Browsing by Author "Martins, Filomena"
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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.
- Epidemiology of Clostridioides difficile Infection in Portugal: A Retrospective, Observational Study of Hospitalized PatientsPublication . Nazareth, Cláudia; Leitão, Inês; Reis, Ernestina; Inácio, Hugo; Martins, Filomena; Ramalheira, Elmano; Cunha, Flávia; Santos, Carla; Lino, Sara; Moreira, Hugo; Kruptsala, Nadiya; Santos, Andrea; Paixão, Laura; Pássaro, Leonor; Oleastro, MónicaIntroduction: Clostridioides difficile is the main cause of healthcare-associated diarrhea in Europe and North America. The aim of this study was to characterize the epidemiology and clinical burden of Clostridioides difficile infection among hospitalized patients in Portugal. Material and methods: Retrospective study conducted in six public hospital centers in Portugal. All primary Clostridioides difficile infection episodes and related recurrences occurring in 2017, as well as episodes developing two to eight weeks after the last episode diagnosed in that year, were documented. The National Reference Laboratory (National Institute of Health Dr. Ricardo Jorge) provided national surveillance data on Clostridioides difficile infection. Results: A total of 385 inpatients with at least one primary episode diagnosed in 2017 were included. Most patients were aged over 70 years-old (73.2%). The included patients developed 451 episodes during the observation period. Approximately 44% of primary episodes were community-associated. Most episodes (94.9%) occurred in patients with one or more risk factors, with recent antibiotic exposure being particularly common (86.0%). All-cause in-hospital mortality was 19.5%, being significantly higher in patients aged over 65 years-old versus those aged 18 to 64 years-old (22.4% vs 7.8%, respectively). Over 50 different ribotypes were observed among 206 Clostridioides difficile strains received by the National Reference Laboratory. Conclusion: In Portugal, hospitalized patients with Clostridioides difficile infection are mostly older patients presenting risk factors for the development of this infection, particularly recent antibiotic exposure. Mortality is disproportionately high among the older population. Community-associated Clostridioides difficile infection is common among inpatients with this infection.
- 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.
- Genomic Study of a Clostridium difficile Multidrug Resistant Outbreak-Related Clone Reveals Novel Determinants of ResistancePublication . Isidro, Joana; Menezes, Juliana; Serrano, Mónica; Borges, Vítor; Paixão, Pedro; Mimoso, Margarida; Martins, Filomena; Toscano, Cristina; Santos, Andrea; Henriques, Adriano O.; Oleastro, MónicaBackground: Clostridium difficile infection (CDI) is prevalent in healthcare settings. The emergence of hypervirulent and antibiotic resistant strains has led to an increase in CDI incidence and frequent outbreaks. While the main virulence factors are the TcdA and TcdB toxins, antibiotic resistance is thought to play a key role in the infection by and dissemination of C. difficile. Methods: A CDI outbreak involving 12 patients was detected in a tertiary care hospital, in Lisbon, which extended from January to July, with a peak in February, in 2016. The C. difficile isolates, obtained from anaerobic culture of stool samples, were subjected to antimicrobial susceptibility testing with Etest®strips against 11 antibiotics, determination of toxin genes profile, PCR-ribotyping, multilocus variable-number tandem-repeat analysis (MLVA) and whole genome sequencing (WGS). Results: Of the 12 CDI cases detected, 11 isolates from 11 patients were characterized. All isolates were tcdA -/tcdB + and belonged to ribotype 017, and showed high level resistance to clindamycin, erythromycin, gentamicin, imipenem, moxifloxacin, rifampicin and tetracycline. The isolates belonged to four genetically related MLVA types, with six isolates forming a clonal cluster. Three outbreak isolates, each from a different MLVA type, were selected for WGS. Bioinformatics analysis showed the presence of several antibiotic resistance determinants, including the Thr82Ile substitution in gyrA, conferring moxifloxacin resistance, the substitutions His502Asn and Arg505Lys in rpoB for rifampicin resistance, the tetM gene, associated with tetracycline resistance, and two genes encoding putative aminoglycoside-modifying enzymes, aadE and aac(6')-aph(2″). Furthermore, a not previously described 61.3 kb putative mobile element was identified, presenting a mosaic structure and containing the genes ermG, mefA/msrD and vat, associated with macrolide, lincosamide and streptogramins resistance. A substitution found in a class B penicillin-binding protein, Cys721Ser, is thought to contribute to imipenem resistance. Conclusion: We describe an epidemic, tcdA -/tcdB +, multidrug resistant clone of C. difficile from ribotype 017 associated with a hospital outbreak, providing further evidence that the lack of TcdA does not impair the infectious potential of these strains. We identified several determinants of antimicrobial resistance, including new ones located in mobile elements, highlighting the importance of horizontal gene transfer in the pathogenicity and epidemiological success of C. difficile.
