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Particle physics experiments rely extensively on computing and data services, making e-infrastructure an integral part of the research collaboration. Constructing and operating distributed computing can however be challenging for a smaller-scale collaboration. The Light Dark Matter eXperiment (LDMX) is a planned small-scale accelerator-based experiment to search for dark matter in the sub-GeV mass region. Finalizing the design of the detector relies on Monte-Carlo simulation of expected physics processes. A distributed computing pilot project was proposed to better utilize available resources at the collaborating institutes, and to improve scalability and reproducibility. This paper outlines the chosen lightweight distributed solution, presenting requirements, the component integration steps, and the experiences using a pilot system for tests with large-scale simulations. The system leverages existing technologies wherever possible, minimizing the need for software development, and deploys only non-intrusive components at the participating sites. The pilot proved that integrating existing components can dramatically reduce the effort needed to build and operate a distributed e-infrastructure, making it attainable even for smaller research collaborations.

Biofilm formation is a critical step in the pathogenesis of difficult-to-treat Gram-positive bacterial infections. We identified that YajC, a conserved membrane protein in bacteria, plays a role in biofilm formation of the clinically relevant Enterococcus faecium strain E1162. Deletion of yajC conferred significantly impaired biofilm formation in vitro and was attenuated in a rat endocarditis model. Mass spectrometry analysis of supernatants of washed Δ yajC cells revealed increased amounts in cytoplasmic and cell-surface-located proteins, including biofilm-associated proteins, suggesting that proteins on the surface of the yajC mutant are only loosely attached. In Streptococcus mutans YajC has been identified in complex with proteins of two cotranslational membrane protein-insertion pathways; the signal recognition particle (SRP)-SecYEG-YajC-YidC1 and the SRP-YajC-YidC2 pathway, but its function is unknown. In S. mutans mutation of yidC1 and yidC2 resulted in impaired protein insertion in the cell membrane and secretion in the supernatant. The E. faecium genome contains all homologous genes encoding for the cotranslational membrane protein-insertion pathways. By combining the studies in S. mutans and E. faecium , we propose that YajC is involved in the stabilization of the SRP-SecYEG-YajC-YidC1 and SRP-YajC-Yid2 pathway or plays a role in retaining proteins for proper docking to the YidC insertases for translocation in and over the membrane., Competing Interests: The authors declare noncompeting interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Enterococcus faecium is an opportunistic pathogen able to colonize the intestines of hospitalized patients. This initial colonization is an important step in the downstream pathogenesis, which includes outgrowth of the intestinal microbiota and potential infection of the host. The impact of intestinal overgrowth on host-enterococcal interactions is not well understood. We therefore applied a RNAseq approach in order to unravel the transcriptional dynamics of E. faecium upon co-culturing with human derived colonic epithelium. Co-cultures of colonic epithelium with a hospital-associated vancomycin resistant (vanA-type) E. faecium (VRE) showed that VRE resided on top of the colonic epithelium when analyzed by microscopy. RNAseq revealed that exposure to the colonic epithelium resulted in upregulation of 238 VRE genes compared to the control condition, including genes implicated in pili expression, conjugation (plasmid_2), genes related to sugar uptake, and biofilm formation (chromosome). In total, 260 were downregulated, including the vanA operon located on plasmid_3. Pathway analysis revealed an overall switch in metabolism to amino acid scavenging and reduction. In summary, our study demonstrates that co-culturing of VRE with human colonic epithelium promotes an elaborate gene response in VRE, enhancing our insight in host- E. faecium interactions, which might facilitate the design of novel anti-infectivity strategies., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Importance: Colonization with extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-PE) often precedes infections and is therefore considered as a great threat for public health. Here, we studied the gut microbiome dynamics in eight index patients colonized with ESBL-PE after hospital discharge and the impact of exposure to this index patient on the gut microbiome dynamics of their household contacts. We showed that the microbiome composition from index patients is different from their household contacts upon hospital discharge and that, in some of the index patients, their microbiome composition over time shifted toward the composition of their household contacts. In contrast, household contacts showed a stable microbiome composition over time irrespective of low-level extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-Ec) or extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL-Kp) gut colonization, suggesting that, in healthy microbiomes, colonization resistance is able to prevent ESBL-PE expansion., Competing Interests: The authors declare no conflict of interest.

