María Ángeles Tormo-Mas

Pseudomonas aeruginosa is a high-priority pathogen responsible for up to 23% of infections in intensive care units, yet current diagnostic methods remain hindered by multiday delays or high technical complexity. In this study, we report a rapid and highly sensitive biosensor based on nanoporous anodic alumina (NAA) capped with a specific oligonucleotide molecular gate targeting the phzA2 gene. The S3 biosensor achieved a limit of detection (LOD) of 0.153 ng μL^-1 for genomic DNA and an exceptional 28 CFU mL^-1 for direct bacterial detection without the need for prior DNA extraction. Validation in 63 clinical urine samples demonstrated a sensitivity of 91.67% and a specificity of 94.87% (AUC: 0.961, p-value < 0.0001). Furthermore, the sensor was integrated into Lateral Flow Assay (LFA), enabling the discrimination of positive and negative clinical samples in only 1 min with 100% specificity and 90.9% sensitivity. These results, combined with a one-year stability at 4 °C, position this gated-nanodevice as a robust and low-cost tool for Point-of-care (POC) diagnosis of P. aeruginosa infections.

Background/Objectives: Orthopaedic infections associated with implant surgery remain a major public health concern, often caused by bacterial colonization of implant surfaces. Staphylococcus epidermidis is among the most common pathogens involved. Developing antimicrobial bone implants that prevent infection without compromising bone regeneration is therefore essential. This study investigates the antimicrobial and osteointegrative performance of calcium phosphate (CaP) materials functionalized with vanillin, an essential oil component with known antimicrobial properties. Methods: Commercial CaP regenerative materials were covalently coated with vanillin. Antibacterial activity was evaluated against Staphylococcus epidermidis RP62A using viability assays. In vivo osseointegration was assessed in New Zealand female rabbits implanted with vanillin-coated and uncoated CaP scaffolds. Results: Vanillin-functionalized CaP scaffolds exhibited strong bactericidal activity at 24 h and bacteriostatic effects at 48 h at a concentration of 10 mg/mL. In vivo analyses showed no significant differences in osseointegration between vanillin-coated implants and control CaP materials. Conclusions: Vanillin-functionalized CaP materials maintain a high safety profile without impairing bone integration, supporting their potential use in clinical applications.

Prosthetic joint infection represents a problem that worsens the patient’s quality of life and produces an economic impact on health systems. We report the anodization of Ti6Al4V coupons obtained by electron beam melting to produce a nanostructured surface. Anodization at 10 V produced TiO₂ nanopores with a diameter in the range of 15–20 nm. Thereafter, Ag nanoparticles (AgNPs) were deposited in three different ways to provide antibacterial functionality to the coatings: electrochemically, thermally, and chemically. The electrochemical method did not provide good coverage of AgNPs. At 0.1 V of synthesis potential, cubic, octahedral, and truncated octahedral Ag crystals were obtained. The thermal method provided a good distribution of AgNPs but it damaged the TiO₂ nanostructure. The chemical method showed the best distribution of AgNPs over the anodized surface and preserved the anodized nanostructure. For this reason, the chemical method was selected to perform further studies. Ag⁺ release was monitored in simulated body fluid at 37 °C, reaching 1.86 mg Ag⁺/L after 42 days. The antibacterial coating showed excellent antibacterial activity and inhibited biofilm formation for Staphylococcus epidermidis RP62A and Staphylococcus aureus V329 strains (lethality > 99.9% for both bacteria and assays).

