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Hosoi Y, Kawanishi M, Harada S, Kumakawa M, Matsuda M, Sekiguchi H. The Prevalence and the Underlying Mechanisms of Fosfomycin Resistance of Escherichia coli and Salmonella spp. Among Cattle in Japan. Int J Mol Sci 2024; 25:13723. [PMID: 39769485 PMCID: PMC11676364 DOI: 10.3390/ijms252413723] [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: 11/26/2024] [Revised: 12/20/2024] [Accepted: 12/20/2024] [Indexed: 01/11/2025] Open
Abstract
To investigate fosfomycin resistance rates in cattle across Japan, we carried out susceptibility tests. To identify the genes contributing to fosfomycin resistance, we performed whole-genome sequencing on the fosfomycin-resistant strains. Escherichia coli were sampled from healthy cattle (n = 292, combined total from 2017, 2020, 2021, and 2022) and diseased cattle (n = 73, from 2021 to 2022). Salmonella spp. were obtained from diseased cattle (n = 74 from 2021 to 2022). These samples originated from different and non-duplicated farms. The MICs to fosfomycin were measured using an agar dilution method with a breakpoint of 256 μg/mL. We conducted whole-genome sequencing with a MiSeq, followed by in silico analysis of the acquired draft genomes. The resistance rates were 0.3% (95% CI [0-1.9%]), 6.8% (95% CI [2.3-15.3%]), and 1.4% (95% CI [0-7.3%]). The FosA3 gene was detected in five out of six fosfomycin-resistant E. coli strains and one Salmonella spp. strain. The fosfomycin-resistant Salmonella spp. strain also has a fosA7 gene. One E. coli strain showed resistance to fosfomycin without having the fosA3 gene, and with the mutations of glpT, uhpT, uhpT and ptsI, and with the existence of efflux pumps. The nationwide scale of resistance rates to fosfomycin in E. coli isolated from healthy and diseased cattle and that of Salmonella spp. from diseased cattle were revealed for the first time, and the resistance rates were low. In addition, genes linked to the mechanism of fosfomycin resistance were identified.
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Affiliation(s)
- Yuta Hosoi
- Veterinary AMR Center, National Veterinary Assay Laboratory, Ministry of Agriculture, Forestry and Fisheries, Tokyo 185-8511, Japan
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Gschwind R, Ugarcina Perovic S, Weiss M, Petitjean M, Lao J, Coelho LP, Ruppé E. ResFinderFG v2.0: a database of antibiotic resistance genes obtained by functional metagenomics. Nucleic Acids Res 2023:7173762. [PMID: 37207327 DOI: 10.1093/nar/gkad384] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2023] [Revised: 04/27/2023] [Accepted: 05/02/2023] [Indexed: 05/21/2023] Open
Abstract
Metagenomics can be used to monitor the spread of antibiotic resistance genes (ARGs). ARGs found in databases such as ResFinder and CARD primarily originate from culturable and pathogenic bacteria, while ARGs from non-culturable and non-pathogenic bacteria remain understudied. Functional metagenomics is based on phenotypic gene selection and can identify ARGs from non-culturable bacteria with a potentially low identity shared with known ARGs. In 2016, the ResFinderFG v1.0 database was created to collect ARGs from functional metagenomics studies. Here, we present the second version of the database, ResFinderFG v2.0, which is available on the Center of Genomic Epidemiology web server (https://cge.food.dtu.dk/services/ResFinderFG/). It comprises 3913 ARGs identified by functional metagenomics from 50 carefully curated datasets. We assessed its potential to detect ARGs in comparison to other popular databases in gut, soil and water (marine + freshwater) Global Microbial Gene Catalogues (https://gmgc.embl.de). ResFinderFG v2.0 allowed for the detection of ARGs that were not detected using other databases. These included ARGs conferring resistance to beta-lactams, cycline, phenicol, glycopeptide/cycloserine and trimethoprim/sulfonamide. Thus, ResFinderFG v2.0 can be used to identify ARGs differing from those found in conventional databases and therefore improve the description of resistomes.