The gut microbiome of humans and animals acts as a reservoir of extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC). Dogs are known for having a high prevalence of ESBL-EC in their gut microbiota, although their ESBL-EC carrier status often shifts over time. We hypothesized that the gut microbiome composition of dogs is implicated in ESBL-EC colonization status. Therefore, we assessed whether ESBL-EC carriage in dogs is associated with changes in the gut microbiome and resistome. Fecal samples were collected longitudinally from 57 companion dogs in the Netherlands every 2 weeks for a total of 6 weeks ( n  = 4 samples/dog). Carriage of ESBL-EC was determined through selective culturing and PCR and in line with previous studies, we observed a high prevalence of ESBL-EC carriage in dogs. Using 16s rRNA gene profiling we found significant associations between detected ESBL-EC carriage and an increased abundance of Clostridium sensu stricto 1 , Enterococcus , Lactococcus , and the shared genera of Escherichia -Shigella in the dog microbiome. A resistome capture sequencing approach (ResCap) furthermore, revealed associations between detected ESBL-EC carriage and the increased abundance of the antimicrobial resistance genes: cmlA , dfrA , dhfR , floR , and sul3 . In summary, our study showed that ESBL-EC carriage is associated with a distinct microbiome and resistome composition. IMPORTANCE The gut microbiome of humans and animals is an important source of multidrug resistant pathogens, including beta-lactamase-producing Escherichia coli (ESBL-EC). In this study, we assessed if the carriage of ESBL-EC in dogs was associated with changes in gut composition of bacteria and antimicrobial resistant genes (ARGs). Therefore, stool samples from 57 dogs were collected every 2 weeks for a total of 6 weeks. Sixty eight percent of the dogs carried ESBL-EC during at least one of the time points analyzed. By investigating the gut microbiome and resistome composition, we observed specific changes at time points when dogs were colonized with ESBL-EC compared to time points whenESBL-EC were not detected. In conclusion, our study highlights the importance to study the microbial diversity in companion animals, as gut colonization of particular antimicrobial resistant bacteria might be an indication of a changed microbial composition that is associated with the selection of particular ARGs., Competing Interests: The authors declare no conflict of interest.

The most common resistance mechanism to carbapenems is the production of carbapenemases. In 2021, the Pan American Health Organization warned of the emergence and increase in new carbapenemase combinations in Enterobacterales in Latin America. In this study, we characterized four Klebsiella pneumoniae isolates harboring bla KPC and bla NDM from an outbreak during the COVID-19 pandemic in a Brazilian hospital. We assessed their plasmids' transference ability, fitness effects, and relative copy number in different hosts. The K. pneumoniae BHKPC93 and BHKPC104 strains were selected for whole genome sequencing (WGS) based on their pulsed-field gel electrophoresis profile. The WGS revealed that both isolates belong to ST11, and 20 resistance genes were identified in each isolate, including bla KPC-2 and bla NDM-1 . The bla KPC gene was present on a ~56 Kbp IncN plasmid and the bla NDM-1 gene on a ~102 Kbp IncC plasmid, along with five other resistance genes. Although the bla NDM plasmid contained genes for conjugational transfer, only the bla KPC plasmid conjugated to E. coli J53, without apparent fitness effects. The minimum inhibitory concentrations (MICs) of meropenem/imipenem against BHKPC93 and BHKPC104 were 128/64 and 256/128 mg/L, respectively. Although the meropenem and imipenem MICs against E. coli J53 transconjugants carrying the bla KPC gene were 2 mg/L, this was a substantial increment in the MIC relative to the original J53 strain. The bla KPC plasmid copy number was higher in K. pneumoniae BHKPC93 and BHKPC104 than in E. coli and higher than that of the bla NDM plasmids. In conclusion, two ST11 K. pneumoniae isolates that were part of a hospital outbreak co-harbored bla KPC-2 and bla NDM-1 . The bla KPC -harboring IncN plasmid has been circulating in this hospital since at least 2015, and its high copy number might have contributed to the conjugative transfer of this particular plasmid to an E. coli host. The observation that the bla KPC -containing plasmid had a lower copy number in this E. coli strain may explain why this plasmid did not confer phenotypic resistance against meropenem and imipenem.

Bacillus Calmette-Guerin (BCG) vaccination has been hypothesized to reduce severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, severity, and/or duration via trained immunity induction. Health care workers (HCWs) in nine Dutch hospitals were randomized to BCG or placebo vaccination (1:1) in March and April 2020 and followed for 1 year. They reported daily symptoms, SARS-CoV-2 test results, and health care-seeking behavior via a smartphone application, and they donated blood for SARS-CoV-2 serology at two time points. A total of 1,511 HCWs were randomized and 1,309 analyzed (665 BCG and 644 placebo). Of the 298 infections detected during the trial, 74 were detected by serology only. The SARS-CoV-2 incidence rates were 0.25 and 0.26 per person-year in the BCG and placebo groups, respectively (incidence rate ratio, 0.95; 95% confidence interval, 0.76 to 1.21; P = 0.732). Only three participants required hospitalization for SARS-CoV-2. The proportions of participants with asymptomatic, mild, or moderate infections and the mean infection durations did not differ between randomization groups. In addition, unadjusted and adjusted logistic regression and Cox proportional hazards models showed no differences between BCG and placebo vaccination for any of these outcomes. The percentage of participants with seroconversion (7.8% versus 2.8%; P = 0.006) and mean SARS-CoV-2 anti-S1 antibody concentration (13.1 versus 4.3 IU/mL; P = 0.023) were higher in the BCG than placebo group at 3 months but not at 6 or 12 months postvaccination. BCG vaccination of HCWs did not reduce SARS-CoV-2 infections nor infection duration or severity (ranging from asymptomatic to moderate). In the first 3 months after vaccination, BCG vaccination may enhance SARS-CoV-2 antibody production during SARS-CoV-2 infection. IMPORTANCE While several BCG trials in adults were conducted during the 2019 coronavirus disease epidemic, our data set is the most comprehensive to date, because we included serologically confirmed infections in addition to self-reported positive SARS-CoV-2 test results. We also collected data on symptoms for every day during the 1-year follow-up period, which enabled us to characterize infections in detail. We found that BCG vaccination did not reduce SARS-CoV-2 infections nor infection duration or severity but may have enhanced SARS-CoV-2 antibody production during SARS-CoV-2 infection in the first 3 months after vaccination. These results are in agreement with other BCG trials that reported negative results (but did not use serological endpoints), except for two trials in Greece and India that reported positive results but had few endpoints and included endpoints that were not laboratory confirmed. The enhanced antibody production is in agreement with prior mechanistic studies but did not translate into protection from SARS-CoV-2 infection.

