial resistant character of the studied CRBSI-driving pathogens. The results suggest that biofilms allow interaction and adaptation of microorganisms belonging to different kingdoms (Bacteria and Fungi). Adaptation might affect virulence in a transitory or permanent fashion, with potential impact on microorganisms’ potential to cause CRBSI.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; José Borrego, MariaObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990-2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Distribution of Chlamydia trachomatis ompA-genotypes over three decades in PortugalPublication . Lodhia, Zohra; Cordeiro, Dora; Correia, Cristina; João, Inês; Carreira, Teresa; Vieira, Luís; Nunes, Alexandra; Ferreira, Rita; Schäfer, Sandra; Aliyeva, Elzara; Portugal, Clara; Monge, Isabel; Pessanha, Maria Ana; Toscano, Cristina; Côrte-Real, Rita; Antunes, Marília; Gomes, Joao Paulo; Borges, Vítor; Borrego, Maria JoséObjectives: Chlamydia trachomatis is classified into 15 major genotypes, A to L3, based on the diversity of ompA gene. Here, we evaluated and characterised the distribution and diversity of ompA-genotypes over 32 years (1990–2021) in Portugal. Methods: The collection of the Portuguese National Reference Laboratory for Sexually Transmitted Infections includes 5824 C. trachomatis-positive samples that were successfully ompA-genotyped between 1990 and 2021. An in-depth analysis of ompA-genotypes distribution across the years, as well as by biological sex, age and anatomical site of infection was performed. Results: ompA-genotype E was consistently the most frequently detected across the years, with a median frequency of 34.6%, followed by D/Da (17.6%), F (14.3%) and G (10.7%). The prevalence of lymphogranuloma venereum (LGV) genotypes (mostly L2, 62.0%, followed by L2b, 32.1%) increased since 2016, reaching the highest value in 2019 (20.9%). LGV, G and Da genotypes were associated with biological sex, specifically with being male, and were the most frequent among anorectal specimens (37.7%, 19.4% and 17.7%, respectively). Notably, LGV ompA-genotypes represented 38.9% of the male anorectal specimens since 2016, and were also detected among oropharynx and urogenital samples. ompA-genotype E was the most frequently detected at the oropharynx (28.6%) and urogenital (33.9%) sites during the study period, followed by D/Da (17.4%) and F (16.0%) in the urogenital specimens, and by G (26.1%) and D/Da (25.7%) in oropharynx specimens. Our data also highlight the emergence of the recombinant L2b/D-Da strain since 2017 (representing between 2.0% and 15.5% of LGV cases per year) and the non-negligible detection of ompA-genotype B in urogenital and anorectal specimens. Conclusions: This study provides a comprehensive landscape of C. trachomatis molecular surveillance in Portugal, highlighting the continued relevance of ompA-genotyping as a complement to rapid LGV-specific detection tests. It also contributes to a deeper understanding of C. trachomatis epidemiology, diversity and pathogenicity.
