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Dvorak J, Fojtík L, Adámková L, Vlkova K, Studentova V, Chudejova K, Geigerová L, Volny M, Novak P, Hrabak J, Pompach P. Proof-of-concept MALDI-TOF-MS assay for the detection of Toxin B enzymatic activity in Clostridioides difficile infection. Microbiol Spectr 2025; 13:e0245324. [PMID: 40162757 PMCID: PMC12054005 DOI: 10.1128/spectrum.02453-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2024] [Accepted: 03/12/2025] [Indexed: 04/02/2025] Open
Abstract
Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometers have become an integral part of all modern clinical microbiology laboratories. They serve as the key tool for pathogen identification and antibiotic resistance determination. However, certain limiting conditions must be fulfilled. The pathogen cannot be tested directly from the sample and requires the cultivation of a pure colony, which means that the standard protocol takes additional time, workforce, and consumables. The testing protocol is also more complicated when it comes to anaerobes. In our work, we focused on the functional detection of Clostridioides difficile, an important nosocomial human pathogen that is responsible for diarrhea and can lead to life-threatening colitis, as a model diagnostic problem. The virulence of C. difficile is mainly caused by two toxins, Toxin A and Toxin B. Established diagnostic methods, including nucleic acid amplification testing methods and immunoassays, detect the presence of the microorganism or the presence and concentration of the toxins, with limited ability to gauge infection severity based on the actual biochemical activity of the toxins and thus their potency to cause harm. This work presents proof-of-concept assays that indirectly determine the toxin activity in the human stool, a very complex matrix sample, using the natural RhoA protein as substrate. The RhoA protein substrate was recombinantly prepared with biotin tag modification, which allows its attachment to the NeutrAvidin MALDI chips. In the assay, the RhoA substrate anchored on the MALDI chip undergoes enzymatic glycosylation when exposed to the Toxin B in the stool sample, and the reaction product is then detected by MALDI-TOF mass spectrometry directly from the MALDI chip. The entire assay, from sampling to final mass spectrometry detection, was performed in situ, on the NeutrAvidin MALDI chip, which was prepared by unique surface modification technology also described in this work. The assay was successfully tested for the detection of Toxin B in a cohort of patient samples as well as in cell culture of C. difficile. IMPORTANCE The diagnostics of Clostridioides difficile infection is usually based on the identification of the bacterial pathogen and/or on the detection of the Toxins A and B. Due to the variance in Toxins A and B activity across species, the toxin concentration determined by standard methods does not necessarily correlate with the severity of the disease. Assays that would target toxins' enzymatic activity are not routinely used because the requirements are unsuitable for clinical laboratories. In this study, we demonstrate a new approach that determines the presence and potency of Toxin B indirectly by determining its enzymatic activity rather than its concentration. This is performed by detecting mass difference due to glycosylation of RhoA substrate by Toxin B, which is then determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presented proof-of-concept assay thus offers the possibility to quickly determine the activity of C. difficile toxins directly in the stool samples without pathogen cultivation.
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Affiliation(s)
- Josef Dvorak
- Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
- Department of Biochemistry, Charles University, Prague, Czechia
| | - Lukáš Fojtík
- Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
- Department of Biochemistry, Charles University, Prague, Czechia
| | - Ljubina Adámková
- Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
| | - Katerina Vlkova
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
- Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
| | - Vendula Studentova
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
- Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
| | - Katerina Chudejova
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
- Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
| | - Lenka Geigerová
- Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
| | - Michael Volny
- Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
- Department of Analytical Chemistry, University of Chemistry and Technology, Prague, Czechia
| | - Petr Novak
- Institute of Microbiology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
| | - Jaroslav Hrabak
- Biomedical Center, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
- Department of Microbiology, Faculty of Medicine in Pilsen, Charles University, Pilsen, Czechia
| | - Petr Pompach
- Department of Biochemistry, Charles University, Prague, Czechia
- Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Vestec, Czechia
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Dost I, Abdel-Glil M, Persson S, Conza KL, Oleastro M, Alves F, Maurischat S, Scholtzek A, Mazuet C, Diancourt L, Tenson T, Schmoock G, Neubauer H, Schwarz S, Seyboldt C. Genomic study of European Clostridioides difficile ribotype 002/sequence type 8. Microb Genom 2024; 10:001270. [PMID: 39051872 PMCID: PMC11316560 DOI: 10.1099/mgen.0.001270] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Accepted: 06/21/2024] [Indexed: 07/27/2024] Open
Abstract
