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Sebastian PJ, Schlesener C, Byrne BA, Miller M, Smith W, Batac F, Goertz CEC, Weimer BC, Johnson CK. Antimicrobial resistance of Vibrio spp. from the coastal California system: discordance between genotypic and phenotypic patterns. Appl Environ Microbiol 2025; 91:e0180824. [PMID: 39898660 PMCID: PMC11921324 DOI: 10.1128/aem.01808-24] [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] [Received: 09/23/2024] [Accepted: 12/14/2024] [Indexed: 02/04/2025] Open
Abstract
Antimicrobial resistance in Vibrio species poses risks to both human and marine mammal health. Whole genome sequencing of Vibrio spp. can be utilized to screen for antimicrobial resistance genes and allelic variants to provide mechanistic insights in ways that PCR screening and phenotypic interpretation cannot. Our goals were to (i) characterize antimicrobial resistance patterns of Vibrio spp. pathogens isolated from southern sea otters (Enhydra lutris nereis), northern sea otters (Enhydra lutris kenyoni), and environmental samples from the central California coast using whole genome sequencing, and (ii) compare the presence of antimicrobial resistance genes with phenotypic interpretation from antibiotic susceptibility testing. Unexpectedly, genomic classification identified an understudied species, Vibrio diabolicus, in sea otter and environmental isolates that were previously identified as Vibrio alginolyticus. A total of 489 Vibrio spp. isolates were sequenced, and frequently detected antimicrobial resistance genes included multidrug efflux pumps and genes associated with resistance to ß-lactams and tetracyclines. Genes associated with resistance to fluoroquinolones, aminoglycosides, chloramphenicol, and sulfonamides were uncommon. Sea otter isolates were phenotypically susceptible to tetracycline despite carrying genes tet34 and tet35. Both between- and within-species variations in ampicillin resistance were observed despite the ubiquitous presence of blaCARB genes in V. alginolyticus, V. diabolicus, and Vibrio parahaemolyticus. Discordance between phenotypic and genotypic ampicillin resistance was especially noted for V. parahaemolyticus and was partially attributed to the allelic variation of the blaCARB genes. Tetracyclines and fluoroquinolones, but not ß-lactams, are likely to be effective treatments for vibriosis in sea otters. IMPORTANCE Vibriosis (infection with non-cholera Vibrio spp.) is the most common seafood-borne illness globally, with major impacts on public health, food security, and wildlife health. Potential treatments of antimicrobial-resistant Vibrio spp. in humans, aquaculture, and marine wildlife rehabilitation are complicated by current diagnostic challenges regarding bacterial species identification and interpretation of antimicrobial resistance patterns. Unexpected detection of previously misidentified Vibrio diabolicus in sea otters suggests that a broader taxonomic group of Vibrio infect sea otters than previously described. We also determined that the presence of ß-lactamase genes alone in sea otter isolates does not necessarily correlate with an ampicillin-resistant phenotype, likely due to deleterious amino acid substitutions in certain blaCARB alleles. Continued monitoring of Vibrio spp. phenotypes and genotypes in sea otters is warranted to observe biologically relevant changes in antimicrobial resistance.
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Affiliation(s)
- Peter J. Sebastian
- EpiCenter for Disease Dynamics, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
- Karen C. Drayer Wildlife Health Center, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
| | - Cory Schlesener
- Department of Population Health and Reproduction, 100K Pathogen Genome Project, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
| | - Barbara A. Byrne
- Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
| | - Melissa Miller
- Karen C. Drayer Wildlife Health Center, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
- Marine Wildlife Veterinary Care and Research Center, California Department of Fish and Wildlife, Santa Cruz, California, USA
| | - Woutrina Smith
- Karen C. Drayer Wildlife Health Center, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
| | - Francesca Batac
- Marine Wildlife Veterinary Care and Research Center, California Department of Fish and Wildlife, Santa Cruz, California, USA
| | | | - Bart C. Weimer
- Department of Population Health and Reproduction, 100K Pathogen Genome Project, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
| | - Christine K. Johnson
- EpiCenter for Disease Dynamics, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
- Karen C. Drayer Wildlife Health Center, One Health Institute, School of Veterinary Medicine, University of California, Davis, Davis, California, USA
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2
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Jago MJ, Soley JK, Denisov S, Walsh CJ, Gifford DR, Howden BP, Lagator M. High-throughput method characterizes hundreds of previously unknown antibiotic resistance mutations. Nat Commun 2025; 16:780. [PMID: 39824824 PMCID: PMC11742677 DOI: 10.1038/s41467-025-56050-2] [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: 05/10/2024] [Accepted: 01/07/2025] [Indexed: 01/20/2025] Open
Abstract
A fundamental obstacle to tackling the antimicrobial resistance crisis is identifying mutations that lead to resistance in a given genomic background and environment. We present a high-throughput technique - Quantitative Mutational Scan sequencing (QMS-seq) - that enables quantitative comparison of which genes are under antibiotic selection and captures how genetic background influences resistance evolution. We compare four E. coli strains exposed to ciprofloxacin, cycloserine, or nitrofurantoin and identify 812 resistance mutations, many in genes and regulatory regions not previously associated with resistance. We find that multi-drug and antibiotic-specific resistance are acquired through categorically different types of mutations, and that minor genotypic differences significantly influence evolutionary routes to resistance. By quantifying mutation frequency with single base pair resolution, QMS-seq informs about the underlying mechanisms of resistance and identifies mutational hotspots within genes. Our method provides a way to rapidly screen for resistance mutations while assessing the impact of multiple confounding factors.
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Affiliation(s)
- Matthew J Jago
- Division of Evolution, Infection and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK
| | - Jake K Soley
- Division of Evolution, Infection and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK
- Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia
| | - Stepan Denisov
- Division of Evolution, Infection and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK
| | - Calum J Walsh
- Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia
| | - Danna R Gifford
- Division of Evolution, Infection and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK
| | - Benjamin P Howden
- Department of Microbiology and Immunology, University of Melbourne, at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, 3000, Australia
- Centre for Pathogen Genomics, University of Melbourne, Melbourne, VIC, 3000, Australia
| | - Mato Lagator
- Division of Evolution, Infection and Genomic Sciences, School of Biological Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, M13 9PL, UK.
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3
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Hetman BM, Pearl DL, Reid-Smith R, Parmley EJ, Taboada EN. An epidemiological framework for improving the accuracy of whole-genome sequence-based antimicrobial resistance surveillance in Salmonella. Can J Microbiol 2025; 71:1-17. [PMID: 40101256 DOI: 10.1139/cjm-2024-0090] [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] [Indexed: 03/20/2025]
Abstract
Whole-genome sequence-based surveillance of bacteria for determinants of antimicrobial resistance (AMR) promises many advantages over traditional, wet-lab approaches. However, adjustments to parameters used to identify genetic determinants from sequencing data can affect results and interpretation of the important determinants in circulation. Using a dataset of whole-genome sequences from 1633 isolates of Salmonella Heidelberg and S. Kentucky collected from surveillance of Canadian poultry production, we queried the genomic data using an in silico AMR detection tool, StarAMR, applying a range of parameter values required for the detection pipeline to test for differences in detection accuracy. We compared the results from each iteration to phenotypic antimicrobial susceptibility results, and generated estimates of sensitivity and specificity using regression models that controlled for the effects of multiple sampling events and variables, and interactions between covariates. Results from our analyses revealed small, yet significant effects of the input parameters on the sensitivity and specificity of the AMR detection tool, and these effects differed based on the serovar and drug class in question. Findings from this study may have implications for the incorporation of whole-genome sequence-based approaches to the surveillance of AMR determinants in bacteria sampled from food products and animals related to food production.
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Affiliation(s)
- Benjamin M Hetman
- Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
- Office of Public Health Field Services and Training, Center for Emergency Preparedness, Public Health Agency of Canada, Winnipeg, MB, Canada
| | - David L Pearl
- Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
| | - Richard Reid-Smith
- Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
- Centre for Foodborne, Environmental and Zoonotic Infectious Diseases, Public Health Agency of Canada, Guelph, ON, Canada
| | - E Jane Parmley
- Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, ON, Canada
| | - Eduardo N Taboada
- National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada
- Department of Food and Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, Canada
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4
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Jia H, Li X, Zhuang Y, Wu Y, Shi S, Sun Q, He F, Liang S, Wang J, Draz MS, Xie X, Zhang J, Yang Q, Ruan Z. Neural network-based predictions of antimicrobial resistance phenotypes in multidrug-resistant Acinetobacter baumannii from whole genome sequencing and gene expression. Antimicrob Agents Chemother 2024; 68:e0144624. [PMID: 39540735 PMCID: PMC11619347 DOI: 10.1128/aac.01446-24] [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] [Received: 09/29/2024] [Accepted: 10/22/2024] [Indexed: 11/16/2024] Open
Abstract
Whole genome sequencing (WGS) potentially represents a rapid approach for antimicrobial resistance genotype-to-phenotype prediction. However, the challenge still exists to predict fully minimum inhibitory concentrations (MICs) and antimicrobial susceptibility phenotypes based on WGS data. This study aimed to establish an artificial intelligence-based computational approach in predicting antimicrobial susceptibilities of multidrug-resistant Acinetobacter baumannii from WGS and gene expression data. Antimicrobial susceptibility testing (AST) was performed using the broth microdilution method for 10 antimicrobial agents. In silico multilocus sequence typing (MLST), antimicrobial resistance genes, and phylogeny based on cgSNP and cgMLST strategies were analyzed. High-throughput qPCR was performed to measure the expression level of antimicrobial resistance (AMR) genes. Most isolates exhibited a high level of resistance to most of the tested antimicrobial agents, with the majority belonging to the IC2/CC92 lineage. Phylogenetic analysis revealed undetected transmission events or local outbreaks. The percentage agreements between AMR phenotype and genotype ranged from 70.08% to 89.96%, with the coefficient of agreement (κ) extending from 0.025 and 0.881. The prediction of AST employed by deep neural network models achieved an accuracy of up to 98.64% on the testing data set. Additionally, several linear regression models demonstrated high prediction accuracy, reaching up to 86.15% within an error range of one gradient, indicating a linear relationship between certain gene expressions and the corresponding antimicrobial MICs. In conclusion, neural network-based predictions could be used as a tool for the surveillance of antimicrobial resistance in multidrug-resistant A. baumannii.
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Affiliation(s)
- Huiqiong Jia
- Department of Laboratory Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, China
| | - Xinyang Li
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
| | - Yilu Zhuang
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
| | - Yuye Wu
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
| | - Shasha Shi
- Department of Laboratory Medicine, Wuyi First People’s Hospital, Jinhua, China
| | - Qingyang Sun
- Department of Clinical Laboratory, No. 903 Hospital of PLA Joint Logistic Support Force, Hangzhou, China
| | - Fang He
- Laboratory Medicine Center, Department of Clinical Laboratory, Zhejiang Provincial People’s Hospital (Affiliated People’s Hospital, Hangzhou Medical College), Hangzhou, China
| | - Shanyan Liang
- Department of Clinical Laboratory, Ningbo No.2 Hospital, Ningbo, China
| | - Jianfeng Wang
- Department of Respiratory and Critical Care Medicine, Zhejiang Provincial Hospital of Chinese Medicine, Hangzhou, China
| | - Mohamed S. Draz
- Department of Medicine, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA
- Department of Biomedical Engineering, Cleveland Clinic, Cleveland, Ohio, USA
| | - Xinyou Xie
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
| | - Jun Zhang
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
| | - Qing Yang
- Department of Laboratory Medicine, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Clinical In Vitro Diagnostic Techniques of Zhejiang Province, Hangzhou, China
| | - Zhi Ruan
- Department of Clinical Laboratory, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou, China
- Key Laboratory of Precision Medicine in Diagnosis and Monitoring Research of Zhejiang Province, Hangzhou, China
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5
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Gupta YD, Bhandary S. Artificial Intelligence for Understanding Mechanisms of Antimicrobial Resistance and Antimicrobial Discovery. ARTIFICIAL INTELLIGENCE AND MACHINE LEARNING IN DRUG DESIGN AND DEVELOPMENT 2024:117-156. [DOI: 10.1002/9781394234196.ch5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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Yang Y, Xie S, He F, Xu Y, Wang Z, Ihsan A, Wang X. Recent development and fighting strategies for lincosamide antibiotic resistance. Clin Microbiol Rev 2024; 37:e0016123. [PMID: 38634634 PMCID: PMC11237733 DOI: 10.1128/cmr.00161-23] [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: 04/19/2024] Open
Abstract
SUMMARYLincosamides constitute an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. However, due to the misuse of lincosamide and co-selection pressure, the resistance to lincosamide has become a serious concern. It is urgently needed to carefully understand the phenomenon and mechanism of lincosamide resistance to effectively prevent and control lincosamide resistance. To date, six mobile lincosamide resistance classes, including lnu, cfr, erm, vga, lsa, and sal, have been identified. These lincosamide resistance genes are frequently found on mobile genetic elements (MGEs), such as plasmids, transposons, integrative and conjugative elements, genomic islands, and prophages. Additionally, MGEs harbor the genes that confer resistance not only to antimicrobial agents of other classes but also to metals and biocides. The ultimate purpose of discovering and summarizing bacterial resistance is to prevent, control, and combat resistance effectively. This review highlights four promising strategies, including chemical modification of antibiotics, the development of antimicrobial peptides, the initiation of bacterial self-destruct program, and antimicrobial stewardship, to fight against resistance and safeguard global health.
