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Purpose: This study compared the results of the new Sysmex PA-100 AST System, a point-of-care analyser, with routine microbiology for the detection of urinary tract infections (UTI) and performance of antimicrobial susceptibility tests (AST) directly from urine., Methods: Native urine samples from 278 female patients with suspected uncomplicated UTI were tested in the Sysmex PA-100 and with reference methods of routine microbiology: urine culture for bacteriuria and disc diffusion for AST., Results: The analyser delivered bacteriuria results in 15 min and AST results within 45 min. Sensitivity and specificity for detection of microbiologically confirmed bacteriuria were 84.0% (89/106; 95% CI: 75.6-90.4%) and 99.4% (155/156; 95% CI: 96.5-100%), respectively, for bacterial species within the analyser specifications. These are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterococcus faecalis and Staphylococcus saprophyticus, which are common species causing uncomplicated UTI. Overall categorical agreement (OCA) for AST results for the five antimicrobials tested in the Sysmex PA-100 (amoxicillin/clavulanic acid, ciprofloxacin, fosfomycin, nitrofurantoin and trimethoprim) ranged from 85.4% (70/82; 95%CI: 75.9-92.2%) for ciprofloxacin to 96.4% (81/84; 95% CI: 89.9-99.3%) for trimethoprim. The Sysmex PA-100 provided an optimal treatment recommendation in 218/278 cases (78.4%), against 162/278 (58.3%) of clinical decisions., Conclusion: This first clinical evaluation of the Sysmex PA-100 in a near-patient setting demonstrated that the analyser delivers phenotypic AST results within 45 min, which could enable rapid initiation of the correct targeted treatment with no further adjustment needed. The Sysmex PA-100 has the potential to significantly reduce ineffective or unnecessary antibiotic prescription in patients with UTI symptoms., (© 2024. The Author(s).)

Early detection of disseminating vancomycin-resistant Enterococcus faecium (VREfm) in ICU wards is crucial for outbreak identification and the implementation of prompt infection control measures. Genotypic methods like pulsed-field gel electrophoresis (PFGE) and whole-genome sequencing (WGS) are costly and time-consuming, hindering rapid response due to batch dependency. Fourier-transform infrared spectroscopy (FT-IR) offers the potential for real-time outbreak detection and reliable strain typing. We utilized FT-IR to identify clonal VREfm dissemination and compared its performance to PFGE and WGS. Between February through October 2023, an unusually high number of VREfm were recovered at a tertiary hospital in Barcelona. Isolates were examined for antimicrobial susceptibility, carriage of vanA/vanB genes and clonality was also studied using FT-IR, PFGE, and WGS. Routine FT-IR inspections revealed recurring VREfm clustering during the outbreak's initial weeks. In total, 104 isolates were recovered from 75 patients and from multiple wards. However, only one isolate was recovered from an environmental sample, suggesting the absence of environmental reservoirs. An ST80 vancomycin-resistant ( vanA ) E. faecium strain was the main strain responsible for the outbreak, although a few additional VREfm strains were also identified, all belonging to CC17. PFGE and cgMLST (WGS) yielded identical clustering results to FT-IR, and WGS confirmed vanA/vanB gene carriage in all VREfm isolates. Infection control measures led to a rapid decline in VREfm isolates, with no isolates detected in November. FT-IR spectroscopy offers rapid turnaround times, sensitivity, and reproducibility, comparable to standard typing methods. It proved as an effective tool for monitoring VREfm dissemination and early outbreak detection.

Humoral immunity in COVID-19 includes antibodies (Abs) targeting spike (S) and nucleocapsid (N) SARS-CoV-2 proteins. Antibody levels are known to correlate with disease severity, but titers are poorly reported in mild or asymptomatic cases. Here, we analyzed the titers of IgA and IgG against SARS-CoV-2 proteins in samples from 200 unvaccinated Hospital Workers (HWs) with mild COVID-19 at two time points after infection. We analyzed the relationship between Ab titers and patient characteristics, clinical features, and evolution over time. Significant differences in IgG and IgA titers against N, S1 and S2 proteins were found when samples were segregated according to time T1 after infection, seroprevalence at T1, sex and age of HWs and symptoms at infection. We found that IgM + samples had higher titers of IgG against N antigen and IgA against S1 and S2 antigens than IgM - samples. There were significant correlations between anti-S1 and S2 Abs. Interestingly, IgM + patients with dyspnea had lower titers of IgG and IgA against N, S1 and S2 than those without dyspnea. Comparing T1 and T2, we found that IgA against N, S1 and S2 but only IgG against certain Ag decreased significantly. In conclusion, an association was established between Ab titers and the development of infection symptoms., (© 2024. The Author(s).)

Introduction. The absence of a gold-standard methodology for the microbiological diagnosis of urinary tract infections (UTI) has led to insufficient standardization of criteria for the interpretation of results and processing methods, particularly incubation time and culture media. Hypothesis. 48-hour incubation time period and use of blood agar enhances the sensitivity of microorganisms isolated significantly. Aim. To determine the sensitivity of blood agar and Brilliance UTI chromogenic agar, incubating for different periods (24-48 hours), for the detection of positive urine cultures. Methodoloy. Comparisons were made between all possible combinations of media and incubation times. As the gold-standard reference, we used the routine methodology of our laboratory, which involves prior screening with available clinical data, flow cytometry, sediment analysis and/or Gram staining. Screened samples were then cultured on blood agar and chromogenic agar and incubated for 48 hours. Also, based on the results of Gram staining, additional media were added in selected cases. Results. The most significant difference was found between chromogenic agar incubated for 24 hours and blood agar incubated for 48 hours, with the latter method allowing the recovery of 10.14 % more microorganisms ( P < 0.0001). Furthermore, the value of performing Gram staining to guide processing was demonstrated, as it avoided the loss of at least 5.14 % of isolates. Conclusions. At least in urological and nephrological patients it is essential to include enriched culture media (blood agar) or to extend the incubation times due to the improvement of the diagnostic sensitivity of urine cultures. Gram staining also can help detect the presence of fastidious microorganisms or mixed infections, indicating whether rich and/or selective media should be included to enhance the diagnostic sensitivity of cultures. If this methodology is not followed, it should be noted that besides fastidious species, fastidious strains of Escherichia coli, Proteus mirabilis, Pseudomonas aerugniosa and Stenotrophomonas maltophilia will also be missed.

Background: Intravitreal injections are a common ophthalmologic procedure. While infections following these injections are rare, they can lead to endophthalmitis, with potentially serious consequences. Various methods have been proposed to prevent endophthalmitis, including the use of antisepsis and antibiotics in patient preparation., Purpose: To evaluate the antiseptic efficacy of aqueous chlorhexidine (CHX) and povidone-iodine (PI) when used alone and in combination with lidocaine gel (LG) in vitro., Methods: Two independent experimental trials were conducted. The first trial determined the minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of CHX and PI against six bacterial strains. The second trial evaluated the bactericidal efficacy of the antiseptic agents (CHX 0.1% and PI 5%) and their combination with LG against the same bacterial strains., Results: CHX was more effective than PI in reducing the number of colonies forming units (cfus) of the tested bacteria. The order in which the antiseptic and LG were administered affected their effectiveness, with CHX administered before LG resulting in greater reduction of bacterial growth., Conclusions: CHX 0.1% is more effective than PI 5% as an antiseptic agent. Application of CHX and PI prior to the use of lidocaine gel results in a more effective reduction of microorganisms., (© 2024. The Author(s).)

