Subinhibitory Concentrations of Clinically-Relevant Antimicrobials Affect Resistance-Nodulation-Division Family Promoter Activity in Acinetobacter baumannii

Efflux pumps contribute to multidrug resistance in Acinetobacter baumannii due to their ability to expel a wide variety of structurally unrelated compounds. This study aimed to characterize the effect of subinhibitory concentrations of clinically-relevant antibiotics and disinfectants on the promoter activity of members of the Resistance-Nodulation-Division (RND) family in A. baumannii. The promoter regions from three RND efflux pumps (AdeABC, AdeFGH and AdeIJK) and the AdeRS regulatory system from three different A. baumannii strains (ATCC 17961, ATCC 17978, and ATCC 19606) were cloned into a luciferase reporter system (pLPV1Z). Promoter activity was quantitatively assessed in both exponential and stationary phase cultures after exposure to subinhibitory concentrations of four antibiotics from different classes (rifampicin, meropenem, tigecycline and colistin) and two disinfectants (ethanol and chlorhexidine). Subinhibitory concentrations of the compounds tested had variable effects on promoter activity that were highly dependent on the A. baumannii strain, the compound tested and the growth phase. Fold changes in AdeABC promoter activity ranged from 1.97 to 113.7, in AdeFGH from -5.6 to 1.13, in AdeIJK from -2.5 to 2, and in AdeRS from -36.2 to -1.32. Taken together, these results indicate that subinhibitory concentrations of clinically-relevant antibiotics and disinfectants affect the promoter activity of RND family members in A. baumannii in a strain and growth phase dependent manner. These results may have important implications for the treatment of infections caused by A. baumannii.AT is supported by the Garantía Juvenil Program of the Comunidad Autonoma de Madrid and ML-S is supported by the Sara Borrell Program of the Instituto de Salud Carlos III. MJM is supported by grants from the Instituto de Salud Carlos III (MP 516/19 and MPY 380/18).S

Cross-Recognition of SARS-CoV-2 B-Cell Epitopes with Other Betacoronavirus Nucleoproteins

The B and T lymphocytes of the adaptive immune system are important for the control of most viral infections, including COVID-19. Identification of epitopes recognized by these cells is fundamental for understanding how the immune system detects and removes pathogens, and for antiviral vaccine design. Intriguingly, several cross-reactive T lymphocyte epitopes from SARS-CoV-2 with other betacoronaviruses responsible for the common cold have been identified. In addition, antibodies that cross-recognize the spike protein, but not the nucleoprotein (N protein), from different betacoronavirus have also been reported. Using a consensus of eight bioinformatic methods for predicting B-cell epitopes and the collection of experimentally detected epitopes for SARS-CoV and SARS-CoV-2, we identified four surface-exposed, conserved, and hypothetical antigenic regions that are exclusive of the N protein. These regions were analyzed using ELISA assays with two cohorts: SARS-CoV-2 infected patients and pre-COVID-19 samples. Here we describe four epitopes from SARS-CoV-2 N protein that are recognized by the humoral response from multiple individuals infected with COVID-19, and are conserved in other human coronaviruses. Three of these linear surface-exposed sequences and their peptide homologs in SARS-CoV-2 and HCoV-OC43 were also recognized by antibodies from pre-COVID-19 serum samples, indicating cross-reactivity of antibodies against coronavirus N proteins. Different conserved human coronaviruses (HCoVs) cross-reactive B epitopes against SARS-CoV-2 N protein are detected in a significant fraction of individuals not exposed to this pandemic virus. These results have potential clinical implications.This research was supported by grants from COV20_00679 (MPY 222-20), to M.J.M., MPY 509/19 to A.J.M.-G. and MPY 388/18 to D.L. of “Acción Estratégica en Salud” from the ISCIII.S

Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients

Erratum for Brief Research Report: Virus-Specific Humoral Immunity at Admission Predicts the Development of Respiratory Failure in Unvaccinated SARS-CoV-2 Patients. Tajuelo A, Carretero O, García-Ríos E, López-Siles M, Cano O, Vázquez M, Más V, Rodríguez-Goncer I, Lalueza A, López-Medrano F, Juan RS, Fernández-Ruiz M, Aguado JM, McConnell MJ, Pérez-Romero P. Front Immunol. 2022 Apr 25;13:878812. doi: 10.3389/fimmu.2022.878812. eCollection 2022. PMID: 35547738 Free PMC article.Introduction: There is robust evidence indicating that the SARS-CoV-2-specific humoral response is associated with protection against severe disease. However, relatively little data exist regarding how the humoral immune response at the time of hospital admission correlates with disease severity in unimmunized patients. Our goal was toidentify variables of the humoral response that could potentially serve as prognostic markers for COVID-19 progressionin unvaccinated SARS-CoV-2 patients. Methods: A prospective cross-sectional study was carried out in a cohort of 160 unimmunized, adult COVID-19 patients from the Hospital Universitario 12Octubre. Participants were classified into four clinical groups based on disease severity: non-survivors with respiratory failure (RF), RF survivors, patients requiring oxygen therapy and those not receiving oxygen therapy. Serum samples were taken on admission and IgM, IgG, IgG subclass antibody titers were determined by ELISA, and neutralizing antibody titersusing a surrogate neutralization assay. The differences in the antibody titers between groups and the association between the clinical and analytical characteristics of the patients and the antibody titers were analyzed. Results: Patients that developed RF and survived had IgM titers that were 2-fold higher than non-survivors (p = 0.001), higher levels of total IgG than those who developed RF and succumbed to infection (p< 0.001), and than patients who required oxygen therapy (p< 0.05), and had 5-fold higher IgG1 titers than RF non-survivors (p< 0.001) and those who needed oxygen therapy (p< 0.001), and 2-fold higher than patients that did not require oxygen therapy during admission (p< 0.05). In contrast, RF non-survivorshad the lowest neutralizing antibodylevels, which were significantly lower compared those with RF that survived (p = 0.03). A positive correlation was found between IgM, total IgG, IgG1 and IgG3 titers and neutralizing antibody titers in the total cohort (p ≤ 0.0036). Conclusions: We demonstrate that patients with RF that survived infection had significantly higher IgM, IgG, IgG1 and neutralizing titers compared to patients with RF that succumb to infection, suggesting that using humoral response variables could be used as a prognostic marker for guiding the clinical management of unimmunized patients admitted to the hospital for SARS-CoV-2 infection.This work was supported by Mutua Madrileña Foundation (2020/0056) “Plan Nacional de I+D+I” and Instituto de Salud Carlos III (COVID-19 Research Call COV20/00181 and COV20_00679), Subdirección General de Redes y Centros de Investigación Cooperativa, Spanish Ministry of Science and Innovation, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016) - co-financed by the European Development Regional Fund (EDRF) and the European Social Fund (ESF) "A way to achieve Europe- The ESF invests in your future". Red de Enfermedades Infecciosas (CIBERINFEC), CB21/13/00079. EG-R is supported by the Sara Borrell Program (CD18CIII/00007), MLS is supported by the Sara Borrell Program (CD17CIIII/00017), Instituto de Salud Carlos III, Ministerio de Ciencia, Innovación y Universidades., and AT is supported by the Garantía Juvenil Program of the Comunidad Autonoma de Madrid. IRG holds a research training contract “Río Hortega” (CM19/00163) and MFR a research contract “Miguel Servet” (CP18/00073), both from the Instituto de Salud Carlos III, Spanish Ministry of Science and Innovation.S

Ecophysiology and philogeny of Faecalibacterium prausnitzii in healthy and diseased gut. Application in Inflamatory Bowel Disease diagnostics

