| 1. |
- Berglund, Fanny, et al.
(author)
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Comprehensive screening of genomic and metagenomic data reveals a large diversity of tetracycline resistance genes
- 2020
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In: Microbial genomics. - : Microbiology Society. - 2057-5858. ; 6:11
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Journal article (peer-reviewed)abstract
- Tetracyclines are broad-spectrum antibiotics used to prevent or treat a variety of bacterial infections. Resistance is often mediated through mobile resistance genes, which encode one of the three main mechanisms: active efflux, ribosomal target protection or enzymatic degradation. In the last few decades, a large number of new tetracycline-resistance genes have been discovered in clinical settings. These genes are hypothesized to originate from environmental and commensal bacteria, but the diversity of tetracycline-resistance determinants that have not yet been mobilized into pathogens is unknown. In this study, we aimed to characterize the potential tetracycline resistome by screening genomic and metagenomic data for novel resistance genes. By using probabilistic models, we predicted 1254 unique putative tetracycline resistance genes, representing 195 gene families (<70% amino acid sequence identity), whereof 164 families had not been described previously. Out of 17 predicted genes selected for experimental verification, 7 induced a resistance phenotype in an Escherichia coli host. Several of the predicted genes were located on mobile genetic elements or in regions that indicated mobility, suggesting that they easily can be shared between bacteria. Furthermore, phylogenetic analysis indicated several events of horizontal gene transfer between bacterial phyla. Our results also suggested that acquired efflux pumps originate from proteobacterial species, while ribosomal protection genes have been mobilized from Firmicutes and Actinobacteria. This study significantly expands the knowledge of known and putatively novel tetracycline resistance genes, their mobility and evolutionary history. The study also provides insights into the unknown resistome and genes that may be encountered in clinical settings in the future.
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| 2. |
- Berglund, Fanny, et al.
(author)
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Evidence for wastewaters as environments where mobile antibiotic resistance genes emerge
- 2023
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 6
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Journal article (peer-reviewed)abstract
- The emergence and spread of mobile antibiotic resistance genes (ARGs) in pathogens have become a serious threat to global health. Still little is known about where ARGs gain mobility in the first place. Here, we aimed to collect evidence indicating where suchinitial mobilizationevents of clinically relevant ARGs may have occurred. We found that the majority of previously identified origin species did not carry the mobilizing elements that likely enabled intracellular mobility of the ARGs, suggesting a necessary interplay between different bacteria. Analyses of a broad range of metagenomes revealed that wastewaters and wastewater-impacted environments had by far the highest abundance of both origin species and corresponding mobilizing elements. Most origin species were only occasionally detected in other environments. Co-occurrence of origin species and corresponding mobilizing elements were rare in human microbiota. Our results identify wastewaters and wastewater-impacted environments as plausible arenas for the initial mobilization of resistance genes.
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| 3. |
- Ebmeyer, Stefan, 1990, et al.
(author)
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A framework for identifying the recent origins of mobile antibiotic resistance genes
- 2021
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 4:1
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Journal article (peer-reviewed)abstract
- Since the introduction of antibiotics as therapeutic agents, many bacterial pathogens have developed resistance to antibiotics. Mobile resistance genes, acquired through horizontal gene transfer, play an important role in this process. Understanding from which bacterial taxa these genes were mobilized, and whether their origin taxa share common traits, is critical for predicting which environments and conditions contribute to the emergence of novel resistance genes. This knowledge may prove valuable for limiting or delaying future transfer of novel resistance genes into pathogens. The literature on the origins of mobile resistance genes is scattered and based on evidence of variable quality. Here, we summarize, amend and scrutinize the evidence for 37 proposed origins of mobile resistance genes. Using state-of-the-art genomic analyses, we supplement and evaluate the evidence based on well-defined criteria. Nineteen percent of reported origins did not fulfill the criteria to confidently assign the respective origin. Of the curated origin taxa, >90% have been associated with infection in humans or domestic animals, some taxa being the origin of several different resistance genes. The clinical emergence of these resistance genes appears to be a consequence of antibiotic selection pressure on taxa that are permanently or transiently associated with the human/domestic animal microbiome. Ebmeyer and colleagues developed a genomic framework for identification and scrutiny of the origins of antibiotic resistance genes. Using data scoured from the literature and publicly available genomes, their results indicate that only 81% of previously reported origins are valid, and that the majority of resistance genes of which the origin is known to date emerged in taxa that have been associated with infection in humans and domesticated animals.