The endometrial microbiota composition may be associated with implantation success. However, a 'core' composition has not yet been defined. This exploratory study analysed the endometrial microbiota by 16S rRNA sequencing (V1-V2 region) of 141 infertile women whose first IVF/ICSI cycle failed and compared the microbiota profiles of women with and without a live birth within 12 months of follow-up, and by infertility cause and type. Lactobacillus was the most abundant genus in the majority of samples. Women with a live birth compared to those without had significantly higher Lactobacillus crispatus relative abundance (RA) (p = 0.029), and a smaller proportion of them had ≤ 10% L. crispatus RA (42.1% and 70.4%, respectively; p = 0.015). A smaller proportion of women in the male factor infertility group had ≤ 10% L. crispatus RA compared to women in the unexplained and other infertility causes groups combined (p = 0.030). Women with primary infertility compared to secondary infertility had significantly higher L. crispatus RA (p = 0.004); lower proportions of them had ≤ 10% L. crispatus RA (p = 0.009) and > 10% Gardnerella vaginalis RA (p = 0.019). In conclusion, IVF/ICSI success may be associated with L. crispatus RA and secondary infertility with endometrial dysbiosis, more often than primary infertility. These hypotheses should be tested in rigorous well-powered longitudinal studies., (© 2023. The Author(s).)

Diet is an important determinant of the human gut microbiome. Here, we analyzed fecal metagenomes of Dutch adults following omnivorous, pescatarian, vegan, and vegetarian diets. We compared the taxonomic composition of individuals from our study with publicly available gut metagenomes from westernized and non-westernized societies. We observed that, despite long-term transition to diets rich in plant fibers (vegan or vegetarian), the microbiomes of these were typical of westernized populations, and similar in composition to omnivores. Although there were no major differences in metabolic modules, we identified differences in the species that contributed to particular functions, such as carbohydrate degradation and short-chain fatty acid metabolism. Overall, this study shows functional redundancy of the microbiomes among westernized populations, which is independent of long-term individual dietary habits. IMPORTANCE Diet is an important modulator of the human gut microbiome, which is susceptible to increased consumption of plant fibers in vegan or vegetarian lifestyles. To investigate this, we compared the gut microbiome of Dutch adults following omnivorous, pescatarian, vegan and vegetarian diets. We did not observe major differences in the gut microbiome composition and function between individuals with different dietary habits. However, we observed differences in the species that contribute to the core functions of the gut microbiome. Our study thus emphasizes the need to better understand the species-specific functional changes associated with dietary habits in the human gut microbiome.

Air pollution from livestock farms is known to affect respiratory health of patients with chronic obstructive pulmonary disease (COPD). The mechanisms behind this relationship, however, remain poorly understood. We hypothesise that air pollutants could influence respiratory health through modulation of the airway microbiome. Therefore, we studied associations between air pollution exposure and the oropharyngeal microbiota (OPM) composition of COPD patients and controls in a livestock-dense area. Oropharyngeal swabs were collected from 99 community-based (mostly mild) COPD cases and 184 controls (baseline), and after 6 and 12 weeks. Participants were non-smokers or former smokers. Annual average livestock-related outdoor air pollution at the home address was predicted using dispersion modelling. OPM composition was analysed using 16S rRNA-based sequencing in all baseline samples and 6-week and 12-week repeated samples of 20 randomly selected subjects (n = 323 samples). A random selection of negative control swabs, taken every sampling day, were also included in the downstream analysis. Both farm-emitted endotoxin and PM 10 levels were associated with increased OPM richness in COPD patients (p < 0.05) but not in controls. COPD case-control status was not associated with community structure, while correcting for known confounders (multivariate PERMANOVA p > 0.05). However, members of the genus Streptococcus were more abundant in COPD patients (Benjamini-Hochberg adjusted p < 0.01). Moderate correlation was found between ordinations of 20 subjects analysed at 0, 6, and 12 weeks (Procrustes r = 0.52 to 0.66; p < 0.05; Principal coordinate analysis of Bray-Curtis dissimilarity), indicating that the OPM is relatively stable over a 12 week period and that a single sample sufficiently represents the OPM. Air pollution from livestock farms is associated with OPM richness of COPD patients, suggesting that the OPM of COPD patients is susceptible to alterations induced by exposure to air pollutants., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The human gut microbiome plays a central role in health and disease. Environmental factors, such as lifestyle and diet, are known to shape the gut microbiome as well as the reservoir of resistance genes that these microbes harbour; the resistome. In this study we assessed whether long-term dietary habits within a single geographical region (the Netherlands) impact the human gut resistome. Faecal samples from Dutch omnivores, pescatarians, vegetarians and vegans were analysed by metagenomic shotgun sequencing (MSS) (n = 149) and resistome capture sequencing approach (ResCap) (n = 64). Among all diet groups, 119 and 145 unique antibiotic resistance genes (ARGs) were detected by MSS or ResCap, respectively. Five or fifteen ARGs were shared between all diet groups, based on MSS and ResCap, respectively. The total number of detected ARGs by MSS or ResCap was not significantly different between the groups. MSS also revealed that vegans have a distinct microbiome composition, compared to other diet groups. Vegans had a lower abundance of Streptococcus thermophilus and Lactococcus lactis compared to pescatarians and a lower abundance of S. thermophilus when compared to omnivores. In summary, our study showed that long-term dietary habits are not associated with a specific resistome signature., (© 2022. The Author(s).)

