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Hughes, Diarmaid
Publications (10 of 130) Show all publications
Juhas, M., Widlake, E., Teo, J., Huseby, D. L., Tyrrell, J. M., Polikanov, Y. S., . . . Hobbie, S. N. (2019). In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii. Journal of Antimicrobial Chemotherapy, 74(4), 944-952
Open this publication in new window or tab >>In vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii
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2019 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 74, no 4, p. 944-952Article in journal (Refereed) Published
Abstract [en]

Objectives: Widespread antimicrobial resistance often limits the availability of therapeutic options to only a few last-resort drugs that are themselves challenged by emerging resistance and adverse side effects. Apramycin, an aminoglycoside antibiotic, has a unique chemical structure that evades almost all resistance mechanisms including the RNA methyltransferases frequently encountered in carbapenemase-producing clinical isolates. This study evaluates the in vitro activity of apramycin against multidrug-, carbapenem- and aminoglycoside-resistant Enterobacteriaceae and Acinetobacter baumannii, and provides a rationale for its superior antibacterial activity in the presence of aminoglycoside resistance determinants.

Methods: A thorough antibacterial assessment of apramycin with 1232 clinical isolates from Europe, Asia, Africa and South America was performed by standard CLSI broth microdilution testing. WGS and susceptibility testing with an engineered panel of aminoglycoside resistance-conferring determinants were used to provide a mechanistic rationale for the breadth of apramycin activity.

Results: MIC distributions and MIC90 values demonstrated broad antibacterial activity of apramycin against Escherichia coli, Klebsiella pneumoniae, Enterobacter spp., Morganella morganii, Citrobacter freundii, Providencia spp., Proteus mirabilis, Serratia marcescens and A. baumannii. Genotypic analysis revealed the variety of aminoglycoside-modifying enzymes and rRNA methyltransferases that rendered a remarkable proportion of clinical isolates resistant to standard-of-care aminoglycosides, but not to apramycin. Screening a panel of engineered strains each with a single well-defined resistance mechanism further demonstrated a lack of cross-resistance to gentamicin, amikacin, tobramycin and plazomicin.

Conclusions: Its superior breadth of activity renders apramycin a promising drug candidate for the treatment of systemic Gram-negative infections that are resistant to treatment with other aminoglycoside antibiotics.

Place, publisher, year, edition, pages
OXFORD UNIV PRESS, 2019
National Category
Infectious Medicine
Identifiers
urn:nbn:se:uu:diva-382394 (URN)10.1093/jac/dky546 (DOI)000463812100014 ()30629184 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 115583
Available from: 2019-04-25 Created: 2019-04-25 Last updated: 2019-04-25Bibliographically approved
Brandis, G., Cao, S. & Hughes, D. (2019). Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella. BIO-PROTOCOL, 9(3), Article ID e3159.
Open this publication in new window or tab >>Measuring Homologous Recombination Rates between Chromosomal Locations in Salmonella
2019 (English)In: BIO-PROTOCOL, ISSN 2331-8325, Vol. 9, no 3, article id e3159Article in journal (Refereed) Published
Abstract [en]

Homologous recombination between two similar DNA molecules, plays an important role in the repair of double-stranded DNA breaks. Recombination can occur between two sister chromosomes, or between two locations of similar sequence identity within the same chromosome. The assay described here is designed to measure the rate of homologous recombination between two locations with sequence similarity within the same bacterial chromosome. For this purpose, a selectable/counter-selectable genetic cassette is inserted into one of the locations and homologous recombination repair rates are measured as a function of recombinational removal of the inserted cassette. This recombinational repair process is called gene conversion, non-reciprocal recombination. We used this method to measure the recombination rates between genes within gene families and to study the stability of mobile genetic elements inserted into members of gene families.

Place, publisher, year, edition, pages
BIO-PROTOCOL, 2019
Keywords
Homologous recombination, Homologous recombination rate(s), DNA repair, Double stranded DNA breaks, Salmonella, Gene families
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-377594 (URN)10.21769/BioProtoc.3159 (DOI)000458033700009 ()
Funder
Swedish Research Council, 2016-04449Swedish Research Council, 2017-03953Carl Tryggers foundation , CTS16: 194Carl Tryggers foundation , CTS17: 204
Available from: 2019-03-01 Created: 2019-03-01 Last updated: 2019-03-07Bibliographically approved
Brandis, G., Cao, S. & Hughes, D. (2019). Operon Concatenation Is an Ancient Feature That Restricts the Potential to Rearrange Bacterial Chromosomes. Molecular biology and evolution, 36(9), 1990-2000
Open this publication in new window or tab >>Operon Concatenation Is an Ancient Feature That Restricts the Potential to Rearrange Bacterial Chromosomes
2019 (English)In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 36, no 9, p. 1990-2000Article in journal (Refereed) Published
Abstract [en]

