Structural and functional analysis of the key enzyme responsible for the degradation of ochratoxin A in the Alcaligenes genus.

The potential to degrade ochratoxin A (OTA), a highly poisonous mycotoxin, was investigated in cultures from Alcaligenes-type strains. Genome sequence analyses from different Alcaligenes species have permitted us to demonstrate a direct, causal link between the gene coding a known N-acyl-L-amino acid amidohydrolase from A. faecalis (AfOTH) and the OTA-degrading activity of this bacterium. In agreement with this finding, we found the gene coding AfOTH in two additional species included in the Alcaligenes genus, namely, A. pakistanensis, and A. aquatilis, which also degraded OTA. Notably, A. faecalis subsp. faecalis DSM 30030T was able to transform OTα, the product of OTA hydrolysis. AfOTH from A. faecalis subsp. phenolicus DSM 16503T was recombinantly over-produced and enzymatically characterized. AfOTH is a Zn2+-containing metalloenzyme that possesses structural features and conserved residues identified in the M20D family of enzymes. AfOTH is a tetramer in solution that shows both aminoacylase and carboxypeptidase activities. Using diverse potential substrates, namely, N-acetyl-L-amino acids and carbobenzyloxy-L-amino acids, a marked preference towards C-terminal Phe and Tyr residues could be deduced. The structural basis for this specificity has been determined by in silico molecular docking analyses. The amidase activity of AfOTH on C-terminal Phe residues structurally supports its OTA and OTB degradation activity.

A new and promiscuous α/ß hydrolase from Acinetobacter tandoii DSM 14970 T inactivates the mycotoxin ochratoxin A.

The presence of ochratoxin A (OTA) in food and feed represents a serious concern since it raises severe health implications. Bacterial strains of the Acinetobacter genus hydrolyse the amide bond of OTA yielding non-toxic OTα and L-ß-phenylalanine; in particular, the carboxypeptidase PJ15_1540 from Acinetobacter sp. neg1 has been identified as an OTA-degrading enzyme. Here, we describe the ability to transform OTA of cell-free protein extracts from Acinetobacter tandoii DSM 14970 T, a strain isolated from sludge plants, and also report on the finding of a new and promiscuous α/ß hydrolase (ABH), with close homologs highly distributed within the Acinetobacter genus. ABH from A. tandoii (AtABH) exhibited amidase activity against OTA and OTB mycotoxins, as well as against several carboxypeptidase substrates. The predicted structure of AtABH reveals an α/ß hydrolase core composed of a parallel, six-stranded ß-sheet, with a large cap domain similar to the marine esterase EprEst. Further biochemical analyses of AtABH reveal that it is an efficient esterase with a similar specificity profile as EprEst. Molecular docking studies rendered a consistent OTA-binding mode. We proposed a potential procedure for preparing new OTA-degrading enzymes starting from promiscuous α/ß hydrolases based on our results. KEY POINTS: • AtABH is a promiscuous αß hydrolase with both esterase and amidohydrolase activities • AtABH hydrolyses the amide bond of ochratoxin A rendering nontoxic OTα • Promiscuous αß hydrolases are a possible source of new OTA-degrading enzymes.

Food phenolics and Lactiplantibacillus plantarum.

Phenolic compounds are important constituents of plant food products. These compounds play a key role in food characteristics such as flavor, astringency and color. Lactic acid bacteria are naturally found in raw vegetables, being Lactiplantibacillus plantarum the most commonly used commercial starter for the fermentation of plant foods. Hence, the metabolism of phenolic compounds of L. plantarum has been a subject of study in recent decades. Such studies confirm that L. plantarum, in addition to presenting catalytic capacity to transform aromatic alcohols and phenolic glycosides, exhibits two main differentiated metabolic routes that allow the biotransformation of dietary hydroxybenzoic and hydroxycinnamic acid-derived compounds. These metabolic pathways lead to the production of new compounds with new biological and organoleptic properties. The described metabolic pathways involve the action of specialized esterases, decarboxylases and reductases that have been identified through genetic analysis and biochemically characterized. The purpose of this review is to provide a comprehensive and up-to-date summary of the current knowledge of the metabolism of food phenolics in L. plantarum.