Candida auris is an emerging, multidrug-resistant yeast that causes systemic infections, mainly in hospitalized or immunosuppressed patients. This pathogen has a high mortality and morbidity rate. This study aims to evaluate the antifungal potential of micafungin (MICA) encapsulated in a nanoemulsion (NEM) against four clades of C. auris and other non-C. auris species. The antifungal potential of MICA and NEM was evaluated by determining mature biofilm inhibition (0.78–50 µg/mL). The antifungal activities of MICA and NEM (5.92 mg/Kg) were evaluated using an in vivo model of Galleria mellonella. The results showed that NEM intensified the antibiofilm action of MICA, especially in 48 h mature biofilms. In vivo results displayed a higher effectiveness of NEM against all clades of C. auris tested, inhibiting the fungal load in the hemolymph and tissues of G. mellonella with a difference of 3 log10. In addition, C. auris infection caused granulomas surrounded by hemocytes, mainly at the lower and upper ends. Conversely, C. albicans developed pseudohyphae, biofilms, filaments, and chlamydospores. In conclusion, encapsulation of MICA in a nanoemulsion enhances its antifungal activity against mature biofilms of C. auris. This strategy may be considered a therapeutic approach for the control of infections and the dissemination of this new global health threat.

Candida auris is an emerging yeast of worldwide interest due to its antifungal resistance and mortality rates. The aim of this study was to analyse the in vitro and in vivo antifungal activity of a nanoemulsion loaded with amphotericin B (NEA) against planktonic cells and biofilm of C. auris clinical isolates belonging to four different clades. In vivo assays were performed using the Galleria mellonella model to analyse antifungal activity and histopathological changes. The in vitro results showed that NEA exhibited better antifungal activity than free amphotericin B (AmB) in both planktonic and sessile cells, with >31% inhibition of mature biofilm. In the in vivo assays, NEA demonstrated superior antifungal activity in both haemolymph and tissue. NEA reduced the fungal load in the haemolymph more rapidly and with more activity in the first 24 h after infection. The histological analysis of infected larvae revealed clusters of yeast, immune cells, melanisation, and granulomas. In conclusion, NEA significantly improved the in vitro and in vivo antifungal activity of AmB and could be considered a promising therapy for C. auris infections.

In this work, fluorogenic probes based on oligonucleotide capped nanoporous anodic alumina films are developed for specific and sensitive detection of human papilloma virus (HPV) DNA. The probe consists of anodic alumina nanoporous films loaded with the fluorophore rhodamine B (RhB) and capped with oligonucleotides bearing specific base sequences complementary to genetic material of different high‐risk (hr) HPV types. Synthesis protocol is optimized for scale up production of sensors with high reproducibility. The sensors' surfaces are characterized by scanning electron microscopy (HR‐FESEM) and atomic force microscopy (AFM) and their atomic composition is determined by energy dispersive X‐ray spectroscopy (EDXS). Oligonucleotide molecules onto nanoporous films block the pores and avoid diffusion of RhB to the liquid phase. Pore opening is produced when specific DNA of HPV is present in the medium, resulting in RhB delivery, that is detected by fluorescence measurements. The sensing assay is optimized for reliable fluorescence signal reading. Nine different sensors are synthesized for specific detection of 14 different hr‐HPV types in clinical samples with very high sensitivity (100%) and high selectivity (93–100%), allowing rapid screening of virus infections with very high negative predictive values (100%).

The COVID‐19 pandemic, which began in 2019, has highlighted the importance of testing and tracking infected individuals as a means of mitigating the spread of the virus. In this context, the development of sensitive and rapid methods for the detection of SARS‐CoV‐2, the virus responsible for COVID‐19, is crucial. Herein, a biosensor based on oligonucleotide‐gated nanomaterials for the specific detection of SARS‐CoV‐2 spike protein is presented. The sensing system consists of a nanoporous anodic alumina disk loaded with the fluorescent indicator rhodamine B and capped with a DNA aptamer that selectively binds the SARS‐CoV‐2 spike protein. The system is initially evaluated using pseudotype virus systems based on vesicular stomatitis virus carrying different SARS‐CoV‐2 S‐proteins on their surface. When the pseudotype virus is present, the cap of the solid is selectively removed, triggering the release of the dye from the pore voids to the medium. The nanodevice demonstrated its ability to detect pseudotype virus concentrations as low as 7.5·10³ PFU mL. In addition, the nanodevice is tested on nasopharyngeal samples from individuals suspected of having COVID‐19.