The last common ancestor of the Gammaproteobacteria carried an important 40-kb chromosome section encoding 51 proteins of the transcriptional and translational machinery. These genes were organized into eight contiguous operons (rrnB-tufB-secE-rpoBC-str-S10-spc-alpha). Over 2 Gy of evolution, in different lineages, some of the operons became separated by multigene insertions. Surprisingly, in many Enterobacteriaceae, much of the ancient organization is conserved, indicating a strong selective force on the operons to remain colinear. Here, we show for one operon pair, tufB-secE in Salmonella, that an interruption of contiguity significantly reduces growth rate. Our data show that the tufB-secE operons are concatenated by an interoperon terminator-promoter overlap that plays a significant role regulating gene expression. Interrupting operon contiguity interferes with this regulation, reducing cellular fitness. Six operons of the ancestral chromosome section remain contiguous in Salmonella (tufB-secE-rpoBC and S10-spc-alpha) and, strikingly, each of these operon pairs is also connected by an interoperon terminator-promoter overlap. Accordingly, we propose that operon concatenation is an ancient feature that restricts the potential to rearrange bacterial chromosomes and can select for the maintenance of a colinear operon organization over billions of years.

Keywords
tufA, tufB, inversion, promoter-terminator overlap
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-397120 (URN)10.1093/molbev/msz129 (DOI)000493043800012 ()31132113 (PubMedID)
Funder
Swedish Research Council, 2016-04449Swedish Research Council, 2017-03953Carl Tryggers foundation , CTS16:194Carl Tryggers foundation , CTS17:204
Available from: 2019-11-26 Created: 2019-11-26 Last updated: 2019-11-26Bibliographically approved
Szałaj, N., Lu, L., Benediktsdottir, A., Zamaratski, E., Cao, S., Olanders, G., . . . Brandt, P. (2018). Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.. European Journal of Medicinal Chemistry, 157, 1346-1360
Open this publication in new window or tab >>Boronic ester-linked macrocyclic lipopeptides as serine protease inhibitors targeting Escherichia coli type I signal peptidase.
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2018 (English)In: European Journal of Medicinal Chemistry, ISSN 0223-5234, E-ISSN 1768-3254, Vol. 157, p. 1346-1360Article in journal (Refereed) Published
Abstract [en]

Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC50 = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 μg/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing off-target effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.

Keywords
Antibacterial lipopeptides, Bacterial type I signal peptidase, Escherichia coli type I signal peptidase (EcLepB), P2–P1′ boronic ester-linked macrocycles
National Category
Medicinal Chemistry
Research subject
Infectious Diseases
Identifiers
urn:nbn:se:uu:diva-362335 (URN)10.1016/j.ejmech.2018.08.086 (DOI)000447480000096 ()30196059 (PubMedID)
Funder
Swedish Research Council, 2014-6711Swedish Research Council, 2015-05406Swedish Research Council, 2017-03953
Available from: 2018-10-03 Created: 2018-10-03 Last updated: 2018-12-11Bibliographically approved
Brandis, G., Cao, S. & Hughes, D. (2018). Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella. Molecular Microbiology, 108(6), 697-710
Open this publication in new window or tab >>Co-evolution with recombination affects the stability of mobile genetic element insertions within gene families of Salmonella
2018 (English)In: Molecular Microbiology, ISSN 0950-382X, E-ISSN 1365-2958, Vol. 108, no 6, p. 697-710Article in journal (Refereed) Published
Abstract [en]

Bacteria can have multiple copies of a gene at separate locations on the same chromosome. Some of these gene families, including tuf (translation elongation factor EF-Tu) and rrl (ribosomal RNA), encode functions critically important for bacterial fitness. Genes within these families are known to evolve in concert using homologous recombination to transfer genetic information from one gene to another. This mechanism can counteract the detrimental effects of nucleotide sequence divergence over time. Whether such mechanisms can also protect against the potentially lethal effects of mobile genetic element insertion is not well understood. To address this we constructed two different length insertion cassettes to mimic mobile genetic elements and inserted these into various positions of the tuf and rrl genes. Wemeasured rates of recombinational repair that removed the inserted cassette and studied the underlying mechanism. Our results indicate that homologous recombination can protect the tuf and rrl genes from inactivation by mobile genetic elements, but forinsertions within shorter gene sequences the efficiency of repair is very low. Intriguingly, we found that physical distance separating genes on the chromosome directly affects the rate of recombinational repair suggesting that relative location will influence the ability of homologous recombination to maintain homogeneity.

