Validación de una nueva estrategia para la identificación de variantes de SARS-CoV-2 mediante secuenciación del gen espiga por Sanger / Validation of a new strategy for the identification of SARS-CoV-2 variants by sequencing the spike gene by Sanger

Introducción: La aparición de múltiples variantes del SARS-CoV-2 durante la pandemia de COVID-19 es motivo de gran preocupación mundial. Hasta el momento, su análisis se ha centrado principalmente en la secuenciación de nueva generación. Sin embargo, esta técnica es costosa y requiere equipos sofisticados, largos tiempos de procesamiento y personal técnico altamente cualificado con experiencia en bioinformática. Para contribuir al análisis de variantes de interés y de preocupación, aumentar la capacidad diagnóstica y procesar muestras para realizar vigilancia genómica, proponemos una metodología rápida y fácil de aplicar, basada en la secuenciación Sanger de 3 fragmentos del gen que codifica para la proteína espiga. Métodos: Se secuenciaron 15 muestras positivas para SARS-CoV-2 con un valor de umbral de ciclo inferior a 25 por metodologías Sanger y secuenciación de nueva generación. Los datos obtenidos fueron analizados en las plataformas Nextstrain y PANGO Lineages. Resultados: Ambas metodologías permitieron identificar las variantes de interés reportadas por la OMS. Se identificaron 2 muestras como alfa, 3 gamma, una delta, tres mu, una ómicron y 5 cepas cercanas al aislado inicial del virus Wuhan-Hu-1. Según el análisis in silico, también se pueden detectar mutaciones clave para identificar y clasificar otras variantes no evaluadas en el estudio. Conclusión: Los diferentes linajes de interés y preocupación de SARS-CoV-2 se clasifican de forma rápida, ágil y fiable con la metodología de secuenciación de Sanger.(AU)

Introduction: The emergence of multiple variants of SARS-CoV-2 during the COVID-19 pandemic is of great world concern. Until now, their analysis has mainly focused on next-generation sequencing. However, this technique is expensive and requires sophisticated equipment, long processing times, and highly qualified technical personnel with experience in bioinformatics. To contribute to the analysis of variants of interest and variants of concern, increase the diagnostic capacity, and process samples to carry out genomic surveillance, we propose a quick and easy methodology to apply, based on Sanger sequencing of 3 gene fragments that code for protein spike. Methods: Fifteen positive samples for SARS-CoV-2 with a cycle threshold below 25 were sequenced by Sanger and next-generation sequencing methodologies. The data obtained were analyzed on the Nextstrain and PANGO Lineages platforms. Results: Both methodologies allowed the identification of the variants of interest reported by the WHO. Two samples were identified as Alpha, 3 Gamma, one Delta, 3 Mu, one Omicron, and 5 strains were close to the initial Wuhan-Hu-1 virus isolate. According to in silico analysis, key mutations can also be detected to identify and classify other variants not evaluated in the study. Conclusion: The different SARS-CoV-2 lineages of interest and concern are classified quickly, agilely, and reliably with the Sanger sequencing methodology.(AU)

From degrader to producer: reversing the gallic acid metabolism of Pseudomonas putida KT2440 / De degradador a productor: revirtiendo el metabolismo del ácido gálico de Pseudomonas putida KT2440

Gallic acid is a powerful antioxidant with multiple therapeutic applications, usually obtained from the acidic hydrolysis of tannins produced by many plants. As this process generates a considerable amount of toxic waste, the use of tannases or tannase-producing microorganisms has become a greener alternative over the last years. However, their high costs still impose some barriers for industrial scalability, requiring solutions that could be both greener and cost-effective. Since Pseudomonas putida KT2440 is a powerful degrader of gallic acid, its metabolism offers pathways that can be engineered to produce it from cheap and renewable carbon sources, such as the crude glycerol generated in biodiesel units. In this study, a synthetic operon with the heterologous genes aroG4, quiC and pobA* was developed and expressed in P. putida, based on an in silico analysis of possible metabolic routes, resulting in no production. Then, the sequences pcaHG and galTAPR were deleted from the genome of this strain to avoid the degradation of gallic acid and its main intermediate, the protocatechuic acid. This mutant was transformed with the vector containing the synthetic operon and was finally able to convert glycerol into gallic acid. Production assays in shaker showed a final concentration of 346.7 ± 0.004 mg L−1 gallic acid after 72 h.(AU)