The acquisition of antibiotic resistance (ABR) genes via horizontal gene transfer (HGT) is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of ABR. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of ABR. We analysed approximately 40 000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens, including Enterococcus , Staphylococcus , Acinetobacter and Pseudomonas . We modelled the association between CRISPR-Cas and indicators of HGT, and found that pathogens with a CRISPR-Cas system were less likely to carry ABR genes than those lacking this defence system. Analysis of the mobile genetic elements (MGEs) targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of ABR. These results suggest a potential 'immunocompromised' state for multidrug-resistant strains that may be exploited in tailored interventions that rely on MGEs, such as phages or phagemids, to treat infections caused by bacterial pathogens. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Common Variable Immunodeficiency (CVID) and X-linked agammaglobulinemia (XLA) are primary antibody deficiencies characterized by hypogammaglobulinemia and recurrent infections, which can lead to structural airway disease (AD) and interstitial lung disease (ILD). We investigated associations between serum IgA, oropharyngeal microbiota composition and severity of lung disease in these patients. In this cross-sectional multicentre study we analyzed oropharyngeal microbiota composition of 86 CVID patients, 12 XLA patients and 49 healthy controls (HC) using next-generation sequencing of the 16S rRNA gene. qPCR was used to estimate bacterial load. IgA was measured in serum. High resolution CT scans were scored for severity of AD and ILD. Oropharyngeal bacterial load was increased in CVID patients with low IgA ( p = 0.013) and XLA ( p = 0.029) compared to HC. IgA status was associated with distinct beta (between-sample) diversity ( p = 0.039), enrichment of ( Allo)prevotella , and more severe radiographic lung disease ( p = 0.003), independently of recent antibiotic use. AD scores were positively associated with Prevotella, Alloprevotella , and Selenomonas , and ILD scores with Streptococcus and negatively with Rothia . In clinically stable patients with CVID and XLA, radiographic lung disease was associated with IgA deficiency and expansion of distinct oropharyngeal bacterial taxa. Our findings highlight IgA as a potential driver of upper respiratory tract microbiota homeostasis., (Copyright © 2020 Berbers, Mohamed Hoesein, Ellerbroek, van Montfrans, Dalm, van Hagen, Paganelli, Viveen, Rogers, de Jong, Uh, Willems and Leavis.)

Various species of the intestinal microbiota have been associated with the development of colorectal cancer 1,2 , but it has not been demonstrated that bacteria have a direct role in the occurrence of oncogenic mutations. Escherichia coli can carry the pathogenicity island pks, which encodes a set of enzymes that synthesize colibactin 3 . This compound is believed to alkylate DNA on adenine residues 4,5 and induces double-strand breaks in cultured cells 3 . Here we expose human intestinal organoids to genotoxic pks + E. coli by repeated luminal injection over five months. Whole-genome sequencing of clonal organoids before and after this exposure revealed a distinct mutational signature that was absent from organoids injected with isogenic pks-mutant bacteria. The same mutational signature was detected in a subset of 5,876 human cancer genomes from two independent cohorts, predominantly in colorectal cancer. Our study describes a distinct mutational signature in colorectal cancer and implies that the underlying mutational process results directly from past exposure to bacteria carrying the colibactin-producing pks pathogenicity island.

The aim of this study was to determine whether dietary intervention influenced luminal Ca 2+ levels and Enterococcus faecium gut colonization in mice. For this purpose, mice fed semi-synthetic food AIN93 were compared to mice fed AIN93-low calcium (LC). Administration of AIN93-LC resulted in lower luminal Ca 2+ levels independent of the presence of E. faecium. Furthermore, E. faecium gut colonization was reduced in mice fed AIN93-LC based on culture, and which was in concordance with a reduction of Enterococcaceae in microbiota analysis. In conclusion, diet intervention might be a strategy for controlling gut colonization of E. faecium, an important opportunistic nosocomial pathogen., (© 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.)

