Computerized restriction endonuclease analysis compared with O-serotype and phage type in the epidemiologic fingerprinting of Pseudomonas aeruginosa strains

ObjectiveTo assess restriction endonuclease analysis (REA) of chromosomal DNA using SalI enzyme, low-concentration (0.4%) agarose gels and digitalized data management of the REA patterns obtained for the typing of clinical Pseudomonas aeruginosa isolates.MethodsA group of 67 clinical unrelated isolates from 10 Spanish hospitals was used to study the discriminatory power, reproducibility and typeability of REA typing.ResultsA SalI REA pattern consisted of a variety (1–10) of restriction bands in the range between 12.2 and 48.5 kb and an unresolvable smear of low-molecular-weight bands. Forty different SalI REA patterns with an index of discrimination of 0.979 were obtained. Low typeability (91.04%) was the major limitation of REA typing. Analysis of blinded subcultures of eight Pseudomonas aeruginosa strains showed the reproducibility of REA typing to be 87.5%. Combined phenotypic typing (O-serotyping and phage typing) performed on the same group of strains showed comparable discrimination but much lower reproducibility. Isolates selected from five clusters of nosocomial infections in hospitals in the UK were typed by REA typing, and the results show high agreement when compared with conventional phenotypic typing methods in distinguishing between strains.ConclusionsThese data underline the usefulness of REA typing enhanced with digitalized data management for the epidemiologic subtyping of clinical Pseudomonas aeruginosa isolates

Un anticuerpo monoclonal específico frente al antígeno H:1,2 de Salmonella enterica serotipo Typhimurium que presenta reactividad cruzada con Salmonella enterica serotipo 4,5,12:i:-, línea celular productora, y su uso.

Esta invención se refiere a un anticuerpo monoclonal específico frente al antígeno H:1,2 presente en Salmonella entérica serotipo Typhimurium con reactividad cruzada frente al serotipo 4,5,12:i:-, una línea celular murina productora de dicho anticuerpo monoclonal y la aplicación de dicho anticuerpo monoclonal mediante técnicas inmunológicas para reconocer antígenos asociados a flagelo específicos de Salmonella enterica serotipo Typhimurium y Salmonella enterica serotipo 4,5,12:i:-.REIVINDICACIONES: 1. Un procedimiento para la obtención de un anticuerpo monoclonal, fragmento o variante del mismo caracterizado porque comprende las siguientes etapas: a) inmunizar un animal con el antígeno flagelar H:1, 2 aislado; b) inmortalizar los linfocitos B procedentes de los ratones inmunizados en el paso anterior, mediante su fusión con células de mieloma para dar lugar a un hibridoma; c) aislamiento de aquellos clones de hibridomas que secretan anticuerpos monoclonales reactivos frente al antígeno flagelar H:1, 2; d) cultivo de dichos clones celulares y posterior obtención del anticuerpo monoclonal de interés, fragmento o variante del mismo, caracterizado porque dicho anticuerpo monoclonal, fragmento o variante del mismo reacciona específicamente frente al antígeno flagelar H:1, 2 presente en Salmonella enterica serotipo Typhimurium y de forma cruzada con el serotipo 4, 5, 12:i:-. 2. Un anticuerpo monoclonal, fragmento o variante del mismo obtenido según el procedimiento de la reivindicación 1 caracterizado porque reacciona específicamente frente al antígeno flagelar H:1, 2 presente en Salmonella enterica serotipo Typhimurium y de forma cruzada con el serotipo 4, 5, 12:i:-. 3. Un anticuerpo monoclonal, fragmento o variante del mismo según la reivindicación 2, caracterizado porque el animal inmunizado para su obtención está seleccionado entre: conejo, cabra y ratón. 4. Un anticuerpo monoclonal, fragmento o variante del mismo según la reivindicación 2, caracterizado porque dicho anticuerpo posee un isotipo seleccionado entre: IgG, e IgM. 5. Un anticuerpo monoclonal, fragmento o variante del mismo según una de las reivindicaciones 2 a 4, caracterizado porque el hibridoma aislado en el paso c) de su procedimiento de obtención es la línea celular 23D4, ECACC no. 05122301. 6. Un anticuerpo monoclonal, fragmento o variante del mismo según la reivindicación 5, caracterizado porque dicho anticuerpo es murino. 7. Un anticuerpo monoclonal, fragmento o variante del mismo según una de las reivindicaciones 5 o 6, caracterizado porque dicho anticuerpo es del isotipo IgM. 8. Un anticuerpo monoclonal, fragmento o variante del mismo según una de las reivindicaciones 2 a 7, caracterizado porque dicho anticuerpo monoclonal se encuentra en sobrenadante, ascites o purificado. 9. Un fragmento de un anticuerpo monoclonal según una cualquiera de las reivindicaciones 2 a 8 caracterizado porque está seleccionado entre: un fragmento Fab, un fragmento F (ab') 2 y un fragmento Fv. 10. Una línea celular capaz de producir un anticuerpo monoclonal que reacciona específicamente frente al antígeno H:1, 2 presente en el serotipo Typhimurium y de forma cruzada con el serotipo 4, 5, 12:i:- de Salmonella enterica definido en las reivindicaciones 2 a 8 caracterizada porque dicha línea celular es la línea 23D4, ECACC no. 05122301. 11. Uso de un anticuerpo, fragmento o variante del mismo según una de las reivindicaciones 2 a 9, para detectar la presencia de los serotipos Typhimurium o 4, 5, 12:i:- de Salmonella enterica en una muestra. 12. Uso de un anticuerpo, fragmento o variante del mismo según la reivindicación 11, caracterizado porque dicha muestra está seleccionada entre una muestra alimentaria y una muestra clínica. 13. Uso de un anticuerpo, fragmento o variante del mismo según una de las reivindicaciones 11 u 12, caracterizado porque dicha muestra clínica está seleccionada entre sangre, suero, plasma y orina. 14. Uso de un anticuerpo monoclonal, un fragmento o variante del mismo según una de las reivindicaciones 11 a 13, caracterizado porque dicha detección se realiza mediante un ensayo seleccionado entre: ELISA, Western-blot y Dot-blot.Cuando una patente se hace internacional, se puede encontrar en el idioma de cada país en que se ha solicitado. En Espacenet se tiene acceso a los documentos en cada idioma.Universidad del País Vasco; Instituto de Salud Carlos IIISolicitud de patent

