Targeted Metagenomic Databases Provide Improved Analysis of Microbiota Samples.

We report on Moonbase, an innovative pipeline that builds upon the established tools of MetaPhlAn and Kraken2, enhancing their capabilities for more precise taxonomic detection and quantification in diverse microbial communities. Moonbase enhances the performance of Kraken2 mapping by providing an efficient method for constructing project-specific databases. Moonbase was evaluated using synthetic metagenomic samples and compared against MetaPhlAn3 and generalized Kraken2 databases. Moonbase significantly improved species precision and quantification, outperforming marker genes and generalized databases. Construction of a phylogenetic tree from 16S genome data in Moonbase allowed for the incorporation of UniFrac-type phylogenetic information into diversity calculations of samples. We demonstrated that the resulting analysis increased statistical power in distinguishing microbial communities. This study highlights the continual evolution of metagenomic tools with the goal of improving metagenomic analysis and highlighting the potential of the Moonbase pipeline.

Hospital and urban wastewaters shape the matrix and active resistome of environmental biofilms.

Understanding the dynamics of antibiotic resistance gene (ARG) transfer and dissemination in natural environments remains challenging. Biofilms play a crucial role in bacterial survival and antimicrobial resistance (AMR) dissemination in natural environments, particularly in aquatic systems. This study focused on hospital and urban wastewater (WW) biofilms to investigate the potential for ARG dissemination through mobile genetic elements (MGEs). The analysis included assessing the biofilm extracellular polymeric substances (EPS), microbiota composition as well as metatranscriptomic profiling of the resistome and mobilome. We produced both in vitro and in situ biofilms and performed phenotypic and genomic analyses. In the in vitro setup, untreated urban and hospital WW was used to establish biofilm reactors, with ciprofloxacin added as a selective agent at minimal selective concentration. In the in situ setup, biofilms were developed directly in hospital and urban WW pipes. We first showed that a) the composition of EPS differed depending on the growth environment (in situ and in vitro) and the sampling origin (hospital vs urban WW) and that b) ciprofloxacin impacted the composition of the EPS. The metatranscriptomic approach showed that a) expression of several ARGs and MGEs increased upon adding ciprofloxacin for biofilms from hospital WW only and b) that the abundance and type of plasmids that carried individual or multiple ARGs varied depending on the WW origins of the biofilms. When the same plasmids were present in both, urban and hospital WW biofilms, they carried different ARGs.  We showed that hospital and urban wastewaters shaped the structure and active resistome of environmental biofilms, and we confirmed that hospital WW is an important hot spot for the dissemination and selection of antimicrobial resistance. Our study provides a comprehensive assessment of WW biofilms as crucial hotspots for ARG transfer. Hospital WW biofilms exhibited distinct characteristics, including higher eDNA abundance and expression levels of ARGs and MGEs, highlighting their role in antimicrobial resistance dissemination. These findings emphasize the importance of understanding the structural, ecological, functional, and genetic organization of biofilms in anthropized environments and their contribution to antibiotic resistance dynamics.

Identification of the active mechanism of aminoglycoside entry in V. cholerae through characterization of sRNA ctrR, regulating carbohydrate utilization and transport.

The possible active entry of aminoglycosides in bacterial cells has been debated since the development of this antibiotic family. Here we report the identification of their active transport mechanism in Vibrio species. We combined genome-wide transcriptional analysis and fitness screens to identify alterations driven by treatment of V. cholerae with sub-minimum inhibitory concentrations (sub-MIC) of the aminoglycoside tobramycin. RNA-seq data showed downregulation of the small non-coding RNA ncRNA586 during such treatment, while Tn-seq revealed that inactivation of this sRNA was associated with improved fitness in the presence of tobramycin. This sRNA is located near sugar transport genes and previous work on a homologous region in Vibrio tasmaniensis suggested that this sRNA stabilizes gene transcripts for carbohydrate transport and utilization, as well as phage receptors. The role for ncRNA586, hereafter named ctrR, in the transport of both carbohydrates and aminoglycosides, was further investigated. Flow cytometry on cells treated with a fluorescent aminoglycoside confirmed the role of ctrR and of carbohydrate transporters in differential aminoglycoside entry. Despite sequence diversity, ctrR showed functional conservation across the Vibrionales. This system in directly modulated by carbon sources, suggesting regulation by carbon catabolite repression, a widely conserved mechanism in Gram-negative bacteria, priming future research on aminoglycoside uptake by sugar transporters in other bacterial species.

Microbial diversity in the vaginal microbiota and its link to pregnancy outcomes.