- 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.
- Prospective observational study of health care associated infections with central venous catheters in three tertiary hospitals in Lisbon, PortugalPublication . Nascimento, Maria; Faria, Isabel; Pessanha, Maria Ana; Duarte, Aida; Martins, Filomena; Jordão, LuísaHealthcare Associated Infection (HAI) are a major public health problem affecting 4 million patients per year in Europe and contribute increase the costs associated with healthcare. Cathether related bloodstream infections (CRBSI) are an important group of HAIs registering high incidence (87%) and mortality rates (23%) particulary in intensive care units (ICUs). In this work, an observational prospective study aiming at documenting the occurrence of CRBSI in three hospitals in Lisbon was performed. The study has been ongoing for 2 years and have identified 40 cases of CRBSI. The etiological agent, health unit, patient and central venous catheter (CVC) were characterized. The majority of the CRBSI (92.5%) have as etiological agent a bacterium resistant to at least one antibiotic (28/37); however, 3 cases (7.5%) of Candida spp. infections were identified. Two cases of infection are due to 2 bacteria. It should be noted that 67% of the infections are caused by staphylococci, of which 55.6% are coagulase negative (14 S. epidermidis and 1 S. haemolyticus) and 44.4% coag positive (12 S. aureus). It was not possible to associate a particular microorganism with one of the 3 hospitals. This may be due, in part, to sample dimension. The distribution of cases according to the inpatient service was as follows: intensive care units (35%), medical specialties (32.5%), surgical specialties (17%), general surgery (10%) and internal medicine (5%). The adults (mean age 58 years: 14/40) and the elderly (mean age 74 years: 22/40) were the age groups with the highest number of cases. The remainder were recorded in newborns (<1 month: 2/40) and young adults (2/40). More than half of the CVCs were inserted into the patient's jugular and remained there for less than 4 weeks (75%). The results obtained demonstrate that coagulase negative staphylococci are major players in CRBSI and antibiotic resistance. Cases due to more than one etiological agent may be associated with colonization of CVCs by biofilms. This aspect is being investigated by researching these structures in the CVCs. The collection of more data and its statistical treatment will allow to establish / exclude possible relationships between the different factors that determine the establishment of HAI. This, together with a greater knowledge of the etiological agents resulting from the sequencing of its genome, will increase our knowledge and, in the long term, establish more effective methods of control and prevention of HAIs.
- Prospective observational study on the role of catheter colonization and multidrug-resistance associated with catheter-related bloodstream infectionsPublication . Pinto, Miguel; Borges, Vitor; Nascimento, Maria; Martins, Filomena; Pessanha, Maria Ana; Faria, Isabel; Rodrigues, Joao; Matias, Rui; Joao Paulo, Gomes; Jordao, LuisaBackground: Central venous catheter (CVC)-related bloodstream infection (CRBSI) is a huge public health consern with considerable impact on mortality and health costs. The emergence of antimicrobial resistant microorganisms associated or not with CVC colonization by biofilms makes the treatment of CRBSI even more challeging. Methods:A 3-year observational study enrolling 3 tertiary hospitals located in Lisbon (Portugal) was designed to identify the major etiological agent of 58 CRBSI, their ability to colonize CVCs and their antimicrobial resistance profiles. Etiological agents of CRBSI were idebtified by VITEK-2. Whole-genome sequencing was used to confirm CRBSI by the most prevalent etiological agents and characteriza their resistome. CVC's colonization (namely by biofilm assembly) was monitored by scanning electron microscopy. Results: Staphylococci were the most prevalent causative agent (36/58, 62%), with S. aureus and coagulase negative S. epidermidis accounting for 24.1% and 36.2% of CRBSIs, respectively. Comparative genomic analysis of CVCs/hemoculture pairs of isolates revealed genomic matches for 35/36 pairs and a good correlation between antibiotic susceptibility phenotype and the presence of antimicrobials resistance genetic determinants. CVCs colonization was observed mainly in the catheter lumen and presented different phenotypes ranging from isolated attached microorganisms to mature biofilms. The latest phenotype, mature biofilms of S. epidermidis and S. aureus were found for 50.0% and 48.6% of the CVCs, respectively. Nevertheless, no statistical significant association was established between biofilm assembly and CRBSI highlighting the need for further studies to elucidate biofilms' role on this HAI. Conclusion: WGS proved to be a valuable tool to confirm CRBSI. Despite staphylococci biofilms identification on a considerable number of CVCs, no statistically significant association was found between CRBSI and biofilms.