A novel SPE-UHPLC-DAD method for the determination of fumagillin produced by Aspergillus fumigatus in cell culture media

[EN]Fumagillin is a biomolecule produced by Aspergillus fumigatus that is gaining relevance due to its connection with invasive aspergillosis. The determination of this molecule might help to understand the propagation of this disease and study its use as a potential biomarker. In spite of the interest of fumagillin in microbiological research, no quantitative method has been developed so far for its determination in cell culture media. In this work, the first validated method for the quantitative analysis of fumagillin in RPMI-1640 is presented. The sample treatment consists of a mixed-mode anion exchange Solid Phase Extraction that effectively removes potential interferences and offered a recovery of 83 ± 7%. The analysis was carried out by Ultra High Performance Liquid Chromatography coupled to Diode Array Detection at 336 nm. The method fulfilled the validation criteria established by EMA and FDA guidelines for bioanalysis (selectivity, carry over, linearity, accuracy, precision, dilution integrity and stability) and offers a limit of quantitation (25 μg·L−1) suitable for its intended use. Indeed, the method was satisfactorily applied to the quantification of the fumagillin produced by three strains of Aspergillus fumigatus with different toxin production capacity.This research was funded by University of the Basque Country (UPV/EHU) (project GIU19/068 and project COLAB20/11) and by Basque Government (grant number IT1362-19). XGP was funded by Basque Government

Molecular and cellular responses of the pathogenic fungus Lomentospora prolificans to the antifungal drug voriconazole

The filamentous fungus Lomentospora (Scedosporium) prolificans is an emerging opportunistic pathogen associated with fatal infections in patients with disturbed immune function. Unfortunately, conventional therapies are hardly of any use against this fungus due to its intrinsic resistance. Therefore, we performed an integrated study of the L. prolificans responses to the first option to treat these mycoses, namely voriconazole, with the aim of unveiling mechanisms involved in the resistance to this compound. To do that, we used a wide range of techniques, including fluorescence and electron microscopy to study morphological alterations, ion chromatography to measure changes in cell-wall carbohydrate composition, and proteomics-based techniques to identify the proteins differentially expressed under the presence of the drug. Significantly, we showed drastic changes occurring in cell shape after voriconazole exposure, L. prolificans hyphae being shorter and wider than under control conditions. Interestingly, we proved that the architecture and carbohydrate composition of the cell wall had been modified in the presence of the drug. Specifically, L. prolificans constructed a more complex organelle with a higher presence of glucans and mannans. In addition to this, we identified several differentially expressed proteins, including Srp1 and heat shock protein 70 (Hsp70), as the most overexpressed under voriconazole-induced stress conditions. The mechanisms described in this study, which may be directly related to L. prolificans antifungal resistance or tolerance, could be used as targets to improve existing therapies or to develop new ones in order to successfully eliminate these mycoses.This work has been supported by grants (GIU15/36 and UFI11/25) from the UPV/EHU. AP was supported by a predoctoral fellowship from the UPV/EHU, and IB and AA were supported by predoctoral fellowships from the Basque Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Cyclophilin and enolase are the most prevalent conidial antigens of Lomentospora prolificans recognized by healthy human salivary IgA and cross-react with Aspergillus fumigatus