Clostridioides difficile has significant clinical importance as a leading cause of healthcare-associated infections, with symptoms ranging from mild diarrhoea to severe colitis, and possible life-threatening complications. C. difficile ribotype (RT) 002, mainly associated with MLST sequence type (ST) 8, is one of the most common RTs found in humans. This study aimed at investigating the genetic characteristics of 537 C. difficile genomes of ST8/RT002. To this end, we sequenced 298 C. difficile strains representing a new European genome collection, with strains from Germany, Denmark, France and Portugal. These sequences were analysed against a global dataset consisting of 1,437 ST8 genomes available through Enterobase. Our results showed close genetic relatedness among the studied ST8 genomes, a diverse array of antimicrobial resistance (AMR) genes and the presence of multiple mobile elements. Notably, the pangenome analysis revealed an open genomic structure. ST8 shows relatively low overall variation. Thus, clonal isolates were found across different One Health sectors (humans, animals, environment and food), time periods, and geographical locations, suggesting the lineage's stability and a universal environmental source. Importantly, this stability did not hinder the acquisition of AMR genes, emphasizing the adaptability of this bacterium to different selective pressures. Although only 2.4 % (41/1,735) of the studied genomes originated from non-human sources, such as animals, food, or the environment, we identified 9 cross-sectoral core genome multilocus sequence typing (cgMLST) clusters. Our study highlights the importance of ST8 as a prominent lineage of C. difficile with critical implications in the context of One Health. In addition, these findings strongly support the need for continued surveillance and investigation of non-human samples to gain a more comprehensive understanding of the epidemiology of C. difficile.
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Affiliation(s)
- Ines Dost
- Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Straße 96a, 07743 Jena, Germany
| | - Mostafa Abdel-Glil
- Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Straße 96a, 07743 Jena, Germany
| | - Søren Persson
- Statens Serum Institut, Dept. Bacteria, Parasites and Fungi, Unit of Foodborne Infections, Artillerivej 5, 2300 Copenhagen, Denmark
| | - Karen Loaiza Conza
- Statens Serum Institut, Dept. Bacteria, Parasites and Fungi, Unit of Foodborne Infections, Artillerivej 5, 2300 Copenhagen, Denmark
| | - Mónica Oleastro
- National Reference Laboratory of Gastrointestinal Infections, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), 1649-016 Lisbon, Portugal
| | - Frederico Alves
- National Reference Laboratory of Gastrointestinal Infections, Department of Infectious Diseases, National Institute of Health Doutor Ricardo Jorge (INSA), 1649-016 Lisbon, Portugal
- Chief Scientific Office, European Food Safety Authority (EFSA), Parma, Italy
| | - Sven Maurischat
- German Federal Institute for Risk Assessment, Department Biological Safety, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany
| | - Anissa Scholtzek
- German Federal Institute for Risk Assessment, Department Biological Safety, Max-Dohrn-Str. 8-10, 10589 Berlin, Germany
| | - Christelle Mazuet
- Institut Pasteur, Université Paris Cité, Centre National de Référence Bactéries anaérobies et Botulisme, F-75015 Paris, France
| | - Laure Diancourt
- Institut Pasteur, Université Paris Cité, Centre National de Référence Bactéries anaérobies et Botulisme, F-75015 Paris, France
| | - Tanel Tenson
- Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
| | - Gernot Schmoock
- Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Straße 96a, 07743 Jena, Germany
| | - Heinrich Neubauer
- Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Straße 96a, 07743 Jena, Germany
| | - Stefan Schwarz
- Institute of Microbiology and Epizootics, Centre for Infection Medicine, School of Veterinary Medicine, Freie Universität Berlin, 14163 Berlin, Germany
- Veterinary Centre for Resistance Research (TZR), Freie Universität Berlin, 14163 Berlin, Germany
| | - Christian Seyboldt
- Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Naumburger Straße 96a, 07743 Jena, Germany
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3
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Nath A, Bhattacharjee R, Nandi A, Sinha A, Kar S, Manoharan N, Mitra S, Mojumdar A, Panda PK, Patro S, Dutt A, Ahuja R, Verma SK, Suar M. Phage delivered CRISPR-Cas system to combat multidrug-resistant pathogens in gut microbiome. Biomed Pharmacother 2022; 151:113122. [PMID: 35594718 DOI: 10.1016/j.biopha.2022.113122] [Citation(s) in RCA: 19] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2022] [Revised: 05/08/2022] [Accepted: 05/10/2022] [Indexed: 11/02/2022] Open
Abstract
The Host-microbiome interactions that exist inside the gut microbiota operate in a synergistic and abnormal manner. Additionally, the normal homeostasis and functioning of gut microbiota are frequently disrupted by the intervention of Multi-Drug Resistant (MDR) pathogens. CRISPR-Cas (CRISPR-associated protein with clustered regularly interspersed short palindromic repeats) recognized as a prokaryotic immune system has emerged as an effective genome-editing tool to edit and delete specific microbial genes for the expulsion of bacteria through bactericidal action. In this review, we demonstrate many functioning CRISPR-Cas systems against the anti-microbial resistance of multiple pathogens, which infiltrate the gastrointestinal tract. Moreover, we discuss the advancement in the development of a phage-delivered CRISPR-Cas system for killing a gut MDR pathogen. We also discuss a combinatorial approach to use bacteriophage as a delivery system for the CRISPR-Cas gene for targeting a pathogenic community in the gut microbiome to resensitize the drug sensitivity. Finally, we discuss engineered phage as a plausible potential option for the CRISPR-Cas system for pathogenic killing and improvement of the efficacy of the system.
