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Journal articleMcKenney PT, Yan J, Vaubourgeix J, et al., 2019,
Intestinal bile acids induce a morphotype switch in vancomycin-resistant enterococcus that facilitates intestinal colonization
, Cell Host and Microbe, Vol: 25, Pages: 695-705.e5, ISSN: 1931-3128Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation.
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Journal articleSchuster C, Howard S, Grundling A, 2019,
Use of the counter selectable marker PheS* for genome engineering in Staphylococcus aureus
, Microbiology, Vol: 165, Pages: 572-584, ISSN: 1350-0872The gold standard tocreate gene deletions in Staphylococcus aureusis by homologous 16recombination using allelic exchange plasmids with a temperature sensitive origin of replication. A knockout vectorthat containsregions of homologyis first integrated into thechromosome of S. aureus by a single crossover event selected for at high temperatures (non-permissive for plasmid replication) and antibiotic selection. Next, the second crossover event is encouraged by 20growth without antibiotic selection at lowtemperature, leading at a certain frequencyto the excision of the plasmid and deletionof the gene of interest.To detector encourage plasmid loss, either a beta-galactosidase screening method or more typically, a counter selection step is used. We here present the adaption of the counter selectable markerpheS*, coding for a mutated subunit of the phenylalanine-tRNA-synthetase for use in S. aureus. The PheS* protein variant allows for the incorporation of the toxic phenylalanine amino acid analoguepara-chlorophenylalanine(PCPA)into proteins and the addition of 20-40 m M PCPAto rich medialeads to a drasticgrowth reductionof S. aureus and supplementing chemically defined medium with 2.5-5 mM PCPA to a complete growth inhibition. Using the new allelic exchange plasmid pIMAY*, wedeletedthe magnesium transporter gene mgtEin S. aureusUSA300 LAC*(SAUSA300_0910/ SAUSA300_RS04895) and RN4220(SAOUHSC_00945) and demonstrate that cobalt toxicity in S. aureusis mainly mediated by the presence of MgtE.This new plasmid will aidto efficiently and easily create gene knockouts in S. aureus.
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Journal articleHaag AF, Fitzgerald JR, Penades JR, 2019,
Staphylococcus aureus in Animals
, Microbiology Spectrum, Vol: 7, Pages: 1-19, ISSN: 2165-0497Staphylococcus aureus is a mammalian commensal and opportunistic pathogen that colonizes niches such as skin, nares and diverse mucosal membranes of about 20-30% of the human population. S. aureus can cause a wide spectrum of diseases in humans and both methicillin-sensitive and methicillin-resistant strains are common causes of nosocomial- and community-acquired infections. Despite the prevalence of literature characterising staphylococcal pathogenesis in humans, S. aureus is a major cause of infection and disease in a plethora of animal hosts leading to a significant impact on public health and agriculture. Infections in animals are deleterious to animal health, and animals can act as a reservoir for staphylococcal transmission to humans.Host-switching events between humans and animals and amongst animals are frequent and have been accentuated with the domestication and/or commercialisation of specific animal species. Host-switching is typically followed by subsequent adaptation through acquisition and/or loss of mobile genetic elements such as phages, pathogenicity islands and plasmids as well as further host-specific mutations allowing it to expand into new host populations.In this chapter, we will be giving an overview of S. aureus in animals, how this bacterial species was, and is, being transferred to new host species and the key elements thought to be involved in its adaptation to new ecological host niches. We will also highlight animal hosts as a reservoir for the development and transfer of antimicrobial resistance determinants.