Abundance, distribution, and expression of nematicidal crystal protein genes in Bacillus thuringiensis strains from diverse habitats / Abundancia, distribución y expresión de genes de proteínas cristalinas nematicidas en cepas de Bacillus thuringiensis de diversos hábitats

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that accumulates pesticidal proteins (Cry and Cyt) in parasporal crystals. Proteins from the Cry5, App6 (formerly Cry6), Cry12, Cry13, Cry14, Cry21, and Xpp55 (formerly Cry55) families have been identified as toxic to nematodes. In this study, a total of 846 Bt strains belonging to four collections were analyzed to determine the diversity and distribution of the Bt Cry nematicidal protein genes. We analyzed their presence by PCR, and positives were confirmed by sequencing. As a result, 164 Bt isolates (20%) contained at least one gene coding for nematicidal Cry proteins. The cry5 and cry21 genes were enriched in collection 1 and were often found together in the same strain. Differently, in collection 4, obtained from similar habitats but after 10 years, cry14 was the gene most frequently found. In collection 2, cry5 and app6 were the most abundant genes, and collection 3 had a low incidence of any of these genes. The results point to high variability in the frequencies of the studied genes depending on the timing, geographical origins, and sources. The occurrence of cry1A, cry2, and cry3 genes was also analyzed and showed that the nematicidal Cry protein genes were frequently accompanied by cry1A + cry2. The expression of the genes was assessed by mass spectrometry showing that only 14% of the positive strains produced nematicidal proteins. To our knowledge, this is the first comprehensive screening that examines the presence and expression of genes from the seven known Bt Cry nematicidal families.(AU)

Characterization of kefir yeasts with antifungal capacity against Aspergillus species / Caracterización de levaduras de kéfir con capacidad antifúngica frente a especies de Aspergillus

Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them—belonging to S. cerevisiae and P. kudriavzevii species—generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.(AU)

Epidemiology, molecular characterization, and drug resistance of IncHI5 plasmids from Enterobacteriaceae / Epidemiología, caracterización molecular y farmacorresistencia de plásmidos IncHI5 de Enterobacteriaceae

The increasingly frequent occurence of IncHI5 plasmids has attracted worldwide attention. The aim of this study was to perform an in-depth bioinformatics analysis to determine the genetic characteristics and global distribution of all IncHI5 plasmids. The geographic distribution and epidemiology of all IncHI5 plasmids from GenBank were analyzed based on relevant literature reports and background information from the National Center for Biotechnology Information (NCBI). Detailed annotation of antibiotic resistance genes was performed. A total of 65 IncHI5 plasmid genomes were collected in GenBank. All IncHI5 plasmids were carried by Enterobacteriaceae, of which Klebsiella pneumoniae accounted for the largest proportion (50%, 33/65). The host bacterium of IncHI5 plasmids was mainly isolated from Homo Sapiens (81%, 53/65). All strains carrying IncHI5 plasmids were mainly distributed in China (83%, 54/65). Evolutionary analysis can divide IncHI5 plasmids into two groups, namely Groups I/II, of which Group II was more widely distributed worldwide. This study showed that Enterobacteriaceae, especially Klebsiella, was the main host for IncHI5 plasmid. Almost all IncHI5 plasmids carried multiple types of antibiotic resistance genes, related to Tn1696 or Tn6535. The IncHI5 plasmids should be of continuing interest as good repositories for antibiotic resistance genes.(AU)

Antiviral activity of nano-monocaprin against Phi6 as a surrogate for SARS-CoV-2 / Actividad antiviral de nano-monocaprina contra Phi6 como sustituto del SARS-CoV-2

The COVID-19 pandemic involving SARS-CoV-2 has raised interest in using antimicrobial lipid formulations to inhibit viral entry into their host cells or to inactivate them. Lipids are a part of the innate defense mechanism against pathogens. Here, we evaluated the use of nano-monocaprin (NMC) in inhibiting enveloped (phi6) and unenveloped (MS2) bacteriophages. NMC was prepared using the sonochemistry technique. Size and morphology analysis revealed the formation of ~ 8.4 ± 0.2-nm NMC as measured by dynamic light scattering. We compared the antiviral activity of NMC with molecular monocaprin (MMC) at 0.5 mM and 2 mM concentrations against phi6, which we used as a surrogate for SARS-CoV-2. The synthesized NMC exhibited 50% higher antiviral activity against phi6 than MMC at pH 7 using plaque assay. NMC inactivated phi6 stronger at pH 4 than at pH 7. To determine if NMC is toxic to mammalian cells, we used MTS assay to assess its IC50 for HPDE and HeLa cell lines, which were ~ 203 and 221 µM, respectively. NMC may be used for prophylactic application either as a drop or spray since many viruses enter the human body through the mucosal lining of the nose, eyes, and lungs.(AU)

