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Multidrug-resistant Enterococcus faecium strains represent a major concern due to their ability to thrive in diverse environments and cause life-threatening infections. While antimicrobial resistance and virulence mechanisms have been extensively studied, the contribution of bacteriocins to E. faecium 's adaptability remains poorly explored. E. faecium , within the Bacillota phylum, is a prominent bacteriocin producer. Here, we developed a tailored database of 76 Bacillota bacteriocins (217 sequences, including 40 novel bacteriocins) and applied it to uncover bacteriocin distribution patterns in 997 quality-filtered E. faecium and Enterococcus lactis (former E. faecium clade B) genomes. Curated using computational pipelines and literature mining, our database demonstrates superior precision versus leading public tools in identifying diverse bacteriocins. Distinct bacteriocin profiles emerged between E. faecium and E. lactis , highlighting species-specific adaptations. E. faecium strains from hospitalized patients were significantly enriched in bacteriocins as enterocin A and bacteriocins 43 (or T8), AS5, and AS11. These bacteriocin genes were strongly associated with antibiotic resistance, particularly vancomycin and ampicillin, and Inc18 rep 2_pRE25-derivative plasmids, classically associated with vancomycin resistance transposons. Such bacteriocin arsenal likely enhances the adaptability and competitive fitness of E. faecium in the nosocomial environment. By combining a novel tailored database, whole-genome sequencing, and epidemiological data, our work elucidates meaningful connections between bacteriocin determinants, antimicrobial resistance, mobile genetic elements, and ecological origins in E. faecium and provides a framework for elucidating bacteriocin landscapes in other organisms. Characterizing species- and strain-level differences in bacteriocin profiles may reveal determinants of ecological adaptation, and translating these discoveries could further inform strategies to exploit bacteriocins against high-risk clones., Importance: This work significantly expands the knowledge on the understudied bacteriocin diversity in opportunistic enterococci, revealing their contribution in the adaptation to different environments. It underscores the importance of placing increased emphasis on genetic platforms carrying bacteriocins as well as on cryptic plasmids that often exclusively harbor bacteriocins since bacteriocin production can significantly contribute to plasmid maintenance, potentially facilitating their stable transmission across generations. Further characterization of strain-level bacteriocin landscapes could inform strategies to combat high-risk clones. Overall, these insights provide a framework for unraveling the therapeutic and biotechnological potential of bacteriocins., Competing Interests: The authors declare no conflict of interest.

Serratia marcescens is an opportunistic pathogen historically associated with sudden outbreaks in intensive care units (ICUs) and the spread of carbapenem-resistant genes. However, the ecology of S. marcescens populations in the hospital ecosystem remains largely unknown. We combined epidemiological information of 1,432 Serratia spp. isolates collected from sinks of a large ICU that underwent demographic and operational changes (2019-2021) and 99 non-redundant outbreak/non-outbreak isolates from the same hospital (2003-2019) with 165 genomic data. These genomes were grouped into clades (1-4) and subclades (A and B) associated with distinct species: Serratia nematodiphila (1A), S. marcescens (1B), Serratia bockelmannii (2A), Serratia ureilytica (2B), S. marcescens / Serratia nevei (3), and S. nevei (4A and 4B). They may be classified into an S. marcescens complex (SMC) due to the similarity between/within subclades (average nucleotide identity >95%-98%), with clades 3 and 4 predominating in our study and publicly available databases. Chromosomal AmpC β-lactamase with unusual basal-like expression and prodigiosin-lacking species contrasted classical features of Serratia . We found persistent and coexisting clones in sinks of subclades 4A (ST92 and ST490) and 4B (ST424), clonally related to outbreak isolates carrying bla VIM-1 or bla OXA-48 on prevalent IncL/pB77-CPsm plasmids from our hospital since 2017. The distribution of SMC populations in ICU sinks and patients reflects how Serratia species acquire, maintain, and enable plasmid evolution in both "source" (permanent, sinks) and "sink" (transient, patients) hospital patches. The results contribute to understanding how water sinks serve as reservoirs of Enterobacterales clones and plasmids that enable the persistence of carbapenemase genes in healthcare settings, potentially leading to outbreaks and/or hospital-acquired infections.IMPORTANCEThe "hospital environment," including sinks and surfaces, is increasingly recognized as a reservoir for bacterial species, clones, and plasmids of high epidemiological concern. Available studies on Serratia epidemiology have focused mainly on outbreaks of multidrug-resistant species, overlooking local longitudinal analyses necessary for understanding the dynamics of opportunistic pathogens and antibiotic-resistant genes within the hospital setting. This long-term genomic comparative analysis of Serratia isolated from the ICU environment with isolates causing nosocomial infections and/or outbreaks within the same hospital revealed the coexistence and persistence of Serratia populations in water reservoirs. Moreover, predominant sink strains may acquire highly conserved and widely distributed plasmids carrying carbapenemase genes, such as the prevalent IncL-pB77-CPsm (pOXA48), persisting in ICU sinks for years. The work highlights the relevance of ICU environmental reservoirs in the endemicity of certain opportunistic pathogens and resistance mechanisms mainly confined to hospitals., Competing Interests: The authors declare no conflict of interest.

Background: The implications of the gut microbial communities in the immune response against parasites and gut motility could explain the differences in clinical manifestations and treatment responses found in patients with chronic Chagas disease., Methodology/principal Findings: In this pilot prospective cross-sectional study, we included 80 participants: 29 with indeterminate CD (ICD), 16 with cardiac CD (CCD), 15 with digestive CD (DCD), and 20 controls without CD. Stool was collected at the baseline visit and faecal microbial community structure DNA was analyzed by whole genome sequencing. We also performed a comprehensive dietary analysis. Ninety per cent (72/80) of subjects were of Bolivian origin with a median age of 47 years (IQR 39-54) and 48.3% (29/60) had received benznidazole treatment. There were no substantial differences in dietary habits between patients with CD and controls. We identified that the presence or absence of CD explained 5% of the observed microbiota variability. Subjects with CD exhibited consistent enrichment of Parabacteroides spp, while for Enterococcus hirae, Lactobacillus buchneri and Megamonas spp, the effect was less clear once excluded the outliers values. Sex, type of visceral involvement and previous treatment with benznidazole did not appear to have a confounding effect on gut microbiota structure. We also found that patients with DCD showed consistent Prevotella spp enrichment., Conclusions: We found a detectable effect of Chagas disease on overall microbiota structure with several potential disease biomarkers, which warrants further research in this field. The analysis of bacterial diversity could prove to be a viable target to improve the prognosis of this prevalent and neglected disease., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Pérez-Molina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Extrachromosomal elements of bacterial cells such as plasmids are notorious for their importance in evolution and adaptation to changing ecology. However, high-resolution population-wide analysis of plasmids has only become accessible recently with the advent of scalable long-read sequencing technology. Current typing methods for the classification of plasmids remain limited in their scope which motivated us to develop a computationally efficient approach to simultaneously recognize novel types and classify plasmids into previously identified groups. Here, we introduce mge-cluster that can easily handle thousands of input sequences which are compressed using a unitig representation in a de Bruijn graph. Our approach offers a faster runtime than existing algorithms, with moderate memory usage, and enables an intuitive visualization, classification and clustering scheme that users can explore interactively within a single framework. M ge-cluster platform for plasmid analysis can be easily distributed and replicated, enabling a consistent labelling of plasmids across past, present, and future sequence collections. We underscore the advantages of our approach by analysing a population-wide plasmid data set obtained from the opportunistic pathogen Escherichia coli , studying the prevalence of the colistin resistance gene mcr-1.1 within the plasmid population, and describing an instance of resistance plasmid transmission within a hospital environment., (© The Author(s) 2023. Published by Oxford University Press on behalf of NAR Genomics and Bioinformatics.)

