Emerging mutant populations of Listeria monocytogenes EGD-e under selective pressure of Thymbra capitata essential oil question its use in food preservation

Due to their excellent antimicrobial properties, essential oils (EO) have been proposed as potential preservatives for certain kinds of foods, such as dairy products. However, the occurrence of pathogenic populations that are resistant to EOs could pose a health risk. This report seeks to assess the emergence of resistant populations in Listeria monocytogenes EGD-e growth at 37 °C under selective pressure of Thymbra capitata EO (TCO), to characterise their resistance in laboratory media, and to identify their genotypic changes, as well as to evaluate the resistance in skimmed milk. TCO cyclic treatment allowed the isolation of two L. monocytogenes EGD-e resistant strains against the EO: LmSTCO by sublethal doses (75 µL/L TCO) and LmLTCO by lethal doses (300 µL/L TCO) after 20 and 30 cycles, respectively. Both strains displayed an increase of the minimum inhibitory and bactericidal concentration against TCO and a higher survival rate after lethal treatments than the wild-type strain (LmWT). Growth kinetics revealed a better adaptation of LmSTCO in presence of TCO, while LmLTCO grew more slowly compared to LmWT, even in the absence of the antimicrobial. Moreover, a slight increase in cross-resistance to antibiotics was observed: LmSTCO to β-lactams and LmLTCO to a series of broad-spectrum antibiotics. The genomic study revealed one sole nucleotide change in LmSTCO located in plsC gene codifying an enzyme involved in the production of phosphatidic acid, a precursor in cell membrane synthesis. Five genetic variations were found in LmLTCO: among them, the deletion of an ATP-synthesis system involved in slowing bacterial growth. Inhibition and inactivation assays in skimmed milk confirmed the increased resistance of both strains, thereby indicating a safety risk in case these strains emerge in the food chain. These results strongly suggest that the occurrence of such resistances should be taken into account in order to ensure the efficacy of natural antimicrobials in the design of food preservation strategies

Salmonella enterica serovar Typhimurium genetic variants isolated after lethal treatment with Thymbra capitata essential oil (TCO) showed increased resistance to TCO in milk

The high prevalence of Salmonella enterica in milk poses a risk of considerable concern in the preservation of certain dairy products, mainly those elaborated from raw milk. Essential oils (EOs) have been proposed as a promising food preservative for such products due to their strong antimicrobial properties. Additionally, these natural antimicrobials have been shown to be effective against multi-drug resistant strains. They can thus also be utilized to prevent the dissemination of antimicrobial resistances (AMR). However, recent evidence of the development of bacterial resistance under EO treatments may call their use into question. This study sought to assess the emergence of antimicrobial resistant genetic variants of S. enterica serovar Typhimurium from survivors after cyclic exposure to lethal doses (>5 log10 cycles of inactivation) of Thymbra capitata EO (TCO), in order to evaluate the impact that it could have on milk preservation, to ascertain whether cross-resistance to antibiotics occurs, and to identify the genomic changes responsible for their phenotype. Isolated strains by TCO (SeTCO) showed a two-fold increase in minimum inhibitory and bactericide concentrations (MIC and MBC) of TCO compared to Salmonella enterica serovar Typhimurium wild-type strain (SeWT) in laboratory growth medium, as well as a greater adaptation and growth rate in the presence of the EOs and a higher survival to TCO treatments in buffers of pH 4.0 and 7.0. The increased resistance of SeTCO was confirmed in skimmed milk: 300 μL/L TCO reduced only 1 log10 cycle of SeTCO population, whereas it inactivated more than 5 log10 cycles in SeWT. Moreover, SeTCO showed an increased cross-resistance against aminoglycosides, quinolones and tetracyclines. Whole genome sequencing revealed 5 mutations in SeTCO: 2 in genes involved in O-antigens synthesis (rfbV and rfbX), 2 in genes related to adaptation to the growing medium (trkA and glpK), and 1 in a redox-sensitive transcriptional regulator (soxR). The phenotypic characterization of a constructed SeWT strain with mutant soxRSeTCO demonstrated that the mutation of soxR was the main cause of the increased resistance and tolerance observed in SeTCO against TCO and antibiotics. The emergence of resistant strains against EOs might jeopardize their use as food preservatives. Further studies will thus be required to determine under which conditions such resistant strains might occur, and to assess the food risk they may pose, as well as to ascertain their impact on the spread of AMR

Carvacrol selective pressure allows the occurrence of genetic resistant variants of Listeria monocytogenes EGD-e

