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Karimi M, Ghorbani A, Niazi A, Rostami M, Tahmasebi A. CRISPR-Cas13a as a next-generation tool for rapid and precise plant RNA virus diagnostics. PLANT METHODS 2025; 21:83. [PMID: 40490780 DOI: 10.1186/s13007-025-01401-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/13/2025] [Accepted: 05/29/2025] [Indexed: 06/11/2025]
Abstract
Plant viruses are among the most serious threats to global agriculture, causing significant yield losses and jeopardizing food security. Identifying these viruses is crucial to prevent widespread crop damage and ensure effective management. CRISPR-Cas13a, a subtype of the RNA-targeting Cas13 family, has emerged as a transformative tool in molecular diagnostics, specifically tailored to detect these plant RNA viruses with unparalleled precision. Unlike traditional methods such as ELISA and RT-PCR, which are often limited by sensitivity, equipment dependency, and long processing times, Cas13a offers exceptional specificity and attomolar-level sensitivity. Its RNA-guided collateral cleavage mechanism allows signal amplification, making it particularly suitable for field-deployable diagnostics. Recent advances in Cas13 engineering, including compact variants such as Cas13bt3 and Cas13Y, have further improved its delivery efficiency and minimized immune responses, enhancing its agricultural applications. Integration with amplification methods like LAMP and innovative biosensor platforms like graphene-based and electrochemical systems further enhances its diagnostic potential. While challenges remain, including off-target effects, reagent stability, and scalability, innovations in CRISPR RNA (crRNA) design, reagent encapsulation, and microfluidic technologies are actively addressing these barriers. CRISPR-Cas13a represents a cutting-edge solution for rapid, accurate, and accessible plant virus diagnostics, providing a powerful safeguard for crop yields and global food security.
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Affiliation(s)
- Marzieh Karimi
- Institute of Biotechnology, Shiraz University, Shiraz, Iran
| | - Abozar Ghorbani
- Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Karaj, Iran.
| | - Ali Niazi
- Institute of Biotechnology, Shiraz University, Shiraz, Iran.
| | - Mahsa Rostami
- Nuclear Agriculture Research School, Nuclear Science and Technology Research Institute (NSTRI), Karaj, Iran
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El-Son MAM, Elbahnaswy S, Khormi MA, Aborasain AM, Abdelhaffez HH, Zahran E. Harnessing the fish gut microbiome and immune system to enhance disease resistance in aquaculture. FISH & SHELLFISH IMMUNOLOGY 2025; 163:110394. [PMID: 40350102 DOI: 10.1016/j.fsi.2025.110394] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/24/2025] [Revised: 05/03/2025] [Accepted: 05/05/2025] [Indexed: 05/14/2025]
Abstract
The increasing global reliance on aquaculture is challenged by disease outbreaks, exacerbated by antibiotic resistance, and environmental stressors. Traditional strategies, such as antibiotic treatments and chemical interventions, are becoming less effective, necessitating a shift toward microbiota-based disease control. The fish gut microbiome is a key determinant of immune homeostasis and pathogen resistance. However, previous reviews lack integration of microbiome engineering, machine learning, and next-generation sequencing in fish health strategies. This review encompasses recent advancements in microbiome research, including dietary strategies such as prebiotics, probiotics, synbiotics, and phytogenic feed additives. It synthesizes the latest metagenomic insights, microbiota modulation techniques, and AI-driven disease prediction models. It presents a novel conceptual framework for disease control using microbiome-based approaches in aquaculture. Additionally, we explore emerging methodologies, including microbiota transplantation and synthetic probiotics, to develop precision microbiome interventions. By bridging existing knowledge gaps, this review provides actionable insights into sustainable aquaculture practices through microbiome-driven disease resistance.
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Affiliation(s)
- Mai A M El-Son
- Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
| | - Samia Elbahnaswy
- Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt
| | - Mohsen A Khormi
- Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Kingdom of Saudi Arabia
| | - Ali M Aborasain
- Department of Biology, College of Science, Jazan University, P.O. Box 114, Jazan 45142, Kingdom of Saudi Arabia
| | - Hanan H Abdelhaffez
- Department of Cell and Tissues, Faculty of Veterinary Medicine, Assiut University, Assiut 20 71526, Egypt
| | - Eman Zahran
- Department of Aquatic Animal Medicine, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt
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3
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Sherry NL, Lee JYH, Giulieri SG, Connor CH, Horan K, Lacey JA, Lane CR, Carter GP, Seemann T, Egli A, Stinear TP, Howden BP. Genomics for antimicrobial resistance-progress and future directions. Antimicrob Agents Chemother 2025; 69:e0108224. [PMID: 40227048 PMCID: PMC12057382 DOI: 10.1128/aac.01082-24] [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] [Indexed: 04/15/2025] Open
Abstract
Antimicrobial resistance (AMR) is a critical global public health threat, with bacterial pathogens of primary concern. Pathogen genomics has revolutionized the study of bacterial pathogens and provided deep insights into the mechanisms and dissemination of AMR, with the precision of whole-genome sequencing informing better control strategies. However, generating actionable data from genomic surveillance and diagnostic efforts requires integration at the public health and clinical interface that goes beyond academic efforts to identify resistance mechanisms, undertake post hoc analyses of outbreaks, and share data after research publications. In addition to timely genomics data, consideration also needs to be given to epidemiological sampling frames, analysis, and reporting mechanisms that meet International Organization for Standardization (ISO) standards and generation of reports that are interpretable and actionable for public health and clinical "end-users." Importantly, ensuring all countries have equitable access to data and technology is critical, through timely data sharing following the FAIR principles (findable, accessible, interoperable, and re-usable). In this review, we describe (i) advances in genomic approaches for AMR research and surveillance to understand emergence, evolution, and transmission of AMR and the key requirements to enable this work and (ii) discuss emerging and future applications of genomics at the clinical and public health interface, including barriers to implementation. Harnessing advances in genomics-enhanced AMR research and embedding robust and reproducible workflows within clinical and public health practice promises to maximize the impact of pathogen genomics for AMR globally in the coming decade.
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Affiliation(s)
- Norelle L. Sherry
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- WHO Collaborating Centre for Antimicrobial Resistance, Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Department of Infectious Diseases and Immunology, Austin Health, Heidelberg, Victoria, Australia
| | - Jean Y. H. Lee
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Department of Infectious Diseases, Monash Health, Clayton, Victoria, Australia
| | - Stefano G. Giulieri
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Victorian Infectious Diseases Service, Doherty Institute for Infection and Immunity, The Royal Melbourne Hospital, , Melbourne, Victoria, Australia
| | - Christopher H. Connor
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - Kristy Horan
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - Jake A. Lacey
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - Courtney R. Lane
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- WHO Collaborating Centre for Antimicrobial Resistance, Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
| | - Glen P. Carter
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - Torsten Seemann
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
| | - Adrian Egli
- Institute of Medical Microbiology, University of Zurich, Zurich, Switzerland
| | - Timothy P. Stinear
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
| | - Benjamin P. Howden
- Microbiological Diagnostic Unit Public Health Laboratory, Department of Microbiology and Immunology, University of Melbourne at the Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- WHO Collaborating Centre for Antimicrobial Resistance, Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia
- Department of Infectious Diseases and Immunology, Austin Health, Heidelberg, Victoria, Australia
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, Victoria, Australia
- Microbiology Department, Royal Melbourne Hospital, Melbourne, Victoria, Australia
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Sallah YH, Bratti VF, Rafinejad-Farahani B, Jayasekar Zurn S, Johnson S, Crestani AS, Dacoregio MI, Majeed H, Fazelzad R, Pabani A, Wilson BE, Favorito FM, de Moraes FY, Sung L, Martei YM, Rodin D. Antimicrobial resistance in patients with haematological malignancies: a scoping review. Lancet Oncol 2025; 26:e242-e252. [PMID: 40318656 DOI: 10.1016/s1470-2045(25)00079-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2024] [Revised: 02/03/2025] [Accepted: 02/10/2025] [Indexed: 05/07/2025]
Abstract
Antimicrobial resistance (AMR) is a substantial global health threat. Patients with haematological malignancies have an increased risk of AMR infection due to disease-related and treatment-related immunosuppression. This scoping review searched four bibliographic databases from Jan 1, 2000, to Dec 7, 2023, for publications on AMR bacterial infections in patients with haematological malignancies and identified 274 eligible articles. AMR prevalence data extraction focused on WHO bacterial priority pathogens. The prevalence of AMR bacterial infections from seven WHO priority pathogens in patients with haematological malignancies was 35% (95% CI 30-40; I2 99·4%). The most frequent AMR infections reported were bloodstream infections, with the highest reported AMR pathogens in third-generation cephalosporin-resistant Enterobacterales (pooled prevalence rate 44% [95% CI 23-64; I2 99·8%]), meticillin-resistant Staphylococcus aureus (43% [31-54; I2 95·9%]), and vancomycin-resistant enterococci (41% [26-56; I2 96·2%]). 53 (65%) of the 81 studies that reported mortality showed higher mortality rates associated with AMR infections. 168 (61%) studies were conducted in high-income countries, with no studies published from the WHO Africa region, revealing a substantial data gap from low-income and middle-income regions. Future efforts should prioritise standardised reporting measures, robust surveillance, antimicrobial stewardship, and well designed clinical trials, particularly in under-represented regions, to mitigate the effect of AMR on cancer care.
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Affiliation(s)
- Ya Haddy Sallah
- Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, NY, USA
| | - Vanessa F Bratti
- Department of Public Health Sciences, Queen's University, Kingston, ON, Canada; Global Cancer Program, Toronto, ON, Canada
| | | | | | - Sonali Johnson
- Union for International Cancer Control, Geneva, Switzerland
| | | | | | - Haris Majeed
- Institute of Medical Science, University of Toronto, Toronto, ON, Canada
| | | | - Aliyah Pabani
- Sidney Kimmel Comprehensive Cancer Center, Johns Hopkins University, Baltimore, MD, USA
| | | | | | - Fabio Ynoe de Moraes
- Department of Oncology, Division of Radiation Oncology, Queen's University, Kingston, ON, Canada
| | - Lillian Sung
- The Hospital for Sick Children, Toronto, ON, Canada
| | - Yehoda M Martei
- Department of Medicine, Hematology-Oncology Division, University of Pennsylvania, PA, USA
| | - Danielle Rodin
- Global Cancer Program, Toronto, ON, Canada; Radiation Medicine Program, Toronto, ON, Canada; Department of Radiation Oncology, University of Toronto, Toronto, ON, Canada.
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5
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Torshizi Esfahani A, Zafarjafarzadeh N, Vakili F, Bizhanpour A, Mashaollahi A, Karimi Kordestani B, Baratinamin M, Mohammadpour S. Gut microbiome in colorectal cancer: metagenomics from bench to bedside. JNCI Cancer Spectr 2025; 9:pkaf026. [PMID: 40045177 DOI: 10.1093/jncics/pkaf026] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2024] [Revised: 12/27/2024] [Accepted: 02/27/2025] [Indexed: 05/27/2025] Open
Abstract
Colorectal cancer (CRC) is a major global health challenge. Emerging research highlights the pivotal role of the gut microbiota in influencing CRC risk, progression, and treatment response. Metagenomic approaches, especially high-throughput shotgun sequencing, have provided unprecedented insights into the intricate connections between the gut microbiome and CRC. By enabling comprehensive taxonomic and functional profiling, metagenomics has revealed microbial signatures, activities, and biomarkers associated with colorectal tumorigenesis. Furthermore, metagenomics has shown a potential to guide patient stratification, predict treatment outcomes, and inform microbiome-targeted interventions. Despite remaining challenges in multi-omics data integration, taxonomic gaps, and validation across diverse cohorts, metagenomics has propelled our comprehension of the intricate gut microbiome-CRC interplay. This review underscores the clinical relevance of microbial signatures as potential diagnostic and prognostic tools in CRC. Furthermore, it discusses personalized treatment strategies guided by this omics' approach.
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Affiliation(s)
- Amir Torshizi Esfahani
- Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Nikta Zafarjafarzadeh
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Fatemeh Vakili
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Anahita Bizhanpour
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Amirhesam Mashaollahi
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Bita Karimi Kordestani
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Mahdieh Baratinamin
- Department of Cellular and Molecular Biology, Faculty of Advanced Science and Technology, Medical Sciences, Islamic Azad University Tehran, Tehran, Iran
| | - Somayeh Mohammadpour
- Basic and Molecular Epidemiology of Gastrointestinal Disorders Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
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6
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Chen X, Liu R, Wang W, Liu Y, Sun J, Shao H, McMinn A, Wang M, Liang Y. Isolation, complete characterization and phylogeography of the first bacteriophage against Vibrio neocaledonicus, which encodes a pyruvate phosphate dikinase and represents a novel viral family. Microb Genom 2025; 11:001403. [PMID: 40294083 PMCID: PMC12038007 DOI: 10.1099/mgen.0.001403] [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: 11/30/2024] [Accepted: 03/27/2025] [Indexed: 04/30/2025] Open
Abstract
Vibrio are widely distributed in aquatic environments and are major pathogens commonly found in aquaculture environments, playing a significant role in human production activities and maintaining ecological stability. Here, a novel phage, vB_VneS_J26, which infects Vibrio neocaledonicus, was isolated from coastal seawater in Qingdao, China. Transmission electron microscopy revealed that vB_VneS_J26 exhibits siphovirus morphotype, with a linear double-stranded DNA genome of 82,477 bp in length and G+C content of 45.11 mol%, encoding 122 putative ORFs. Three auxiliary metabolic genes related to carbon metabolism and host cell redox processes were identified, including a pyruvate phosphate dikinase, which catalyses the reversible conversion between phosphoenolpyruvate and pyruvate and is rarely detected in viruses. Whole-genome phylogenetic and comparative genomic analyses suggested that vB_VneS_J26 represents a potential novel viral family, comprising six isolated vibriophages, proposed as Modirecodeviridae. Phylogeographic analysis indicated that Modirecodeviridae is primarily distributed in epipelagic and mesopelagic zones of the Arctic and temperate tropical oceans.