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| 4. |
- Ebmeyer, Stefan, 1990, et al.
(author)
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CMY-1/MOX-family AmpC β-lactamases MOX-1, MOX-2 and MOX-9 were mobilized independently from three Aeromonas species.
- 2019
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In: The Journal of antimicrobial chemotherapy. - : Oxford University Press (OUP). - 1460-2091 .- 0305-7453. ; 74:5, s. 1202-1206
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Journal article (peer-reviewed)abstract
- To investigate the origin of CMY-1/MOX-family β-lactamases.Publicly available genome assemblies were screened for CMY-1/MOX genes. The loci of CMY-1/MOX genes were compared with respect to synteny and nucleotide identity, and subjected to phylogenetic analysis.The chromosomal ampC genes of several Aeromonas species were highly similar to known mobile CMY-1/MOX variants. Annotation and sequence comparison revealed nucleotide identities >98% and conserved syntenies between MOX-1-, MOX-2- and MOX-9-associated mobile sequences and the chromosomal Aeromonas sanarellii, Aeromonas caviae and Aeromonas media ampC loci. Furthermore, the phylogenetic analysis showed that MOX-1, MOX-2 and MOX-9 formed three distinct monophyletic groups with the chromosomal ampC genes of A. sanarellii, A. caviae and A. media, respectively.Our findings show that three CMY-1/MOX-family β-lactamases were mobilized independently from three Aeromonas species and hence shine new light on the evolution and emergence of mobile antibiotic resistance genes.
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| 5. |
- Ebmeyer, Stefan, 1990, et al.
(author)
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GEnView: a gene-centric, phylogeny-based comparative genomics pipeline for bacterial genomes and plasmids
- 2022
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In: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 38:6, s. 1727-1728
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Journal article (peer-reviewed)abstract
- Comparing genomic loci of a given bacterial gene across strains and species can provide insights into their evolution, including information on e.g. acquired mobility, the degree of conservation between different taxa or indications of horizontal gene transfer events. While thousands of bacterial genomes are available to date, there is no software that facilitates comparisons of individual gene loci for a large number of genomes. GEnView (Genetic Environment View) is a Python-based pipeline for the comparative analysis of gene-loci in a large number of bacterial genomes, providing users with automated, taxon-selective access to the >800.000 genomes and plasmids currently available in the NCBI Assembly and RefSeq databases, and is able to process local genomes that are not deposited at NCBI, enabling searches for genomic sequences and to analyze their genetic environments through the interactive visualization and extensive metadata files created by GEnView.
