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Background: Mycobacterium tuberculosis is a slow-growing bacterium, which could delay its diagnosis and, therefore, promote the spread of the disease. Whole-genome sequencing allows us to obtain the complete drug-resistance profile of the strain; however, bacterial cultivation of clinical samples, along with complex processing, is required. Methods: In this work, we explore AmpliSeq, an amplicon-based enrichment method for preparing libraries for targeted next-generation sequencing, to identify lineage and drug resistance directly from clinical samples. Results: In our study, 111 clinical samples were tested. The lineage was identified in 100% of the culture-derived samples (52/52), in 95% of the smear (BK)-positive clinical samples (38/40) and in 42.1% of the BK-negative clinical samples (8/19). The drug-resistance profile was accurately identified in all but 11 samples, in which some phenotypic and genotypic discrepancies were found. In this respect, our panels were not exact in the detection of streptomycin resistance for isolates derived from clinical samples, as an extremely high number of SNPs in the rrs and rrl genes were detected due to cross-contamination. Conclusion: This technique has demonstrated high sensitivity in obtaining the drug-resistance profile of the isolates, as even those samples with DNA concentrations below the detection limit of Qubit produced a result. AmpliSeq technology is cheaper than whole-genome sequencing, easy to perform by laboratory technicians and applicable to any microorganism using the Ion Torrent platform.

Objective Serratia marcescens is a Gram-negative bacterium that is found in hospital environments and commonly associated with outbreaks in neonatal units. One S. marcescens isolate was detected from a bloodstream culture from a neonate in our hospital that was followed by an outbreak. The aim of this study was to describe the molecular epidemiology of a S. marcescens outbreak in the neonatal unit. Methods In order to investigate the outbreak, weekly surveillance rectal swabs were submitted for culture from all patients admitted in this unit from August to September 2018. Environmental samples were obtained from potential sources in September 2018. Typing of isolates was performed by pulsed field gel electrophoresis (PFGE). In addition, we studied the in vitro activity of chlorhexidine against S. marcescens. Results During this period, 146 infants were hospitalised in our neonatal unit, of which 16 patients had a S. marcescens-positive sample. A total of 36 environmental surveillance samples were collected, and one sample from a stethoscope from an incubator of a colonized baby was positive for S. marcescens. All the 18 isolates, including the isolate from the stethoscope, belonged to a single PFGE cluster. We found that very low concentrations of chlorhexidine, even with application times close to 0 achieved significant reductions in the amount of S. marcescens. Conclusion A unique clone of S. marcescens caused this outbreak, including isolates from patients and from one stethoscope. The outbreak was controlled with the early implementation of specific control measures.

Natural Killer (NK) cells have shown great promise as a viable cell-based cancer immunotherapy. We have developed a protocol that produces expanded and highly activated NK (eNK) cells from healthy human donors that have been shown to be effective alone and in combination with monoclonal antibodies in a variety of hematological malignancies. The eNK cells showed a dramatic increase in cytolytic capacity after their expansion and activation using IL-2, IL-15, and 721.221 feeder cells. In order to elucidate the underlying mechanisms behind the changes, mRNAseq and miRNA arrays were performed on mRNA and miRNA samples, respectively, extracted from several NK expansions, taken at Day 0 (before activation) and Day 20 (after activation and expansion). Bioinformatic analysis was performed to identify differential gene expression changes and their downstream effects. Based on the genes with greatest differential expression, we quantified their corresponding proteins using flow cytometry, western blots, and ELISAs. Results reveal several discrete changes in the transcriptome that are reflected in changes in protein expression levels within the eNK cells. We conclude that the increased cytolytic capacity of eNK cells is due to the synergistic effect of variations in different genes responsible for target recognition, effector function, cell cycle progression and cell survival. These data can be used in the future to produce better eNK cells as treatment for hematological malignancies. Citation Format: Chantal Reina-Ortiz, Pilar Mozas Alonso, Alberto Cebollada, David Ovelleiro, Luis Alberto Anel Bernal. Transcriptomic changes experienced by activated and expanded NK cells for their use in the treatment of hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2755.