Role of the first WHO mutation catalogue in the diagnosis of antibiotic resistance in Mycobacterium tuberculosis in the Valencia Region, Spain: a retrospective genomic analysis.

BACKGROUND: In June, 2021, WHO published the most complete catalogue to date of resistance-conferring mutations in Mycobacterium tuberculosis. Here, we aimed to assess the performance of genome-based antimicrobial resistance prediction using the catalogue and its potential for improving diagnostics in a real low-burden setting. METHODS: In this retrospective population-based genomic study M tuberculosis isolates were collected from 25 clinical laboratories in the low-burden setting of the Valencia Region, Spain. Culture-positive tuberculosis cases reported by regional public health authorities between Jan 1, 2014, and Dec 31, 2016, were included. The drug resistance profiles of these isolates were predicted by the genomic identification, via whole-genome sequencing (WGS), of the high-confidence resistance-causing variants included in the catalogue and compared with the phenotype. We determined the minimum inhibitory concentration (MIC) of the isolates with discordant resistance profiles using the resazurin microtitre assay. FINDINGS: WGS was performed on 785 M tuberculosis complex culture-positive isolates, and the WGS resistance prediction sensitivities were: 85·4% (95% CI 70·8-94·4) for isoniazid, 73·3% (44·9-92·2) for rifampicin, 50·0% (21·1-78·9) for ethambutol, and 57·1% (34·0-78·2) for pyrazinamide; all specificities were more than 99·6%. Sensitivity values were lower than previously reported, but the overall pan-susceptibility accuracy was 96·4%. Genotypic analysis revealed that four phenotypically susceptible isolates carried mutations (rpoB Leu430Pro and rpoB Ile491Phe for rifampicin and fabG1 Leu203Leu for isoniazid) known to give borderline resistance in standard phenotypic tests. Additionally, we identified three putative resistance-associated mutations (inhA Ser94Ala, katG Leu48Pro, and katG Gly273Arg for isoniazid) in samples with substantially higher MICs than those of susceptible isolates. Combining both genomic and phenotypic data, in accordance with the WHO diagnostic guidelines, we could detect two new multidrug-resistant cases. Additionally, we detected 11 (1·6%) of 706 isolates to be monoresistant to fluoroquinolone, which had been previously undetected. INTERPRETATION: We showed that the WHO catalogue enables the detection of resistant cases missed in phenotypic testing in a low-burden region, thus allowing for better patient-tailored treatment. We also identified mutations not included in the catalogue, relevant at the local level. Evidence from this study, together with future updates of the catalogue, will probably lead in the future to the partial replacement of culture testing with WGS-based drug susceptibility testing in our setting. FUNDING: European Research Council and the Spanish Ministerio de Ciencia.

Dual 6Pß-Galactosidase/6Pß-Glucosidase GH1 Family for Lactose Metabolism in the Probiotic Bacterium Lactiplantibacillus plantarum WCFS1.

Intestinal lactic acid bacteria can help alleviate lactose maldigestion by promoting lactose hydrolysis in the small intestine. This study shows that protein extracts from probiotic bacterium Lactiplantibacillus plantarum WCFS1 possess two metabolic pathways for lactose metabolism, involving ß-galactosidase (ß-gal) and 6Pß-galactosidase (6Pß-gal) activities. As L. plantarum WCFS1 genome lacks a putative 6Pß-gal gene, the 11 GH1 family proteins, in which their 6Pß-glucosidase (6Pß-glc) activity was experimentally demonstrated,, were assayed for 6Pß-gal activity. Among them, only Lp_3525 (Pbg9) also exhibited a high 6Pß-gal activity. The sequence comparison of this dual 6Pß-gal/6Pß-glc GH1 protein to previously described dual GH1 proteins revealed that L. plantarum WCFS1 Lp_3525 belonged to a new group of dual 6Pß-gal/6Pß-glc GH1 proteins, as it possessed conserved residues and structural motifs mainly present in 6Pß-glc GH1 proteins. Finally, Lp_3525 exhibited, under intestinal conditions, an adequate 6Pß-gal activity with possible relevance for lactose maldigestion management.