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| 6. |
- Ebmeyer, Stefan, 1990
(author)
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On the Origins of mobile Antibiotic Resistance Genes : A comparative genomics approach
- 2021
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Doctoral thesis (other academic/artistic)abstract
- Mobile antibiotic resistance genes (ARGs), transferable between bacterial cells, are major contributors to the antibiotic resistance crisis we are facing today. From which organisms pathogens acquired these genes is mostly unknown, yet knowledge about their origin is needed in order to limit the emergence and spread of novel ARGs in the future. Increasing the number of known origins of mobile resistance genes would allow us to investigate potential patterns that may hint towards the conditions that potentially promote the emergence of mobile ARGs. This thesis aims to identify from which taxa ARGs have been mobilized into pathogens, so that this knowledge may aid mitigations to limit the emergence of novel ARGs in the future. We used comparative genomic methods on the large amount of publicly available sequenced bacterial genomes in order to identify bacterial taxa from which certain ARGs have been mobilized (paper I-IV). A literature review and the development of a computational pipeline (paper VI) to compare hundreds of genomic loci allowed us to scrutinize previously reported origins and analyze patterns among to-date identified ARG origins (paper V). In this thesis, we have identified the recent origins of PER-type class A beta-lactamases as Pararheinheimera spp. (Paper I), the recent origins of CMY-1/MOX-1, MOX-2 and MOX-9 class C beta-lactamases as Aeromonas sanarellii, Aeromonas caviae and Aeromonas media respectively (Paper II), the recent origin of FOX-type class C beta-lactamases as Aeromonas allosaccharophila (Paper III), and the recent origin of GPC-1/BKC-1 carbapenemases as Shinella spp (Paper IV). In paper V, based on the amended and curated data from the literature, five criteria allowing for the confident identification of recent origins of mobile ARGs were identified. Of all recent origins identified on species level, all were Proteobacteria, >90% were identified as potential pathogens of humans and/or domestic animals, none of them known antibiotic producers themselves. However, all curated recent origins account for only about 4% of known mobile ARGs, indicating that environmental bacteria may represent a significant source of resistance genes. Finally, Paper VI presents a bioinformatics pipeline, GEnView, for comparative genomic analysis of gene loci among hundreds of genomes, developed throughout this thesis. This thesis further elucidates the recent origins of several mobile resistance genes, identifies previously unrecognized patterns about their emergence and provides other researchers with the tools to investigate the origins of other resistance genes. This knowledge may prove valuable to guide future efforts trying to mitigate the emergence of additional ARGs in the clinics.
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| 7. |
- Ebmeyer, Stefan, et al.
(author)
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PER extended-spectrum β-lactamases originate from Pararheinheimera spp
- 2019
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 1872-7913 .- 0924-8579. ; 53:2, s. 158-164
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Journal article (peer-reviewed)abstract
- To investigate the origin of PER extended-spectrum β-lactamases, publicly available sequence databases were searched for bla PER-like genes. Three genomes from Pararheinheimera, a genus associated with water and soil environments, were found to carry bla PER-like genes but lacked the ISCR1/ISPa12/ISPa13 insertion sequences commonly associated with bla PER in clinical isolates. Sequence analysis revealed 78–96% nucleotide identity and conserved synteny between the clinical mobile genetic elements (MGEs) encoding bla PER-1 and the bla PER locus in the Pararheinheimera genomes. Notably, bla PER genes were only identified in 3 of 21 Pararheinheimera and Rheinheimera genomes, whereas the genetic environment of bla PER genes as found in clinical MGEs was conserved in all Pararheinheimera and Rheinheimera genomes. These findings indicate that bla PER genes were likely acquired by a branch of the Pararheinheimera genus long before the antibiotic era. Later, bla PER genes were mobilised, likely through the involvement of insertion sequences, from one or several Pararheinheimera species, allowing their dissemination into human pathogens.
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| 8. |
- Ebmeyer, Stefan, 1990, et al.
(author)
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The mobile FOX AmpC beta-lactamases originated in Aeromonas allosaccharophila
- 2019
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 1872-7913 .- 0924-8579. ; 54:6, s. 798-802
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Journal article (peer-reviewed)abstract
- Objective: It is important to understand the origins of antibiotic resistance genes so that risks associated with the emergence of novel resistance genes can be assessed and managed. The chromosomal ampC gene (CAV-1) of Aeromonas caviae (A. caviae) has been reported as the origin of mobile FOX cephalosporinases. The recent identification of A. caviae as the origin of MOX-2 cephalosporinases and the comparably great sequence divergence between FOX and MOX genes makes it unlikely that both genes arose from the same species. Therefore, this study investigated the origin of FOX cephalosporinases using large-scale genomics. Methods: Publicly available genomes and plasmids were searched for FOX-like genes. Synteny and nucleotide identities of the identified FOX-like genes and their genetic environments were compared and a phylogenetic tree was generated. Results: FOX-like genes were identified in > 230 Aeromonas genomes and in 46 Enterobacteriaceae isolates. Analysis of the genomic context of CAV-1 revealed a truncated insertion sequence directly upstream of the ampC gene. The chromosomal ampCs of A. caviae (n = 31) were 75–78% identical to CAV-1. In contrast, CAV-1, mobile FOX genes and their context were 95–98% similar to the chromosomal ampC-locus of Aeromonas allosaccharophila (A. allosaccharophila) (n = 6). The A. allosaccharophila ampCs formed a monophyletic branch with mobile FOX genes, whereas the A. caviae ampCs clustered with mobile MOX genes. Conclusions: These findings show that FOX cephalosporinases originate not in A. caviae, as previously reported, but in A. allosaccharophila, which is a fish pathogen. This finding agrees with the hypothesis that antibiotic use in aquaculture could have contributed to the emergence of FOX genes in human pathogens.