Essential oils and their constituents, such as carvacrol, are potential food preservatives because of their great antimicrobial properties. However, the long-term effects of these compounds are unknown and raise the question of whether resistance to these antimicrobials could emerge. This work aims to evaluate the occurrence of genetic resistant variants (RVs) in Listeria monocytogenes EGD-e by exposure to carvacrol. Two protocols were performed for the RVs selection: (a) by continuous exposure to sublethal doses, where LmSCar was isolated, and (b) by reiterative exposure to short lethal treatments of carvacrol, where LmLCar was isolated. Both RVs showed an increase in carvacrol resistance. Moreover, LmLCar revealed an increased cross-resistance to heat treatments at acid conditions and to ampicillin. Whole-genome sequencing identified two single nucleotide variations in LmSCar and three non-silent mutations in LmLCar. Among them, those located in the genes encoding the transcriptional regulators RsbT (in LmSCar) and ManR (in LmLCar) could contribute to their increased carvacrol resistance. These results provide information regarding the mode of action of this antimicrobial and support the importance of knowing how RVs appear. Further studies are required to determine the emergence of RVs in food matrices and their impact on food safety

Phenotypic and genotypic comparison of antimicrobial-resistant variants of Escherichia coli and Salmonella Typhimurium isolated from evolution assays with antibiotics or commercial products based on essential oils

On account of the widespread development and propagation of antimicrobial-resistant (AMR) bacteria, essential oils (EOs) have emerged as potential alternatives to antibiotics. However, as already observed for antibiotics, recent studies have raised concerns regarding the potential emergence of resistant variants (RVs) to EOs. In this study, we assessed the emergence of RVs in Escherichia coli and Salmonella enterica Typhimurium after evolution assays under extended exposure to subinhibitory doses of two commercial EOs (AEN and COLIFIT) as well as to two antibiotics (amoxicillin and colistin). Phenotypic characterization of RVs from evolution assays with commercial EOs yielded no relevant increases in the minimum inhibitory concentration (MIC) of E. coli and did not even modify MIC values in S. Typhimurium. Conversely, RVs of E. coli and S. Typhimurium isolated from evolution assays with antibiotics showed increased resistance. Genotypic analysis demonstrated that resistance to commercial EOs was associated with enhanced protection against oxidative stress and redirection of cell energy toward efflux activity, while resistance to antibiotics was primarily linked to modifications in the cell binding sites of antibiotics. These findings suggest that AEN and COLIFIT could serve as safe alternatives to antibiotics in combating the emergence and dissemination of antimicrobial resistance within the agrifood system

Circulating immune biomarkers in peripheral blood correlate with clinical outcomes in advanced breast cancer

Identification of the different elements intervening at the tumor microenvironment seems key to explain clinical evolution in several tumor types. In this study, a set of immune biomarkers (myeloid derived suppressor cells, regulatory T cells, and OX40 + and PD-1 + T lymphocytes counts) in peripheral blood of patients diagnosed with advanced breast cancer were analyzed along of first line antineoplastic therapy. Subsequently, a comparison between groups with clinical benefit versus progression of disease and with a healthy women cohort was executed. Results reflected that patients showed higher basal levels of myeloid derived suppressor cells (35.43, IR = 180.73 vs 17.53, IR = 16.96 cells/μl; p = 0.001) and regulatory T cells (32.05, IR = 29.84 vs 22.61, IR = 13.57 cells/μl; p = 0.001) in comparison with healthy women. Furthermore, an increase in the number of activated T lymphocytes (expressing OX40), a decrease of immune inhibitory cells (MDSCs and Tregs) and inhibited T lymphocytes (expressing PD-1) were observed along the treatment in patients with clinical benefit (p ≤ 0.001). The opposite trend was observed in the case of disease progression. These findings suggest that some critical immune elements can be easily detected and measured in peripheral blood, which open a new opportunity for translational research, as they seem to be correlated with clinical evolution, at least in ABC.Fundación Progreso y Salud. Junta de Andalucía (PI-0502-2014 FPS-2014