Non-redundant functionality of Lactiplantibacillus plantarum phospho-ß-glucosidases revealed by carbohydrate utilization signatures associated to pbg2 and pbg4 gene mutants.

AIM: To increase our knowledge on the functionality of 6-phospho-ß-glucosidases linked to phosphoenolpyruvate-dependent phosphotransferase systems (PTS) that are encountered in high redundancy in the Lactiplantibacillus plantarum WCFS1 genome. METHODS AND RESULTS: Two L. plantarum WCFS1 gene mutants that lacked one of the 6-phospho-ß-glucosidases, ∆pbg2 (or ∆lp_0906) or ∆pbg4 (or ∆lp_2777) were constructed and the metabolic impact of these mutations assessed by high-throughput phenotyping (Omnilog). The ∆pbg2 mutant displayed a reduced metabolic performance, having lost the capacity to utilize 20 out of 57 carbon (C)-sources used by the wild-type strain. Conversely, the ∆pbg4 mutant conserved the capacity to metabolize most of the C-sources preferred by the wild type strain. This mutant utilized 56 C-sources albeit the range of substrates used and hence its metabolic profiling differed from that of the WCFS1 strain. The ∆pbg2 mutant notably reduced or abolished the capacity to metabolize substrates related to pentose and glucoronate interconversions and was unable to assimilate fatty acids or nucleosides as sole C-sources for growth. The ∆pbg4 mutant acquired the capacity to utilize efficiently glycogen, indicating an efficient supply of glucose from this source. CONCLUSION: Lactiplantibacillus plantarum gene mutants that lack individual 6-phospho-ß-glucosidases display very different carbohydrate utilization signatures showing that these enzymes can be crucial to determine the capacity of L. plantarum to consume different C-sources and hence for the nutrition and physiology of this microorganism.

The salicylate 1,2-dioxygenase from Pseudaminobacter salicylatoxidans DSM 6986T is a bifunctional enzyme that inactivates the mycotoxin ochratoxin A by a novel amidohydrolase activity.

The salicylate 1,2-dioxygenase from the bacterium Pseudaminobacter salicylatoxidans DSM 6986T (PsSDO) is a versatile metalloenzyme that participates in the aerobic biodegradation of aromatic compounds, such as gentisates and salicylates. Surprisingly, and unrelated to this metabolic role, it has been reported that PsSDO may transform the mycotoxin ochratoxin A (OTA), a molecule that appears in numerous food products that results in serious biotechnological concern. In this work, we show that PsSDO, together with its dioxygenase activity, behaves as an amidohydrolase with a marked specificity for substrates containing a C-terminal phenylalanine residue, similar to OTA, although its presence is not an absolute requirement. This side chain would establish aromatic stacking interactions with the indole ring of Trp104. PsSDO hydrolysed the amide bond of OTA rendering the much less toxic ochratoxin α and L-ß-phenylalanine. The binding mode of OTA and of a diverse set of synthetic carboxypeptidase substrates these substrates have been characterized by molecular docking simulations, which has permitted us to propose a catalytic mechanism of hydrolysis by PsSDO that, similarly to metallocarboxypeptidases, assumes a water-induced pathway following a general acid/base mechanism in which the side chain of Glu82 would provide the solvent nucleophilicity required for the enzymatic reaction. Since the PsSDO chromosomal region, absent in other Pseudaminobacter strains, contained a set of genes present in conjugative plasmids, it could have been acquired by horizontal gene transfer, probably from a Celeribacter strain.

The burden of non-SARS-CoV2 viral lower respiratory tract infections in hospitalized children in Barcelona (Spain): A long-term, clinical, epidemiologic and economic study.

