Biofilm-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.

The application of whole genome sequencing of Mycobacterium tuberculosis directly on clinical samples has been investigated as a means to avoid the time-consuming need for culture isolation that can lead to a potential prolonged suboptimal antibiotic treatment. We aimed to provide a proof-of-concept regarding the application of the molecular capture of M. tuberculosis genomes directly from positive sputum samples as an approach for epidemiological and drug susceptibility predictions. Smear-positive sputum samples (n = 100) were subjected to the SureSelectXT HS Target Enrichment protocol (Agilent Technologies, Santa Clara, CA, USA) and whole-genome sequencing analysis. A higher number of reads on target were obtained for higher smear grades samples (i.e., 3+ followed by 2+). Moreover, 37 out of 100 samples showed ≥90% of the reference genome covered with at least 10-fold depth of coverage (27, 9, and 1 samples were 3+, 2+, and 1+, respectively). Regarding drug-resistance/susceptibility prediction, for 42 samples, ≥90% of the >9000 hits that are surveyed by TB-profiler were detected. Our results demonstrated that M. tuberculosis genome capture and sequencing directly from clinical samples constitute a potential valid backup approach for phylogenetic inferences and resistance prediction, essentially in settings when culture is not routinely performed or for samples that fail to grow.

This study extensively analyzed campylobacteriosis surveillance in Portugal from 2009 to 2021, aiming to investigate demographic shifts, seasonal variations, and antimicrobial resistance (AMR) within Campylobacter isolates. Surveillance network and sentinel laboratory-based system data revealed a substantial under-notification of campylobacteriosis cases, suggesting an underestimated disease burden. Notification rates exhibited a paradigm shift, with a notable prevalence among the pediatric population, particularly in children aged 1–4 years, diverging from European reports. Additionally, an emerging trend of Campylobacter infections in younger adults (15–44 years) was observed. The study unveiled a unique seasonal distribution of cases, defying typical summer peaks seen elsewhere. AMR analysis revealed high resistance to ciprofloxacin and tetracycline, in both C. jejuni (93.7% and 79.2%, respectively) and C. coli (96.5% and 93.2%, respectively), stable throughout the studied period (2013–2021). C. coli exhibited significantly higher resistance to erythromycin, gentamicin, ampicillin and ertapenem compared to C. jejuni (p < 0.001). Multilocus Sequence Typing (MLST) data demonstrated the distribution of resistance markers across diverse sequence types, challenging the notion of a clonal origin for multidrug-resistant isolates. In conclusion, the study highlights the need for enhanced surveillance and raises concerns about alarming AMR levels, recommending the implementation of whole-genome sequencing (WGS)-based surveillance for a deeper comprehension of disease patterns and an evolving AMR landscape.

The Mycobacterium abscessus complex (MABC) is an emerging, difficult to treat, multidrug-resistant nontuberculous mycobacteria responsible for a wide spectrum of infections and associated with an increasing number of cases worldwide. Dominant circulating clones (DCCs) of MABC have been genetically identified as groups of strains associated with higher prevalence, higher levels of antimicrobial resistance, and worse clinical outcomes. To date, little is known about the genomic characteristics of MABC species circulating in Portugal. Here, we examined the genetic diversity and antimicrobial resistance profiles of 30 MABC strains isolated between 2014 and 2022 in Portugal. The genetic diversity of circulating MABC strains was assessed through a gene-by-gene approach (wgMLST), allowing their subspecies differentiation and the classification of isolates into DCCs. Antimicrobial resistance profiles were defined using phenotypic, molecular, and genomic approaches. The majority of isolates were resistant to at least two antimicrobials, although a poor correlation between phenotype and genotype data was observed. Portuguese genomes were highly diverse, and data suggest the existence of MABC lineages with potential international circulation or cross-border transmission. This study highlights the genetic diversity and antimicrobial resistance profile of circulating MABC isolates in Portugal while representing the first step towards the implementation of a genomic-based surveillance system for MABC at the Portuguese NIH.

Hepatitis C virus (HCV) is associated with liver damage and an increased progression rate to cirrhosis and hepatocellular carcinoma. In Portugal, it is prevalent in vulnerable populations such as injection drug users (IDU). HCV is characterized by a high intra-host variability, and the selecting driving forces could select variants containing resistance-associated substitutions (RAS) that reduce treatment effectiveness. The main goal of this study was to analyze the sequence variation of NS5A in treatment-naïve IDU. The epidemiological and clinical status of hepatitis C were analyzed, and samples were sequenced by Sanger and Next-Generation sequencing (NGS) to assess RAS and confirm HCV subtypes. Phylogenetic classification was concordant: 1a, 52.4%; 1b, 10.7%; 3a, 20.2%; 4a, 8.3%; 4d, 7.1%; and one 2k/1b recombinant. A 1a/3a mixed infection was detected by NGS. RAS were found in 34.5% (29/84) of samples using Sanger sequencing, while in 42.9% (36/84) using NGS. In sequences from subtypes 1a and 1b, RAS K24R, M28V, Q30H/R, H58D/P/Q/R, and RAS L31M and P58S were detected, respectively. In subtype 3a, RAS A30S/T, Y93H and polymorphisms in position 62 were identified. Additionally, RAS P58L was detected in genotype 4. The strategy used for the molecular survey of baseline HCV resistance is of particular importance to achieve treatment effectiveness and contribute to the elimination of hepatitis C.