Place, publisher, year, edition, pages
WILEY, 2018
National Category
Genetics Microbiology Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-358073 (URN)10.1111/mmi.13959 (DOI)000434978300008 ()29603442 (PubMedID)
Funder
Swedish Research Council, 2016-04449Swedish Research Council, 2017-03953Carl Tryggers foundation , CTS16:194Carl Tryggers foundation , CTS17:204
Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2018-08-30Bibliographically approved
Garoff, L., Huseby, D. L., Praski Alzrigat, L. & Hughes, D. (2018). Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli. Journal of Antimicrobial Chemotherapy, 73(12), 3285-3292
Open this publication in new window or tab >>Effect of aminoacyl-tRNA synthetase mutations on susceptibility to ciprofloxacin in Escherichia coli
2018 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, no 12, p. 3285-3292Article in journal (Refereed) Published
Abstract [en]

Background: Chromosomal mutations that reduce ciprofloxacin susceptibility in Escherichia coli characteristically map to drug target genes (gyrAB and parCE), and genes encoding regulators of the AcrAB-TolC efflux pump. Mutations in RNA polymerase can also reduce susceptibility, by up-regulating the MdtK efflux pump.

Objectives: We asked whether mutations in additional chromosomal gene classes could reduce susceptibility to ciprofloxacin.

Methods: Experimental evolution, complemented by WGS analysis, was used to select and identify mutations that reduce susceptibility to ciprofloxacin. Transcriptome analysis, genetic reconstructions, susceptibility measurements and competition assays were used to identify significant genes and explore the mechanism of resistance.

Results: Mutations in three different aminoacyl-tRNA synthetase genes (leuS, aspS and thrS) were shown to re- duce susceptibility to ciprofloxacin. For two of the genes (leuS and aspS) the mechanism was partially dependent on RelA activity. Two independently selected mutations in leuS (Asp162Asn and Ser496Pro) were studied in most detail, revealing that they induce transcriptome changes similar to a stringent response, including up-regulation of three efflux-associated loci (mdtK, acrZ and ydhJK). Genetic analysis showed that reduced susceptibility depended on the activity of these loci. Broader antimicrobial susceptibility testing showed that the leuS mutations also reduce susceptibility to additional classes of antibiotics chloramphenicol, rifampicin, mecillinam, ampicillin and trimethoprim).

Conclusions: The identification of mutations in multiple tRNA synthetase genes that reduce susceptibility to ciprofloxacin and other antibiotics reveals the existence of a large mutational target that could contribute to re- sistance development by up-regulation of an array of efflux pumps.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-361197 (URN)10.1093/jac/dky356 (DOI)000452916600009 ()30239743 (PubMedID)
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449Swedish Research Council, 2017-03593
Available from: 2018-09-21 Created: 2018-09-21 Last updated: 2019-01-10Bibliographically approved
Jeannot, F., Taillier, T., Despeyroux, P., Renard, S., Rey, A., Mourez, M., . . . Bacqué, E. (2018). Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones. Journal of Medicinal Chemistry, 61(8), 3565-3581
Open this publication in new window or tab >>Imidazopyrazinones (IPYs): Non-Quinolone Bacterial Topoisomerase Inhibitors Showing Partial Cross-Resistance with Quinolones
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2018 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 61, no 8, p. 3565-3581Article in journal (Refereed) Published
Abstract [en]

In our quest for new antibiotics able to address the growing threat of multidrug resistant infections caused by Gram-negative bacteria, we have investigated an unprecedented series of non-quinolone bacterial topoisomerase inhibitors from the Sanofi patrimony, named IPYs for imidazopyrazinones, as part of the Innovative Medicines Initiative (IMI) European Gram Negative Antibacterial Engine (ENABLE) organization. Hybridization of these historical compounds with the quinazolinediones, a known series of topoisomerase inhibitors, led us to a novel series of tricyclic IPYs that demonstrated potential for broad spectrum activity, in vivo efficacy, and a good developability profile, although later profiling revealed a genotoxicity risk. Resistance studies revealed partial cross-resistance with fluoroquinolones (FQs) suggesting that IPYs bind to the same region of bacterial topoisomerases as FQs and interact with at least some of the keys residues involved in FQ binding.