BACKGROUND: Viral lower respiratory tract infections (LRTI) are the leading cause of hospitalization in children. In Catalonia (Spain), information is scarce about the burden of viral LRTIs in paediatric hospitalizations. The aim of this study is to describe epidemiological, clinical, virological and economic features of paediatric hospitalizations due to viral LRTI. METHODS: From October 2012 to December 2020, children aged <16 years admitted to a tertiary paediatric hospital in Catalonia (Spain) with confirmed viral LRTI were included in the study. Virus seasonality, prevalence, age and sex distribution, clinical characteristics, hospital costs and bed occupancy rates were determined. RESULTS: A total of 3,325 children were included (57.17% male, 9.44% with comorbidities) accounting for 4056 hospitalizations (32.47% ≤ 12 months): 53.87% with wheezing/asthma, 37.85% with bronchiolitis and 8.28% with pneumonia. The most common virus was respiratory syncytial virus (RSV) (52.59%). Influenza A was associated with pneumonia (odds ratio [OR] 7.75) and caused longer hospitalizations (7 ± 31.58 days), while RSV was associated with bronchiolitis (OR 6.62) and was the most frequent reason for admission to the paediatric intensive care unit (PICU) (11.23%) and for respiratory support (78.76%). Male sex, age ≤12 months, chronic conditions and bronchiolitis significantly increased the odds of PICU admission. From October to May, viral LRTIs accounted for 12.36% of overall hospital bed days. The total hospitalization cost during the study period was €16,603,415. CONCLUSIONS: Viral LRTIs are an important cause of morbidity, hospitalization and PICU admission in children. The clinical burden is associated with significant bed occupancy and health-care costs, especially during seasonal periods.

Deciphering the Myrosinase-like Activity of Lactiplantibacillus plantarum WCFS1 among GH1 Family Glycoside Hydrolases.

The hydrolysis of plant glucosinolates by myrosinases (thioglucosidases) originates metabolites with chemopreventive properties. In this study, the ability to hydrolyze the glucosinolate sinigrin by cultures or protein extracts of Lactiplantibacillus plantarum WCFS1 was assayed. This strain possesses myrosinase-like activity as sinigrin was partly hydrolyzed by induced cultures but not by protein extracts. The 11 glycoside hydrolase GH1 family proteins, annotated as 6-phospho-ß-glucosidases, were the proteins most similar to plant myrosinases. The activity of these proteins was assayed against sinigrin and synthetic glucosides. As expected, none of the proteins assayed possessed myrosinase activity against sinigrin or the synthetic ß-thio-glucoside derivative or against the ß-glucoside. However, all 11 proteins were active on the phosphorylated-ß-glucoside derivative. Moreover, only eight of these proteins were active on phospho-ß-thioglucose. These results supported that, in L. plantarum WCFS1, glucosinolates may undergo previous phosphorylation, and GH1 proteins are the glycosidases involved in the hydrolysis of phosphorylated glucosinolates.

Incidence, Hospitalization, Mortality and Risk Factors of COVID-19 in Long-Term Care Residential Homes for Patients with Chronic Mental Illness.

Long-term care residential homes (LTCRH) for patients with chronic mental illness have suffered the enormous impact of COVID-19. This study aimed to estimate incidence, hospitalization, mortality, and risk factors of COVID-19 to prevent future epidemics. From March 2020 to January 2021 and before vaccination anti-SARS-CoV-2 begins, cumulate incidence rate (CIR), hospitalization rate (HR), mortality rate (MR), and risk factors of COVID-19 in the 11 LTCRH of two Health Departments of Castellon (Spain) were studied by epidemiological surveillance and an ecological design. Laboratory tests confirmed COVID-19 cases, and multilevel Poisson regression models were employed. All LTCRH participated and comprised 346 residents and 482 staff. Residents had a mean age of 47 years, 40% women, and suffered 75 cases of COVID-19 (CIR = 21.7%), five hospitalizations (HR = 1.4%), and two deaths (MR = 0.6%) with 2.5% fatality-case. Staff suffered 74 cases of the disease (CIR = 15.4%), one hospitalization (HR = 0.2%), and no deaths were reported. Risk factors associated with COVID-19 incidence in residents were private ownership, severe disability, residents be younger, CIR in municipalities where LTCRH was located, CIR in staff, and older age of the facilities. Conclusion: COVID-19 incidence could be prevented by improving infection control in residents and staff and modernizing facilities with increased public ownership.