Purpose: The study of the immunocompetent airways immune response may provide important information to improve the therapeutic efficacy against Lomentospora (Scedosporium) prolificans. So, this study aimed to identify the most prevalent conidial antigens of this multiresistant fungus recognized by healthy human salivary immunoglobulin A, and to study their expression and cross-reactivity with other fungal species. Experimental design: Twenty saliva from immunocompetent donors were used to detect and identify the immunoreactive proteins by 2D immunoblotting and LC-MS/MS. Moreover, anti-Aspergillus antibodies were purified to study their cross-reactivity. Results: Ten proteins of L. prolificans conidia showed reactivity with more than 50% of the saliva samples. Among them, cyclophilin and enolase were the most prevalent antigens recognized by 85 and 80% of the samples, respectively. These enzymes were also identified on the cell wall surface of L. prolificans and on the immunomes of Scedosporium apiospermum and Scedosporium aurantiacum. Additionally, they showed cross-reactivity with the most common pathogenic filamentous fungus Aspergillus fumigatus. Conclusion and clinical relevance: These results show that the immunocompetent immune response might offer a pan-fungal recognition of conserved antigens such as enolase and cyclophilins, making them potential candidates for study as therapeutic targets.This work has been partially supported by several grants (EHUA13/14, UFI11/25, GIU15/36) from the University of the Basque Country (UPV/EHU). I. B. and A. A. were supported by a fellowship from the Basque Government, and Aize Pellon was supported by a fellowship from the UPV/EHU

The Host Immune Response to Scedosporium/Lomentospora

Infections caused by the opportunistic pathogens Scedosporium/Lomentospora are on the rise. This causes problems in the clinic due to the difficulty in diagnosing and treating them. This review collates information published on immune response against these fungi, since an understanding of the mechanisms involved is of great interest in developing more effective strategies against them. Scedosporium/Lomentospora cell wall components, including peptidorhamnomannans (PRMs), α-glucans and glucosylceramides, are important immune response activators following their recognition by TLR2, TLR4 and Dectin-1 and through receptors that are yet unknown. After recognition, cytokine synthesis and antifungal activity of different phagocytes and epithelial cells is species-specific, highlighting the poor response by microglial cells against L. prolificans. Moreover, a great number of Scedosporium/Lomentospora antigens have been identified, most notably catalase, PRM and Hsp70 for their potential medical applicability. Against host immune response, these fungi contain evasion mechanisms, inducing host non-protective response, masking fungal molecular patterns, destructing host defense proteins and decreasing oxidative killing. In conclusion, although many advances have been made, many aspects remain to be elucidated and more research is necessary to shed light on the immune response to Scedosporium/Lomentospora.This research was funded by the Basque Government, grant number IT1362-19. L.M.-S., M.A., and L.A.-F. were funded by the Basque Government

Study of antifungal agent caspofungin adsorption to laboratory materials

[EN] Treatment of invasive fungal infections with Caspofungin is used as the first-line antifungal agents. The minimum inhibitory concentration value is a test which indicates the degree of sensitivity of a strain regarding a drug. However, no value of minimum inhibitory concentration for caspofungin is available because very variable value is obtained. In this work, we study the link with the adsorption phenomenon of CSF previously described in literature and the lack of minimum inhibitory concentration value. A systematic study of the impact of different parameters on CSF adsorption is reported. The effect of the nature of container material, the aqueous solution pH and the organic solvent proportion was studied. In addition, the possibility of using a coating agent to minimize the adsorption was assayed and evaluated. Results obtained showed the importance of the material used during the manipulation of CSF. The use of acidic pH aqueous solution or the addition of acetonitrile or methanol proportions (50 % and 70 %, respectively) were found efficient to avoid adsorption of CSF on glassware material, which is the relevant strategy for analytical samples of caspofungin. The treatment of HPLC glass vials and 96-well plates with N-(2-aminoethyl)-3-aminopropyltrimethoxysilane reduced the adsorption. The significant adsorption observed in this work especially with plastic materials, questions the results obtained before in different assays and explained the absence of MIC value.The authors thank University of the Basque Country (UPV/EHU) (Project GIU19/068 and Project COLAB20/11) and Basque Government (grant number IT1362-19) for financial support. B. Uribe thanks UPV/EHU for the pre-doctoral fellowship in co-supervision with the University of Bordeaux. X. Guruceaga thanks the Basque Government for his predoctoral grant