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Affiliation(s)
- Yingying Yang
- National Reference Laboratory of Veterinary Drug Residues (HZAU), Huazhong Agricultural University, Wuhan, Hubei, China
- MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Shiyu Xie
- MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Fangjing He
- MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China
| | - Yindi Xu
- Institute of Animal Husbandry Research, Henan Academy of Agricultural Sciences, Zhengzhou, Henan, China
| | - Zhifang Wang
- Institute of Animal Husbandry Research, Henan Academy of Agricultural Sciences, Zhengzhou, Henan, China
| | - Awais Ihsan
- Department of Biosciences, COMSATS University Islamabad, Sahiwal campus, Islamabad, Pakistan
| | - Xu Wang
- National Reference Laboratory of Veterinary Drug Residues (HZAU), Huazhong Agricultural University, Wuhan, Hubei, China
- MAO Key Laboratory for Detection of Veterinary Drug Residues, Huazhong Agricultural University, Wuhan, Hubei, China
- MOA Laboratory for Risk Assessment of Quality and Safety of Livestock and Poultry Products, Huazhong Agricultural University, Wuhan, Hubei, China
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7
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Cimen C, Bathoorn E, Loeve AJ, Fliss M, Berends MS, Nagengast WB, Hamprecht A, Voss A, Lokate M. Uncovering the spread of drug-resistant bacteria through next-generation sequencing based surveillance: transmission of extended-spectrum β-lactamase-producing Enterobacterales by a contaminated duodenoscope. Antimicrob Resist Infect Control 2024; 13:31. [PMID: 38459544 PMCID: PMC10924313 DOI: 10.1186/s13756-024-01386-5] [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: 11/02/2023] [Accepted: 03/03/2024] [Indexed: 03/10/2024] Open
Abstract
Contamination of duodenoscopes is a significant concern due to the transmission of multidrug-resistant organisms (MDROs) among patients who undergo endoscopic retrograde cholangiopancreatography (ERCP), resulting in outbreaks worldwide. In July 2020, it was determined that three different patients, all had undergone ERCP with the same duodenoscope, were infected. Two patients were infected with blaCTX-M-15 encoding Citrobacter freundii, one experiencing a bloodstream infection and the other a urinary tract infection, while another patient had a bloodstream infection caused by blaSHV-12 encoding Klebsiella pneumoniae. Molecular characterization of isolates was available as every ESBL-producing isolate undergoes Next-Generation Sequencing (NGS) for comprehensive genomic analysis in our center. After withdrawing the suspected duodenoscope, we initiated comprehensive epidemiological research, encompassing case investigations, along with a thorough duodenoscope investigation. Screening of patients who had undergone ERCP with the implicated duodenoscope, as well as a selection of hospitalized patients who had ERCP with a different duodenoscope during the outbreak period, led to the discovery of three additional cases of colonization in addition to the three infections initially detected. No microorganisms were detected in eight routine culture samples retrieved from the suspected duodenoscope. Only after destructive dismantling of the duodenoscope, the forceps elevator was found to be positive for blaSHV-12 encoding K. pneumoniae which was identical to the isolates detected in three patients. This study highlights the importance of using NGS to monitor the transmission of MDROs and demonstrates that standard cultures may fail to detect contaminated medical equipment such as duodenoscopes.
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Affiliation(s)
- Cansu Cimen
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands
- Institute for Medical Microbiology and Virology, University of Oldenburg, Oldenburg, Germany
| | - Erik Bathoorn
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands
| | - Arjo J Loeve
- Department of Biomechanical Engineering, Faculty of Mechanical Engineering, Delft University of Technology, Delft, The Netherlands
| | - Monika Fliss
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands
| | - Matthijs S Berends
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands
- Certe Medical Diagnostics and Advice Foundation, Department of Medical Epidemiology, Groningen, The Netherlands
| | - Wouter B Nagengast
- Department of Gastroenterology, University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands
| | - Axel Hamprecht
- Institute for Medical Microbiology and Virology, University of Oldenburg, Oldenburg, Germany
| | - Andreas Voss
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands
| | - Mariëtte Lokate
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, Hanzeplein 1, 9700RB, Groningen, The Netherlands.
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Vieira ADA, Piccoli BC, Y Castro TR, Casarin BC, Tessele LF, Martins RCR, Schwarzbold AV, Trindade PDA. Pipeline validation for the identification of antimicrobial-resistant genes in carbapenem-resistant Klebsiella pneumoniae. Sci Rep 2023; 13:15189. [PMID: 37709838 PMCID: PMC10502106 DOI: 10.1038/s41598-023-42154-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Accepted: 09/06/2023] [Indexed: 09/16/2023] Open
Abstract
Antimicrobial-resistant Klebsiella pneumoniae is a global threat to healthcare and an important cause of nosocomial infections. Antimicrobial resistance causes prolonged treatment periods, high mortality rates, and economic impacts. Whole Genome Sequencing (WGS) has been used in laboratory diagnosis, but there is limited evidence about pipeline validation to parse generated data. Thus, the present study aimed to validate a bioinformatics pipeline for the identification of antimicrobial resistance genes from carbapenem-resistant K. pneumoniae WGS. Sequences were obtained from a publicly available database, trimmed, de novo assembled, mapped to the K. pneumoniae reference genome, and annotated. Contigs were submitted to different tools for bacterial (Kraken2 and SpeciesFinder) and antimicrobial resistance gene identification (ResFinder and ABRicate). We analyzed 201 K. pneumoniae genomes. In the bacterial identification by Kraken2, all samples were correctly identified, and in SpeciesFinder, 92.54% were correctly identified as K. pneumoniae, 6.96% erroneously as Pseudomonas aeruginosa, and 0.5% erroneously as Citrobacter freundii. ResFinder found a greater number of antimicrobial resistance genes than ABRicate; however, many were identified more than once in the same sample. All tools presented 100% repeatability and reproducibility and > 75% performance in other metrics. Kraken2 was more assertive in recognizing bacterial species, and SpeciesFinder may need improvements.
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Affiliation(s)
- Andressa de Almeida Vieira
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil
| | - Bruna Candia Piccoli
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil
| | - Thaís Regina Y Castro
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil
| | - Bruna Campestrini Casarin
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil
| | - Luiza Funck Tessele
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil
| | - Roberta Cristina Ruedas Martins
- Laboratório de Parasitologia Médica (LIM-46), Departamento de Doenças Infecciosas e Parasitárias, Instituto de Medicina Tropical da Universidade de São Paulo, Faculdade de Medicina da Universidade de São Paulo, São Paulo, 01246-903, Brazil
| | | | - Priscila de Arruda Trindade
- Laboratório de Biologia Molecular e Bioinformática Aplicada à Microbiologia Clínica, Programa de Pós-Graduação em Ciências Farmacêuticas, Universidade Federal de Santa Maria, Santa Maria, 97105-900, Brazil.
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9
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Lakshmanan D, Ramasamy D, Subramanyam V, Saravanan SK. Mobile colistin resistance (mcr) genes and recent developments in colistin resistance detection. Lett Appl Microbiol 2023; 76:ovad102. [PMID: 37673673 DOI: 10.1093/lambio/ovad102] [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: 06/20/2023] [Revised: 08/17/2023] [Accepted: 09/05/2023] [Indexed: 09/08/2023]
Abstract
The peptide antibiotic colistin has been reserved as a last resort antibiotic treatment option for cases where other antibiotics including carbapenems have failed. Recent emergence of colistin resistance and discovery of mobile colistin resistance (mcr) genes, which encode the cell wall modifying phosphoethanolamine transferase enzyme, complicates the issue. The mcr genes have been associated with conjugative plasmids and can be horizontally transferred between different bacterial species. The global spread of mcr genes has been extensively documented and this warrants surveillance of the resistance genes in the community. However, susceptibility testing of colistin is fraught with practical challenges owing to the chemical nature of the drug and multiple mechanisms of resistance. Although broth microdilution is the current gold standard for colistin susceptibility testing, the method poses technical challenges. Hence, alternative detection methods for screening colistin resistance are the need of the hour. Several methods have been studied in the recent times to address this issue. In this review, we discuss some of the recent developments in the detection of colistin resistance.
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Affiliation(s)
- Divya Lakshmanan
- Mahatma Gandhi Medical Advanced Research Institute (MGMARI), Sri Balaji Vidyapeeth (Deemed-to-be-University), Pillayarkuppam, Pondicherry 607042, India
| | - Dhamodharan Ramasamy
- Mahatma Gandhi Medical Advanced Research Institute (MGMARI), Sri Balaji Vidyapeeth (Deemed-to-be-University), Pillayarkuppam, Pondicherry 607042, India
| | - Veni Subramanyam
- Mahatma Gandhi Medical Advanced Research Institute (MGMARI), Sri Balaji Vidyapeeth (Deemed-to-be-University), Pillayarkuppam, Pondicherry 607042, India
| | - Suresh Kumar Saravanan
- Mahatma Gandhi Medical Preclinical Research Centre (MGMPRC), Sri Balaji Vidyapeeth (Deemed-to-be-University), Pillayarkuppam, Pondicherry 607402, India
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10
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Zarras C, Iosifidis E, Simitsopoulou M, Pappa S, Kontou A, Roilides E, Papa A. Neonatal Bloodstream Infection with Ceftazidime-Avibactam-Resistant blaKPC-2-Producing Klebsiella pneumoniae Carrying blaVEB-25. Antibiotics (Basel) 2023; 12:1290. [PMID: 37627710 PMCID: PMC10451261 DOI: 10.3390/antibiotics12081290] [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: 06/25/2023] [Revised: 07/23/2023] [Accepted: 08/03/2023] [Indexed: 08/27/2023] Open
Abstract
BACKGROUND Although ceftazidime/avibactam (CAZ/AVI) has become an important option for treating adults and children, no data or recommendations exist for neonates. We report a neonatal sepsis case due to CAZ/AVI-resistant blaKPC-2-harboring Klebsiella pneumoniae carrying blaVEB-25 and the use of a customized active surveillance program in conjunction with enhanced infection control measures. METHODS The index case was an extremely premature neonate hospitalized for 110 days that had been previously treated with multiple antibiotics. Customized molecular surveillance was implemented at hospital level and enhanced infection control measures were taken for early recognition and prevention of outbreak. Detection and identification of blaVEB-25 was performed using next-generation sequencing. RESULTS This was the first case of a bloodstream infection caused by KPC-producing K. pneumoniae that was resistant to CAZ/AVI without the presence of a metalo-β-lactamase in the multiplex PCR platform in a neonate. All 36 additional patients tested (12 in the same NICU and 24 from other hospital departments) carried wild-type blaVEB-1 but they did not harbor blaVEB-25. CONCLUSION The emergence of blaVEB-25 is signal for the horizontal transfer of plasmids at hospital facilities and it is of greatest concern for maintaining a sharp vigilance for the surveillance of novel resistance mechanisms. Molecular diagnostics can guide appropriate antimicrobial therapy and the early implementation of infection control measures against antimicrobial resistance.
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Affiliation(s)
- Charalampos Zarras
- Microbiology Department, Hippokration Hospital, 54642 Thessaloniki, Greece;
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (S.P.); (A.P.)
| | - Elias Iosifidis
- Infectious Disease Unit, 3rd Department of Pediatrics, School of Medicine, Faculty of Health Sciences, Hippokration Hospital, 54642 Thessaloniki, Greece; (M.S.); (E.R.)
- Basic and Translational Research Unit, Special Unit for Biomedical Research and Education, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Maria Simitsopoulou
- Infectious Disease Unit, 3rd Department of Pediatrics, School of Medicine, Faculty of Health Sciences, Hippokration Hospital, 54642 Thessaloniki, Greece; (M.S.); (E.R.)
- Basic and Translational Research Unit, Special Unit for Biomedical Research and Education, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Styliani Pappa
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (S.P.); (A.P.)
| | - Angeliki Kontou
- 1st Department of Neonatology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece;
| | - Emmanuel Roilides
- Infectious Disease Unit, 3rd Department of Pediatrics, School of Medicine, Faculty of Health Sciences, Hippokration Hospital, 54642 Thessaloniki, Greece; (M.S.); (E.R.)
- Basic and Translational Research Unit, Special Unit for Biomedical Research and Education, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece
| | - Anna Papa
- Department of Microbiology, School of Medicine, Faculty of Health Sciences, Aristotle University of Thessaloniki, 54124 Thessaloniki, Greece; (S.P.); (A.P.)
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Vázquez L, Srednik ME, Rodríguez J, Flórez AB, Mayo B. Antibiotic Resistance/Susceptibility Profiles of Staphylococcus equorum Strains from Cheese, and Genome Analysis for Antibiotic Resistance Genes. Int J Mol Sci 2023; 24:11657. [PMID: 37511416 PMCID: PMC10380560 DOI: 10.3390/ijms241411657] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Revised: 07/14/2023] [Accepted: 07/17/2023] [Indexed: 07/30/2023] Open
Abstract
In food, bacteria carrying antibiotic resistance genes could play a prominent role in the spread of resistance. Staphylococcus equorum populations can become large in a number of fermented foods, yet the antibiotic resistance properties of this species have been little studied. In this work, the resistance/susceptibility (R/S) profile of S. equorum strains (n = 30) from cheese to 16 antibiotics was determined by broth microdilution. The minimum inhibitory concentration (MIC) for all antibiotics was low in most strains, although higher MICs compatible with acquired genes were also noted. Genome analysis of 13 strains showed the S. equorum resistome to be composed of intrinsic mechanisms, acquired mutations, and acquired genes. As such, a plasmidic cat gene providing resistance to chloramphenicol was found in one strain; this was able to provide resistance to Staphylococcus aureus after electroporation. An msr(A) polymorphic gene was identified in five strains. The Mrs(A) variants were associated with variable resistance to erythromycin. However, the genetic data did not always correlate with the phenotype. As such, all strains harbored a polymorphic fosB/fosD gene, although only one acquired copy was associated with strong resistance to fosfomycin. Similarly, a plasmid-associated blaR1-blaZI operon encoding a penicillinase system was identified in five ampicillin- and penicillin G-susceptible strains. Identified genes not associated with phenotypic resistance further included mph(C) in two strains and norA in all strains. The antibiotic R/S status and gene content of S. equorum strains intended to be employed in food systems should be carefully determined.
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Affiliation(s)
- Lucía Vázquez
- Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain
- Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain
| | - Mariela E Srednik
- Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA
| | - Javier Rodríguez
- Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain
- Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain
| | - Ana Belén Flórez
- Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain
- Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain
| | - Baltasar Mayo
- Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA), Consejo Superior de Investigaciones Científicas (CSIC), Paseo Río Linares s/n, 33300 Villaviciosa, Spain
- Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Avenida de Roma s/n, 33011 Oviedo, Spain
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Kumari K, Sharma PK, Shikha S, Singh RP. Molecular characterization and in-depth genome analysis of Enterobacter sp. S-16. Funct Integr Genomics 2023; 23:245. [PMID: 37460717 DOI: 10.1007/s10142-023-01161-6] [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: 03/14/2023] [Revised: 04/20/2023] [Accepted: 06/27/2023] [Indexed: 07/20/2023]
Abstract
Enterobacter species are considered to be an opportunistic human pathogen owing to the existence of antibiotic-resistant strains and drug resides; however, the detailed analysis of the antibiotic resistance and virulence features in environmental isolates is poorly characterized. Here, in the study, we characterized the biochemical characteristics, and genome, pan-genome, and comparative genome analyses of an environmental isolate Enterobacter sp. S-16. The strain was identified as Enterobacter spp. by using 16S rRNA gene sequencing. To unravel genomic features, whole genome of Enterobacter sp. S-16 was sequenced using a hybrid assembly approach and genome assembly was performed using the Unicycler tool. The assembled genome contained the single conting size 5.3 Mbp, GC content 55.43%, and 4500 protein-coding genes. The genome analysis revealed the various gene clusters associated with virulence, antibiotic resistance, type VI secretion system (T6SS), and many stress tolerant genes, which may provide important insight for adapting to changing environment conditions. Moreover, different metabolic pathways were identified that potentially contribute to environmental survival. Various hydrolytic enzymes and motility functions equipped the strain S-16 as an active colonizer. The genome analysis confirms the presence of carbohydrate-active enzymes (CAZymes), and non-enzymatic carbohydrate-binding modules (CBMs) involved in the hydrolysis of complex carbohydrate polymers. Moreover, the pan-genome analysis provides detailed information about the core genes and shared genes with the closest related Enterobacter species. The present study is the first report showing the presence of YdhE/NorM in Enterobacter spp. Thus, the elucidation of genome sequencing will increase our understanding of the pathogenic nature of environmental isolate, supporting the One Health Concept.