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| 9. |
- Ebmeyer, Stefan, 1990, et al.
(author)
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The PER extended-spectrum beta-lactamases originate from Pararheinheimera sp.
- 2019
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In: International journal of antimicrobial agents. - : Elsevier BV. - 1872-7913 .- 0924-8579. ; 53:2, s. 158-64
-
Journal article (peer-reviewed)abstract
- To investigate the origin of the PER extended-spectrum beta-lactamase, publicly available sequence databases were searched for PER-like genes. Three genomes from Pararheinheimera, a genus associated with water and soil environments, were found to carry PER-like genes, but lacked the ISCR1/ISPa12/ISPa13 insertion sequences (IS) commonly associated with PER in clinical isolates. Sequence analysis revealed 78-96% nucleotide identity and conserved synteny between the clinical mobile genetic elements (MGEs) encoding PER-1 and the PER locus in the Pararheinheimera genomes. Notably, PER genes were only identified in 3 of 21 Pararheinheimera and Rheinheimera genomes, whereas the genetic environment of PER genes as found in clinical MGEs was conserved in all Pararheinheimera and Rheinheimera genomes. These findings indicate that PER genes were likely acquired by a branch of the Pararheinheimera genus long before the antibiotic era. Later, PER genes were mobilized, likely through involvement of IS, from one or several Pararheinheimera species, allowing their dissemination into human pathogens.
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| 10. |
- Kieffer, Nicolas, et al.
(author)
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Evidence for Pseudoxanthomonas mexicana as the recent origin of the blaAIM-1 carbapenemase gene
- 2022
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In: International Journal of Antimicrobial Agents. - : Elsevier BV. - 0924-8579. ; 59:4
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Journal article (peer-reviewed)abstract
- Objectives: Elucidating the recent evolutionary history of clinically important antibiotic resistance genes may inform measures to delay the future emergence of additional resistance genes in clinics. This study investigated the recent origin of blaAIM-1, a metallo-β-lactamase gene found in Pseudomonas aeruginosa, and the possible role of ISCR15 in its mobilisation and transfer into clinical species. Methods: Comparative genomics were used to identify the recent origin of blaAIM. Mobilisation attempts were performed under different conditions by cloning ISCR15 and the blaAIM-1-like gene in Escherichia coli. Results: Several blaAIM-1 homologues were identified in the Pseudoxanthomonas genus, with conserved synteny of the locus between species and absence of elements associated with mobility. The closest AIM-1 homologue (97.7% amino acid identity) was found in a Pseudoxanthomonas mexicana (P. mexicana) strain. Cloning the blaAIM-like gene in Escherichia coli resulted in high resistance towards carbapenems. While blaAIM-1 is surrounded by ISCR15 elements in clinical strains, in vitro experiments failed to demonstrate their role as mobilising elements. Conclusions: This study presents evidence that P. mexicana, an environmental species occasionally associated with infections, is the origin of the B3 metallo-β-lactamase AIM-1. The presence of terIS, a plausible recognition site for ISCR15, in other parts of the P. mexicana genome suggests a more complex and yet not understood mobilisation mechanism.