Klebsiella pneumoniae sequence type 14 (ST14) and ST15 caused outbreaks of CTX-M-15 and/or carbapenemase producers worldwide, but their phylogeny and global dynamics remain unclear. We clarified the evolution of K. pneumoniae clonal group 14 (CG14) and CG15 by analyzing the capsular locus (KL), resistome, virulome, and plasmidome of public genomes ( n  = 481) and de novo sequences ( n  = 9) representing main sublineages circulating in Portugal. CG14 and CG15 evolved independently within 6 main subclades defined according to the KL and the accessory genome. The CG14 ( n  = 65) clade was structured in two large monophyletic subclades, CG14-I (KL2, 86%) and CG14-II (KL16, 14%), whose emergences were dated to 1932 and 1911, respectively. Genes encoding extended-spectrum β-lactamase (ESBL), AmpC, and/or carbapenemases were mostly observed in CG14-I (71% versus 22%). CG15 clade ( n  = 170) was segregated into subclades CG15-IA (KL19/KL106, 9%), CG15-IB (variable KL types, 6%), CG15-IIA (KL24, 43%) and CG15-IIB (KL112, 37%). Most CG15 genomes carried specific GyrA and ParC mutations and emerged from a common ancestor in 1989. CTX-M-15 was especially prevalent in CG15 (68% CG15 versus 38% CG14) and in CG15-IIB (92%). Plasmidome analysis revealed 27 predominant plasmid groups (PG), including particularly pervasive and recombinant F-type ( n  = 10), Col ( n  = 10), and new plasmid types. While bla CTX-M-15 was acquired multiple times by a high diversity of F-type mosaic plasmids, other antibiotic resistance genes (ARGs) were dispersed by IncL ( bla OXA-48 ) or IncC ( bla CMY/TEM-24 ) plasmids. We first demonstrate an independent evolutionary trajectory for CG15 and CG14 and how the acquisition of specific KL, quinolone-resistance determining region (QRDR) mutations (CG15), and ARGs in highly recombinant plasmids could have shaped the expansion and diversification of particular subclades (CG14-I and CG15-IIA/IIB). IMPORTANCE Klebsiella pneumoniae represents a major threat in the burden of antibiotic resistance (ABR). Available studies to explain the origin, the diversity, and the evolution of certain ABR K. pneumoniae populations have mainly been focused on a few clonal groups (CGs) using phylogenetic analysis of the core genome, the accessory genome being overlooked. Here, we provide unique insights into the phylogenetic evolution of CG14 and CG15, two poorly characterized CGs which have contributed to the global dissemination of genes responsible for resistance to first-line antibiotics such as β-lactams. Our results point out an independent evolution of these two CGs and highlight the existence of different subclades structured by the capsular type and the accessory genome. Moreover, the contribution of a turbulent flux of plasmids (especially multireplicon F type and Col) and adaptive traits (antibiotic resistance and metal tolerance genes) to the pangenome reflect the exposure and adaptation of K. pneumoniae under different selective pressures., Competing Interests: The authors declare no conflict of interest.

Aim: To identify alterations in steroid metabolism in patients with nonfunctioning adrenal incidentalomas (NFAIs) through the analysis of their urinary steroid profile (USP)., Methods: Cross-sectional study with one study group (NFAIs, cortisol post dexamethasone suppression test [DST] ≤ 1.8 µg/dl [49.7 nmol/L]) and 2 control groups: patients with autonomous cortisol secretion (ACS group, cortisol post-DST > 1.8 µg/dl (49.7 nmol/L) and patients without adrenal tumours (healthy-adrenal group). Twenty-four-hour urine collections for USP measurement (total and free fraction of 51 24 h-urine specimens) were obtained from 73 participants (24 with NFAIs, 24 without AIs, and 25 with ACS). USP was determined by gas chromatography coupled to mass spectrometry. Patients of the three groups were matched according to sex, age (±5 years-old) and body mass index (±5 kg/m 2 )., Results: Compared to healthy-adrenal controls, patients with NFAIs had a lower excretion of androgen metabolites (230.5 ± 190.12 vs. 388.7 ± 328.58 µg/24 h, p = .046) and a higher excretion of urinary free cortisol (UFC) (54.3 ± 66.07 vs. 25.4 ± 11.16 µg/24 h, p = .038). UFC was above the reference range in 20.8% of patients in the NFAI, compared to 0% in the healthy-adrenal group (p = .018). Patients with ACS had a higher prevalence of hypertension, dyslipidemia, and diabetes than patients with NFAIs or the control group. A lower excretion of androgen metabolites (218.4 ± 204.24 vs. 231 ± 190 µg/24 h, p = .041) and a nonsignificant higher excretion of glucocorticoid metabolites (2129.6 ± 1195.96 vs. 1550.8 ± 810.03 µg/24 h, p = .180) was found in patients with ACS compared to patients with NFAIs., Conclusion: NFAIs seem to secrete a subtle, yet clinically relevant, excess of glucocorticoids. Future studies are needed to confirm our findings; and to identify metabolic alterations associated with an increased cardiometabolic risk., (© 2022 John Wiley & Sons Ltd.)

Background: Patients with acute myocardial infarction (MI) are at high risk of upcoming events, in particular heart failure (HF), but reliable stratification methods are lacking. Our goal was to evaluate the potential role of circulating miRNAs as prognostic biomarkers in patients presenting with MI., Methods and Results: We conducted a prospective study among 311 consecutive patients hospitalized with MI (65% ST-segment elevation MI & median age of 55 years) with long-term follow-up. An initial screening was conducted to select candidate miRNAs, with subsequent study of 14 candidate miRNAs. The primary outcome was the composite of hospital admission for HF or cardiovascular death. During a mean follow-up of 2.1 years miR-21-5p, miR-23a-3p, miR27b-3p, miR-122-5p, miR210-3p, and miR-221-3p reliably predicted the primary outcome. Multivariate Cox regression analyses highlighted that miR-210-3p [hazard ratio (HR) 2.65 per 1 SD increase, P < 0.001], miR-23a-3p (HR 2.11 per 1 SD increase, P < 0.001), and miR-221-3p (HR 2.03 per 1 SD increase, P < 0.001) were able to accurately predict the primary outcome, as well as cardiovascular death, HF hospitalizations, and long-term New York Heart Association (NYHA) functional class. These three miRNAs clearly improved the performance of multivariate clinical models: ΔC-statistic = 0.10 [95% confidence interval (CI), 0.03-0.17], continuous net reclassification index = 34.8% (95%CI, 5.8-57.4%), and integrated discrimination improvement (P < 0.001)., Conclusions: This is the largest study evaluating the prognostic value of circulating miRNAs for HF-related events among patients with MI. We show that several miRNAs predict HF hospitalizations, cardiovascular mortality, and poor long-term NYHA status and improve current risk prediction methods., (© 2022 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

This work aimed to evaluate the predatory activity of Bdellovibrio bacteriovorus 109J on clinical isolates of Pseudomonas aeruginosa selected from well-characterized collections of cystic fibrosis (CF) lung colonization (n = 30) and bloodstream infections (BSI) (n = 48) including strains selected by genetic lineage (frequent and rare sequence types), antibiotic resistance phenotype (susceptible and multidrug-resistant isolates), and colony phenotype (mucoid and non-mucoid isolates). The intraspecies predation range (I-PR) was defined as the proportion of susceptible strains within the entire collection. In contrast, the predation efficiency (PE) is the ratio of viable prey cells remaining after predation compared to the initial inoculum. I-PR was significantly higher for CF (67%) than for BSI P. aeruginosa isolates (35%) probably related to an environmental origin of CF strains whereas invasive strains are more adapted to humans. I-PR correlation with bacterial features such as mucoid morphotype, genetic background, or antibiotic susceptibility profile was not detected. To test the possibility of increasing I-PR of BSI isolates, a polyhydroxyalkanoate depolymerase deficient B. bacteriovorus bd2637 mutant was used. Global median I-PR and PE values remained constant for both predators, but 31.2% of 109J-resistant isolates were susceptible to the mutant, and 22.9% of 109J-susceptible isolates showed resistance to predation by the mutant, pointing to a predator-prey specificity process. The potential use of predators in the clinical setting should be based on the determination of the I-PR for each species, and the PE of each particular target strain., (© 2022. The Author(s).)