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Affiliation(s)
- Rémi Gschwind
- University of Paris Cité, INSERM UMR 1137 IAME, F-75018Paris, France
| | - Svetlana Ugarcina Perovic
- Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai200433, China
| | - Maja Weiss
- Research Group for Genomic Epidemiology, Technical University of Denmark, Kgs, Lyngby 2800, Denmark
| | - Marie Petitjean
- University of Paris Cité, INSERM UMR 1137 IAME, F-75018Paris, France
| | - Julie Lao
- University of Paris Cité, INSERM UMR 1137 IAME, F-75018Paris, France
| | - Luis Pedro Coelho
- Institute of Science and Technology for Brain-Inspired Intelligence, Fudan University, Shanghai200433, China
| | - Etienne Ruppé
- University of Paris Cité, INSERM UMR 1137 IAME, F-75018Paris, France
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Li XH, Huang YY, Lu LM, Zhao LJ, Luo XK, Li RJ, Dai YY, Qin C, Huang YQ, Chen H. Early genetic diagnosis of clarithromycin resistance in Helicobacter pylori. World J Gastroenterol 2021; 27:3595-3608. [PMID: 34239272 PMCID: PMC8240046 DOI: 10.3748/wjg.v27.i24.3595] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/02/2021] [Revised: 04/13/2021] [Accepted: 05/21/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The drug resistance rate of clinical Helicobacter pylori (H. pylori) isolates has increased. However, the mechanism of drug resistance remains unclear. In this study, drug-resistant H. pylori strains were isolated from different areas and different populations of Chinese for genomic analysis.
AIM To investigate drug-resistant genes in H. pylori and find the genes for the early diagnosis of clarithromycin resistance.
METHODS Three drug-resistant H. pylori strains were isolated from patients with gastritis in Bama County, China. Minimal inhibitory concentrations of clarithromycin, metronidazole, and levofloxacin were determined and complete genome sequencing was performed with annotation. Hp1181 and hp1184 genes were found in these strains and then detected by reverse transcription polymerase chain reaction. The relationships between hp1181 or hp1184 and clarithromycin resistance were ascertained with gene mutant and drug-resistant strains. The homology of the strains with hp26695 was assessed through complete genome detection and identification. Differences in genome sequences, gene quantity, and gene characteristics were detected amongst the three strains. Prediction and analysis of the function of drug-resistant genes indicated that the RNA expression of hp1181 and hp1184 increased in the three strains, which was the same in the artificially induced clarithromycin-resistant bacteria. After gene knockout, the drug sensitivity of the strains was assessed.
RESULTS The strains showing a high degree of homology with hp26695, hp1181, and hp1184 genes were found in these strains; the expression of the genes hp1184 and hp1181 was associated with clarithromycin resistance.
CONCLUSION Hp1181 and hp1184 mutations may be the earliest and most persistent response to clarithromycin resistance, and they may be the potential target genes for the diagnosis, prevention, and treatment of clarithromycin resistance.
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Affiliation(s)
- Xiao-Hua Li
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Yong-Yi Huang
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Lin-Ming Lu
- Department of Pathology, Wannan Medical College, Wuhu 241002, Anhui Province, China
| | - Li-Juan Zhao
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Xian-Ke Luo
- Department of Gastroenterology, National Hospital of Guangxi Zhuang Autonomous Region, Nanning Guangxi Zhuang Autonomous Region, 530001, China
| | - Ru-Jia Li
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Yuan-Yuan Dai
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Chun Qin
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Yan-Qiang Huang
- Research Center for the Prevention and Treatment of Drug Resistant Microbial Infection, Youjiang Medical University for Nationalities, Baise 533000, Guangxi Zhuang Autonomous Region, China
| | - Hao Chen
- Department of Pathology, Wannan Medical College, Wuhu 241002, Anhui Province, China
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Lima AH, Silva JR, Alves C, Lameira J. QM/MM Study of the Fosfomycin Resistance Mechanism Involving FosB Enzyme. ACS OMEGA 2021; 6:12507-12512. [PMID: 34056400 PMCID: PMC8154160 DOI: 10.1021/acsomega.1c00096] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/06/2021] [Accepted: 03/12/2021] [Indexed: 06/01/2023]
Abstract
Multidrug-resistant organisms contain antibiotic-modifying enzymes that facilitate resistance to a variety of antimicrobial compounds. Particularly, the fosfomycin (FOF) drug can be structurally modified by several FOF-modifying enzymes before it reaches the biological target. Among them, FosB is an enzyme that utilizes l-cysteine or bacillithiol in the presence of a divalent metal to open the epoxide ring of FOF and, consequently, inactivate the drug. Here, we have used hybrid quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulations to explore the mechanism of the reaction involving FosB and FOF. The calculated free-energy profiles show that the cost to open the epoxide ring of FOF at the C2 atom is ∼3.0 kcal/mol higher than that at the C1 atom. Besides, our QM/MM MD results revealed the critical role of conformation change of Cys9 and Asn50 to release the drug from the active site. Overall, the present study provides insights into the mechanism of FOF-resistant proteins.
