Browsing by Author "Bajanca-Lavado, Paula"
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- Antibody Binding and Complement-Mediated Killing of Invasive Haemophilus influenzae Isolates from Spain, Portugal, and the NetherlandsPublication . Dudukina, Elena; de Smit, Laura; Verhagen, Giel J.A.; van de Ende, Arie; Marimón, José María; Bajanca-Lavado, Paula; Ardanuy, Carmen; Marti, Sara; de Jonge, Marien I.; Langereis, Jeroen D.Haemophilus influenzae is a Gram-negative bacterium that can be classified into typeable (types a through f) and nontypeable (NTHi) groups. This opportunistic pathogen asymptomatically colonizes the mucosal epithelium of the upper respiratory tract, from where it spreads to other neighboring regions, potentially leading to disease. Infection with NTHi can cause otitis media, sinusitis, conjunctivitis, exacerbations of chronic obstructive pulmonary disease, and pneumonia, but it is increasingly causing invasive disease, including bacteremia and meningitis. Invasive NTHi strains are more resistant to complement-mediated killing. However, the mechanisms of complement resistance have never been studied in large numbers of invasive NTHi strains. In this study, we determined the relationship between binding of IgG or IgM and the bacterial survival in normal human serum for 267 invasive H. influenzae strains from Spain, Portugal, and the Netherlands, of which the majority (200 [75%]) were NTHi. NTHi bacteria opsonized with high levels of IgM had the lowest survival in human serum. IgM binding to the bacterial surface, but not IgG binding, was shown to be associated with complement-mediated killing of NTHi strains. We conclude that evasion of IgM binding by NTHi strains increases survival in blood, thereby potentially contributing to their ability to cause severe invasive diseases.
- Comparative pangenome analysis of capsulated Haemophilus influenzae serotype f highlights their high genomic stabilityPublication . Gonzalez-Diaz, Aida; Carrera-Salinas, Anna; Pinto, Miguel; Cubero, Meritxell; van der Ende, Arie; Langereis, Jeroen D.; Domínguez, M. Ángeles; Ardanuy, Carmen; Bajanca-Lavado, Paula; Marti, SaraHaemophilus influenzae is an opportunistic pathogen adapted to the human respiratory tract. Non-typeable H. influenzae are highly heterogeneous, but few studies have analysed the genomic variability of capsulated strains. This study aims to examine the genetic diversity of 37 serotype f isolates from the Netherlands, Portugal, and Spain, and to compare all capsulated genomes available on public databases. Serotype f isolates belonged to CC124 and shared few single nucleotide polymorphisms (SNPs) (n = 10,999), but a high core genome (> 80%). Three main clades were identified by the presence of 75, 60 and 41 exclusive genes for each clade, respectively. Multi-locus sequence type analysis of all capsulated genomes revealed a reduced number of clonal complexes associated with each serotype. Pangenome analysis showed a large pool of genes (n = 6360), many of which were accessory genome (n = 5323). Phylogenetic analysis revealed that serotypes a, b, and f had greater diversity. The total number of SNPs in serotype f was significantly lower than in serotypes a, b, and e (p < 0.0001), indicating low variability within the serotype f clonal complexes. Capsulated H. influenzae are genetically homogeneous, with few lineages in each serotype. Serotype f has high genetic stability regardless of time and country of isolation.