In this PhD thesis Faecalibacterium prausnitzii populations of patients with gut disease and healthy individuals have been characterized. First, isolates from healthy volunteers have been phenotypically characterised, which has allowed to gain insight into the physiology of this species. A possible link between F. prausnitzii sensitivity to changes in gut physicochemical conditions and its disappearance in a diseased gut has been revealed. Second, molecular studies on F. prausnitzii populations have allowed to define two phylogroups within this species, and to describe the diversity of phylotypes in healthy individuals and in patients with intestinal disease. The phylotypes specifically compromised in patients suffering some gut disorders have been identified. Finally, new molecular tools for the detection and quantification of this species and its phylogroups have been designed. Their usefulness to be implemented as complementary molecular tools for the diagnosis and prognosis of intestinal diseases has been determined.En aquesta tesi doctoral s'ha estudiat la població de Faecalibacterium prausnitzii de pacients amb malalties intestinals i individus sans. En primer lloc, es va realitzar una caracterització fenotípica d'aíllats d'aqueta espècie obtinguts d'individus sans, el que ha permès adquirir coneixement sobre la fisiologia d'aquesta espècie. S'ha evidenciat una possible relació entre la sensibilitat de F.prausnitzii a canvis en les condicions fisicoquímiques de l'intestí i la seva desaparició en un intestí malalt. En segon lloc, s'han realitzat estudis moleculars de les poblacions de F. prausnitzii. Això ha permès definir dos filogrups dins d'aquesta espècie, i descriure la diversitat de filotips en individus sans i pacients amb malalties intestinals.Per primera vegada, s'han identificat els filotips especificament compromesos en pacients que pateixen determinades malalties intestinals. Per últim, s’han dissenyat eines moleculars per a la detecció i quantificació d'aquesta espècie i els seus filogrups. S’ha determinat la utilitat d’aquestes eines moleculars per al suport al diagnòstic o prognòstic de malalties intestinals

Abundància i persistència de Faecalibacteriun prausnitzii en la Malaltia de Crohn

Faecalibacterium prausnitzii és un del bacteris anaeròbis més abundants entre les espècies comensals del tracte intestinal humà sa. Aquesta espècie és una de les principals productores de butirat a l'intestí (que és la principal font d’energia per als colonòcits), però també s'ha suggerit que pot produir compostos antiinflamatoris i intervenir en la regulació de vàries rutes metabòliques de l’hoste. F. prausnitzii és un bacteri difícil de cultivar, ja que presenta una elevada sensibilitat a l'oxigen i presenta uns requeriments nutricionals molt exigents, el que ha compromès considerablement el nombre d’estudis basats en aïllats d’aquesta espècie. No obstant això, en els darrers anys l’interès en aquest bacteri està creixent ja que s’ha evidenciat que les poblacions de F. prausnitzii són variables en diferents grups d'edat i que es veuen reduïdes en certs trastorns intestinals com ara la malaltia inflamatòria intestinal i el càncer colorectal. L’objectiu d'aquest treball ha estat aprofundir en el rol que desenvolupa F. prausnitzii com un dels principals bacteris comensals del tracte intestinal humà. En primer lloc, s’ha dissenyat, optimitzat i validat un nou mètode molecular per determinar l’abundància d’aquesta espècie en mostres del tracte gastrointesinal, i s’ha demostrat la seva possible aplicació per ajudar al diagnòstic de la malaltia de Crohn. En segon lloc, s’ha dut a terme un estudi de les característiques filogenètiques i fenotípiques dels aïllats de F. Prausnitzii disponibles en l'actualitat a fi de coneixre’n millor la diversitat genètica i fenotípica i dilucidar quins factors són crucials en comprometre la població d’aquest bacteri en un intestí malalt. L’anàlisi de les soques ha revelat que F. prausnitzii inclou Principalment dos filogrups, nutricionalment versàtils i molt sensibles a canvis en les condicions ecològiques que pot patir l’intestí de l’hoste sota certes malalties intestinals. En conclusió, els resultat obtinguts en aquest estudi mostren que F. prausnitzii és una espècie ben establerta al còlon sa, amb una elvada versatilitat metabòlica ja que és capaç d’ interactuar amb carbohidrats de diferent estructura i complexitat. S’ha corroborat que aquest microorganisme seria un bon indicador de salut intestinal ja que la seva abundància es veu significativament reduida en pacients amb malaltia de Crohn. Aquests resultats concorden amb els obtinguts per proves fisiològiques que mostren una elevada sensibilitat de l’espècie a determinades condicions relacionades amb malalties intestinals. Estudis futurs s’orientaran a comprendre millor quins factros derrivats de la interacció amb l’hoste també determinen la persistència d’aquesta espècie en un intestí sa o malalt.Faecalibacterium prausnitzii is one of the most abundant anaerobic commensal bacteria in the healthy human large intestine. F. prausnitzii is one of the major butyrate producers in the gut, but it has also been suggested that it can produce other anti-inflammatory compounds and regulate many host metabolic pathways. F. prausnitzii has been shown to be a fastidious bacterium given its high oxygen sensitivity and nutritional requirements, so little attention has been paid to this species since its firstly isolated. However, there is an increasing interest concerning this species, since it has been recently reported that F. prausnitzii populations are variable in different age groups and also could be reduced in certain intestinal disorders such as Inflammatory Bowel Diseases and colorectal cancer. Therefore, the main aim of this research project is to deepen in this bacterium as one of the main gut human commensal bacterium. Firstly, we developed a new molecular method to monitor this species abundance in Inflammatory Bowel Diseases, further elucidating its applicability to assist the diagnostic of Crohn’s disease. Secondly, an study of the currently available isolates phylogenetical and phenotypical characteristics has been performed in order to better know F. prausnitzii genetic and phenotypic diversity and elucidate which factors are crucially compromising this commensal bacterium population in a diseased gut. Our data demonstrates that F. prausnitzii could mainly be divided in two phylogroups, being both nutritionally versatile and highly sensitive to changes in gut ecological conditions that may occur under certain intestinal disorders. In conclusion, the results derived from this work suggest that F. prausnitzii is a well established species in the colon, highly versatile due to its ability to grow on carbohydrates of different origin and structure. It has been corroborated that this species can be a good indicator of healthy intestinal microbial community given its significantly lower abundance in Crohn’s disease patients. These results are in agreement with the physiological data that revealed little tolerance by this bacterium to changes in the gut environmental conditions that frequently happen under intestinal diseases. Future studies will be addressed to explore which host derived factors may be also compromising F. prausnitzii persistence in a healthy or diseased intestine