Incidence, Mortality, and Risk Factors of COVID-19 in Nursing Homes.

During the period from March 2020 to January 2021, we performed an analysis of incidence, mortality, and risk factors of COVID-19 in nursing homes (NHs) in two health departments (HDs) of Castellon (Spain) 2021 through epidemiological surveillance and an ecological design. Laboratory-confirmed COVID-19 cases, cumulative incidence rate (CIR), and mortality rate (MR) of 27 NHs were collected. Information of residents, staff, and facilities was obtained by questionnaire. Multilevel Poisson regression models were applied. All NHs in the HDs participated with 2229 residents (median: 83 years old, 67.3% women) and 1666 staff. Among residents, 815 cases (CIR: 34.8 per 100) and 202 deaths (MR: 8.7 per 100, case fatality 21.0%) were reported and, among staff, 296 cases (CIR: 19.2 per 100) without deaths. Residents' CIR and MR increased with staff CIR, age of the building, residents/staff ratios, occupancy rate, and crowding index; CIR increased with private NH ownership, large NH size, large urban area, and the percentage of women residents; and MR was associated with residents' severe disabilities. In conclusion, several risk factors of COVID-19 incidence and mortality can be prevented by improving infection and quality controls, ameliorating residents/staff ratios, improving structural facilities, and increasing NH public ownership to avoid new outbreaks.

Population-based sequencing of Mycobacterium tuberculosis reveals how current population dynamics are shaped by past epidemics.

Transmission is a driver of tuberculosis (TB) epidemics in high-burden regions, with assumed negligible impact in low-burden areas. However, we still lack a full characterization of transmission dynamics in settings with similar and different burdens. Genomic epidemiology can greatly help to quantify transmission, but the lack of whole genome sequencing population-based studies has hampered its application. Here, we generate a population-based dataset from Valencia region and compare it with available datasets from different TB-burden settings to reveal transmission dynamics heterogeneity and its public health implications. We sequenced the whole genome of 785 Mycobacterium tuberculosis strains and linked genomes to patient epidemiological data. We use a pairwise distance clustering approach and phylodynamic methods to characterize transmission events over the last 150 years, in different TB-burden regions. Our results underscore significant differences in transmission between low-burden TB settings, i.e., clustering in Valencia region is higher (47.4%) than in Oxfordshire (27%), and similar to a high-burden area as Malawi (49.8%). By modeling times of the transmission links, we observed that settings with high transmission rate are associated with decades of uninterrupted transmission, irrespective of burden. Together, our results reveal that burden and transmission are not necessarily linked due to the role of past epidemics in the ongoing TB incidence, and highlight the need for in-depth characterization of transmission dynamics and specifically tailored TB control strategies.

Production and Digestibility Studies of ß-Galactosyl Xylitol Derivatives Using Heterogeneous Catalysts of LacA ß-Galactosidase from Lactobacillus Plantarum WCFS1.