- Detection of the Hmw adhesins in clinical Haemophilus influenzae isolates from bacteraemic patients and association with biofilm formationPublication . Bosh, Ana; González, Aida; Carrera-Salinas, Anna; Cubero, Meritxell; Marimón, José María; Bajanca-Lavado, Paula; Ardanuy, Carmen; Marti, SaraBackground: Non-typeable Haemophilus influenzae (NTHi) forms part of the normal nasopharyngeal microbiota in humans, but it is also an opportunistic pathogen causing respiratory infections and bacteraemia. Recently, high molecular weight (HMW1) proteins have been identified as a key factor for cell invasion, a feature implicated in persistence during chronic infection(a). Our aims were to identify the different allelic variants of the HMW adhesin and, given the characteristics of these surface proteins on bacterial adhesion capacity, the second objective was to check if their presence could be related to biofilm formation. Materials/Methods: A collection of 89 strains isolated from patients with bacteraemia from Spain and Portugal in the 2013-2014 period were used in this study. Strains were genotyped by PFGE (SmaI) and analyzed with the FingerPrinting software (BioRad). The allelic variants of the hmw gene (Hi375 and Hi86-028NP) encoding the high molecular weight adhesins Hmw1/Hmw2 were identified by PCR amplification. Biofilm formation was performed in a static biofilm assay with crystal violet staining. Statistical analysis was performed using the GraphPad Prism 5 software. Results: Forty-eight NTHi isolates (54%) were positive for the hmw gene. Only the allelic variants of the Hi375 strain could be identified, among them, one strain (1/48, 2%) had hmw-1A, 33 (69%) had hmw-2A and 14 (29%) had both allelic variants, hmw-1A and hmw-2A. Biofilm formation showed great diversity among the studied strains with OD¬570 values ranging between 0.06 and 1.4. Forty-three strains (48.3%) were classified as high biofilm formers and the remaining 46 strains (51.7%) were low biofilm formers. An inverse relationship was found between the presence of hmw genes and in vitro biofilm formation. The invasive NTHi clinical isolates presented high genetic diversity by PFGE, with no strain clustering observed linked to the presence of hmw genes or to biofilm formation. Conclusion: The allelic variants of the H. influenzae strain 375, especially the hmw-2A gene, were more commonly found among invasive NTHi clinical isolates, which despite having an important role on intracellular invasion, were not linked to in vitro biofilm formation. (a)Mell, JC et al. (2016). PLoS Pathogens 12: e1005576.
- Epidemiological analysis of invasive Haemophilus influenzae clinical isolates obtanied from Portugal, Spain and the NetherlandsPublication . Raeven, Elisabeth A.M.; González, Aida; van der Ende, Arie; Liñares, Josefina; Marimón, José María; Bajanca-Lavado, Paula; Langereis, Jeroen D.; Ardanuy, Carmen; Martí, SaraBackground. Haemophilus influenzae is a human-restricted pathogen that forms part of the normal nasopharyngeal microbiota. The introduction of the H. influenzae serotype b vaccine has drastically decreased the number of bacteremia cases caused by H. influenzae serotype b (Hib). Conversely, the cases of non-typeable H. influenzae (NTHi) bacteremia have increased substantially. Therefore, we aimed to perform an epidemiological study comparing invasive H. influenzae clinical isolates from three European countries. Material & Methods. Clinical isolates were obtained from two southern European countries, Spain (Hospital de Bellvitge, n=44; Hospital de Donostia, n=18) and Portugal (n=55), and a northern country, the Netherlands (n=146) between 2013 and 2015. The clinical source of the samples was blood (n=250), cerebrospinal fluid (n=4) and pleural effusion (n=9). Capsular serotyping was done by PCR and genotyping by PFGE (SmaI), followed by FingerPrinting analysis. Antimicrobial susceptibility was tested by disk diffusion and microdilution against Ampicillin (AP), Tetracycline (TC), Chloramphenicol (CL), Ciprofloxacin (CP) and Trimethoprim/Sulfamethoxazole (T/S). Results. Overall, NTHi were the most prevalent isolates (201/263, 76%), followed by Hib (38/263, 14%), Hif (16/263, 6%) and other capsulated H. influenzae (Hia=3; Hid=1; Hie=4). By countries, NTHi was also the major pathogen identified in Spain (82%), Portugal (80%) and the Netherlands (73%), while Hib was slightly more frequent in the Netherlands (27/146, 18%) and Portugal (7/55, 13%) than in Spain (4/62, 6%). PFGE clustering identified high diversity among the NTHi strains, although some strains from different countries were found to be highly related (14 clusters of two or three strains). Hib were grouped together in four main clusters including isolates from different countries: Cluster I (5 strains Netherlands; 4 strains Portugal), Cluster II (18 strains Netherlands, 2 strains Portugal; 1 strain Spain), Cluster III (5 strains Netherlands), Cluster IV (1 strain Portugal; 1 strain Spain).