The vaginal microbiota refers to the microorganisms that reside in the vagina. These microorganisms contribute significantly to a woman's reproductive and general health. A healthy vaginal microbiota is typically a low-diversity environment with a predominance of lactic acid-producing Lactobacillus species. Factors such as antibiotic use, sexual activity, and hormonal changes can disrupt the balance of the vaginal microbiota, leading to conditions such as bacterial vaginosis. The composition of the vaginal microbiota changes and takes on added importance during pregnancy, serving as a barrier against infection for both mother and fetus. Despite the importance of the microorganisms that colonize the vagina, details of how changes in composition and diversity can impact pregnancy outcomes is poorly understood. This is especially true for woman with a high prevalence of Gardnerella vaginalis. Here we report on a diverse cohort of 749 women, enrolled in the InSPIRe cohort, during their final trimester of pregnancy. We show that Lactobacilli, including L. crispatus are important in maintaining low diversity, and that depletion in this critical community is linked with preterm delivery. We further demonstrate that it is overall diversity of the vaginal microbiota, not specific species, which provides the best indicator of risk.

Precision Medicine and Novel Therapeutic Strategies in Detection and Treatment of Cancer: Highlights from the 58th IACR Annual Conference.

Innovation in both detection and treatment of cancer is necessary for the constant improvement in therapeutic strategies, especially in patients with novel or resistant variants of cancer. Cancer mortality rates have declined by almost 30% since 1991, however, depending on the cancer type, acquired resistance can occur to varying degrees. To combat this, researchers are looking towards advancing our understanding of cancer biology, in order to inform early detection, and guide novel therapeutic approaches. Through combination of these approaches, it is believed that a more complete and thorough intervention on cancer can be achieved. Here, we will discuss the advances and approaches in both detection and treatment of cancer, presented at the 58th Irish Association for Cancer Research (IACR) annual conference.

Genome-wide identification of genes required for alternative peptidoglycan cross-linking in Escherichia coli revealed unexpected impacts of ß-lactams.

The D,D-transpeptidase activity of penicillin-binding proteins (PBPs) is the well-known primary target of ß-lactam antibiotics that block peptidoglycan polymerization. ß-lactam-induced bacterial killing involves complex downstream responses whose causes and consequences are difficult to resolve. Here, we use the functional replacement of PBPs by a ß-lactam-insensitive L,D-transpeptidase to identify genes essential to mitigate the effects of PBP inactivation by ß-lactams in actively dividing bacteria. The functions of the 179 conditionally essential genes identified by this approach extend far beyond L,D-transpeptidase partners for peptidoglycan polymerization to include proteins involved in stress response and in the assembly of outer membrane polymers. The unsuspected effects of ß-lactams include loss of the lipoprotein-mediated covalent bond that links the outer membrane to the peptidoglycan, destabilization of the cell envelope in spite of effective peptidoglycan cross-linking, and increased permeability of the outer membrane. The latter effect indicates that the mode of action of ß-lactams involves self-promoted penetration through the outer membrane.

Complete Genome and Methylome Analysis of the Box-Shaped Halophilic Archaeon Haloarcula sinaiiensis ATCC 33800.

The genome of halophilic archaeon Haloarcula sinaiiensis ATCC 33800 was sequenced and assembled and comprises seven replicons. Four m6A and one m4C modified motifs and their responsible methyltransferase genes have been identified in the genome by single-molecule real-time (SMRT) sequencing and bioinformatic analysis.

Protein supplementation during an energy-restricted diet induces visceral fat loss and gut microbiota amino acid metabolism activation: a randomized trial.

Interactions between diet and gut microbiota are critical regulators of energy metabolism. The effects of fibre intake have been deeply studied but little is known about the impact of proteins. Here, we investigated the effects of high protein supplementation (Investigational Product, IP) in a double blind, randomised placebo-controled intervention study (NCT01755104) where 107 participants received the IP or an isocaloric normoproteic comparator (CP) alongside a mild caloric restriction. Gut microbiota profiles were explored in a patient subset (n = 53) using shotgun metagenomic sequencing. Visceral fat decreased in both groups (IP group: - 20.8 ± 23.2 cm2; CP group: - 14.5 ± 24.3 cm2) with a greater reduction (p < 0.05) with the IP supplementation in the Per Protocol population. Microbial diversity increased in individuals with a baseline low gene count (p < 0.05). The decrease in weight, fat mass and visceral fat mass significantly correlated with the increase in microbial diversity (p < 0.05). Protein supplementation had little effects on bacteria composition but major differences were seen at functional level. Protein supplementation stimulated bacterial amino acid metabolism (90% amino-acid synthesis functions enriched with IP versus 13% in CP group (p < 0.01)). Protein supplementation alongside a mild energy restriction induces visceral fat mass loss and an activation of gut microbiota amino-acid metabolism.Clinical trial registration: NCT01755104 (24/12/2012). https://clinicaltrials.gov/ct2/show/record/NCT01755104?term=NCT01755104&draw=2&rank=1 .