Background: Risk stratification of COVID-19 patients is fundamental to improving prognosis and selecting the right treatment. We hypothesized that a combination of lung ultrasound (LUZ-score), biomarkers (sST2), and clinical models (PANDEMYC score) could be useful to improve risk stratification. Methods: This was a prospective cohort study designed to analyze the prognostic value of lung ultrasound, sST2, and PANDEMYC score in COVID-19 patients. The primary endpoint was in-hospital death and/or admission to the intensive care unit. The total length of hospital stay, increase of oxygen flow, or escalated medical treatment during the first 72 h were secondary endpoints. Results: a total of 144 patients were included; the mean age was 57.5 ± 12.78 years. The median PANDEMYC score was 243 (52), the median LUZ-score was 21 (10), and the median sST2 was 53.1 ng/mL (30.9). Soluble ST2 showed the best predictive capacity for the primary endpoint (AUC = 0.764 (0.658–0.871); p = 0.001), towards the PANDEMYC score (AUC = 0.762 (0.655–0.870); p = 0.001) and LUZ-score (AUC = 0.749 (0.596–0.901); p = 0.002). Taken together, these three tools significantly improved the risk capacity (AUC = 0.840 (0.727–0.953); p = 0.001). Conclusions: The PANDEMYC score, lung ultrasound, and sST2 concentrations upon admission for COVID-19 are independent predictors of intra-hospital death and/or the need for admission to the ICU for mechanical ventilation. The combination of these predictive tools improves the predictive power compared to each one separately. The use of decision trees, based on multivariate models, could be useful in clinical practice. © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Lineage 4/X-family of Mycobacterium tuberculosis is not very notorious, except for the CDC1551 strain. One strain of this family, named Ara50, caused one of the largest tuberculosis outbreaks of the Aragon region, Spain, during the 1990s and remained until 2018. These X-strains are characterised by high transmissibility and by carrying a low copy number of IS6110 in their genomes. Epidemiological data of the 61 patients consisted of inmates, HIV seropositives, intravenous drug users and the homeless. The application of whole-genome sequencing (WGS) to 36 out of 61 isolates, selected by IS6110-RFLP, allowed to confirm 32 as recent transmissions. We found 10 SNPs in genes considered as virulence factors, five of them specific of this strain. WGS identified three sub-clusters (CLSs). The largest one, sub-CLS 1, included 10 cases. Seven of them shared a SNP in the mce3C gene, considered a virulence factor gene. Sub-CLS 2 involved familiar cases, and no link was known for sub-CLS 3. Finally, the strain showed efficacy in latency as a confirmed epidemiological link was established between two cases, with 6 years of distance in their diagnosis. This outbreak study combined epidemiological and molecular analyses in order to elucidate tuberculosis transmission.

International audience; AbstractField studies on Salmonella infection in suckling piglets are scarce due to the intrinsic difficulties of collecting proper samples (i.e. tonsils or mesenteric lymph nodes), and most of them rely on the analysis of rectal swabs that limit their accuracy. We used 495 slaughtered 4-weeks-old male piglets intended for human consumption from 5 Salmonella-seropositive breeding farms to collect gastrointestinal packages and perform a thorough detection of Salmonella on mesenteric lymph nodes and intestinal content. The overall prevalence of both infection and shedding was high (≈ 36%) indicating that piglets played an active role in Salmonella maintenance in the farms. Major serotypes found in piglets included 4,[5],12:i: (35.4%), Rissen (17.1%), Derby (10.9%) and Bovismorbificans (10.3%). In most of the infected animals (72.8%) the same serotype was found in mesenteric lymph nodes and feces. Significant higher ELISA OD% values were found in meat juice samples from non-infected piglets compared to infected ones (median OD% of 12.0 and 17.3, respectively; P = 0.002) suggesting some protective effect of sow’s colostrum. Salmonella was also isolated from feces from weaned sows contemporary of the slaughtered piglets, and 89% of the serotypes identified in sows were also detected in piglets. Pulsed field gel electrophoresis analyses showed that 75% of the piglet isolates that were compared to those of sows were related to them, suggesting the circulation of Salmonella strains between sows and piglets. It appears that improving piglet colostrum intake along with the reduction of the shedding in sows may favor the control of Salmonella infection in breeding farms.

This paper describes the application of whole-genome sequencing (WGS) to investigate an outbreak of Mycobacterium tuberculosis occurring in Aragon, Spain, where strains have been submitted to genotyping since 2004. The responsible outbreak strain appeared in our region first in 2014 and it spread to 14 patients in the following three years. WGS found low variability between the isolates with none of the SNPs differences detected more than once, all of which were attributed to a recent transmission. Although two ambiguous bases linked two cases with those who presented the SNP in the same position, the establishment of a definitive transmission route was not possible. The epidemiological data supported the existence of a super-spreader, probably responsible for the majority of the cases involved since there was a two-year delay in diagnoses among cases. This fact would also help explaining the low variability found. The index case was not identified, possibly because it was not diagnosed in Aragon. In addition WGS characterised the strain as a Linage 4.3.3/LAM family and corroborated the susceptibility to anti-tuberculosis drugs observed by the clinical laboratories. This work shows the need to have epidemiological data to support the genomic data in order to clarify the evolution of tuberculosis outbreaks.

