| 1. |
- Berglund, Fanny, 1988, et al.
(författare)
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Identification and reconstruction of novel antibiotic resistance genes from metagenomes
- 2019
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Ingår i: Microbiome. - : Springer Science and Business Media LLC. - 2049-2618. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundEnvironmental and commensal bacteria maintain a diverse and largely unknown collection of antibiotic resistance genes (ARGs) that, over time, may be mobilized and transferred to pathogens. Metagenomics enables cultivation-independent characterization of bacterial communities but the resulting data is noisy and highly fragmented, severely hampering the identification of previously undescribed ARGs. We have therefore developed fARGene, a method for identification and reconstruction of ARGs directly from shotgun metagenomic data.ResultsfARGene uses optimized gene models and can therefore with high accuracy identify previously uncharacterized resistance genes, even if their sequence similarity to known ARGs is low. By performing the analysis directly on the metagenomic fragments, fARGene also circumvents the need for a high-quality assembly. To demonstrate the applicability of fARGene, we reconstructed -lactamases from five billion metagenomic reads, resulting in 221 ARGs, of which 58 were previously not reported. Based on 38 ARGs reconstructed by fARGene, experimental verification showed that 81% provided a resistance phenotype in Escherichia coli. Compared to other methods for detecting ARGs in metagenomic data, fARGene has superior sensitivity and the ability to reconstruct previously unknown genes directly from the sequence reads.ConclusionsWe conclude that fARGene provides an efficient and reliable way to explore the unknown resistome in bacterial communities. The method is applicable to any type of ARGs and is freely available via GitHub under the MIT license.
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| 2. |
- Berglund, Fanny, 1988, et al.
(författare)
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Identification of 76 novel B1 metallo-beta-lactamases through large-scale screening of genomic and metagenomic data
- 2017
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Ingår i: Microbiome. - : Springer Science and Business Media LLC. - 2049-2618. ; 5:1, s. 134-134
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Tidskriftsartikel (refereegranskat)abstract
- Background: Metallo-beta-lactamases are bacterial enzymes that provide resistance to carbapenems, the most potent class of antibiotics. These enzymes are commonly encoded on mobile genetic elements, which, together with their broad substrate spectrum and lack of clinically useful inhibitors, make them a particularly problematic class of antibiotic resistance determinants. We hypothesized that there is a large and unexplored reservoir of unknown metallo-beta-lactamases, some of which may spread to pathogens, thereby threatening public health. The aim of this study was to identify novel metallo-beta-lactamases of class B1, the most clinically important subclass of these enzymes. Results: Based on a new computational method using an optimized hidden Markov model, we analyzed over 10,000 bacterial genomes and plasmids together with more than 5 terabases of metagenomic data to identify novel metallo-beta-lactamase genes. In total, 76 novel genes were predicted, forming 59 previously undescribed metallo-beta-lactamase gene families. The ability to hydrolyze imipenem in an Escherichia coli host was experimentally confirmed for 18 of the 21 tested genes. Two of the novel B1 metallo-beta-lactamase genes contained atypical zinc-binding motifs in their active sites, which were previously undescribed for metallo-beta-lactamases. Phylogenetic analysis showed that B1 metallo-beta-lactamases could be divided into five major groups based on their evolutionary origin. Our results also show that, except for one, all of the previously characterized mobile B1 beta-lactamases are likely to have originated from chromosomal genes present in Shewanella spp. and other Proteobacterial species. Conclusions: This study more than doubles the number of known B1 metallo-beta-lactamases. The findings have further elucidated the diversity and evolutionary history of this important class of antibiotic resistance genes and prepare us for some of the challenges that may be faced in clinics in the future.
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| 3. |
- Berglund, Fanny, 1988
(författare)
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New antibiotic resistance genes and their diversity
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antibiotic resistance is increasing worldwide and is considered a severe threat to public health. Often, antibiotic resistance is caused by antibiotic resistance genes, of which many are hypothesized to have been transferred into human pathogens from environmental bacteria. It is, therefore, of great importance to explore bacterial communities to identify new antibiotic resistance genes before they reach clinical settings. The six papers presented in this thesis aim to identify new antibiotic resistance genes in large genomic and metagenomic datasets and to place them in an evolutionary context. In Paper I, a new method for the identification and reconstruction of new antibiotic resistance genes directly from fragmented metagenomic data was developed and was shown to outperform other methods significantly. In Papers II and III, novel genes of the clinically important class metallo-β-lactamases were identified. By analyzing metagenomes and bacterial genomes, 96 novel putative metallo-β-lactamase genes were predicted. In Paper IV, the diversity and phylogeny of the metallo-β-lactamases were further investigated. The results showed that the genes mainly clustered based on the taxonomy of the host species and that many of the mobile metallo-β-lactamases potentially were mobilized from species of the phylum Proteobacteria. In Paper V, the aim was to identify new genes providing resistance to the antibiotic class tetracyclines. A total of 195 gene families were predicted, of which 164 were new putative tetracycline resistance genes. Finally, in Paper VI, we searched for and predicted 20 novel putative quinolone resistance (qnr) genes from a large amount of metagenomic data. Throughout the thesis, a total of 54 novel genes have been functionally verified in Escherichia coli, of which 37 expressed the predicted phenotype. The results of this thesis provide deeper insights into the diversity and evolutionary history of three major classes of antibiotic resistance genes. It also provides new methodologies for efficient and reliable identification of new resistance genes in genomic and metagenomic data.