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Affiliation(s)
- Kiran Kumari
- Department of Bioengineering and Biotechnology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, Pin 835215, India
| | - Parva Kumar Sharma
- Department of Plant Sciences and Landscape Architecture, University of Maryland, College Park, MD, 20742, USA
| | - Shweta Shikha
- Shyama Prasad Mukherjee University, Ranchi, Jharkhand, India
| | - Rajnish Prakash Singh
- Department of Bioengineering and Biotechnology, Birla Institute of Technology, Mesra, Ranchi, Jharkhand, Pin 835215, India.
- Department of Biotechnology, Jaypee Institute of Information Technology, Noida, India.
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Myintzaw P, Pennone V, McAuliffe O, Begley M, Callanan M. Association of Virulence, Biofilm, and Antimicrobial Resistance Genes with Specific Clonal Complex Types of Listeria monocytogenes. Microorganisms 2023; 11:1603. [PMID: 37375105 DOI: 10.3390/microorganisms11061603] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2023] [Revised: 06/14/2023] [Accepted: 06/15/2023] [Indexed: 06/29/2023] Open
Abstract
Precise classification of foodborne pathogen Listeria monocytogenes is a necessity in efficient foodborne disease surveillance, outbreak detection, and source tracking throughout the food chain. In this study, a total of 150 L. monocytogenes isolates from various food products, food processing environments, and clinical sources were investigated for variations in virulence, biofilm formation, and the presence of antimicrobial resistance genes based on their Whole-Genome Sequences. Clonal complex (CC) determination based on Multi-Locus Sequence Typing (MLST) revealed twenty-eight CC-types including eight isolates representing novel CC-types. The eight isolates comprising the novel CC-types share the majority of the known (cold and acid) stress tolerance genes and are all genetic lineage II, serogroup 1/2a-3a. Pan-genome-wide association analysis by Scoary using Fisher's exact test identified eleven genes specifically associated with clinical isolates. Screening for the presence of antimicrobial and virulence genes using the ABRicate tool uncovered variations in the presence of Listeria Pathogenicity Islands (LIPIs) and other known virulence genes. Specifically, the distributions of actA, ecbA, inlF, inlJ, lapB, LIPI-3, and vip genes across isolates were found to be significantly CC-dependent while the presence of ami, inlF, inlJ, and LIPI-3 was associated with clinical isolates specifically. In addition, Roary-derived phylogenetic grouping based on Antimicrobial-Resistant Genes (AMRs) revealed that the thiol transferase (FosX) gene was present in all lineage I isolates, and the presence of the lincomycin resistance ABC-F-type ribosomal protection protein (lmo0919_fam) was also genetic-lineage-dependent. More importantly, the genes found to be specific to CC-type were consistent when a validation analysis was performed with fully assembled, high-quality complete L. monocytogenes genome sequences (n = 247) extracted from the National Centre for Biotechnology Information (NCBI) microbial genomes database. This work highlights the usefulness of MLST-based CC typing using the Whole-Genome Sequence as a tool in classifying isolates.
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Affiliation(s)
- Peter Myintzaw
- Department of Biological Sciences, Munster Technological University, Bishopstown, T12 P928 Cork, Ireland
| | - Vincenzo Pennone
- Teagasc Food Research Centre, Moorepark, Fermoy, Co., P61 C996 Cork, Ireland
| | - Olivia McAuliffe
- Teagasc Food Research Centre, Moorepark, Fermoy, Co., P61 C996 Cork, Ireland
| | - Máire Begley
- Department of Biological Sciences, Munster Technological University, Bishopstown, T12 P928 Cork, Ireland
| | - Michael Callanan
- Department of Biological Sciences, Munster Technological University, Bishopstown, T12 P928 Cork, Ireland
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Avershina E, Khezri A, Ahmad R. Clinical Diagnostics of Bacterial Infections and Their Resistance to Antibiotics-Current State and Whole Genome Sequencing Implementation Perspectives. Antibiotics (Basel) 2023; 12:781. [PMID: 37107143 PMCID: PMC10135054 DOI: 10.3390/antibiotics12040781] [Citation(s) in RCA: 17] [Impact Index Per Article: 8.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2023] [Revised: 03/19/2023] [Accepted: 04/13/2023] [Indexed: 04/29/2023] Open
Abstract
Antimicrobial resistance (AMR), defined as the ability of microorganisms to withstand antimicrobial treatment, is responsible for millions of deaths annually. The rapid spread of AMR across continents warrants systematic changes in healthcare routines and protocols. One of the fundamental issues with AMR spread is the lack of rapid diagnostic tools for pathogen identification and AMR detection. Resistance profile identification often depends on pathogen culturing and thus may last up to several days. This contributes to the misuse of antibiotics for viral infection, the use of inappropriate antibiotics, the overuse of broad-spectrum antibiotics, or delayed infection treatment. Current DNA sequencing technologies offer the potential to develop rapid infection and AMR diagnostic tools that can provide information in a few hours rather than days. However, these techniques commonly require advanced bioinformatics knowledge and, at present, are not suited for routine lab use. In this review, we give an overview of the AMR burden on healthcare, describe current pathogen identification and AMR screening methods, and provide perspectives on how DNA sequencing may be used for rapid diagnostics. Additionally, we discuss the common steps used for DNA data analysis, currently available pipelines, and tools for analysis. Direct, culture-independent sequencing has the potential to complement current culture-based methods in routine clinical settings. However, there is a need for a minimum set of standards in terms of evaluating the results generated. Additionally, we discuss the use of machine learning algorithms regarding pathogen phenotype detection (resistance/susceptibility to an antibiotic).
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Affiliation(s)
- Ekaterina Avershina
- Department of Biotechnology, Inland Norway University of Applied Sciences, Holsetgata, 222317 Hamar, Norway
| | - Abdolrahman Khezri
- Department of Biotechnology, Inland Norway University of Applied Sciences, Holsetgata, 222317 Hamar, Norway
| | - Rafi Ahmad
- Department of Biotechnology, Inland Norway University of Applied Sciences, Holsetgata, 222317 Hamar, Norway
- Institute of Clinical Medicine, Faculty of Health Science, UiT The Arctic University of Norway, Hansine Hansens veg, 189019 Tromsø, Norway
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15
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Artificial Intelligence for Antimicrobial Resistance Prediction: Challenges and Opportunities towards Practical Implementation. Antibiotics (Basel) 2023; 12:antibiotics12030523. [PMID: 36978390 PMCID: PMC10044311 DOI: 10.3390/antibiotics12030523] [Citation(s) in RCA: 23] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Revised: 03/01/2023] [Accepted: 03/03/2023] [Indexed: 03/08/2023] Open
Abstract
Antimicrobial resistance (AMR) is emerging as a potential threat to many lives worldwide. It is very important to understand and apply effective strategies to counter the impact of AMR and its mutation from a medical treatment point of view. The intersection of artificial intelligence (AI), especially deep learning/machine learning, has led to a new direction in antimicrobial identification. Furthermore, presently, the availability of huge amounts of data from multiple sources has made it more effective to use these artificial intelligence techniques to identify interesting insights into AMR genes such as new genes, mutations, drug identification, conditions favorable to spread, and so on. Therefore, this paper presents a review of state-of-the-art challenges and opportunities. These include interesting input features posing challenges in use, state-of-the-art deep-learning/machine-learning models for robustness and high accuracy, challenges, and prospects to apply these techniques for practical purposes. The paper concludes with the encouragement to apply AI to the AMR sector with the intention of practical diagnosis and treatment, since presently most studies are at early stages with minimal application in the practice of diagnosis and treatment of disease.
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[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. Bundesgesundheitsblatt Gesundheitsforschung Gesundheitsschutz 2023; 66:75-83. [PMID: 36547697 PMCID: PMC9773680 DOI: 10.1007/s00103-022-03622-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2022] [Accepted: 11/03/2022] [Indexed: 12/24/2022]
Abstract
BACKGROUND In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.
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Maciel-Guerra A, Baker M, Hu Y, Wang W, Zhang X, Rong J, Zhang Y, Zhang J, Kaler J, Renney D, Loose M, Emes RD, Liu L, Chen J, Peng Z, Li F, Dottorini T. Dissecting microbial communities and resistomes for interconnected humans, soil, and livestock. THE ISME JOURNAL 2023; 17:21-35. [PMID: 36151458 PMCID: PMC9751072 DOI: 10.1038/s41396-022-01315-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 13.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Revised: 08/26/2022] [Accepted: 09/01/2022] [Indexed: 12/24/2022]
Abstract
A debate is currently ongoing as to whether intensive livestock farms may constitute reservoirs of clinically relevant antimicrobial resistance (AMR), thus posing a threat to surrounding communities. Here, combining shotgun metagenome sequencing, machine learning (ML), and culture-based methods, we focused on a poultry farm and connected slaughterhouse in China, investigating the gut microbiome of livestock, workers and their households, and microbial communities in carcasses and soil. For both the microbiome and resistomes in this study, differences are observed across environments and hosts. However, at a finer scale, several similar clinically relevant antimicrobial resistance genes (ARGs) and similar associated mobile genetic elements were found in both human and broiler chicken samples. Next, we focused on Escherichia coli, an important indicator for the surveillance of AMR on the farm. Strains of E. coli were found intermixed between humans and chickens. We observed that several ARGs present in the chicken faecal resistome showed correlation to resistance/susceptibility profiles of E. coli isolates cultured from the same samples. Finally, by using environmental sensing these ARGs were found to be correlated to variations in environmental temperature and humidity. Our results show the importance of adopting a multi-domain and multi-scale approach when studying microbial communities and AMR in complex, interconnected environments.
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Affiliation(s)
- Alexandre Maciel-Guerra
- grid.4563.40000 0004 1936 8868School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD UK
| | - Michelle Baker
- grid.4563.40000 0004 1936 8868School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD UK
| | - Yue Hu
- grid.4563.40000 0004 1936 8868School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD UK
| | - Wei Wang
- grid.464207.30000 0004 4914 5614NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021 People’s Republic of China
| | - Xibin Zhang
- grid.508175.eNew Hope Liuhe Co., Ltd., Laboratory of Feed and Livestock and Poultry Products Quality & Safety Control, Ministry of Agriculture, Beijing 100102 and Weifang Heshengyuan Food Co. Ltd., Weifang, 262167 People’s Republic of China
| | - Jia Rong
- grid.508175.eNew Hope Liuhe Co., Ltd., Laboratory of Feed and Livestock and Poultry Products Quality & Safety Control, Ministry of Agriculture, Beijing 100102 and Weifang Heshengyuan Food Co. Ltd., Weifang, 262167 People’s Republic of China
| | - Yimin Zhang
- grid.440622.60000 0000 9482 4676College of Food Science and Engineering, Shandong Agricultural University, Tai’an, Shandong 271018 People’s Republic of China
| | - Jing Zhang
- grid.464207.30000 0004 4914 5614NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021 People’s Republic of China
| | - Jasmeet Kaler
- grid.4563.40000 0004 1936 8868School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD UK
| | - David Renney
- Nimrod Veterinary Products Limited, 2, Wychwood Court, Cotswold Business Village, Moreton-in-Marsh, GL56 0JQ UK
| | - Matthew Loose
- grid.4563.40000 0004 1936 8868DeepSeq, School of Life Sciences, Queens Medical Centre, University of Nottingham, Nottingham, NG7 2UH UK
| | - Richard D. Emes
- grid.4563.40000 0004 1936 8868School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD UK
| | - Longhai Liu
- grid.508175.eNew Hope Liuhe Co., Ltd., Laboratory of Feed and Livestock and Poultry Products Quality & Safety Control, Ministry of Agriculture, Beijing 100102 and Weifang Heshengyuan Food Co. Ltd., Weifang, 262167 People’s Republic of China
| | - Junshi Chen
- grid.464207.30000 0004 4914 5614NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021 People’s Republic of China
| | - Zixin Peng
- NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021, People's Republic of China.
| | - Fengqin Li
- NHC Key Laboratory of Food Safety Risk Assessment, China National Center for Food Safety Risk Assessment, Beijing, 100021, People's Republic of China.
| | - Tania Dottorini
- School of Veterinary Medicine and Science, University of Nottingham, College Road, Sutton Bonington, Leicestershire, LE12 5RD, UK.
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Integrated surveillance of extended-spectrum beta-lactamase (ESBL)-producing Salmonella and Escherichia coli from humans and animal species raised for human consumption in Canada from 2012 to 2017. Epidemiol Infect 2022; 151:e14. [PMID: 36698196 PMCID: PMC9990382 DOI: 10.1017/s0950268822001509] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Resistance to beta-lactam antimicrobials caused by extended-spectrum beta-lactamase (ESBL)-producing organisms is a global health concern. The objectives of this study were to (1) summarise the prevalence of potential ESBL-producing Escherichia coli (ESBL-EC) and Salmonella spp. (ESBL-SA) isolates from agrifood and human sources in Canada from 2012 to 2017, and (2) describe the distribution of ESBL genotypes among these isolates. All data were obtained from the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS). CIPARS analysed samples for the presence of ESBLs through phenotypic classification and identified beta-lactamase genes (blaTEM, blaSHV, blaCTX, blaOXA, blaCMY-2) using polymerase chain reaction (PCR) and whole genome sequencing (WGS). The prevalence of PCR-confirmed ESBL-EC in agrifood samples ranged from 0.5% to 3% across the surveillance years, and was detected most frequently in samples from broiler chicken farms. The overall prevalence of PCR-confirmed ESBL-SA varied between 1% and 4% between 2012 and 2017, and was most frequently detected in clinical isolates from domestic cattle. The TEM-CMY2 gene combination was the most frequently detected genotype for both ESBL-EC and ESBL-SA. The data suggest that the prevalence of ESBL-EC and ESBL-SA in Canada was low (i.e. <5%), but ongoing surveillance is needed to detect emerging or changing trends.
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Yee R, Simner PJ. Next-Generation Sequencing Approaches to Predicting Antimicrobial Susceptibility Testing Results. Clin Lab Med 2022; 42:557-572. [PMID: 36368782 DOI: 10.1016/j.cll.2022.09.011] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Affiliation(s)
- Rebecca Yee
- Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Meyer B1-193, 600 North Wolfe Street, Baltimore, MD 21287-7093, USA
| | - Patricia J Simner
- Division of Medical Microbiology, Department of Pathology, Johns Hopkins University School of Medicine, Meyer B1-193, 600 North Wolfe Street, Baltimore, MD 21287-7093, USA.