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Journal articleRebollo-Ramirez S, Larrouy-Maumus G, 2019,
NaCl triggers the CRP-dependent increase of cAMP in Mycobacterium tuberculosis
, Tuberculosis, Vol: 116, Pages: 8-16, ISSN: 1472-9792The second messenger 3′,5′-cyclic adenosine monophosphate (3′,5′-cAMP) has been shown to be involved in the regulation of many biological processes ranging from carbon catabolite repression in bacteria to cell signalling in eukaryotes. In mycobacteria, the role of cAMP and the mechanisms utilized by the bacterium to adapt to and resist immune and pharmacological sterilization remain poorly understood. Among the stresses encountered by bacteria, ionic and non-ionic osmotic stresses are among the best studied. However, in mycobacteria, the link between ionic osmotic stress, particularly sodium chloride, and cAMP has been relatively unexplored. Using a targeted metabolic analysis combined with stable isotope tracing, we show that the pathogenic Mycobacterium tuberculosis but not the opportunistic pathogen Mycobacterium marinum nor the non-pathogenic Mycobacterium smegmatis responds to NaCl stress via an increase in intracellular cAMP levels. We further showed that this increase in cAMP is dependent on the cAMP receptor protein and in part on the threonine/serine kinase PnkD, which has previously been associated with the NaCl stress response in mycobacteria.
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Journal articleMcCarthy AJ, Birchenough GMH, Taylor PW, 2019,
Loss of Trefoil Factor 2 Sensitizes Rat Pups to Systemic Infection with the Neonatal Pathogen Escherichia coli K1
, Infection and Immunity, Vol: 87, ISSN: 0019-9567<jats:title>ABSTRACT</jats:title><jats:p>Gastrointestinal (GI) colonization of 2-day-old (P2) rat pups with <jats:named-content content-type="genus-species">Escherichia coli</jats:named-content> K1 results in translocation of the colonizing bacteria across the small intestine, bacteremia, and invasion of the meninges, with animals frequently succumbing to lethal infection. Infection, but not colonization, is strongly age dependent; pups become progressively less susceptible to infection over the P2-to-P9 period. Colonization leads to strong downregulation of the gene encoding trefoil factor 2 (Tff2), preventing maturation of the protective mucus barrier in the small intestine. Trefoil factors promote mucosal homeostasis. We investigated the contribution of Tff2 to protection of the neonatal rat from <jats:named-content content-type="genus-species">E. coli</jats:named-content> K1 bacteremia and tissue invasion. Deletion of <jats:italic>tff2</jats:italic>, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, sensitized P9 pups to <jats:named-content content-type="genus-species">E. coli</jats:named-content> K1 bacteremia. There were no differences between <jats:italic>tff2<jats:sup>−/</jats:sup></jats:italic><jats:sup>−</jats:sup> homozygotes and the wild type with regard to the dynamics of GI colonization. Loss of the capacity to elaborate Tff2 did not impact GI tract integrity or the thickness of the small-intestinal mucus layer but, in contrast to P9 wild-type pups, enabled <jats:named-content content-type="genus-species">E. coli</jats:named-content> K1 bacteria to gain access to epithelial surfaces in the distal region of the small intestine and exploit an intracellular route across the epithelial monolayer to enter the blood circulation via the mesenteric lymphatic system. Al
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Journal articleMcDonald JA, Perez JL, Mullish BH, et al., 2019,
Mo1953 – Growth inhibition of clostridioides difficile by short and medium chain fatty acids
, Gastroenterology, Vol: 156, Pages: S-898-S-898, ISSN: 0016-5085 -
Journal articleAllegretti JR, Hurtado J, Carrellas M, et al., 2019,
7 – The icon study: Inflammatory bowel disease and recurrent clostridium difficile infection: Outcomes after fecal microbiota transplantation
, Gastroenterology, Vol: 156, Pages: S-2-S-3, ISSN: 0016-5085 -
Journal articleAllegretti JR, Kassam Z, Chiang AL, et al., 2019,
621 – Fecal microbiota transplantation for the treatment of obesity: A randomized, placebo-controlled pilot trial