EL AVANCE DE LA INVESTIGACIÓN EN ESTRATEGIA EMPRESARIAL. UN ANÁLISIS COMPARADO ESPAÑA – CASTILLA Y LEÓN

El objetivo del trabajo es considerar el desarrollo alcanzado por el conocimiento en estrategia empresarial en Castilla y León y su posición en el conjunto nacional, ofreciendo una visión complementaria a la de anteriores estudios. Para dicho análisis se han considerado, durante la década de los 90, congresos y revistas de alcance regional y nacional. Los resultados obtenidos nos muestran la necesidad de contar con más foros de encuentro en Castilla y León y con medios especializados en el campo de la estrategia. Además, los investigadores castellano-leoneses parecen mostrar un interés poco frecuente por la ejecución estratégica y por los aspectos de delimitación teórica, un tanto marginados en el conjunto nacional. El objetivo del trabajo es considerar el desarrollo alcanzado por el conocimiento en estrategia empresarial en Castilla y León y su posición en el conjunto nacional, ofreciendo una visión complementaria a la de anteriores estudios. Para dicho análisis se han considerado, durante la década de los 90, congresos y revistas de alcance regional y nacional. Los resultados obtenidos nos muestran la necesidad de contar con más foros de encuentro en Castilla y León y con medios especializados en el campo de la estrategia. Además, los investigadores castellano-leoneses parecen mostrar un interés poco frecuente por la ejecución estratégica y por los aspectos de delimitación teórica, un tanto marginados en el conjunto nacional.

Clinical implications of changing thyroglobulin and antithyroglobulin antibodies analytical methods in the follow-up of patients with differentiated thyroid carcinoma

Background and aims: Patients’ response to treatment in differentiated thyroid cancer (DTC) is classified according to serum thyroglobulin concentrations (Tg), usually using the American Thyroid Association guidelines and considering potential interfering anti-thyroglobulin antibodies (Ab-Tg). We aim to evaluate the clinical implications of changing Tg and Ab-Tg quantification method. Material and methods: Tg and Ab-Tg were quantified in 82 serum samples (60 from DTC patients) by Elecsys and Access immunoassays. Results: Elecsys immunoassay rendered higher values of Tg than Access: mean bias 5.03 ng/mL (95%CI:- 14.14–24.21). In DTC patients, there was an almost perfect agreement for response classification (kappa index = 0.833). Discrepancies appeared in patients with undetermined response, with a more tendency to subclassification with Access. Ab-Tg showed a poor correlation (r = 0.5394). When Elecsys cut-off was reduced to 43 IU/ mL, agreement for positive/negative classification improved from a kappa index of 0.607 to 0.650. Prospective study with personalized follow-up showed that only 6.3% of Tg results required an analytical confirmation, being confirmed 93% of them. Conclusions: Despite the biases observed, clinical impact of an analytical change is minimal in patients’ management. However, cautious and personalized follow-up period after the change is still mandatory, especially in patients with Tg levels between 0.2 and 1 ng/mL

Circulating myeloid-derived suppressor cells and regulatory T cells as immunological biomarkers in refractory/relapsed diffuse large B-cell lymphoma: translational results from the R2-GDP-GOTEL trial

Background The search for immunological markers with ability of predicting clinical outcome is a priority in lymphomas, and in cancer in general. It is well known that some immunomodulatory cells, such as myeloid derived suppressor cells (MDSCs) or regulatory T cells (Tregs), are recruited by tumors, jeopardizing antitumor immunosurveillance. In this work, we have studied blood levels of these immunosuppressive cells in patients with relapsed/refractory diffuse large B-cell lymphoma (R/R DLBCL), prior to and along the course of the experimental rituximab, gemcitabine, dexamethasone, and cisplatin (R2- GDP) schedule, as a translational substudy of the R2-GDP GOTEL trial (EudraCT Number: 2014-001620-29), which included lenalidomide as an immunomodulator. Methods Blood samples were taken before treatment, at cycle 3 and end of induction. Samples were analyzed by flow cytometry. Non-parametric tests were used. Mann-Whitney U test was used to compare basal cells distributions, and Wilcoxon test was considered to compare cells distribution at different times. Spearman test was performed to measure the degree of association between cell populations. Results In this study, MDSC and Treg circulating concentration was found increased in all patients compared with a healthy control group and decreased after treatment only in patients with longest overall survival (>24 months), reaching the levels of the healthy group. Likewise, the number of inhibited T lymphocytes expressing Programmed Death-1 (PD-1) were increased in peripheral blood from patients and decreased on the treatment, whereas activated T lymphocytes increased after therapy in those with better overall survival. Conclusions In conclusion, blood concentration of MDSCs and Treg cells may be good prognostic markers for overall survival after 2 years in R/R DLBCL. These results point to a possible role of these elements in the immunosuppression of these patients, as assessed by the circulating activated and inhibited T lymphocytes, and therefore, they may be considered as therapeutic targets in DLBCL

Highly accurate whole-genome imputation of SARS-CoV-2 from partial or low-quality sequences