The synthesis of ß-galactosyl xylitol derivatives using immobilized LacA ß-galactosidase from Lactobacillus plantarum WCFS1 is presented. These compounds have the potential to replace traditional sugars by their properties as sweetener and taking the advantages of a low digestibility. The enzyme was immobilized on different supports, obtaining immobilized preparations with different activity and stability. The immobilization on agarose-IDA-Zn-CHO in the presence of galactose allowed for the conserving of 78% of the offered activity. This preparation was 3.8 times more stable than soluble. Since the enzyme has polyhistidine tags, this support allowed the immobilization, purification and stabilization in one step. The immobilized preparation was used in synthesis obtaining two main products and a total of around 68 g/L of ß-galactosyl xylitol derivatives and improving the synthesis/hydrolysis ratio by around 30% compared to that of the soluble enzyme. The catalyst was recycled 10 times, preserving an activity higher than 50%. The in vitro intestinal digestibility of the main ß-galactosyl xylitol derivatives was lower than that of lactose, being around 6 and 15% for the galacto-xylitol derivatives compared to 55% of lactose after 120 min of digestion. The optimal amount immobilized constitutes a very useful tool to synthetize ß-galactosyl xylitol derivatives since it can be used as a catalyst with high yield and being recycled for at least 10 more cycles.

A structurally unique Fusobacterium nucleatum tannase provides detoxicant activity against gallotannins and pathogen resistance.

Colorectal cancer pathogenesis and progression is associated with the presence of Fusobacterium nucleatum and the reduction of acetylated derivatives of spermidine, as well as dietary components such as tannin-rich foods. We show that a new tannase orthologue of F. nucleatum (TanBFnn ) has significant structural differences with its Lactobacillus plantarum counterpart affecting the flap covering the active site and the accessibility of substrates. Crystallographic and molecular dynamics analysis revealed binding of polyamines to a small cavity that connects the active site with the bulk solvent which interact with catalytically indispensable residues. As a result, spermidine and its derivatives, particularly N8 -acetylated spermidine, inhibit the hydrolytic activity of TanBFnn and increase the toxicity of gallotannins to F. nucleatum. Our results support a model in which the balance between the detoxicant activity of TanBFnn and the presence of metabolic inhibitors can dictate either conducive or unfavourable conditions for the survival of F. nucleatum.

Establishment and Persistence of Glycopeptide-Resistant Enterococcus faecium ST80 and ST117 Clones Within a Health Care Facility Located in a Low-Prevalence Geographical Region.

Vancomycin-resistant Enterococcus faecium (VREfm) is one of the most important nosocomial pathogens with limited therapeutic alternatives. In this study, we followed the trends of VREfm and E. faecium causing bloodstream infections (BSIs) in a Spanish hospital, from 2011 to 2020. During this period, 832 E. faecium strains were isolated and 121 (14.5%) were vancomycin resistant. Nineteen of 101 BSIs (18.8%) caused by E. faecium were due to VREfm. The number of BSI-producing E. faecium isolates increased significantly over the past 5 years, with the percentage of invasive VREfm isolates being substantially higher than the average values in Europe and especially in Spain (<3%). VREfm isolates recovered in 2018 (28) and BSI-producing isolates from 2019 (3) and 2020 (2) were molecularly characterized. All were positive for vanA and belonged to sequence type (ST) 80 (28) or ST117 (5), within clonal complex 17. The isolates were only susceptible to linezolid, although most of them were also susceptible (dose dependent) to daptomycin. We report for the first time the establishment and persistence of the VREfm ST80 and ST117 clones in a Spanish hospital. The spread and establishment of hospital-adapted, multidrug-resistant VREfm clones in health care settings are cause for concern and may precede an increment in the BSIs caused by them.

[Comparison of postvaccinal antibodies to SARS-CoV-2 in asymptomatic cases, symptomatic cases, and non-cases in a cohort of general hospital workers]. / Comparación de anticuerpos postvacunales al SARS-CoV-2 en casos asintomáticos, casos sintomáticos, y no casos de una cohorte de trabajadores de un hospital general.

