Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Dispersal history of SARS-CoV-2 in Galicia, Spain.

The dynamics of SARS-CoV-2 transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the USA became increasingly significant. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures.

Outbreak of Fusarium solani Meningitis in Immunocompetent Persons Associated With Neuraxial Blockade in Durango, Mexico, 2022-2023.

Background: Fungal meningitis can be associated with epidural anesthesia procedures. Fusariosis is a rare infection typically affecting immunocompromised patients and rarely causes meningitis. During 2022-2023, public health officials responded to a large outbreak of Fusarium solani meningitis associated with epidural anesthesia in Durango, Mexico. Methods: The public health response and epidemiological and clinical features of patients affected by this outbreak were described. Coordinated actions were addressed to identify the etiological agent, determine its drug susceptibility, develop diagnostic tests, and implement clinical and epidemiological protocols. Retrospective analyses of clinical variables and outcomes were performed to determine association with better patient survival. Results: A total of 1801 persons exposed to epidural anesthesia were identified, of whom 80 developed meningitis. Fusarium solani was found in 3 brain biopsies and showed susceptibility to voriconazole and amphotericin B. After F solani polymerase chain reaction (PCR) implementation, 57 patients with meningitis were PCR-screened, and 31 (38.8%) had a positive result. Most patients were female (95%), and cesarean section was the most common surgical procedure (76.3%). The case fatality rate was 51.3% (41 patients) and the median hospitalization duration was 39.5 days (interquartile range, 18-86 days). Seventy-one patients (88.8%) received voriconazole/amphotericin B and 64 subjects (80%) additionally received steroids. Cox regression analysis showed an increased lethality risk in patients who received antifungal treatment after 5 days (hazard ratio, 2.1 [95% confidence interval, 1.01-4.48], P < .05). Conclusions: The F solani meningitis outbreak in Durango was an unprecedented medical challenge. Timely treatment and effective healthcare management were associated with better survival outcomes.

Discovery and Characterization of the Fully Decorated Nocardiosis-Associated Polyketide Natural Product.

The genomes of 40 strains of Nocardia, most of which were associated with life-threatening human infections, encode a highly conserved assembly line polyketide synthase designated as the NOCAP (NOCardiosis-Associated Polyketide) synthase, whose product structure has been previously described. Here we report the structure and inferred biosynthetic pathway of the fully decorated glycolipid natural product. Its structure reveals a fully substituted benzaldehyde headgroup harboring an unusual polyfunctional tail and an O-linked disaccharide comprising a 3-α-epimycarose and 2-O-methyl-α-rhamnose whose installation requires flavin monooxygenase-dependent hydroxylation of the polyketide product. Production of the fully decorated glycolipid was verified in cultures of two patient-derived Nocardia species. In both E. coli and Nocardia spp., the glycolipid was only detected in culture supernatants, consistent with data from genetic knockout experiments implicating roles for two dedicated proteins in installing the second sugar substituent only after the monoglycosyl intermediate is exported across the bacterial cell membrane. With the NOCAP product in hand, the stage is set for investigating the evolutionary benefit of this polyketide biosynthetic pathway for Nocardia strains capable of infecting human hosts.

Identificación directa con MALDI-TOF desde el frasco de hemocultivo a partir de subcultivos con incubación corta. Estudio multicéntrico / Direct identification with MALDI-TOF from the blood culture bottle from subcultures with short incubation. Multicenter study / Identificação direta com MALDI-TOF do frasco de hemocultura de subculturas com incubação curta. Estudo multicêntrico

Resumen El objetivo de este trabajo fue evaluar el rendimiento de la identificación realizada con MALDI-TOF a partir de la incubación de 3-5 h de subcultivos de hemocultivos positivos monomicrobianos que se comparó con la obtenida con la incubación de 24 h de los mismos. En dos hospitales se utilizó el sistema Vitek-MS (bioMérieux, Francia) y en uno el sistema Micro- Flex LT (Bruker, Daltonics). A partir de la incubación corta, MALDI-TOF identificó correctamente a 5/5 de las levaduras, a 91,1% (153/168) de las bacterias gram positivas, a 96,7% (119/123) de los bacilos gram negativos y a 93,6% (277/296) del total de cepas. La identificación por medio de MALDI-TOF a partir de una corta incubación de los subcultivos de los hemocultivos en medio sólidos es un método práctico, sencillo y confiable.