, Gastroenterology, Vol: 156, Pages: S-129-S-129, ISSN: 0016-5085 -
Journal articleHegade VS, Pechlivanis A, McDonald JA, et al., 2019,
Autotaxin, bile acid profile and effect of IBAT inhibition in primary biliary cholangitis patients with pruritus
, Liver International, Vol: 39, Pages: 967-975, ISSN: 1478-3223BACKGROUND &AIMS: Pruritus is a common symptom in patients with primary biliary cholangitis (PBC) for which ileal bile acid transporter (IBAT) inhibition is emerging as a potential therapy. We explored the serum metabonome and gut microbiota profile in PBC patients with pruritus and investigated the effect of GSK2330672, an IBAT inhibitor. METHODS: We studied fasting serum bile acids (BAs), autotaxin and faecal microbiota in 22 PBC patients with pruritus at baseline and after two weeks of GSK2330672 treatment. Control group included 31 asymptomatic PBC patients and 18 healthy volunteers. BA profiling was done by ultraperformance liquid chromatography coupled to a mass spectrometry (UPLC-MS). Faecal microbiomes were analysed by 16S ribosomal RNA gene sequencing. RESULTS: In PBC patients with pruritus serum levels of total and glyco-conjugated primary BAs and autotaxin were significantly elevated. Autotaxin activity correlated significantly with tauro- and glyco-conjugated cholic acid (CA) and chenodeoxycholic acid (CDCA), both at baseline and after GSK2330672. GSK2330672 significantly reduced autotaxin and all tauro- and glyco- conjugated BAs and increased faecal levels of CA (p=0.048) and CDCA (p=0.027). Gut microbiota of PBC patients with pruritus was similar to control groups. GSK2330672 increased relative abundance of Firmicutes (p=0.033) and Clostridia (p=0.04) and decreased Bacteroidetes (p=0.033) and Bacteroidia (p=0.04). CONCLUSIONS: Pruritus in PBC does not show a distinct gut bacterial profile but is associated with elevated serum bile acid and autotaxin levels which decrease after IBAT inhibition. In cholestatic pruritus, a complex interplay between BAs and ATX is likely and may be modified by IBAT inhibition. This article is protected by copyright. All rights reserved.
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Journal articleRoy RB, Sambou B, Uhia I, et al., 2019,
An auto-luminescent fluorescent BCG whole blood assay to enable evaluation of paediatric mycobacterial responses using minimal blood volumes
, Frontiers in Pediatrics, Vol: 7, ISSN: 2296-2360Introduction: Understanding protective human immunity against mycobacteria is critical to developing and evaluating new vaccines against tuberculosis. Children are the most susceptible population to infection, disease, and death from tuberculosis, but also have the strongest evidence of BCG-inducible protection. Limited amounts of blood can be obtained for research purposes in paediatrics and therefore there is a need for high-yield, low-volume, human immunology assays.Methods: We transformed BCG Danish with plasmids encoding luciferase full operon derived from Photorhabdus luminescens together with Green Fluorescent Protein and antibiotic selection markers. We characterised the luminescent and fluorescent properties of this recombinant BCG strain (BCG-GFP-LuxFO) using a luminometer and flow cytometry and developed a paediatric whole blood in vitro infection model.Results: Luminescence of BCG-GFP-LuxFO correlated with optical density (Spearman Rank Correlation coefficient r = 0.985, p < 0.0001) and colony forming units (CFUs) in liquid culture medium (r = 0.971, p < 0.0001). Fluorescence of BCG-GFP-LuxFO in paediatric whole blood was confirmed by flow cytometry in granulocytes and monocytes 1 h following infection. Luminescence of BCG-GFP-LuxFO in whole blood corresponded with CFUs (r = 0.7123, p < 0.0001).Conclusion: The BCG-GFP-LuxFO assay requires 225 μL whole blood per sample, from which serial luminescence measurements can be obtained, together with biochemical analysis of supernatants and cellular assay applications using its fluorescent properties. This offers the opportunity to study human-mycobacterial interactions using multiple experimental modalities with only minimal blood volumes. It is therefore a valuable method for investigating paediatric immunity to tuberculosis.
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