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Affiliation(s)
- Anderson H. Lima
- Laboratório de Planejamento
e Desenvolvimento de Fármacos, Instituto de Ciências
Exatas e Naturais, Universidade Federal
do Pará, Rua Augusto Corrêa, 01, 66075-110, Belém, Pará, Brasil
| | - José Rogério
A. Silva
- Laboratório de Planejamento
e Desenvolvimento de Fármacos, Instituto de Ciências
Exatas e Naturais, Universidade Federal
do Pará, Rua Augusto Corrêa, 01, 66075-110, Belém, Pará, Brasil
| | - Cláudio
Nahum Alves
- Laboratório de Planejamento
e Desenvolvimento de Fármacos, Instituto de Ciências
Exatas e Naturais, Universidade Federal
do Pará, Rua Augusto Corrêa, 01, 66075-110, Belém, Pará, Brasil
| | - Jerônimo Lameira
- Laboratório de Planejamento
e Desenvolvimento de Fármacos, Instituto de Ciências
Exatas e Naturais, Universidade Federal
do Pará, Rua Augusto Corrêa, 01, 66075-110, Belém, Pará, Brasil
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Willms IM, Grote M, Kocatürk M, Singhoff L, Kraft AA, Bolz SH, Nacke H. Novel Soil-Derived Beta-Lactam, Chloramphenicol, Fosfomycin and Trimethoprim Resistance Genes Revealed by Functional Metagenomics. Antibiotics (Basel) 2021; 10:antibiotics10040378. [PMID: 33916668 PMCID: PMC8066302 DOI: 10.3390/antibiotics10040378] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 03/30/2021] [Accepted: 03/30/2021] [Indexed: 11/16/2022] Open
Abstract
Antibiotic resistance genes (ARGs) in soil are considered to represent one of the largest environmental resistomes on our planet. As these genes can potentially be disseminated among microorganisms via horizontal gene transfer (HGT) and in some cases are acquired by clinical pathogens, knowledge about their diversity, mobility and encoded resistance spectra gained increasing public attention. This knowledge offers opportunities with respect to improved risk prediction and development of strategies to tackle antibiotic resistance, and might help to direct the design of novel antibiotics, before further resistances reach hospital settings or the animal sector. Here, metagenomic libraries, which comprise genes of cultivated microorganisms, but, importantly, also those carried by the uncultured microbial majority, were screened for novel ARGs from forest and grassland soils. We detected three new beta-lactam, a so far unknown chloramphenicol, a novel fosfomycin, as well as three previously undiscovered trimethoprim resistance genes. These ARGs were derived from phylogenetically diverse soil bacteria and predicted to encode antibiotic inactivation, antibiotic efflux, or alternative variants of target enzymes. Moreover, deduced gene products show a minimum identity of ~21% to reference database entries and confer high-level resistance. This highlights the vast potential of functional metagenomics for the discovery of novel ARGs from soil ecosystems.
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Dos Santos DFK, Istvan P, Quirino BF, Kruger RH. Functional Metagenomics as a Tool for Identification of New Antibiotic Resistance Genes from Natural Environments. MICROBIAL ECOLOGY 2017; 73:479-491. [PMID: 27709246 DOI: 10.1007/s00248-016-0866-x] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/18/2016] [Accepted: 09/19/2016] [Indexed: 05/26/2023]
Abstract
Antibiotic resistance has become a major concern for human and animal health, as therapeutic alternatives to treat multidrug-resistant microorganisms are rapidly dwindling. The problem is compounded by low investment in antibiotic research and lack of new effective antimicrobial drugs on the market. Exploring environmental antibiotic resistance genes (ARGs) will help us to better understand bacterial resistance mechanisms, which may be the key to identifying new drug targets. Because most environment-associated microorganisms are not yet cultivable, culture-independent techniques are essential to determine which organisms are present in a given environmental sample and allow the assessment and utilization of the genetic wealth they represent. Metagenomics represents a powerful tool to achieve these goals using sequence-based and functional-based approaches. Functional metagenomic approaches are particularly well suited to the identification new ARGs from natural environments because, unlike sequence-based approaches, they do not require previous knowledge of these genes. This review discusses functional metagenomics-based ARG research and describes new possibilities for surveying the resistome in environmental samples.