Abstract The objective of this work was to evaluate the performance of the identification carried out with MALDI-TOF from the 3-5 h incubation of subcultures of monomicrobial positive blood cultures that was compared with that obtained with the 24 h incubation of the same subcultures. The Vitek-MS system (bioMérieux, France) was used in two hospitals and the Micro-Flex LT system (Bruker, Daltonics) in one. With a short incubation, MALDI-TOF correctly identified 5/5 of the yeasts, 91.1% (153/168) of the gram-positive bacteria, 96.7% (119/123) of the gram-negative bacilli and 93.6% (277/296) of the total strains. Identification by means of MALDI-TOF with a short incubation of subcultures of blood cultures in solid media is a practical, simple and reliable method.

Resumo O objetivo deste trabalho foi avaliar o desempenho da identificação realizada com MALDI-TOF a partir de 3 a 5 h de incubação de subculturas de hemoculturas positivas monomicrobianas que foi comparada com a obtida com a incubação de 24 h das mesmas. O sistema Vitek-MS (bioMérieux, França) foi utilizado em dois hospitais e o sistema Micro-Flex LT (Bruker, Daltonics) em um. A partir da incubação curta, o MALDI-TOF identificou corretamente 5/5 das leveduras, 91,1% (153/168) das bactérias gram positivas, 96,7% (119/123) dos bacilos gram-negativos e 93,6% (277/296) das cepas totais. A identificação por meio de MALDI-TOF a partir de uma incubação curta das subculturas das hemoculturas em meio sólido é um método prático, simples e confiável.

[Optimization in the detection of bacteremia: Integrated use of FilmArray (BCID2) and MALDI-TOF]. / Optimización en la detección de bacteriemias. Uso integrado de FilmArray (BCID2) y MALDI-TOF.

The usefulness of the combined use of MALDI-TOF MS from a subculture with 3-5h of incubation and the BCID2 panel (FilmArray) for the identification of microorganisms from positive blood cultures and its importance in the adjustment of antimicrobial therapy was analyzed. Overall identification with BCID2 was 90.4% (142/157) and with Maldi-TOF MS 83.4% (131/157) (p=0.0858); in 23 polymicrobial episodes (47 strains), the BCID2 panel identified 45 (95.7%) and MALDI-TOF MS 24 (51.1%) (p<0.0000). BCID2 detected the presence of the resistance genes mecA/C (n=16), blaKPC (n=8); blaCTX-M (n=17), blaNDM (n=8), blaOXA-48 (n=1), and vanA/B (n=2). The median time to report a result was 2.0h for BCID2 and 4.0h for MALDI-TOF MS (p<0.0000). Of 124 episodes analyzed, the rapid result of BCID2 led to 82.3% (102/124) therapeutic changes.

Chorioamnionitis secondary to Ureaplasma parvum infection: a case report.

Objectives: Ureaplasma species are the most frequently isolated microorganisms in cases of spontaneous preterm labor, premature rupture of the membranes, or chorioamnionitis. Case presentation: A woman at 28+6 weeks of gestation with no apparent history of interest presented at the hospital with contractions. Upon suspicion of chorioamnionitis, the patient was admitted for a low segment transverse cesarean section, which was completed without any complications. The patient was discharged at 7 days. The newborn remained stable and showed no clinical signs of infection. However, on suspicion of chorioamnionitis, empirical treatment with intravenous ampicillin (2 g every 6 h) and gentamicin (5 mg/kg once daily) was initiated. Samples of pharyngeal/tonsillar, ear, and anal/rectal exudates were collected. At 24 h, all samples were positive for Ureaplasma parvum. Empirical treatment was suspended, and treatment with intravenous azithromycin was initiated (12 mg once daily). Endocervical and placental exudates were also positive for U. parvum. Fifty-two days after birth, the newborn was discharged. Conclusions: The relationship between Ureaplasma spp. colonization and perinatal disease seem to be clear. However, the high frequency of vaginal Ureaplasma spp. colonization and high rates of term labor among pregnant women with this colonization make further studies necessary.