Background: Community-acquired (CA) and healthcare-associated (HCA) infections caused by carbapenemase-producing Enterobacterales (CPE) are not well characterized. The objective was to provide detailed information about the clinical and molecular epidemiological features of nosocomial, HCA and CA infections caused by carbapenemase-producing Klebsiella pneumoniae (CP-Kp) and Escherichia coli (CP-Ec)., Methods: A prospective cohort study was performed in 59 Spanish hospitals from February to March 2019, including the first 10 consecutive patients from whom CP-Kp or CP-Ec were isolated. Patients were stratified according to acquisition type. A multivariate analysis was performed to identify the impact of acquisition type in 30-day mortality., Results: Overall, 386 patients were included (363 [94%] with CP-Kp and 23 [6%] CP-Ec); in 296 patients (76.3%), the CPE was causing an infection. Acquisition was CA in 31 (8.0%) patients, HCA in 183 (47.4%) and nosocomial in 172 (48.3%). Among patients with a HCA acquisition, 100 (54.6%) had been previously admitted to hospital and 71 (38.8%) were nursing home residents. Urinary tract infections accounted for 19/23 (82.6%), 89/130 (68.5%) and 42/143 (29.4%) of CA, HCA and nosocomial infections, respectively. Overall, 68 infections (23%) were bacteremia (8.7%, 17.7% and 30.1% of CA, HCA and nosocomial, respectively). Mortality in infections was 28% (13%, 14.6% and 42.7% of CA, HCA and nosocomial, respectively). Nosocomial bloodstream infections were associated with increased odds for mortality (adjusted OR, 4.00; 95%CI 1.21-13.19)., Conclusions: HCA and CA infections caused by CPE are frequent and clinically significant. This information may be useful for a better understanding of the epidemiology of CPE., (© 2024. The Author(s).)

Genome-wide association studies (GWAS) have identified more than 200 common genetic variants independently associated with colorectal cancer (CRC) risk, but the causal variants and target genes are mostly unknown. We sought to fine-map all known CRC risk loci using GWAS data from 100,204 cases and 154,587 controls of East Asian and European ancestry. Our stepwise conditional analyses revealed 238 independent association signals of CRC risk, each with a set of credible causal variants (CCVs), of which 28 signals had a single CCV. Our cis-eQTL/mQTL and colocalization analyses using colorectal tissue-specific transcriptome and methylome data separately from 1299 and 321 individuals, along with functional genomic investigation, uncovered 136 putative CRC susceptibility genes, including 56 genes not previously reported. Analyses of single-cell RNA-seq data from colorectal tissues revealed 17 putative CRC susceptibility genes with distinct expression patterns in specific cell types. Analyses of whole exome sequencing data provided additional support for several target genes identified in this study as CRC susceptibility genes. Enrichment analyses of the 136 genes uncover pathways not previously linked to CRC risk. Our study substantially expanded association signals for CRC and provided additional insight into the biological mechanisms underlying CRC development., (© 2024. The Author(s).)

Background: High red meat and/or processed meat consumption are established colorectal cancer risk factors. We conducted a genome-wide gene-environment (GxE) interaction analysis to identify genetic variants that may modify these associations., Methods: A pooled sample of 29,842 colorectal cancer cases and 39,635 controls of European ancestry from 27 studies were included. Quantiles for red meat and processed meat intake were constructed from harmonized questionnaire data. Genotyping arrays were imputed to the Haplotype Reference Consortium. Two-step EDGE and joint tests of GxE interaction were utilized in our genome-wide scan., Results: Meta-analyses confirmed positive associations between increased consumption of red meat and processed meat with colorectal cancer risk [per quartile red meat OR = 1.30; 95% confidence interval (CI) = 1.21-1.41; processed meat OR = 1.40; 95% CI = 1.20-1.63]. Two significant genome-wide GxE interactions for red meat consumption were found. Joint GxE tests revealed the rs4871179 SNP in chromosome 8 (downstream of HAS2); greater than median of consumption ORs = 1.38 (95% CI = 1.29-1.46), 1.20 (95% CI = 1.12-1.27), and 1.07 (95% CI = 0.95-1.19) for CC, CG, and GG, respectively. The two-step EDGE method identified the rs35352860 SNP in chromosome 18 (SMAD7 intron); greater than median of consumption ORs = 1.18 (95% CI = 1.11-1.24), 1.35 (95% CI = 1.26-1.44), and 1.46 (95% CI = 1.26-1.69) for CC, CT, and TT, respectively., Conclusions: We propose two novel biomarkers that support the role of meat consumption with an increased risk of colorectal cancer., Impact: The reported GxE interactions may explain the increased risk of colorectal cancer in certain population subgroups., (©2023 American Association for Cancer Research.)

Background: Transcriptome-wide association studies have been successful in identifying candidate susceptibility genes for colorectal cancer (CRC). To strengthen susceptibility gene discovery, we conducted a large transcriptome-wide association study and an alternative splicing transcriptome-wide association study in CRC using improved genetic prediction models and performed in-depth functional investigations., Methods: We analyzed RNA-sequencing data from normal colon tissues and genotype data from 423 European descendants to build genetic prediction models of gene expression and alternative splicing and evaluated model performance using independent RNA-sequencing data from normal colon tissues of the Genotype-Tissue Expression Project. We applied the verified models to genome-wide association studies (GWAS) summary statistics among 58 131 CRC cases and 67 347 controls of European ancestry to evaluate associations of genetically predicted gene expression and alternative splicing with CRC risk. We performed in vitro functional assays for 3 selected genes in multiple CRC cell lines., Results: We identified 57 putative CRC susceptibility genes, which included the 48 genes from transcriptome-wide association studies and 15 genes from splicing transcriptome-wide association studies, at a Bonferroni-corrected P value less than .05. Of these, 16 genes were not previously implicated in CRC susceptibility, including a gene PDE7B (6q23.3) at locus previously not reported by CRC GWAS. Gene knockdown experiments confirmed the oncogenic roles for 2 unreported genes, TRPS1 and METRNL, and a recently reported gene, C14orf166., Conclusion: This study discovered new putative susceptibility genes of CRC and provided novel insights into the biological mechanisms underlying CRC development., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Genome-wide association studies have identified thousands of loci associated with common diseases and traits. However, a large fraction of heritability remains unexplained. Epigenetic modifications, such as the observed in DNA methylation have been proposed as a mechanism of intergenerational inheritance. To investigate the potential contribution of DNA methylation to the missing heritability, we analysed the methylomes of four healthy trios (two parents and one offspring) using whole genome bisulphite sequencing. Of the 1.5 million CpGs (19%) with over 20% variability between parents in at least one family and compatible with a Mendelian inheritance pattern, only 3488 CpGs (0.2%) lacked correlation with any SNP in the genome, marking them as potential sites for intergenerational epigenetic inheritance. These markers were distributed genome-wide, with some preference to be located in promoters. They displayed a bimodal distribution, being either fully methylated or unmethylated, and were often found at the boundaries of genomic regions with high/low GC content. This analysis provides a starting point for future investigations into the missing heritability of simple and complex traits., (© 2023. The Author(s).)