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Affiliation(s)
- Xin Chen
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
| | - Ruize Liu
- Department of Intensive Care Unit, Qingdao Hospital, University of Health and Rehabilitation Sciences (Qingdao Municipal Hospital), Qingdao, PR China
| | - Wei Wang
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
| | - Yundan Liu
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
| | - Jianhua Sun
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
- Haide College, Ocean University of China, Qingdao, PR China
| | - Hongbing Shao
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
- UMT-OUC Joint Centre for Marine Studies, Qingdao, PR China
| | - Andrew McMinn
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
- Institute for Marine and Antarctic Studies, University of Tasmania, Hobart, TAS, Australia
| | - Min Wang
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
- Haide College, Ocean University of China, Qingdao, PR China
- UMT-OUC Joint Centre for Marine Studies, Qingdao, PR China
| | - Yantao Liang
- College of Marine Life Sciences, Institute of Evolution and Marine Biodiversity, MOE Key Laboratory of Evolution and Marine Biodiversity, Frontiers Science Center for Deep Ocean Multispheres and Earth System, Center for Ocean Carbon Neutrality, Ocean University of China, Qingdao, PR China
- UMT-OUC Joint Centre for Marine Studies, Qingdao, PR China
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7
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Thangavel M, Sneha MJX, Mani I, Surendrababu A, Rajapriya P, Arulselvan P, Alarfaj AA, Thangavelu I, Pandi M. Diversity of Endophytic Fungi in Plant Species: Traditional vs. High-Throughput Sequencing Approaches. Chem Biodivers 2025:e202402792. [PMID: 39995022 DOI: 10.1002/cbdv.202402792] [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: 10/29/2024] [Revised: 02/07/2025] [Accepted: 02/07/2025] [Indexed: 02/26/2025]
Abstract
The plant microbiome significantly impacts plant life, with fungi playing a crucial role in shaping interactions and classifications. Advances in cultivation technologies have refined fungal classification, and research highlights the vital connection between endophytic fungi and their plant hosts. The present study employs morphological and phylogenetic techniques, predicting the Internal Transcribed Spacer 2 (ITS2) secondary structure and using next-generation sequencing (NGS) data to detect fungal endophytes in plant leaves via both traditional and conventional approaches. The research area, with its hot semi-arid environment and red and black soils, supports drought-resistant plants like Senna auriculata, Ziziphus mauritiana, and Catunaregam spinosa, known for their medicinal properties. These plants, rich in antioxidants, play a vital role in traditional medicine and highlight the region's rich ethno-botanical heritage. The culture-dependent study on the foliage yielded a total of 17 isolates from S. auriculata and 16 each from both C. spinosa and Z. mauritiana. The most common genera, Alternaria and Nigrospora, account for 18.3% of all isolated endophytic fungi. Three plants were colonized with Nigrospora and Lasiodiplodia, and their morphotypes were determined using ITS2 secondary structure prediction. Recent ecological studies highlight unculturable taxa, or dark taxa, where many species cannot sporulate or be cultured, emphasizing the need for High - Throughput Sequencing (HTS) approaches. The study gathered 68,791 reads from S. auriculata with 101 operational taxonomic units (OTUs), 58,620 from C. spinosa with 219 OTUs, and 66,087 from Z. mauritiana with 193 OTUs, with the majority of OTUs related to Colletotrichum (69%) and a minimum of Myrmaecium (2%). A total of 49 fungal isolates were obtained from traditional methods, whereas 513 fungal OTUs were retrieved through HTS methods, confirming the presence of a highly abundant fungus population in plant samples. The study reveals that using the ITS short amplicon sequencing technique provides distinct insights into endophytic fungal communities in three plant samples. In conclusion, analyzing plant fungal components using a combination of culture-dependent and culture-independent techniques may be a novel strategy.
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Affiliation(s)
- Myithili Thangavel
- Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
| | - Michael Joe Xavier Sneha
- Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
| | - Israel Mani
- Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
| | - Akash Surendrababu
- Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
| | - Pandy Rajapriya
- Department of Zoology, M.S.S. Wakf Board College, Madurai, Tamil Nadu, India
| | - Palanisamy Arulselvan
- Department of Biochemistry, Karpagam Academy of Higher Education (Deemed to be University), Coimbatore, Tamil Nadu, India
| | - Abdullah A Alarfaj
- Department of Botany and Microbiology, College of Science, King Saud University, Riyadh, Saudi Arabia
| | | | - Mohan Pandi
- Department of Molecular Microbiology, School of Biotechnology, Madurai Kamaraj University, Madurai, Tamil Nadu, India
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Srinivas M, Walsh CJ, Crispie F, O'Sullivan O, Cotter PD, van Sinderen D, Kenny JG. Evaluating the efficiency of 16S-ITS-23S operon sequencing for species level resolution in microbial communities. Sci Rep 2025; 15:2822. [PMID: 39843557 PMCID: PMC11754871 DOI: 10.1038/s41598-024-83410-7] [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: 05/17/2024] [Accepted: 12/13/2024] [Indexed: 01/24/2025] Open
Abstract
Rapid advancements in long-read sequencing have facilitated species-level microbial profiling through full-length 16S rRNA sequencing (~ 1500 bp), and more notably, by the newer 16S-ITS-23S ribosomal RNA operon (RRN) sequencing (~ 4500 bp). RRN sequencing is emerging as a superior method for species resolution, exceeding the capabilities of short-read and full-length 16S rRNA sequencing. However, being in its early stages of development, RRN sequencing has several underexplored or understudied elements, highlighting the need for a critical and thorough examination of its methodologies. Key areas that require detailed analysis include understanding how primer pairs, sequencing platforms, and classifiers and databases affect the accuracy of species resolution achieved through RRN sequencing. Our study addresses these gaps by evaluating the effect of primer pairs using four RRN primer combinations, and that of sequencing platforms by employing PacBio and Oxford Nanopore Technologies (ONT) systems. Furthermore, two classification methods (Minimap2 and OTU clustering), in combination with four RRN reference databases (MIrROR, rrnDB, and two versions of GROND) were compared to identify consistent and accurate classification methods with RRN sequencing. Here we demonstrate that RRN primer pair choice and sequencing platform do not substantially bias taxonomic profiles for most of the tested mock communities, while classification methods significantly impact the accuracy of species-level assignments. Of the classification methods tested, Minimap2 classifier in combination with the GROND database most consistently provided accurate species-level classification across the communities tested, irrespective of sequencing platform.
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Affiliation(s)
- Meghana Srinivas
- Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland
- School of Microbiology, University College Cork, Cork, Ireland
- APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - Calum J Walsh
- Doherty Applied Microbial Genomics, Department of Microbiology & Immunology, The University of Melbourne at the Peter Doherty Institute for Infection & Immunity, 792 Elizabeth Street, Melbourne, VIC, 3000, Australia
| | - Fiona Crispie
- Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland
- APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - Orla O'Sullivan
- Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland
- APC Microbiome Ireland, University College Cork, Cork, Ireland
- VistaMilk SFI Research Centre, Cork, Ireland
| | - Paul D Cotter
- Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland
- APC Microbiome Ireland, University College Cork, Cork, Ireland
- VistaMilk SFI Research Centre, Cork, Ireland
| | - Douwe van Sinderen
- School of Microbiology, University College Cork, Cork, Ireland
- APC Microbiome Ireland, University College Cork, Cork, Ireland
| | - John G Kenny
- Teagasc Food Research Centre, Fermoy, Co. Cork, Ireland.
- APC Microbiome Ireland, University College Cork, Cork, Ireland.
- VistaMilk SFI Research Centre, Cork, Ireland.
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9
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Shah AA, Mirza R, Sattar A, Khan Y, Khan SA. "Unveiling onychomycosis: Pathogenesis, diagnosis, and innovative treatment strategies". Microb Pathog 2025; 198:107111. [PMID: 39522833 DOI: 10.1016/j.micpath.2024.107111] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2024] [Revised: 10/17/2024] [Accepted: 11/07/2024] [Indexed: 11/16/2024]
Abstract
Onychomycosis, a widespread fungal nail infection, manifests as discoloration, thickening, and detachment of nails, often affecting the surrounding skin. While dermatophytes were historically considered the primary causative agents, recent studies reveal a rise in non-dermatophyte mold (NDM) infections, particularly in warmer climates. Dermatophytes dominate toenail infections, while yeasts and molds also contribute to fingernail infections, with certain molds like Fusarium spp. and Scytalidium spp being notable culprits. Diagnostic challenges arise from elevated false-negative rates in conventional methods like microscopy and culture, particularly with NDM infections. Histology and polymerase chain reaction (PCR) offers higher accuracy, albeit requiring multiple confirmations due to contamination risks. Treatment options encompass oral antifungals with higher cure rates but significant side effects and topical treatments with milder side effects but inferior efficacy. Several ongoing research aims to enhance transungual delivery through various approaches for the treatment of onychomycosis. Recurrence rates underscore the importance of prompt treatment, footwear hygiene, and preventive measures like topical treatments to mitigate the risk of reinfection. Understanding the evolving fungal landscape in onychomycosis is critical for effective management and recurrence prevention strategies.
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Affiliation(s)
- Amjad Ali Shah
- Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
| | - Rashna Mirza
- Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
| | - Ariba Sattar
- Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
| | - Yousaf Khan
- Department of Chemistry, Faculty of Natural Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
| | - Shahid Ali Khan
- Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad, Pakistan.
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10
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Bhatt P, Li Y, Xagoraraki I. Genomic mapping of wastewater bacteriophage may predict potential bacterial pathogens infecting the community. THE SCIENCE OF THE TOTAL ENVIRONMENT 2024; 955:176834. [PMID: 39396796 DOI: 10.1016/j.scitotenv.2024.176834] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2024] [Revised: 09/14/2024] [Accepted: 10/07/2024] [Indexed: 10/15/2024]
Abstract
Most existing wastewater surveillance studies that focus on viruses have identified a large fraction of bacteriophages. Identifying bacteria by considering bacteriophage-host interactions is a novel method for detecting bacterial pathogens circulating in a community, using wastewater surveillance. This study aims to identify human-related bacterial pathogens in municipal wastewater collected in metro Detroit, using high-throughput sequencing and bioinformatics. Untreated municipal wastewater samples were collected on August 11, 2020, and bacteriophages were concentrated using the VIRus ADsorption-ELution (VIRADEL) method. Bacteriophage-related contigs in samples ranged from 15.53 % to 18.91 %, with 2477 classified and 8853 unclassified contigs. Most identified bacteriophages were from Caudoviricetes and Malgrandaviricetes classes belonging to 19 families. Hosts of bacteriophages were predicted with the PhaBOX (CHERRY) tool. The results indicated that out of the 2477 classified phages, 2373 were associated with known bacterial hosts. Also, out of 8853 unclassified bacteriophages, 8421 were associated with known bacterial hosts, and the remaining 432 were with unknown bacterial hosts. Among all bacteriophage-associated hosts, 399 were identified as pathogenic bacteria at the species level. Approximately, 85 % of the identified pathogenic bacteria are reported to be associated with human diseases. Genome quality assessments showed that 15 bacteriophages had nearly complete genomes, which were further analyzed to understand bacteriophage-bacteria interactions in wastewater. Identified hosts of these complete-genome phages included human pathogens such as Salmonella enterica, Bacillus cereus, Achromobacter xylosoxidans, and Escherichia coli. The S. enterica bacteriophage (k141_1005294) genomic map was annotated, and responsible open reading frames (ORFs) were characterized to illustrate bacteriophage behavior during infection of pathogenic bacteria in untreated wastewater. To the best of our knowledge, this is the first attempt to characterize human bacterial pathogens in wastewater through bacteriophage-pathogen interactions. Novel bioinformatic approaches enhance pathogen detection and improve the understanding of community wastewater microbiomes.
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Affiliation(s)
- Pankaj Bhatt
- Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.
| | - Yabing Li
- Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA
| | - Irene Xagoraraki
- Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA
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11
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Stewart RD, Oluwalana-Sanusi AE, Munzeiwa WA, Magoswana L, Chaukura N. Profiling the bacterial microbiome diversity and assessing the potential to detect antimicrobial resistance bacteria in wastewater in Kimberley, South Africa. Sci Rep 2024; 14:26867. [PMID: 39500921 PMCID: PMC11538266 DOI: 10.1038/s41598-024-76466-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2024] [Accepted: 10/14/2024] [Indexed: 11/08/2024] Open
Abstract
Wastewater treatment plants (WWTPs) are hotspots for pathogens, and can facilitate horizontal gene transfer, potentially releasing harmful genetic material and antimicrobial resistance genes into the environment. Little information exists on the composition and behavior of microbes in WWTPs, especially in developing countries. This study used environmental DNA (eDNA) techniques to examine the microbiome load of wastewater from WWTPs. The DNA was isolated from wastewater samples collected from the treatment trains of three WWTPs in Kimberley, South Africa, and the microbial diversity and composition was compared through 16 S rRNA gene sequencing. The microbes detected were of the Kingdom Bacteria, and of these, 48.27% were successfully identified to genus level. The majority of reads from the combined bacterial data fall within the class Gammaproteobacteria, which is known to adversely impact ecological and human health. Arcobacteraceae constituted 19% of the bacterial reads, which is expected as this family is widespread in aquatic environments. Interestingly, the most abundant bacterial group was Bacteroides, which contain a variety of antibiotic-resistant members. Overall, various antibiotic-resistant taxa were detected in the wastewater, indicating a concerning level of antibiotic resistance within the bacterial community. Therefore, eDNA analysis can be a valuable tool in monitoring and assessing the bacterial microbiome in wastewater, thus providing important information for the optimization and improvement of wastewater treatment systems and mitigate public health risks.