Microscopía vs. biología molecular en el diagnóstico de infecciones protozoarias intestinales, ¿es el momento del cambio? / Microscopy vs. molecular biology in the diagnosis of intestinal protozoal infections, is it time for a change?

Introducción: El examen microscópico de las parasitosis intestinales, a partir de las heces concentradas del paciente, presenta una menor sensibilidad si se compara con las técnicas de diagnóstico molecular. Por ello, el objetivo del presente estudio ha sido comparar ambas técnicas, así como evaluar si existe correlación entre el examen microscópico y los ciclos umbrales (Ct) obtenidos para Blastocystis hominis. Material y métodos: Estudio retrospectivo de las muestras recibidas en el laboratorio de Microbiología durante septiembre de 2021. Se realizó la prueba de concentración MiniParasep SF® para la visualización microscópica y posteriormente se realizó la PCR con el panel Seegene AllplexTM Parasite Assay. Resultados: Un 27% (n=74) de las muestras fueron positivas por diagnóstico molecular, con un total de 87 parásitosdetectados. El 53% (n=39) fueron mujeres con una edad media de 47 ± 24 años. En el 76% (n=56) de los casos el servicio de procedencia fue Atención Primaria. El parásito hallado con más frecuencia fue B. hominis, 85% (n=64), seguido de Dientamoeba fragilis 20% (n=15) y Giardia lamblia 11% (n=8). En 13 casos se detectaron co-infección por dos parásitos (en 6 casos B. hominis + D. fragilis, y en 7 casos B. hominis + G. lamblia). En el diagnóstico microscópico se obtuvo un 9,5% (n=26) de positividad. El parásito hallado con más frecuencia fue B. hominis,84% (n=23), seguido de G. lamblia se visualizó en tres casos por microscopía. D. fragilis no se visualizó en ningún caso. En una muestra se observó coinfección de B. hominis + G. lamblia. Conclusiones: Las técnicas para el diagnóstico molecular de las parasitosis intestinales son rápidas, fiables y más sensibles que las técnicas microscópicas, mejorando el diagnóstico microbiológico y la calidad asistencial. (AU)

Introduction: Microscopic examination of the intestinal parasites, from the patient’s concentrated feces, has a lower sensitivity when compared to molecular diagnostic techniques. Therefore, the objective of this study has been to compare both techniques, as well as to evaluate whether there is a correlation between the microscopic examination and the threshold cycles (Ct) obtained for Blastocystis hominis. Material and methods: Retrospective study of the samples received in the Microbiology laboratory during September 2021. The MiniParasep SF® concentration test was performed for microscopic visualization and then PCR was performed with the Seegene AllplexTM Parasite Assay panel. Results: A 27% (n=74) of the samples were positive by molecular diagnosis, with a total of 87 parasites detected. 53% (n=39) were women with a mean age of 47 ± 24 years. In 76% (n=56) of the cases the service of origin was Primary Care. The most frequently found parasite was B. hominis, 85% (n=64), followed by Dientamoeba fragilis 20% (n=15) and Giardia lamblia 11% (n=8). Co-infection by two parasites was detected in 13 cases (B. hominis + D. fragilis in 6 cases, and B. hominis + G. lamblia in 7 cases). In the microscopic diagnosis, 9.5% (n=26) positivity was obtained. The most frequently found parasite was B. hominis, 84% (n=23), followed by G. lamblia, which was seen in three cases by microscopy. D. fragilis was not seen in any case. Coinfection of B. hominis + G. lamblia was observed in one sample. Conclusions: Techniques for molecular diagnosis of intestinal parasites are fast, reliable and more sensitive than microscopic techniques, improving microbiological diagnosis and quality of care. (AU)

Biosynthesis of Isonitrile Lipopeptide Metallophores from Pathogenic Mycobacteria.

Isonitrile lipopeptides (INLPs) are known to be related to the virulence of pathogenic mycobacteria by mediating metal transport, but their biosynthesis remains obscure. In this work, we use in vitro biochemical assays, site-directed mutagenesis, chemical synthesis, and spectroscopy techniques to scrutinize the activity of core enzymes required for INLP biosynthesis in mycobacteria. Compared to environmental Streptomyces, pathogenic Mycobacterium employ a similar chemical logic and enzymatic machinery in INLP biosynthesis, differing mainly in the fatty-acyl chain length, which is controlled by multiple enzymes in the pathway. Our in-depth study on the non-heme iron(II) and α-ketoglutarate-dependent dioxygenase for isonitrile generation, including Rv0097 from Mycobacterium tuberculosis (Mtb), demonstrates that it recognizes a free-standing small molecule substrate, different from the recent hypothesis that a carrier protein is required for Rv0097 in Mtb. A key residue in Rv0097 is further identified to dictate the varied fatty-acyl chain length specificity between Streptomyces and Mycobacterium.