Analysis of the bacterial community from a 16S rRNA gene sequencing technologies requires comparing the reads to a reference database. The challenging task involved in annotation relies on the currently available tools and 16S rRNA databases: SILVA, Greengenes and RDP. A successful annotation depends on the quality of the database. For instance, Greengenes and RDP have not been updated since 2013 and 2016, respectively. In addition, the nature of 16S sequencing technologies (short reads) focuses mainly on the V3-V4 hypervariable region sequencing and hinders the species assignment, in contrast to whole shotgun metagenome sequencing. Here, we combine the results of three standard protocols for 16S rRNA amplicon annotation that utilize homology-based methods, and we propose a new re-annotation strategy to enlarge the percentage of amplicon sequence variants (ASV) classified up to the species level. Following the pattern (reference) method: DADA2 pipeline and SILVA v.138.1 reference database classification (Basic Protocol 1), our method maps the ASV sequences to custom nucleotide BLAST with the SILVA v.138.1 (Basic Protocol 2), and to the 16S database of Bacteria and Archaea of NCBI RefSeq Targeted Loci Project databases (Basic Protocol 3). This new re-annotation workflow was tested in 16S rRNA amplicon data from 156 human fecal samples. The proposed new strategy achieved an increase of nearly eight times the proportion of ASV classified at the species level in contrast to the reference method for the database used in the present research. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Sample inference and taxonomic profiling through DADA2 algorithm. Basic Protocol 2: Custom BLASTN database creation and ASV taxonomical assignment. Basic Protocol 3: ASV taxonomical assignment using NCBI RefSeq Targeted Loci Project database. Basic Protocol 4: Definitive selection of lineages among the three methods., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Disk diffusion is a well standardized method that provides reliable categorical results to guide antimicrobial therapy in numerous types of infections. Based on the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST), which are widely implemented in Spain, the Spanish Antibiogram Committee (COESANT) has drawn up recommendations for antimicrobial selection by the disk diffusion technique, including selective reporting and its use for the detection of resistance mechanisms. Factors affecting disk diffusion results, along with advantages and shortcomings of the method, are also discussed., (Copyright © 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

Molecular crosstalk, the dialogue between different cell types, is attracting more attention in cancer research. On the one hand, the communication between tumor and non-tumor cells in the microenvironment or between different tumor clones has influential consequences for the progression and spread of tumors and response to treatment. On the other hand, novel techniques such as single-cell sequencing or spatial transcriptomics provide detailed information that needs to be interpreted. TALKIEN: crossTALK IntEraction Network is a simple and intuitive online R/shiny application to visualize molecular crosstalk information through the construction and analysis of a protein-protein interaction network. Taking two or more lists of genes or proteins as input, which are representative of cell lineages, TALKIEN extracts information about ligand-receptor interactions, builds a network and analyzes it using systems biology techniques such as centrality measures and component analysis, among others. Moreover, it expands the network displaying pathways downstream receptors. The application allows users to select different graphical layouts, performs functional analysis and gives information about drugs targeting receptors. In conclusion, TALKIEN allows users to detect ligand-receptor interactions generating new in silico predictions of cell-cell communication thus providing a translational rationale for future experiments. It is freely available at https://www.odap-ico.org/talkien.

Objective: Reduced diversity at Human Leukocyte Antigen (HLA) loci may adversely affect the host's ability to recognize tumor neoantigens and subsequently increase disease burden. We hypothesized that increased heterozygosity at HLA loci is associated with a reduced risk of developing colorectal cancer (CRC)., Methods: We imputed HLA class I and II four-digit alleles using genotype data from a population-based study of 5,406 cases and 4,635 controls from the Molecular Epidemiology of Colorectal Cancer Study (MECC). Heterozygosity at each HLA locus and the number of heterozygous genotypes at HLA class -I ( A , B , and C ) and HLA class -II loci ( DQB1 , DRB1 , and DPB1 ) were quantified. Logistic regression analysis was used to estimate the risk of CRC associated with HLA heterozygosity. Individuals with homozygous genotypes for all loci served as the reference category, and the analyses were adjusted for sex, age, genotyping platform, and ancestry. Further, we investigated associations between HLA diversity and tumor-associated T cell repertoire features, as measured by tumor infiltrating lymphocytes (TILs; N=2,839) and immunosequencing (N=2,357)., Results: Individuals with all heterozygous genotypes at all three class I genes had a reduced odds of CRC (OR: 0.74; 95% CI: 0.56-0.97, p = 0.031). A similar association was observed for class II loci, with an OR of 0.75 (95% CI: 0.60-0.95, p = 0.016). For class-I and class-II combined, individuals with all heterozygous genotypes had significantly lower odds of developing CRC (OR: 0.66, 95% CI: 0.49-0.87, p = 0.004) than those with 0 or one heterozygous genotype. HLA class I and/or II diversity was associated with higher T cell receptor (TCR) abundance and lower TCR clonality, but results were not statistically significant., Conclusion: Our findings support a heterozygote advantage for the HLA class-I and -II loci, indicating an important role for HLA genetic variability in the etiology of CRC., Competing Interests: GI: receives/received research funding from Myriad Genetics and Laboratories. JG: consultant for Guardant Health. VM: owns stock in Aniling. SG: Co-founder of Brogent International LLC with equity. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tsai, Qu, Bonner, Sanz-Pamplona, Lindsey, Melas, McDonnell, Idos, Walker, Tsang, Da Silva, Moratalla-Navarro, Maoz, Rennert, Kast, Greenson, Moreno, Rennert, Gruber and Schmit.)

The Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) presents in this document a series of recommendations intending to unify how cumulative antibiogram reports must be made in Clinical Microbiology Spanish laboratories. This article is based on the information included in the Clinical Microbiology Procedure No. 51, «Preparation of cumulative reports on antimicrobial susceptibility» of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), published in 2014. The recommendations also include the modifications in the definition of clinical interpretive categories recently published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) in 2019. Its final objective is to establish a homogeneous way of preparing these summaries to compare results from different centers or aggregate the information from these in order to carry out an adequate local or even national surveillance regarding the evolution of antimicrobial susceptibility., (Copyright © 2022 Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. Published by Elsevier España, S.L.U. All rights reserved.)

We aimed to identify and validate a set of miRNAs that could serve as a prognostic signature useful to determine the recurrence risk for patients with COAD. Small RNAs from tumors of 100 stage II, untreated, MSS colon cancer patients were sequenced for the discovery step. For this purpose, we built an miRNA score using an elastic net Cox regression model based on the disease-free survival status. Patients were grouped into high or low recurrence risk categories based on the median value of the score. We then validated these results in an independent sample of stage II microsatellite stable tumor tissues, with a hazard ratio of 3.24, (CI 95% = 1.05-10.0) and a 10-year area under the receiver operating characteristic curve of 0.67. Functional analysis of the miRNAs present in the signature identified key pathways in cancer progression. In conclusion, the proposed signature of 12 miRNAs can contribute to improving the prediction of disease relapse in patients with stage II MSS colorectal cancer, and might be useful in deciding which patients may benefit from adjuvant chemotherapy.

Colorectal cancer (CRC) is a leading cause of mortality worldwide. We conducted a genome-wide association study meta-analysis of 100,204 CRC cases and 154,587 controls of European and east Asian ancestry, identifying 205 independent risk associations, of which 50 were unreported. We performed integrative genomic, transcriptomic and methylomic analyses across large bowel mucosa and other tissues. Transcriptome- and methylome-wide association studies revealed an additional 53 risk associations. We identified 155 high-confidence effector genes functionally linked to CRC risk, many of which had no previously established role in CRC. These have multiple different functions and specifically indicate that variation in normal colorectal homeostasis, proliferation, cell adhesion, migration, immunity and microbial interactions determines CRC risk. Crosstissue analyses indicated that over a third of effector genes most probably act outside the colonic mucosa. Our findings provide insights into colorectal oncogenesis and highlight potential targets across tissues for new CRC treatment and chemoprevention strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Campylobacter, a major cause of food-borne gastroenteritis worldwide, colonize the gastrointestinal tract of a wide range of animals, being birds the main reservoir. The mechanisms involved in the interaction of Campylobacter with the different hosts are poorly understood. The cytolethal distending toxin, encoded in the cdtABC operon, is considered a pivotal virulence factor during human infection. Differences in the prevalence of cdtABC genes in Campylobacter isolates from three distinct origins (wild birds, broiler chickens and humans) prompted us to further characterize their allelic variability. The sequence of cdtABC is highly conserved among broiler and human isolates. A high diversity of cdtABC alleles was found among wild bird isolates, including several alleles that do not produce any functional CDT. These results suggest that specific variants of the cdtABC operon might define the host range of specific Campylobacter jejuni isolates. Moreover, our data indicate that PCR methodology is inaccurate to characterize the prevalence of the cdt genes, since negative PCR detection can be the result of divergences in the sequence used for primer design rather than indicating the absence of a specific gene., (© 2022 The Authors. Zoonoses and Public Health published by Wiley-VCH GmbH.)