Healthy development of the gut microbiome provides long-term health benefits. Children raised in countries with high infectious disease burdens are frequently exposed to diarrhoeal pathogens and antibiotics, which perturb gut microbiome assembly. A recent cluster-randomized trial leveraging >4,000 child observations in Dhaka, Bangladesh, found that automated water chlorination of shared taps effectively reduced child diarrhoea and antibiotic use. In this substudy, we leveraged stool samples collected from 130 children 1 year after chlorine doser installation to examine differences between treatment and control children's gut microbiota. Water chlorination was associated with increased abundance of several bacterial genera previously linked to improved gut health; however, we observed no effects on the overall richness or diversity of taxa. Several clinically relevant antibiotic resistance genes were relatively more abundant in the gut microbiome of treatment children, possibly due to increases in Enterobacteriaceae. While further studies on the long-term health impacts of drinking chlorinated water would be valuable, we conclude that access to chlorinated water did not substantially impact child gut microbiome development in this setting, supporting the use of chlorination to increase global access to safe drinking water., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

While changes in microbiome composition have been associated with HIV, the effect of diet and its potential impact on inflammation remains unclear. Methods: Twenty-seven people living with HIV (PWH) on antiretroviral therapy (ART) were studied. A comprehensive dietary analysis was performed and two types of dietary patterns were determined. We explored the associations of each dietary pattern with gut microbiota and plasma inflammatory biomarkers. Results: We appreciated two dietary patterns, Mediterranean-like (MEL) and one Western-like (WEL). Compared to participants with the WEL pattern, participants with MEL pattern showed higher abundance of Lachnospira (p-value = 0.02) and lower levels of the inflammatory biomarkers D-dimer (p-value = 0.050) and soluble TNF-alpha receptor 2 (sTNFR2) (p-value = 0.049). Men who have sex with men (MSM) with MEL pattern had lower abundance of Erysipelotrichaceae (p-value < 0.001) and lower levels of D-dimer (p-value = 0.026) than MSM with WEL pattern. Conclusion: MEL pattern favours Lachnospira abundance, and protects against Erysipelotrichaceae abundance and higher levels of the inflammatory biomarkers D-dimer and sTNFR2, precursors of inflammatory processes in HIV-infected patients. Our study contributes to understanding the determinants of a healthier diet and its connections with gut microbiota and inflammation.

Detection of mixed Mycobacterium tuberculosis (MTB) infections is essential, particularly when resistance mutations are present in minority bacterial populations that may affect patients' disease evolution and treatment. Whole-genome sequencing (WGS) has extended the amount of key information available for the diagnosis of MTB infection, including the identification of mixed infections. Having genomic information at diagnosis for early intervention requires carrying out WGS directly on the clinical samples. However, few studies have been successful with this approach due to the low representation of MTB DNA in sputa. In this study, we evaluated the ability of a strategy based on specific MTB DNA enrichment by using a newly designed capture platform (MycoCap) to detect minority variants and mixed infections by WGS on controlled mixtures of MTB DNAs in a simulated sputum genetic background. A pilot study was carried out with 12 samples containing 98% of a DNA pool from sputa of patients without MTB infection and 2% of MTB DNA mixtures at different proportions. Our strategy allowed us to generate sequences with a quality equivalent to those obtained from culture: 62.5× depth coverage and 95% breadth coverage (for at least 20× reads). Assessment of minority variant detection was carried out by manual analysis and allowed us to identify heterozygous positions up to a 95:5 ratio. The strategy also automatically distinguished mixed infections up to a 90:10 proportion. Our strategy efficiently captures MTB DNA in a nonspecific genetic background, allows detection of minority variants and mixed infections, and is a promising tool for performing WGS directly on clinical samples. IMPORTANCE We present a new strategy to identify mixed infections and minority variants in Mycobacterium tuberculosis by whole-genome sequencing. The objective of the strategy is the direct detection in patient sputum; in this way, minority populations of resistant strains can be identified at the time of diagnosis, facilitating identification of the most appropriate treatment for the patient from the first moment. For this, a platform for capturing M. tuberculosis-specific DNA was designed to enrich the clinical sample and obtain quality sequences.

This is a longitudinal study comprising 649 Escherichia coli isolates representing all 7,165 E. coli bloodstream infection (BSI) episodes recorded in a hospital (1996 to 2016). Strain analysis included clonal identification (phylogenetic groups/subgroups, STc131 subclades, pulsed-field gel electrophoresis [PFGE], and whole-genome sequencing [WGS]), antibiotic susceptibility (13 antibiotics), and virulence-associated genes (VAGs; 29 genes). The incidence of E. coli BSI increased from 1996 to 2016 (5.5 to 10.8 BSI episodes/1,000 hospitalizations, average 7 to 8/1,000). B2 isolates predominate (53%), with subgroups B2-I (STc131), B2-II, B2-IX, and B2-VI representing 25%, 25%, 14%, and 9%, respectively. Intertwined waves of community-acquired (CA) plus health care-associated and community-onset health care-associated (HCA) and hospital-acquired (HA) episodes of both B2 and non-B2 phylogroups occurred. A remarkable increase was observed only for B2-I-STc131 (C1/C2 subclades), with oscillations for other B2 subgroups and phylogroups throughout the years. Epidemic and persistent clones (comprising isolates with highly similar/identical PFGE types and genomes differing in 6 to 173 single nucleotide polymorphisms [SNPs]) of B2-I (STc131), B2-II (STc73), B2-III (STc127), B2-IX (STc95), and B2-VI (STc12) were recovered from different patients, most at hospital admission, for long periods (2 to 17 years), and extended-spectrum beta-lactamase (ESBL) producers or resistance to ciprofloxacin in B2 isolates was almost restricted to B2-I (STc131) subclade C. STc131 contributed to increasing the B2 rates but only transiently altered the E. coli population structure. The increase of E. coli BSI was determined by waves of CA+HCA BSI episodes that predate the waves of HA BSI. Besides the risk of hospital transmission that led to temporal increases in BSI, this study suggests that E. coli populations/clones from community-based healthy individuals may occasionally have an epidemic structure and provide a source of transmissible strains influencing the HA BSI incidence. IMPORTANCE Sepsis is the third leading cause of mortality in Western countries and one of the Global Health Threats recognized by the WHO since 2017. Despite Escherichia coli constituting the most common cause of bloodstream infections (BSI), its epidemiology is not fully understood, in part due to the scarcity of local and longitudinal studies. Our work analyzes the long-term dynamics of E. coli causing bacteremia in a single institution and reveals waves of different clonal lineages that emerge periodically and successfully spread afterward in both the community and hospitals. Because the origin of E. coli bloodstream infections is the gut, the microbiota of healthy individuals might occasionally have an epidemic structure, providing a source of E. coli strains to influence the incidence of hospital BSI. The study complements previous fractionated observations focusing on specific E. coli lineages or antibiotic-resistant isolates in the last decades and helps to understand the epidemiology of E. coli BSI and the dynamics of pandemic clones.