What is essential remains invisible to the eyes? Blood pressure cuffs colonized by bacterial diversity / ¿Qué es lo esencial permanece invisible a los ojos? Esfigmomanómetros colonizados por diversidad bacteriana

Using sphygmomanometers to measure blood pressure is a common practice in the healthcare context. The disinfection and maintenance of these devices is essential in clinical practice to prevent the proliferation of microorganisms. The aim of this study was to determine the presence of pathogenic microorganisms in sphygmomanometer cuffs in the clinical setting. A cross-sectional study was carried out. Five types of healthcare centers, selected through convenience sampling, participated in this study. Samples were collected from the inside of sphygmomanometer cuffs, and labeled and delivered to the laboratory for analysis. The samples were incubated in an oven at 35.5 °C for 24 h. A total CFU count was carried out on the plates that were cataloged as positive. Colonies that showed growth were identified using the matrix-assisted laser desorption/ionization-mass spectrometry technology. Of the total sample, (N = 372), 69.1% were positive and were isolated. In 30.9% (n = 115), no bacterial development was found within 48 h. A total of 257 microorganisms were found. The mean number of colony-forming units was 29.62 (SD = 32.33). The socio-health centers had the highest amount of bacterial contamination in the cuffs. In regards to the type of microorganisms, 31.5% (n = 81) found were Bacillus cereus, followed by 26.8% (n = 69) of Staphylococcus hominis and 9.7% (n = 25) were Pantoea agglomerans, among others. Statistically significant differences were found between the type of microorganism and the hours elapsed since the last disinfection (X2(19) = 44.582; p = 0.001). Statistically significant differences were found between the time elapsed since the last disinfection and the type of sphygmomanometer (X2 (2) = 117.752; p = 0.000). Despite the fact that most hospitals and health centers have established infection control policies and protocols, the results of this study indicate the presence of pathogenic microorganisms in blood pressure cuffs in the clinical setting.(AU)

Co-diet supplementation of low density polyethylene and honeybee wax did not influence the core gut bacteria and associated enzymes of Galleria mellonella larvae (Lepidoptera: Pyralidae) / La suplementación en la dieta conjunta de polietileno de baja densidad y cera de abeja no influyó en las bacterias intestinales centrales y las enzimas asociadas de las larvas de Galleria mellonella (Lepidoptera: Pyralidae)

The current plastic pollution throughout the world is a rising concern that demands the optimization of biodegradation processes. One avenue for this is to identify plastic-degrading bacteria and associated enzymes from the gut bacteria of insect models such as Tenebrio molitor, Plodia interpunctella or Galleria mellonella that have the ability to ingest and rapidly degrade polyethylene. Therefore, this study takes part in understanding the role of the gut bacteria by investigating G. mellonella as a biological model feeding with a diet based on honeybee wax mixed or not with low-density polyethylene. Gut microbiome was analyzed by high throughput 16S rRNA sequencing, and Enterococcaceae and Oxalobacteraceae were found to be the major bacterial families. Compared to the control, the supplementation of low-density polyethylene did not cause significant modification of the bacterial microbiota at community and taxa levels, suggesting bacterial microbiome resilience. The bacterial proteome analysis of gut contents was encouraging for the identification of plastic degrading enzymes such as the phenylacetaldehyde dehydrogenase which participate in styrene degradation. This study allowed a better characterization of the gut bacteria of G. mellonella and provided a basis for the further study of biodegradation of polyethylene based on the bacterial microbiota from insect guts.(AU)

In situ bioremediation of petroleum hydrocarbon–contaminated soil: isolation and application of a Rhodococcus strain / Biorremediación in situ de suelos contaminados con hidrocarburos de petróleo: aislamiento y aplicación de una cepa de Rhodococcus