The insertion Sequence IS6110, only present in the pathogens of the Mycobacterium tuberculosis Complex (MTBC), has been the gold-standard epidemiological marker for TB for more than 25 years, but biological implications of IS6110 transposition during MTBC adaptation to humans remain elusive. By studying 2,236 clinical isolates typed by IS6110-RFLP and covering the MTBC, we remarked a lineage-specific content of IS6110 being higher in modern globally distributed strains. Once observed the IS6110 distribution in the MTBC, we selected representative isolates and found a correlation between the normalized expression of IS6110 and its abundance in MTBC chromosomes. We also studied the molecular regulation of IS6110 transposition and we found a synergistic action of two post-transcriptional mechanisms: a -1 ribosomal frameshift and a RNA pseudoknot which interferes translation. The construction of a transcriptionally active transposase resulted in 20-fold increase of the transposition frequency. Finally, we examined transposition in M. bovis and M. tuberculosis during laboratory starvation and in a mouse infection model of TB. Our results shown a higher transposition in M. tuberculosis, that preferably happens during TB infection in mice and after one year of laboratory culture, suggesting that IS6110 transposition is dynamically adapted to the host and to adverse growth conditions., Author summary Since the pioneering discovery of transposition by Barbara McClintock in eukaryotes and later in prokaryotes by Robert W. Hedges and Alan E. Jacob, it has become clear the key role of mobile genetics elements in chromosome remodelling, microbial evolution and host adaptation. The insertion sequence IS6110 is widely recognized for its utility in TB diagnosis and epidemiology because it is only present in the M. tuberculosis Complex (MTBC) and its transposition provides an excellent chromosomal polymorphic variability allowing the study of recent TB transmission. This inherent feature of IS6110 leads us to hypothesize that IS6110 plays a crucial role during the TB infectious cycle. However, the biological significance of IS6110 has been hindered by its almost exclusive use as an epidemiological marker. Here, we study the regulatory mechanisms and the distribution of IS6110 in the different MTBC lineages. We discuss the potential biological implications of IS6110, that is much more than an excellent TB epidemiological tool. Since IS6110 could play an important role in the adaptation of MTBC to the host, this study opens new avenues to decipher the biological roles of IS6110 in TB pathogenesis.

Multidrug-resistant Mycobacterium tuberculosis strain diversity in Ibero-America was examined by comparing extant genotype collections in national or state tuberculosis networks. To this end, genotypes from over 1000 patients with multidrug-resistant tuberculosis diagnosed from 2004 through 2008 in Argentina, Brazil, Chile, Colombia, Venezuela and Spain were compared in a database constructed ad hoc. Most of the 116 clusters identified by IS 6110 restriction fragment length polymorphism were small and restricted to individual countries. The three largest clusters, of 116, 49 and 25 patients, were found in Argentina and corresponded to previously documented locally-epidemic strains. Only 13 small clusters involved more than one country, altogether accounting for 41 patients, of whom 13 were, in turn, immigrants from Latin American countries different from those participating in the study (Peru, Ecuador and Bolivia). Most of these international clusters belonged either to the emerging RD Rio LAM lineage or to the Haarlem family of M. tuberculosis and four were further split by country when analyzed with spoligotyping and rifampin resistance-conferring mutations, suggesting that they did not represent ongoing transnational transmission events. The Beijing genotype accounted for 1.3% and 10.2% of patients with multidrug-resistant tuberculosis in Latin America and Spain, respectively, including one international cluster of two cases. In brief, Euro-American genotypes were widely predominant among multidrug-resistant M. tuberculosis strains in Ibero-America, reflecting closely their predominance in the general M. tuberculosis population in the region, and no evidence was found of acknowledged outbreak strains trespassing country borders.