Motivation: We present the Pangenome Analysis Toolkit (PATO) designed to simultaneously analyze thousands of genomes using a desktop computer. The tool performs common tasks of pangenome analysis such as core-genome definition and accessory genome properties and includes new features that help characterize population structure, annotate pathogenic features and create gene sharedness networks. PATO has been developed in R to integrate with the large set of tools available for genetic, phylogenetic and statistical analysis in this environment., Results: PATO can perform the most demanding bioinformatic analyses in minutes with an accuracy comparable to state-of-the-art software but 20-30× times faster. PATO also integrates all the necessary functions for the complete analysis of the most common objectives in microbiology studies. Finally, PATO includes the necessary tools for visualizing the results and can be integrated with other analytical packages available in R., Availabilityand Implementation: The source code for PATO is freely available at https://github.com/irycisBioinfo/PATO under the GPLv3 license., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Background: Vancomycin resistance is mostly associated with Enterococcus faecium due to Tn1546-vanA located on narrow- and broad-host plasmids of various families. This study's aim was to analyse the effects of acquiring Tn1546-carrying plasmids with proven epidemicity in different bacterial host backgrounds., Methods: Widespread Tn1546-carrying plasmids of different families RepA&#95;N (n = 5), Inc18 (n = 4) and/or pHTβ (n = 1), and prototype plasmids RepA&#95;N (pRUM) and Inc18 (pRE25, pIP501) were analysed. Plasmid transferability and fitness cost were assessed using E. faecium (GE1, 64/3) and Enterococcus faecalis (JH2-2/FA202/UV202) recipient strains. Growth curves (Bioscreen C) and Relative Growth Rates were obtained in the presence/absence of vancomycin. Plasmid stability was analysed (300 generations). WGS (Illumina-MiSeq) of non-evolved and evolved strains (GE1/64/3 transconjugants, n = 49) was performed. SNP calling (Breseq software) of non-evolved strains was used for comparison., Results: All plasmids were successfully transferred to different E. faecium clonal backgrounds. Most Tn1546-carrying plasmids and Inc18 and RepA&#95;N prototypes reduced host fitness (-2% to 18%) while the cost of Tn1546 expression varied according to the Tn1546-variant and the recipient strain (9%-49%). Stability of Tn1546-carrying plasmids was documented in all cases, often with loss of phenotypic resistance and/or partial plasmid deletions. SNPs and/or indels associated with essential bacterial functions were observed on the chromosome of evolved strains, some of them linked to increased fitness., Conclusions: The stability of E. faecium Tn1546-carrying plasmids in the absence of selective pressure and the high intra-species conjugation rates might explain the persistence of vancomycin resistance in E. faecium populations despite the significant burden they might impose on bacterial host strains., (© The Author(s) 2021. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The adaptation to the different biotic and abiotic factors of wine fermentation has led to the accumulation of numerous genomic hallmarks in Saccharomyces cerevisiae wine strains. IRC7, a gene encoding a cysteine-S-β-lyase enzyme related volatile thiols production in wines, has two alleles: a full-length allele (IRC7 F ) and a mutated one (IRC7 S ), harbouring a 38 bp-deletion. Interestingly, IRC7 S -encoding a less active enzyme - appears widespread amongst wine populations. Studying the global distribution of the IRC7 S allele in different yeast lineages, we confirmed its high prevalence in the Wine clade and demonstrated a minority presence in other domesticated clades (Wine-PDM, Beer and Bread) while it is completely missing in wild clades. Here, we show that IRC7 S -homozygous (HS) strains exhibited both fitness and competitive advantages compared with IRC7 F -homozygous (HF) strains. There are some pieces of evidence of the direct contribution of the IRC7 S allele to the outstanding behaviour of HS strains (i.e., improved response to oxidative stress conditions and higher tolerance to high copper levels); however, we also identified a set of sequence variants with significant co-occurrence patterns with the IRC7 S allele, which can be co-contributing to the fitness and competitive advantages of HS strains in wine fermentations., (© 2021 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

Enterococcus faecalis is a commensal and nosocomial pathogen, which is also ubiquitous in animals and insects, representing a classical generalist microorganism. Here, we study E. faecalis isolates ranging from the pre-antibiotic era in 1936 up to 2018, covering a large set of host species including wild birds, mammals, healthy humans, and hospitalised patients. We sequence the bacterial genomes using short- and long-read techniques, and identify multiple extant hospital-associated lineages, with last common ancestors dating back as far as the 19th century. We find a population cohesively connected through homologous recombination, a metabolic flexibility despite a small genome size, and a stable large core genome. Our findings indicate that the apparent hospital adaptations found in hospital-associated E. faecalis lineages likely predate the "modern hospital" era, suggesting selection in another niche, and underlining the generalist nature of this nosocomial pathogen.

Changes in the microbiota have been linked to persistent inflammation during treated HIV infection. In this pilot double-blind study, we study 30 HIV-infected subjects on antiretroviral therapy (ART) with a CD4/CD8 ratio < 1 randomized to either weekly fecal microbiota capsules or placebo for 8 weeks. Stool donors were rationally selected based on their microbiota signatures. We report that fecal microbiota transplantation (FMT) is safe, not related to severe adverse events, and attenuates HIV-associated dysbiosis. FMT elicits changes in gut microbiota structure, including significant increases in alpha diversity, and a mild and transient engraftment of donor's microbiota during the treatment period. The greater engraftment seems to be achieved by recent antibiotic use before FMT. The Lachnospiraceae and Ruminococcaceae families, which are typically depleted in people with HIV, are the taxa more robustly engrafted across time-points. In exploratory analyses, we describe a significant amelioration in the FMT group in intestinal fatty acid-binding protein (IFABP), a biomarker of intestinal damage that independently predicts mortality. Gut microbiota manipulation using a non-invasive and safe strategy of FMT delivery is feasible and deserves further investigation. Trial number: NCT03008941.

Bacterial plasmids harboring antibiotic resistance genes are critical in the spread of antibiotic resistance. It is known that plasmids differ in their kinetic values, i.e., conjugation rate, segregation rate by copy number incompatibility with related plasmids, and rate of stochastic loss during replication. They also differ in cost to the cell in terms of reducing fitness and in the frequency of compensatory mutations compensating plasmid cost. However, we do not know how variation in these values influences the success of a plasmid and its resistance genes in complex ecosystems, such as the microbiota. Genes are in plasmids, plasmids are in cells, and cells are in bacterial populations and microbiotas, which are inside hosts, and hosts are in human communities at the hospital or the community under various levels of cross-colonization and antibiotic exposure. Differences in plasmid kinetics might have consequences on the global spread of antibiotic resistance. New membrane computing methods help to predict these consequences. In our simulation, conjugation frequency of at least 10 -3 influences the dominance of a strain with a resistance plasmid. Coexistence of different antibiotic resistances occurs if host strains can maintain two copies of similar plasmids. Plasmid loss rates of 10 -4 or 10 -5 or plasmid fitness costs of ≥0.06 favor plasmids located in the most abundant species. The beneficial effect of compensatory mutations for plasmid fitness cost is proportional to this cost at high mutation frequencies (10 -3 to 10 -5 ). The results of this computational model clearly show how changes in plasmid kinetics can modify the entire population ecology of antibiotic resistance in the hospital setting., (Copyright © 2020 Campos et al.)