Dynamics of extended-spectrum beta-lactamase-producing Enterobacterales colonization in long-term carriers following travel abroad.

Travel to tropical regions is associated with high risk of acquiring extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) that are typically cleared in less than 3 months following return. The conditions leading to persistent carriage that exceeds 3 months in some travellers require investigation. Whole-genome sequencing (Illumina MiSeq) was performed on the 82 ESBL-E isolates detected upon return and 1, 2, 3, 6 and 12 months later from the stools of 11 long-term (>3 months) ESBL-E carriers following travel abroad. One to five different ESBL Escherichia coli strains were detected per traveller upon return, and this diminished to one after 3 months. Long-term carriage was due to the presence of the same ESBL E. coli strain, for more than 3 months, in 9 out of 11 travellers, belonging to epidemic sequence type complexes (STc 10, 14, 38, 69, 131 and 648). The mean carriage duration of strains belonging to phylogroups B2/D/F, associated with extra-intestinal virulence, was higher than that for commensal-associated A/B1/E phylogroups (3.5 vs 0.5 months, P=0.021). Genes encoding iron capture systems (fyuA, irp), toxins (senB, sat), adhesins (flu, daaF, afa/nfaE, pap, ecpA) and colicin (cjrA) were more often present in persistent strains than in transient ones. Single-nucleotide polymorphism (SNP) analysis in persistent strains showed a maximum divergence of eight SNPs over 12 months without signs of adaptation. Genomic plasticity was observed during the follow-up with the loss or gain of mobile genetic elements such as plasmids, integrons and/or transposons that may contain resistance genes at different points in the follow-up. Long-term colonization of ESBL-E following travel is primarily due to the acquisition of E. coli strains belonging to epidemic clones and harbouring 'virulence genes', allowing good adaptation to the intestinal microbiota.

Alteration of the gut fecal microbiome in children living with HIV on antiretroviral therapy in Yaounde, Cameroon.

Multiple factors, such as immune disruption, prophylactic co-trimoxazole, and antiretroviral therapy, may influence the structure and function of the gut microbiome of children infected with HIV from birth. In order to understand whether HIV infection altered gut microbiome and to relate changes in microbiome structure and function to immune status, virological response and pediatric ART regimens, we characterized the gut microbiome of 87 HIV-infected and 82 non-exposed HIV-negative children from Yaounde, a cosmopolitan city in Cameroon. We found that children living with HIV had significantly lower alpha diversity in their gut microbiome and altered beta diversity that may not be related to CD4+ T cell count or viral load. There was an increased level of Akkermansia and Faecalibacterium genera and decreased level of Escherichia and other Gamma proteobacteria in children infected with HIV, among other differences. We noted an effect of ethnicity/geography on observed gut microbiome composition and that children on ritonavir-boosted protease inhibitor (PI/r)-based ART had gut microbiome composition that diverged more from HIV-negative controls compared to those on non-nucleoside reverse-transcriptase inhibitors-based ART. Further studies investigating the role of this altered gut microbiome in increased disease susceptibility are warranted for individuals who acquired HIV via mother-to-child transmission.

Hospital discharges in urban sanitation systems: Long-term monitoring of wastewater resistome and microbiota in relationship to their eco-exposome.

Wastewaters (WW) are important sources for the dissemination of antimicrobial resistance (AMR) into the environment. Hospital WW (HWW) contain higher loads of micro-pollutants and AMR markers than urban WW (UWW). Little is known about the long-term dynamics of H and U WW and the impact of their joined treatment on the general burden of AMR. Here, we characterized the resistome, microbiota and eco-exposome signature of 126 H and U WW samples treated separately for three years, and then mixed, over one year. Multi-variate analysis and machine learning revealed a robust signature for each WW with no significant variation over time before mixing, and once mixed, both WW closely resembled Urban signatures. We demonstrated a significant impact of pharmaceuticals and surfactants on the resistome and microbiota of H and U WW. Our results present considerable targets for AMR related risk assessment of WW.

Preclinical evaluation of a novel triple-acting PIM/PI3K/mTOR inhibitor, IBL-302, in breast cancer.