- Epidemiology and genetic variability of respiratory syncytial virus in Portugal, 2014-2018Publication . Sáez-López, Emma; Cristóvão, Paula; Costa, Inês; Pechirra, Pedro; Conde, Patrícia; Guiomar, Raquel; Peres, Maria João; Viseu, Regina; Lopes, Paulo; Soares, Vânia; Vale, Fátima; Fonseca, Patricia; Freitas, Ludivina; Alves, Jose; Pessanha, Maria Ana; Toscano, Cristina; Mota-Vieira, Luísa; Veloso, Rita Cabral; Côrte-Real, Rita; Branquinho, Paula; Pereira‑Vaz, João; Rodrigues, Fernando; Cunha, Mário; Martins, Luís; Mota, Paula; Couto, Ana Rita; Bruges-Armas, Jácome; Almeida, Sofia; Rodrigues, Débora; Portuguese Laboratory Network for the Diagnosis of Influenza InfectionIntroduction: Respiratory syncytial virus (RSV) is associated with substantial morbidity and mortality since it is a predominant viral agent causing respiratory tract infections in infants, young children and the elderly. Considering the availability of the RSV vaccines in the coming years, molecular understanding in RSV is necessary. Objective: The objective of the present study was to describe RSV epidemiology and genotype variability in Portugal during the 2014/15-2017/18 period. Material and methods: Epidemiological data and RSV-positive samples from patients with a respiratory infection were collected through the non-sentinel and sentinel influenza surveillance system (ISS). RSV detection, subtyping in A and B, and sequencing of the second hypervariable region (HVR2) of G gene were performed by molecular methods. Phylogenetic trees were generated using the Neighbor-Joining method and p-distance model on MEGA 7.0. Results: RSV prevalence varied between the sentinel (2.5%, 97/3891) and the non-sentinel ISS (20.7%, 3138/16779), being higher (P < 0.0001) among children aged <5 years. Bronchiolitis (62.9%, 183/291) and influenza-like illness (24.6%, 14/57) were associated (P < 0.0001) with RSV laboratory confirmation among children aged <6 months and adults ≥65 years, respectively. The HVR2 was sequenced for 562 samples. RSV-A (46.4%, 261/562) and RSV-B (53.6%, 301/562) strains clustered mainly to ON1 (89.2%, 233/261) and BA9 (92%, 277/301) genotypes, respectively, although NA1 and BA10 were also present until 2015/2016. Conclusion: The sequence and phylogenetic analysis reflected the relatively high diversity of Portuguese RSV strains. BA9 and ON1 genotypes, which have been circulating in Portugal since 2010/2011 and 2011/2012 respectively, predominated during the whole study period.
- Estudo observacional prospetivo de infeções associadas a cuidados de saúde relacionadas com cateteres venosos centrais em três hospitais terciários de LisboaPublication . Duarte, Maria; Faria, Isabel; Pessanha, Maria Ana; Duarte, Aida; Martins, Filomena; Jordão, LuísaAs Infeção Associada aos Cuidados de Saúde (IACS) constituem um importante problema de saúde publica que afeta 4 milhões de pacientes/ano na Europa e contribuiu para o aumento dos gastos em saúde. As infeções da corrente sanguínea associadas a cateteres venosos centrais (ICSCVC) são um grupo particularmente importante de IACS, com maior incidência (87%) nas unidades de cuidados intensivos (UCI) onde são responsáveis por 23% dos óbitos. Este estudo observacional prospetivo, visa documentar a ocorrência de ICVCVC, está em curso há 2 anos e até à data identificou 40 casos. O agente etiológico (identificação e perfil de resistência aos antimicrobianos), unidade de saúde (dimensão, serviço), paciente (idade, proveniência, antibioterapia) e CVC (local de inserção, tempo de permanência no paciente, material) foram caraterizados.