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Affiliation(s)
- Ross D Stewart
- Department of Biological and Agricultural Sciences, Sol Plaatje University, Kimberley, 8301, South Africa
| | - Abimbola E Oluwalana-Sanusi
- Department of Physical and Earth Sciences, Sol Plaatje University, Kimberley, 8301, South Africa
- Centre for Global Change, Sol Plaatje University, Kimberley, 8301, South Africa
| | - Wisdom A Munzeiwa
- Department of Physical and Earth Sciences, Sol Plaatje University, Kimberley, 8301, South Africa
| | - Luvo Magoswana
- National Herbarium, South African National Biodiversity Institute, Private Bag X101, Pretoria, South Africa
| | - Nhamo Chaukura
- Department of Physical and Earth Sciences, Sol Plaatje University, Kimberley, 8301, South Africa.
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12
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Manyatsi TS, Lin YH, Jou YT. The isolation and identification of Bacillus velezensis ZN-S10 from vanilla (V. planifolia), and the microbial distribution after the curing process. Sci Rep 2024; 14:16339. [PMID: 39014002 PMCID: PMC11252412 DOI: 10.1038/s41598-024-66753-z] [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: 02/22/2024] [Accepted: 07/03/2024] [Indexed: 07/18/2024] Open
Abstract
The market value of vanilla beans (Vanilla planifolia) is constantly increasing due to their natural aroma and flavor properties that improve after a curing process, where bacteria colonization plays a critical role. However, a few publications suggest that bacteria play a role in the curing process. Hence, this study aimed to isolate Bacillus sp. that could be used for fermenting V. planifolia while analyzing their role in the curing process. Bacillus velezensis ZN-S10 identified with 16S rRNA sequencing was isolated from conventionally cured V. planifolia beans. A bacteria culture solution of B. velezensis ZN-S10 (1 mL of 1 × 107 CFU mL-1) was then coated on 1 kg of non-cured vanilla pods that was found to ferment and colonize vanilla. PCA results revealed distinguished bacterial communities of fermented vanilla and the control group, suggesting colonization of vanilla. Phylogenetic analysis showed that ZN-S10 was the dominant Bacillus genus member and narrowly correlated to B. velezensis EM-1 and B. velezensis PMC206-1, with 78% and 73% similarity, respectively. The bacterial taxonomic profiling of cured V. planifolia had a significant relative abundance of Firmicutes, Proteobacteria, Cyanobacteria, Planctomycetes, and Bacteroidetes phyla according to the predominance. Firmicutes accounted for 55% of the total bacterial sequences, suggesting their colonization and effective fermentation roles in curing vanilla.
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Affiliation(s)
- Thabani Sydney Manyatsi
- Department of Tropical Agriculture and International Cooperation, National Pingtung University of Science and Technology, Neipu Shuefu Road 1, 91201, Pingtung, Taiwan
| | - Yu-Hsin Lin
- Department of Biological Science and Technology, National Pingtung University of Science and Technology, Neipu Shuefu Road 1, 91201, Pingtung, Taiwan
| | - Ying-Tzy Jou
- Department of Biological Science and Technology, National Pingtung University of Science and Technology, Neipu Shuefu Road 1, 91201, Pingtung, Taiwan.
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13
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Li J, Wang C, Zhao S, Qi L, Yu J, Hu X, Chen L, Sun Y, Wang D, Jiang Y, Du Y. Custom-Designed Probes for the Accurate Determination of Epidermal Growth Factor Receptor Mutations and Their Allelic Configuration. Anal Chem 2024; 96:10056-10063. [PMID: 38832555 DOI: 10.1021/acs.analchem.4c01771] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/05/2024]
Abstract
The identification of single nucleotide polymorphisms (SNPs) is of paramount importance for disease diagnosis and clinical prognostication. In the context of nonsmall cell lung cancer (NSCLC), the emergence of resistance mutations, exemplified by the epidermal growth factor receptor (EGFR) T790 M and C797S, is intricately linked to the therapeutic efficacy of EGFR tyrosine kinase inhibitors (EGFR-TKIs). Herein, a highly efficient and specific SNP detection platform for T790 M and C797S mutations has been engineered through the integration of an asymmetric polymerase chain reaction (PCR) and an ingeniously tailored four-way junction (4WJ) probe. Notably, a molecular beacon (MB) probe was judiciously designed to discern the allelic configuration of these mutations. The administration of first- and third-generation EGFR-TKIs demonstrates therapeutic efficacy solely when the mutations are in the trans configuration, characterized by a low fluorescence signal. In contrast, significant fluorescence by the MB probe is indicative of the C797S mutation being in a cis arrangement with T790M, thereby rendering the cells refractory to the therapeutic interventions of both first- and third-generation EGFR-TKIs. The assay is capable of concurrently detecting two point-mutations and ascertaining their allelic positions in a single test within 1.5 h, enhancing both efficiency and simplicity. It also exhibits high accuracy in the identification of clinical samples, offering promising implications for therapeutic guidelines. By enabling tailored treatment plans based on specific genetic profiles, our approach not only advances the precision of NSCLC treatment strategies but also marks a significant contribution to personalized medicine.
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Affiliation(s)
- Jiaqi Li
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
| | - Chang Wang
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
| | - Songchen Zhao
- Department of Medical Oncology, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai 200000, China
| | - Lijuan Qi
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
| | - Jingyuan Yu
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
| | - Xintong Hu
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130022, China
| | - Liguo Chen
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130022, China
| | - Yi Sun
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
| | - Duo Wang
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130022, China
| | - Yanfang Jiang
- Key Laboratory of Organ Regeneration and Transplantation of the Ministry of Education, Genetic Diagnosis Center, The First Hospital of Jilin University, Changchun 130022, China
| | - Yan Du
- State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, China
- School of Applied Chemistry and Engineering, University of Science and Technology of China, Hefei 230026, China
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14
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Spatafora G, Li Y, He X, Cowan A, Tanner ACR. The Evolving Microbiome of Dental Caries. Microorganisms 2024; 12:121. [PMID: 38257948 PMCID: PMC10819217 DOI: 10.3390/microorganisms12010121] [Citation(s) in RCA: 57] [Impact Index Per Article: 57.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2023] [Revised: 12/28/2023] [Accepted: 01/04/2024] [Indexed: 01/24/2024] Open
Abstract
Dental caries is a significant oral and public health problem worldwide, especially in low-income populations. The risk of dental caries increases with frequent intake of dietary carbohydrates, including sugars, leading to increased acidity and disruption of the symbiotic diverse and complex microbial community of health. Excess acid production leads to a dysbiotic shift in the bacterial biofilm composition, demineralization of tooth structure, and cavities. Highly acidic and acid-tolerant species associated with caries include Streptococcus mutans, Lactobacillus, Actinomyces, Bifidobacterium, and Scardovia species. The differences in microbiotas depend on tooth site, extent of carious lesions, and rate of disease progression. Metagenomics and metatranscriptomics not only reveal the structure and genetic potential of the caries-associated microbiome, but, more importantly, capture the genetic makeup of the metabolically active microbiome in lesion sites. Due to its multifactorial nature, caries has been difficult to prevent. The use of topical fluoride has had a significant impact on reducing caries in clinical settings, but the approach is costly; the results are less sustainable for high-caries-risk individuals, especially children. Developing treatment regimens that specifically target S. mutans and other acidogenic bacteria, such as using nanoparticles, show promise in altering the cariogenic microbiome, thereby combatting the disease.
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Affiliation(s)
- Grace Spatafora
- Biology and Program in Molecular Biology and Biochemistry, Middlebury College, Middlebury, VT 05753, USA
| | - Yihong Li
- Department of Public and Ecosystem Health, Cornell University, Ithaca, NY 14853, USA;
| | - Xuesong He
- ADA-Forsyth Institute, Cambridge, MA 02142, USA;
| | - Annie Cowan
- The Mayo Clinic College of Medicine and Science, Rochester, MN 55905, USA
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15
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Gieroń M, Żarnowiec P, Zegadło K, Gmiter D, Czerwonka G, Kaca W, Kręcisz B. Loop-Mediated Isothermal Amplification of DNA (LAMP) as an Alternative Method for Determining Bacteria in Wound Infections. Int J Mol Sci 2023; 25:411. [PMID: 38203582 PMCID: PMC10778741 DOI: 10.3390/ijms25010411] [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: 11/04/2023] [Revised: 12/23/2023] [Accepted: 12/25/2023] [Indexed: 01/12/2024] Open
Abstract
The increasing number of patients with chronic wounds requires the development of quick and accurate diagnostics methods. One of the key and challenging aspects of treating ulcers is to control wound infection. Early detection of infection is essential for the application of suitable treatment methods, such as systemic antibiotics or other antimicrobial agents. Clinically, the most frequently used method for detecting microorganisms in wounds is through a swab and culture on appropriate media. This test has major limitations, such as the long bacterial growth time and the selectivity of bacterial growth. This article presents an overview of molecular methods for detecting bacteria in wounds, including real-time polymerase chain reaction (rtPCR), quantitative polymerase chain reaction (qPCR), genotyping, next-generation sequencing (NGS), and loop-mediated isothermal amplification (LAMP). We focus on the LAMP method, which has not yet been widely used to detect bacteria in wounds, but it is an interesting alternative to conventional detection methods. LAMP does not require additional complicated equipment and provides the fastest detection time for microorganisms (approx. 30 min reaction). It also allows the use of many pairs of primers in one reaction and determination of up to 15 organisms in one sample. Isothermal amplification of DNA is currently the easiest and most economical method for microbial detection in wound infection. Direct visualization of the reaction with dyes, along with omitting DNA isolation, has increased the potential use of this method.
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Affiliation(s)
- Monika Gieroń
- Faculty of Medicine, Jan Kochanowski University in Kielce, 25-369 Kielce, Poland; (M.G.); (B.K.)
- Dermatology Department, Provincial General Hospital, 25-317 Kielce, Poland
| | - Paulina Żarnowiec
- Department of Microbiology, Institute of Biology, Jan Kochanowski University in Kielce, 25-406 Kielce, Poland; (P.Ż.); (K.Z.); (D.G.); (W.K.)
| | - Katarzyna Zegadło
- Department of Microbiology, Institute of Biology, Jan Kochanowski University in Kielce, 25-406 Kielce, Poland; (P.Ż.); (K.Z.); (D.G.); (W.K.)
| | - Dawid Gmiter
- Department of Microbiology, Institute of Biology, Jan Kochanowski University in Kielce, 25-406 Kielce, Poland; (P.Ż.); (K.Z.); (D.G.); (W.K.)
| | - Grzegorz Czerwonka
- Department of Microbiology, Institute of Biology, Jan Kochanowski University in Kielce, 25-406 Kielce, Poland; (P.Ż.); (K.Z.); (D.G.); (W.K.)
| | - Wiesław Kaca
- Department of Microbiology, Institute of Biology, Jan Kochanowski University in Kielce, 25-406 Kielce, Poland; (P.Ż.); (K.Z.); (D.G.); (W.K.)
| | - Beata Kręcisz
- Faculty of Medicine, Jan Kochanowski University in Kielce, 25-369 Kielce, Poland; (M.G.); (B.K.)
- Dermatology Department, Provincial General Hospital, 25-317 Kielce, Poland
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16
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Roume H, Mondot S, Saliou A, Le Fresne-Languille S, Doré J. Multicenter evaluation of gut microbiome profiling by next-generation sequencing reveals major biases in partial-length metabarcoding approach. Sci Rep 2023; 13:22593. [PMID: 38114587 PMCID: PMC10730622 DOI: 10.1038/s41598-023-46062-7] [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: 02/27/2023] [Accepted: 10/27/2023] [Indexed: 12/21/2023] Open
Abstract
Next-generation sequencing workflows, using either metabarcoding or metagenomic approaches, have massively contributed to expanding knowledge of the human gut microbiota, but methodological bias compromises reproducibility across studies. Where these biases have been quantified within several comparative analyses on their own, none have measured inter-laboratory reproducibility using similar DNA material. Here, we designed a multicenter study involving seven participating laboratories dedicated to partial- (P1 to P5), full-length (P6) metabarcoding, or metagenomic profiling (MGP) using DNA from a mock microbial community or extracted from 10 fecal samples collected at two time points from five donors. Fecal material was collected, and the DNA was extracted according to the IHMS protocols. The mock and isolated DNA were then provided to the participating laboratories for sequencing. Following sequencing analysis according to the laboratories' routine pipelines, relative taxonomic-count tables defined at the genus level were provided and analyzed. Large variations in alpha-diversity between laboratories, uncorrelated with sequencing depth, were detected among the profiles. Half of the genera identified by P1 were unique to this partner and two-thirds of the genera identified by MGP were not detected by P3. Analysis of beta-diversity revealed lower inter-individual variance than inter-laboratory variances. The taxonomic profiles of P5 and P6 were more similar to those of MGP than those obtained by P1, P2, P3, and P4. Reanalysis of the raw sequences obtained by partial-length metabarcoding profiling, using a single bioinformatic pipeline, harmonized the description of the bacterial profiles, which were more similar to each other, except for P3, and closer to the profiles obtained by MGP. This study highlights the major impact of the bioinformatics pipeline, and primarily the database used for taxonomic annotation. Laboratories need to benchmark and optimize their bioinformatic pipelines using standards to monitor their effectiveness in accurately detecting taxa present in gut microbiota.
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Affiliation(s)
- Hugo Roume
- Université Paris-Saclay, INRAE, MetaGenoPolis, 78350, Jouy-en-Josas, France
- Discovery & Front End Innovation, Lesaffre Institute of Science & Technology, Lesaffre International, 101 rue de Menin, 59700, Marcq-en-Barœul, France
| | - Stanislas Mondot
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France
| | - Adrien Saliou
- BIOASTER, Microbiology Technology Institute, 40 Avenue Tony Garnier, 69007, Lyon, France
| | | | - Joël Doré
- Université Paris-Saclay, INRAE, MetaGenoPolis, 78350, Jouy-en-Josas, France.