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Al-Omari AW, Ramadan Matter I, Hussein Almola A. Molecular study of Enterobacteriaceae bacterium isolated from dishwashers. BIONATURA 2022. [DOI: 10.21931/rb/2022.07.03.28] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022] Open
Abstract
The Enterobacteriaceae family is considered one of the medically essential families in which there is a continuous change in classification, and new species are added to it. In this study, we obtained a novel strain registered in the NCBI under Enterobacteriaceae bacterium strain PRL 4-2, with the accession number MW540823, and the isolate considered unclassified Enterobacteriaceae. The isolate was obtained from dishwashers, which is a mine for many new species and strains due to the unique environmental conditions of this habitat, fluctuation in temperature, use of high temperatures, utilization of cleaning materials as well as humidity and the presence of organic materials, especially in case of neglect of cleaning. The isolate was characterized by its slow growth on culture media and its ability to form biofilms and possess some virulence factors. Its resistance to antibiotics was also studied, as it showed resistance to the antibiotics used in this study. The oddness for this strain is that it showed a different diagnosis when using VITEK (The VITEK 2 system has everything healthcare laboratories need for fast, accurate microbial identification and antibiotic susceptibility testing.) compared to diagnosis using 16S rRNA. As for the taxonomic tree, the closest species was Enterobacter sp. Strain 188. This study supplements a few other studies of this novel species, isolated from different environments. These researches can be integrated to give an itinerary for other studies on the new species and their different capabilities.
Keywords. Enterobacteriaceae bacterium, dishwasher, Enterobacteriaceae bacterium PRL-2, Enterobacter sp., biofilm.
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Affiliation(s)
- Aisha W. Al-Omari
- Department of Biology, College of Sciences, University of Mosul, Mosul, Iraq
| | | | - Alaa Hussein Almola
- Department of Biology, College of Sciences, University of Mosul, Mosul, Iraq
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Wang H, Jia C, Li H, Yin R, Chen J, Li Y, Yue M. Paving the way for precise diagnostics of antimicrobial resistant bacteria. Front Mol Biosci 2022; 9:976705. [PMID: 36032670 PMCID: PMC9413203 DOI: 10.3389/fmolb.2022.976705] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2022] [Accepted: 07/19/2022] [Indexed: 12/26/2022] Open
Abstract
The antimicrobial resistance (AMR) crisis from bacterial pathogens is frequently emerging and rapidly disseminated during the sustained antimicrobial exposure in human-dominated communities, posing a compelling threat as one of the biggest challenges in humans. The frequent incidences of some common but untreatable infections unfold the public health catastrophe that antimicrobial-resistant pathogens have outpaced the available countermeasures, now explicitly amplified during the COVID-19 pandemic. Nowadays, biotechnology and machine learning advancements help create more fundamental knowledge of distinct spatiotemporal dynamics in AMR bacterial adaptation and evolutionary processes. Integrated with reliable diagnostic tools and powerful analytic approaches, a collaborative and systematic surveillance platform with high accuracy and predictability should be established and implemented, which is not just for an effective controlling strategy on AMR but also for protecting the longevity of valuable antimicrobials currently and in the future.
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Affiliation(s)
- Hao Wang
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
| | - Chenhao Jia
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
- Hainan Institute of Zhejiang University, Sanya, China
| | - Hongzhao Li
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
- Hainan Institute of Zhejiang University, Sanya, China
| | - Rui Yin
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
| | - Jiang Chen
- Department of Microbiology, Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China
- *Correspondence: Jiang Chen, ; Yan Li, ; Min Yue,
| | - Yan Li
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
- Hainan Institute of Zhejiang University, Sanya, China
- Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Hangzhou, China
- *Correspondence: Jiang Chen, ; Yan Li, ; Min Yue,
| | - Min Yue
- Institute of Preventive Veterinary Sciences & Department of Veterinary Medicine, Zhejiang University College of Animal Sciences, Hangzhou, China
- Hainan Institute of Zhejiang University, Sanya, China
- Zhejiang Provincial Key Laboratory of Preventive Veterinary Medicine, Hangzhou, China
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, National Clinical Research Center for Infectious Diseases, National Medical Center for Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, China
- *Correspondence: Jiang Chen, ; Yan Li, ; Min Yue,
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22
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Aljeldah MM. Antimicrobial Resistance and Its Spread Is a Global Threat. Antibiotics (Basel) 2022; 11:antibiotics11081082. [PMID: 36009948 PMCID: PMC9405321 DOI: 10.3390/antibiotics11081082] [Citation(s) in RCA: 97] [Impact Index Per Article: 32.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2022] [Revised: 07/20/2022] [Accepted: 07/27/2022] [Indexed: 02/07/2023] Open
Abstract
Antimicrobial resistance (AMR) is a challenge to human wellbeing the world over and is one of the more serious public health concerns. AMR has the potential to emerge as a serious healthcare threat if left unchecked, and could put into motion another pandemic. This establishes the need for the establishment of global health solutions around AMR, taking into account microdata from different parts of the world. The positive influences in this regard could be establishing conducive social norms, charting individual and group behavior practices that favor global human health, and lastly, increasing collective awareness around the need for such action. Apart from being an emerging threat in the clinical space, AMR also increases treatment complexity, posing a real challenge to the existing guidelines around the management of antibiotic resistance. The attribute of resistance development has been linked to many genetic elements, some of which have complex transmission pathways between microbes. Beyond this, new mechanisms underlying the development of AMR are being discovered, making this field an important aspect of medical microbiology. Apart from the genetic aspects of AMR, other practices, including misdiagnosis, exposure to broad-spectrum antibiotics, and lack of rapid diagnosis, add to the creation of resistance. However, upgrades and innovations in DNA sequencing technologies with bioinformatics have revolutionized the diagnostic industry, aiding the real-time detection of causes of AMR and its elements, which are important to delineating control and prevention approaches to fight the threat.
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Affiliation(s)
- Mohammed M Aljeldah
- Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, University of Hafr Al Batin, Hafar al-Batin 31991, Saudi Arabia
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23
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Priyamvada P, Debroy R, Anbarasu A, Ramaiah S. A comprehensive review on genomics, systems biology and structural biology approaches for combating antimicrobial resistance in ESKAPE pathogens: computational tools and recent advancements. World J Microbiol Biotechnol 2022; 38:153. [PMID: 35788443 DOI: 10.1007/s11274-022-03343-z] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 06/21/2022] [Indexed: 12/11/2022]
Abstract
In recent decades, antimicrobial resistance has been augmented as a global concern to public health owing to the global spread of multidrug-resistant strains from different ESKAPE pathogens. This alarming trend and the lack of new antibiotics with novel modes of action in the pipeline necessitate the development of non-antibiotic ways to treat illnesses caused by these isolates. In molecular biology, computational approaches have become crucial tools, particularly in one of the most challenging areas of multidrug resistance. The rapid advancements in bioinformatics have led to a plethora of computational approaches involving genomics, systems biology, and structural biology currently gaining momentum among molecular biologists since they can be useful and provide valuable information on the complex mechanisms of AMR research in ESKAPE pathogens. These computational approaches would be helpful in elucidating the AMR mechanisms, identifying important hub genes/proteins, and their promising targets together with their interactions with important drug targets, which is a crucial step in drug discovery. Therefore, the present review aims to provide holistic information on currently employed bioinformatic tools and their application in the discovery of multifunctional novel therapeutic drugs to combat the current problem of AMR in ESKAPE pathogens. The review also summarizes the recent advancement in the AMR research in ESKAPE pathogens utilizing the in silico approaches.
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Affiliation(s)
- P Priyamvada
- Medical and Biological Computing Laboratory, School of Biosciences and Technology (SBST), Vellore Institute of Technology (VIT), 632014, Vellore, India.,Department of Bio-Sciences, SBST, VIT, 632014, Vellore, India
| | - Reetika Debroy
- Medical and Biological Computing Laboratory, School of Biosciences and Technology (SBST), Vellore Institute of Technology (VIT), 632014, Vellore, India.,Department of Bio-Medical Sciences, SBST, VIT, 632014, Vellore, India
| | - Anand Anbarasu
- Medical and Biological Computing Laboratory, School of Biosciences and Technology (SBST), Vellore Institute of Technology (VIT), 632014, Vellore, India.,Department of Biotechnology, SBST, VIT, 632014, Vellore, India
| | - Sudha Ramaiah
- Medical and Biological Computing Laboratory, School of Biosciences and Technology (SBST), Vellore Institute of Technology (VIT), 632014, Vellore, India. .,Department of Bio-Sciences, SBST, VIT, 632014, Vellore, India. .,School of Biosciences and Technology VIT, 632014, Vellore, Tamil Nadu, India.
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24
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Zhou L, Lopez Rodas A, Llangarí LM, Romero Sandoval N, Cooper P, Sadiq ST. Single gene targeted nanopore sequencing enables simultaneous identification and antimicrobial resistance detection of sexually transmitted infections. PLoS One 2022; 17:e0262242. [PMID: 35061780 PMCID: PMC8782522 DOI: 10.1371/journal.pone.0262242] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 12/20/2021] [Indexed: 11/18/2022] Open
Abstract
OBJECTIVES To develop a simple DNA sequencing test for simultaneous identification and antimicrobial resistance (AMR) detection of multiple sexually transmitted infections (STIs). METHODS Real-time PCR (qPCR) was initially performed to identify Neisseria gonorrhoeae (NG), Chlamydia trachomatis (CT), Mycoplasma genitalium (MG) and Trichomonas vaginalis (TV) infections among a total of 200 vulvo-vaginal swab samples from female sex workers in Ecuador. qPCR positive samples plus qPCR negative controls for these STIs were subjected to single gene targeted PCR MinION-nanopore sequencing using the smartphone operated MinIT. RESULTS Among 200 vulvo-vaginal swab samples 43 were qPCR positive for at least one of the STIs. Single gene targeted nanopore sequencing generally yielded higher pathogen specific read counts in qPCR positive samples than qPCR negative controls. Of the 26 CT, NG or MG infections identified by qPCR, 25 were clearly distinguishable from qPCR negative controls by read count. Discrimination of TV qPCR positives from qPCR negative controls was poorer as many had low pathogen loads (qPCR cycle threshold >35) which produced few specific reads. Real-time AMR profiling revealed that 3/3 NG samples identified had gyrA mutations associated with fluoroquinolone resistance, 2/10 of TV had mutations related to metronidazole resistance, while none of the MG samples possessed 23S rRNA gene mutations contributing to macrolide resistance. CONCLUSIONS Single gene targeted nanopore sequencing for diagnosing and simultaneously identifying key antimicrobial resistance markers for four common genital STIs shows promise. Further work to optimise accuracy, reduce costs and improve speed may allow sustainable approaches for managing STIs and emerging AMR in resource poor and laboratory limited settings.
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Affiliation(s)
- Liqing Zhou
- Applied Diagnostic Research and Evaluation Unit, Institute for Infection and Immunity, St George’s, University of London, London, United Kingdom
| | - Andrea Lopez Rodas
- School of Medicine, Faculty of Health and Life Sciences, Universidad Internacional del Ecuador, Quito, Ecuador
| | - Luz Marina Llangarí
- School of Medicine, Faculty of Health and Life Sciences, Universidad Internacional del Ecuador, Quito, Ecuador
| | - Natalia Romero Sandoval
- School of Medicine, Faculty of Health and Life Sciences, Universidad Internacional del Ecuador, Quito, Ecuador
- Grups de Recerca d’Amèrica i Àfrica Llatines, GRAAL, Barcelona, Spain
| | - Philip Cooper
- Applied Diagnostic Research and Evaluation Unit, Institute for Infection and Immunity, St George’s, University of London, London, United Kingdom
- School of Medicine, Faculty of Health and Life Sciences, Universidad Internacional del Ecuador, Quito, Ecuador
| | - Syed Tariq Sadiq
- Applied Diagnostic Research and Evaluation Unit, Institute for Infection and Immunity, St George’s, University of London, London, United Kingdom
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25
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Forde BM, De Oliveira DMP, Falconer C, Graves B, Harris PNA. Strengths and caveats of identifying resistance genes from whole genome sequencing data. Expert Rev Anti Infect Ther 2021; 20:533-547. [PMID: 34852720 DOI: 10.1080/14787210.2022.2013806] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/19/2022]
Abstract
INTRODUCTION Antimicrobial resistance (AMR) continues to present major challenges to modern healthcare. Recent advances in whole-genome sequencing (WGS) have made the rapid molecular characterization of AMR a realistic possibility for diagnostic laboratories; yet major barriers to clinical implementation exist. AREAS COVERED We describe and compare short- and long-read sequencing platforms, typical components of bioinformatics pipelines, tools for AMR gene detection and the relative merits of read- or assembly-based approaches. The challenges of characterizing mobile genetic elements from genomic data are outlined, as well as the complexities inherent to the prediction of phenotypic resistance from WGS. Practical obstacles to implementation in diagnostic laboratories, the critical role of quality control and external quality assurance, as well as standardized reporting standards are also discussed. Future directions, such as the application of machine-learning and artificial intelligence algorithms, linked to clinically meaningful outcomes, may offer a new paradigm for the clinical application of AMR prediction. EXPERT OPINION AMR prediction from WGS data presents an exciting opportunity to advance our capacity to comprehensively characterize infectious pathogens in a rapid manner, ultimately aiming to improve patient outcomes. Collaborative efforts between clinicians, scientists, regulatory bodies and healthcare administrators will be critical to achieve the full promise of this approach.