- Infeção sanguinea associada a cateteres venosos centrais: um estudo observacional prospetivoPublication . Faria, Isabel; Pessanha, Maria Ana; Duarte, Aida; Martins, Filomena; Jordao, LuisaNos últimos anos, as infeções associadas aos cuidados de saúde (HAIs) têm sido referenciadas como um importante problema de saúde pública. A infeção sanguínea associada aos cateteres venosos centrais (CRBSI) é uma HAIs responsável por elevadas taxas de morbilidade e mortalidade em pacientes críticos; ou seja, pacientes internados em unidades de cuidados intensivos. Além do risco de vida para o paciente, estas infeções têm impacto económico contribuindo para aumentar os custos associados ao internamento. No último relatório divulgado pelo ECDC o Staphylococcus spp foi identificado como o principal agente etiológico de CRBSI, sendo os Staphylococcus coagulase negativo (como S. epidermidis) e S. aureus responsáveis por 25,3% e 12,1% dos casos, respetivamente. A formação de biofilmes nos cateteres venosos centrais (CVC) constitui um fator de risco adicional. Uma vez que as infeções associadas a biofilmes são mais resistentes ao tratamento com antibióticos, bem como à própria resposta imunitária do hospodeiro pode-se evoluir para uma condição crónica. O presente estudo tem como objetivos documentar a ocorrência de CRBSIs, determinar quais são os seus agentes etiológicos mais frequentes e o papel desempenhado pela formação de biofilmes nos CVCs no estabelecimento e progressão destas infeções. Para tal, foi desenhado um estudo observacional prospetivo que está a decorrer desde março de 2017 num centro hospitalar localizado em Lisboa, Portugal. Os resultados preliminares aqui apresentados são referentes às amostras colhidas entre março e novembro de 2017. Neste período foram recolhidos 41 CVCs com cultura positiva. Em 19 casos o mesmo microrganismo foi isolado simultaneamente do CVC e da hemocultura confirmando a existência de uma CRBSI. Os microrganismos isolados foram os seguintes: S. aureus (n=8), S. epidermidis (n=5), Enterobacter (n=2), K. pneumoniae (n=2), P. aeruginosa (n=1) e C. parapsilosis (n=1). Estes resultados estão de acordo com os publicados pelo ECDC que identificam o S. aureus e o S. epidermidis como principais agentes etiológicos. A média de idades dos casos de CRBSI é de 65 anos confirmando a maior propensão de indivíduos com o sistema imunitário diminuído para contraírem este tipo de infeção. Numa próxima fase iremos analisar, por microscopia eletrónica de varrimento os CVC, para determinar se existem biofilmes nos mesmos e averiguar a possibilidade de existir uma infeção associada aos biofilmes. Uma caracterização detalhada do microrganismo isolado do sangue e do CVC será também efetuada de forma a identificar eventuais fatores que predisponham para a ocorrência deste tipo de infeções.
- Insights on catheter-related bloodstream infections: a prospective observational study on the catheter colonization and multi-drug resistancePublication . Pinto, Miguel; Borges, Vítor; Nascimento, Maria; Martins, Filomena; Pessanha, Maria Ana; Faria, Isabel; Rodrigues, João; Matias, Rui; Gomes, João Paulo; Jordao, LuisaBackground: Central venous catheter-related bloodstream infection (CRBSI) is a huge public health concern with considerable impact on mortality and health costs. Aim: A three-year observational study enrolling three tertiary hospitals located in Lisbon, Portugal, was designed to identify the major aetiological agents of CRBSI, their ability to colonize central venous catheters and their antimicrobial resistance profiles. Methods: Aetiological agents of CRBSI were identified by Vitek 2. Whole-genome sequencing was used to confirm CRBSI by the most prevalent aetiological agents and characterize their resistome. Central venous catheter colonization (namely by biofilm assembly) was monitored by scanning electron microscopy. Findings: Staphylococci were the most prevalent causative agent (36/58, 62.0%), with S. aureus and coagulase-negative S. epidermidis accounting for 24.1% and 36.2% of CRBSIs, respectively. Fifty-nine of 72 staphylococci isolates were meticillin resistant. Comparative genomic analysis of central venous catheters/haemoculture pairs of isolates revealed genomic matches for 35 of 36 pairs and a good correlation between antibiotic susceptibility phenotype and the presence of antimicrobial resistance genetic determinants. Biofilms were present on 48.6% of the central venous catheters; nevertheless, no statistically significant association was established between biofilm assembly and CRBSI, and the presence/absence of ica operon and agr groups did not correlate with biofilm phenotypes, highlighting the need for further studies to elucidate biofilms' role on this healthcare-associated infection. Conclusion: Whole-genome sequencing was shown to be a valuable tool to confirm CRBSI. Although more than 42.3% of the central venous catheters were colonized by staphylococci, no statistically significant association was found between CRBSI and biofilms.