- Identification of polysaccharide capsules among extensively drug-resistant genitourinary Haemophilus parainfluenzae isolatesPublication . González-Díaz, Aida; Tubau, Fe; Pinto, Miguel; Sierra, Yanik; Cubero, Meritxell; Càmara, Jordi; Ayats, Josefina; Bajanca-Lavado, Paula; Ardanuy, Carmen; Marti, SaraThe human commensal Haemophilus parainfluenzae is emerging as an opportunistic multidrug-resistant pathogen. The objectives of this work were to characterise a new capsular operon of extensively drug-resistant (XDR) H. parainfluenzae clinical isolates and study their resistance mechanisms using whole-genome sequencing. All strains were resistant to: ß-lactams, via amino acid changes in PBP3 (S385T, I442F, V511A, N526K and V562I); quinolones, by alterations in GyrA (S84F and D88Y) and ParC (S84F and S138T); chloramphenicol, through the presence of catS; macrolides, via the presence of mel and mef(E)-carrying MEGA element; and tetracycline, through the presence of tet(M) and/or tet(B). Phylogenetic analysis revealed high genomic diversity when compared to the H. parainfluenzae genomes available on the NCBI, the isolates from this study being closely related to the Swiss XDR AE-2096513. A full capsular operon showing homology to that of H. influenzae was identified, in accordance with the observation of a capsular structure by TEM. This study describes for the first time a capsular operon in H. parainfluenzae, a major determinant of pathogenicity that may contribute to increased virulence in XDR clinical isolates. Moreover, phylogenetic analysis suggests the possible spread of an XDR-encapsulated strain in Europe.
- A osteomelite crónica pode ser o precedente de uma infeção por Corynebacterium diphtheriaePublication . Faustino, Joana; Milheiro Silva, Tiago; Lameiras Campagnolo, João; Bajanca-Lavado, Paula; Gouveia, CatarinaIntrodução: A infeção por C. diphtheriae foi virtualmente eliminada de muitos países desenvolvidos, contudo é endémica na áfrica subsariana. A difteria cutânea é caracterizada por feridas crónicas não cicatrizantes, precedidas habitualmente por traumatismo. Caso Clínico: Rapariga de 11 anos, natural e residente na Guiné, internada na Guiné aos 10 anos por fratura exposta do úmero esquerdo secundária a queda da própria altura. Aos 11 anos por dor, impotência funcional e fístula do úmero esquerdo e anca direita é transferida para Portugal. Analiticamente sem leucocitose, PCR 1,2 mg/L, VS 25 mm/h, HIV e IGRA negativos. TC Osteoarticular evidenciava osteomielite crónica multifocal, antiga fratura do colo cirúrgico do úmero esquerdo. Exame cultural do exsudado da ferida do úmero positivo para C. diphtheriae, S. aureus meticilina-sensivel e S. pyogenes. Foi excluída produção de toxina e presença de C. diphtheriae no exsudado faríngeo. Submetida a limpeza cirúrgica da ferida e fistulectomia. Iniciou terapêutica com penicilina, flucloxacilina e rifampicina. Conclusão: As infeções da pele por C. diphtheriae originam uma rápida resposta do sistema imunitário do hospedeiro, diminuindo a probabilidade de infeção faríngea. Contudo, constituem reservatórios para infetar indivíduos suscetíveis, reforçando a importância de um diagnóstico e início de terapêutica precoce.