Estimada Sra. Directora: Agradecemos a los doctores Rujittika Mungmunpuntipantip y Viroj Wiwanitkit su interés por nuestra publicación(1) en la carta en la que se subrayan la importancia de los casos asintomáticos de COVID-19 en la evaluación de los efectos de la vacunación anti-SARS-CoV-2(2). Compartimos con ellos que las personas asintomáticas al COVID-19 pueden presentar unos niveles de anticuerpos anti-SARS-CoV-2 IgG-S mayores que las personas sin historia previa de COVID-19, y que pueden diferir en las reacciones a la vacunación, considerando que la prevalencia de COVID-19 asintomáticos se ha descrito como elevada(3).  En nuestra cohorte de trabajadores del Hospital General Universitario de Castellón, se detectaron 5 casos de COVID-19 asintomáticos (CA), incluyendo los dos seguimientos realizados(1,4), y 20 casos presentaron síntomas de COVID-19 (CS), con un total de 25 casos con confirmación por el laboratorio, 20 % tasa de asintomáticos (5/25). En la tabla 1 se recogen las características de los CA, CS, y de los participantes que no habían sufrido la enfermedad. Los CA eran más jóvenes que los otros 2 grupos, y la proporción de varones era significativamente mayor (p=0,027). En cuanto a los anticuerpos Anti-SARS-CoV-2 IgG-S al mes de la vacunación, los niveles de los CS fueron mayores que los de CA, y de los no casos, siendo estos últimos los que tuvieron significativamente menores niveles (p<0,001). Sin embargo, a los 8 meses de la vacunación la caída de IgG-S fue general, y los niveles de IgG-S eran mayores en los CA que en los CS y en los no casos (p<0,001). Los niveles de IgG-S considerados como protectores ? 4160 UA/ml, eran mayores en los CA y CS que el de los no casos (p=0,001). Si bien, el declive era similar en los tres grupos (p=0,084). Los síntomas y los efectos secundarios de las dos dosis de vacuna Pfizer-BioNTech no presentaron diferencias significativas entre los grupos. Estos resultados son coincidentes con númerosos estudios, en los que se constata que los casos de COVID-19 presentan niveles más elevados de IgG-S que las personas que no han sufrido la enfermedad(5), y se apreció que en valores medio no se alcanzaron los niveles de IgG-S protectores. De aquí la importancia de disponer de marcadores más efectivos de la situación de protección de la personas vacunadas tanto si han sufrido la enfermedad como sino. Además de los anticuerpos neutralizantes, la determinación de la inmunidad celular podría ser muy conveniente para conocer los niveles de protección.

[Vaccine response to SARS-CoV-2 in hospital workers]. / Respuesta vacunal frente a SARS-CoV-2 en trabajadores de un hospital.

INTRODUCTION: The aim of this study was to measure anti-SARS-CoV-2 immunity of hospital workers after a completed 2-dose Pfizer-BionTech vaccination, and to examine factors potentially associated with immunity status. Side effects of the vaccine were also studied. METHOD: This was a cross-sectional study of a representative sample of General University Hospital of Castellon workers, vaccinated with two doses in January and February 2021. We measured IgG antibodies against protein N (IgG-NP), IgM against protein S (IgM-S), and quantitative levles of IgG against protein S (IgG-Quant) one month after the last dose. We obtained information on demographic, risk factors, and vaccine side effects via a self-completed questionnaire. For the statistical analysis we used multiple regression models. RESULTS: Two hundred seventy-five workers participated (96.8%, 275/284). Positive IgG-Quant, IgM-S, and IgG-NP were 99.6%, 14.9% and 4.4%, respectively. Adjusted IgG-Quant levels increased significantly with obesity, nonsmoking status, positive IgM-S, and/or IgG-NP. The prevalence of IgM-S was higher in males, and associated with the same factors as those for IgG-Quant. Among those with a history of COVD-19 infection, 42.9% did not have IgG-NP. Overall 86.5% of participants had side effects, which were associated with positive IgG-NP, high IgG-Quant levels, younger age, and being female. CONCLUSIONS: All but one participant developed immunity. Those who had suffered from COVID-19 infection had higher antibody levels. A high proportion of participants had mild secondary effects, especially those with previous COVID-19 infection.