Special Issue: Vaccines against Antibiotic-Resistant Bacteria: From Bench to Bedside

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Identification and Analysis of Unstructured, Linear B-Cell Epitopes in SARS-CoV-2 Virion Proteins for Vaccine Development.

The efficacy of SARS-CoV-2 nucleic acid-based vaccines may be limited by proteolysis of the translated product due to anomalous protein folding. This may be the case for vaccines employing linear SARS-CoV-2 B-cell epitopes identified in previous studies since most of them participate in secondary structure formation. In contrast, we have employed a consensus of predictors for epitopic zones plus a structural filter for identifying 20 unstructured B-cell epitope-containing loops (uBCELs) in S, M, and N proteins. Phylogenetic comparison suggests epitope switching with respect to SARS-CoV in some of the identified uBCELs. Such events may be associated with the reported lack of serum cross-protection between the 2003 and 2019 pandemic strains. Incipient variability within a sample of 1639 SARS-CoV-2 isolates was also detected for 10 uBCELs which could cause vaccine failure. Intermediate stages of the putative epitope switch events were observed in bat coronaviruses in which additive mutational processes possibly facilitating evasion of the bat immune system appear to have taken place prior to transfer to humans. While there was some overlap between uBCELs and previously validated SARS-CoV B-cell epitopes, multiple uBCELs had not been identified in prior studies. Overall, these uBCELs may facilitate the development of biomedical products for SARS-CoV-2.This research was supported by Acción Estratégica en Salud from the ISCIII, grants MPY 380/18, MPY388/18, and MPY 509/19. A.C.-L. is the recipient of a Comunidad de Madrid contract by the ISCIII. M.L.-S. is therecipient of a Sara Borrell contract by the ISCIII. A.J.M.-G. is the recipient of a Miguel Servet contract by the ISCIIIS