- Université Paris-Saclay, INRAE, AgroParisTech, Micalis Institute, 78350, Jouy-en-Josas, France.
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17
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Al-Shuhaib MBS, Hashim HO. Mastering DNA chromatogram analysis in Sanger sequencing for reliable clinical analysis. J Genet Eng Biotechnol 2023; 21:115. [PMID: 37955813 PMCID: PMC10643650 DOI: 10.1186/s43141-023-00587-6] [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: 10/04/2023] [Accepted: 11/06/2023] [Indexed: 11/14/2023]
Abstract
BACKGROUND Sanger dideoxy sequencing is vital in clinical analysis due to its accuracy, ability to analyze genetic markers like SNPs and STRs, capability to generate reliable DNA profiles, and its role in resolving complex clinical cases. The precision and robustness of Sanger sequencing contribute significantly to the scientific basis of clinical investigations. Though the reading of chromatograms seems to be a routine step, many errors conducted in PCR may lead to consequent limitations in the readings of AGCT peaks. These errors are possibly associated with improper DNA amplification and its subsequent interpretation of DNA sequencing files, such as noisy peaks, artifacts, and confusion between double-peak technical errors, heterozygosity, and double infection potentials. Thus, it is not feasible to read nucleic acid sequences without giving serious attention to these technical problems. To ensure the accuracy of DNA sequencing outcomes, it is also imperative to detect and rectify technical challenges that may lead to misinterpretation of the DNA sequence, resulting in errors and incongruities in subsequent analyses. SHORT CONCLUSION This overview sheds light on prominent technical concerns that can emerge prior to and during the interpretation of DNA chromatograms in Sanger sequencing, along with offering strategies to address them effectively. The significance of identifying and tackling these technical limitations during the chromatogram analysis is underscored in this review. Recognizing these concerns can aid in enhancing the quality of downstream analyses for Sanger sequencing results, which holds notable improvement in accuracy, reliability, and ability to provide crucial genetic information in clinical analysis.
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Affiliation(s)
- Mohammed Baqur S Al-Shuhaib
- Department of Animal Production, College of Agriculture, Al-Qasim Green University, Al-Qasim 8, Babil, 51001, Iraq.
| | - Hayder O Hashim
- Department of Clinical Laboratory Sciences, College of Pharmacy, University of Babylon, Babil, 51001, Iraq
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18
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Lema NK, Gemeda MT, Woldesemayat AA. Recent Advances in Metagenomic Approaches, Applications, and Challenge. Curr Microbiol 2023; 80:347. [PMID: 37733134 DOI: 10.1007/s00284-023-03451-5] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/30/2022] [Accepted: 08/20/2023] [Indexed: 09/22/2023]
Abstract
Advances in metagenomics analysis with the advent of next-generation sequencing have extended our knowledge of microbial communities as compared to conventional techniques providing advanced approach to identify novel and uncultivable microorganisms based on their genetic information derived from a particular environment. Shotgun metagenomics involves investigating the DNA of the entire community without the requirement of PCR amplification. It provides access to study all genes present in the sample. On the other hand, amplicon sequencing targets taxonomically important marker genes, the analysis of which is restricted to previously known DNA sequences. While sequence-based metagenomics is used to analyze DNA sequences directly from the environment without the requirement of library construction and with limited identification of novel genes and products that can be complemented by functional genomics, function-based metagenomics requires fragmentation and cloning of extracted metagenome DNA in a suitable host with subsequent functional screening and sequencing clone for detection of a novel gene. Although advances were made in metagenomics, different challenges arise. This review provides insight into advances in the metagenomic approaches combined with next-generation sequencing, their recent applications highlighting the emerging ones, such as in astrobiology, forensic sciences, and SARS-CoV-2 infection diagnosis, and the challenges associated. This review further discusses the different types of metagenomics and outlines advancements in bioinformatics tools and their significance in the analysis of metagenomic datasets.
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Affiliation(s)
- Niguse K Lema
- Department of Biotechnology, College of Biological and Chemical Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia
- Biotechnology and Bioprocess Center of Excellence, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia
- Department of Biotechnology, Arba Minch University, Arba Minch, Ethiopia
| | - Mesfin T Gemeda
- Department of Biotechnology, College of Biological and Chemical Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia
- Biotechnology and Bioprocess Center of Excellence, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia
| | - Adugna A Woldesemayat
- Department of Biotechnology, College of Biological and Chemical Engineering, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia.
- Biotechnology and Bioprocess Center of Excellence, Addis Ababa Science and Technology University, Addis Ababa, Ethiopia.
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19
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Kimura H, Hayashi Y, Kitagawa M, Yoshizaki M, Saito K, Harada K, Okayama K, Miura Y, Kimura R, Shirai T, Fujita K, Machida S, Ito K, Kurosawa I. Pathogen Profiles in Outpatients with Non-COVID-19 during the 7th Prevalent Period of COVID-19 in Gunma, Japan. Microorganisms 2023; 11:2142. [PMID: 37763986 PMCID: PMC10536078 DOI: 10.3390/microorganisms11092142] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 08/17/2023] [Accepted: 08/19/2023] [Indexed: 09/29/2023] Open
Abstract
The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1-7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.
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Affiliation(s)
- Hirokazu Kimura
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
- Advanced Medical Science Research Center, Gunma Paz University Research Institute, Shibukawa-shi 377-0008, Gunma, Japan; (R.K.); (T.S.); (K.I.)
| | - Yuriko Hayashi
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
| | - Masanari Kitagawa
- Project Department, Takara Bio Inc., Kusatsu-shi 525-0058, Shiga, Japan; (M.K.); (M.Y.); (K.S.)
| | - Miwa Yoshizaki
- Project Department, Takara Bio Inc., Kusatsu-shi 525-0058, Shiga, Japan; (M.K.); (M.Y.); (K.S.)
| | - Kensuke Saito
- Project Department, Takara Bio Inc., Kusatsu-shi 525-0058, Shiga, Japan; (M.K.); (M.Y.); (K.S.)
| | - Kazuhiko Harada
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
- Kurosawa Hospital, Takasaki-shi 370-1203, Gunma, Japan; (S.M.); (I.K.)
| | - Kaori Okayama
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
| | - Yusuke Miura
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
| | - Ryusuke Kimura
- Advanced Medical Science Research Center, Gunma Paz University Research Institute, Shibukawa-shi 377-0008, Gunma, Japan; (R.K.); (T.S.); (K.I.)
- Department of Bacteriology, Graduate School of Medicine, Gunma University, Maebashi-shi 371-8514, Gunma, Japan
| | - Tatsuya Shirai
- Advanced Medical Science Research Center, Gunma Paz University Research Institute, Shibukawa-shi 377-0008, Gunma, Japan; (R.K.); (T.S.); (K.I.)
| | - Kiyotaka Fujita
- Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki-shi 370-0006, Gunma, Japan; (Y.H.); (K.H.); (K.O.); (Y.M.); (K.F.)
| | - Suguru Machida
- Kurosawa Hospital, Takasaki-shi 370-1203, Gunma, Japan; (S.M.); (I.K.)
| | - Kazuto Ito
- Advanced Medical Science Research Center, Gunma Paz University Research Institute, Shibukawa-shi 377-0008, Gunma, Japan; (R.K.); (T.S.); (K.I.)
- Kurosawa Hospital, Takasaki-shi 370-1203, Gunma, Japan; (S.M.); (I.K.)
| | - Isao Kurosawa
- Kurosawa Hospital, Takasaki-shi 370-1203, Gunma, Japan; (S.M.); (I.K.)
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20
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Charette SJ. Aeromonas salmonicida: Genomics, Taxonomy, Diversity, Pathogenesis, Treatments and Beyond. Microorganisms 2023; 11:1189. [PMID: 37317163 DOI: 10.3390/microorganisms11051189] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/31/2023] [Accepted: 04/04/2023] [Indexed: 06/16/2023] Open
Abstract
For a long time, the bacterial species Aeromonas salmonicida seemed to be limited to a regrouping of psychrophilic subspecies that infect fish, particularly salmonids [...].
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Affiliation(s)
- Steve J Charette
- Département de Biochimie, de Microbiologie et de Bio-Informatique, Faculté des Sciences et de Génie, Université Laval, Quebec City, QC G1V 0A6, Canada
- Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, QC G1V 0A6, Canada
- Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec, Quebec City, QC G1V 4G5, Canada
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21
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Rezapour A, Souresrafil A, Barzegar M, Sheikhy-Chaman M, Tatarpour P. Economic evaluation of next-generation sequencing techniques in diagnosis of genetic disorders: A systematic review. Clin Genet 2023; 103:513-528. [PMID: 36808726 DOI: 10.1111/cge.14313] [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: 09/28/2022] [Revised: 02/12/2023] [Accepted: 02/14/2023] [Indexed: 02/23/2023]
Abstract
In recent years, massively parallel sequencing or next generation sequencing (NGS) has considerably changed both the research and diagnostic fields, and rapid developments have led to the combination of NGS techniques in clinical practice, ease of analysis, and detection of genetic mutations. This article aimed at reviewing the economic evaluation studies of the NGS techniques in the diagnosis of genetic diseases. In this systematic review, scientific databases (PubMed, EMBASE, Web of Science, Cochrane, Scopus, and CEA registry) were searched from 2005 to 2022 to identify the related literature on the economic evaluation of NGS techniques in the diagnosis of genetic diseases. Full-text reviews and data extraction were all performed by two independent researchers. The quality of all the articles included in this study was evaluated using the Checklist of Quality of Health Economic Studies (QHES). Out of 20 521 screened abstracts, 36 studies met the inclusion criteria. The mean score of the QHES checklist for the studies was 0.78 (high quality). Seventeen studies were conducted based on modeling. Cost-effectiveness analysis, cost-utility analysis, and cost-minimization analysis were done in 26 studies, 13 studies, and 1 study, respectively. Based on the available evidence and findings, exome sequencing, which is one of the NGS techniques, could have the potential to be used as a cost-effective genomic test to diagnose children with suspected genetic diseases. The results of the present study support the cost-effectiveness of exome sequencing in diagnosing suspected genetic disorders. However, the use of exome sequencing as a first- or second-line diagnostic test is still controversial. Most studies have been conducted in high-income countries, and research on the cost-effectiveness of NGS methods is recommended in low- and middle-income countries.
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Affiliation(s)
- Aziz Rezapour
- Health Management and Economics Research Center, Health Management Research Institute, Iran University of Medical Sciences, Tehran, Iran
| | - Aghdas Souresrafil
- Department of Health Services and Health Promotion, School of Health, Occupational Environment Research Center, Rafsanjan University of Medical Sciences, Rafsanjan, Iran
| | - Mohammad Barzegar
- Department of English Language, School of Health Management and Information Sciences, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammadreza Sheikhy-Chaman
- Department of Health Economics, School of Health Management and Information Sciences, Iran University of Medical Sciences, Tehran, Iran
| | - Parvin Tatarpour
- School of Health Management and Information Sciences, Iran University of Medical Sciences, Tehran, Iran
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22
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Xiao X, Liu S, Deng H, Song Y, Zhang L, Song Z. Advances in the oral microbiota and rapid detection of oral infectious diseases. Front Microbiol 2023; 14:1121737. [PMID: 36814562 PMCID: PMC9939651 DOI: 10.3389/fmicb.2023.1121737] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/12/2022] [Accepted: 01/13/2023] [Indexed: 02/09/2023] Open
Abstract
Several studies have shown that the dysregulation of the oral microbiota plays a crucial role in human health conditions, such as dental caries, periodontal disease, oral cancer, other oral infectious diseases, cardiovascular diseases, diabetes, bacteremia, and low birth weight. The use of traditional detection methods in conjunction with rapidly advancing molecular techniques in the diagnosis of harmful oral microorganisms has expanded our understanding of the diversity, location, and function of the microbiota associated with health and disease. This review aimed to highlight the latest knowledge in this field, including microbial colonization; the most modern detection methods; and interactions in disease progression. The next decade may achieve the rapid diagnosis and precise treatment of harmful oral microorganisms.
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Affiliation(s)
- Xuan Xiao
- Department of Oral Mucosa, Shanghai Stomatological Hospital, Fudan University, Shanghai, China,Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Shanghai Stomatological Hospital, Fudan University, Shanghai, China
| | - Shangfeng Liu
- Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Shanghai Stomatological Hospital, Fudan University, Shanghai, China
| | - Hua Deng
- Translational Medicine Center, Guangdong Women and Children Hospital, Guangzhou, China
| | - Yuhan Song
- Department of Oral Mucosa, Shanghai Stomatological Hospital, Fudan University, Shanghai, China,Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Shanghai Stomatological Hospital, Fudan University, Shanghai, China
| | - Liang Zhang
- Translational Medicine Center, Guangdong Women and Children Hospital, Guangzhou, China,Liang Zhang,
| | - Zhifeng Song
- Department of Oral Mucosa, Shanghai Stomatological Hospital, Fudan University, Shanghai, China,Shanghai Key Laboratory of Craniomaxillofacial Development and Diseases, Shanghai Stomatological Hospital, Fudan University, Shanghai, China,*Correspondence: Zhifeng Song,
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23
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Sherry NL, Horan KA, Ballard SA, Gonҫalves da Silva A, Gorrie CL, Schultz MB, Stevens K, Valcanis M, Sait ML, Stinear TP, Howden BP, Seemann T. An ISO-certified genomics workflow for identification and surveillance of antimicrobial resistance. Nat Commun 2023; 14:60. [PMID: 36599823 DOI: 10.1038/s41467-022-35713-4] [Citation(s) in RCA: 49] [Impact Index Per Article: 24.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2022] [Accepted: 12/21/2022] [Indexed: 01/05/2023] Open
Abstract
Realising the promise of genomics to revolutionise identification and surveillance of antimicrobial resistance (AMR) has been a long-standing challenge in clinical and public health microbiology. Here, we report the creation and validation of abritAMR, an ISO-certified bioinformatics platform for genomics-based bacterial AMR gene detection. The abritAMR platform utilises NCBI's AMRFinderPlus, as well as additional features that classify AMR determinants into antibiotic classes and provide customised reports. We validate abritAMR by comparing with PCR or reference genomes, representing 1500 different bacteria and 415 resistance alleles. In these analyses, abritAMR displays 99.9% accuracy, 97.9% sensitivity and 100% specificity. We also compared genomic predictions of phenotype for 864 Salmonella spp. against agar dilution results, showing 98.9% accuracy. The implementation of abritAMR in our institution has resulted in streamlined bioinformatics and reporting pathways, and has been readily updated and re-verified. The abritAMR tool and validation datasets are publicly available to assist laboratories everywhere harness the power of AMR genomics in professional practice.