Linezolid-resistant Enterococcus faecalis (LREfs) carrying optrA are increasingly reported globally from multiple sources, but we lack a comprehensive analysis of human and animal optrA -LREfs strains. To assess if optrA is dispersed in isolates with varied genetic backgrounds or with common genetic features, we investigated the phylogenetic structure, genetic content [antimicrobial resistance (AMR), virulence, prophages, plasmidome] and optrA -containing platforms of 27 publicly available optrA -positive E. faecalis genomes from different hosts in seven countries. At the genome-level analysis, an in-house database with 64 virulence genes was tested for the first time. Our analysis showed a diversity of clones and adaptive gene sequences related to a wide range of genera from Firmicutes . Phylogenies of core and accessory genomes were not congruent, and at least PAI-associated and prophage genes contribute to such differences. Epidemiologically unrelated clones (ST21, ST476-like and ST489) obtained from human clinical and animal hosts in different continents over eight years (2010-2017) could be phylogenetically related (3-126 SNPs difference). optrA was located on the chromosome within a Tn 6674 -like element ( n =10) or on medium-size plasmids (30-60 kb; n =14) belonging to main plasmid families (RepA&#95;N/Inc18/Rep&#95;3). In most cases, the immediate gene vicinity of optrA was generally identical in chromosomal (Tn 6674 ) or plasmid ( impB-fexA-optrA ) backbones. Tn 6674 was always inserted into the same ∆ radC integration site and embedded in a 32 kb chromosomal platform common to strains from different origins (patients, healthy humans, and animals) in Europe, Africa, and Asia during 2012-2017. This platform is conserved among hundreds of E. faecalis genomes and proposed as a chromosomal hotspot for optrA integration. The finding of optrA in strains sharing common adaptive features and genetic backgrounds across different hosts and countries suggests the occurrence of common and independent genetic events occurring in distant regions and might explain the easy de novo generation of optrA -positive strains. It also anticipates a dramatic increase of optrA carriage and spread with a serious impact on the efficacy of linezolid for the treatment of Gram-positive infections.

The first documented nosocomial outbreak caused by Serratia marcescens in Spain occurred in 1969 at the neonatal intensive care unit (NICU) of the tertiary La Paz Children's Hospital in Madrid, Spain, and based on the available phenotyping techniques at this time, it was considered as a monoclonal outbreak. Only 47 years later, another S. marcescens outbreak of an equivalent dimension occurred at the same NICU. The aim of the present study was to study isolates from these historical and contemporary outbreaks by phenotypic analysis and whole-genome sequencing techniques and to position these strains along with 444 publicly available S. marcescens genomes, separately comparing core genome and accessory genome contents. Clades inferred by both approaches showed high correlation, indicating that core and accessory genomes seem to evolve in the same manner for S. marcescens. Nine S. marcescens clusters were identified, and isolates were grouped in two of them according to sampling year. One exception was isolate 13F-69, the most genetically distant strain, located in a different cluster. Categorical functions in the annotated accessory genes of both collections were preserved among all isolates. No significant differences in frequency of insertion sequences in historical (0.18-0.20)-excluding the outlier strain-versus contemporary isolates (0.11-0.19) were found despite the expected resting effect. The most dissimilar isolate, 13F-69, contains a highly preserved plasmid previously described in Bordetella bronchiseptica. This strain exhibited a few antibiotic resistance genes not resulting in a resistant phenotype, suggesting the value of gene down expression in adaptation to long-term starvation., (Copyright © 2020 Saralegui, Ponce-Alonso, Pérez-Viso, Moles Alegre, Escribano, Lázaro-Perona, Lanza, de Pipaón, Rodríguez, Baquero and del Campo.)

Escherichia coli is present in multiple hosts and environmental compartments as a normal inhabitant, temporary or persistent colonizer, and as a pathogen. Transmission of E. coli between hosts and with the environment is considered to occur more often in areas with poor sanitation. We performed whole-genome comparative analyses on 60 E. coli isolates from soils and fecal sources (cattle, chickens, and humans) in households in rural Bangladesh. Isolates from household soils were in multiple branches of the reconstructed phylogeny, intermixed with isolates from fecal sources. Pairwise differences between all strain pairs were large (minimum, 189 single nucleotide polymorphisms [SNPs]), suggesting high diversity and heterogeneous origins of the isolates. The presence of multiple virulence and antibiotic resistance genes is indicative of the risk that E. coli from soil and feces represent for the transmission of variants that pose potential harm to people. Analysis of the accessory genomes of the Bangladeshi E. coli relative to E. coli genomes available in NCBI identified a common pool of accessory genes shared among E. coli isolates in this geographic area. Together, these findings indicate that in rural Bangladesh, a high level of E. coli in soil is likely driven by contributions from multiple and diverse E. coli sources (human and animal) that share an accessory gene pool relatively unique to previously published E. coli genomes. Thus, interventions to reduce environmental pathogen or antimicrobial resistance transmission should adopt integrated One Health approaches that consider heterogeneous origins and high diversity to improve effectiveness and reduce prevalence and transmission. IMPORTANCE Escherichia coli is reported in high levels in household soil in low-income settings. When E. coli reaches a soil environment, different mechanisms, including survival, clonal expansion, and genetic exchange, have the potential to either maintain or generate E. coli variants with capabilities of causing harm to people. In this study, we used whole-genome sequencing to identify that E. coli isolates collected from rural Bangladeshi household soils, including pathogenic and antibiotic-resistant variants, are diverse and likely originated from multiple diverse sources. In addition, we observed specialization of the accessory genome of this Bangladeshi E. coli compared to E. coli genomes available in current sequence databases. Thus, to address the high level of pathogenic and antibiotic-resistant E. coli transmission in low-income settings, interventions should focus on addressing the heterogeneous origins and high diversity., (Copyright © 2020 Montealegre et al.)

Mobile Genetic Elements (MGE) play essential roles in adaptive bacterial evolution, facilitating genetic exchange for extrachromosomal DNA, especially antibiotic resistance genes and virulence factors. For this reason, high-throughput next-generation sequencing of bacteria is of great relevance, especially for clinical pathogenic bacteria. Accurate identification of MGE from whole-genome sequencing (WGS) datasets is one of the major challenges, still hindered by methodological limitations and high sequencing costs.This chapter encompasses the protocol used for plasmid reconstruction by applying the PLACNETw methodology, from raw reads to assembled plasmids and chromosome. PLACNETw is a graphical user-friendly interface to visualize and reconstruct MGE from short-read WGS datasets. No bioinformatic background or sophisticated computational resources are required and high precision and sensitivity are achieved.

AcCNET (Accessory genome Constellation Network) is a Perl application to determine the variation of content in any genomic unit (e.g., bacterial genomes, plasmids, and transposons) within large datasets. It provides functional information that can be handled and visualized interactively as a bipartite network. In this chapter, we explain how to perform a statistical analysis of AcCNET data.

Antibiotic resistance is a field in which the concept of One Health can best be illustrated. One Health is based on the definition of communication spaces among diverse environments. Antibiotic resistance is encoded by genes, however, these genes are propagated in mobile genetic elements (MGEs), circulating among bacterial species and clones that are integrated into the multiple microbiotas of humans, animals, food, sewage, soil, and water environments, the One Health microbiosphere. The dynamics and evolution of antibiotic resistance depend on the communication networks linking all these ecological, biological, and genetic entities. These communications occur by environmental overlapping and merging, a critical issue in countries with poor sanitation, but also favored by the homogenizing power of globalization. The overwhelming increase in the population of highly uniform food animals has contributed to the parallel increase in the absolute size of their microbiotas, consequently enhancing the possibility of microbiome merging between humans and animals. Microbial communities coalescence might lead to shared microbiomes in which the spread of antibiotic resistance (of human, animal, or environmental origin) is facilitated. Intermicrobiome communication is exerted by shuttle bacterial species (or clones within species) belonging to generalist taxa, able to multiply in the microbiomes of various hosts, including humans, animals, and plants. Their integration into local genetic exchange communities fosters antibiotic resistance gene flow, following the channels of accessory genome exchange among bacterial species. These channels delineate a topology of gene circulation, including dense clusters of species with frequent historical and recent exchanges. The ecological compatibility of these species, sharing the same niches and environments, determines the exchange possibilities. In summary, the fertility of the One Health approach to antibiotic resistance depends on the progress of understanding multihierarchical systems, encompassing communications among environments (macro/microaggregates), among microbiotas (communities), among bacterial species (clones), and communications among MGEs., (Copyright © 2019 Baquero, Coque, Martínez, Aracil-Gisbert and Lanza.)