Identification of differences in gene expression implicated in the Adherent-Invasive Escherichia coli phenotype during in vitro infection of intestinal epithelial cells

IntroductionAdherent-invasive Escherichia coli (AIEC) is strongly associated with the pathogenesis of Crohn’s disease (CD). However, no molecular markers currently exist for AIEC identification. This study aimed to identify differentially expressed genes (DEGs) between AIEC and non-AIEC strains that may contribute to AIEC pathogenicity and to evaluate their utility as molecular markers.MethodsComparative transcriptomics was performed on two closely related AIEC/non-AIEC strain pairs during Intestine-407 cell infection. DEGs were quantified by RT-qPCR in the same RNA extracts, as well as in 14 AIEC and 23 non-AIEC strains to validate the results across a diverse strain collection. Binary logistical regression was performed to identify DEGs whose quantification could be used as AIEC biomarkers.ResultsComparative transcriptomics revealed 67 differences in expression between the two phenotypes in the strain pairs, 50 of which (81.97%) were corroborated by RT-qPCR. When explored in the whole strain collection, 29 DEGs were differentially expressed between AIEC and non-AIEC phenotypes (p-value &lt; 0.042), and 42 genes between the supernatant fraction of infected cell cultures and the cellular fraction containing adhered and intracellular bacteria (p-value &lt; 0.049). Notably, six DEGs detected in the strain collection were implicated in arginine biosynthesis and five in colanic acid synthesis. Furthermore, two biomarkers based on wzb and cueR gene expression were proposed with an accuracy of ≥ 85% in our strain collection.DiscussionThis is the first transcriptomic study conducted using AIEC-infected cell cultures. We have identified several genes that may be involved in AIEC pathogenicity, two of which are putative biomarkers for identification

A debat : la lectura al segle XXI

La lectura al segle XXI és un tema que per als bibliotecaris inclou molts aspectes apassionants i difícils d'aglutinar en una hora i mitja. En aquesta ocasió, hem volgut parlar no només amb bibliotecaris, sinó també amb altres actors involucrats en aquest sector. El resultat ha estat un debat ric i divers, que ha abastat aspectes com el comportament del lector, l'accés a la informació, els canvis en la lectura, el llibre electrònic o el paper de les institucions i la societat.La lectura en el siglo XXI es un tema que para los bibliotecarios incluye muchos aspectos apasionantes y difíciles de aglutinar en una hora y media. En esta ocasión, hemos querido hablar no sólo con bibliotecarios, sino también con otros actores involucrados en este sector. El resultado ha sido un debate rico y diverso, que ha abarcado aspectos como el comportamiento del lector, el acceso a la información, los cambios en la lectura, el libro electrónico o el papel de las instituciones y la sociedad.Reading in the XXI century is an exciting and challenging subject for librarians. This time, we wanted to speak not only with librarians, but also with other stakeholders. The result has been a rich and diverse discussion, which covered aspects such as reader behavior, access to information, changes in reading, e-books or the role of institutions and society

Prevalence, Abundance, and Virulence of Adherent-Invasive Escherichia coli in Ulcerative Colitis, Colorectal Cancer, and Coeliac Disease

Adherent-invasive E. coli (AIEC) has largely been implicated in the pathogenesis of Crohn's disease (CD). E. coli strains with similar genetic backgrounds and virulence genes profiles have been associated with other intestinal disorders, such as ulcerative colitis (UC), colorectal cancer (CRC), and coeliac disease (CeD), but the role of AIEC in these diseases remains unexplored. We aimed to assess the distribution, abundance, and pathogenic features of AIEC in UC, CRC, and CeD.This work was funded by the Spanish Ministry of Education and Science through projects SAF2010-15896, SAF2013-43284-P, and SAF2107-82261-P (MINECO/AEI/FEDER/UE) and the grant AGL2017-88801-P from the Spanish Ministry of Science and Innovation (MICINN, Spain). MLS is a Serra Húnter Fellow.Peer reviewedSAF2010-15896, SAF2013-43284-P, and SAF2107-82261-P (MINECO/AEI/FEDER/UE) and the grant AGL2017-88801-