The proviral integration of Moloney virus (PIM) family of protein kinases are overexpressed in many haematological and solid tumours. PIM kinase expression is elevated in PI3K inhibitor-treated breast cancer samples, suggesting a major resistance pathway for PI3K inhibitors in breast cancer, potentially limiting their clinical utility. IBL-302 is a novel molecule that inhibits both PIM and PI3K/AKT/mTOR signalling. We thus evaluated the preclinical activity of IBL-302, in a range of breast cancer models. Our results demonstrate in vitro efficacy of IBL-302 in a range of breast cancer cell lines, including lines with acquired resistance to trastuzumab and lapatinib. IBL-302 demonstrated single-agent, anti-tumour efficacy in suppression of pAKT, pmTOR and pBAD in the SKBR-3, BT-474 and HCC-1954 HER2+/PIK3CA-mutated cell lines. We have also shown the in vivo single-agent efficacy of IBL-302 in the subcutaneous BT-474 and HCC-1954 xenograft model in BALB/c nude mice. The combination of trastuzumab and IBL-302 significantly increased the anti-proliferative effect in HER2+ breast cancer cell line, and matched trastuzumab-resistant line, relative to testing either drug alone. We thus believe that the novel PIM and PI3K/mTOR inhibitor, IBL-302, represents an exciting new potential treatment option for breast cancer, and that it should be considered for clinical investigation.

Dynamic insights on transcription initiation and RNA processing during bacterial adaptation.

Transcription initiation and RNA processing govern gene expression and enable bacterial adaptation by reshaping the RNA landscape. The aim of this study was to simultaneously observe these two fundamental processes in a transcriptome responding to an environmental signal. A controlled σE system in E. coli was coupled to our previously described tagRNA-seq method to yield process kinetics information. Changes in transcription initiation frequencies (TIF) and RNA processing frequencies (PF) were followed using 5' RNA tags. Changes in TIF showed a binary increased/decreased pattern that alternated between transcriptionally activated and repressed promoters, providing the bacterial population with transcriptional oscillation. PF variation fell into three categories of cleavage activity: (i) constant and independent of RNA levels, (ii) increased once RNA has accumulated, and (iii) positively correlated to changes in TIF. This work provides a comprehensive and dynamic view of major events leading to transcriptomic reshaping during bacterial adaptation. It unveils an interplay between transcription initiation and the activity of specific RNA cleavage sites. This study utilized a well-known genetic system to analyze fundamental processes and can serve as a blueprint for comprehensive studies that exploit the RNA metabolism to decipher and understand bacterial gene expression control.

Quantitative analysis of workplace morale in academic librarians and the impact of direct supervisors on workplace morale.

Many studies have attempted to measure the workplace morale of academic librarians through related variables such as job satisfaction. Other studies have taken a qualitative or phenomenological approach to understanding workplace morale. This exploratory study has two objectives: (1) to quantitatively measure the workplace morale of academic librarians to set an initial baseline measure for future research and (2) to analyze the variables that impact academic librarian morale with an emphasis on the variables that academic library supervisors directly influence. Findings suggest the workplace morale of academic librarians is predicted by several variables within a supervisor's influence including feedback, work autonomy, and supervisor qualities such as communication, transparency, and empowering subordinates. Outside of a small negative relationship to age and work experience, demographics were largely found to not be a predictor of workplace morale. This study also evaluated workplace morale as a predictor of turnover intention in academic librarians. There was a strong statistically significant negative correlation between workplace morale and the turnover intention of academic librarians.

RadD Contributes to R-Loop Avoidance in Sub-MIC Tobramycin.

We have previously identified Vibrio cholerae mutants in which the stress response to subinhibitory concentrations of aminoglycoside is altered. One gene identified, VC1636, encodes a putative DNA/RNA helicase, recently named RadD in Escherichia coli Here we combined extensive genetic characterization and high-throughput approaches in order to identify partners and molecular mechanisms involving RadD. We show that double-strand DNA breaks (DSBs) are formed upon subinhibitory tobramycin treatment in the absence of radD and recBCD and that formation of these DSBs can be overcome by RNase H1 overexpression. Loss of RNase H1, or of the transcription-translation coupling factor EF-P, is lethal in the radD deletion mutant. We propose that R-loops are formed upon sublethal aminoglycoside treatment, leading to the formation of DSBs that can be repaired by the RecBCD homologous recombination pathway, and that RadD counteracts such R-loop accumulation. We discuss how R-loops that can occur upon translation-transcription uncoupling could be the link between tobramycin treatment and DNA break formation.IMPORTANCE Bacteria frequently encounter low concentrations of antibiotics. Active antibiotics are commonly detected in soil and water at concentrations much below lethal concentration. Although sub-MICs of antibiotics do not kill bacteria, they can have a major impact on bacterial populations by contributing to the development of antibiotic resistance through mutations in originally sensitive bacteria or acquisition of DNA from resistant bacteria. It was shown that concentrations as low as 100-fold below the MIC can actually lead to the selection of antibiotic-resistant cells. We seek to understand how bacterial cells react to such antibiotic concentrations using E. coli, the Gram-negative bacterial paradigm, and V. cholerae, the causative agent of cholera. Our findings shed light on the processes triggered at the DNA level by antibiotics targeting translation, how damage occurs, and what the bacterial strategies are to respond to such DNA damage.