Bariatric surgery in morbid obesity, either through sleeve gastrectomy (SG) or Roux-Y gastric bypass (RYGB), leads to sustainable weight loss, improvement of metabolic disorders and changes in intestinal microbiota. Yet, the relationship between changes in gut microbiota, weight loss and surgical procedure remains incompletely understood. We determined temporal changes in microbiota composition in 45 obese patients undergoing crash diet followed by SG (n = 22) or RYGB (n = 23). Intestinal microbiota composition was determined before intervention (baseline, S1), 2 weeks after crash diet (S2), and 1 week (S3), 3 months (S4) and 6 months (S5) after surgery. Relative to S1, the microbial diversity index declined at S2 and S3 (p < 0.05), and gradually returned to baseline levels at S5. Rikenellaceae relative abundance increased and Ruminococcaceae and Streptococcaceae abundance decreased at S2 (p < 0.05). At S3, Bifidobacteriaceae abundance decreased, whereas those of Streptococcaceae and Enterobacteriaceae increased (p < 0.05). Increased weight loss between S3-S5 was not associated with major changes in microbiota composition. No significant differences appeared between both surgical procedures. In conclusion, undergoing a crash diet and bariatric surgery were associated with an immediate but temporary decline in microbial diversity, with immediate and permanent changes in microbiota composition, independent of surgery type.

Enterococcus faecium has undergone a transition to a multidrug-resistant nosocomial pathogen. The population structure of E. faecium is characterized by a sharp distinction of clades, where the hospital-adapted lineage is primarily responsible for bacteremia. So far, factors that were identified in hospital-adapted strains and that promoted pathogenesis of nosocomial E. faecium mainly play a role in adherence and biofilm production, while less is known about factors contributing to survival in blood. This study identified a gene cluster, which includes genes encoding bacterial Toll/interleukin-1 receptor- (TIR-) domain-containing proteins (TirEs). The cluster was found to be unique to nosocomial strains and to be located on a putative mobile genetic element of phage origin. The three genes within the cluster appeared to be expressed as an operon. Expression was detected in bacterial culture media and in the presence of human blood. TirEs are released into the bacterial supernatant, and TirE2 is associated with membrane vesicles. Furthermore, the tirE -gene cluster promotes bacterial proliferation in human blood, indicating that TirE may contribute to the pathogenesis of bacteremia.

Human innate immunity employs cellular and humoral mechanisms to facilitate rapid killing of invading bacteria. The direct killing of bacteria by human serum is attributed mainly to the activity of the complement system, which forms pores in Gram-negative bacteria. Although Gram-positive bacteria are considered resistant to killing by serum, we uncover here that normal human serum effectively kills Enterococcus faecium Comparison of a well-characterized collection of commensal and clinical E. faecium isolates revealed that human serum specifically kills commensal E. faecium strains isolated from normal gut microbiota but not clinical isolates. Inhibitor studies show that the human group IIA secreted phospholipase A2 (hGIIA), but not complement, is responsible for killing of commensal E. faecium strains in human normal serum. This is remarkable since the hGIIA concentration in "noninflamed" serum was considered too low to be bactericidal against Gram-positive bacteria. Mechanistic studies showed that serum hGIIA specifically causes permeabilization of commensal E. faecium membranes. Altogether, we find that a normal concentration of hGIIA in serum effectively kills commensal E. faecium and that resistance of clinical E. faecium to hGIIA could have contributed to the ability of these strains to become opportunistic pathogens in hospitalized patients., (Copyright © 2018 American Society for Microbiology.)

Industrial areas on estuarine systems are commonly affected by heavy metals, affecting all local biota. Random Amplified Polymorphic DNA (RAPD) was used to evaluate genetic diversity of Ucides cordatus at mangroves in southeastern Brazil (Juréia, J; São Vicente, SV; and Cubatão, C), with distinct pollution levels by metals. The genetic diversity of this species was compared with concentrations of metals (Cd, Pb, Cu, Cr and Hg) in the environment. A pollution gradient was confirmed (SV>C>J), with low levels detected in water, except for mercury in SV. All metals in the sediment samples were below Threshold Effect Level (TEL), without an apparent biological risk to the biota. Genetic distance was very similar between J and C, with SV occurring as an out-group. RAPD was a powerful tool to investigate the effect of metal pollution on genetic diversity of this mangrove crab, and to evaluate the conservation status of the mangrove ecosystem., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Enterococcus faecium is a multidrug-resistant (MDR) nosocomial pathogen causing significant morbidity in debilitated patients. New antimicrobials are needed to treat antibiotic-resistant E. faecium infections in hospitalised patients. E. faecium incorporates lipoteichoic acid (LTA) (1,3-polyglycerol-phosphate linked to glycolipid) in its cell wall. The small-molecule inhibitor 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] specifically blocks the activity of Staphylococcus aureus LtaS synthase, which polymerises 1,3-glycerolphosphate into LTA polymers. Here we characterised the effects of the small-molecule inhibitor 1771 on the growth of E. faecium isolates, alone (28 strains) or in combination with the antibiotics vancomycin, daptomycin, ampicillin, gentamicin or linezolid (15 strains), and on biofilm formation (16 strains). Inhibition of LTA synthesis at the surface of the cell by compound 1771 in combination with current antibiotic therapy abrogates enterococcal growth in vitro but does not affect mature E. faecium biofilms. Targeting LTA synthesis may provide new possibilities to treat MDR E. faecium infections., (Copyright © 2017 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.)