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Affiliation(s)
- Arijit Nath
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | - Rahul Bhattacharjee
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | - Aditya Nandi
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | - Adrija Sinha
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | - Sulagna Kar
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | | | - Shirsajit Mitra
- KaviKrishna Laboratory, Indian Institute of Technology, Guwahati, Assam, India
| | - Abhik Mojumdar
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India
| | - Pritam Kumar Panda
- Condensed Matter Theory Group, Materials Theory Division, Department of Physics and Astronomy, Uppsala University, Box 516, SE-751 20 Uppsala, Sweden
| | - Swadheena Patro
- KIIT School of Dental Sciences, KIIT University. Bhubaneswar 751024, Odisha
| | - Ateet Dutt
- Instituto de Investigaciones en Materiales, UNAM, CDMX, Mexico
| | - Rajeev Ahuja
- Condensed Matter Theory Group, Materials Theory Division, Department of Physics and Astronomy, Uppsala University, Box 516, SE-751 20 Uppsala, Sweden.
| | - Suresh K Verma
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India; Condensed Matter Theory Group, Materials Theory Division, Department of Physics and Astronomy, Uppsala University, Box 516, SE-751 20 Uppsala, Sweden.
| | - Mrutyunjay Suar
- KIIT School of Biotechnology, KIIT University, Bhubaneswar 751024, Odisha, India.
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Smits WK, Roseboom AM, Corver J. Plasmids of Clostridioides difficile. Curr Opin Microbiol 2021; 65:87-94. [PMID: 34775173 DOI: 10.1016/j.mib.2021.10.016] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 10/20/2021] [Accepted: 10/23/2021] [Indexed: 12/16/2022]
Abstract
Plasmids are ubiquitous in the bacterial world. In many microorganisms, plasmids have been implicated in important aspects of bacterial physiology and contribute to horizontal gene transfer. In contrast, knowledge on plasmids of the enteropathogen Clostridioides difficile is limited, and there appears to be no phenotypic consequence to carriage of many of the identified plasmids. Emerging evidence suggests, however, that plasmids are common in C. difficile and may encode functions relevant to pathogenesis, such as antimicrobial resistance and toxin production. Here, we review our current knowledge about the abundance, functions and clinical relevance of plasmids in C. difficile.
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Affiliation(s)
- Wiep Klaas Smits
- Experimental Bacteriology Group, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands; Centre for Microbial Cell Biology, Leiden, The Netherlands; Leiden University Center for Infectious Diseases (LU-CID), Leiden, The Netherlands.