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Affiliation(s)
| | - Paula Istvan
- Departamento de Biologia Celular, Universidade de Brasília, Brasília, DF, Brazil
| | - Betania Ferraz Quirino
- Embrapa-Agroenergia, Brasília, DF, Brazil
- Universidade Católica de Brasília, Genomic Sciences and Biotechnology Program, Brasília, DF, Brazil
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Lima AH, dos Santos AM, Alves CN, Lameira J. Computed insight into a peptide inhibitor preventing the induced fit mechanism of MurA enzyme fromPseudomonas aeruginosa. Chem Biol Drug Des 2016; 89:599-607. [DOI: 10.1111/cbdd.12882] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2016] [Revised: 08/16/2016] [Accepted: 09/29/2016] [Indexed: 11/28/2022]
Affiliation(s)
- Anderson H. Lima
- Laboratório de Planejamento e Desenvolvimento de Fármacos; Instituto de Ciências Exatas e Naturais; Universidade Federal do Pará; Belém PA Brasil
| | - Alberto M. dos Santos
- Laboratório de Planejamento e Desenvolvimento de Fármacos; Instituto de Ciências Exatas e Naturais; Universidade Federal do Pará; Belém PA Brasil
| | - Cláudio Nahum Alves
- Laboratório de Planejamento e Desenvolvimento de Fármacos; Instituto de Ciências Exatas e Naturais; Universidade Federal do Pará; Belém PA Brasil
| | - Jerônimo Lameira
- Laboratório de Planejamento e Desenvolvimento de Fármacos; Instituto de Ciências Exatas e Naturais; Universidade Federal do Pará; Belém PA Brasil
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Tai YT, Foong CP, Najimudin N, Sudesh K. Discovery of a new polyhydroxyalkanoate synthase from limestone soil through metagenomic approach. J Biosci Bioeng 2015; 121:355-64. [PMID: 26467694 DOI: 10.1016/j.jbiosc.2015.08.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2015] [Revised: 08/10/2015] [Accepted: 08/11/2015] [Indexed: 11/16/2022]
Abstract
PHA synthase (PhaC) is the key enzyme in the production of biodegradable plastics known as polyhydroxyalkanoate (PHA). Nevertheless, most of these enzymes are isolated from cultivable bacteria using traditional isolation method. Most of the microorganisms found in nature could not be successfully cultivated due to the lack of knowledge on their growth conditions. In this study, a culture-independent approach was applied. The presence of phaC genes in limestone soil was screened using primers targeting the class I and II PHA synthases. Based on the partial gene sequences, a total of 19 gene clusters have been identified and 7 clones were selected for full length amplification through genome walking. The complete phaC gene sequence of one of the clones (SC8) was obtained and it revealed 81% nucleotide identity to the PHA synthase gene of Chromobacterium violaceum ATCC 12472. This gene obtained from uncultured bacterium was successfully cloned and expressed in a Cupriavidus necator PHB(-)4 PHA-negative mutant resulting in the accumulation of significant amount of PHA. The PHA synthase activity of this transformant was 64 ± 12 U/g proteins. This paper presents a pioneering study on the discovery of phaC in a limestone area using metagenomic approach. Through this study, a new functional phaC was discovered from uncultured bacterium. Phylogenetic classification for all the phaCs isolated from this study has revealed that limestone hill harbors a great diversity of PhaCs with activities that have not yet been investigated.
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Affiliation(s)
- Yen Teng Tai
- School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
| | - Choon Pin Foong
- School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
| | - Nazalan Najimudin
- School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia
| | - Kumar Sudesh
- School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang, Malaysia; Centre for Chemical Biology, Universiti Sains Malaysia, 11800 Penang, Malaysia.
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