The genus Bradyrhizobium comprises bacteria with the ability to form nitrogen-fixing symbioses with legumes. They are of great interest in agriculture, as well as for the production of biopolymers such as polyhydroxyalkanoates. Here, we report the draft genome assembly of Bradyrhizobium elkanii Tn phoA 33 comprising 9 Mb, 1,124 contigs, and 9,418 open reading frames., (Copyright © 2017 Lopes et al.)

Neisseria meningitidis is a Gram-negative bacterium that resides as a commensal in the upper respiratory tract of humans, but occasionally, it invades the host and causes sepsis and/or meningitis. The bacterium can produce eight autotransporters, seven of which have been studied to some detail. The remaining one, AutB, has not been characterized yet. Here, we show that the autB gene is broadly distributed among pathogenic Neisseria spp. The gene is intact in most meningococcal strains. However, its expression is prone to phase variation due to slipped-strand mispairing at AAGC repeats located within the DNA encoding the signal sequence and is switched off in the vast majority of these strains. Moreover, various genetic disruptions prevent autB expression in most of the strains in which the gene is in phase indicating a strong selection against AutB synthesis. We observed that autB is expressed in two of the strains examined and that AutB is secreted and exposed at the cell surface. Functionality assays revealed that AutB synthesis promotes biofilm formation and delays the passage of epithelial cell layers in vitro . We hypothesize that this autotransporter is produced during the colonization process only in specific niches to facilitate microcolony formation, but its synthesis is switched off probably to evade the immune system and facilitate human tissue invasion.

Enterococcus faecium is a common cause of nosocomial infections, of which infective endocarditis is associated with substantial mortality. In this study, we used a microarray-based transposon mapping (M-TraM) approach to evaluate a rat endocarditis model and identified a gene, originally annotated as "fruA" and renamed "bepA," putatively encoding a carbohydrate phosphotransferase system (PTS) permease (biofilm and endocarditis-associated permease A [BepA]), as important in infective endocarditis. This gene is highly enriched in E. faecium clinical isolates and absent in commensal isolates that are not associated with infection. Confirmation of the phenotype was established in a competition experiment of wild-type and a markerless bepA mutant in a rat endocarditis model. In addition, deletion of bepA impaired biofilm formation in vitro in the presence of 100% human serum and metabolism of β-methyl-D-glucoside. β-glucoside metabolism has been linked to the metabolism of glycosaminoglycans that are exposed on injured heart valves, where bacteria attach and form vegetations. Therefore, we propose that the PTS permease BepA is directly implicated in E. faecium pathogenesis., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.)

Additive manufacturing (3D printing) has enabled fabrication of geometrically complex and fully interconnected porous biomaterials with huge surface areas that could be used for biofunctionalization to achieve multifunctional biomaterials. Covering the huge surface area of such porous titanium with nanotubes has been already shown to result in improved bone regeneration performance and implant fixation. In this study, we loaded TiO2 nanotubes with silver antimicrobial agents to equip them with an additional biofunctionality, i.e., antimicrobial behavior. An optimized anodizing protocol was used to create nanotubes on the entire surface area of direct metal printed porous titanium scaffolds. The nanotubes were then loaded by soaking them in three different concentrations (i.e., 0.02, 0.1, and 0.5 M) of AgNO3 solution. The antimicrobial behavior and cell viability of the developed biomaterials were assessed. As far as the early time points (i.e., up to 1 day) are concerned, the biomaterials were found to be extremely effective in preventing biofilm formation and decreasing the number of planktonic bacteria particularly for the middle and high concentrations of silver ions. Interestingly, nanotubes not loaded with antimicrobial agents also showed significantly smaller numbers of adherent bacteria at day 1, which may be attributed to the bactericidal effect of high aspect ratio nanotopographies. The specimens with the highest concentrations of antimicrobial agents adversely affected cell viability at day 1, but this effect is expected to decrease or disappear in the following days as the rate of release of silver ions was observed to markedly decrease within the next few days. The antimicrobial effects of the biomaterials, particularly the ones with the middle and high concentrations of antimicrobial agents, continued until 2 weeks. The potency of the developed biomaterials in decreasing the number of planktonic bacteria and hindering the formation of biofilms make them promising candidates for combating peri-operative implant-associated infections.

Enterococcus faecium is a commensal of the mammalian gastrointestinal tract, but is also found in non-enteric environments where it can grow between 10 °C and 45 °C. E. faecium has recently emerged as a multi-drug resistant nosocomial pathogen. We hypothesized that genes involved in the colonization and infection of mammals exhibit temperature-regulated expression control and we therefore performed a transcriptome analysis of the clinical isolate E. faecium E1162, during mid-exponential growth at 25 °C and 37 °C. One of the genes that exhibited differential expression between 25 °C and 37 °C, was predicted to encode a peptidoglycan-anchored surface protein. The N-terminal domain of this protein is unique to E. faecium and closely related enterococci, while the C-terminal domain is homologous to the Streptococcus agalactiae surface protein BibA. This region of the protein contains proline-rich repeats, leading us to name the protein PrpA for proline-rich protein A. We found that PrpA is a surface-exposed protein which is most abundant during exponential growth at 37 °C in E. faecium E1162. The heterologously expressed and purified N-terminal domain of PrpA was able to bind to the extracellular matrix proteins fibrinogen and fibronectin. In addition, the N-terminal domain of PrpA interacted with both non-activated and activated platelets.