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Affiliation(s)
- Brian M Forde
- University of Queensland, Faculty of Medicine, Uq Centre for Clinical Research, Royal Brisbane and Woman's Hospital, Herston, Australia
| | - David M P De Oliveira
- University of Queensland, Faculty of Science, School of Chemistry and Molecular Biosciences, St Lucia, Australia
| | - Caitlin Falconer
- University of Queensland, Faculty of Medicine, Uq Centre for Clinical Research, Royal Brisbane and Woman's Hospital, Herston, Australia
| | - Bianca Graves
- Herston Infectious Disease Institute, Royal Brisbane & Women's Hospital, Herston, Australia
| | - Patrick N A Harris
- University of Queensland, Faculty of Medicine, Uq Centre for Clinical Research, Royal Brisbane and Woman's Hospital, Herston, Australia.,Herston Infectious Disease Institute, Royal Brisbane & Women's Hospital, Herston, Australia.,Central Microbiology, Pathology Queensland, Royal Brisbane & Women's Hospital, Herston, Australia
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26
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Arredondo-Alonso S, Pöntinen AK, Cléon F, Gladstone RA, Schürch AC, Johnsen PJ, Samuelsen Ø, Corander J. A high-throughput multiplexing and selection strategy to complete bacterial genomes. Gigascience 2021; 10:giab079. [PMID: 34891160 PMCID: PMC8673558 DOI: 10.1093/gigascience/giab079] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2021] [Revised: 09/29/2021] [Accepted: 11/12/2021] [Indexed: 12/27/2022] Open
Abstract
BACKGROUND Bacterial whole-genome sequencing based on short-read technologies often results in a draft assembly formed by contiguous sequences. The introduction of long-read sequencing technologies permits those contiguous sequences to be unambiguously bridged into complete genomes. However, the elevated costs associated with long-read sequencing frequently limit the number of bacterial isolates that can be long-read sequenced. Here we evaluated the recently released 96 barcoding kit from Oxford Nanopore Technologies (ONT) to generate complete genomes on a high-throughput basis. In addition, we propose an isolate selection strategy that optimizes a representative selection of isolates for long-read sequencing considering as input large-scale bacterial collections. RESULTS Despite an uneven distribution of long reads per barcode, near-complete chromosomal sequences (assembly contiguity = 0.89) were generated for 96 Escherichia coli isolates with associated short-read sequencing data. The assembly contiguity of the plasmid replicons was even higher (0.98), which indicated the suitability of the multiplexing strategy for studies focused on resolving plasmid sequences. We benchmarked hybrid and ONT-only assemblies and showed that the combination of ONT sequencing data with short-read sequencing data is still highly desirable (i) to perform an unbiased selection of isolates for long-read sequencing, (ii) to achieve an optimal genome accuracy and completeness, and (iii) to include small plasmids underrepresented in the ONT library. CONCLUSIONS The proposed long-read isolate selection ensures the completion of bacterial genomes that span the genome diversity inherent in large collections of bacterial isolates. We show the potential of using this multiplexing approach to close bacterial genomes on a high-throughput basis.
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Affiliation(s)
- Sergio Arredondo-Alonso
- Department of Biostatistics, University of Oslo, 0317, Oslo, Norway
- Parasites and Microbes, Wellcome Sanger Institute, Cambridgeshire CB10 1RQ, UK
| | - Anna K Pöntinen
- Department of Biostatistics, University of Oslo, 0317, Oslo, Norway
| | - François Cléon
- Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, 9037, Tromsø, Norway
| | | | - Anita C Schürch
- Department of Medical Microbiology, UMC Utrecht, 3584 CX, Utrecht, the Netherlands
| | - Pål J Johnsen
- Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, 9037, Tromsø, Norway
| | - Ørjan Samuelsen
- Department of Pharmacy, Faculty of Health Sciences, UiT The Arctic University of Norway, 9037, Tromsø, Norway
- Norwegian National Advisory Unit on Detection of Antimicrobial Resistance, Department of Microbiology and Infection Control, University Hospital of North Norway, 9038, Tromsø, Norway
| | - Jukka Corander
- Department of Biostatistics, University of Oslo, 0317, Oslo, Norway
- Parasites and Microbes, Wellcome Sanger Institute, Cambridgeshire CB10 1RQ, UK
- Department of Mathematics and Statistics, Helsinki Institute of Information Technology (HIIT), FI-00014 University of Helsinki, 02130, Espoo, Helsinki, Finland
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27
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Kekre M, Arevalo SA, Valencia MF, Lagrada ML, Macaranas PKV, Nagaraj G, Oaikhena AO, Olorosa AM, Aanensen DM. Integrating Scalable Genome Sequencing Into Microbiology Laboratories for Routine Antimicrobial Resistance Surveillance. Clin Infect Dis 2021; 73:S258-S266. [PMID: 34850836 PMCID: PMC8634525 DOI: 10.1093/cid/ciab796] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Antimicrobial resistance (AMR) is considered a global threat, and novel drug discovery needs to be complemented with systematic and standardized epidemiological surveillance. Surveillance data are currently generated using phenotypic characterization. However, due to poor scalability, this approach does little for true epidemiological investigations. There is a strong case for whole-genome sequencing (WGS) to enhance the phenotypic data. To establish global AMR surveillance using WGS, we developed a laboratory implementation approach that we applied within the NIHR Global Health Research Unit (GHRU) on Genomic Surveillance of Antimicrobial Resistance. In this paper, we outline the laboratory implementation at 4 units: Colombia, India, Nigeria, and the Philippines. The journey to embedding WGS capacity was split into 4 phases: Assessment, Assembly, Optimization, and Reassessment. We show that on-boarding WGS capabilities can greatly enhance the real-time processing power within regional and national AMR surveillance initiatives, despite the high initial investment in laboratory infrastructure and maintenance. Countries looking to introduce WGS as a surveillance tool could begin by sequencing select Global Antimicrobial Resistance Surveillance System (GLASS) priority pathogens that can demonstrate the standardization and impact genome sequencing has in tackling AMR.
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Affiliation(s)
- Mihir Kekre
- Centre for Genomic Pathogen Surveillance, Big Data Institute, University of Oxford, Old Road Campus, Oxford, United Kingdom
- Wellcome Genome Campus, Hinxton, United Kingdom
| | - Stefany Alejandra Arevalo
- Colombian Integrated Program for Antimicrobial Resistance Surveillance—Coipars, CI Tibaitatá, Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA), Tibaitatá–Mosquera, Cundinamarca, Colombia
| | - María Fernanda Valencia
- Colombian Integrated Program for Antimicrobial Resistance Surveillance—Coipars, CI Tibaitatá, Corporación Colombiana de Investigación Agropecuaria (AGROSAVIA), Tibaitatá–Mosquera, Cundinamarca, Colombia
| | | | | | - Geetha Nagaraj
- Central Research Laboratory, Kempegowda Institute of Medical Sciences, Bengaluru, India
| | - Anderson O Oaikhena
- Department of Pharmaceutical Microbiology, University of Ibadan, Ibadan, Nigeria
| | | | - David M Aanensen
- Centre for Genomic Pathogen Surveillance, Big Data Institute, University of Oxford, Old Road Campus, Oxford, United Kingdom
- Wellcome Genome Campus, Hinxton, United Kingdom
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28
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Antibiotic Susceptibility Profiles and Frequency of Resistance Genes in Clinical Shiga Toxin-Producing Escherichia coli Isolates from Michigan over a 14-Year Period. Antimicrob Agents Chemother 2021; 65:e0118921. [PMID: 34424041 DOI: 10.1128/aac.01189-21] [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] [Indexed: 11/20/2022] Open
Abstract
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen that contributes to over 250,000 infections in the United States each year. Because antibiotics are not recommended for STEC infections, resistance in STEC has not been widely researched despite an increased likelihood for the transfer of resistance genes from STEC to opportunistic pathogens residing within the same microbial community. From 2001 to 2014, 969 STEC isolates were collected from Michigan patients. Antibiotic susceptibility profiles to clinically relevant antibiotics were determined using disc diffusion, while epidemiological data were used to identify factors associated with resistance. Whole-genome sequencing was used for serotyping, examining genetic relatedness, and identifying genetic determinants and mechanisms of resistance in the non-O157 isolates. Increasing frequencies of resistance to at least one antibiotic were observed over the 14 years (P = 0.01). While the non-O157 serogroups were more commonly resistant than O157 (odds ratio, 2.4; 95% confidence interval,1.43 to 4.05), the frequency of ampicillin resistance among O157 isolates was significantly higher in Michigan than the national average (P = 0.03). Genomic analysis of 321 non-O157 isolates uncovered 32 distinct antibiotic resistance genes (ARGs). Although mutations in genes encoding resistance to ciprofloxacin and ampicillin were detected in four isolates, most of the horizontally acquired ARGs conferred resistance to aminoglycosides, β-lactams, sulfonamides, and/or tetracycline. This study provides insight into the mechanisms of resistance in a large collection of clinical non-O157 STEC isolates and demonstrates that antibiotic resistance among all STEC serogroups has increased over time, prompting the need for enhanced surveillance.
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29
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Coolen JPM, Jamin C, Savelkoul PHM, Rossen JWA, Wertheim HFL, Matamoros SP, van Alphen LB, On Behalf Of Sig Bioinformatics In Medical Microbiology Nl Consortium. Centre-specific bacterial pathogen typing affects infection-control decision making. Microb Genom 2021; 7. [PMID: 34356004 PMCID: PMC8549354 DOI: 10.1099/mgen.0.000612] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Whole-genome sequencing is becoming the de facto standard for bacterial outbreak surveillance and infection prevention. This is accompanied by a variety of bioinformatic tools and needs bioinformatics expertise for implementation. However, little is known about the concordance of reported outbreaks when using different bioinformatic workflows. In this multi-centre proficiency testing among 13 major Dutch healthcare-affiliated centres, bacterial whole-genome outbreak analysis was assessed. Centres who participated obtained two randomized bacterial datasets of Illumina sequences, a Klebsiella pneumoniae and a Vancomycin-resistant Enterococcus faecium, and were asked to apply their bioinformatic workflows. Centres reported back on antimicrobial resistance, multi-locus sequence typing (MLST), and outbreak clusters. The reported clusters were analysed using a method to compare landscapes of phylogenetic trees and calculating Kendall–Colijn distances. Furthermore, fasta files were analysed by state-of-the-art single nucleotide polymorphism (SNP) analysis to mitigate the differences introduced by each centre and determine standardized SNP cut-offs. Thirteen centres participated in this study. The reported outbreak clusters revealed discrepancies between centres, even when almost identical bioinformatic workflows were used. Due to stringent filtering, some centres failed to detect extended-spectrum beta-lactamase genes and MLST loci. Applying a standardized method to determine outbreak clusters on the reported de novo assemblies, did not result in uniformity of outbreak-cluster composition among centres.
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Affiliation(s)
- Jordy P M Coolen
- Department of Medical Microbiology and Radboudumc Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Casper Jamin
- Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands
| | - Paul H M Savelkoul
- Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands.,Department of Medical Microbiology & Infection Control, Amsterdam University Medical Centers, location VUmc, Amsterdam, The Netherlands
| | - John W A Rossen
- Department of Medical Microbiology and Infection Prevention, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.,Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT, USA
| | - Heiman F L Wertheim
- Department of Medical Microbiology and Radboudumc Center for Infectious Diseases, Radboud University Medical Center, Nijmegen, The Netherlands
| | - Sébastien P Matamoros
- Department of Medical Microbiology & Infection Control, Amsterdam University Medical Centers, location VUmc, Amsterdam, The Netherlands
| | - Lieke B van Alphen
- Department of Medical Microbiology, Care and Public Health Research Institute (CAPHRI), Maastricht University Medical Center+, Maastricht, The Netherlands
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30
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Applications of Machine Learning to the Problem of Antimicrobial Resistance: an Emerging Model for Translational Research. J Clin Microbiol 2021; 59:e0126020. [PMID: 33536291 DOI: 10.1128/jcm.01260-20] [Citation(s) in RCA: 80] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023] Open
Abstract
Antimicrobial resistance (AMR) remains one of the most challenging phenomena of modern medicine. Machine learning (ML) is a subfield of artificial intelligence that focuses on the development of algorithms that learn how to accurately predict outcome variables using large sets of predictor variables that are typically not hand selected and are minimally curated. Models are parameterized using a training data set and then applied to a test data set on which predictive performance is evaluated. The application of ML algorithms to the problem of AMR has garnered increasing interest in the past 5 years due to the exponential growth of experimental and clinical data, heavy investment in computational capacity, improvements in algorithm performance, and increasing urgency for innovative approaches to reducing the burden of disease. Here, we review the current state of research at the intersection of ML and AMR with an emphasis on three domains of work. The first is the prediction of AMR using genomic data. The second is the use of ML to gain insight into the cellular functions disrupted by antibiotics, which forms the basis for understanding mechanisms of action and developing novel anti-infectives. The third focuses on the application of ML for antimicrobial stewardship using data extracted from the electronic health record. Although the use of ML for understanding, diagnosing, treating, and preventing AMR is still in its infancy, the continued growth of data and interest ensures it will become an important tool for future translational research programs.
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31
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Probst K, Nurjadi D, Heeg K, Frede AM, Dalpke AH, Boutin S. Molecular Detection of Carbapenemases in Enterobacterales: A Comparison of Real-Time Multiplex PCR and Whole-Genome Sequencing. Antibiotics (Basel) 2021; 10:antibiotics10060726. [PMID: 34208657 PMCID: PMC8233969 DOI: 10.3390/antibiotics10060726] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2021] [Revised: 06/11/2021] [Accepted: 06/15/2021] [Indexed: 11/16/2022] Open
Abstract
Carbapenem-resistant Enterobacterales are a growing problem in healthcare systems worldwide. While whole-genome sequencing (WGS) has become a powerful tool for analyzing transmission and possible outbreaks, it remains laborious, and the limitations in diagnostic workflows are not well studied. The aim of this study was to compare the performance of WGS and real-time multiplex PCR (RT-qPCR) for diagnosing carbapenem-resistant Enterobacterales. In this study, we analyzed 92 phenotypically carbapenem-resistant Enterobacterales, sent to the University Hospital Heidelberg in 2019, by the carbapenem inactivation method (CIM) and compared WGS and RT-qPCR as genotypic carbapenemase detection methods. In total, 80.4% of the collected isolates were identified as carbapenemase producers. For six isolates, discordant results were recorded for WGS, PCR and CIM, as the carbapenemase genes were initially not detected by WGS. A reanalysis using raw reads, rather than assembly, highlighted a coverage issue with failure to detect carbapenemases located in contigs with a coverage lower than 10×, which were then discarded. Our study shows that multiplex RT-qPCR and CIM can be a simple alternative to WGS for basic surveillance of carbapenemase-producing Enterobacterales. Using WGS in clinical workflow has some limitations, especially regarding coverage and sensitivity. We demonstrate that antimicrobial resistance gene detection should be performed on the raw reads or non-curated draft genome to increase sensitivity.
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Affiliation(s)
- Katja Probst
- Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Heidelberg, Germany; (D.N.); (K.H.); (A.-M.F.); (S.B.)
- Correspondence: ; Tel.: +49-6221-56-36420; Fax: +49-6221-56-5857
| | - Dennis Nurjadi
- Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Heidelberg, Germany; (D.N.); (K.H.); (A.-M.F.); (S.B.)
| | - Klaus Heeg
- Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Heidelberg, Germany; (D.N.); (K.H.); (A.-M.F.); (S.B.)
| | - Anne-Marie Frede
- Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Heidelberg, Germany; (D.N.); (K.H.); (A.-M.F.); (S.B.)
| | - Alexander H. Dalpke
- Institute of Medical Microbiology and Virology, University Hospital Carl Gustav Carus, 01307 Dresden, Germany;
| | - Sébastien Boutin
- Department of Infectious Diseases, Medical Microbiology and Hygiene, University Hospital Heidelberg, 69120 Heidelberg, Germany; (D.N.); (K.H.); (A.-M.F.); (S.B.)