Microcins are low-molecular-weight, ribosomally produced, highly stable, bacterial-inhibitory molecules involved in competitive, and amensalistic interactions between Enterobacteriaceae in the intestine. These interactions take place in a highly complex chemical landscape, the intestinal eco-active chemosphere, composed of chemical substances that positively or negatively influence bacterial growth, including those originated from nutrient uptake, and those produced by the action of the human or animal host and the intestinal microbiome. The contribution of bacteria results from their effect on the host generated molecules, on food and digested food, and organic substances from microbial origin, including from bacterial degradation. Here, we comprehensively review the main chemical substances present in the human intestinal chemosphere, particularly of those having inhibitory effects on microorganisms. With this background, and focusing on Enterobacteriaceae , the most relevant human pathogens from the intestinal microbiota, the microcin's history and classification, mechanisms of action, and mechanisms involved in microcin's immunity (in microcin producers) and resistance (non-producers) are reviewed. Products from the chemosphere likely modulate the ecological effects of microcin activity. Several cross-resistance mechanisms are shared by microcins, colicins, bacteriophages, and some conventional antibiotics, which are expected to produce cross-effects. Double-microcin-producing strains (such as microcins MccM and MccH47) have been successfully used for decades in the control of pathogenic gut organisms. Microcins are associated with successful gut colonization, facilitating translocation and invasion, leading to bacteremia, and urinary tract infections. In fact, Escherichia coli strains from the more invasive phylogroups (e.g., B2) are frequently microcinogenic. A publicly accessible APD3 database http://aps.unmc.edu/AP/ shows particular genes encoding microcins in 34.1% of E . coli strains (mostly MccV, MccM, MccH47, and MccI47), and much less in Shigella and Salmonella (<2%). Some 4.65% of Klebsiella pneumoniae are microcinogenic (mostly with MccE492), and even less in Enterobacter or Citrobacter (mostly MccS). The high frequency and variety of microcins in some Enterobacteriaceae indicate key ecological functions, a notion supported by their dominance in the intestinal microbiota of biosynthetic gene clusters involved in the synthesis of post-translationally modified peptide microcins., (Copyright © 2019 Baquero, Lanza, Baquero, del Campo and Bravo-Vázquez.)

The intestinal microbiota is considered to be a major reservoir of antibiotic resistance determinants (ARDs) that could potentially be transferred to bacterial pathogens via mobile genetic elements. Yet, this assumption is poorly supported by empirical evidence due to the distant homologies between known ARDs (mostly from culturable bacteria) and ARDs from the intestinal microbiota. Consequently, an accurate census of intestinal ARDs (that is, the intestinal resistome) has not yet been fully determined. For this purpose, we developed and validated an annotation method (called pairwise comparative modelling) on the basis of a three-dimensional structure (homology comparative modelling), leading to the prediction of 6,095 ARDs in a catalogue of 3.9 million proteins from the human intestinal microbiota. We found that the majority of predicted ARDs (pdARDs) were distantly related to known ARDs (mean amino acid identity 29.8%) and found little evidence supporting their transfer between species. According to the composition of their resistome, we were able to cluster subjects from the MetaHIT cohort (n = 663) into six resistotypes that were connected to the previously described enterotypes. Finally, we found that the relative abundance of pdARDs was positively associated with gene richness, but not when subjects were exposed to antibiotics. Altogether, our results indicate that the majority of intestinal microbiota ARDs can be considered intrinsic to the dominant commensal microbiota and that these genes are rarely shared with bacterial pathogens.

Introduction: endoscopic ultrasonography (EUS) elastography is considered a useful tool for the evaluation of solid pancreatic lesions (SPL)., Objective: The aim of our study was to evaluate the diagnostic performance of elastography in patients with SPL., Material and Methods: A prospective, cross-sectional study was performed at the Rebagliati Hospital between July 2017 and June 2018. Patients with a diagnosis of SPL and echoendoscopic study, elastography and FNA were included. Qualitative and quantitative elastography: strain ratio (SR) and strain histogram, were performed and analyzed with histopathological results. The diagnostic accuracy of EUS elastography in detecting malignancy was calculated using receiver operating curve analysis. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy for the detection of malignancy were calculated., Results: Out of 832 EUS examinations performed, 46 patients with SPL (mean age, 64.6 years; 29 women) were included in the study. Pancreatic adenocarcinoma was diagnosed in 36 cases. In qualitative elastography, score 3 was most frequent (n = 39, 84.8%) with sensitivity, specificity and accuracy of 88.9%, 30% and 76.1%, respectively, for predict adenocarcinoma. A strain ratio of 15 or higher (100% sensitivity, 66.7% specificity and 97.8% accuracy) and a histogram of less than 49 (66.7% sensitivity, 97.6% specificity and 95.6% accuracy) predicts malignancy in SPL, with area under a ROC curve of 0.941 (95% CI, 0.82 - 1.0)., Conclusions: EUS elastography provides information to predict the malignant nature of the pancreatic lesion. In our study, the elastographic detection of a score 3, SR≥15 or a histogram <49 predicts the presence of malignancy in LSP.

Next-Generation Sequencing (NGS) technologies are expected to play a crucial role in the surveillance of infectious diseases, with their unprecedented capabilities for the characterisation of genetic information underlying the virulence and antimicrobial resistance (AMR) properties of microorganisms.  In the implementation of any novel technology for regulatory purposes, important considerations such as harmonisation, validation and quality assurance need to be addressed.  NGS technologies pose unique challenges in these regards, in part due to their reliance on bioinformatics for the processing and proper interpretation of the data produced.  Well-designed benchmark resources are thus needed to evaluate, validate and ensure continued quality control over the bioinformatics component of the process.  This concept was explored as part of a workshop on "Next-generation sequencing technologies and antimicrobial resistance" held October 4-5 2017.   Challenges involved in the development of such a benchmark resource, with a specific focus on identifying the molecular determinants of AMR, were identified. For each of the challenges, sets of unsolved questions that will need to be tackled for them to be properly addressed were compiled. These take into consideration the requirement for monitoring of AMR bacteria in humans, animals, food and the environment, which is aligned with the principles of a "One Health" approach., Competing Interests: Competing interests: JOG receives some research funding from Oxford Nanopore Technologies. ER is consultant for Pathoquest.

Background: Antimicrobial resistance is a major global health challenge. Metagenomics allows analyzing the presence and dynamics of "resistomes" (the ensemble of genes encoding antimicrobial resistance in a given microbiome) in disparate microbial ecosystems. However, the low sensitivity and specificity of available metagenomic methods preclude the detection of minority populations (often present below their detection threshold) and/or the identification of allelic variants that differ in the resulting phenotype. Here, we describe a novel strategy that combines targeted metagenomics using last generation in-solution capture platforms, with novel bioinformatics tools to establish a standardized framework that allows both quantitative and qualitative analyses of resistomes., Methods: We developed ResCap, a targeted sequence capture platform based on SeqCapEZ (NimbleGene) technology, which includes probes for 8667 canonical resistance genes (7963 antibiotic resistance genes and 704 genes conferring resistance to metals or biocides), and 2517 relaxase genes (plasmid markers) and 78,600 genes homologous to the previous identified targets (47,806 for antibiotics and 30,794 for biocides or metals). Its performance was compared with metagenomic shotgun sequencing (MSS) for 17 fecal samples (9 humans, 8 swine). ResCap significantly improves MSS to detect "gene abundance" (from 2.0 to 83.2%) and "gene diversity" (26 versus 14.9 genes unequivocally detected per sample per million of reads; the number of reads unequivocally mapped increasing up to 300-fold by using ResCap), which were calculated using novel bioinformatic tools. ResCap also facilitated the analysis of novel genes potentially involved in the resistance to antibiotics, metals, biocides, or any combination thereof., Conclusions: ResCap, the first targeted sequence capture, specifically developed to analyze resistomes, greatly enhances the sensitivity and specificity of available metagenomic methods and offers the possibility to analyze genes related to the selection and transfer of antimicrobial resistance (biocides, heavy metals, plasmids). The model opens the possibility to study other complex microbial systems in which minority populations play a relevant role.