Objective: We constructed epilepsy multimorbidity networks to study associations with chronic conditions, and co-prescriptions and drug-disease networks to assess potential interactions. We conducted a population-based study in Catalonia, Spain, with electronic files of 3,135,948 adult patients with multimorbidity, 32,625 of them with epilepsy (active diagnosis any time during 2006-2017). We constructed epilepsy comorbidity networks using logistic regression models from odds ratio estimates adjusted by age, sex, and comorbidities with R software and generated trajectories to study the progression of epilepsy. We constructed drug-disease and co-prescription networks using mixed models with repeated measures adjusting by age, sex, and period with chronic prescription invoiced data. Comorbidity more frequently preceding epilepsy included cerebrovascular accident (OR: 3.59), congenital anomalies (2.18), and multiple sclerosis (1.33); and following epilepsy: dementia (1.91), personality disorder (1.59), alcohol abuse (1.22), and Parkinson (1.21). Mental retardation (13.08), neurological cancer (8.49), benign neoplasm (4.69), infections (3.14), and psychosis (1.58) might precede or not epilepsy. A common progression was to schizophrenia, dementia, and other neurological diseases (mainly cerebral palsy and other degenerative diseases of nervous system). Co-prescription associations with major-moderate potential interactions were 54% for carbamazepine, 61% phenytoin, 53% phenobarbital, and 32% valproate. Major potential interactions were with antipsychotic, anxiolytic, opioid, cardiovascular, and other anti-seizure medications (ASMs). The most frequent comorbidities of epilepsy were congenital, cerebrovascular, and neurological and psychiatric conditions. High comorbidity and co-prescription with potential interactions can increase the complexity of care of patients with epilepsy., (© 2022. Fondazione Società Italiana di Neurologia.)

Colonomics is a multi-omics dataset that includes 250 samples: 50 samples from healthy colon mucosa donors and 100 paired samples from colon cancer patients (tumor/adjacent). From these samples, Colonomics project includes data from genotyping, DNA methylation, gene expression, whole exome sequencing and micro-RNAs (miRNAs) expression. It also includes data from copy number variation (CNV) from tumoral samples. In addition, clinical data from all these samples is available. The aims of the project were to explore and integrate these datasets to describe colon cancer at molecular level and to compare normal and tumoral tissues. Also, to improve screening by finding biomarkers for the diagnosis and prognosis of colon cancer. This project has its own website including four browsers allowing users to explore Colonomics datasets. Since generated data could be reuse for the scientific community for exploratory or validation purposes, here we describe omics datasets included in the Colonomics project as well as results from multi-omics layers integration., (© 2022. The Author(s).)

The gut microbiome is a potential modifiable risk factor for colorectal cancer (CRC). We re-analyzed all eight previously published stool sequencing data and conducted an MWAS meta-analysis. We used cross-validated LASSO predictive models to identify a microbiome signature for predicting the risk of CRC and precancerous lesions. These models were validated in a new study, Colorectal Cancer Screening (COLSCREEN), including 156 participants that were recruited in a CRC screening context. The MWAS meta-analysis identified 95 bacterial species that were statistically significantly associated with CRC (FDR < 0.05). The LASSO CRC predictive model obtained an area under the receiver operating characteristic curve (aROC) of 0.81 (95%CI: 0.78−0.83) and the validation in the COLSCREEN dataset was 0.75 (95%CI: 0.66−0.84). This model selected a total of 32 species. The aROC of this CRC-trained model to predict precancerous lesions was 0.52 (95%CI: 0.41−0.63). We have identified a signature of 32 bacterial species that have a good predictive accuracy to identify CRC but not precancerous lesions, suggesting that the identified microbes that were enriched or depleted in CRC are merely a consequence of the tumor. Further studies should focus on CRC as well as precancerous lesions with the intent to implement a microbiome signature in CRC screening programs.

Background: Colorectal cancer has high incidence and associated mortality worldwide. Screening programs are recommended for men and women over 50. Intermediate screens such as fecal immunochemical testing (FIT) select patients for colonoscopy with suboptimal sensitivity. Additional biomarkers could improve the current scenario., Methods: We included 2,893 individuals with a positive FIT test. They were classified as cases when a high-risk lesion for colorectal cancer was detected after colonoscopy, whereas the control group comprised individuals with low-risk or no lesions. 65 colorectal cancer risk genetic variants were genotyped. Polygenic risk score (PRS) and additive models for risk prediction incorporating sex, age, FIT value, and PRS were generated., Results: Risk score was higher in cases compared with controls [per allele OR = 1.04; 95% confidence interval (CI), 1.02-1.06; P < 0.0001]. A 2-fold increase in colorectal cancer risk was observed for subjects in the highest decile of risk alleles (≥65), compared with those in the first decile (≤54; OR = 2.22; 95% CI, 1.59-3.12; P < 0.0001). The model combining sex, age, FIT value, and PRS reached the highest accuracy for identifying patients with a high-risk lesion [cross-validated area under the ROC curve (AUROC): 0.64; 95% CI, 0.62-0.66]., Conclusions: This is the first investigation analyzing PRS in a two-step colorectal cancer screening program. PRS could improve current colorectal cancer screening, most likely for higher at-risk subgroups. However, its capacity is limited to predict colorectal cancer risk status and should be complemented by additional biomarkers., Impact: PRS has capacity for risk stratification of colorectal cancer suggesting its potential for optimizing screening strategies alongside with other biomarkers., (©2022 The Authors; Published by the American Association for Cancer Research.)

Background: Few long-term reports have been published on the epidemiology of respiratory viruses despite their frequent involvement in extremely common infections. The aim here was to determine the frequency and distribution of respiratory viruses in a temperate climate area (Barcelona, Spain) throughout a 24-year period., Methods: We collected data on all respiratory viruses detected from 1997 to 2020 in our institution. Clinical specimens were analyzed mainly by conventional techniques, and molecular techniques were also used., Results: Of the 59,579 specimens analyzed, 21,382 (35.9%) were positive for at least one virus. The number of positive samples during cold months was significantly higher than in warm months. Respiratory virus infections were detected in patients of all ages, above all in children under 3 years of age, who were most frequently infected with the respiratory syncytial virus, whereas Influenza A virus predominated in the other groups, especially in adults. A clear demographic and seasonal pattern was established for some viruses. Circulation of other respiratory viruses during the FLUAV H1N1pdm09 and SARS-CoV-2 pandemics was observed., Conclusions: This long-term study provides new knowledge about the prevalence of respiratory viruses in a Mediterranean region. Throughout the study period, the frequency of some viruses remained constant, whereas others varied with the year. A clear demographic and seasonal pattern was established for some viruses. Patients suffering from severe respiratory infections should be examined for a range of respiratory viruses regardless of gender, age, or season., (© 2022 The Authors. Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