- Translational Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), Heidelberg University Hospital, 69120 Heidelberg, Germany
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Gil-Gil T, Ochoa-Sánchez LE, Baquero F, Martínez JL. Antibiotic resistance: Time of synthesis in a post-genomic age. Comput Struct Biotechnol J 2021; 19:3110-3124. [PMID: 34141134 PMCID: PMC8181582 DOI: 10.1016/j.csbj.2021.05.034] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2021] [Revised: 05/13/2021] [Accepted: 05/20/2021] [Indexed: 12/20/2022] Open
Abstract
Antibiotic resistance has been highlighted by international organizations, including World Health Organization, World Bank and United Nations, as one of the most relevant global health problems. Classical approaches to study this problem have focused in infected humans, mainly at hospitals. Nevertheless, antibiotic resistance can expand through different ecosystems and geographical allocations, hence constituting a One-Health, Global-Health problem, requiring specific integrative analytic tools. Antibiotic resistance evolution and transmission are multilayer, hierarchically organized processes with several elements (from genes to the whole microbiome) involved. However, their study has been traditionally gene-centric, each element independently studied. The development of robust-economically affordable whole genome sequencing approaches, as well as other -omic techniques as transcriptomics and proteomics, is changing this panorama. These technologies allow the description of a system, either a cell or a microbiome as a whole, overcoming the problems associated with gene-centric approaches. We are currently at the time of combining the information derived from -omic studies to have a more holistic view of the evolution and spread of antibiotic resistance. This synthesis process requires the accurate integration of -omic information into computational models that serve to analyse the causes and the consequences of acquiring AR, fed by curated databases capable of identifying the elements involved in the acquisition of resistance. In this review, we analyse the capacities and drawbacks of the tools that are currently in use for the global analysis of AR, aiming to identify the more useful targets for effective corrective interventions.
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Affiliation(s)
- Teresa Gil-Gil
- Centro Nacional de Biotecnología, CSIC, Darwin 3, 28049 Madrid, Spain
| | | | - Fernando Baquero
- Department of Microbiology, Hospital Universitario Ramón y Cajal (IRYCIS), Madrid, Spain
- CIBER en Epidemiología y Salud Pública (CIBER-ESP), Madrid, Spain
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Kim DW, Cha CJ. Antibiotic resistome from the One-Health perspective: understanding and controlling antimicrobial resistance transmission. Exp Mol Med 2021; 53:301-309. [PMID: 33642573 PMCID: PMC8080597 DOI: 10.1038/s12276-021-00569-z] [Citation(s) in RCA: 144] [Impact Index Per Article: 36.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/04/2020] [Revised: 12/17/2020] [Accepted: 12/21/2020] [Indexed: 01/31/2023] Open
Abstract
The concept of the antibiotic resistome was introduced just over a decade ago, and since then, active resistome studies have been conducted. In the present study, we describe the previously established concept of the resistome, which encompasses all types of antibiotic resistance genes (ARGs), and the important findings from each One-Health sector considering this concept, thereby emphasizing the significance of the One-Health approach in understanding ARG transmission. Cutting-edge research methodologies are essential for deciphering the complex resistome structure in the microbiomes of humans, animals, and the environment. Based on the recent achievements of resistome studies in multiple One-Health sectors, future directions for resistome research have been suggested to improve the understanding and control of ARG transmission: (1) ranking the critical ARGs and their hosts; (2) understanding ARG transmission at the interfaces of One-Health sectors; (3) identifying selective pressures affecting the emergence, transmission, and evolution of ARGs; and (4) elucidating the mechanisms that allow an organism to overcome taxonomic barriers in ARG transmission.
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Affiliation(s)
- Dae-Wi Kim
- grid.411545.00000 0004 0470 4320Division of Life Sciences, Jeonbuk National University, Jeonju, 54896 Republic of Korea
| | - Chang-Jun Cha
- grid.254224.70000 0001 0789 9563Department of Systems Biotechnology and Center for Antibiotic Resistome, Chung-Ang University, Anseong, 17546 Republic of Korea
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The History of Colistin Resistance Mechanisms in Bacteria: Progress and Challenges. Microorganisms 2021; 9:microorganisms9020442. [PMID: 33672663 PMCID: PMC7924381 DOI: 10.3390/microorganisms9020442] [Citation(s) in RCA: 59] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2021] [Revised: 02/17/2021] [Accepted: 02/18/2021] [Indexed: 12/13/2022] Open
Abstract
Since 2015, the discovery of colistin resistance genes has been limited to the characterization of new mobile colistin resistance (mcr) gene variants. However, given the complexity of the mechanisms involved, there are many colistin-resistant bacterial strains whose mechanism remains unknown and whose exploitation requires complementary technologies. In this review, through the history of colistin, we underline the methods used over the last decades, both old and recent, to facilitate the discovery of the main colistin resistance mechanisms and how new technological approaches may help to improve the rapid and efficient exploration of new target genes. To accomplish this, a systematic search was carried out via PubMed and Google Scholar on published data concerning polymyxin resistance from 1950 to 2020 using terms most related to colistin. This review first explores the history of the discovery of the mechanisms of action and resistance to colistin, based on the technologies deployed. Then we focus on the most advanced technologies used, such as MALDI-TOF-MS, high throughput sequencing or the genetic toolbox. Finally, we outline promising new approaches, such as omics tools and CRISPR-Cas9, as well as the challenges they face. Much has been achieved since the discovery of polymyxins, through several innovative technologies. Nevertheless, colistin resistance mechanisms remains very complex.
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Lüth S, Deneke C, Kleta S, Al Dahouk S. Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes. Microb Genom 2021; 7:mgen000491. [PMID: 33275089 PMCID: PMC8115905 DOI: 10.1099/mgen.0.000491] [Citation(s) in RCA: 16] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2020] [Accepted: 11/21/2020] [Indexed: 12/26/2022] Open
Abstract
Where classical epidemiology has proven to be inadequate for surveillance and control of foodborne pathogens, molecular epidemiology, using genomic typing methods, can add value. However, the analysis of whole genome sequencing (WGS) data varies widely and is not yet fully harmonised. We used genomic data on 494 Listeria monocytogenes isolates from ready-to-eat food products and food processing environments deposited in the strain collection of the German National Reference Laboratory to compare various procedures for WGS data analysis and to evaluate compatibility of results. Two different core genome multilocus sequence typing (cgMLST) schemes, different reference genomes in single nucleotide polymorphism (SNP) analysis and commercial as well as open-source software were compared. Correlation of allele distances from the different cgMLST approaches was high, ranging from 0.97 to 1, and unified thresholds yielded higher clustering concordance than scheme-specific thresholds. The number of detected SNP differences could be increased up to a factor of 3.9 using a specific reference genome compared with a general one. Additionally, specific reference genomes improved comparability of SNP analysis results obtained using different software tools. The use of a closed or a draft specific reference genome did not make a difference. The harmonisation of WGS data analysis will finally guarantee seamless data exchange, but, in the meantime, knowledge on threshold values that lead to comparable clustering of isolates by different methods may improve communication between laboratories. We therefore established a translation code between commonly applied cgMLST and SNP methods based on optimised clustering concordances. This code can work as a first filter to identify WGS-based typing matches resulting from different methods, which opens up a new perspective for data exchange and thereby accelerates time-critical analyses, such as in outbreak investigations.
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Affiliation(s)
- Stefanie Lüth
- National Reference Laboratory for Listeria monocytogenes, German Federal Institute for Risk Assessment, Department of Biological Safety, Berlin, Germany
- Institute of Biology, Freie Universität Berlin, Department of Biology, Chemistry and Pharmacy, Berlin, Germany
| | - Carlus Deneke
- Study Centre for Genome Sequencing and Analysis, German Federal Institute for Risk Assessment, Department of Biological Safety, Berlin, Germany
| | - Sylvia Kleta
- National Reference Laboratory for Listeria monocytogenes, German Federal Institute for Risk Assessment, Department of Biological Safety, Berlin, Germany
| | - Sascha Al Dahouk
- National Reference Laboratory for Listeria monocytogenes, German Federal Institute for Risk Assessment, Department of Biological Safety, Berlin, Germany
- RWTH Aachen University Hospital, Department of Internal Medicine, Aachen, Germany
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Confronting Ceftolozane-Tazobactam Susceptibility in Multidrug-Resistant Enterobacterales Isolates and Whole-Genome Sequencing Results (STEP Study). Int J Antimicrob Agents 2020; 57:106259. [PMID: 33310115 DOI: 10.1016/j.ijantimicag.2020.106259] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2020] [Revised: 10/05/2020] [Accepted: 11/28/2020] [Indexed: 01/04/2023]
Abstract
Ceftolozane-tazobactam (C/T) is frequently used for infections caused by multidrug-resistant (MDR)-Enterobacterales isolates. Whole-genome sequencing (WGS, Illumina-Hiseq 4000/NovaSeq 6000, OGC, UK) was used to study the population structure, the resistome and the virulome of C/T-susceptible and -resistant MDR Escherichia spp. (n=30) and Klebsiella spp. (n=78) isolates, recovered from lower respiratory, intra-abdominal and urinary tract infections of ICU patients from 11 Portuguese Hospitals (STEP study, 2017-2018). Minimum inhibitory concentrations (MICs) were determined (ISO-broth microdilution, breakpoints EUCAST-2020). In Escherichia spp., a weak concordance between the phenotypic and the WGS method (P=0.051) was observed in the carbapenemase detection (3/30) [blaVIM-2 (2/3), blaKPC-3 (1/3)]; VIM-2-Escherichia coli isolates were C/T-susceptible and only the KPC-3-Escherichia marmotae producer showed C/T-resistance. Overall, CTX-M-15-E. coli-ST131-O25:H4-H30-Rx (11/30) was the most frequent subclone, followed by CTX-M-27-E. coli-ST131-O25:H4-H30 (4/4). Moreover, a wide resistome and virulome were detected in all E. coli isolates. Among Klebsiella spp. isolates [K. pneumoniae (67/78), K. aerogenes (7/78), K. oxytoca (2/78), K. variicola (2/78)], concordance (P<0.001) was observed between the phenotypic and the genomic carbapenemase detection (21/78) [blaKPC-3 (14/21), blaOXA-48 (3/21), blaOXA-181 (3/21)]. A high correlation between C/T-resistance and carbapenemase detection was established (P<0.05). Overall, a high clonal diversity was observed, mainly in KPC-3-producing K. pneumoniae isolates. An extensive resistome was detected in Klebsiella spp. isolates, whereas virulence determinants were mostly identified in carbapenemase producers (P<0.001). WGS is a powerful tool for typing characterization and microbiological study of MDR-Enterobacterales pathogens. Furthermore, carbapenemase genes are associated with C/T-resistance in Klebsiella spp., but other mechanisms might also be involved.
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Diallo OO, Baron SA, Abat C, Colson P, Chaudet H, Rolain JM. Antibiotic resistance surveillance systems: A review. J Glob Antimicrob Resist 2020; 23:430-438. [PMID: 33176216 DOI: 10.1016/j.jgar.2020.10.009] [Citation(s) in RCA: 74] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/23/2019] [Revised: 04/16/2020] [Accepted: 10/08/2020] [Indexed: 12/12/2022] Open
Abstract
OBJECTIVES Epidemiological surveillance is one critical approach to estimate and fight the burden of antibiotic resistance (AR). Here we summarise the characteristics of surveillance systems devoted to the surveillance of AR worldwide and published in the literature. METHODS We performed a systematic review of the literature available on PubMed from January 2007 to July 2019 (12.5 years). The keywords ('surveillance system' OR 'laboratory-based surveillance' OR 'syndromic surveillance' OR 'sentinel surveillance' OR 'integrated surveillance' OR 'population-based surveillance') AND ('antibiotic resistance' OR 'antimicrobial resistance') were used. This research was completed with AR monitoring systems available on websites. RESULTS We identified 71 AR surveillance systems described by 90 publications from 35 countries, including 64 (90.1%) national and 7 (9.9%) multinational surveillance systems. Two regions accounted for ∼72% of systems: European region (37; 52.1%) and Region of the Americas (14; 19.7%). Fifty-three focused on AR surveillance in humans, 12 studied both humans and animals, and 6 focused only on animals. The two most common bacterial species reported were Staphylococcus aureus (42; 59.2%) and Escherichia coli (39; 54.9%). Of the 71 AR surveillance systems, 20 (28.2%) used prevalence as an indicator, 3 (4.2%) used incidence and 7 (9.9%) used both. Methicillin-resistant S. aureus (MRSA), vancomycin-resistant Enterococcus spp., S. aureus and Streptococcus pneumoniae, penicillin-resistant S. pneumoniae, and extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant E. coli and Klebsiella pneumoniae were monitored. CONCLUSIONS Our results showed heterogeneous surveillance systems. A 'One Health' approach is needed to monitor AR, with reference to the WHO Global Action Plan.
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Affiliation(s)
- Ousmane Oumou Diallo
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix-Marseille Univ., Institut de Recherche Pour le Développement (IRD), Assistance Publique-Hôpitaux de Marseille (AP-HM), Microbes Evolution Phylogeny and Infections (MEPHI), 19-21 Boulevard Jean Moulin, 13005 Marseille, France
| | - Sophie Alexandra Baron
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix-Marseille Univ., Institut de Recherche Pour le Développement (IRD), Assistance Publique-Hôpitaux de Marseille (AP-HM), Microbes Evolution Phylogeny and Infections (MEPHI), 19-21 Boulevard Jean Moulin, 13005 Marseille, France
| | - Cédric Abat
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France
| | - Philippe Colson
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix-Marseille Univ., Institut de Recherche Pour le Développement (IRD), Assistance Publique-Hôpitaux de Marseille (AP-HM), Microbes Evolution Phylogeny and Infections (MEPHI), 19-21 Boulevard Jean Moulin, 13005 Marseille, France
| | - Hervé Chaudet
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix-Marseille Univ., Institut de Recherche Pour le Développement (IRD), Assistance Publique-Hôpitaux de Marseille (AP-HM), Vecteurs-Infections Tropicales et Méditerranéennes (VITROME), 19-21 Boulevard Jean Moulin, 13005 Marseille, France
| | - Jean-Marc Rolain
- IHU Méditerranée Infection, 19-21 Boulevard Jean Moulin, 13005 Marseille, France; Aix-Marseille Univ., Institut de Recherche Pour le Développement (IRD), Assistance Publique-Hôpitaux de Marseille (AP-HM), Microbes Evolution Phylogeny and Infections (MEPHI), 19-21 Boulevard Jean Moulin, 13005 Marseille, France.