| | - Anna Maria Roseboom
- Experimental Bacteriology Group, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
| | - Jeroen Corver
- Experimental Bacteriology Group, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands; Centre for Microbial Cell Biology, Leiden, The Netherlands; Leiden University Center for Infectious Diseases (LU-CID), Leiden, The Netherlands
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5
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Kartalidis P, Skoulakis A, Tsilipounidaki K, Florou Z, Petinaki E, Fthenakis GC. Clostridioides difficile as a Dynamic Vehicle for the Dissemination of Antimicrobial-Resistance Determinants: Review and In Silico Analysis. Microorganisms 2021; 9:microorganisms9071383. [PMID: 34202117 PMCID: PMC8307371 DOI: 10.3390/microorganisms9071383] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/24/2021] [Revised: 06/21/2021] [Accepted: 06/22/2021] [Indexed: 01/11/2023] Open
Abstract
The present paper is divided into two parts. The first part focuses on the role of Clostridioides difficile in the accumulation of genes associated with antimicrobial resistance and then the transmission of them to other pathogenic bacteria occupying the same human intestinal niche. The second part describes an in silico analysis of the genomes of C. difficile available in GenBank, with regard to the presence of mobile genetic elements and antimicrobial resistance genes. The diversity of the C. difficile genome is discussed, and the current status of resistance of the organisms to various antimicrobial agents is reviewed. The role of transposons associated with antimicrobial resistance is appraised; the importance of plasmids associated with antimicrobial resistance is discussed, and the significance of bacteriophages as a potential shuttle for antimicrobial resistance genes is presented. In the in silico study, 1101 C. difficile genomes were found to harbor mobile genetic elements; Tn6009, Tn6105, CTn7 and Tn6192, Tn6194 and IS256 were the ones more frequently identified. The genes most commonly harbored therein were: ermB, blaCDD, vanT, vanR, vanG and vanS. Tn6194 was likely associated with resistance to erythromycin, Tn6192 and CTn7 with resistance to the β-lactams and vancomycin, IS256 with resistance to aminoglycoside and Tn6105 to vancomycin.
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Affiliation(s)
- Philip Kartalidis
- Department of Clinical and Laboratory Research, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41110 Larissa, Greece; (P.K.); (A.S.); (K.T.); (Z.F.); (E.P.)
| | - Anargyros Skoulakis
- Department of Clinical and Laboratory Research, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41110 Larissa, Greece; (P.K.); (A.S.); (K.T.); (Z.F.); (E.P.)
| | - Katerina Tsilipounidaki
- Department of Clinical and Laboratory Research, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41110 Larissa, Greece; (P.K.); (A.S.); (K.T.); (Z.F.); (E.P.)
| | - Zoi Florou
- Department of Clinical and Laboratory Research, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41110 Larissa, Greece; (P.K.); (A.S.); (K.T.); (Z.F.); (E.P.)
| | - Efthymia Petinaki
- Department of Clinical and Laboratory Research, Faculty of Medicine, School of Health Sciences, University of Thessaly, 41110 Larissa, Greece; (P.K.); (A.S.); (K.T.); (Z.F.); (E.P.)
| | - George C. Fthenakis
- Veterinary Faculty, University of Thessaly, 43100 Karditsa, Greece
- Correspondence:
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Marsh JW, Pacey MP, Ezeonwuka C, Ohm SL, Snyder D, Cooper VS, Harrison LH, Doi Y, Mustapha MM. Clostridioides difficile: a potential source of NpmA in the clinical environment. J Antimicrob Chemother 2020; 74:521-523. [PMID: 30295814 DOI: 10.1093/jac/dky420] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Jane W Marsh
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Marissa P Pacey
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Center for Innovative Antimicrobial Therapy, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Chinelo Ezeonwuka
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Sara L Ohm
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Dan Snyder
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Vaughn S Cooper
- Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Lee H Harrison
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
| | - Yohei Doi
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Center for Innovative Antimicrobial Therapy, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Department of Microbiology, Fujita Health University, Aichi, Japan
| | - Mustapha M Mustapha
- Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Microbial Genomic Epidemiology Laboratory (MiGEL), University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.,Center for Innovative Antimicrobial Therapy, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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7
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Plasmid-mediated metronidazole resistance in Clostridioides difficile. Nat Commun 2020; 11:598. [PMID: 32001686 PMCID: PMC6992631 DOI: 10.1038/s41467-020-14382-1] [Citation(s) in RCA: 83] [Impact Index Per Article: 16.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Accepted: 12/24/2019] [Indexed: 12/17/2022] Open
Abstract
Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L−1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections. Cases of C. difficile (CD) resistant to metronidazole have been reported but the mechanism remains enigmatic. Here the authors identify a plasmid, which correlates with metronidazole resistance status in a large international collection of CD isolates, and demonstrate that the plasmid can confer metronidazole resistance.