Background: Knowing how long SARS-CoV-2-positive individuals can remain infective is crucial for the design of infection prevention and control strategies. Viral culture is the gold standard for detecting an active-replicative virus and evaluating its infectious potential., Objective: To assess the correlation of SARS-CoV-2 infectivity with the number of days from symptom onset and the Ct value, using culture as a reference method. Also, to describe a detailed protocol for SARS-CoV-2 culture and immunofluorescence confirmation based on our experience with other respiratory viruses., Study Design: 100 consecutive respiratory samples positive for SARS-CoV-2 by RT-PCR from different subjects were inoculated into VERO E6 cells., Results: Viral isolation was successful in 58% of samples. The median number of days from symptom onset for culture-positive samples was 2, and 15 for culture-negative samples. Six positive cultures were obtained in patients ≥14 days after symptom onset, all of whom were immunocompromised or with severe COVID-19. The mean Ct value was 12.64 units higher in culture-negative than in culture-positive samples. The probability of successfully isolating SARS-CoV-2 in samples with a Ct value <22 was 100%, decreasing to 3.1% when >27., Conclusions: Our findings show a significant positive correlation between the probability of isolating SARS-CoV-2 in culture, fewer days of symptoms and a lower RT-PCR Ct value. SARS-CoV-2 infectivity lasts no more than 14 days from symptom onset in immunocompetent individuals. In contrast, in immunocompromised patients or those with severe COVID-19 infectivity may remain after 14 days. Ct value <22 always indicates infectivity., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: CARB-ES-19 is a comprehensive, multicenter, nationwide study integrating whole-genome sequencing (WGS) in the surveillance of carbapenemase-producing K. pneumoniae (CP-Kpn) and E. coli (CP-Eco) to determine their incidence, geographical distribution, phylogeny, and resistance mechanisms in Spain., Methods: In total, 71 hospitals, representing all 50 Spanish provinces, collected the first 10 isolates per hospital (February to May 2019); CPE isolates were first identified according to EUCAST (meropenem MIC > 0.12 mg/L with immunochromatography, colorimetric tests, carbapenem inactivation, or carbapenem hydrolysis with MALDI-TOF). Prevalence and incidence were calculated according to population denominators. Antibiotic susceptibility testing was performed using the microdilution method (EUCAST). All 403 isolates collected were sequenced for high-resolution single-nucleotide polymorphism (SNP) typing, core genome multilocus sequence typing (cgMLST), and resistome analysis., Results: In total, 377 (93.5%) CP-Kpn and 26 (6.5%) CP-Eco isolates were collected from 62 (87.3%) hospitals in 46 (92%) provinces. CP-Kpn was more prevalent in the blood (5.8%, 50/853) than in the urine (1.4%, 201/14,464). The cumulative incidence for both CP-Kpn and CP-Eco was 0.05 per 100 admitted patients. The main carbapenemase genes identified in CP-Kpn were bla OXA-48 (263/377), bla KPC-3 (62/377), bla VIM-1 (28/377), and bla NDM-1 (12/377). All isolates were susceptible to at least two antibiotics. Interregional dissemination of eight high-risk CP-Kpn clones was detected, mainly ST307/OXA-48 (16.4%), ST11/OXA-48 (16.4%), and ST512-ST258/KPC (13.8%). ST512/KPC and ST15/OXA-48 were the most frequent bacteremia-causative clones. The average number of acquired resistance genes was higher in CP-Kpn (7.9) than in CP-Eco (5.5)., Conclusion: This study serves as a first step toward WGS integration in the surveillance of carbapenemase-producing Enterobacterales in Spain. We detected important epidemiological changes, including increased CP-Kpn and CP-Eco prevalence and incidence compared to previous studies, wide interregional dissemination, and increased dissemination of high-risk clones, such as ST307/OXA-48 and ST512/KPC-3., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cañada-García, Moure, Sola-Campoy, Delgado-Valverde, Cano, Gijón, González, Gracia-Ahufinger, Larrosa, Mulet, Pitart, Rivera, Bou, Calvo, Cantón, González-López, Martínez-Martínez, Navarro, Oliver, Palacios-Baena, Pascual, Ruiz-Carrascoso, Vila, Aracil, Pérez-Vázquez, Oteo-Iglesias and the GEMARA/GEIRAS-SEIMC/REIPI CARB-ES-19 Study Group.)

Campylobacter jejuni is a foodborne pathogen causing bacterial gastroenteritis, with the highest incidence reported in Europe. The prevalence of antibiotic resistance in C. jejuni , as well as in many other bacterial pathogens, has increased over the last few years. In this report, we describe the presence of a plasmid in a multi-drug-resistant C. jejuni strain isolated from a gastroenteritis patient. Mating experiments demonstrated the transference of this genetic element (pCjH01) among C. jejuni by plasmid conjugation. The pCjH01 plasmid was sequenced and assembled, revealing high similarity (97% identity) with pTet, a described tetracycline resistance encoding plasmid. pCjH01 (47.7 kb) is a mosaic plasmid composed of a pTet backbone that has acquired two discrete DNA regions. Remarkably, one of the acquired sequences carried an undescribed variant of the aadE-sat4-aphA-3 gene cluster, providing resistance to at least kanamycin and gentamycin. Aside from the antibiotic resistance genes, the cluster also carries genes coding for putative regulators, such as a sigma factor of the RNA polymerase and an antisigma factor. Homology searches suggest that Campylobacter exchanges genetic material with distant G-positive bacterial genera.

Background and Aims: Genome-wide association studies [GWAS] for inflammatory bowel disease [IBD] have identified 240 risk variants. However, the benefit of understanding the genetic architecture of IBD remains to be exploited. Transcriptome-wide association studies [TWAS] associate gene expression with genetic susceptibility to disease, providing functional insight into risk loci. In this study, we integrate relevant datasets for IBD and perform a TWAS to nominate novel genes implicated in IBD genetic susceptibility., Methods: We applied elastic net regression to generate gene expression prediction models for the University of Barcelona and University of Virginia RNA sequencing project [BarcUVa-Seq] and correlated expression and disease association research [CEDAR] datasets. Together with Genotype-Tissue Expression project [GTEx] data, and GWAS results from about 60 000 individuals, we employed Summary-PrediXcan and Summary-MultiXcan for single and joint analyses of TWAS results, respectively., Results: BarcUVa-Seq TWAS revealed 39 novel genes whose expression in the colon is associated with IBD genetic susceptibility. They included expression markers for specific colon cell types. TWAS meta-analysis including all tissues/cell types provided 186 novel candidate susceptibility genes. Additionally, we identified 78 novel susceptibility genes whose expression is associated with IBD exclusively in immune (N = 19), epithelial (N = 25), mesenchymal (N = 22) and neural (N = 12) tissue categories. Associated genes were involved in relevant molecular pathways, including pathways related to known IBD therapeutics, such as tumour necrosis factor signalling., Conclusion: These findings provide insight into tissue-specific molecular processes underlying IBD genetic susceptibility. Associated genes could be candidate targets for new therapeutics and should be prioritized in functional studies., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Tobacco smoke and red/processed meats are well-known risk factors for colorectal cancer (CRC). Most research has focused on studies of normal colon biopsies in epidemiologic studies or treatment of CRC cell lines in vitro . These studies are often constrained by challenges with accuracy of self-report data or, in the case of CRC cell lines, small sample sizes and lack of relationship to normal tissue at risk. In an attempt to address some of these limitations, we performed a 24-hour treatment of a representative carcinogens cocktail in 37 independent organoid lines derived from normal colon biopsies. Machine learning algorithms were applied to bulk RNA-sequencing and revealed cellular composition changes in colon organoids. We identified 738 differentially expressed genes in response to carcinogens exposure. Network analysis identified significantly different modules of co-expression, that included genes related to MSI-H tumor biology, and genes previously implicated in CRC through genome-wide association studies. Our study helps to better define the molecular effects of representative carcinogens from smoking and red/processed meat in normal colon epithelial cells and in the etiology of the MSI-H subtype of CRC, and suggests an overlap between molecular mechanisms involved in inherited and environmental CRC risk., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Devall et al.)