- Probe-based metagenomic pathogen detection: advancing laboratory capacity for complex diagnosisPublication . Ferreira, Rita; Coelho, Luís; Santos, João Dourado; Sobral, Daniel; Isidro, Joana; Mixão, Verónica; Pinto, Miguel; Nunes, Alexandra; Borrego, Maria José; Lopo, Sílvia; Oleastro, Mónica; Sousa, Rita; Palminha, Paula; Veríssimo, Cristina; Gargaté, Maria João; Guiomar, Raquel; Cordeiro, Rita; Macedo, Rita; Bajanca-Lavado, Paula; Paixão, Paulo; Duarte, Sílvia; Vieira, Luís; Borges, Vítor; Gomes, João PauloProbe-based pathogen enrichment, followed by NGS, is a promising tool for complex diagnosis, overcoming traditional challenges of shotgun metagenomics, namely small microbial/human genetic material ratio and demanding computational resources. Here, we assessed the combined detection performance of two Illumina probe-based panels, the Respiratory and the Urinary Pathogen ID panels (RPIP and UPIP), using 99 clinical samples of 15 different matrices (e.g., cerebrospinal fluid, plasma, serum, urine, swabs, biopsies, etc.) available from Portuguese National Reference Laboratories. This sample set involved 114 "PCR-positive hits" (Ct values range of 9.7-41.3; median of 28.4) for 52 non-redundant human pathogens. For a more detailed bioinformatics assessment, as a complement of the Illumina turnkey solution (Explify), we applied an extended version of our INSaFLU-TELEVIR(+) metagenomics pipeline. Whereas Explify analyses resulted in an initial detection frequency of 73.7% (84/114), the subsequent application of INSaFLU-TELEVIR(+), including taxonomic classification followed by confirmatory read mapping, enabled an overall detection proportion of 79.8% (91/114) of the PCR-positive hits. This translated into a detection rate increment from 54.3% (19/35) to 65.7% (23/35) for bacteria, and from 85.3% (58/68) to 89.7% (61/68) for viruses. The implemented workflow was also very satisfactory for samples with qPCR Ct values above 30, with an overall detection frequency of 71.8% (28/39) when compared with the 92.0% (46/50) observed for those with Ct ≤ 30. In summary, this study validated and established a pioneering approach at the Portuguese National Institute of Health to support clinicians in complex diagnosis, contributing to advance diagnostic capabilities toward a more informed clinical decision and potential improvement of infectious disease outcomes.
- Whole-genome-based characterization of invasive Haemophilus influenzae isolates from a pre- and post-vaccine era in PortugalPublication . Gonzalez Diaz, Aida; Pinto, Miguel; Bettencourt, Célia; Duarte, Silvia; Marti, Sara; Gomes, João Paulo; Bajanca-Lavado, PaulaIntroduction: Haemophilus influenzae (Hi) is responsible for severe invasive infections in both adults and children. Since the introduction in the year 2000 of the Hib vaccine, the incidence of disease has substantially declined, even though it doesn’t protect against non-typeable Hi (NTHi) isolates. Although not all NTHi are pathogenic, these are known to possess important virulence factors to promote colonization and host cells interactions, ultimately leading to disease. The application of WGS technology allows the uncovering of Hi population structure, including novel insights into its genomic features. Aims: This study aims to fully characterize, by WGS, Hi isolates from a pre- and post-vaccine era, from 1992 to 2015, in Portugal. Materials and Methods: Ninety invasive Hi isolates from the Portuguese NIH collection were selected for WGS. More than half were NTHi (63.3%) and 32.2% of the strains belong to a pre-vaccine era. Genomes were assembled and both sequence type (ST) and serotype were determined by PCR and confirmed in silico. A core-single nucleotide polymorphism-based phylogenetic tree was reconstructed to analyze overall genomic diversity between strains. Strains were further characterized by identifying the presence and genetic profile of genes related to antibiotic resistance and virulence factors, namely genes involved in adherence, host immune evasion, iron acquisition and lipooligosaccharides (LOS). Results: Preliminary results show high ST heterogeneity among NTHi, contrasting with the homogeneity of ST for Hib strains (all ST6, except one). Core-SNP-based analysis revealed that all strains were distinguishable by more than 140,000 single nucleotide variant sites, with a highest genetic diversity observed between NTHi (overall ~35,000 nucleotide differences). Interestingly, although all Hib segregated together, the ST282 Hib strain possessed a distinct genome profile, diverging by ~17,200 nucleotide differences from ST6, while these overall diverged between them by ~2,480. Differential presence of important virulence factors was observed among strains, namely for hia/hsf, hmw1/hmw2, hap and iga, with distinct genomic profiles observed between strains, requiring in-depth analysis. Curiously, 90% of NTHi had the lgtA LOS-coding gene which was absent in all Hib. Additionally, five genes coding for other LOS were found to be simultaneously present or absent among NTHi strains, most belonging to a post-vaccine era, indicating a potential cluster of circulating strains. Conclusions: Overall, we expect that the integrative analysis of all Hi isolates will strengthen the characterization of the genomic features in pre- and post-vaccine era, ultimately contributing to the understanding of the scenario of strains circulating in Portugal throughout more than 20 years.