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8
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Wu Y, Yang L, Li WG, Zhang WZ, Liu ZJ, Lu JX. Microevolution within ST11 group Clostridioides difficile isolates through mobile genetic elements based on complete genome sequencing. BMC Genomics 2019; 20:796. [PMID: 31666016 PMCID: PMC6822371 DOI: 10.1186/s12864-019-6184-1] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2019] [Accepted: 10/15/2019] [Indexed: 02/08/2023] Open
Abstract
BACKGROUND Clade 5 Clostridioides difficile diverges significantly from the other clades and is therefore, attracting increasing attention due its great heterogeneity. In this study, we used third-generation sequencing techniques to sequence the complete whole genomes of three ST11 C. difficile isolates, RT078 and another two new ribotypes (RTs), obtained from three independent hospitalized elderly patients undergoing antibiotics treatment. Mobile genetic elements (MGEs), antibiotic-resistance, drug resistance genes, and virulent-related genes were analyzed and compared within these three isolates. RESULTS Isolates 10,010 and 12,038 carried a distinct deletion in tcdA compared with isolate 21,062. Furthermore, all three isolates had identical deletions and point-mutations in tcdC, which was once thought to be a unique characteristic of RT078. Isolate 21,062 (RT078) had a unique plasmid, different numbers of transposons and genetic organization, and harboring special CRISPR spacers. All three isolates retained high-level sensitivity to 11 drugs and isolate 21,062 (RT078) carried distinct drug-resistance genes and loss of numerous flagellum-related genes. CONCLUSIONS We concluded that capillary electrophoresis based PCR-ribotyping is important for confirming RT078. Furthermore, RT078 isolates displayed specific MGEs, indicating an independent evolutionary process. In the further study, we could testify these findings with more RT078 isolates of divergent origins.
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Affiliation(s)
- Yuan Wu
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. .,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
| | - Lin Yang
- BGI-Shen zhen, main building, Beishan industry zone, Yan tian District, Shenzhen, China
| | - Wen-Ge Li
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Wen Zhu Zhang
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Zheng Jie Liu
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China
| | - Jin-Xing Lu
- State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, China. .,Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou, China.
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9
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Kociolek LK, Ozer EA, Gerding DN, Hecht DW, Patel SJ, Hauser AR. Whole-genome analysis reveals the evolution and transmission of an MDR DH/NAP11/106 Clostridium difficile clone in a paediatric hospital. J Antimicrob Chemother 2019; 73:1222-1229. [PMID: 29342270 DOI: 10.1093/jac/dkx523] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2017] [Accepted: 12/13/2017] [Indexed: 02/07/2023] Open
Abstract
Background Clostridium difficile strain DH/NAP11/106, a relatively antibiotic-susceptible strain, is now the most common cause of C. difficile infection (CDI) among adults in the USA. Objectives To identify mechanisms underlying the evolution and transmission of an MDR DH/NAP11/106 clone. Methods WGS (Illumina MiSeq), restriction endonuclease analysis (REA) and antibiotic susceptibility testing were performed on 134 C. difficile isolates collected from paediatric patients with CDI over a 2 year period. Results Thirty-one of 134 (23%) isolates were REA group DH. Pairwise single-nucleotide variant (SNV) analyses identified a DH clone causing seven instances of CDI in two patients. During the 337 days between the first and second CDI, Patient 1 (P1) received 313 days of antibiotic therapy. Clindamycin and rifaximin resistance, and reduced vancomycin susceptibility (MIC 0.5-2 mg/L), were newly identified in the relapsed isolate. This MDR clone was transmitted to Patient 2 (P2) while P1 and P2 received care in adjacent private rooms. P1 and P2 each developed two additional CDI relapses. Comparative genomics analyses demonstrated SNVs in multiple antibiotic resistance genes, including rpoB (rifaximin resistance), gyrB and a gene encoding PBP; gyrB and PBP mutations did not consistently confer a resistance phenotype. The clone also acquired a 46 000 bp genomic element, likely a conjugative plasmid, which contained ermB (clindamycin resistance). The element shared 99% identity with the genomic sequence of Faecalibacterium prausnitzii, an enteric commensal. Conclusions These data highlight the emergence of MDR in C. difficile strain DH/NAP11/106 through multiple independent mechanisms probably as a consequence of profound antibiotic pressure.