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| 4. |
- Boulund, Fredrik, 1985, et al.
(författare)
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Computational discovery and functional validation of novel fluoroquinolone resistance genes in public metagenomic data sets
- 2017
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 18:1, s. Art 682-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fluoroquinolones are broad-spectrum antibiotics used to prevent and treat a wide range of bacterial infections. Plasmid-mediated qnr genes provide resistance to fluoroquinolones in many bacterial species and are increasingly encountered in clinical settings. Over the last decade, several families of qnr genes have been discovered and characterized, but their true prevalence and diversity still remain unclear. In particular, environmental and host-associated bacterial communities have been hypothesized to maintain a large and unknown collection of qnr genes that could be mobilized into pathogens. Results: In this study we used computational methods to screen genomes and metagenomes for novel qnr genes. In contrast to previous studies, we analyzed an almost 20-fold larger dataset comprising almost 13 terabases of sequence data. In total, 362,843 potential qnr gene fragments were identified, from which 611 putative qnr genes were reconstructed. These gene sequences included all previously described plasmid-mediated qnr gene families. Fifty-two of the 611 identified qnr genes were reconstructed from metagenomes, and 20 of these were previously undescribed. All of the novel qnr genes were assembled from metagenomes associated with aquatic environments. Nine of the novel genes were selected for validation, and six of the tested genes conferred consistently decreased susceptibility to ciprofloxacin when expressed in Escherichia coli. Conclusions: The results presented in this study provide additional evidence for the ubiquitous presence of qnr genes in environmental microbial communities, expand the number of known qnr gene variants and further elucidate the diversity of this class of resistance genes. This study also strengthens the hypothesis that environmental bacterial communities act as sources of previously uncharacterized qnr genes.
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| 5. |
- Lund, David, 1994, et al.
(författare)
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Extensive screening reveals previously undiscovered aminoglycoside resistance genes in human pathogens
- 2023
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Ingår i: Communications Biology. - 2399-3642. ; 6:1
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Tidskriftsartikel (refereegranskat)abstract
- Antibiotic resistance is a growing threat to human health, caused in part by pathogens accumulating antibiotic resistance genes (ARGs) through horizontal gene transfer. New ARGs are typically not recognized until they have become widely disseminated, which limits our ability to reduce their spread. In this study, we use large-scale computational screening of bacterial genomes to identify previously undiscovered mobile ARGs in pathogens. From ~1 million genomes, we predict 1,071,815 genes encoding 34,053 unique aminoglycoside-modifying enzymes (AMEs). These cluster into 7,612 families (<70% amino acid identity) of which 88 are previously described. Fifty new AME families are associated with mobile genetic elements and pathogenic hosts. From these, 24 of 28 experimentally tested AMEs confer resistance to aminoglycoside(s) in Escherichia coli, with 17 providing resistance above clinical breakpoints. This study greatly expands the range of clinically relevant aminoglycoside resistance determinants and demonstrates that computational methods enable early discovery of potentially emerging ARGs.
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| 6. |
- Lund, David, 1994, et al.
(författare)
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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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Ingår i: Microbial Genomics. - : Microbiology Society. - 2057-5858. ; 8:1
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Tidskriftsartikel (refereegranskat)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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| 7. |
- Marathe, Nachiket, et al.
(författare)
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Sewage effluent from an Indian hospital harbors novel carbapenemases and integron-borne antibiotic resistance genes
- 2019
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Ingår i: Microbiome. - : Springer Science and Business Media LLC. - 2049-2618. ; 7:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Hospital wastewaters contain fecal material from a large number of individuals, of which many are undergoing antibiotic therapy. It is, thus, plausible that hospital wastewaters could provide opportunities to find novel carbapenemases and other resistance genes not yet described in clinical strains. Our aim was therefore to investigate the microbiota and antibiotic resistome of hospital effluent collected from the city of Mumbai, India, with a special focus on identifying novel carbapenemases. Results: Shotgun metagenomics revealed a total of 112 different mobile antibiotic resistance gene types, conferring resistance against almost all classes of antibiotics. Beta-lactamase genes, including encoding clinically important carbapenemases, such as NDM, VIM, IMP, KPC, and OXA-48, were abundant. NDM (0.9% relative abundance to 16S rRNA genes) was the most common carbapenemase gene, followed by OXA-58 (0.84% relative abundance to 16S rRNA genes). Among the investigated mobile genetic elements, class 1 integrons (11% relative abundance to 16S rRNA genes) were the most abundant. The genus Acinetobacter accounted for as many as 30% of the total 16S rRNA reads, with A. baumannii accounting for an estimated 2.5%. High throughput sequencing of amplified integron gene cassettes identified a novel functional variant of an IMP-type (proposed IMP-81) carbapenemase gene (eight aa substitutions) along with recently described novel resistance genes like sul4 and bla RSA1. Using a computational hidden Markov model, we detected 27 unique metallo-beta-lactamase (MBL) genes in the shotgun data, of which nine were novel subclass B1 genes, one novel subclass B2, and 10 novel subclass B3 genes. Six of the seven novel MBL genes were functional when expressed in Escherichia coli. Conclusion: By exploring hospital wastewater from India, our understanding of the diversity of carbapenemases has been extended. The study also demonstrates that the microbiota of hospital wastewater can serve as a reservoir of novel resistance genes, including previously uncharacterized carbapenemases with the potential to spread further.
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