Reporters for Single-Cell Analysis of Colicin Ib Expression in Salmonella enterica Serovar Typhimurium

Colicins are toxins that mediate interference competition in microbial ecosystems. They serve as a "common good" for the entire producer population but are synthesized by only few members which pay the costs of colicin production. We have previously shown that production of colicin Ib (cib),a group B colicin, confers a competitive advantage to Salmonella enterica serovar Typhimurium (S. Tm) over commensal E. coli strains. Here, we studied regulation of S. Tm cib expression at the single cell level. Comparative analysis of a single-and a multicopy gfp-reporter for the colicin Ib promoter (Pcib) revealed that the latter yielded optimal signal intensity for a diverse range of applications. We further validated this reporter and showed that gfp expression correlated well with colicin Ib (ColIb) protein levels in individual cells. Pcib is negatively controlled by two repressors, LexA and Fur. Only a small fraction of S. Tm expressed cib under non-inducing conditions. We studied Pcib activity in response to mitomycin C mediated DNA damage and iron limitation. Both conditions, if applied individually, lead to an increase in the fraction of GFP(+) S. Tm, albeit an overall low fluorescence intensity. When both conditions were applied simultaneously, the majority of S. Tm turned GFP(+) and displayed high fluorescence intensity. Thus, both repressors individually confine cib expression to a subset of the population. Taken together, we provide the first thorough characterization of a conventional gfp-reporter to study regulation of a group B colicin at the single cell level. This reporter will be useful to further investigate the costs and benefits of ColIb production in human pathogenic S. Tm and analyze cib expression under environmental conditions encountered in the mammalian gut

Usability of rectal swabs for microbiome sampling in a cohort study of hematological and oncological patients

Objectives Large-scale clinical studies investigating associations between intestinal microbiota signatures and human diseases usually rely on stool samples. However, the timing of repeated stool sample collection cannot be predefined in longitudinal settings. Rectal swabs, being straightforward to obtain, have the potential to overcome this drawback. Therefore, we assessed the usability of rectal swabs for microbiome sampling in a cohort of hematological and oncological patients. Study design We used a pipeline for intestinal microbiota analysis from deep rectal swabs which was established and validated with test samples and negative controls. Consecutively, a cohort of patients from hematology and oncology wards was established and weekly deep rectal swabs taken during their admissions and re-admissions. Results Validation of our newly developed pipeline for intestinal microbiota analysis from rectal swabs revealed consistent and reproducible results. Over a period of nine months, 418 rectal swabs were collected longitudinally from 41 patients. Adherence to the intended sampling protocol was 97%. After DNA extraction, sequencing, read pre-processing and filtering of chimeric sequences, 405 of 418 samples (96.9%) were eligible for further analyses. Follow-up samples and those taken under current antibiotic exposure showed a significant decrease in alpha diversity as compared to baseline samples. Microbial domination occurred most frequently by Enterococcaceae (99 samples, 24.4%) on family level and Enterococcus (90 samples, 22.2%) on genus level. Furthermore, we noticed a high abundance of potential skin commensals in 99 samples (24.4%). Summary Deep rectal swabs were shown to be reliable for microbiome sampling and analysis, with practical advantages related to high sampling adherence, easy timing, transport and storage. The relatively high abundance of putative skin commensals in this patient cohort may be of potential interest and should be further investigated. Generally, previous findings on alpha diversity dynamics obtained from stool samples were confirmed

Phylogenetic congruence and ecological coherence in terrestrial Thaumarchaeota

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. Acknowledgements We would like to thank Dr Robert Griffith/CEH for providing DNA from soil samples and Dr Anthony Travis for his help with BioLinux. Sequencing was performed in NERC platform in Liverpool. CG-R was funded by a NERC fellowship NE/J019151/1. CQ was funded by a MRC fellowship (MR/M50161X/1) as part of the cloud infrastructure for microbial genomics consortium (MR/L015080/1).Peer reviewedPublisher PD

Patterns of Gene Flow Define Species of Thermophilic Archaea

A genomic view of speciation in Archaea shows higher rates of gene flow within coexisting microbial species than between them

Scalable Reporter Assays to Analyze the Regulation of stx2 Expression in Shiga Toxin-Producing Enteropathogens

Stx2 is the major virulence factor of EHEC and is associated with an increased risk for HUS in infected patients. The conditions influencing its expression in the intestinal tract are largely unknown. For optimal management and treatment of infected patients, the identification of environmental conditions modulating Stx2 levels in the human gut is of central importance. In this study, we established a set of chromosomal stx2 reporter assays. One system is based on superfolder GFP (sfGFP) using a T7 polymerase/T7 promoter-based amplification loop. This reporter can be used to analyze stx2 expression at the single-cell level using FACSs and fluorescence microscopy. The other system is based on the cytosolic release of the Gaussia princeps luciferase (gluc). This latter reporter proves to be a highly sensitive and scalable reporter assay that can be used to quantify reporter protein in the culture supernatant. We envision that this new set of reporter tools will be highly useful to comprehensively analyze the influence of environmental and host factors, including drugs, small metabolites and the microbiota, on Stx2 release and thereby serve the identification of risk factors and new therapies in Stx-mediated pathologies

Evaluation of a Novel CLIA Monotest Assay for the Detection of Anti-Hepatitis E Virus-IgG and IgM: A Retrospective Comparison with a Line Blot and an ELISA