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Affiliation(s)
- Larry K Kociolek
- Ann & Robert H. Lurie Children's Hospital of Chicago, 225 E. Chicago Ave, Chicago, IL 60611, USA.,Northwestern University Feinberg School of Medicine, 320 E. Superior St, Chicago, IL 60611, USA
| | - Egon A Ozer
- Northwestern University Feinberg School of Medicine, 320 E. Superior St, Chicago, IL 60611, USA
| | - Dale N Gerding
- Loyola University Chicago Stritch School of Medicine, 2160 S. 1st Ave, Maywood, IL 60153, USA.,Edward Hines, Jr. Veterans Administration Hospital, 5000 S. 5th Ave, Hines, IL 60141, USA
| | - David W Hecht
- Loyola University Chicago Stritch School of Medicine, 2160 S. 1st Ave, Maywood, IL 60153, USA.,Loyola University Medical Center, 2160 S. 1st Ave, Maywood, IL 60153, USA
| | - Sameer J Patel
- Ann & Robert H. Lurie Children's Hospital of Chicago, 225 E. Chicago Ave, Chicago, IL 60611, USA.,Northwestern University Feinberg School of Medicine, 320 E. Superior St, Chicago, IL 60611, USA
| | - Alan R Hauser
- Northwestern University Feinberg School of Medicine, 320 E. Superior St, Chicago, IL 60611, USA
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Revitt-Mills SA, Vidor CJ, Watts TD, Lyras D, Rood JI, Adams V. Virulence Plasmids of the Pathogenic Clostridia. Microbiol Spectr 2019; 7:10.1128/microbiolspec.gpp3-0034-2018. [PMID: 31111816 PMCID: PMC11257192 DOI: 10.1128/microbiolspec.gpp3-0034-2018] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/29/2018] [Indexed: 12/12/2022] Open
Abstract
The clostridia cause a spectrum of diseases in humans and animals ranging from life-threatening tetanus and botulism, uterine infections, histotoxic infections and enteric diseases, including antibiotic-associated diarrhea, and food poisoning. The symptoms of all these diseases are the result of potent protein toxins produced by these organisms. These toxins are diverse, ranging from a multitude of pore-forming toxins to phospholipases, metalloproteases, ADP-ribosyltransferases and large glycosyltransferases. The location of the toxin genes is the unifying theme of this review because with one or two exceptions they are all located on plasmids or on bacteriophage that replicate using a plasmid-like intermediate. Some of these plasmids are distantly related whilst others share little or no similarity. Many of these toxin plasmids have been shown to be conjugative. The mobile nature of these toxin genes gives a ready explanation of how clostridial toxin genes have been so widely disseminated both within the clostridial genera as well as in the wider bacterial community.
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Affiliation(s)
- Sarah A Revitt-Mills
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
| | - Callum J Vidor
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
| | - Thomas D Watts
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
| | - Dena Lyras
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
| | - Julian I Rood
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
| | - Vicki Adams
- Infection and Immunity Program, Biomedicine Discovery Institute and Department of Microbiology, Monash University, Clayton, Victoria 3800, Australia
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11
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Generation of Markerless Deletions in the Nosocomial Pathogen Clostridium difficile by Induction of DNA Double-Strand Breaks. Appl Environ Microbiol 2019; 85:AEM.02055-18. [PMID: 30478235 PMCID: PMC6344619 DOI: 10.1128/aem.02055-18] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2018] [Accepted: 11/17/2018] [Indexed: 02/08/2023] Open
Abstract
Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen. Clostridium difficile is an important nosocomial pathogen associated with potentially fatal disease induced by the use of antibiotics. Genetic characterization of such clinically important bacteria is often hampered by lack of availability of suitable tools. Here, we describe the use of I-SceI to induce DNA double-strand breaks, which increase the frequency of allelic exchange and enable the generation of markerless deletions in C. difficile. The usefulness of the system is illustrated by the deletion of genes encoding putative AddAB homologues. The ΔaddAB mutants are sensitive to ultraviolet light and the antibiotic metronidazole, indicating a role in homologous recombination and the repair of DNA breaks. Despite the impairment in recombination, the mutants are still proficient for induction of the SOS response. In addition, deletion of the fliC gene, and subsequent complementation, reveals the importance of potential regulatory elements required for expression of a downstream gene encoding the flagellin glycosyltransferase. IMPORTANCE Most sequenced bacterial genomes contain genes encoding proteins of unknown or hypothetical function. To identify a phenotype for mutations in such genes, deletion is the preferred method for mutagenesis because it reduces the likelihood of polar effects, although it does not eliminate the possibility. Allelic exchange to produce deletions is dependent on the length of homologous regions used to generate merodiploids. Shorter regions of homology resolve at lower frequencies. The work presented here demonstrates the utility of inducing DNA double-strand breaks to increase the frequency of merodiploid resolution in Clostridium difficile. Using this approach, we reveal the roles of two genes, encoding homologues of AddAB, in survival following DNA damage. The method is readily applicable to the production of deletions in C. difficile and expands the toolbox available for genetic analysis of this important anaerobic pathogen.