Due to low consumption and high efficiency, in situ microbial remediation of petroleum hydrocarbons (PHs)-contaminated sites in in-service petrochemical enterprises has attracted more and more attention. In this study, a degrading strain was isolated from oil depot–contaminated soil with soil extract (PHs) as the sole carbon source, identified and named Rhodococcus sp. OBD-3. Strain OBD-3 exhibited wide adaptability and degradability over a wide range of temperatures (15–37 °C), pH (6.0–9.0), and salinities (1–7% NaCl) to degrade 60.6–86.6% of PHs. Under extreme conditions (15 °C and 3–7% salinity), PHs were degraded by 60.6 ± 8.2% and more than 82.1% respectively. In OBD-3, the alkane monooxygenase genes alkB1 and alkB2 (GenBank accession numbers: MZ688386 and MZ688387) were found, which belonged to Rhodococcus by sequence alignment. Moreover, strain OBD-3 was used in lab scale remediation in which the contaminated soil with OBD-3 was isolated as the remediation object. The PHs were removed at 2,809 ± 597 mg/kg within 2 months, and the relative abundances of Sphingobium and Pseudomonas in soil increased more than fivefold. This study not only established a system for the isolation and identification of indigenous degrading strains that could efficiently degrade pollutants in the isolated environment but also enabled the isolated degrading strains to have potential application prospects in the in situ bioremediation of PHs-contaminated soils.(AU)

A simple double differential centrifugation-wash procedure to rapidly obtain bacterial identification and direct antimicrobial susceptibility testing from positive blood cultures / Método sencillo basado en una doble centrifugación diferencial para obtener una identificación bacteriana y sensibilidad a antibióticos rápida a partir de hemocultivos positivos

Introduction: This study proposes a simple and rapid method for both bacterial identification and direct antimicrobial susceptibility testing (AST) by using MALDI-TOF and a double differential centrifugation-wash procedure from positive blood cultures. Methods: Fifty-two positive blood cultures (37 gramnegative bacilli and 15 grampositive cocci) were studied by two methods for identification and AST: a reference method, and the rapid MALDI-TOF method obtaining a purified pellet by using a double differential centrifugation procedure. Results : A total of 1101 MIC values (mg/l) were interpreted according to EUCAST clinical breakpoints and compared using the two methods simultaneously. Discrepancies in 81 MIC values (7.35%) were detected. By analyzing standard parameters, we obtained 98.28% essential agreement and 92.65% categorical agreement considering all isolates tested. Conclusion: This method provides rapid bacterial identification and AST, offering definitive results 24–48h earlier than the conventional method (p<0.001) and improving the turnaround time in blood culture diagnostics, especially in laboratories without 24-h on-call.(AU)

Introducción: Este trabajo propone un método sencillo, rápido y barato de identificación bacteriana y sensibilidad antibiótica utilizando MALDI-TOF y una doble centrifugación diferencial a partir de hemocultivo positivo. Métodos: Se estudiaron 52 hemocultivos positivos (37 bacilos gramnegativos y 15 cocos grampositivos). Se compararon 2 métodos: un método convencional de identificación y determinación de sensibilidad a antibióticos automatizada partiendo de colonia crecida, y un método rápido utilizando MALDI-TOF, caracterizado por la obtención de un pellet purificado procedente de un hemocultivo positivo, mediante un procedimiento basado en una doble centrifugación diferencial. Resultados: Se analizaron y compararon 1.101 valores de CMI (mg/l) de acuerdo con los criterios establecidos por EUCAST y obtenidos por ambos métodos. Se detectaron discrepancias en 81 valores de CMI (7,35%). Considerando todos los aislados, la concordancia esencial fue del 98,28% y la concordancia categórica del 92,65%. Conclusión: Este método proporciona resultados de identificación y sensibilidad a antibióticos definitivos 24-48h antes que un método convencional (p<0,001), mejorando el tiempo de respuesta en el diagnóstico microbiológico de bacteriemias, especialmente en laboratorios sin servicio de guardias de 24h.(AU)

Bajo riesgo de contagio ambiental por SARS-CoV-2 en espacios no sanitarios / Low risk of environmental contagion by SARS-CoV-2 in non-sanitary spaces