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Affiliation(s)
- Norelle L Sherry
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
- Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia
- Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Australia
| | - Kristy A Horan
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Susan A Ballard
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Anders Gonҫalves da Silva
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Claire L Gorrie
- Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Australia
| | - Mark B Schultz
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Kerrie Stevens
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Mary Valcanis
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Michelle L Sait
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
| | - Timothy P Stinear
- Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Australia
| | - Benjamin P Howden
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia.
- Department of Infectious Diseases, Austin Health, Heidelberg, Victoria, Australia.
- Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Australia.
| | - Torsten Seemann
- Microbiological Diagnostic Unit Public Health Laboratory (MDU-PHL), Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Victoria, Australia
- Department of Microbiology & Immunology, University of Melbourne at the Peter Doherty Institute for Infection & Immunity, Melbourne, Australia
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24
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Chaudhari HG, Prajapati S, Wardah ZH, Raol G, Prajapati V, Patel R, Shati AA, Alfaifi MY, Elbehairi SEI, Sayyed RZ. Decoding the microbial universe with metagenomics: a brief insight. Front Genet 2023; 14:1119740. [PMID: 37197021 PMCID: PMC10183756 DOI: 10.3389/fgene.2023.1119740] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2022] [Accepted: 04/14/2023] [Indexed: 05/19/2023] Open
Abstract
A major part of any biological system on earth involves microorganisms, of which the majority are yet to be cultured. The conventional methods of culturing microbes have given fruitful outcomes yet have limitations. The curiosity for better understanding has led to the development of culture-independent molecular methods that help push aside the roadblocks of earlier methods. Metagenomics unifies the scientific community in search of a better understanding of the functioning of the ecosystem and its component organisms. This approach has opened a new paradigm in advanced research. It has brought to light the vast diversity and novelty among microbial communities and their genomes. This review focuses on the development of this field over time, the techniques and analysis of data generated through sequencing platforms, and its prominent interpretation and representation.
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Affiliation(s)
- Hiral G. Chaudhari
- Shri Alpesh N. Patel PG Institute of Science and Research, Sardar Patel University, Anand, Gujarat, India
| | - Shobha Prajapati
- Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India
| | - Zuhour Hussein Wardah
- Shri Alpesh N. Patel PG Institute of Science and Research, Sardar Patel University, Anand, Gujarat, India
| | - Gopal Raol
- Shri R. P. Arts, Shri K.B. Commerce, and Smt. BCJ Science College, Khambhat, Gujarat, India
| | - Vimalkumar Prajapati
- Division of Microbial and Environmental Biotechnology, Aspee Shakilam Biotechnology Institute, Navsari Agricultural University, Surat, Gujarat, India
- *Correspondence: Vimalkumar Prajapati,
| | - Rajesh Patel
- Department of Biosciences, Veer Narmad South Gujarat University, Surat, Gujarat, India
| | - Ali A. Shati
- Biology Department, Faculty of Science, King Khalid University, Abha, Saudi Arabia
| | - Mohammad Y. Alfaifi
- Biology Department, Faculty of Science, King Khalid University, Abha, Saudi Arabia
| | | | - R. Z. Sayyed
- Department of Microbiology, PSGVP Mandal's S I Patil Arts, G B Patel Science and STKV Sangh Commerce College, Shahada, India
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25
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Achromobacter spp. prevalence and adaptation in cystic fibrosis lung infection. Microbiol Res 2022; 263:127140. [DOI: 10.1016/j.micres.2022.127140] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2022] [Revised: 07/11/2022] [Accepted: 07/20/2022] [Indexed: 11/30/2022]
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26
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Dong L, Zhang Z, Zhu B, Li S, He Y, Lou Y, Li P, Zheng H, Tian Z, Ma X. Research on safety and compliance of imported microbial inoculants using high-throughput sequencing. Front Med (Lausanne) 2022; 9:963988. [PMID: 36213630 PMCID: PMC9532531 DOI: 10.3389/fmed.2022.963988] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Accepted: 08/22/2022] [Indexed: 01/09/2023] Open
Abstract
Microbial inoculants are widely used in wastewater treatment, soil remediation, and biological control. Safety and compliance for active constituents are considered to be the most important measures of imported microbial inoculants. Microbial inoculants composition was commonly identified by phenotypic culture, which is time-consuming and labor intense with occasionally false negative results provided, and can only be tested for specific species. High-throughput sequencing (HTS), known for its non-targeted detection of unknown species composition in samples, is suitable for composition consistency identification and biosafety analysis of imported microbial inoculants. In this study, the application of HTS for microflora distribution and resistance gene was verified in microbial inoculants for environmental protection and then applicated in imported microbial inoculants. Both Illumina- and Nanopore-based HTS methods identified the same dominant bacterial species successfully in the imported microbial inoculants. The main component of bacterial species was Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Enterococcus faecium, and further confirmed with traditional methods. The antibiotic resistance genes Bacillus subtilis mprF, bcrA, blt, lmrB, rphB, tet(L), tmrB, vmlR, ykkC, and ykkD were detected in all samples. Our results indicated that HTS processes the application potential to identify the active ingredients of microbial inoculants. Therefore, rapid and accurate identification of the microbial compositions in microbial formulation products is of high importance for port biosafety supervision.
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Affiliation(s)
- Lin Dong
- School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, China
| | - Zilong Zhang
- Shanghai International Travel Healthcare Center, Shanghai, China
| | - Biyun Zhu
- School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, China
| | - Shenwei Li
- Shanghai International Travel Healthcare Center, Shanghai, China
| | - Yan He
- School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, China
| | - Yating Lou
- Shanghai International Travel Healthcare Center, Shanghai, China
| | - Ping Li
- Shanghai International Travel Healthcare Center, Shanghai, China
| | - Huajun Zheng
- Shanghai-MOST Key Laboratory of Health and Disease Genomics, Chinese National Human Genome Center at Shanghai, Shanghai Institute for Biomedical and Pharmaceutical Technologies, Shanghai, China
| | - Zhengan Tian
- Shanghai International Travel Healthcare Center, Shanghai, China
- *Correspondence: Zhengan Tian,
| | - Xia Ma
- School of Perfume and Aroma Technology, Shanghai Institute of Technology, Shanghai, China
- Xia Ma,
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27
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Ibal JC, Park YJ, Park MK, Lee J, Kim MC, Shin JH. Review of the Current State of Freely Accessible Web Tools for the Analysis of 16S rRNA Sequencing of the Gut Microbiome. Int J Mol Sci 2022; 23:10865. [PMID: 36142775 PMCID: PMC9501225 DOI: 10.3390/ijms231810865] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2022] [Revised: 09/13/2022] [Accepted: 09/15/2022] [Indexed: 11/16/2022] Open
Abstract
Owing to the emergence and improvement of high-throughput technology and the associated reduction in costs, next-generation sequencing (NGS) technology has made large-scale sampling and sequencing possible. With the large volume of data produced, the processing and downstream analysis of data are important for ensuring meaningful results and interpretation. Problems in data analysis may be encountered if researchers have little experience in using programming languages, especially if they are clinicians and beginners in the field. A strategy for solving this problem involves ensuring easy access to commercial software and tools. Here, we observed the current status of free web-based tools for microbiome analysis that can help users analyze and handle microbiome data effortlessly. We limited our search to freely available web-based tools and identified MicrobiomeAnalyst, Mian, gcMeta, VAMPS, and Microbiome Toolbox. We also highlighted the various analyses that each web tool offers, how users can analyze their data using each web tool, and noted some of their limitations. From the abovementioned list, gcMeta, VAMPS, and Microbiome Toolbox had several issues that made the analysis more difficult. Over time, as more data are generated and accessed, more users will analyze microbiome data. Thus, the availability of free and easily accessible web tools can enable the easy use and analysis of microbiome data, especially for those users with less experience in using command-line interfaces.
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Affiliation(s)
- Jerald Conrad Ibal
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
| | - Yeong-Jun Park
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
| | - Min-Kyu Park
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
- Department of Applied Biosciences, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
| | - Jooeun Lee
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
| | - Min-Chul Kim
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
- Department of Applied Biosciences, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
| | - Jae-Ho Shin
- NGS Core Facility, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
- Department of Applied Biosciences, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
- Department of Integrative Biotechnology, Kyungpook National University, Daehak-ro 80, Daegu 41566, Korea
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28
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Osek J, Lachtara B, Wieczorek K. Listeria monocytogenes in foods-From culture identification to whole-genome characteristics. Food Sci Nutr 2022; 10:2825-2854. [PMID: 36171778 PMCID: PMC9469866 DOI: 10.1002/fsn3.2910] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2022] [Revised: 04/06/2022] [Accepted: 04/19/2022] [Indexed: 12/03/2022] Open
Abstract
Listeria monocytogenes is an important foodborne pathogen, which is able to persist in the food production environments. The presence of these bacteria in different niches makes them a potential threat for public health. In the present review, the current information on the classical and alternative methods used for isolation and identification of L. monocytogenes in food have been described. Although these techniques are usually simple, standardized, inexpensive, and are routinely used in many food testing laboratories, several alternative molecular-based approaches for the bacteria detection in food and food production environments have been developed. They are characterized by the high sample throughput, a short time of analysis, and cost-effectiveness. However, these methods are important for the routine testing toward the presence and number of L. monocytogenes, but are not suitable for characteristics and typing of the bacterial isolates, which are crucial in the study of listeriosis infections. For these purposes, novel approaches, with a high discriminatory power to genetically distinguish the strains during epidemiological studies, have been developed, e.g., whole-genome sequence-based techniques such as NGS which provide an opportunity to perform comparison between strains of the same species. In the present review, we have shown a short description of the principles of microbiological, alternative, and modern methods of detection of L. monocytogenes in foods and characterization of the isolates for epidemiological purposes. According to our knowledge, similar comprehensive papers on such subject have not been recently published, and we hope that the current review may be interesting for research communities.
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Affiliation(s)
- Jacek Osek
- Department of Hygiene of Food of Animal OriginNational Veterinary Research InstitutePuławyPoland
| | - Beata Lachtara
- Department of Hygiene of Food of Animal OriginNational Veterinary Research InstitutePuławyPoland
| | - Kinga Wieczorek
- Department of Hygiene of Food of Animal OriginNational Veterinary Research InstitutePuławyPoland
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29
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Microbiota in Periodontitis: Advances in the Omic Era. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2022; 1373:19-43. [DOI: 10.1007/978-3-030-96881-6_2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
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30
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Neubert K, Zuchantke E, Leidenfrost RM, Wünschiers R, Grützke J, Malorny B, Brendebach H, Al Dahouk S, Homeier T, Hotzel H, Reinert K, Tomaso H, Busch A. Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures. BMC Genomics 2021; 22:822. [PMID: 34773979 PMCID: PMC8590783 DOI: 10.1186/s12864-021-08115-x] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2021] [Accepted: 10/12/2021] [Indexed: 02/08/2023] Open
Abstract
Background We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation “short-read” and third-generation “long-read” sequencing methods. Results We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a “long-read first” approach. Conclusions Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-021-08115-x.
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Affiliation(s)
- Kerstin Neubert
- Department of Mathematics and Computer Science, Algorithmic Bioinformatics, Freie Universität Berlin, Institute of Computer Science, Takustr. 9, 14195, Berlin, Germany.,German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany
| | - Eric Zuchantke
- Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07749, Jena, Germany
| | - Robert Maximilian Leidenfrost
- Department of Biotechnology and Chemistry, Mittweida University of Applied Sciences, Technikumplatz 17a, 09648, Mittweida, Germany
| | - Röbbe Wünschiers
- Department of Biotechnology and Chemistry, Mittweida University of Applied Sciences, Technikumplatz 17a, 09648, Mittweida, Germany
| | - Josephine Grützke
- German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany
| | - Burkhard Malorny
- German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany
| | - Holger Brendebach
- German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany
| | - Sascha Al Dahouk
- German Federal Institute for Risk Assessment, Diedersdorfer Weg 1, 12277, Berlin, Germany
| | - Timo Homeier
- Friedrich-Loeffler-Institut, Institute of Epidemiology, Südufer, 10 17493, Greifswald, Insel Riems, Germany
| | - Helmut Hotzel
- Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07749, Jena, Germany
| | - Knut Reinert
- Department of Mathematics and Computer Science, Algorithmic Bioinformatics, Freie Universität Berlin, Institute of Computer Science, Takustr. 9, 14195, Berlin, Germany
| | - Herbert Tomaso
- Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07749, Jena, Germany
| | - Anne Busch
- Friedrich-Loeffler-Institut, Institute of Bacterial Infections and Zoonoses, Naumburger Str. 96a, 07749, Jena, Germany. .,Department of Anaesthesiology and Intensive Care Medicine, University Hospital Jena, Jena, Germany.