The strong bactericidal action of silver nanoparticles (AgNPs) is usually limited by their degree of aggregation. Deposition of AgNPs onto a graphene oxide (GO) surface to generate GO-Ag hybrids has been shown to be an effective method of controlling these aggregation problems. In this sense, a novel carboxylated graphene oxide–silver nanoparticle (GOCOOH-Ag) material has been synthesized, and their antibacterial and biofilm formation inhibitions have been studied. AgNPs decorating the GOCOOH surface achieved an average size of 6.74 ± 0.25 nm, which was smaller than that of AgNPs deposited onto the GO surface. In addition, better distribution of AgNPs was achieved using carboxylated material. It is important to highlight the main role of the carboxylic groups in the nucleation and growth of the AgNPs that decorate the GO-based material surface. In vitro antibacterial activity and antibiofilm-forming action were tested against Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli). Both GO-Ag and GOCOOH-Ag reduced bacterial growth, analyzed by time–kill curves. However, the minimum inhibitory concentration and the minimum bactericidal concentration of GOCOOH-Ag were lower than those of GO-Ag for all strains studied, indicating that GOCOOH-Ag has better antibacterial activity. In addition, both nanomaterials prevent biofilm formation, with a higher reduction of biofilm mass and cell viability in the presence of GOCOOH-Ag. The carboxylation functionalization in GO-based materials can be applied to improve the bactericidal and antibiofilm-forming action of the AgNPs.

Antibiotic resistance is one of the major challenges that humankind shall face in the short term. (Bacterio)phage therapy is a valuable therapeutic alternative to antibiotics and, although the concept is almost as old as the discovery of phages, its wide application was hindered in the West by the discovery and development of antibiotics in the mid-twentieth century. However, research on phage therapy is currently experiencing a renaissance due to the antimicrobial resistance problem. Some countries are already adopting new ad hoc regulations to favor the short-term implantation of phage therapy in clinical practice. In this regard, the Phage Therapy Work Group from FAGOMA (Spanish Network of Bacteriophages and Transducing Elements) recently contacted the Spanish Drugs and Medical Devices Agency (AEMPS) to promote the regulation of phage therapy in Spain. As a result, FAGOMA was asked to provide a general view on key issues regarding phage therapy legislation. This review comes as the culmination of the FAGOMA initiative and aims at appropriately informing the regulatory debate on phage therapy.

Candida auris has globally emerged as a multidrug-resistant fungus linked to healthcare-associated outbreaks. There is still limited evidence on its virulence, pathogenicity determinants, and complex host–pathogen interactions. This study analyzes the in vivo fungal behaviour, immune response, and host–pathogen interactions upon C. auris infection compared to C. albicans and C. parapsilosis in G. mellonella. This was performed by immunolabelling fungal structures and larval plasmatocytes and using a quantitative approach incorporating bioinformatic morphometric techniques into the study of microbial pathogenesis. C. auris presents a remarkably higher immunogenic activity than expected at its moderate degree of tissue invasion. It induces a greater inflammatory response than C. albicans and C. parapsilosis at the expense of plasmatocyte nodule formation, especially in non-aggregative strains. It specifically invades the larval respiratory system, in a pattern not previously observed in other Candida species, and presents inter-phenotypic tissue tropism differences. C. auris filaments in vivo less frequently than C. albicans or C. parapsilosis mostly through pseudohyphal growth. Filamentation might not be a major pathogenic determinant in C. auris, as less virulent aggregative phenotypes form pseudohyphae to a greater extent. C. auris has important both interspecific and intraspecific virulence and phenotype heterogeneity, with aggregative phenotypes of C. auris sharing characteristics with low pathogenic species such as C. parapsilosis. Our work suggests that C. auris owns an important morphogenetic plasticity that distinguishes it from other yeasts of the genus. Routine phenotypic identification of aggregative or non-aggregative phenotypes should be performed in the clinical setting as it may impact patient management.