Unlabelled: The microbiota of the mammalian gastrointestinal tract is a complex ecosystem of bacterial communities that continuously interact with the mucosal immune system. In a healthy host, the mucosal immune system maintains homeostasis in the intestine and prevents invasion of pathogenic bacteria, a phenomenon termed colonization resistance. Antibiotics create dysbiosis of microbiota, thereby decreasing colonization resistance and facilitating infections caused by antibiotic-resistant bacteria. Here we describe how cephalosporin antibiotics create dysbiosis in the mouse large intestine, allowing intestinal outgrowth of antimicrobial-resistant Enterococcus faecium. This is accompanied by a reduction of the mucus-associated gut microbiota layer, colon wall, and Muc-2 mucus layer. E. faecium agglutinates intraluminally in an extracellular matrix consisting of secretory IgA (sIgA), polymeric immunoglobulin receptor (pIgR), and epithelial cadherin (E-cadherin) proteins, thereby maintaining spatial segregation of E. faecium from the intestinal wall. Addition of recombinant E-cadherin and pIgR proteins or purified IgA to enterococci in vitro mimics agglutination of E. faecium in vivo. Also, the Ca(2+) levels temporarily increased by 75% in feces of antibiotic-treated mice, which led to deformation of E-cadherin adherens junctions between colonic intestinal epithelial cells and release of E-cadherin as an extracellular matrix entrapping E. faecium. These findings indicate that during antibiotic-induced dysbiosis, the intestinal epithelium stays separated from an invading pathogen through an extracellular matrix in which sIgA, pIgR, and E-cadherin are colocalized. Future mucosal vaccination strategies to control E. faecium or other opportunistic pathogens may prevent multidrug-resistant infections, hospital transmission, and outbreaks., Importance: Infections with antibiotic-resistant enterococci are an emerging worldwide problem because enterococci are resistant to most of the antibiotics used in hospitals. During antibiotic treatment, the normal bacteria are replaced by resistant enterococci within the gut, from which they can spread and cause infections. We studied antibiotic-mediated intestinal proliferation of multidrug-resistant Enterococcus faecium and the effects on intestinal architecture. We demonstrated that antibiotics allow proliferation of E. faecium in the gut, alter the mucus-associated gut bacterial layer, and reduce the colon wall, mucus thickness, and amount of Muc-2 protein. E. faecium is agglutinated in the intestine in a matrix consisting of host molecules. We hypothesize that this matrix maintains a segregation of E. faecium from the epithelium. Understanding the processes that occur in the gut during antibiotic treatment may provide clues for future mucosal vaccination strategies to control E. faecium or other multidrug-resistant opportunistic pathogens, thereby preventing infections, hospital transmission, and outbreaks., (Copyright © 2015 Hendrickx et al.)

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Nowadays, Enterococcus faecium is one of the leading nosocomial pathogens worldwide. Strains causing clinical infections or hospital outbreaks are enriched in the enterococcal surface protein (Esp) encoding ICEEfm1 mobile genetic element. Previous studies showed that Esp is involved in biofilm formation, endocarditis and urinary tract infections. In this study, we characterized the role of the putative AraC type of regulator (locus tag EfmE1162&#95;2351), which we renamed ebrB and which is, based on the currently available whole genome sequences, always located upstream of the esp gene, and studied its role in Esp surface exposure during growth. A markerless deletion mutant of ebrB resulted in reduced esp expression and complete abolishment of Esp surface exposure, while Esp cell-surface exposure was restored when this mutant was complemented with an intact copy of ebrB. This demonstrates a role for EbrB in esp expression. However, during growth, ebrB expression levels did not change over time, while an increase in esp expression at both RNA and protein level was observed during mid-log and late-log phase. These results indicate the existence of a secondary regulation system for esp, which might be an unknown quorum sensing system as the enhanced esp expression seems to be cell density dependent. Furthermore, we determined that esp is part of an operon of at least 3 genes putatively involved in biofilm formation. A semi-static biofilm model revealed reduced biofilm formation for the EbrB deficient mutant, while dynamics of biofilm formation using a flow cell system revealed delayed biofilm formation in the ebrB mutant. In a mouse intestinal colonization model the ebrB mutant was less able to colonize the gut compared to wild-type strain, especially in the small intestine. These data indicate that EbrB positively regulates the esp operon and is implicated in biofilm formation and intestinal colonization.