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Belibasakis GN, Lund BK, Krüger Weiner C, Johannsen B, Baumgartner D, Manoil D, Hultin M, Mitsakakis K. Healthcare Challenges and Future Solutions in Dental Practice: Assessing Oral Antibiotic Resistances by Contemporary Point-Of-Care Approaches. Antibiotics (Basel) 2020; 9:E810. [PMID: 33202544 PMCID: PMC7696509 DOI: 10.3390/antibiotics9110810] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2020] [Revised: 11/12/2020] [Accepted: 11/13/2020] [Indexed: 12/17/2022] Open
Abstract
Antibiotic resistance poses a global threat, which is being acknowledged at several levels, including research, clinical implementation, regulation, as well as by the World Health Organization. In the field of oral health, however, the issue of antibiotic resistances, as well as of accurate diagnosis, is underrepresented. Oral diseases in general were ranked third in terms of expenditures among the EU-28 member states in 2015. Yet, the diagnosis and patient management of oral infections, in particular, still depend primarily on empiric means. On the contrary, on the global scale, the field of medical infections has more readily adopted the integration of molecular-based systems in the diagnostic, patient management, and antibiotic stewardship workflows. In this perspective review, we emphasize the clinical significance of supporting in the future antibiotic resistance screening in dental practice with novel integrated and point-of-care operating tools that can greatly support the rapid, accurate, and efficient administration of oral antibiotics.
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Affiliation(s)
- Georgios N. Belibasakis
- Department of Dental Medicine, Karolinska Institutet, Alfred Nobels allè 8, 14104 Stockholm, Sweden; (B.K.L.); (C.K.W.); (D.M.); (M.H.)
| | - Bodil K. Lund
- Department of Dental Medicine, Karolinska Institutet, Alfred Nobels allè 8, 14104 Stockholm, Sweden; (B.K.L.); (C.K.W.); (D.M.); (M.H.)
- Department of Clinical Dentistry, University of Bergen, 5009 Bergen, Norway
- Department of Oral and Maxillofacial Surgery, Haukeland University Hospital, 5021 Bergen, Norway
| | - Carina Krüger Weiner
- Department of Dental Medicine, Karolinska Institutet, Alfred Nobels allè 8, 14104 Stockholm, Sweden; (B.K.L.); (C.K.W.); (D.M.); (M.H.)
- Department of Oral and Maxillofacial Surgery, Folktandvården Stockholm, Eastman Institutet, 11324 Stockholm, Sweden
| | - Benita Johannsen
- Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
| | - Desirée Baumgartner
- Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
| | - Daniel Manoil
- Department of Dental Medicine, Karolinska Institutet, Alfred Nobels allè 8, 14104 Stockholm, Sweden; (B.K.L.); (C.K.W.); (D.M.); (M.H.)
| | - Margareta Hultin
- Department of Dental Medicine, Karolinska Institutet, Alfred Nobels allè 8, 14104 Stockholm, Sweden; (B.K.L.); (C.K.W.); (D.M.); (M.H.)
| | - Konstantinos Mitsakakis
- Hahn-Schickard, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
- Laboratory for MEMS Applications, IMTEK—Department of Microsystems Engineering, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany;
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Imchen M, Moopantakath J, Kumavath R, Barh D, Tiwari S, Ghosh P, Azevedo V. Current Trends in Experimental and Computational Approaches to Combat Antimicrobial Resistance. Front Genet 2020; 11:563975. [PMID: 33240317 PMCID: PMC7677515 DOI: 10.3389/fgene.2020.563975] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 09/01/2020] [Indexed: 12/12/2022] Open
Abstract
A multitude of factors, such as drug misuse, lack of strong regulatory measures, improper sewage disposal, and low-quality medicine and medications, have been attributed to the emergence of drug resistant microbes. The emergence and outbreaks of multidrug resistance to last-line antibiotics has become quite common. This is further fueled by the slow rate of drug development and the lack of effective resistome surveillance systems. In this review, we provide insights into the recent advances made in computational approaches for the surveillance of antibiotic resistomes, as well as experimental formulation of combinatorial drugs. We explore the multiple roles of antibiotics in nature and the current status of combinatorial and adjuvant-based antibiotic treatments with nanoparticles, phytochemical, and other non-antibiotics based on synergetic effects. Furthermore, advancements in machine learning algorithms could also be applied to combat the spread of antibiotic resistance. Development of resistance to new antibiotics is quite rapid. Hence, we review the recent literature on discoveries of novel antibiotic resistant genes though shotgun and expression-based metagenomics. To decelerate the spread of antibiotic resistant genes, surveillance of the resistome is of utmost importance. Therefore, we discuss integrative applications of whole-genome sequencing and metagenomics together with machine learning models as a means for state-of-the-art surveillance of the antibiotic resistome. We further explore the interactions and negative effects between antibiotics and microbiomes upon drug administration.
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Affiliation(s)
- Madangchanok Imchen
- Department of Genomic Science, School of Biological Sciences, Central University of Kerala, Kasaragod, India
| | - Jamseel Moopantakath
- Department of Genomic Science, School of Biological Sciences, Central University of Kerala, Kasaragod, India
| | - Ranjith Kumavath
- Department of Genomic Science, School of Biological Sciences, Central University of Kerala, Kasaragod, India
| | - Debmalya Barh
- Centre for Genomics and Applied Gene Technology, Institute of Integrative Omics and Applied Biotechnology, Purba Medinipur, India
| | - Sandeep Tiwari
- Laboratório de Genética Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
| | - Preetam Ghosh
- Department of Computer Science, Virginia Commonwealth University, Richmond, VA, United States
| | - Vasco Azevedo
- Laboratório de Genética Celular e Molecular, Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
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Whole-genome sequencing as part of national and international surveillance programmes for antimicrobial resistance: a roadmap. BMJ Glob Health 2020; 5:e002244. [PMID: 33239336 PMCID: PMC7689591 DOI: 10.1136/bmjgh-2019-002244] [Citation(s) in RCA: 64] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/20/2019] [Revised: 03/18/2020] [Accepted: 03/27/2020] [Indexed: 12/26/2022] Open
Abstract
The global spread of antimicrobial resistance (AMR) and lack of novel alternative treatments have been declared a global public health emergency by WHO. The greatest impact of AMR is experienced in resource-poor settings, because of lack of access to alternative antibiotics and because the prevalence of multidrug-resistant bacterial strains may be higher in low-income and middle-income countries (LMICs). Intelligent surveillance of AMR infections is key to informed policy decisions and public health interventions to counter AMR. Molecular surveillance using whole-genome sequencing (WGS) can be a valuable addition to phenotypic surveillance of AMR. WGS provides insights into the genetic basis of resistance mechanisms, as well as pathogen evolution and population dynamics at different spatial and temporal scales. Due to its high cost and complexity, WGS is currently mainly carried out in high-income countries. However, given its potential to inform national and international action plans against AMR, establishing WGS as a surveillance tool in LMICs will be important in order to produce a truly global picture. Here, we describe a roadmap for incorporating WGS into existing AMR surveillance frameworks, including WHO Global Antimicrobial Resistance Surveillance System, informed by our ongoing, practical experiences developing WGS surveillance systems in national reference laboratories in Colombia, India, Nigeria and the Philippines. Challenges and barriers to WGS in LMICs will be discussed together with a roadmap to possible solutions.
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Hernández-García M, García-Fernández S, García-Castillo M, Bou G, Cercenado E, Delgado-Valverde M, Mulet X, Pitart C, Rodríguez-Lozano J, Tormo N, López-Mendoza D, Díaz-Regañón J, Cantón R. WGS characterization of MDR Enterobacterales with different ceftolozane/tazobactam susceptibility profiles during the SUPERIOR surveillance study in Spain. JAC Antimicrob Resist 2020; 2:dlaa084. [PMID: 34223039 PMCID: PMC8210196 DOI: 10.1093/jacamr/dlaa084] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2020] [Accepted: 09/03/2020] [Indexed: 11/13/2022] Open
Abstract
Objectives To analyse by WGS the ceftolozane/tazobactam (C/T) resistance mechanisms in Escherichia coli and Klebsiella spp. isolates recovered from complicated intra-abdominal and urinary tract infections in patients from Spanish ICUs (SUPERIOR surveillance study, 2016-17). Methods The clonal relatedness, the resistome and the virulome of 45 E. coli and 43 Klebsiella spp. isolates with different C/T susceptibility profiles were characterized. Results In E. coli, two (C/T susceptible) carbapenemase producers (VIM-2-CC23, OXA-48-ST38) were detected. The most relevant clone was ST131-B2-O25:H4-H30 (17/45), particularly the CTX-M-15-ST131-H30-Rx sublineage (15/17). ST131 strains were mainly C/T susceptible (15/17) and showed an extensive virulome. In non-ST131 strains (28/45), CTX-M enzymes [CTX-M-14 (8/24); CTX-M-15 (6/24); CTX-M-1 (3/24); CTX-M-32 (2/24)] were found in different clones. C/T resistance was detected in non-clonal E. coli isolates (13%, 6/45) with ESBL (4/6) and non-ESBL (2/6) genotypes. Among Klebsiella spp., Klebsiella pneumoniae (42/43) and Klebsiella michiganensis (1/43) species were identified; 42% (18/43) were carbapenemase producers and 58% showed a C/T resistance phenotype (25/43). OXA-48-ST11 (12/18), OXA-48-ST392 (2/18), OXA-48-ST15 (2/18), NDM-1-ST101 (1/18) and OXA-48+VIM-2-ST15 (1/18) isolates were found, all C/T resistant. Correlation between carbapenemase detection and resistance to C/T was demonstrated (P < 0.001). In non-carbapenemase-producing K. pneumoniae (25/43), C/T resistance (28%, 7/25) was detected in ESBL (3/7) and AmpC (2/7) producers. Overall, an extensive virulome was found and was correlated with carbapenemase carriage (P < 0.001) and C/T resistance (P < 0.05), particularly in OXA-48-ST11 strains (P < 0.05). Conclusions Prediction of antimicrobial susceptibility profiles using WGS is challenging. Carbapenemase-encoding genes are associated with C/T resistance in K. pneumoniae, but other resistance mechanisms might be additionally involved.
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Affiliation(s)
| | | | | | - Germán Bou
- Servicio de Microbiología, Hospital Universitario A Coruña, A Coruña, Spain
| | - Emilia Cercenado
- Servicio de Microbiología Clínica y Enfermedades Infecciosas, Hospital General Universitario Gregorio Marañón, Madrid, Spain
| | - Mercedes Delgado-Valverde
- UGC Enfermedades Infecciosas, Microbiología Clínica y Medicina Preventiva, Instituto de Biomedicina de Sevilla (IBIS), Hospital Universitario Virgen Macarena/CSIC/Universidad de Sevilla, Sevilla, Spain
| | - Xavier Mulet
- Servicio de Microbiología, Hospital Universitario Son Espases, Palma de Mallorca, Spain
| | - Cristina Pitart
- Laboratorio de Microbiología. Hospital Clínic i Provincial, Barcelona, Spain
| | - Jesús Rodríguez-Lozano
- Servicio de Microbiología, Hospital Universitario Marqués de Valdecilla, Santander, Spain
| | - Nuria Tormo
- Servicio de Microbiología, Consorcio Hospital General Universitario de Valencia, Valencia, Spain
| | | | | | - Rafael Cantón
- Servicio de Microbiología, Hospital Ramón y Cajal-IRYCIS, Madrid, Spain
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The Evolving Role of the Clinical Microbiology Laboratory in Identifying Resistance in Gram-Negative Bacteria: An Update. Infect Dis Clin North Am 2020; 34:659-676. [PMID: 33011047 DOI: 10.1016/j.idc.2020.08.001] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
The evolution of resistance to antimicrobial agents in gram-negatives has challenged the role of the clinical microbiology laboratory to implement new methods for their timely detection. Recent development has enabled the use of novel methods for more rapid pathogen identification, antimicrobial susceptibility testing, and detection of resistance markers. Commonly used methods improve the rapidity of resistance detection from both cultured bacteria and specimens. This review focuses on the commercially available systems available together with their technical performance and possible clinical impact.
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Zhang L, Fu Y, Han X, Xu Q, Weng S, Yan B, Liu L, Hua X, Chen Y, Yu Y. Phenotypic Variation and Carbapenem Resistance Potential in OXA-499-Producing Acinetobacter pittii. Front Microbiol 2020; 11:1134. [PMID: 32582088 PMCID: PMC7296048 DOI: 10.3389/fmicb.2020.01134] [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: 01/28/2020] [Accepted: 05/05/2020] [Indexed: 12/17/2022] Open
Abstract
Acinetobacter pittii is increasingly recognized as a clinically important species. Here, we identified a carbapenem-non-resistant A. pittii clinical isolate, A1254, harboring blaOXA–499, blaOXA–826, and blaADC–221. The blaOXA–499 genetic environment in A1254 was identical to that of another OXA-499-producing, but carbapenem-resistant, A. pittii isolate, YMC2010/8/T346, indicating the existence of phenotypic variation among OXA-499-producing A. pittii strains. Under imipenem-selective pressure, the A1254 isolate developed resistance to carbapenems in 60 generations. Two carbapenem-resistant mutants (CAB009 and CAB010) with mutations in the blaOXA–499 promoter region were isolated from two independently evolved populations (CAB001 and CAB004). The CAB009 mutant, with a mutation at position −14 (A to G), exhibited a four-fold higher carbapenem minimum inhibitory concentration (MIC) and a 4.53 ± 0.19 log2 fold change higher expression level of blaOXA–499 than the ancestor strain, A1254. The other mutant, CAB010, with a mutation at position −42 (G to A), showed a two-fold higher carbapenem MIC and a 1.65 ± 0.25 log2 fold change higher blaOXA–499 expression level than the ancestor strain. The blaOXA–499 gene and its promoter region were amplified from the wild-type strain and two mutant isolates and then individually cloned into the pYMAb2-Hygr vector and expressed in Acinetobacter baumannii ATCC 17978, A. pittii LMG 1035, and A. pittii A1254. All the transformed strains were resistant to carbapenem, irrespective of whether they harbored the initial or an evolved promoter sequence, and transformed strains expressing the promoter from the most resistant mutant, CAB009, showed the highest carbapenem MICs, with values of 32–64 μg/ml for imipenem and 128 μg/ml for meropenem. RNA sequencing was performed to confirm the contribution of blaOXA–499 to the development of carbapenem resistance. Although the CAB009 and CAB010 transcriptional patterns were different, blaOXA–499 was the only differentially expressed gene shared by the two mutants. Our results indicate that carbapenem-non-resistant Acinetobacter spp. strains carrying blaOXA genes have the potential to develop carbapenem resistance and need to be further investigated and monitored to prevent treatment failure due to the development of resistance.