Conjunctivitis is a frequent ocular disorder caused by human adenoviruses (HAdVs). Only a few of the 45 HAdV-D species are associated with epidemic keratoconjunctivitis, including HAdV-D8. Nosocomial outbreaks due to HAdV-D8 have been rarely described, because keratoconjunctivitis cases are clinically diagnosed and treated without having to characterize the causative agent. Moreover, molecular typing is tedious when using classical techniques. In this study, a hospital outbreak of conjunctivitis caused by HAdV-D8 was characterized using the recently developed whole-genome sequencing (WGS) method. Of the 363 patients attending the Ophthalmology Department between July 13 and August 13, 2018, 36 may have acquired intrahospital conjunctivitis. Also, 11 of 22 samples sent to the Virology section were selected for WGS analysis. The WGS results revealed that 10 out of 11 HAdV-D8 strains were closely related. The remaining strain (Case 28) was more similar to a strain from an outbreak in Germany obtained from a public sequence database. WGS results showed that outbreak HAdV-D8 strains had a minimum percentage of identity of 94.3%. WGS is useful in a clinical setting, because it avoids carrying out viral culture or specific polymerase chain reaction sequencing. The public availability of sequence reads makes it easier to compare clusters in circulation. In conclusion, WGS can play an important role in standard routines to describe viral outbreaks., (© 2021 Wiley Periodicals LLC.)

Bacteriophages are present in fluids from cirrhosis patients. However, their effect on the immune response is unknown. In this work, we explore the role of phages in the phenotype, function, and cytokine production of monocytes. We stimulated healthy monocytes with five different butanol-purified phage suspensions infective for Gram-negative and Gram-positive bacteria. We studied the expression of the monocyte markers involved in lipopolysaccharide recognition (LPS; CD14), antigen presentation (HLA-DR) and co-stimulation (CD86), and the concentration of induced cytokines (TNF-α, IFN-α, and IL-10) by phages. To confirm the direct role of phages without the interference of contaminating soluble LPS in phage suspensions, polymyxin B was added to the cell cultures. Phagocytosis experiments were assessed by flow cytometry using labeled phage suspensions. We observed that butanol-purified phages reduced the surface levels of CD14 and CD86 in monocytes and increased the secreted levels of TNF-α and IL-10 compared with the control sample containing only butanol buffer. All phage suspensions showed downregulation of HLA-DR expression but only Staphylococcus aureus phage contaminated with Escherichia coli reached statistical significance. The addition of polymyxin B did not restore the monocytic response induced by phages, suggesting that the effect was not caused by the presence of LPS. Monocytes were able to phagocyte phages in a dose- and time-dependent manner. To conclude, the phagocytosis of butanol-purified phages altered the phenotype and cytokine production of monocytes suggesting they become tolerogenic.

Some Aeromonas species, potentially pathogenic for humans, are known to express up to three different classes of chromosomal β-lactamases, which may become hyperproduced and cause treatment failure. The aim of this study was to assess the utility of these species-specific β-lactamase genes as phylogenetic markers using whole-genome sequencing data. Core-genome alignments were generated for 36 Aeromonas genomes from seven different species and scanned for antimicrobial resistance genes. Core-genome alignment confirmed the MALDI-TOF identification of most of the isolates and re-identified an A. hydrophila isolate as A. dhakensis . Three (B, C and D) of the four Ambler classes of β-lactamase genes were found in A. sobria, A. allosacharophila, A. hydrophila and A. dhakensis ( bla CphA , bla AmpC and bla OXA ). A. veronii only showed class-B- and class-D-like matches ( bla CphA and bla OXA ), whereas those for A. media, A. rivipollensis and A. caviae were class C and D ( bla CMY , bla MOX and bla OXA427 ). The phylogenetic tree derived from concatenated sequences of β-lactamase genes successfully clustered each species. Some isolates also had resistance to sulfonamides, quinolones and aminoglycosides. Whole-genome sequencing proved to be a useful method to identify Aeromonas at the species level, which led to the unexpected identification of A. dhakensis and A . rivipollensis and revealed the resistome of each isolate.

Purpose: Multimorbidity impacts quality of life. We constructed hypothyroidism comorbidity networks to identify positive and negative associations with other prevalent diseases., Methods: We analyzed data of 285 342 patients with hypothyroidism from 3 135 948 adults with multimorbidity in a population-based study in Catalonia, Spain, (period: 2006-2017). We constructed hypothyroidism comorbidity networks using logistic regression models, adjusted by age and sex, and for men and women separately. We considered relevant associations those with odds ratios (OR) >1.2 or <0.8 and P value < 1e-5 to identify coexistence greater (or smaller) than the expected by the prevalence of diseases. Multivariate models considering comorbidities were used to further adjust OR values., Results: The conditions associated included larynx cancer (adjusted OR: 2.48), congenital anomalies (2.26), thyroid cancer (2.13), hyperthyroidism (1.66), vitamin B12/folate deficiency anemia (1.57), and goiter (1.56). The network restricted to men had more connections (mental, cardiovascular, and neurological) and stronger associations with thyroid cancer (7.26 vs 2.55), congenital anomalies (5.11 vs 2.13), hyperthyroidism (4.46 vs 1.69), larynx cancer (3.55 vs 1.67), and goiter (3.94 vs 1.64). After adjustment for comorbidities, OR values were more similar in men and women. The strongest negative associations after adjusting for comorbidities were with HIV/AIDS (OR: 0.71) and tobacco abuse (0.77)., Conclusions: Networks show direct and indirect hypothyroidism multimorbidity associations. The strongest connections were thyroid and larynx cancer, congenital anomalies, hyperthyroidism, anemia, and goiter. Negative associations included HIV/AIDS and tobacco abuse. The network restricted to men had more and stronger associations, but not after adjusting for comorbidities, suggesting important indirect interactions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background & Aims: The association of genetic variation with tissue-specific gene expression and alternative splicing guides functional characterization of complex trait-associated loci and may suggest novel genes implicated in disease. Here, our aims were as follows: (1) to generate reference profiles of colon mucosa gene expression and alternative splicing and compare them across colon subsites (ascending, transverse, and descending), (2) to identify expression and splicing quantitative trait loci (QTLs), (3) to find traits for which identified QTLs contribute to single-nucleotide polymorphism (SNP)-based heritability, (4) to propose candidate effector genes, and (5) to provide a web-based visualization resource., Methods: We collected colonic mucosal biopsy specimens from 485 healthy adults and performed bulk RNA sequencing. We performed genome-wide SNP genotyping from blood leukocytes. Statistical approaches and bioinformatics software were used for QTL identification and downstream analyses., Results: We provided a complete quantification of gene expression and alternative splicing across colon subsites and described their differences. We identified thousands of expression and splicing QTLs and defined their enrichment at genome-wide regulatory regions. We found that part of the SNP-based heritability of diseases affecting colon tissue, such as colorectal cancer and inflammatory bowel disease, but also of diseases affecting other tissues, such as psychiatric conditions, can be explained by the identified QTLs. We provided candidate effector genes for multiple phenotypes. Finally, we provided the Colon Transcriptome Explorer web application., Conclusions: We provide a large characterization of gene expression and splicing across colon subsites. Our findings provide greater etiologic insight into complex traits and diseases influenced by transcriptomic changes in colon tissue., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Surgical antimicrobial prophylaxis (SAP) is important for the prevention of prosthetic joint infections (PJIs) and must be effective against the microorganisms most likely to contaminate the surgical site. Our aim was to compare different SAP regimens (cefazolin, cefuroxime, or vancomycin, alone or combined with gentamicin) in patients undergoing total knee (TKA) and hip (THA) arthroplasty. In this preclinical exploratory analysis, we analyzed the results of intraoperative sample cultures, the ratio of plasma antibiotic levels to the minimum inhibitory concentrations (MICs) for bacteria isolated at the surgical wound and ATCC strains, and serum bactericidal titers (SBT) against the same microorganisms. A total of 132 surgical procedures (68 TKA, 64 THA) in 128 patients were included. Cultures were positive in 57 (43.2%) procedures (mostly for coagulase-negative staphylococci and Cutibacterium spp.); the rate was lower in the group of patients receiving combination SAP (adjusted OR 0.475, CI95% 0.229-0.987). The SAP regimens evaluated achieved plasma levels above the MICs in almost all of intraoperative isolates (93/94, 98.9%) and showed bactericidal activity against all of them (SBT range 1:8-1:1024), although SBTs were higher in patients receiving cefazolin and gentamicin-containing regimens. The potential clinical relevance of these findings in the prevention of PJIs remains to be determined.