Summary: PLACNET is a graph-based tool for reconstruction of plasmids from next generation sequence pair-end datasets. PLACNET graphs contain two types of nodes (assembled contigs and reference genomes) and two types of edges (scaffold links and homology to references). Manual pruning of the graphs is a necessary requirement in PLACNET, but this is difficult for users without solid bioinformatic background. PLACNETw, a webtool based on PLACNET, provides an interactive graphic interface, automates BLAST searches, and extracts the relevant information for decision making. It allows a user with domain expertise to visualize the scaffold graphs and related information of contigs as well as reference sequences, so that the pruning operations can be done interactively from a personal computer without the need for additional tools. After successful pruning, each plasmid becomes a separate connected component subgraph. The resulting data are automatically downloaded by the user., Availability and Implementation: PLACNETw is freely available at https://castillo.dicom.unican.es/upload/., Contact: delacruz@unican.es., Supplementary Information: A tutorial video and several solved examples are available at https://castillo.dicom.unican.es/placnetw&#95;video/ and https://castillo.dicom.unican.es/examples/., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com)

The emergence of nosocomial infections by multidrug-resistant sequence type 117 (ST117) Enterococcus faecium has been reported in several European countries. ST117 has been detected in Spanish hospitals as one of the main causes of bloodstream infections. We analyzed genome variations of ST117 strains isolated in Madrid and describe the first ST117 closed genome sequences., (Copyright © 2017 Tedim et al.)

Background: Rhodococcus jostii RHA1 and other actinobacteria accumulate triglycerides (TAG) under nutrient starvation. This property has an important biotechnological potential in the production of sustainable oils., Results: To gain insight into the metabolic pathways involved in TAG accumulation, we analysed the transcriptome of R jostii RHA1 under nutrient-limiting conditions. We correlate these physiological conditions with significant changes in cell physiology. The main consequence was a global switch from catabolic to anabolic pathways. Interestingly, the Entner-Doudoroff (ED) pathway was upregulated in detriment of the glycolysis or pentose phosphate pathways. ED induction was independent of the carbon source (either gluconate or glucose). Some of the diacylglycerol acyltransferase genes involved in the last step of the Kennedy pathway were also upregulated. A common feature of the promoter region of most upregulated genes was the presence of a consensus binding sequence for the cAMP-dependent CRP regulator., Conclusion: This is the first experimental observation of an ED shift under nutrient starvation conditions. Knowledge of this switch could help in the design of metabolomic approaches to optimize carbon derivation for single cell oil production.

AcCNET (Accessory genome Constellation Network) is a Perl application that aims to compare accessory genomes of a large number of genomic units, both at qualitative and quantitative levels. Using the proteomes extracted from the analysed genomes, AcCNET creates a bipartite network compatible with standard network analysis platforms. AcCNET allows merging phylogenetic and functional information about the concerned genomes, thus improving the capability of current methods of network analysis. The AcCNET bipartite network opens a new perspective to explore the pangenome of bacterial species, focusing on the accessory genome behind the idiosyncrasy of a particular strain and/or population., Availability and Implementation: AcCNET is available under GNU General Public License version 3.0 (GPLv3) from http://sourceforge.net/projects/accnet CONTACT: : valfernandez.vf@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online., (© The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Antibiotic resistance is a relevant problem for human health that requires global approaches to establish a deep understanding of the processes of acquisition, stabilization, and spread of resistance among human bacterial pathogens. Since natural (nonclinical) ecosystems are reservoirs of resistance genes, a health-integrated study of the epidemiology of antibiotic resistance requires the exploration of such ecosystems with the aim of determining the role they may play in the selection, evolution, and spread of antibiotic resistance genes, involving the so-called resistance mobilome. High-throughput sequencing techniques allow an unprecedented opportunity to describe the genetic composition of a given microbiome without the need to subculture the organisms present inside. However, bioinformatic methods for analyzing this bulk of data, mainly with respect to binning each resistance gene with the organism hosting it, are still in their infancy. Here, we discuss how current genomic methodologies can serve to analyze the resistance mobilome and its linkage with different bacterial genomes and metagenomes. In addition, we describe the drawbacks of current methodologies for analyzing the resistance mobilome, mainly in cases of complex microbiotas, and discuss the possibility of implementing novel tools to improve our current metagenomic toolbox., (© 2016 New York Academy of Sciences.)

Objectives: To investigate the population structure of Enterococcus faecium causing bloodstream infections (BSIs) in a tertiary Spanish hospital with low glycopeptide resistance, and to enhance our knowledge of the dynamics of emergence and spread of high-risk clonal complexes., Methods: All available E. faecium causing BSIs (n = 413) in our hospital (January 1995-May 2015) were analysed for antibiotic susceptibility (CLSI), putative virulence traits (PCR, esp, hyl Efm ) and clonal relationship (SmaI-PFGE, MLST evaluated by goeBURST and BAPS)., Results: The increased incidence of BSIs caused by enterococci [2.3‰ of attended patients (inpatients and outpatients) in 1996 to 3.0‰ in 2014] significantly correlated with the increase in BSIs caused by E. faecium (0.33‰ of attended patients in 1996 to 1.3‰ in 2014). The BSIs Enterococcus faecalis:E. faecium ratio changed from 5:1 in 1996 to 1:1 in 2014. During the last decade an increase in E. faecium BSIs episodes in cancer patients (10.9% in 1995-2005 and 37.1% in 2006-15) was detected. Ampicillin-susceptible E. faecium (ASEfm; different STs/BAPS) and ampicillin-resistant E. faecium (AREfm; ST18/ST17-BAPS 3.3a) isolates were recovered throughout the study. Successive waves of BAPS 2.1a-AREfm (ST117, ST203 and ST80) partially replaced ASEfm and ST18-AREfm since 2006., Conclusions: Different AREfm clones (belonging to BAPS 2.1a and BAPS 3.3a) consistently isolated during the last decade from BSIs might be explained by a continuous and dense colonization (favouring both invasion and cross-transmission) of hospitalized patients. High-density colonization by these clones is probably enhanced in elderly patients by heavy and prolonged antibiotic exposure, particularly in oncological patients., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Ampicillin resistance has greatly contributed to the recent dramatic increase of a cluster of human adapted Enterococcus faecium lineages (ST17, ST18, and ST78) in hospital-based infections. Changes in the chromosomal pbp5 gene have been associated with different levels of ampicillin susceptibility, leading to protein variants (designated as PBP5 C-types to keep the nomenclature used in previous works) with diverse degrees of reduction in penicillin affinity. Our goal was to use a comparative genomics approach to evaluate the relationship between the diversity of PBP5 among E. faecium isolates of different phylogenomic groups as well as to assess the pbp5 transferability among isolates of disparate clonal lineages. The analyses of 78 selected E. faecium strains as well as published E. faecium genomes, suggested that the diversity of pbp5 mirrors the phylogenomic diversification of E. faecium . The presence of identical PBP5 C-types as well as similar pbp5 genetic environments in different E. faecium lineages and clones from quite different geographical and environmental origin was also documented and would indicate their horizontal gene transfer among E. faecium populations. This was supported by experimental assays showing transfer of large (≈180-280 kb) chromosomal genetic platforms containing pbp5 alleles, ponA (transglycosilase) and other metabolic and adaptive features, from E. faecium donor isolates to suitable E. faecium recipient strains. Mutation profile analysis of PBP5 from available genomes and strains from this study suggests that the spread of PBP5 C-types might have occurred even in the absence of a significant ampicillin resistance phenotype. In summary, genetic platforms containing pbp5 sequences were stably maintained in particular E. faecium lineages, but were also able to be transferred among E. faecium clones of different origins, emphasizing the growing risk of further spread of ampicillin resistance in this nosocomial pathogen.