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Affiliation(s)
- Linyue Zhang
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Ying Fu
- Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China.,Department of Clinical Laboratory, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China
| | - Xinhong Han
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Qingye Xu
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Shanshan Weng
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Biyong Yan
- Department of Laboratory Medicine, The Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China
| | - Lilin Liu
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Xiaoting Hua
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Yan Chen
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
| | - Yunsong Yu
- Department of Infectious Diseases, Sir Run Run Shaw Hospital, College of Medicine, Zhejiang University, Hangzhou, China.,Key Laboratory of Microbial Technology and Bioinformatics of Zhejiang Province, Hangzhou, China
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A Retrospective Whole-Genome Sequencing Analysis of Carbapenem and Colistin-Resistant Klebsiella Pneumoniae Nosocomial Strains Isolated during an MDR Surveillance Program. Antibiotics (Basel) 2020; 9:antibiotics9050246. [PMID: 32408565 PMCID: PMC7277725 DOI: 10.3390/antibiotics9050246] [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] [Received: 04/13/2020] [Revised: 05/03/2020] [Accepted: 05/06/2020] [Indexed: 12/02/2022] Open
Abstract
Multidrug-resistant Klebsiella pneumoniae (MDR Kp), in particular carbapenem-resistant Kp (CR-Kp), has become endemic in Italy, where alarming data have been reported on the spread of colistin-resistant CR-Kp (CRCR-Kp). During the period 2013–2014, 27 CRCR-Kp nosocomial strains were isolated within the Modena University Hospital Policlinico (MUHP) multidrug resistance surveillance program. We retrospectively investigated these isolates by whole-genome sequencing (WGS) analysis of the resistome, virulome, plasmid content, and core single nucleotide polymorphisms (cSNPs) in order to gain insights into their molecular epidemiology. The in silico WGS analysis of the resistome revealed the presence of genes, such as blaKPC, related to the phenotypically detected resistances to carbapenems. Concerning colistin resistance, the plasmidic genes mcr1–9 were not detected, while known and new genetic variations in mgrB, phoQ, and pmrB were found. The virulome profile revealed the presence of type-3 fimbriae, capsular polysaccharide, and iron acquisition system genes. The detected plasmid replicons were classified as IncFIB(pQil), IncFIB(K), ColRNAI, IncX3, and IncFII(K) types. The cSNPs genotyping was consistent with the multi locus sequence typing (MLST) and with the distribution of mutations related to colistin resistance genes. In a nosocomial drug resistance surveillance program, WGS proved to be a useful tool for elucidating the spread dynamics of CRCR-Kp nosocomial strains and could help to limit their diffusion.
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Hamidian M, Nigro SJ. Emergence, molecular mechanisms and global spread of carbapenem-resistant Acinetobacter baumannii. Microb Genom 2020; 5. [PMID: 31599224 PMCID: PMC6861865 DOI: 10.1099/mgen.0.000306] [Citation(s) in RCA: 195] [Impact Index Per Article: 39.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/18/2023] Open
Abstract
Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of high levels of resistance to many antibiotics, particularly those considered to be last-resort antibiotics, such as carbapenems. Although alterations in the efflux pump and outer membrane proteins can cause carbapenem resistance, the main mechanism is the acquisition of carbapenem-hydrolyzing oxacillinase-encoding genes. Of these, oxa23 is by far the most widespread in most countries, while oxa24 and oxa58 appear to be dominant in specific regions. Historically, much of the global spread of carbapenem resistance has been due to the dissemination of two major clones, known as global clones 1 and 2, although new lineages are now common in some parts of the world. The analysis of all publicly available genome sequences performed here indicates that ST2, ST1, ST79 and ST25 account for over 71 % of all genomes sequenced to date, with ST2 by far the most dominant type and oxa23 the most widespread carbapenem resistance determinant globally, regardless of clonal type. Whilst this highlights the global spread of ST1 and ST2, and the dominance of oxa23 in both clones, it could also be a result of preferential selection of carbapenem-resistant strains, which mainly belong to the two major clones. Furthermore, ~70 % of the sequenced strains have been isolated from five countries, namely the USA, PR China, Australia, Thailand and Pakistan, with only a limited number from other countries. These genomes are a vital resource, but it is currently difficult to draw an accurate global picture of this important superbug, highlighting the need for more comprehensive genome sequence data and genomic analysis.
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Affiliation(s)
- Mohammad Hamidian
- The ithree institute, University of Technology Sydney, Ultimo, NSW 2007, Australia
| | - Steven J Nigro
- Communicable Diseases Branch, Health Protection NSW, St Leonards, NSW 2065, Australia
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Ouyang Y, Li J, Peng Y, Huang Z, Ren Q, Lu J. The Role and Mechanism of Thiol-Dependent Antioxidant System in Bacterial Drug Susceptibility and Resistance. Curr Med Chem 2020; 27:1940-1954. [PMID: 31124420 DOI: 10.2174/0929867326666190524125232] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/04/2018] [Revised: 01/24/2019] [Accepted: 02/21/2019] [Indexed: 12/16/2022]
Abstract
Antibiotics play an irreplaceable role in the prevention and treatment of bacterial infection
diseases. However, because of the improper use of antibiotics, bacterial resistance emerges as a major
challenge of public health all over the world. The small thiol molecules such as glutathione can directly
react and conjugate with some antibiotics, which thus contribute to drug susceptibility and resistance.
Recently, accumulating evidence shows that there is a close link between the antibacterial activities of
some antibiotics and Reactive Oxygen Species (ROS). Thioredoxin and glutathione systems are two
main cellular disulfide reductase systems maintaining cellular ROS level. Therefore, these two thioldependent
antioxidant systems may affect the antibiotic susceptibility and resistance. Microorganisms
are equipped with different thiol-dependent antioxidant systems, which make the role of thioldependent
antioxidant systems in antibiotic susceptibility and resistance is different in various bacteria.
Here we will focus on the review on the advances of the effects of thiol-dependent antioxidant system
in the bacterial antibiotic susceptibility and resistance.
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Affiliation(s)
- Yanfang Ouyang
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Jing Li
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Yi Peng
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Zhijun Huang
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Qiao Ren
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
| | - Jun Lu
- Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education (Southwest University), College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China
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Zhang Z, Li D, Shi X, Zhai Y, Guo Y, Zheng Y, Zhao L, He Y, Chen Y, Wang Z, Su J, Kang Y, Gao Z. Genomic characterization of an emerging Enterobacteriaceae species: the first case of co-infection with a typical pathogen in a human patient. BMC Genomics 2020; 21:297. [PMID: 32293254 PMCID: PMC7156906 DOI: 10.1186/s12864-020-6720-z] [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: 01/20/2020] [Accepted: 04/05/2020] [Indexed: 12/18/2022] Open
Abstract
BACKGROUND Opportunistic pathogens are important for clinical practice as they often cause antibiotic-resistant infections. However, little is documented for many emerging opportunistic pathogens and their biological characteristics. Here, we isolated a strain of extended-spectrum β-lactamase-producing Enterobacteriaceae from a patient with a biliary tract infection. We explored the biological and genomic characteristics of this strain to provide new evidence and detailed information for opportunistic pathogens about the co-infection they may cause. RESULTS The isolate grew very slowly but conferred strong protection for the co-infected cephalosporin-sensitive Klebsiella pneumoniae. As the initial laboratory testing failed to identify the taxonomy of the strain, great perplexity was caused in the etiological diagnosis and anti-infection treatment for the patient. Rigorous sequencing efforts achieved the complete genome sequence of the isolate which we designated as AF18. AF18 is phylogenetically close to a few strains isolated from soil, clinical sewage, and patients, forming a novel species together, while the taxonomic nomenclature of which is still under discussion. And this is the first report of human infection of this novel species. Like its relatives, AF18 harbors many genes related to cell mobility, various genes adaptive to both the natural environment and animal host, over 30 mobile genetic elements, and a plasmid bearing blaCTX-M-3 gene, indicating its ability to disseminate antimicrobial-resistant genes from the natural environment to patients. Transcriptome sequencing identified two sRNAs that critically regulate the growth rate of AF18, which could serve as targets for novel antimicrobial strategies. CONCLUSIONS Our findings imply that AF18 and its species are not only infection-relevant but also potential disseminators of antibiotic resistance genes, which highlights the need for continuous monitoring for this novel species and efforts to develop treatment strategies.
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Affiliation(s)
- Zhao Zhang
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.,Department of Respiratory & Critical Care Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Daixi Li
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.,Department of Respiratory and Critical Care Medicine, Zhongshan Hospital Xiamen University, Xiamen, 361004, Fujian, China
| | - Xing Shi
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.,Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, Beijing, China
| | - Yao Zhai
- University of Technology Sydney, Ultimo, NSW, Australia
| | - Yatao Guo
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.,Department of Respiratory & Critical Care Medicine, the First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China
| | - Yali Zheng
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.,Department of Respiratory, Critical Care and Sleep Medicine, Xiamen University Xiang'an Hospital, Xiamen, Fujian, China
| | - Lili Zhao
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China
| | - Yukun He
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China
| | - Yusheng Chen
- Department of Respiratory & Critical Care Medicine, Fujian Provincial Hospital, Fuzhou, Fujian, China
| | - Zhanwei Wang
- Laboratory Medicine, Peking University People's Hospital, Beijing, China
| | - Jianrong Su
- Department of Clinical Laboratory Center, Beijing Friendship Hospital, Beijing, Beijing, China
| | - Yu Kang
- Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, Beijing, China.
| | - Zhancheng Gao
- Department of Respiratory & Critical Care Medicine, Peking University People's Hospital, Beijing, Beijing, China.
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Baumgartner M, Bayer F, Pfrunder-Cardozo KR, Buckling A, Hall AR. Resident microbial communities inhibit growth and antibiotic-resistance evolution of Escherichia coli in human gut microbiome samples. PLoS Biol 2020; 18:e3000465. [PMID: 32310938 PMCID: PMC7192512 DOI: 10.1371/journal.pbio.3000465] [Citation(s) in RCA: 30] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/09/2019] [Revised: 04/30/2020] [Accepted: 04/02/2020] [Indexed: 01/05/2023] Open
Abstract
Countering the rise of antibiotic-resistant pathogens requires improved understanding of how resistance emerges and spreads in individual species, which are often embedded in complex microbial communities such as the human gut microbiome. Interactions with other microorganisms in such communities might suppress growth and resistance evolution of individual species (e.g., via resource competition) but could also potentially accelerate resistance evolution via horizontal transfer of resistance genes. It remains unclear how these different effects balance out, partly because it is difficult to observe them directly. Here, we used a gut microcosm approach to quantify the effect of three human gut microbiome communities on growth and resistance evolution of a focal strain of Escherichia coli. We found the resident microbial communities not only suppressed growth and colonisation by focal E. coli but also prevented it from evolving antibiotic resistance upon exposure to a beta-lactam antibiotic. With samples from all three human donors, our focal E. coli strain only evolved antibiotic resistance in the absence of the resident microbial community, even though we found resistance genes, including a highly effective resistance plasmid, in resident microbial communities. We identified physical constraints on plasmid transfer that can explain why our focal strain failed to acquire some of these beneficial resistance genes, and we found some chromosomal resistance mutations were only beneficial in the absence of the resident microbiota. This suggests, depending on in situ gene transfer dynamics, interactions with resident microbiota can inhibit antibiotic-resistance evolution of individual species.
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Affiliation(s)
- Michael Baumgartner
- Institute of Integrative Biology, Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
| | - Florian Bayer
- Biosciences, University of Exeter, Penryn, Cornwall, United Kingdom
| | - Katia R. Pfrunder-Cardozo
- Institute of Integrative Biology, Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
| | - Angus Buckling
- Biosciences, University of Exeter, Penryn, Cornwall, United Kingdom
| | - Alex R. Hall
- Institute of Integrative Biology, Department of Environmental Systems Science, ETH Zürich, Zürich, Switzerland
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Production of Mannosylerythritol Lipids (MELs) to be Used as Antimicrobial Agents Against S. aureus ATCC 6538. Curr Microbiol 2020; 77:1373-1380. [PMID: 32123984 PMCID: PMC7334285 DOI: 10.1007/s00284-020-01927-2] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2019] [Accepted: 02/17/2020] [Indexed: 11/12/2022]
Abstract
Antimicrobial resistance (AMR) is a current major health issue, both for the high rates of resistance observed in bacteria that cause common infections and for the complexity of the consequences of AMR. Pathogens like Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Mycobacterium tuberculosis among others are clear examples of antibiotic-resistant threats. Biosurfactants have recently emerged as a potential new generation of anti-adhesive and anti-biofilm agents; mannosylerythritol lipids (MELs) are biosurfactants produced by a range of fungi. A range of structural variants of MELs can be formed and the proportion of each isomer in the fermentation depends on the yeast used, the carbon substrate used for growth and the duration of the fermentation. In order to allow assessment of the possible functions of MELs as antimicrobial molecules, small quantities of MEL were produced by controlled fermentation. Fermentations of the yeast Pseudozyma aphidis using rapeseed oil as a carbon source yielded up to 165 gMELs/kgSubstrate. The MELs formed by this strain was a mixture of MEL-A, MEL-B, MEL-C and MEL-D. The MELs produced were tested against S. aureus ATCC 6538 on pre-formed biofilm and on co-incubation biofilm experiments on silicone discs; showing a disruption of biomass, reduction of the biofilm metabolic activity and a bacteriostatic/bactericidal effect confirmed by a release of oxygen uptake \documentclass[12pt]{minimal}
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\begin{document}$$p_{{{\text{O}}_{2} }}$$\end{document}pO2, the reduction of citrate synthase activity and scanning electron microscopy. The results show that MELs are promising antimicrobial molecules for biomedical technological applications that could be studied in detail in large-scale systems and in conjunction with animal tissue models.
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MinION sequencing of Streptococcus suis allows for functional characterization of bacteria by multilocus sequence typing and antimicrobial resistance profiling. J Microbiol Methods 2019; 169:105817. [PMID: 31881288 DOI: 10.1016/j.mimet.2019.105817] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2019] [Revised: 12/20/2019] [Accepted: 12/23/2019] [Indexed: 01/31/2023]
Abstract
In recent years, high-throughput sequencing has revolutionized disease diagnosis by its powerful ability to provide high resolution genomic information. The Oxford Nanopore MinION sequencer has unparalleled potential as a rapid disease diagnostic tool due to its high mobility, accessibility, and short turnaround time. However, there is a lack of rigorous quality assessment and control processes standardizing the testing on the MinION, which is necessary for incorporation into a diagnostic workflow. Thus, our study examined the use of the MinION sequencer for bacterial whole genome generation and characterization. Using Streptococcus suis as a model, we optimized DNA isolation and treatments to be used for MinION sequencing and standardized de novo assembly to quickly generate a full-length consensus sequence achieving a 99.4% average accuracy. The consensus genomes from MinION sequencing were able to accurately predict the multilocus sequence type in 8 out of 10 samples and identified antimicrobial resistance profiles for 100% of the samples, despite the concern of a high error rate. The inability to unequivocally predict sequence types was due to difficulty in differentiating high identity alleles, which was overcome by applying additional error correction methods to increase consensus accuracy. This manuscript provides methods for the use of MinION sequencing for identification of S. suis genome sequence, sequence type, and antibiotic resistance profile that can be used as a framework for identification and classification of other pathogens.
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