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O’Dea C, Huerlimann R, Masters N, Kuballa A, Veal C, Fisher P, Stratton H, Katouli M. Microbial Diversity Profiling of Gut Microbiota of Macropus giganteus Using Three Hypervariable Regions of the Bacterial 16S rRNA. Microorganisms 2021; 9:microorganisms9081721. [PMID: 34442800 PMCID: PMC8400485 DOI: 10.3390/microorganisms9081721] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2021] [Revised: 07/19/2021] [Accepted: 08/03/2021] [Indexed: 01/04/2023] Open
Abstract
Animal faecal contamination of surface waters poses a human health risk, as they may contain pathogenic bacteria or viruses. Of the numerous animal species residing along surface waterways in Australia, macropod species are a top contributor to wild animals’ faecal pollution load. We characterised the gut microbiota of 30 native Australian Eastern Grey Kangaroos from six geographical regions (five kangaroos from each region) within South East Queensland in order to establish their bacterial diversity and identify potential novel species-specific bacteria for the rapid detection of faecal contamination of surface waters by these animals. Using three hypervariable regions (HVRs) of the 16S rRNA gene (i.e., V1–V3, V3–V4, and V5–V6), for their effectiveness in delineating the gut microbial diversity, faecal samples from each region were pooled and microbial genomic DNA was extracted, sequenced, and analysed. Results indicated that V1-V3 yielded a higher taxa richness due to its larger target region (~480 bp); however, higher levels of unassigned taxa were observed using the V1-V3 region. In contrast, the V3–V4 HVR (~569 bp) attained a higher likelihood of a taxonomic hit identity to the bacterial species level, with a 5-fold decrease in unassigned taxa. There were distinct dissimilarities in beta diversity between the regions, with the V1-V3 region displaying the highest number of unique taxa (n = 42), followed by V3–V4 (n = 11) and V5–V6 (n = 8). Variations in the gut microbial diversity profiles of kangaroos from different regions were also observed, which indicates that environmental factors may impact the microbial development and, thus, the composition of the gut microbiome of these animals.
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Affiliation(s)
- Christian O’Dea
- Genecology Research Centre, School of Health and Sport Sciences, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia; (C.O.); (N.M.); (A.K.)
| | - Roger Huerlimann
- Marine Climate Change Unit, Okinawa Institute of Science and Technology (OIST), 1919-1 Tancha, Onna-son, Okinawa 904-0495, Japan;
| | - Nicole Masters
- Genecology Research Centre, School of Health and Sport Sciences, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia; (C.O.); (N.M.); (A.K.)
| | - Anna Kuballa
- Genecology Research Centre, School of Health and Sport Sciences, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia; (C.O.); (N.M.); (A.K.)
| | - Cameron Veal
- Seqwater, 117 Brisbane Street, Ipswich, QLD 4305, Australia; (C.V.); (P.F.)
| | - Paul Fisher
- Seqwater, 117 Brisbane Street, Ipswich, QLD 4305, Australia; (C.V.); (P.F.)
| | - Helen Stratton
- School of Environment and Science, Griffith University, Nathan, QLD 4111, Australia;
| | - Mohammad Katouli
- Genecology Research Centre, School of Health and Sport Sciences, University of the Sunshine Coast, Maroochydore, QLD 4558, Australia; (C.O.); (N.M.); (A.K.)
- Correspondence: ; Tel.: +61-7-54302845
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Safiabadi Tali SH, LeBlanc JJ, Sadiq Z, Oyewunmi OD, Camargo C, Nikpour B, Armanfard N, Sagan SM, Jahanshahi-Anbuhi S. Tools and Techniques for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2)/COVID-19 Detection. Clin Microbiol Rev 2021; 34:e00228-20. [PMID: 33980687 PMCID: PMC8142517 DOI: 10.1128/cmr.00228-20] [Citation(s) in RCA: 205] [Impact Index Per Article: 51.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory disease coronavirus 2 (SARS-CoV-2), has led to millions of confirmed cases and deaths worldwide. Efficient diagnostic tools are in high demand, as rapid and large-scale testing plays a pivotal role in patient management and decelerating disease spread. This paper reviews current technologies used to detect SARS-CoV-2 in clinical laboratories as well as advances made for molecular, antigen-based, and immunological point-of-care testing, including recent developments in sensor and biosensor devices. The importance of the timing and type of specimen collection is discussed, along with factors such as disease prevalence, setting, and methods. Details of the mechanisms of action of the various methodologies are presented, along with their application span and known performance characteristics. Diagnostic imaging techniques and biomarkers are also covered, with an emphasis on their use for assessing COVID-19 or monitoring disease severity or complications. While the SARS-CoV-2 literature is rapidly evolving, this review highlights topics of interest that have occurred during the pandemic and the lessons learned throughout. Exploring a broad armamentarium of techniques for detecting SARS-CoV-2 will ensure continued diagnostic support for clinicians, public health, and infection prevention and control for this pandemic and provide advice for future pandemic preparedness.
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Affiliation(s)
- Seyed Hamid Safiabadi Tali
- Department of Chemical and Materials Engineering, Gina Cody School of Engineering, Concordia University, Montréal, Québec, Canada
- Department of Mechanical, Industrial, and Aerospace Engineering, Gina Cody School of Engineering, Concordia University, Montréal, Québec, Canada
| | - Jason J LeBlanc
- Department of Pathology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Microbiology and Immunology, Dalhousie University, Halifax, Nova Scotia, Canada
- Department of Medicine (Infectious Diseases), Dalhousie University, Halifax, Nova Scotia, Canada
- Division of Microbiology, Department of Pathology and Laboratory Medicine, Nova Scotia Health, Halifax, Nova Scotia, Canada
| | - Zubi Sadiq
- Department of Chemical and Materials Engineering, Gina Cody School of Engineering, Concordia University, Montréal, Québec, Canada
| | - Oyejide Damilola Oyewunmi
- Department of Chemical and Materials Engineering, Gina Cody School of Engineering, Concordia University, Montréal, Québec, Canada
| | - Carolina Camargo
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
| | - Bahareh Nikpour
- Department of Electrical and Computer Engineering, McGill University, Montréal, Québec, Canada
| | - Narges Armanfard
- Department of Electrical and Computer Engineering, McGill University, Montréal, Québec, Canada
- Mila-Quebec AI Institute, Montréal, Québec, Canada
| | - Selena M Sagan
- Department of Microbiology and Immunology, McGill University, Montréal, Québec, Canada
- Department of Biochemistry, McGill University, Montréal, Québec, Canada
| | - Sana Jahanshahi-Anbuhi
- Department of Chemical and Materials Engineering, Gina Cody School of Engineering, Concordia University, Montréal, Québec, Canada
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Ahmad-Nejad P, Ashavaid T, Vacaflores Salinas A, Huggett J, Harris K, Linder MW, Baluchova K, Steimer W, Payne DA. Current and future challenges in quality assurance in molecular diagnostics. Clin Chim Acta 2021; 519:239-246. [PMID: 33971158 DOI: 10.1016/j.cca.2021.05.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/30/2020] [Revised: 05/04/2021] [Accepted: 05/05/2021] [Indexed: 02/08/2023]
Abstract
The development and performance of molecular genetic assays has required increasingly complex quality assurance in recent years and continues to pose new challenges. Quality management officers, as well as academic and technical personnel are confronted with new molecular genetic parameters, methods, changing regulatory environments, questions regarding appropriate validation, and quality control for these innovative assays that are increasingly applying quantification and/or multiplex formats. Yet, quality assurance and quality control guidelines are still not widely available or in some circumstances have become outdated. For these reasons, the need for solutions to provide test confidence continues to grow. In order to integrate new test procedures into existing quality assurance measures, the ISO 15189 guideline can serve as an orientation. The ISO 15189 guideline describes requirements for medical laboratories and thus includes those performing molecular diagnostics. This article gives an overview of the possibilities and challenges in quality assurance of molecular parameters and shows possible solutions.
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Affiliation(s)
- Parviz Ahmad-Nejad
- Institute for Medicine Laboratory Diagnostics, Centre for Clinical and Translational Research (CCTR), HELIOS University Hospital, Wuppertal, Witten/Herdecke University, Germany.
| | - Tester Ashavaid
- Department of Laboratory Medicine, P.D. Hinduja National Hospital and Medical Research Center, Mumbai, India
| | | | - Jim Huggett
- National Measurement Laboratory (NML) at LGC, Queens Rd, Teddington, TW11 0LY, United Kingdom; School of Biosciences & Medicine, Faculty of Health & Medical Science, University of Surrey, Guildford GU2 7XH, United Kingdom
| | - Kathryn Harris
- Microbiology Department, Great Ormond Street Hospital NHS Foundation Trust, London, UK
| | - Mark W Linder
- Department of Pathology and Laboratory Medicine, University of Louisville School of Medicine, Louisville, KY, USA
| | - Katarina Baluchova
- OncoLab Diagnostics GmbH Technologie- und Forschungszentrum, Viktor-Kaplan-Straße 2, 2700 Wiener Neustadt, Austria
| | - Werner Steimer
- Institute for Clinical Chemistry and Pathobiochemistry, Munich University of Technology, Klinikum rechts der Isar, D-81675 Munich, Germany
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Evenness-Richness Scatter Plots: a Visual and Insightful Representation of Shannon Entropy Measurements for Ecological Community Analysis. mSphere 2021; 6:6/2/e01019-20. [PMID: 33827912 PMCID: PMC8546709 DOI: 10.1128/msphere.01019-20] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
Shannon’s entropy is a popular alpha diversity metric because it estimates both richness and evenness in a single equation. However, since its value is dependent on both those parameters, there is theoretically an infinite number of richness/evenness value combinations translating into the same index score. By decoupling both components measured by Shannon’s entropy, two communities having identical indices can be differentiated by mapping richness and evenness coordinates on a scatter plot. In such graphs, confidence ellipses would allow testing significant differences between groups of samples. Multivariate statistical tests such as permutational multivariate analysis of variance (PERMANOVA) can be performed on distance matrices calculated from richness and evenness coordinates and detect statistically significant differences that would have remained unforeseen otherwise. Therefore, plotting richness and evenness on two-dimensional (2D) graphs gives a more thorough understanding of how alpha diversity differs between groups of samples.
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Johns MS, Petrelli NJ. Microbiome and colorectal cancer: A review of the past, present, and future. Surg Oncol 2021; 37:101560. [PMID: 33848761 DOI: 10.1016/j.suronc.2021.101560] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2020] [Revised: 11/22/2020] [Accepted: 03/28/2021] [Indexed: 12/27/2022]
Abstract
The gastrointestinal tract is home to diverse and abundant microorganisms, collectively referred to as the microbiome. This ecosystem typically contains trillions of microbial cells that play an important role in regulation of human health. The microbiome has been implicated in host immunity, nutrient absorption, digestion, and metabolism. In recent years, researchers have shown that alteration of the microbiome is associated with disease development, such as obesity, inflammatory bowel disease, and cancer. This review discusses the five decades of research into the human microbiome and the development of colorectal cancer - the historical context including experiments that sparked interest, the explosion of research that has occurred in the last decade, and finally the future of testing and treatment.
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Affiliation(s)
- Michael S Johns
- Department of Surgical Oncology, Helen F. Graham Cancer Center, ChristianaCare, Newark, DE, USA.
| | - Nicholas J Petrelli
- Department of Surgical Oncology, Helen F. Graham Cancer Center, ChristianaCare, Newark, DE, USA
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The Role of Enterobacteriaceae in Gut Microbiota Dysbiosis in Inflammatory Bowel Diseases. Microorganisms 2021; 9:microorganisms9040697. [PMID: 33801755 PMCID: PMC8066304 DOI: 10.3390/microorganisms9040697] [Citation(s) in RCA: 173] [Impact Index Per Article: 43.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2021] [Revised: 03/19/2021] [Accepted: 03/23/2021] [Indexed: 12/12/2022] Open
Abstract
Inflammatory bowel diseases (IBDs) are a group of chronic gastrointestinal inflammatory diseases with unknown etiology. There is a combination of well documented factors in their pathogenesis, including intestinal microbiota dysbiosis. The symbiotic microbiota plays important functions in the host, and the loss of beneficial microbes could favor the expansion of microbial pathobionts. In particular, the bloom of potentially harmful Proteobacteria, especially Enterobacteriaceae, has been described as enhancing the inflammatory response, as observed in IBDs. Herein, we seek to investigate the contribution of Enterobacteriaceae to IBD pathogenesis whilst considering the continuous expansion of the literature and data. Despite the mechanism of their expansion still remaining unclear, their expansion could be correlated with the increase in nitrate and oxygen levels in the inflamed gut and with the bile acid dysmetabolism described in IBD patients. Furthermore, in several Enterobacteriaceae studies conducted at a species level, it has been suggested that some adherent-invasive Escherichia coli (AIEC) play an important role in IBD pathogenesis. Overall, this review highlights the pivotal role played by Enterobacteriaceae in gut dysbiosis associated with IBD pathogenesis and progression.