Background Molecular typing of Mycobacterium tuberculosis strains has become a valuable tool in the epidemiology of tuberculosis (TB) by allowing detection of outbreaks, tracking of epidemics, identification of genotypes and transmission events among patients who would have remained undetected by conventional contact investigation. This is the first genetic biodiversity study of M. tuberculosis in Venezuela. Thus, we investigated the genetic patterns of strains isolated in the first survey of anti-tuberculosis drug-resistance realised as part of the Global Project of Anti-tuberculosis Drug Resistance Surveillance (WHO/IUATLD). Results Clinical isolates (670/873) were genotyped by spoligotyping. The results were compared with the international spoligotyping database (SpolDB4). Multidrug resistant (MDR) strains (14/18) were also analysed by IS6110-RFLP assays, and resistance to isoniazid and rifampicin was characterised. Spoligotyping grouped 82% (548/670) of the strains into 59 clusters. Twenty new spoligotypes (SITs) specific to Venezuela were identified. Eight new inter-regional clusters were created. The Beijing genotype was not found. The genetic network shows that the Latin American and Mediterranean family constitutes the backbone of the genetic TB population-structure in Venezuela, responsible of >60% of total TB cases studied. MDR was 0.5% in never treated patients and 13.5% in previously treated patients. Mutations in rpoB gene and katG genes were detected in 64% and 43% of the MDR strains, respectively. Two clusters were found to be identical by the four different analysis methods, presumably representing cases of recent transmission of MDR tuberculosis. Conclusion This study gives a first overview of the M. tuberculosis strains circulating in Venezuela during the first survey of anti-tuberculosis drug-resistance. It may aid in the creation of a national database that will be a valuable support for further studies.

Ninety-six patients with bipolar disorder who attended a lithium clinic were reviewed in a retrospective study. Sleep disorders were studied in 85 depressive episodes. Eighty-one percent of the subjects presented with insomnia; the mixed type being the most frequent (49%) followed by early awakening (25%). The evolution of depression in the patients was compared according to the treatment received for insomnia: sedative antidepressants vs other anxiolytic or hypnotic drugs. Fifteen percent of patients shifted to mania, this group more frequently receiving sedative antidepressants (p < 0.05). Moreover, the patients who had received sedative antidepressants as therapy for insomnia (N = 61) showed a tendency to have a shorter asymptomatic interval before the following relapse (13 months vs 19 months; p = 0.06). In view of these results, we consider that the use of sedative antidepressants as a treatment for insomnia during depressive episodes in bipolar patients could be a factor contributing to worse prognoses; in these cases it appears that the use of other hypnotic drugs would be more advisable.

Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA‐associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA‐based analyses uncover depleted cardiac‐specific processes, among other relevant functions, that are undetected by CA. Twenty BA‐related microRNAs are identified, and two of them highly heart‐enriched that are present in plasma. We describe a microRNA‐gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research., The biological age, measured by transcriptional markers, explains better than the chronological age the transcriptional dynamics of the human aging myocardium. microRNAs that change expression in the aging left ventricle are identified. Such microRNAs potentially regulate genes with cardiac‐specific functions. Cardiac‐enriched microRNAs that increase or decrease with biological age are present in plasma. They are thus candidate biomarkers to represent the biological age of the heart.

To evaluate changes in the molecular epidemiology of tuberculosis (TB) in Zaragoza, Spain, over a decade that has seen large social and health changes, including the attenuation of the human immunodeficiency virus (HIV) epidemic and increased immigration.A population-based molecular study was conducted using standard restriction fragment length polymorphism IS6110 typing that included all patients with bacteriologically confirmed TB living in the Zaragoza area from 2001 to 2004. The current situation was compared with that described in a previous study from 1993 to 1995.A total of 454 Mycobacterium tuberculosis isolates were genotyped; 239 (52.6%) were grouped in 45 clusters composed of 2 to 85 isolates. Independent risk factors for clustering were identified. The main differences with the previous study were the increase of TB cases among immigrants, a decrease in HIV-TB co-infected patients and the occurrence of a large TB outbreak involving 85 patients (M. tuberculosis Zaragoza [MTZ] strain).A change in the epidemiological pattern of TB has been observed in the last years. TB transmission is more common among the Spanish-born population, while foreign birth is significantly less associated with clustering. A single epidemic strain caused 18.7% of all TB cases.

The ESX-1 secreted virulence factor ESAT-6 is one of the major and most well-studied virulence factors of Mycobacterium tuberculosis , given that its inactivation severely attenuates virulent mycobacteria. In this work, we show that clinical isolates of M. tuberculosis produce and secrete larger amounts of ESAT-6 than the widely used M. tuberculosis H37Rv laboratory strain. A search for the genetic polymorphisms underlying this observation showed that whiB6 ( rv3862c ), a gene upstream of the ESX-1 genetic locus that has not previously been found to be implicated in the regulation of the ESX-1 secretory apparatus, presents a unique single nucleotide insertion in its promoter region in strains H37Rv and H37Ra. This polymorphism is not present in any of the other publicly available M. tuberculosis complex genomes or in any of the 76 clinical M. tuberculosis isolates analyzed in our laboratory. We demonstrate that in consequence, the virulence master regulator PhoP downregulates whiB6 expression in H37Rv, while it upregulates its expression in clinical strains. Importantly, reintroduction of the wild-type (WT) copy of whiB6 in H37Rv restored ESAT-6 production and secretion to the level of clinical strains. Hence, we provide clear evidence that in M. tuberculosis —with the exception of the H37Rv strain—ESX-1 expression is regulated by WhiB6 as part of the PhoP regulon, which adds another level of complexity to the regulation of ESAT-6 secretion with a potential role in virulence adaptation.