Objectives: To assess the effect on the functional ambulatory outcome of postoperative joint infection (PJI) cured at the first treatment attempt versus not developing PJI in patients with hip and knee prostheses., Methods: In a single-hospital retrospectively matched cohort study, each patient with PJI between 2007 and 2016 was matched on age, sex, type of prosthesis and year of implantation with two other patients with uninfected arthroplasties. The definition of a PJI cure included infection eradication, no further surgical procedures, no PJI-related mortality and no suppressive antibiotics. Functional ambulatory status evaluated one year after the last surgery was classified into four simple categories: able to walk without assistance, able to walk with one crutch, able to walk with two crutches, and unable to walk. Patients with total hip arthroplasties (THA), total knee arthroplasties (TKA) and partial hip arthroplasty (PHA) were analysed separately., Results: A total of 109 PJI patients (38 TKA, 41 THA, 30 PHA) and 218 non-PJI patients were included. In a model adjusted for clinically relevant variables, PJI was associated with a higher risk of needing an assistive device for ambulation (vs. walking without aid) among THA (adjusted odds ratio (OR) 3.10, 95% confidence interval (95% CI) 1.26-7.57; p = 0.014) and TKA patients (OR 5.40, 95% CI 2.12-13.67; p < 0.001), and with requiring two crutches to walk or being unable to walk (vs. walking unaided or with one crutch) among PHA patients (OR 3.05, 95% CI 1.01-9.20; p = 0.047)., Conclusions: Ambulatory outcome in patients with hip and knee prostheses with postoperative PJI is worse than in patients who do not have PJI.

Aim: Gain insight about the role of DNA methylation in the malignant growth of colon cancer. Patients & methods: Methylation and gene expression from 90 adjacent-tumor paired tissues and 48 healthy tissues were analyzed. Tumor genes whose change in expression was explained by changes in methylation were identified using linear models adjusted for tumor stromal content. Results: No differences in methylation were found between adjacent and healthy tissues, but clear differences were found between adjacent and tumor samples. We identified hypermethylated CpG islands located in promoter regions that drive differential gene expression of transcription factors and their target genes. Conclusion: Changes in methylation of a few genes provoke important changes in gene expression, by expanding the signal through transcription activation/repression.

Klebsiella pneumoniae is the clinically most important species within the genus Klebsiella and, as a result of the continuous emergence of multi-drug resistant (MDR) strains, the cause of severe nosocomial infections. The decline in the effectiveness of antibiotic treatments for infections caused by MDR bacteria has generated particular interest in the study of bacteriophages. In this study, we characterized a total of 40 temperate bacteriophages (prophages) with a genome range of 11.454-84.199 kb, predicted from 16 carbapenemase-producing clinical strains of K. pneumoniae belonging to different sequence types, previously identified by multilocus sequence typing. These prophages were grouped into the three families in the order Caudovirales (27 prophages belonging to the family Myoviridae , 10 prophages belonging to the family Siphoviridae and 3 prophages belonging to the family Podoviridae ). Genomic comparison of the 40 prophage genomes led to the identification of four prophages isolated from different strains and of genome sizes of around 33.3, 36.1, 39.6 and 42.6 kb. These prophages showed sequence similarities (query cover >90 %, identity >99.9 %) with international Microbe Versus Phage (MVP) (http://mvp.medgenius.info/home) clusters 4762, 4901, 3499 and 4280, respectively. Phylogenetic analysis revealed the evolutionary proximity among the members of the four groups of the most frequently identified prophages in the bacterial genomes studied (33.3, 36.1, 39.6 and 42.6 kb), with bootstrap values of 100 %. This allowed the prophages to be classified into three clusters: A, B and C. Interestingly, these temperate bacteriophages did not infect the highest number of strains as indicated by a host-range assay, these results could be explained by the development of superinfection exclusion mechanisms. In addition, bioinformatic analysis of the 40 identified prophages revealed the presence of 2363 proteins. In total, 59.7 % of the proteins identified had a predicted function, mainly involving viral structure, transcription, replication and regulation (lysogenic/lysis). Interestingly, some proteins had putative functions associated with bacterial virulence (toxin expression and efflux pump regulators), phage defence profiles such as toxin-antitoxin modules, an anti-CRISPR/Cas9 protein, TerB protein (from ter ZABCDE operon) and methyltransferase proteins.

The emergence of multidrug resistant (MDR) pathogenic bacteria is jeopardizing the value of antimicrobials, which had previously changed the course of medical science. In this study, we identified endolysins ElyA1 and ElyA2 (GH108-PG3 family), present in the genome of bacteriophages Ab1051Φ and Ab1052Φ, respectively. The muralytic activity of these endolysins against MDR clinical isolates (Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae) was tested using the turbidity reduction assay. Minimal inhibitory concentrations (MICs) of endolysin, colistin and a combination of endolysin and colistin were determined, and the antimicrobial activity of each treatment was confirmed by time kill curves. Endolysin ElyA1 displayed activity against all 25 strains of A. baumannii and P. aeruginosa tested and against 13 out of 17 strains of K. pneumoniae. Endolysin ElyA2 did not display any such activity. The combined antimicrobial activity of colistin and ElyA1 yielded a reduction in the colistin MIC for all strains studied, except K. pneumoniae. These results were confirmed in vivo in G. mellonella survival assays and in murine skin and lung infection models. In conclusion, combining colistin (1/4 MIC) with the new endolysin ElyA1 (350 µg) enhanced the bactericidal activity of colistin in both in vitro and in vivo studies. This will potentially enable reduction of the dose of colistin used in clinical practice.

Background: Staphylococcus epidermidis is a commensal of human skin flora and a frequent causative microorganism in prosthetic joint infections (PJIs). To date, no single marker has been identified to distinguish infecting strains from commensal S. epidermidis populations., Aim: We aimed to find possible genetic markers to distinguish between the two populations., Methods: We analyzed 50 S. epidermidis strains from patients with PJIs, 50 from skin of healthy individuals (commensal strains) and 17 from the surgical field of patients undergoing primary arthroplasty. In these three groups we studied the antimicrobial susceptibility profile, sequence type, biofilm formation, and virulence factors. Strains from the surgical field have not been compared previously with strains from the other two groups., Findings: S. epidermidis strains from PJI patients were significantly more antibiotic resistant than commensal strains and surgical field strains. A wide variety of sequences types was found in commensal and surgical field strains. The predominant sequence type was ST2 and it was only present in PJI strains (44%). Differences in biofilm production did not differ between populations. Virulence genes sdrF and bhp, the complete ica operon, and the insertion sequence IS256 were significantly predominant in PJI strains. In contrast, embp and hld genes and the mobile element ACME were more prevalent in commensal strains. Surgical field strains could be a valid control group to discriminate between infecting and commensal strains., Conclusion: A combination of characteristic features can differentiate between infecting and commensal S. epidermidis strains in PJI, while a single marker cannot., Competing Interests: Declaration of Competing Interest None to declare., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

In this study, the plasmid content of clinical and commensal strains was analyzed and compared. The replicon profile was similar in both populations, except for L, M, A/C, and N (detected only in clinical strains) and HI1 (only in commensal strains). Although I1 and F were the most frequent replicons, only IncI1, sequence type 12 (ST12) was associated with bla CMY-2 in both populations. In contrast, the widespread resistant IncF plasmids were not linked to a single epidemic plasmid., (Copyright © 2020 American Society for Microbiology.)