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37
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Zorba M, Melidou A, Patsatsi A, Poulopoulos A, Gioula G, Kolokotronis A, Minti F. The role of oral microbiome in pemphigus vulgaris. Arch Microbiol 2021; 203:2237-2247. [PMID: 33634320 DOI: 10.1007/s00203-021-02199-5] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 01/27/2021] [Accepted: 02/08/2021] [Indexed: 12/24/2022]
Abstract
While the impact of oral microbiome dysbiosis on autoimmune diseases has been partially investigated, its role on bullous diseases like Pemphigus Vulgaris (PV) is a totally unexplored field. This study aims to present the composition and relative abundance of microbial communities in both healthy individuals and patients with oral PV lesions. Ion Torrent was used to apply deep sequencing of the bacterial 16S rRNA gene to oral smear samples of 15 healthy subjects and 15 patients. The results showed that the most dominant phyla were Firmicutes (55.88% controls-c vs 61.27% patients-p, p value = 0.002), Proteobacteria (9.17%c vs 12.33%p, p value = 0.007) and Fusobacteria (3.39%c vs 4.09%p, p value = 0.03). Alpha diversity showed a significant difference in the number of genera between patients and controls (p value = 0.04). Beta diversity showed statistical differences in the microbial community composition between two groups. Fusobacterium nucleatum, Gemella haemolysans and Parvimonas micra were statistically abundant in patients. We noticed the characteristic fetor coming out of oral PV lesions. Most of anaerobic bacteria responsible for oral halitosis are periopathogenic. Though, only F. nucleatum and P. micra were differentially abundant in our patients. Especially, F. nucleatum has been reported many times as responsible for bad breath. Furthermore, Streptococcus salivarius and Rothia mucilaginosa, species mostly associated with clean breath, were found in relative abundance in the healthy group. Consequently, the distinct malodor observed in PV patients might be attributed either to the abundance of F. nucleatum and P. micra and/or to the lower levels of S. salivarius and R. mucilanginosa in oral lesions.
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Affiliation(s)
- Matina Zorba
- Department of Oral Medicine and Oral Pathology, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece.
| | - Angeliki Melidou
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Aikaterini Patsatsi
- Autoimmune Skin and Bullous Diseases Outpatient Unit of Papageorgiou General Hospital, 2nd Dermatology Department, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Athanasios Poulopoulos
- Department of Oral Medicine and Oral Pathology, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Georgia Gioula
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Alexandros Kolokotronis
- Department of Oral Medicine and Oral Pathology, School of Dentistry, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Fani Minti
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, Thessaloniki, Greece
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Zorba M, Melidou A, Patsatsi A, Ioannou E, Kolokotronis A. The possible role of oral microbiome in autoimmunity. Int J Womens Dermatol 2020; 6:357-364. [PMID: 33898698 PMCID: PMC8060669 DOI: 10.1016/j.ijwd.2020.07.011] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2020] [Revised: 07/15/2020] [Accepted: 07/28/2020] [Indexed: 12/17/2022] Open
Abstract
OBJECTIVE The human microbiome refers to the entire habitat, including microorganisms, their genomes and the surrounding environmental conditions of the microbial ecosystem. When the equilibrium between microbial habitats and host is disturbed, dysbiosis is caused. The oral microbiome (OMB) has been implicated in the manifestation of many intra- and extraoral diseases. Lately, there has been an intense effort to investigate and specify the relationship between microbial complexes, especially that of the oral cavity and intestine and autoimmunity. This study aimed to review the current literature about the possible role of the OMB in the pathogenesis of autoimmune diseases. METHODS We searched for published articles in English indexed in PubMed, Medline, Research Gate and Google Scholar using a search strategy that included terms for oral microbiome, autoimmune diseases, dysbiosis and next-generation sequencing. RESULTS An important number of articles were gathered and used for the description of the possible impact of dysbiosis of OMB in the pathogenesis of Sjögren's syndrome, systemic lupus erythematosus, rheumatoid arthritis, Behcet's disease, Crohn's disease and psoriasis. CONCLUSION This review article draws attention to the relationship between OMB and the triggering of a number of autoimmune diseases. Although this specific topic has been previously reviewed, herein, the authors review recent literature regarding the full list of nosological entities related to the OMB, point out the interaction between the microbiome and sex hormones with regard to their role in autoimmunity and discuss novel and promising therapeutic approaches for systemic autoimmune diseases. Furthermore, the question arises of whether the OMB is associated with oral bullous autoimmune diseases.
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Affiliation(s)
- Matina Zorba
- Department of Oral Medicine and Maxillofacial Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece
| | - Angeliki Melidou
- Department of Microbiology, Faculty of Medicine, Aristotle University of Thessaloniki, Greece
| | - Aikaterini Patsatsi
- Second Dermatology Department of Papageorgiou General Hospital, Faculty of Medicine, Aristotle University of Thessaloniki, Greece
| | - Eleftheria Ioannou
- Department of Biological Applications and Technology, Aristotle University of Thessaloniki, Greece
| | - Alexandros Kolokotronis
- Department of Oral Medicine and Maxillofacial Pathology, School of Dentistry, Aristotle University of Thessaloniki, Greece
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Guerra Maldonado JF, Vincent AT, Chenal M, Veyrier FJ. CAPRIB: a user-friendly tool to study amino acid changes and selection for the exploration of intra-genus evolution. BMC Genomics 2020; 21:832. [PMID: 33243176 PMCID: PMC7690079 DOI: 10.1186/s12864-020-07232-3] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Accepted: 11/17/2020] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND The evolution of bacteria is shaped by different mechanisms such as mutation, gene deletion, duplication, or insertion of foreign DNA among others. These genetic changes can accumulate in the descendants as a result of natural selection. Using phylogeny and genome comparisons, evolutionary paths can be somehow retraced, with recent events being much easier to detect than older ones. For this reason, multiple tools are available to study the evolutionary events within genomes of single species, such as gene composition alterations, or subtler mutations such as SNPs. However, these tools are generally designed to compare similar genomes and require advanced skills in bioinformatics. We present CAPRIB, a unique tool developed in Java that allows to determine the amino acid changes, at the genus level, that correlate with phenotypic differences between two groups of organisms. RESULTS CAPRIB has a user-friendly graphical interface and uses databases in SQL, making it easy to compare several genomes without the need for programming or thorough knowledge in bioinformatics. This intuitive software narrows down a list of amino acid changes that are concomitant with a given phenotypic divergence at the genus scale. Each permutation found by our software is associated with two already described statistical values that indicate its potential impact on the protein's function, helping the user decide which promising candidates to further investigate. We show that CAPRIB is able to detect already known mutations and uncovers many more, and that this tool can be used to question molecular phylogeny. Finally, we exemplify the utility of CAPRIB by pinpointing amino acid changes that coincided with the emergence of slow-growing mycobacteria from their fast-growing counterparts. The software is freely available at https://github.com/BactSymEvol/Caprib . CONCLUSIONS CAPRIB is a new bioinformatics software aiming to make genus-scale comparisons accessible to all. With its intuitive graphical interface, this tool identifies key amino acid changes concomitant with a phenotypic divergence. By comparing fast and slow-growing mycobacteria, we shed light on evolutionary hotspots, such as the cytokinin pathway, that are interesting candidates for further experimentations.
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Affiliation(s)
- Juan F Guerra Maldonado
- Institut national de la recherche scientifique, Centre Armand-Frappier Santé Biotechnologie, Bacterial Symbionts Evolution, Laval, Québec, Canada
| | - Antony T Vincent
- Institut national de la recherche scientifique, Centre Armand-Frappier Santé Biotechnologie, Bacterial Symbionts Evolution, Laval, Québec, Canada
| | - Martin Chenal
- Institut national de la recherche scientifique, Centre Armand-Frappier Santé Biotechnologie, Bacterial Symbionts Evolution, Laval, Québec, Canada
| | - Frederic J Veyrier
- Institut national de la recherche scientifique, Centre Armand-Frappier Santé Biotechnologie, Bacterial Symbionts Evolution, Laval, Québec, Canada.
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Baldan R, Sendi P. Precision Medicine in the Diagnosis and Management of Orthopedic Biofilm Infections. Front Med (Lausanne) 2020; 7:580671. [PMID: 33240905 PMCID: PMC7683765 DOI: 10.3389/fmed.2020.580671] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2020] [Accepted: 10/22/2020] [Indexed: 01/02/2023] Open
Abstract
Orthopedic biofilm infections are difficult to treat and require a multidisciplinary approach to diagnostics and management. Recent advances in the field include methods to disrupt biofilm, sequencing tools, and antibiotic susceptibility tests for bacteria residing in biofilm. The observation of interclonal differences in biofilm properties of the causative microorganisms, together with considerations of comorbidities and polypharmacy in a growing aging population, calls for a personalized approach to treat these infections. In this article, we highlight aspects of precision medicine that may open new perspectives in the diagnosis and management of orthopedic biofilm infections.
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Affiliation(s)
- Rossella Baldan
- Institute for Infectious Diseases, University of Bern, Bern, Switzerland
| | - Parham Sendi
- Institute for Infectious Diseases, University of Bern, Bern, Switzerland.,Centre for Musculoskeletal Infections, University Hospital Basel, Basel, Switzerland.,Division of Infectious Diseases and Hospital Epidemiology, Departments of Medicine and Clinical Research, University Hospital Basel, Basel, Switzerland.,Department of Orthopaedic and Trauma Surgery, University Hospital Basel, Basel, Switzerland
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41
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Vincent AT, Hosseini N, Charette SJ. The Aeromonas salmonicida plasmidome: a model of modular evolution and genetic diversity. Ann N Y Acad Sci 2020; 1488:16-32. [PMID: 33040386 DOI: 10.1111/nyas.14503] [Citation(s) in RCA: 26] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2020] [Revised: 08/30/2020] [Accepted: 09/09/2020] [Indexed: 12/14/2022]
Abstract
High-throughput genomic sequencing has helped to reveal the plasmidome of Aeromonas salmonicida. This literature review provides an overview of A. salmonicida's rich plasmidome by presenting all the plasmids identified so far, addressing their biological importance and the functional links between them. The plasmids of A. salmonicida, especially those bearing antibiotic resistance genes, can provide clues about interactions of this species with other pathogens (animals and humans), as is the case for pRAS3-3432 and Chlamydia suis or pSN254b and Salmonella enterica. In addition to antibiotic resistance, plasmids play an important role in the virulence of A. salmonicida, particularly for the subspecies salmonicida and the plasmid pAsa5, which carries genes for the type-three secretion system, a virulence factor essential for the bacterium. The A. salmonicida plasmidome also has many cryptic plasmids with no known biological function, but which can be used for the acquisition of new genetic elements. Striking examples are pAsa7 and pAsaXII that provide, respectively, resistance to chloramphenicol and formaldehyde and are derivatives of cryptic pAsa2.
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Affiliation(s)
- Antony T Vincent
- Département des Sciences Animales, Faculté des Sciences de l'Agriculture et de l'Alimentation, Université Laval, Quebec City, Quebec, Canada
| | - Nava Hosseini
- Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, Quebec, Canada.,Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec (Hôpital Laval), Quebec City, Quebec, Canada.,Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Quebec City, Quebec, Canada
| | - Steve J Charette
- Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, Université Laval, Quebec City, Quebec, Canada.,Centre de Recherche de l'Institut Universitaire de Cardiologie et de Pneumologie de Québec (Hôpital Laval), Quebec City, Quebec, Canada.,Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie, Université Laval, Quebec City, Quebec, Canada
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Ransom EM, Potter RF, Dantas G, Burnham CAD. Genomic Prediction of Antimicrobial Resistance: Ready or Not, Here It Comes! Clin Chem 2020; 66:1278-1289. [PMID: 32918462 DOI: 10.1093/clinchem/hvaa172] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/27/2020] [Accepted: 07/01/2020] [Indexed: 12/17/2022]
Abstract
BACKGROUND Next-generation sequencing (NGS) technologies are being used to predict antimicrobial resistance. The field is evolving rapidly and transitioning out of the research setting into clinical use. Clinical laboratories are evaluating the accuracy and utility of genomic resistance prediction, including methods for NGS, downstream bioinformatic pipeline components, and the clinical settings in which this type of testing should be offered. CONTENT We describe genomic sequencing as it pertains to predicting antimicrobial resistance in clinical isolates and samples. We elaborate on current methodologies and workflows to perform this testing and summarize the current state of genomic resistance prediction in clinical settings. To highlight this aspect, we include 3 medically relevant microorganism exemplars: Mycobacterium tuberculosis, Staphylococcus aureus, and Neisseria gonorrhoeae. Last, we discuss the future of genomic-based resistance detection in clinical microbiology laboratories. SUMMARY Antimicrobial resistance prediction by genomic approaches is in its infancy for routine patient care. Genomic approaches have already added value to the current diagnostic testing landscape in specific circumstances and will play an increasingly important role in diagnostic microbiology. Future advancements will shorten turnaround time, reduce costs, and improve our analysis and interpretation of clinically actionable results.
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Affiliation(s)
- Eric M Ransom
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
| | - Robert F Potter
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO
| | - Gautam Dantas
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
- The Edison Family Center for Genome Sciences and Systems Biology, Washington University School of Medicine, St. Louis, MO
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO
- Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, MO
| | - Carey-Ann D Burnham
- Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO
- Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO
- Departments of Pediatrics and Medicine, Washington University School of Medicine, St. Louis, MO
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43
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Jamal RB, Shipovskov S, Ferapontova EE. Electrochemical Immuno- and Aptamer-Based Assays for Bacteria: Pros and Cons over Traditional Detection Schemes. SENSORS (BASEL, SWITZERLAND) 2020; 20:E5561. [PMID: 32998409 PMCID: PMC7582323 DOI: 10.3390/s20195561] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Revised: 09/15/2020] [Accepted: 09/23/2020] [Indexed: 01/20/2023]
Abstract
Microbiological safety of the human environment and health needs advanced monitoring tools both for the specific detection of bacteria in complex biological matrices, often in the presence of excessive amounts of other bacterial species, and for bacteria quantification at a single cell level. Here, we discuss the existing electrochemical approaches for bacterial analysis that are based on the biospecific recognition of whole bacterial cells. Perspectives of such assays applications as emergency-use biosensors for quick analysis of trace levels of bacteria by minimally trained personnel are argued.
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Affiliation(s)
| | | | - Elena E. Ferapontova
- Interdisciplinary Nanoscience Center (iNANO), Aarhus University Gustav Wieds Vej 14, DK-8000 Aarhus, Denmark; (R.B.J.); (S.S.)