Nitrogen, Iron, and Zinc Acquisition: Key Nutrients to Aspergillus fumigatus Virulence

Aspergillus fumigatus is a ubiquitous soil decomposer and an opportunistic pathogen that is characterized by its large metabolic machinery for acquiring nutrients from media. Lately, an ever-increasing number of genes involved in fungal nutrition has been associated with its virulence. Of these, nitrogen, iron, and zinc metabolism-related genes are particularly noteworthy, since 78% of them have a direct implication in virulence. In this review, we describe the sensing, uptake and regulation process of the acquisition of these nutrients, the connections between pathways and the virulence-implicated genes. Nevertheless, only 40% of the genes mentioned in this review have been assayed for roles in virulence, leaving a wide field of knowledge that remains uncertain and might offer new therapeutic and diagnostic targets.This research was funded by the Basque Government: grant number IT1362-19. U.P.-C. and S.C.-S. received a predoctoral fellowship from the University of the Basque Country and Basque Government, respectively

Fungal Diversity and Composition of the Continental Solar Saltern in Añana Salt Valley (Spain)

The Añana Salt Valley in Spain is an active continental solar saltern formed 220 million years ago. To date, no fungal genomic studies of continental salterns have been published, although DNA metabarcoding has recently expanded researchers’ ability to study microbial community structures. Accordingly, the aim of this present study was to evaluate fungal diversity using the internal transcribed spacer (ITS) metabarcoding at different locations along the saltern (springs, ponds, and groundwater) to describe the fungal community of this saline environment. A total of 380 fungal genera were detected. The ubiquity of Saccharomyces was observed in the saltern, although other halotolerant and halophilic fungi like Wallemia, Cladosporium, and Trimmatostroma were also detected. Most of the fungi observed in the saltern were saprotrophs. The fungal distribution appeared to be influenced by surrounding conditions, such as the plant and soil contact, cereal fields, and vineyards of this agricultural region.This research was funded by the University of the Basque Country UPV/EHU grant number US19/01 and the Añana Salt Valley Foundation (Specific Agreement between the Añana Salt Valley Foundation and the University of the Basque Country UPV/EHU)

ELISA Test for the Serological Detection of Scedosporium/Lomentospora in Cystic Fibrosis Patients

The detection and diagnosis of the opportunistic fungi Scedosporium spp. and Lomentospora prolificans still relies mainly on low-sensitive culture-based methods. This fact is especially worrying in Cystic Fibrosis (CF) patients in whom these fungal species are frequently isolated and may increase the risk of suffering from an infection or other health problems. Therefore, with the purpose of developing a serologic detection method for Scedosporium/Lomentospora, four different Scedosporium boydii protein extracts (whole cell protein extract, secretome, total cell surface and conidial surface associated proteins) were studied by ELISA to select the most useful for IgG detection in sera from CF patients. The four extracts were able to discriminate the Scedosporium/Lomentospora-infected from Aspergillus-infected and non-infected patients. However, the whole cell protein extract was the one selected, as it was the one with the highest output in terms of protein concentration per ml of fungal culture used, and its discriminatory capacity was the best. The ELISA test developed was then assayed with 212 sera from CF patients and it showed to be able to detect Scedosporium spp. and Lomentospora prolificans with very high sensitivity and specificity, 86%-100% and 93%-99%, respectively, depending on the cut-off value chosen (four values were proposed A(450nm)= 0.5837, A(450nm)= 0.6042, A(450nm)= 0.6404, and A(450nm)= 0.7099). Thus, although more research is needed to reach a standardized method, this ELISA platform offers a rapid, low-cost and easy solution to detect these elusive fungi through minimally invasive sampling, allowing the monitoring of the humoral response to fungal presenceThis research was funded by the Basque Government, grant number IT1362-19. IB, LM-S, and LA-F received a predoctoral fellowship from the Basque Government. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the result