Bacteriophages are abundant in human biomes and therefore in human clinical samples. Although this is usually not considered, they might interfere with the recovery of bacterial pathogens at two levels: 1) by propagating in the enrichment cultures used to isolate the infectious agent, causing the lysis of the bacterial host and 2) by the detection of bacterial genes inside the phage capsids that mislead the presence of the bacterial pathogen. To unravel these interferences, human samples (n = 271) were analyzed and infectious phages were observed in 11% of blood culture, 28% of serum, 45% of ascitic fluid, 14% of cerebrospinal fluid and 23% of urine samples. The genetic content of phage particles from a pool of urine and ascitic fluid samples corresponded to bacteriophages infecting different bacterial genera. In addition, many bacterial genes packaged in the phage capsids, including antibiotic resistance genes and 16S rRNA genes, were detected in the viromes. Phage interference can be minimized applying a simple procedure that reduced the content of phages up to 3 logs while maintaining the bacterial load. This method reduced the detection of phage genes avoiding the interference with molecular detection of bacteria and reduced the phage propagation in the cultures, enhancing the recovery of bacteria up to 6 logs.

Automated antimicrobial susceptibility testing devices are widely implemented in clinical microbiology laboratories in Spain, mainly using EUCAST (European Committee on Antimicrobial Susceptibility Testing) breakpoints. In 2007, a group of experts published recommendations for including antimicrobial agents and selecting concentrations in these systems. Under the patronage of the Spanish Antibiogram Committee (Comité Español del Antibiograma, COESANT) and the Study Group on Mechanisms of Action and Resistance to Antimicrobial Agents (GEMARA) from the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC), and aligned with the Spanish National Plan against Antimicrobial Resistance (PRAN), a group of experts have updated this document. The main modifications from the previous version comprise the inclusion of new antimicrobial agents, adaptation of the ranges of concentrations to cover the EUCAST breakpoints and epidemiological cut-off values (ECOFFs), and the inference of new resistance mechanisms. This proposal should be considered by different manufacturers and users when designing new panels or cards. In addition, recommendations for selective reporting are also included. With this approach, the implementation of EUCAST breakpoints will be easier, increasing the quality of antimicrobial susceptibility testing data and their microbiological interpretation. It will also benefit epidemiological surveillance studies as well as the clinical use of antimicrobials aligned with antimicrobial stewardship programs., (Copyright © 2019 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Campylobacter jejuni causes campylobacteriosis, a bacterial gastroenteritis with high incidence worldwide. Moreover, C. jejuni infection can trigger the polyneuropathic disorder denominated Guillain-Barré syndrome (GBS). The C. jejuni strains that can elicit GBS carry either wlaN or cgtB, coding both genes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide (LOS SIAL ). We described a differential prevalence of the genes wlaN and cgtB in C. jejuni isolates from three different ecological niches: humans, broiler chickens, and wild birds. The distribution of both genes, which is similar between broiler chicken and human isolates and distinct when compared to the wild bird isolates, suggests a host-dependent distribution. Moreover, the prevalence of the wlaN and cgtB genes seems to be restricted to some clonal complexes. Gene sequencing identified the presence of new variants of the G- homopolymeric tract within the wlaN gene. Furthermore, we detected two variants of a G rich region within the cgtB gene, suggesting that, similarly to wlaN , the G-tract in the cgtB gene mediates the phase variation control of cgtB expression. Caco-2 cell invasion assays indicate that there is no evident correlation between the production of LOS SIAL and the ability to invade eukaryotic cells.

Multimorbidity has great impact on health care. We constructed multimorbidity networks in the general population, extracted subnets focused on common chronic conditions and analysed type 2 diabetes mellitus (T2DM) comorbidity network. We used electronic records from 3,135,948 adult people in Catalonia, Spain (539,909 with T2DM), with at least 2 coexistent chronic conditions within the study period (2006-2017). We constructed networks from odds-ratio estimates adjusted by age and sex and considered connections with OR > 1.2 and p-value < 1e-5. Directed networks and trajectories were derived from temporal associations. Interactive networks are freely available in a website with the option to customize characteristics and subnets. The more connected conditions in T2DM undirected network were: complicated hypertension and atherosclerosis/peripheral vascular disease (degree: 32), cholecystitis/cholelithiasis, retinopathy and peripheral neuritis/neuropathy (degree: 31). T2DM has moderate number of connections and centrality but is associated with conditions with high scores in the multimorbidity network (neuropathy, anaemia and digestive diseases), and severe conditions with poor prognosis. The strongest associations from T2DM directed networks were to retinopathy (OR: 23.8), glomerulonephritis/nephrosis (OR: 3.4), peripheral neuritis/neuropathy (OR: 2.7) and pancreas cancer (OR: 2.4). Temporal associations showed the relevance of retinopathy in the progression to complicated hypertension, cerebrovascular disease, ischemic heart disease and organ failure.

Whole-genome sequencing (WGS)-based typing methods have emerged as promising and highly discriminative epidemiological tools. In this study, we combined gene-by-gene allele calling and core genome single nucleotide polymorphism (cgSNP) approaches to investigate the genetic relatedness of a well-characterized collection of OXA-48 - producing Klebsiella pneumoniae isolates. We included isolates from the predominant sequence type ST405 ( n = 31) OXA-48-producing K. pneumoniae clone and isolates from ST101 ( n = 3), ST14 ( n = 1), ST17 ( n = 1), and ST1233 ( n = 1), obtained from eight Catalan hospitals. Core-genome multilocus sequence typing (cgMLST) schemes from Institut Pasteur's BIGSdb-Kp (634 genes) and SeqSphere+ (2,365 genes), and a SeqSphere+ whole-genome MLST (wgMLST) scheme (4,891 genes) were used. Allele differences or allelic mismatches and the genetic distance, as the proportion of allele differences, were used to interpret the results from a gene-by-gene approach, whereas the number of SNPs was used for the cgSNP analysis. We observed between 0-10 and 0-14 allele differences among the predominant ST405 using cgMLST and wgMLST from SeqSphere+, respectively, and <2 allelic mismatches when using Institut Pasteur's BIGSdb-Kp cgMLST scheme. For ST101, we observed 14 and 54 allele differences when using cgMLST and wgMLST SeqSphere+, respectively, and 2-5 allelic mismatches for BIGSdb-Kp cgMLST. A low genetic distance (<0.0035, a previously established threshold for epidemiological link) was generally in concordance with a low number of allele differences (<8) when using the SeqSphere+ cgMLST scheme. The cgSNP analysis showed 6-29 SNPs in isolates with identical allelic SeqSphere+ cgMLST profiles and 16-61 cgSNPs among ST405 isolates. Furthermore, comparison of WGS-based typing results with previously obtained MLST and pulsed-field gel electrophoresis (PFGE) data showed some differences, demonstrating the different molecular principles underlying these techniques. In conclusion, the use of the different WGS-based typing methods that were used to elucidate the genetic relatedness of clonal OXA-48-producing K. pneumoniae all led to the same conclusions. Furthermore, threshold parameters in WGS-based typing methods should be applied with caution and should be used in combination with clinical epidemiological data and population and species characteristics., (Copyright © 2020 Miro, Rossen, Chlebowicz, Harmsen, Brisse, Passet, Navarro, Friedrich and García-Cobos.)