[Background] The current SARS-CoV-2 pandemic has emphasized the utility of viral whole-genome sequencing in the surveillance and control of the pathogen. An unprecedented ongoing global initiative is producing hundreds of thousands of sequences worldwide. However, the complex circumstances in which viruses are sequenced, along with the demand of urgent results, causes a high rate of incomplete and, therefore, useless sequences. Viral sequences evolve in the context of a complex phylogeny and different positions along the genome are in linkage disequilibrium. Therefore, an imputation method would be able to predict missing positions from the available sequencing data.[Results] We have developed the impuSARS application, which takes advantage of the enormous number of SARS-CoV-2 genomes available, using a reference panel containing 239,301 sequences, to produce missing data imputation in viral genomes. ImpuSARS was tested in a wide range of conditions (continuous fragments, amplicons or sparse individual positions missing), showing great fidelity when reconstructing the original sequences, recovering the lineage with a 100% precision for almost all the lineages, even in very poorly covered genomes (<20%).[Conclusions] Imputation can improve the pace of SARS-CoV-2 sequencing production by recovering many incomplete or low-quality sequences that would be otherwise discarded. ImpuSARS can be incorporated in any primary data processing pipeline for SARS-CoV-2 whole-genome sequencing.This work is supported by grant PT17/0009/0006 from the Spanish Ministry of Economy and Competitiveness, COVID-0012–2020 from Consejería de Salud y Familias, Junta de Andalucía, and postdoctoral contract PAIDI2020- DOC_00350 for C.L., from Junta de Andalucía, co-funded by the European Social Fund (FSE) 2014–2020.Peer reviewe

Enterectomía yeyunal revestida con xenoinjerto de pericardio bovino en un modelo canino

The aim of this study was to evaluate the alteration of the cellular components of the peritoneal lavage and to characterize the cicatricial remodelling, at the macro and microscopic level, produced by the experimental use of bovine pericardium xenograft (XPB) not treated as a lining of the end to end-jejunal terminal anastomosis in a canine model. Nine canines between 4 and 6 years of age, 15-30 kg of weight, and both sexes were randomly distributed in three experimental groups (A, B and C for a second celiotomy at 5, 10 and 30 days post-surgery) of three animals each, to which an untreated XPB was placed as a lining of the jejunal enterectomy. The results of the analysis of the cellular components of the peritoneal fluid showed a significant increase in lymphocytes, macrophages and eosinophils 30 days after the surgery and to a lesser extent 10 and 5 days after the intervention. In the macroscopic pathology, none of the groups denoted the presence of leakage, stenosis, abscesses or signs of peritonitis, nor was visualization of contraction or displacement of the XPB, but adhesion of the omentum was evident. The histopathology revealed a reaction to a foreign body of minimal to moderate, but totally compatible with life and almost surpassed 30 days after the surgery. It is concluded that the technique of jejunal anastomosis in canines coated with untreated XPB is viable and effective because it does not generate severe cellular infiltration against possible harmful cellular antigens or calcification processes. In addition, it does not alter the functions of intestinal digestion, secretion and absorption.El estudio tuvo como objetivo evaluar la alteración de los componentes celulares del lavado peritoneal y caracterizar la remodelación cicatricial, a nivel macro y microscópico, producida por el uso experimental del xenoinjerto de pericardio bovino (XPB) no tratado como revestimiento de la anastomosis término-terminal yeyunal en un modelo canino. Se trabajó con nueve caninos entre 4 y 6 años, de 15 a 30 kg de peso, sin discriminar por sexo. Se distribuyeron de forma aleatoria en tres grupos experimentales (A, B y C para una segunda celiotomía a los 5, 10 y 30 días del posperatorio, respectivamente) de tres animales cada uno, a los que se les colocó un XPB no tratado como revestimiento de la enterectomía yeyunal. Los resultados del análisis de los componentes celulares del fluido peritoneal evidenciaron un incremento significativo en linfocitos, macrófagos y eosinófilos a los 30 días del posoperatorio y en menor medida a los 10 y 5 días de la intervención. En la patología macroscópica, ninguno de los grupos denotó presencia de fugas, estenosis o abscesos, o signos de peritonitis ni se visualizó contracción o desplazamiento del XPB, pero fue evidente la adhesión del epiplón. La histopatología reveló una reacción a cuerpo extraño de mínima a moderada, pero totalmente compatible con la vida y casi superada a los 30 días del posoperatorio. Se concluye que la técnica de anastomosis término-terminal yeyunal en caninos revestida con XPB no tratado es viable y efectiva porque no genera infiltración celular severa frente a posibles antígenos celulares nocivos, ni procesos de calcificación; además, no altera las funciones de digestión, secreción y absorción intestinal