[Description of the first case of SARS-CoV-2 reinfection in Galicia (Spain).] / Descripción del primer caso de reinfección por SARS-CoV-2 en Galicia (España).

Since the first reinfection by SARS-CoV-2 was known in August 2020, several cases have been described around the world. We present the first reinfection of an immunocompetent patient confirmed in Galicia (Spain). The clinical-epidemiological information was obtained through an interview with the patient. The microbiological diagnosis was made by PCR of the nasopharyngeal exudate samples, and a serological study was carried out. In addition, a summary of the characteristics of all reinfections identified between 04-01-2021 and 03-10-2021 is presented.

Desde que en Agosto de 2020 se conociese la primera reinfección por SARS-CoV-2, se han descrito varios casos en todo el Mundo. Presentamos el primer caso confirmado en Galicia (España) de reinfección, en una paciente inmunocompetente. La información clínico-epidemiológica se obtuvo mediante entrevista con la paciente. El diagnóstico microbiológico se realizó mediante PCR de las muestras de exudado nasofaríngeo y se realizó estudio serológico. Además, se presenta un resumen de las características de todas las reinfecciones identificadas entre el 04-01-2021 y el 03-10-2021.

Probing the Mechanism of Isonitrile Formation by a Non-Heme Iron(II)-Dependent Oxidase/Decarboxylase.

The isonitrile moiety is an electron-rich functionality that decorates various bioactive natural products isolated from diverse kingdoms of life. Isonitrile biosynthesis was restricted for over a decade to isonitrile synthases, a family of enzymes catalyzing a condensation reaction between l-Trp/l-Tyr and ribulose-5-phosphate. The discovery of ScoE, a non-heme iron(II) and α-ketoglutarate-dependent dioxygenase, demonstrated an alternative pathway employed by nature for isonitrile installation. Biochemical, crystallographic, and computational investigations of ScoE have previously been reported, yet the isonitrile formation mechanism remains obscure. In the present work, we employed in vitro biochemistry, chemical synthesis, spectroscopy techniques, and computational simulations that enabled us to propose a plausible molecular mechanism for isonitrile formation. Our findings demonstrate that the ScoE reaction initiates with C5 hydroxylation of (R)-3-((carboxymethyl)amino)butanoic acid to generate 1, which undergoes dehydration, presumably mediated by Tyr96 to synthesize 2 in a trans configuration. (R)-3-isocyanobutanoic acid is finally generated through radical-based decarboxylation of 2, instead of the common hydroxylation pathway employed by this enzyme superfamily.

Biosynthesis of Isonitrile- and Alkyne-Containing Natural Products.

Natural products are a diverse class of biologically produced compounds that participate in fundamental biological processes such as cell signaling, nutrient acquisition, and interference competition. Unique triple-bond functionalities like isonitriles and alkynes often drive bioactivity and may serve as indicators of novel chemical logic and enzymatic machinery. Yet, the biosynthetic underpinnings of these groups remain only partially understood, constraining the opportunity to rationally engineer biomolecules with these functionalities for applications in pharmaceuticals, bioorthogonal chemistry, and other value-added chemical processes. Here, we focus our review on characterized biosynthetic pathways for isonitrile and alkyne functionalities, their bioorthogonal transformations, and prospects for engineering their biosynthetic machinery for biotechnological applications.

Nanoparticle cellular internalization is not required for RNA delivery to mature plant leaves.