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12
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Johanesen P, Lyras D. Methods for Determining Transfer of Mobile Genetic Elements in Clostridium difficile. Methods Mol Biol 2018; 1476:199-213. [PMID: 27507343 DOI: 10.1007/978-1-4939-6361-4_15] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
Horizontal gene transfer by mobile genetic elements plays an important role in the evolution of bacteria, allowing them to rapidly acquire new traits, including antibiotic resistance. Mobile genetic elements such as conjugative and mobilizable transposons make up a considerable part of the C. difficile genome. While sequence analysis has identified a large number of these elements, experimental analysis is required to demonstrate mobility and function. This chapter describes the experimental methods utilized for determining function and transfer of mobile genetic elements in C. difficile including detection of the circular transfer intermediate and the analysis and confirmation of mobile genetic element transfer to recipient cells.
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Affiliation(s)
- Priscilla Johanesen
- Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Victoria, 3800, Australia
| | - Dena Lyras
- Infection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Microbiology, Monash University, Victoria, 3800, Australia.
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13
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Hargreaves KR, Thanki AM, Jose BR, Oggioni MR, Clokie MRJ. Use of single molecule sequencing for comparative genomics of an environmental and a clinical isolate of Clostridium difficile ribotype 078. BMC Genomics 2016; 17:1020. [PMID: 27964731 PMCID: PMC5154133 DOI: 10.1186/s12864-016-3346-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2016] [Accepted: 11/25/2016] [Indexed: 01/20/2023] Open
Abstract
Background How the pathogen Clostridium difficile might survive, evolve and be transferred between reservoirs within the natural environment is poorly understood. Some ribotypes are found both in clinical and environmental settings. Whether these strains are distinct from each another and evolve in the specific environments is not established. The possession of a highly mobile genome has contributed to the genetic diversity and ongoing evolution of C. difficile. Interpretations of genetic diversity have been limited by fragmented assemblies resulting from short-read length sequencing approaches and by a limited understanding of epigenetic regulation of diversity. To address this, single molecule real time (SMRT) sequencing was used in this study as it produces high quality genome sequences, with resolution of repeat regions (including those found in mobile elements) and can generate data to determine methylation modifications across the sequence (the methylome). Results Chromosomal rearrangements and ribosomal operon duplications were observed in both genomes. The rearrangements occurred at insertion sites within two mobile genetic elements (MGEs), Tn6164 and Tn6293, present only in the M120 and CD105HS27 genomes, respectively. The gene content of these two transposons differ considerably which could impact upon horizontal gene transfer; differences include CDSs encoding methylases and a conjugative prophage only in Tn6164. To investigate mechanisms which could affect MGE transfer, the methylome, restriction modification (RM) and the CRISPR/Cas systems were characterised for each strain. Notably, the environmental isolate, CD105HS27, does not share a consensus motif for m4C methylation, but has one additional spacer when compared to the clinical isolate M120. Conclusions These findings show key differences between the two strains in terms of their genetic capacity for MGE transfer. The carriage of horizontally transferred genes appear to have genome wide effects based on two different methylation patterns. The CRISPR/Cas system appears active although perhaps slow to evolve. Data suggests that both mechanisms are functional and impact upon horizontal gene transfer and genome evolution within C. difficile. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3346-2) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Katherine R Hargreaves
- Department Infection, Immunity and Inflammation, University of Leicester, Leicester, UK. .,Department Microbiology, The Ohio State University, Columbus, OH, USA.
| | - Anisha M Thanki
- Department Infection, Immunity and Inflammation, University of Leicester, Leicester, UK
| | - Bethany R Jose
- Department Infection, Immunity and Inflammation, University of Leicester, Leicester, UK
| | | | - Martha R J Clokie
- Department Infection, Immunity and Inflammation, University of Leicester, Leicester, UK.