Objetivo: Estudiar la presencia de SARS-CoV-2 en superficies (alto, medio y bajo contacto) y aires de espacios no sanitarios pero de elevada afluencia de público para evaluar el riesgo de contagio ambiental. Método: Se ha realizado el análisis de las superficies y de los aires por RT-qPCR para detectar la presencia de SARS-CoV-2. Resultados: Se obtuvieron 394 superficies y 23 muestras de aire de espacios de alta afluencia de personas, como oficinas, centros comerciales y residencias de ancianos. El virus no fue detectado en ninguna de las muestras analizadas. Conclusión: Aunque no podemos concluir rotundamente que no existe un riesgo de infección ambiental por SARS-CoV-2 en espacios no sanitarios, sí podemos afirmar que el riesgo es casi nulo.(AU)

Objective: To study the presence of SARS-CoV-2 on surfaces (high, medium and low contacts) and airs in non-sanitary spaces with high public influx to evaluate the risk of environmental contagion. Method: Surfaces and airs were analysed by RT-qPCR to detect the presence of SARS-CoV-2. Results: A total of 394 surfaces and air samples were obtained from spaces with high public influx such as offices, shopping centres and nursing homes. The virus was not detected in any of the samples analysed. Conclusion: Although we cannot emphatically conclude that there is no risk of environmental infection by SARS-CoV-2 in non-sanitary spaces, we can affirm that the risk is almost non- existent.(AU)

Antimicrobial stewardship in hospitals: Expert recommendation guidance document for activities in specific populations, syndromes and other aspects (PROA-2) from SEIMC, SEFH, SEMPSPGS, SEMICYUC and SEIP / Programas de optimización del uso de antimicrobianos en hospitales: guía de recomendaciones de expertos para actividades en poblaciones específicas, síndromes y otros aspectos (PROA-2) de la SEIMC, SEFH, SEMPSPGS y SEIP

In 2012, The Spanish Societies of Infectious Diseases and Clinical Microbiology (SEIMC), Hospital Pharmacy (SEFH), and Preventive Medicine, Public Health and Healthcare Management (SEMPSGS) lead a consensus document including recommendations for the implementation of antimicrobial stewardship (AMS) programs (AMSP; PROA in Spanish) in acute care hospitals in Spain. While these recommendations were critical for the development of these programs in many centres, there is a need for guidance in the development of AMS activities for specific patient populations, syndromes or other specific aspects which were not included in the previous document or have developed significantly since then. The objective of this expert recommendation guidance document is to review the available information about these activities in these patient populations or circumstances, and to provide guidance recommendations about them. With this objective the SEIMC, SEFH, SEMPSPGS, the Spanish Society of Intensive Care Medicine (SEMICYUC) and the Spanish Pediatric Infectious Disease Society (SEIP) selected a panel of experts who chose the different aspects to include in the document. Because of the lack of high-level evidence in the implementation of the activities, the panel opted to perform a narrative review of the literature for the different topics for which recommendations were agreed by consensus. The document was open to public consultation for the members of these societies for their comments and suggestions, which were reviewed and considered by the panel.(AU)

En 2012, las Sociedades Españolas de Enfermedades Infecciosas y Microbiología Clínica (SEIMC), Farmacia Hospitalaria (SEFH) y Medicina Preventiva, Salud Pública y Gestión Sanitaria (SEMPSPGS) lideraron un documento de consenso que incluía recomendaciones para la implementación de Programas de optimización del uso de antimicrobianos (PROA) en hospitales de agudos en España. Si bien estas recomendaciones fueron críticas para el desarrollo de estos programas en muchos centros, actualmente es necesario establecer unas guías para la implementación de las actividades de los PROA en determinadas poblaciones de pacientes, síndromes clínicos y otros aspectos específicos que no se incluyeron en el documento previo o que desde entonces se han desarrollado significativamente. El objetivo de esta guía de recomendaciones de expertos es revisar la información disponible acerca de esas actividades en estas poblaciones o circunstancias de pacientes y proporcionar unas recomendaciones que sirvan de guía sobre ellas. Con este objetivo, la SEIMC, la SEFH y la SEMPSPGS, así como la Sociedad Española de Medicina Intensiva, Crítica y Unidades Coronarias (SEMICYUC) y la Sociedad Española de Infectología Pediátrica (SEIP), seleccionaron un panel de expertos que eligieron los diferentes aspectos a incluir en el documento. Debido a la ausencia de evidencia de alto nivel en la implementación de las diferentes actividades, el panel optó por realizar una revisión narrativa de la literatura de los diferentes aspectos, en los que las recomendaciones se acordaron por consenso. El documento se abrió para consulta pública a los miembros de estas sociedades para sus comentarios y sugerencias, que fueron revisadas y consideradas por el panel.(AU)