Rapidly growing interest in the nanoparticle-mediated delivery of DNA and RNA to plants requires a better understanding of how nanoparticles and their cargoes translocate in plant tissues and into plant cells. However, little is known about how the size and shape of nanoparticles influence transport in plants and the delivery efficiency of their cargoes, limiting the development of nanotechnology in plant systems. In this study we employed non-biolistically delivered DNA-modified gold nanoparticles (AuNPs) of various sizes (5-20 nm) and shapes (spheres and rods) to systematically investigate their transport following infiltration into Nicotiana benthamiana leaves. Generally, smaller AuNPs demonstrated more rapid, higher and longer-lasting levels of association with plant cell walls compared with larger AuNPs. We observed internalization of rod-shaped but not spherical AuNPs into plant cells, yet, surprisingly, 10 nm spherical AuNPs functionalized with small-interfering RNA (siRNA) were the most efficient at siRNA delivery and inducing gene silencing in mature plant leaves. These results indicate the importance of nanoparticle size in efficient biomolecule delivery and, counterintuitively, demonstrate that efficient cargo delivery is possible and potentially optimal in the absence of nanoparticle cellular internalization. Overall, our results highlight nanoparticle features of importance for transport within plant tissues, providing a mechanistic overview of how nanoparticles can be designed to achieve efficacious biocargo delivery for future developments in plant nanobiotechnology.

Biosynthesis of triacsin featuring an N-hydroxytriazene pharmacophore.

Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing, hydrazine-forming enzyme (Tri28) and a nitrite-utilizing, N-nitrosating enzyme (Tri17). This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.

Rainfall decrease and red deer rutting behaviour: Weaker and delayed rutting activity though higher opportunity for sexual selection.

In the last decades, climate change has caused an increase in mean temperatures and a reduction in average rainfall in southern Europe, which is expected to reduce resource availability for herbivores. Resource availability can influence animals' physical condition and population growth. However, much less is known on its effects on reproductive performance and sexual selection. In this study, we assessed the impact of three environmental factors related to climate change (rainfall, temperature and vegetation index) on Iberian red deer Cervus elaphus hispanicus reproductive timing and sexual behaviour, and their effects on the opportunity for sexual selection in the population. We measured rutting phenology as rut peak date, the intensity of male rutting activity as roaring rate, and the opportunity for sexual selection from the distribution of females among harem holding males in Doñana Biological Reserve (Southwest Spain), from data of daily observations collected during the rut over a period of 25 years. For this study period, we found a trend for less raining and hence poorer environmental conditions, which associated with delayed rutting season and decreased rutting intensity, but that appeared to favour a higher degree of polygyny and opportunity for sexual selection, all these relationships being modulated by population density and sex ratio. This study highlights how climate change (mainly rainfall reduction in this area) can alter the conditions for mating and the opportunity for sexual selection in a large terrestrial mammal.

Biochemical and crystallographic investigations into isonitrile formation by a nonheme iron-dependent oxidase/decarboxylase.

The isonitrile moiety is found in marine sponges and some microbes, where it plays a role in processes such as virulence and metal acquisition. Until recently only one route was known for isonitrile biosynthesis, a condensation reaction that brings together a nitrogen atom of l-Trp/l-Tyr with a carbon atom from ribulose-5-phosphate. With the discovery of ScoE, a mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase from Streptomyces coeruleorubidus, a second route was identified. ScoE forms isonitrile from a glycine adduct, with both the nitrogen and carbon atoms coming from the same glycyl moiety. This reaction is part of the nonribosomal biosynthetic pathway of isonitrile lipopeptides. Here, we present structural, biochemical, and computational investigations of the mechanism of isonitrile formation by ScoE, an unprecedented reaction in the mononuclear Fe(II) α-ketoglutarate-dependent dioxygenase superfamily. The stoichiometry of this enzymatic reaction is measured, and multiple high-resolution (1.45-1.96 Å resolution) crystal structures of Fe(II)-bound ScoE are presented, providing insight into the binding of substrate, (R)-3-((carboxylmethyl)amino)butanoic acid (CABA), cosubstrate α-ketoglutarate, and an Fe(IV)=O mimic oxovanadium. Comparison to a previously published crystal structure of ScoE suggests that ScoE has an "inducible" α-ketoglutarate binding site, in which two residues arginine-157 and histidine-299 move by approximately 10 Å from the surface of the protein into the active site to create a transient α-ketoglutarate binding pocket. Together, data from structural analyses, site-directed mutagenesis, and computation provide insight into the mode of α-ketoglutarate binding, the mechanism of isonitrile formation, and how the structure of ScoE has been adapted to perform this unusual chemical reaction.