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44
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Yazaki E, Kume K, Shiratori T, Eglit Y, Tanifuji G, Harada R, Simpson AGB, Ishida KI, Hashimoto T, Inagaki Y. Barthelonids represent a deep-branching metamonad clade with mitochondrion-related organelles predicted to generate no ATP. Proc Biol Sci 2020; 287:20201538. [PMID: 32873198 PMCID: PMC7542792 DOI: 10.1098/rspb.2020.1538] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
We here report the phylogenetic position of barthelonids, small anaerobic flagellates previously examined using light microscopy alone. Barthelona spp. were isolated from geographically distinct regions and we established five laboratory strains. Transcriptomic data generated from one Barthelona strain (PAP020) were used for large-scale, multi-gene phylogenetic (phylogenomic) analyses. Our analyses robustly placed strain PAP020 at the base of the Fornicata clade, indicating that barthelonids represent a deep-branching metamonad clade. Considering the anaerobic/microaerophilic nature of barthelonids and preliminary electron microscopy observations on strain PAP020, we suspected that barthelonids possess functionally and structurally reduced mitochondria (i.e. mitochondrion-related organelles or MROs). The metabolic pathways localized in the MRO of strain PAP020 were predicted based on its transcriptomic data and compared with those in the MROs of fornicates. We here propose that strain PAP020 is incapable of generating ATP in the MRO, as no mitochondrial/MRO enzymes involved in substrate-level phosphorylation were detected. Instead, we detected a putative cytosolic ATP-generating enzyme (acetyl-CoA synthetase), suggesting that strain PAP020 depends on ATP generated in the cytosol. We propose two separate losses of substrate-level phosphorylation from the MRO in the clade containing barthelonids and (other) fornicates.
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Affiliation(s)
- Euki Yazaki
- Interdisciplinary Theoretical and Mathematical Sciences (iTHEMS), RIKEN, Wako, Saitama, Japan
| | - Keitaro Kume
- Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Takashi Shiratori
- Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.,Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Yana Eglit
- Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.,Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Goro Tanifuji
- Department of Zoology, National Museum of Nature and Science, Ibaraki, Japan
| | - Ryo Harada
- Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Alastair G B Simpson
- Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.,Centre for Comparative Genomics and Evolutionary Bioinformatics, Dalhousie University, Halifax, Nova Scotia, Canada
| | - Ken-Ichiro Ishida
- Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.,Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Tetsuo Hashimoto
- Faculty of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.,Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
| | - Yuji Inagaki
- Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan.,Center for Computational Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan
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45
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Tribolet L, Kerr E, Cowled C, Bean AGD, Stewart CR, Dearnley M, Farr RJ. MicroRNA Biomarkers for Infectious Diseases: From Basic Research to Biosensing. Front Microbiol 2020; 11:1197. [PMID: 32582115 PMCID: PMC7286131 DOI: 10.3389/fmicb.2020.01197] [Citation(s) in RCA: 120] [Impact Index Per Article: 24.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2020] [Accepted: 05/12/2020] [Indexed: 12/19/2022] Open
Abstract
In the pursuit of improved diagnostic tests for infectious diseases, several classes of molecules have been scrutinized as prospective biomarkers. Small (18–22 nucleotide), non-coding RNA transcripts called microRNAs (miRNAs) have emerged as promising candidates with extensive diagnostic potential, due to their role in numerous diseases, previously established methods for quantitation and their stability within biofluids. Despite efforts to identify, characterize and apply miRNA signatures as diagnostic markers in a range of non-infectious diseases, their application in infectious disease has advanced relatively slowly. Here, we outline the benefits that miRNA biomarkers offer to the diagnosis, management, and treatment of infectious diseases. Investigation of these novel biomarkers could advance the use of personalized medicine in infectious disease treatment, which raises important considerations for validating their use as diagnostic or prognostic markers. Finally, we discuss new and emerging miRNA detection platforms, with a focus on rapid, point-of-care testing, to evaluate the benefits and obstacles of miRNA biomarkers for infectious disease.
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Affiliation(s)
- Leon Tribolet
- Health and Biosecurity, Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
| | - Emily Kerr
- Institute for Frontier Materials, Deakin University, Geelong, VIC, Australia
| | - Christopher Cowled
- Health and Biosecurity, Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
| | - Andrew G D Bean
- Health and Biosecurity, Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
| | - Cameron R Stewart
- Health and Biosecurity, Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
| | - Megan Dearnley
- Diagnostics, Surveillance and Response (DSR), Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
| | - Ryan J Farr
- Diagnostics, Surveillance and Response (DSR), Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, VIC, Australia
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46
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Marine RL, Magaña LC, Castro CJ, Zhao K, Montmayeur AM, Schmidt A, Diez-Valcarce M, Ng TFF, Vinjé J, Burns CC, Nix WA, Rota PA, Oberste MS. Comparison of Illumina MiSeq and the Ion Torrent PGM and S5 platforms for whole-genome sequencing of picornaviruses and caliciviruses. J Virol Methods 2020; 280:113865. [PMID: 32302601 PMCID: PMC9119587 DOI: 10.1016/j.jviromet.2020.113865] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Revised: 02/04/2020] [Accepted: 04/06/2020] [Indexed: 02/06/2023]
Abstract
Next-generation sequencing is a powerful tool for virological surveillance. While Illumina® and Ion Torrent® sequencing platforms are used extensively for generating viral RNA genome sequences, there is limited data comparing different platforms. The Illumina MiSeq, Ion Torrent PGM and Ion Torrent S5 platforms were evaluated using a panel of sixteen specimens containing picornaviruses and human caliciviruses (noroviruses and sapoviruses). The specimens were processed, using combinations of three library preparation and five sequencing kits, to assess the quality and completeness of assembled viral genomes, and an estimation of cost per sample to generate the data was calculated. The choice of library preparation kit and sequencing platform was found to impact the breadth of genome coverage and accuracy of consensus viral genomes. The Ion Torrent S5 510 chip runs produced more reads at a lower cost per sample than the highest output Ion Torrent PGM 318 chip run, and generated the highest proportion of reads for enterovirus D68 samples. However, indels at homopolymer regions impacted the accuracy of consensus genome sequences. For lower throughput sequencing runs (i.e., Ion Torrent 510 and Illumina MiSeq Nano V2), the cost per sample was lower on the MiSeq platform, whereas with higher throughput runs (Ion Torrent 530 and Illumina MiSeq V2) there is less of a difference in the cost per sample between the two sequencing platforms ($5.47-$10.25 more per sample for an Ion Torrent 530 chip run when multiplexing 24 samples). These findings suggest that the Ion Torrent S5 and Illumina MiSeq platforms are both viable options for genomic sequencing of RNA viruses, each with specific advantages and tradeoffs.
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Affiliation(s)
- Rachel L Marine
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.
| | - Laura C Magaña
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA
| | - Christina J Castro
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA
| | - Kun Zhao
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | | | | | - Marta Diez-Valcarce
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA; Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA
| | - Terry Fei Fan Ng
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Jan Vinjé
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Cara C Burns
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - W Allan Nix
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - Paul A Rota
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
| | - M Steven Oberste
- Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA
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47
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Santhosh Kumar J, Ramakrishan M, Seethapathy G, Krishna V, Uma Shaanker R, Ravikanth G. DNA barcoding of Momordica species and assessment of adulteration in Momordica herbal products, an anti-diabetic drug. ACTA ACUST UNITED AC 2020. [DOI: 10.1016/j.plgene.2020.100227] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
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48
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Contrasting bacteriome of the hornwort Leiosporoceros dussii in two nearby sites with emphasis on the hornwort-cyanobacterial symbiosis. Symbiosis 2020. [DOI: 10.1007/s13199-020-00680-1] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/27/2022]
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49
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Mas-Lloret J, Obón-Santacana M, Ibáñez-Sanz G, Guinó E, Pato ML, Rodriguez-Moranta F, Mata A, García-Rodríguez A, Moreno V, Pimenoff VN. Gut microbiome diversity detected by high-coverage 16S and shotgun sequencing of paired stool and colon sample. Sci Data 2020; 7:92. [PMID: 32179734 PMCID: PMC7075950 DOI: 10.1038/s41597-020-0427-5] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2019] [Accepted: 02/21/2020] [Indexed: 12/22/2022] Open
Abstract
The gut microbiome has a fundamental role in human health and disease. However, studying the complex structure and function of the gut microbiome using next generation sequencing is challenging and prone to reproducibility problems. Here, we obtained cross-sectional colon biopsies and faecal samples from nine participants in our COLSCREEN study and sequenced them in high coverage using Illumina pair-end shotgun (for faecal samples) and IonTorrent 16S (for paired feces and colon biopsies) technologies. The metagenomes consisted of between 47 and 92 million reads per sample and the targeted sequencing covered more than 300 k reads per sample across seven hypervariable regions of the 16S gene. Our data is freely available and coupled with code for the presented metagenomic analysis using up-to-date bioinformatics algorithms. These results will add up to the informed insights into designing comprehensive microbiome analysis and also provide data for further testing for unambiguous gut microbiome analysis.
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Affiliation(s)
- Joan Mas-Lloret
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain.,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain.,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain
| | - Mireia Obón-Santacana
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain.,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain.,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain
| | - Gemma Ibáñez-Sanz
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain.,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain.,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain.,Gastroenterology Department, Bellvitge University Hospital-IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
| | - Elisabet Guinó
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain.,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain.,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain
| | - Miguel L Pato
- Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Biomedical Research Institute (IDIBELL), Barcelona, Catalonia, Spain
| | - Francisco Rodriguez-Moranta
- Gastroenterology Department, Bellvitge University Hospital-IDIBELL, Hospitalet de Llobregat, Barcelona, Spain
| | - Alfredo Mata
- Digestive System Service, Moisés Broggi Hospital, Sant Joan Despí, Spain
| | - Ana García-Rodríguez
- Endoscopy Unit, Digestive System Service, Viladecans Hospital-IDIBELL, Viladecans, Spain
| | - Victor Moreno
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain. .,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain. .,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain. .,Department of Clinical Sciences, Faculty of Medicine, University of Barcelona, Barcelona, Spain.
| | - Ville Nikolai Pimenoff
- Oncology Data Analytics Program, Catalan Institute of Oncology (ICO), Barcelona, Spain. .,Colorectal Cancer Group, ONCOBELL Program, Bellvitge Institute of Biomedical Research (IDIBELL), Barcelona, Spain. .,Consortium for Biomedical Research in Epidemiology and Public Health (CIBERESP), Barcelona, Spain. .,National Cancer Center Finland (FICAN-MID) and Karolinska Institute, Stockholm, Sweden.
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50
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Ju M, Bai X, Zhang T, Lin Y, Yang L, Zhou H, Chang X, Guan S, Ren X, Li K, Wang Y, Li G. Mutation spectrum of COL1A1/COL1A2 screening by high-resolution melting analysis of Chinese patients with osteogenesis imperfecta. J Bone Miner Metab 2020; 38:188-197. [PMID: 31414283 DOI: 10.1007/s00774-019-01039-3] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2018] [Accepted: 07/24/2019] [Indexed: 11/30/2022]
Abstract
High-resolution melting (HRM) analysis has been shown to be a time-saving method for the screening of genetic variants. To increase the precision of the diagnosis of osteogenesis imperfecta (OI), we used HRM to explore COL1A1/COL1A2 mutations in 87 Chinese OI patients and to perform population-based studies of the relationships between their genotypes and phenotypes. Peripheral blood samples were collected from the 87 non-consanguineous probands. The coding regions and exon boundaries of COL1A1/COL1A2 were detected by HRM and confirmed by Sanger sequencing. The functional effects of mutations were predicted through bioinformatic tools. Mutations were detected in 70.3% of familial cases and 40% of sporadic cases (p < 0.01). Compared with COL1A1 mutations, patients with COL1A2 mutations were more prone to severe phenotypes. Helical mutations (caused by substitution of the glycine within the Gly-X-Y triplet domain) were more likely to occur in patients with type III and IV (p < 0.05). Haploinsufficiency mutations (caused by frameshift, nonsense, and splice-site mutations) appeared more frequently in patients with type I (p < 0.05). Compared with the Sanger sequencing and whole exome sequencing (WES), HRM was found to reduce total costs by 78%- 80% in patients who had a positive HRM separate melting curve. Our findings suggest that HRM would greatly benefit small and understaffed hospitals and laboratories, and would facilitate the accurate diagnosis and early treatment of OI in remote and less developed regions.
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Affiliation(s)
- Mingyan Ju
- Department of Genetics, Tianjin Medical University, Tianjin, 300070, People's Republic of China
| | - Xue Bai
- Department of Medical Laboratory, Tianjin Hospital, Tianjin, 300000, People's Republic of China
| | - Tianke Zhang
- Colorectal Surgery, Tianjin People's Hospital, Tianjin, 300000, People's Republic of China
| | - Yunshou Lin
- School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, People's Republic of China
| | - Li Yang
- School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, People's Republic of China
| | - Huaiyu Zhou
- School of Basic Medical Sciences, Tianjin Medical University, Tianjin, 300070, People's Republic of China
| | - Xiaoli Chang
- Department of Medical Laboratory, Tianjin Hospital, Tianjin, 300000, People's Republic of China
| | - Shizhen Guan
- Department of Medical Laboratory, Tianjin Hospital, Tianjin, 300000, People's Republic of China
| | - Xiuzhi Ren
- Orthopedic Ward III, Wuqing People's Hospital, Tianjin, 300000, People's Republic of China
| | - Keqiu Li
- Department of Genetics, Tianjin Medical University, Tianjin, 300070, People's Republic of China
| | - Yi Wang
- Department of Medical Laboratory, Tianjin Hospital, Tianjin, 300000, People's Republic of China
| | - Guang Li
- Department of Genetics, Tianjin Medical University, Tianjin, 300070, People's Republic of China.
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