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14
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Andersen JM, Shoup M, Robinson C, Britton R, Olsen KEP, Barrangou R. CRISPR Diversity and Microevolution in Clostridium difficile. Genome Biol Evol 2016; 8:2841-55. [PMID: 27576538 PMCID: PMC5630864 DOI: 10.1093/gbe/evw203] [Citation(s) in RCA: 39] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/12/2016] [Indexed: 12/20/2022] Open
Abstract
Virulent strains of Clostridium difficile have become a global health problem associated with morbidity and mortality. Traditional typing methods do not provide ideal resolution to track outbreak strains, ascertain genetic diversity between isolates, or monitor the phylogeny of this species on a global basis. Here, we investigate the occurrence and diversity of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) in C. difficile to assess the potential of CRISPR-based phylogeny and high-resolution genotyping. A single Type-IB CRISPR-Cas system was identified in 217 analyzed genomes with cas gene clusters present at conserved chromosomal locations, suggesting vertical evolution of the system, assessing a total of 1,865 CRISPR arrays. The CRISPR arrays, markedly enriched (8.5 arrays/genome) compared with other species, occur both at conserved and variable locations across strains, and thus provide a basis for typing based on locus occurrence and spacer polymorphism. Clustering of strains by array composition correlated with sequence type (ST) analysis. Spacer content and polymorphism within conserved CRISPR arrays revealed phylogenetic relationship across clades and within ST. Spacer polymorphisms of conserved arrays were instrumental for differentiating closely related strains, e.g., ST1/RT027/B1 strains and pathogenicity locus encoding ST3/RT001 strains. CRISPR spacers showed sequence similarity to phage sequences, which is consistent with the native role of CRISPR-Cas as adaptive immune systems in bacteria. Overall, CRISPR-Cas sequences constitute a valuable basis for genotyping of C. difficile isolates, provide insights into the micro-evolutionary events that occur between closely related strains, and reflect the evolutionary trajectory of these genomes.
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Affiliation(s)
- Joakim M Andersen
- Department of Food, Processing and Nutritional Sciences, North Carolina State University, NC
| | - Madelyn Shoup
- Department of Microbiology and Molecular Genetics, Michigan State University, MI
| | - Cathy Robinson
- Department of Microbiology and Molecular Genetics, Michigan State University, MI
| | - Robert Britton
- Department of Molecular Virology and Microbiology, Center for Metagenomics and Microbiome Research, Baylor College of Medicine, TX
| | - Katharina E P Olsen
- Microbial Competence Centre, Novo Nordisk, Bagsværd, Denmark (Former Employment: Department of Microbiology & Infection Control, Statens Serum Institut, Copenhagen, Denmark)
| | - Rodolphe Barrangou
- Department of Food, Processing and Nutritional Sciences, North Carolina State University, NC
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Abstract
Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota.
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Affiliation(s)
- Wiep Klaas Smits
- Section Experimental Bacteriology, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
| | - Dena Lyras
- Infection and Immunity Program, Monash Biomedicine Discovery Institute, and Department of Microbiology, Monash University, Victoria, Australia
| | - D. Borden Lacy
- Department of Pathology, Microbiology and Immunology, Vanderbilt University Medical Center, and The Veterans Affairs Tennessee Valley Healthcare System, Nashville Tennessee, USA
| | - Mark H. Wilcox
- Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, UK
| | - Ed J. Kuijper
- Section Experimental Bacteriology, Department of Medical Microbiology, Leiden University Medical Center, PO Box 9600, 2300 RC Leiden, The Netherlands
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Schenck LP, Beck PL, MacDonald JA. Gastrointestinal dysbiosis and the use of fecal microbial transplantation in Clostridium difficile infection. World J Gastrointest Pathophysiol 2015; 6:169-180. [PMID: 26600975 PMCID: PMC4644881 DOI: 10.4291/wjgp.v6.i4.169] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/25/2015] [Revised: 08/28/2015] [Accepted: 10/13/2015] [Indexed: 02/06/2023] Open
Abstract
The impact of antibiotics on the human gut microbiota is a significant concern. Antibiotic-associated diarrhea has been on the rise for the past few decades with the increasing usage of antibiotics. Clostridium difficile infections (CDI) have become one of the most prominent types of infectious diarrheal disease, with dramatically increased incidence in both the hospital and community setting worldwide. Studies show that variability in the innate host response may in part impact upon CDI severity in patients. That being said, CDI is a disease that shows the most prominent links to alterations to the gut microbiota, in both cause and treatment. With recurrence rates still relatively high, it is important to explore alternative therapies to CDI. Fecal microbiota transplantation (FMT) and other types of bacteriotherapy have become exciting avenues of treatment for CDI. Recent clinical trials have generated excitement for the use of FMT as a therapeutic option for CDI; however, the exact components of the human gut microbiota needed for protection against CDI have remained elusive. Additional investigations on the effects of antibiotics on the human gut microbiota and subsequent CDI will help reduce the socioeconomic burden of CDI and potentially lead to new therapeutic modalities.
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