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| 11. |
- Kieffer, Nicolas, et al.
(author)
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The Class A Carbapenemases BKC-1 and GPC-1 Both Originate from the Bacterial Genus Shinella
- 2020
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In: Antimicrobial agents and chemotherapy. - 1098-6596. ; 64:12
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Journal article (peer-reviewed)abstract
- Copyright © 2020 Kieffer et al. Comparative genomics identified the environmental bacterial genus Shinella as the most likely origin of the class A carbapenemases BKC-1 and GPC-1. Available sequences and PCR analyses of additional Shinella species revealed homologous β-lactamases showing up to 85.4% and 93.3% amino acid identity to both enzymes, respectively. The genes conferred resistance to β-lactams once expressed in Escherichia coliblaBKC-1 likely evolved from a putative ancestral Shinella gene with higher homology through duplication of a gene fragment.
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| 12. |
- Kraupner, Nadine, et al.
(author)
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Selective concentration for ciprofloxacin resistance in Escherichia coli grown in complex aquatic bacterial biofilms
- 2018
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In: Environment International. - : Elsevier BV. - 0160-4120 .- 1873-6750. ; 116, s. 255-268
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Journal article (peer-reviewed)abstract
- There is concern that antibiotics in the environment can select for and enrich bacteria carrying acquired antibiotic resistance genes, thus increasing the potential of those genes to emerge in a clinical context. A critical question for understanding and managing such risks is what levels of antibiotics are needed to select for resistance in complex bacterial communities. Here, we address this question by examining the phenotypic and genotypic profiles of aquatic communities exposed to ciprofloxacin, also evaluating the within-species selection of resistant E. coli in complex communities. The taxonomic composition was significantly altered at ciprofloxacin exposure concentrations down to 1 μg/L. Shotgun metagenomic analysis indicated that mobile quinolone resistance determinants (qnrD, qnrS and qnrB) were enriched as a direct consequence of ciprofloxacin exposure from 1 μg/L or higher. Only at 5–10 μg/L resistant E.coli increased relative to their sensitive counterparts. These resistant E. coli predominantly harbored non-transferrable, chromosomal triple mutations (gyrA S83 L, D87N and parC S80I), which confer high-level resistance. In a controlled experimental setup such as this, we interpret effects on taxonomic composition and enrichment of mobile quinolone resistance genes as relevant indicators of risk. Hence, the lowest observed effect concentration for resistance selection in complex communities by ciprofloxacin was 1 μg/L and the corresponding no observed effect concentration 0.1 μg/L. These findings can be used to define and implement discharge or surface water limits to reduce risks for selection of antibiotic resistance in the environment. © 2018 Elsevier Ltd
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| 13. |
- Kraupner, Nadine, et al.
(author)
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Selective concentrations for trimethoprim resistance in aquatic environments
- 2020
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In: Environment International. - : Elsevier BV. - 0160-4120 .- 1873-6750. ; 144
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Journal article (peer-reviewed)abstract
- Antibiotic resistance presents a serious and still growing threat to human health. Environmental exposure levels required to select for resistance are unknown for most antibiotics. Here, we evaluated different experimental approaches and ways to interpret effect measures, in order to identify what concentration of trimethoprim that are likely to select for resistance in aquatic environments. When grown in complex biofilms, selection for resistant E. coli increased at 100 mu g/L, whereas there was only a non-significant trend with regards to changes in taxonomic composition within the tested range (0-100 mu g/L). Planktonic co-culturing of 149 different E. coli strains isolated from sewage again confirmed selection at 100 mu g/L. Finally, pairwise competition experiments were performed with engineered E. coli strains carrying different trimethoprim resistance genes (dfr) and their sensitive counterparts. While strains with introduced resistance genes grew slower than the sensitive ones at 0 and 10 mu g/L, a significant reduction in cost was found already at 10 mu g/L. Defining lowest effect concentrations by comparing proportion of resistant strains to sensitive ones at the same time point, rather than to their initial ratios, will reflect the advantage a resistance factor can bring, while ignoring exposure-independent fitness costs. As costs are likely to be highly dependent on the specific environmental and genetic contexts, the former approach might be more suitable as a basis for defining exposure limits with the intention to prevent selection for resistance. Based on the present and other studies, we propose that 1 mu g/L would be a reasonably protective exposure limit for trimethoprim in aquatic environments.