Mutational status of TP53 together with expression of wild-type (wt) IGHV represents the most widely accepted biomarkers, establishing a very poor prognosis in B-cell chronic lymphocytic leukemia (B-CLL) patients. Adoptive cell therapy using allogeneic HLA-mismatched Natural killer (NK) cells has emerged as an effective and safe alternative in the treatment of acute myeloid and lymphoid leukemias that do not respond to traditional therapies. We have described that allogeneic activated NK cells eliminate hematological cancer cell lines with multidrug resistance acquired by mutations in the apoptotic machinery. This effect depends on the activation protocol, being B-lymphoblastoid cell lines (LCLs) the most effective stimulus to activate NK cells. Here, we have further analyzed the molecular determinants involved in allogeneic NK cell recognition and elimination of B-CLL cells, including the expression of ligands of the main NK cell-activating receptors (NKG2D and NCRs) and HLA mismatch. We present preliminary data suggesting that B-CLL susceptibility significantly correlates with HLA mismatch between NK cell donor and B-CLL patient. Moreover, we show that the sensitivity of B-CLL cells to NK cells depends on the prognosis based on TP53 and IGHV mutational status. Cells from patients with worse prognosis (mutated TP53 and wt IGHV) are the most susceptible to activated NK cells. Hence, B-CLL prognosis may predict the efficacy of allogenic activated NK cells, and, thus, NK cell transfer represents a good alternative to treat poor prognosis B-CLL patients who present a very short life expectancy due to lack of effective treatments. All our funders are public or charitable organizations. A grant from Fondation de France (0057921) to MV and DS-M. SUDOE to AA, JP, MV and CV. SAF2011-25390 and SAF2014-54763-C2-1-R to JP. Fondo Social Europeo (FSE) to JP and AA. JP is supported by Fundacion ARAID.

To the Editor: Eleven years of molecular epidemiologic data allowed the Spanish Multidrug-resistant Tuberculosis (MDR TB) Surveillance Network to identify a specific MDR Mycobacterium tuberculosis strain that had been imported into Spain from Equatorial Guinea (1). Our study brings to light the potential dissemination of this strain (named MDR-TBEG) in Equatorial Guinea, a country where little is known about the extent and features of TB or MDR TB. It also highlights that MDR strains can spread across continents, and thus MDR TB’s emergence in any country becomes a global problem. Ten MDR M. tuberculosis isolates obtained from 10 patients from Equatorial Guinea were detected in Spain during 2000 through 2008. Evidence of clonality was found within the 10 isolates because all exhibited identical genetic profiles defined by different molecular epidemiology methods (2,3) and mutations involved in drug resistance (Figure). Notably, none of the remaining 504 MDR isolates in the Spanish database matched SIT177, a spoligotype belonging to the Latin American–Mediterranean 9 (LAM9) subfamily (4). Figure Genetic profile of the multidrug-resistant tuberculosis Equatorial Guinea (MDR-TBEG) strain. RFLP, restriction fragment length polymorphism; SIT, spoligotype international type; LAM, Latin American-Mediterranean; MIRU-VNTR, mycobacterial interspersed ... The data routinely collected for all cases of MDR TB have been previously described (1). All 10 patients in the study were from Equatorial Guinea, a small African country on the Gulf of Guinea with a population of ≈500,000, an MDR TB rate >2.0% (5) of all combined (new and previously treated) TB cases, and an estimated adult HIV prevalence rate of 3.2% (www.who.int/globalatlas/predefinedReports/EFS2008/full/EFS2008_GQ.pdf). The MDR TB isolates were collected within a 9-year period (Technical Appendix): 1 in 2000, 2 in 2001, 3 in 2003, 1 in 2004, 2 in 2007, and 1 in 2008. According to their hospitals of origin, the patients were geographically dispersed in 6 different Spanish cities. We found that the interval between the patients’ arrival in Spain to the initiation of anti-TB treatment was