Fitness Restoration of a Genetically Tractable Enterococcus faecalis V583 Derivative To Study Decoration-Related Phenotypes of the Enterococcal Polysaccharide Antigen.

Commensal and generally harmless in healthy individuals, Enterococcus faecalis causes opportunistic infections in immunocompromised patients. Plasmid-cured E. faecalis strain VE14089, derived from sequenced reference strain V583, is widely used for functional studies due to its improved genetic amenability. Although strain VE14089 has no major DNA rearrangements, with the exception of an ∼20-kb integrated region of pTEF1 plasmid, the strain presented significant growth differences from the V583 reference strain of our collection (renamed VE14002). In the present study, genome sequencing of strain VE14089 identified additional point mutations. Excision of the integrated pTEF1 plasmid region and sequential restoration of wild-type alleles showing nonsilent mutations were performed to obtain the VE18379 reference-derivative strain. Recovery of the growth ability of the restored VE18379 strain at a level similar to that seen with the reference strain points to GreA and Spx as bacterial fitness determinants. Virulence potential in Galleria mellonella and intestinal colonization in mouse demonstrated host adaptation of the VE18379 strain equivalent to VE14002 host adaptation. We further demonstrated that deletion of the 16.8-kb variable region of the epa locus recapitulates the key role of Epa decoration in host adaptation, providing a genetic system to study the role of specific epa-variable regions in host adaptation independently of other genetic variations.IMPORTANCEE. faecalis strain VE14089 was derived from V583 cured of its plasmids. Although VE14089 had no major DNA rearrangements, it presented significant growth and host adaptation differences from the reference strain V583 of our collection. To construct a strain with better fitness, we sequenced the genome of VE14089, identified single nucleotide polymorphisms (SNPs), and repaired the genes that could account for these changes. Using this reference-derivative strain, we provide a novel genetic system to understand the role of the variable region of epa in the enterococcal lifestyle.

Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer.

BACKGROUND: The oncogenic receptor tyrosine kinase (RTK) ERBB2 is known to dimerize with other EGFR family members, particularly ERBB3, through which it potently activates PI3K signalling. Antibody-mediated inhibition of this ERBB2/ERBB3/PI3K axis has been a cornerstone of treatment for ERBB2-amplified breast cancer patients for two decades. However, the lack of response and the rapid onset of relapse in many patients now question the assumption that the ERBB2/ERBB3 heterodimer is the sole relevant effector target of these therapies. METHODS: Through a systematic protein-protein interaction screen, we have identified and validated alternative RTKs that interact with ERBB2. Using quantitative readouts of signalling pathway activation and cell proliferation, we have examined their influence upon the mechanism of trastuzumab- and pertuzumab-mediated inhibition of cell growth in ERBB2-amplified breast cancer cell lines and a patient-derived xenograft model. RESULTS: We now demonstrate that inactivation of ERBB3/PI3K by these therapeutic antibodies is insufficient to inhibit the growth of ERBB2-amplified breast cancer cells. Instead, we show extensive promiscuity between ERBB2 and an array of RTKs from outside of the EGFR family. Paradoxically, pertuzumab also acts as an artificial ligand to promote ERBB2 activation and ERK signalling, through allosteric activation by a subset of these non-canonical RTKs. However, this unexpected activation mechanism also increases the sensitivity of the receptor network to the ERBB2 kinase inhibitor lapatinib, which in combination with pertuzumab, displays a synergistic effect in single-agent resistant cell lines and PDX models. CONCLUSIONS: The interaction of ERBB2 with a number of non-canonical RTKs activates a compensatory signalling response following treatment with pertuzumab, although a counter-intuitive combination of ERBB2 antibody therapy and a kinase inhibitor can overcome this innate therapeutic resistance.

Prediction of the intestinal resistome by a three-dimensional structure-based method.

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.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP).

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes. Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.