Introducción: Evaluar la inmunidad de los trabajadores de un hospital tras haber completado la vacunación Pfizer-BionTech, y su relación con factores individuales. También describir los efectos adversos de la vacuna. Método: Estudio transversal de una muestra de los trabajadores del Hospital General Universitario de Castellón vacunados con dos dosis en enero y febrero de 2021. Un mes después se detectaron: anticuerpos IgG frente a la proteína N (IgG-NP), de IgM frente a la proteína S (IgM-S) y detección cuantitativa de IgG frente a la proteína S (IgG-Quant). Se utilizó un cuestionario para recoger datos demográficos, factores de riesgo y efectos secundarios. En el análisis estadístico se utilizaron modelos de regresión múltiple. Resultados: La participación fue del 96,8% (275/284). Presentaron IgG-Quant el 99,6%, 14,9% IgM-S, y 4,4% IgG-NP. El nivel ajustado de IgG-Quant aumentó significativamente con la obesidad, en no fumadores y con positividad IgM-S y/o IgG-NP. La prevalencia de IgM-S era mayor en varones, y se asociaba con los mismos factores que la IgG-Quant. De los infectados por COVID-19, el 42,9% no presentaron IgG-NP. Un 86,5% sufrió algún efecto secundario que se asoció a tener IgG-NP, mayores niveles de IgG-Quant, y fue más frecuente en jóvenes y mujeres. Conclusiones: Todos los participantes desarrollaron inmunidad humoral excepto uno. Tuvieron mayores niveles de anticuerpos los que habían padecido la COVID-19. Un porcentaje alto desarrolló efectos secundarios leves, más frecuentes en los que habían padecido la enfermedad.

Production of α-rhamnosidases from Lactobacillus plantarum WCFS1 and their role in deglycosylation of dietary flavonoids naringin and rutin.

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable.

The first wave of the COVID-19 epidemic in Spain was associated with early introductions and fast spread of a dominating genetic variant.

The coronavirus disease 2019 (COVID-19) pandemic has affected the world radically since 2020. Spain was one of the European countries with the highest incidence during the first wave. As a part of a consortium to monitor and study the evolution of the epidemic, we sequenced 2,170 samples, diagnosed mostly before lockdown measures. Here, we identified at least 500 introductions from multiple international sources and documented the early rise of two dominant Spanish epidemic clades (SECs), probably amplified by superspreading events. Both SECs were related closely to the initial Asian variants of SARS-CoV-2 and spread widely across Spain. We inferred a substantial reduction in the effective reproductive number of both SECs due to public-health interventions (Re < 1), also reflected in the replacement of SECs by a new variant over the summer of 2020. In summary, we reveal a notable difference in the initial genetic makeup of SARS-CoV-2 in Spain compared with other European countries and show evidence to support the effectiveness of lockdown measures in controlling virus spread, even for the most successful genetic variants.

The commensal bacterium Lactiplantibacillus plantarum imprints innate memory-like responses in mononuclear phagocytes.

Gut microbiota is a constant source of antigens and stimuli to which the resident immune system has developed tolerance. However, the mechanisms by which mononuclear phagocytes, specifically monocytes/macrophages, cope with these usually pro-inflammatory signals are poorly understood. Here, we show that innate immune memory promotes anti-inflammatory homeostasis, using as model strains of the commensal bacterium Lactiplantibacillus plantarum. Priming of monocytes/macrophages with bacteria, especially in its live form, enhances bacterial intracellular survival and decreases the release of pro-inflammatory signals to the environment, with lower production of TNF and higher levels of IL-10. Analysis of the transcriptomic landscape of these cells shows downregulation of pathways associated with the production of reactive oxygen species (ROS) and the release of cytokines, chemokines and antimicrobial peptides. Indeed, the induction of ROS prevents memory-induced bacterial survival. In addition, there is a dysregulation in gene expression of several metabolic pathways leading to decreased glycolytic and respiratory rates in memory cells. These data support commensal microbe-specific metabolic changes in innate immune memory cells that might contribute to homeostasis in the gut.