The phylum Firmicutes is one of the most abundant groups of prokaryotes in the microbiota of humans and animals and includes genera of outstanding relevance in biomedicine, health care, and industry. Antimicrobial drug resistance is now considered a global health security challenge of the 21st century, and this heterogeneous group of microorganisms represents a significant part of this public health issue.The presence of the same resistant genes in unrelated bacterial genera indicates a complex history of genetic interactions. Plasmids have largely contributed to the spread of resistance genes among Staphylococcus, Enterococcus, and Streptococcus species, also influencing the selection and ecological variation of specific populations. However, this information is fragmented and often omits species outside these genera. To date, the antimicrobial resistance problem has been analyzed under a "single centric" perspective ("gene tracking" or "vehicle centric" in "single host-single pathogen" systems) that has greatly delayed the understanding of gene and plasmid dynamics and their role in the evolution of bacterial communities.This work analyzes the dynamics of antimicrobial resistance genes using gene exchange networks; the role of plasmids in the emergence, dissemination, and maintenance of genes encoding resistance to antimicrobials (antibiotics, heavy metals, and biocides); and their influence on the genomic diversity of the main Gram-positive opportunistic pathogens under the light of evolutionary ecology. A revision of the approaches to categorize plasmids in this group of microorganisms is given using the 1,326 fully sequenced plasmids of Gram-positive bacteria available in the GenBank database at the time the article was written.

The three main processes shaping the evolutionary ecology of antibiotic resistance (AbR) involve the emergence, invasion and occupation by antibiotic-resistant genes of significant environments for human health. The process of emergence in complex bacterial populations is a high-frequency, continuous swarming of ephemeral combinatory genetic and epigenetic explorations inside cells and among cells, populations and communities, expanding in different environments (migration), creating the stochastic variation required for evolutionary progress. Invasion refers to the process by which AbR significantly increases in frequency in a given (invaded) environment, led by external invaders local multiplication and spread, or by endogenous conversion. Conversion occurs because of the spread of AbR genes from an exogenous resistant clone into an established (endogenous) bacterial clone(s) colonizing the environment; and/or because of dissemination of particular resistant genetic variants that emerged within an endogenous clonal population. Occupation of a given environment by a resistant variant means a permanent establishment of this organism in this environment, even in the absence of antibiotic selection. Specific interventions on emergence influence invasion, those acting on invasion also influence occupation and interventions on occupation determine emergence. Such interventions should be simultaneously applied, as they are not simple solutions to the complex problem of AbR.

Third-generation cephalosporins are a class of β-lactam antibiotics that are often used for the treatment of human infections caused by Gram-negative bacteria, especially Escherichia coli. Worryingly, the incidence of human infections caused by third-generation cephalosporin-resistant E. coli is increasing worldwide. Recent studies have suggested that these E. coli strains, and their antibiotic resistance genes, can spread from food-producing animals, via the food-chain, to humans. However, these studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these strains. We therefore used whole-genome sequencing (WGS) to study the relatedness of cephalosporin-resistant E. coli from humans, chicken meat, poultry and pigs. One strain collection included pairs of human and poultry-associated strains that had previously been considered to be identical based on Multi-Locus Sequence Typing, plasmid typing and antibiotic resistance gene sequencing. The second collection included isolates from farmers and their pigs. WGS analysis revealed considerable heterogeneity between human and poultry-associated isolates. The most closely related pairs of strains from both sources carried 1263 Single-Nucleotide Polymorphisms (SNPs) per Mbp core genome. In contrast, epidemiologically linked strains from humans and pigs differed by only 1.8 SNPs per Mbp core genome. WGS-based plasmid reconstructions revealed three distinct plasmid lineages (IncI1- and IncK-type) that carried cephalosporin resistance genes of the Extended-Spectrum Beta-Lactamase (ESBL)- and AmpC-types. The plasmid backbones within each lineage were virtually identical and were shared by genetically unrelated human and animal isolates. Plasmid reconstructions from short-read sequencing data were validated by long-read DNA sequencing for two strains. Our findings failed to demonstrate evidence for recent clonal transmission of cephalosporin-resistant E. coli strains from poultry to humans, as has been suggested based on traditional, low-resolution typing methods. Instead, our data suggest that cephalosporin resistance genes are mainly disseminated in animals and humans via distinct plasmids.

Bacterial whole genome sequence (WGS) methods are rapidly overtaking classical sequence analysis. Many bacterial sequencing projects focus on mobilome changes, since macroevolutionary events, such as the acquisition or loss of mobile genetic elements, mainly plasmids, play essential roles in adaptive evolution. Existing WGS analysis protocols do not assort contigs between plasmids and the main chromosome, thus hampering full analysis of plasmid sequences. We developed a method (called plasmid constellation networks or PLACNET) that identifies, visualizes and analyzes plasmids in WGS projects by creating a network of contig interactions, thus allowing comprehensive plasmid analysis within WGS datasets. The workflow of the method is based on three types of data: assembly information (including scaffold links and coverage), comparison to reference sequences and plasmid-diagnostic sequence features. The resulting network is pruned by expert analysis, to eliminate confounding data, and implemented in a Cytoscape-based graphic representation. To demonstrate PLACNET sensitivity and efficacy, the plasmidome of the Escherichia coli lineage ST131 was analyzed. ST131 is a globally spread clonal group of extraintestinal pathogenic E. coli (ExPEC), comprising different sublineages with ability to acquire and spread antibiotic resistance and virulence genes via plasmids. Results show that plasmids flux in the evolution of this lineage, which is wide open for plasmid exchange. MOBF12/IncF plasmids were pervasive, adding just by themselves more than 350 protein families to the ST131 pangenome. Nearly 50% of the most frequent γ-proteobacterial plasmid groups were found to be present in our limited sample of ten analyzed ST131 genomes, which represent the main ST131 sublineages.

To modulate the expression of genes involved in nitrogen assimilation, the cyanobacterial PII-interacting protein X (PipX) interacts with the global transcriptional regulator NtcA and the signal transduction protein PII, a protein found in all three domains of life as an integrator of signals of the nitrogen and carbon balance. PipX can form alternate complexes with NtcA and PII, and these interactions are stimulated and inhibited, respectively, by 2-oxoglutarate, providing a mechanistic link between PII signaling and NtcA-regulated gene expression. Here, we demonstrate that PipX is involved in a much wider interaction network. The effect of pipX alleles on transcript levels was studied by RNA sequencing of S. elongatus strains grown in the presence of either nitrate or ammonium, followed by multivariate analyses of relevant mutant/control comparisons. As a result of this process, 222 genes were classified into six coherent groups of differentially regulated genes, two of which, containing either NtcA-activated or NtcA-repressed genes, provided further insights into the function of NtcA-PipX complexes. The remaining four groups suggest the involvement of PipX in at least three NtcA-independent regulatory pathways. Our results pave the way to uncover new regulatory interactions and mechanisms in the control of gene expression in cyanobacteria.

Nature has endowed cells with powerful nanomotors to accomplish intricate mechanical tasks, such as the macromolecular transport across membranes occurring in cell division, bacterial conjugation, and in a wide variety of secretion systems. These biological motors couple the chemical energy provided by ATP hydrolysis to the mechanical work needed to transport DNA and/or protein effectors. Here, we review what is known about the molecular mechanisms of these membrane-associated machines. Sequence and structural comparison between these ATPases reveal that they share a similar motor domain, suggesting a common evolutionary ancestor. Learning how these machines operate will lead the design of nanotechnology devices with unique applications in medicine and engineering., (Copyright © 2011 Elsevier Ltd. All rights reserved.)