Despite the increasing relevance of Hepatitis E, an emerging disease endemic in developing and with increasing numbers of sporadic cases in industrialized countries, commercial tests are mainly based on batch oriented serological assays. In this retrospective study, we compared a line immunoassay (LIA; recomLine HEV, Mikrogen) and an ELISA (EIA; Anti-Hepatitis E Virus ELISA, Euroimmun) with a novel chemoluminescence immunoassay in a monotest format (CLIA; Hepatitis E VirClia, Vircell). Twenty sera of PCR proven cases of hepatitis E and 68 blood samples serologically pre-characterized were included. Applying the WHO reference standard, the CLIA demonstrated the highest analytical sensitivity for IgG and IgM. The combinations of CLIA/EIA (IgG and IgM) and CLIA/LIA (IgG) measurements showed substantial correlation. Compared to overall antibody detection (seropositivity in ≥2 assays), CLIA correlation was excellent, outperforming LIA (IgM) and EIA (IgG and IgM). Minor IgM cross reactivity in samples of patients with acute EBV infection was observed in all three assays. The CLIA showed good performance in diagnostic samples compared to established LIA and EIA assays. Due to its ready-to-use monotest format, the CLIA allows simple, time- and cost-effective handling of single samples. These qualities make the assay suitable for diagnostics, especially in the emergency setting and for low-throughput laboratories

Influence of the multicopy reporter p^{Pcib gfp} and its derivatives on expression of native <i>S</i>. Tm p2^cib-HA at the single cell level.

<p><i>S</i>. Tm p2 ^{<i>cib-HA</i>} transformed with p^{Pcib gfp} or the derivatives (p^control, p^Pcib, p^<i>gfp</i> and p^{Prpsm gfp}), respectively, were grown in LB (+ampicillin) overnight (12h; stationary phase) or subcultured for 4h in LB (late logarithmic phase) with or without indicated supplements (0.25μg/ml MitC, 100μM DTPA). Bacteria were fixed, permeabilized and intracellular ColIb-HA was detected using HA-specific antiserum and a DyLight 594-conjugated secondary antibody, DNA was stained with DAPI. Bacteria were imaged by confocal microscopy and fluorescence of DAPI, GFP and DyLight 594-conjugate was recorded. Mean fluorescence intensity (MFI) of ColIb-HA-DyLight594 <b>(A,C,E,G,I)</b> and GFP <b>(B,D,F,H,J)</b> as determined by ImageJ is shown. Dots represent MFI of individual objects (bacteria). Bars represent the median and the dotted line the detection limit (background fluorescence of <i>S</i>. Tm^wt p^control). Values indicate the fraction (%) of the population above detection limit (blue). Statistical analysis was done using Kruskal-Wallis test with Dunn's post test (* <i>p</i> < 0.05).</p

Bacteria and plasmids used in this study.

<p>Bacteria and plasmids used in this study.</p

Detection of ColIb-HA within individual bacteria.

<p><b>(A)</b> Schematic of the modified ColIb locus in the strain <i>S</i>. Tm p2^{<i>cib-HA</i>}. <i>S</i>. Tm p2^{<i>cib-HA</i>} p^control and <i>S</i>. Tm^wt p^control were grown in LB supplemented with 100μM DTPA <b>(B)</b> or LB supplemented 0.25μg/ml MitC + 100μM DTPA <b>(C)</b>. Bacteria were fixed, permeabilized and intracellular ColIb-HA was detected using HA-specific antiserum (red). DNA was stained with DAPI (greyscale). Scale bar 25μm.</p

Inflammation Fuels Colicin Ib-Dependent Competition of <i>Salmonella</i> Serovar Typhimurium and <i>E. coli</i> in <i>Enterobacterial</i> Blooms

<div><p>The host's immune system plays a key role in modulating growth of pathogens and the intestinal microbiota in the gut. In particular, inflammatory bowel disorders and pathogen infections induce shifts of the resident commensal microbiota which can result in overgrowth of <i>Enterobacteriaceae</i> (“inflammation-inflicted blooms”). Here, we investigated competition of the human pathogenic <i>Salmonella enterica</i> serovar Typhimurium strain SL1344 (<i>S.</i> Tm) and commensal <i>E. coli</i> in inflammation-inflicted blooms. <i>S.</i> Tm produces colicin Ib (ColIb), which is a narrow-spectrum protein toxin active against related <i>Enterobacteriaceae</i>. Production of ColIb conferred a competitive advantage to <i>S.</i> Tm over sensitive <i>E. coli</i> strains in the inflamed gut. In contrast, an avirulent <i>S.</i> Tm mutant strain defective in triggering gut inflammation did not benefit from ColIb. Expression of ColIb (<i>cib</i>) is regulated by iron limitation and the SOS response. CirA, the cognate outer membrane receptor of ColIb on colicin-sensitive <i>E. coli</i>, is induced upon iron limitation. We demonstrate that growth in inflammation-induced blooms favours expression of both <i>S.</i> Tm ColIb and the receptor CirA, thereby fuelling ColIb dependent competition of <i>S.</i> Tm and commensal <i>E. coli</i> in the gut. In conclusion, this study uncovers a so-far unappreciated role of inflammation-inflicted blooms as an environment favouring ColIb-dependent competition of pathogenic and commensal representatives of the <i>Enterobacteriaceae</i> family.</p></div