Enterococcus faecium is an important multidrug-resistant nosocomial pathogen causing biofilm-mediated infections in patients with medical devices. Insight into E. faecium biofilm pathogenesis is pivotal for the development of new strategies to prevent and treat these infections. In several bacteria, a major autolysin is essential for extracellular DNA (eDNA) release in the biofilm matrix, contributing to biofilm attachment and stability. In this study, we identified and functionally characterized the major autolysin of E. faecium E1162 by a bioinformatic genome screen followed by insertional gene disruption of six putative autolysin genes. Insertional inactivation of locus tag EfmE1162&#95;2692 resulted in resistance to lysis, reduced eDNA release, deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and significant chaining compared to that of the wild type. Therefore, locus tag EfmE1162&#95;2692 was considered the major autolysin in E. faecium and renamed atlAEfm. In addition, AtlAEfm was implicated in cell surface exposure of Acm, a virulence factor in E. faecium, and thereby facilitates binding to collagen types I and IV. This is a novel feature of enterococcal autolysins not described previously. Furthermore, we identified (and localized) autolysin-independent DNA release in E. faecium that contributes to cell-cell interactions in the atlAEfm mutant and is important for cell separation. In conclusion, AtlAEfm is the major autolysin in E. faecium and contributes to biofilm stability and Acm localization, making AtlAEfm a promising target for treatment of E. faecium biofilm-mediated infections. IMPORTANCE Nosocomial infections caused by Enterococcus faecium have rapidly increased, and treatment options have become more limited. This is due not only to increasing resistance to antibiotics but also to biofilm-associated infections. DNA is released in biofilm matrix via cell lysis, caused by autolysin, and acts as a matrix stabilizer. In this study, we identified and characterized the major autolysin in E. faecium, which we designated AtlAEfm. atlAEfm disruption resulted in resistance to lysis, reduced extracellular DNA (eDNA), deficient cell attachment, decreased biofilm, decreased cell wall hydrolysis, and chaining. Furthermore, AtlAEfm is associated with Acm cell surface localization, resulting in less binding to collagen types I and IV in the atlAEfm mutant. We also identified AtlAEfm-independent eDNA release that contributes to cell-cell interactions in the atlAEfm mutant. These findings indicate that AtlAEfm is important in biofilm and collagen binding in E. faecium, making AtlAEfm a promising target for treatment of E. faecium infections.

Shigella flexneri is a Gram-negative bacillus that is responsible for a severe form of dysentery called Shigellosis, which mainly affects children and the elderly in both underdeveloped and developed countries. Pathogenic S. flexneri strains possess a large virulence plasmid that codes for effector proteins that are required for the entry and spread of the bacteria into colonocytes. Among these proteins is the translocator IpaC, which plays an important role in the invasion process; IpaC is implicated in pore formation in the host cell membrane and induces cytoskeletal rearrangements in macrophages and epithelial cells, thereby promoting bacterial entry. The ability of IpaC to insert onto the plasma membrane is due to a large nonpolar region of the protein structure. This characteristic also renders difficulties in recovery and purification when the protein is expressed in E. coli. Several works have considered different methodologies for the improved production and purification of IpaC. Herein, we propose an alternative method that is based on changes in the induction temperature and extraction buffer to facilitate the accumulation of high yields of soluble proteins for their further processing and ultimate use in biotechnological approaches.

Enterococcus faecium has become a nosocomial pathogen of major importance, causing infections that are difficult to treat owing to its multi-drug resistance. In particular, resistance to the β-lactam antibiotic ampicillin has become ubiquitous among clinical isolates. Mutations in the low-affinity penicillin binding protein PBP5 have previously been shown to be important for ampicillin resistance in E. faecium, but the existence of additional resistance determinants has been suggested. Here, we constructed a high-density transposon mutant library in E. faecium and developed a transposon mutant tracking approach termed Microarray-based Transposon Mapping (M-TraM), leading to the identification of a compendium of E. faecium genes that contribute to ampicillin resistance. These genes are part of the core genome of E. faecium, indicating a high potential for E. faecium to evolve towards β-lactam resistance. To validate the M-TraM results, we adapted a Cre-lox recombination system to construct targeted, markerless mutants in E. faecium. We confirmed the role of four genes in ampicillin resistance by the generation of targeted mutants and further characterized these mutants regarding their resistance to lysozyme. The results revealed that ddcP, a gene predicted to encode a low-molecular-weight penicillin binding protein with D-alanyl-D-alanine carboxypeptidase activity, was essential for high-level ampicillin resistance. Furthermore, deletion of ddcP sensitized E. faecium to lysozyme and abolished membrane-associated D,D-carboxypeptidase activity. This study has led to the development of a broadly applicable platform for functional genomic-based studies in E. faecium, and it provides a new perspective on the genetic basis of ampicillin resistance in this organism., Competing Interests: The authors have declared that no competing interests exist.

Enterococcus faecalis and Enterococcus faecium are among the leading causative agents of nosocomial infections and are infamous for their resistance to many antibiotics. They cause difficult-to-treat infections, often originating from biofilm-mediated infections associated with implanted medical devices or endocarditis. Biofilms protect bacteria against antibiotics and phagocytosis, and physical removal of devices or infected tissue is often needed but is frequently not possible. Currently there are no clinically available compounds that disassemble biofilms. In this review we discuss all known structural and regulatory genes involved in enterococcal biofilm formation, the compounds directed against biofilm formation that have been studied, and potentially useful targets for future drugs to treat enterococcal biofilm-associated infections., (Copyright © 2011 Elsevier Ltd. All rights reserved.)