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| 14. |
- Lund, David, 1994, et al.
(author)
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Large-scale characterization of the macrolide resistome reveals high diversity and several new pathogen-associated genes
- 2022
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In: Microbial Genomics. - : Microbiology Society. - 2057-5858. ; 8:1
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Journal article (peer-reviewed)abstract
- Macrolides are broad-spectrum antibiotics used to treat a range of infections. Resistance to macrolides is often conferred by mobile resistance genes encoding Erm methyltransferases or Mph phosphotransferases. New erm and mph genes keep being discovered in clinical settings but their origins remain unknown, as is the type of macrolide resistance genes that will appear in the future. In this study, we used optimized hidden Markov models to characterize the macrolide resistome. Over 16 terabases of genomic and metagenomic data, representing a large taxonomic diversity (11 030 species) and diverse environments (1944 metagenomic samples), were searched for the presence of erm and mph genes. From this data, we predicted 28 340 macrolide resistance genes encoding 2892 unique protein sequences, which were clustered into 663 gene families (<70 % amino acid identity), of which 619 (94 %) were previously uncharacterized. This included six new resistance gene families, which were located on mobile genetic elements in pathogens. The function of ten predicted new resistance genes were experimentally validated in Escherichia coli using a growth assay. Among the ten tested genes, seven conferred increased resistance to erythromycin, with five genes additionally conferring increased resistance to azithromycin, showing that our models can be used to predict new functional resistance genes. Our analysis also showed that macrolide resistance genes have diverse origins and have transferred horizontally over large phylogenetic distances into human pathogens. This study expands the known macrolide resistome more than ten-fold, provides insights into its evolution, and demonstrates how computational screening can identify new resistance genes before they become a significant clinical problem.
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| 15. |
- Rutgersson, Carolin, 1983, et al.
(author)
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Long-term application of Swedish sewage sludge on farmland does not cause clear changes in the soil bacterial resistome
- 2020
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In: Environment International. - : Elsevier BV. - 0160-4120 .- 1873-6750. ; 137
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Journal article (peer-reviewed)abstract
- The widespread practice of applying sewage sludge to arable land makes use of nutrients indispensable for crops and reduces the need for inorganic fertilizer, however this application also provides a potential route for human exposure to chemical contaminants and microbial pathogens in the sludge. A recent concern is that such practice could promote environmental selection and dissemination of antibiotic resistant bacteria or resistance genes. Understanding the risks of sludge amendment in relation to antibiotic resistance development is important for sustainable agriculture, waste treatment and infectious disease management. To assess such risks, we took advantage of an agricultural field trial in southern Sweden, where land used for growing different crops has been amended with sludge every four years since 1981. We sampled raw, semi-digested and digested and stored sludge together with soils from the experimental plots before and two weeks after the most recent amendment in 2017. Levels of selected antimicrobials and bioavailable metals were determined and microbial effects were evaluated using both culture-independent metagenome sequencing and conventional culturing. Antimicrobials or bioavailable metals (Cu and Zn) did not accumulate to levels of concern for environmental selection of antibiotic resistance, and no coherent signs, neither on short or long time scales, of enrichment of antibiotic-resistant bacteria or resistance genes were found in soils amended with digested and stored sewage sludge in doses up to 12 metric tons per hectare. Likewise, only very few and slight differences in microbial community composition were observed after sludge amendment. Taken together, the current study does not indicate risks of sludge amendment related to antibiotic resistance development under the given conditions. Extrapolations should however be done with care as sludge quality and application practices vary between regions. Hence, the antibiotic concentrations and resistance load of the sludge are likely to be higher in regions with larger antibiotic consumption and resistance burden than Sweden.
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