National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-356096 (URN)10.1021/acs.jmedchem.7b01892 (DOI)000431151000021 ()29596745 (PubMedID)
Available from: 2018-07-19 Created: 2018-07-19 Last updated: 2018-07-19Bibliographically approved
Garoff, L., Yadav, K. & Hughes, D. (2018). Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli. Journal of Antimicrobial Chemotherapy, 73(2), 348-352
Open this publication in new window or tab >>Increased expression of Qnr is sufficient to confer clinical resistance to ciprofloxacin in Escherichia coli
2018 (English)In: Journal of Antimicrobial Chemotherapy, ISSN 0305-7453, E-ISSN 1460-2091, Vol. 73, no 2, p. 348-352Article in journal (Refereed) Published
Abstract [en]

Background: Ciprofloxacin, a fluoroquinolone, targets two essential bacterial enzymes, DNA gyrase and topoisomerase IV. Plasmid-borne qnr genes, encoding proteins that protect DNA gyrase and topoisomerase IV from inhibition by fluoroquinolones, contribute to resistance development. However, the presence of a plasmid-borne qnr gene alone is insufficient to confer clinical resistance. Objectives: We asked whether the level of expression of qnr was a limiting factor in its ability to confer clinical resistance and whether expression could be increased without reducing fitness or viability. Methods: qnrB and qnrS were recombineered onto the chromosome of Escherichia coli under the control of constitutive promoters of various strengths. Expression was measured by qPCR, MIC and relative fitness as a function of expression level were determined. Results: For both qnr genes there was a positive relationship between the level of qnr mRNA and the MIC of ciprofloxacin. The highest MICs achieved with qnrB or qnrS as the sole resistance determinant were 0.375 and 1 mg/L, respectively, and were reached at expression levels that did not affect growth rate or viability. The qnrS-mediated MIC is above the EUCAST clinical breakpoint for resistance to ciprofloxacin. In the absence of Lon protease activity, overexpression of qnr genes was associated with high fitness cost, possibly explaining observations of toxicity in other genetic backgrounds. Conclusions: The ability to generate a high MIC without incurring a fitness cost shows that, in an appropriate genetic context, qnrS has the potential to generate clinical resistance to ciprofloxacin in one step.

National Category
Microbiology
Identifiers
urn:nbn:se:uu:diva-361199 (URN)10.1093/jac/dkx375 (DOI)000424144300010 ()29106520 (PubMedID)1460-2091 (Electronic) 0305-7453 (Linking) (ISBN)
Funder
Swedish Research Council, 2013-02904Swedish Research Council, 2016-04449
Available from: 2018-09-21 Created: 2018-09-21 Last updated: 2019-06-24Bibliographically approved
Brandis, G. & Hughes, D. (2018). Mechanisms of fitness cost reduction for rifampicin-resistant strains with deletion or duplication mutations in rpoB.. Scientific Reports, 8, Article ID 17488.
Open this publication in new window or tab >>Mechanisms of fitness cost reduction for rifampicin-resistant strains with deletion or duplication mutations in rpoB.
2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 17488Article in journal (Refereed) Published
Abstract [en]

Rifampicin resistance (Rif(R)) is caused by mutations in rpoB, encoding the beta-subunit of RNA polymerase. Rif(R )mutations generally incur a fitness cost and in resistant isolates are frequently accompanied by compensatory mutations in rpoA, rpoB or rpoC. Previous studies of fitness compensation focused on Rif(R) caused by amino acid substitutions within rpoB. Rif(R) is also caused by deletion and duplication mutations in rpoB but it is not known whether or how such mutants can ameliorate their fitness costs. Using experimental evolution of Salmonella carrying Rif(R) deletion or duplication mutations we identified compensatory amino acid substitution mutations within rpoA, rpoB or rpoC in 16 of 21 evolved lineages. Additionally, we found one lineage where a large deletion was compensated by duplication of adjacent amino acids (possibly to fill the gap within the protein structure), two lineages where mutations occurred outside of rpoABC, and two lineages where a duplication mutant reverted to the wild-type sequence. All but the two revertant mutants maintained the Rif(R) phenotype. These data suggest that amino acid substitution mutations are the major compensatory mechanism regardless of the nature of the primary Rif(R) mutation.

National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-371257 (URN)10.1038/s41598-018-36005-y (DOI)000451748700010 ()30504835 (PubMedID)
Funder
Swedish Research Council, 2016-04449Swedish Research Council, 2017-03953Carl Tryggers foundation , CTS16:194Carl Tryggers foundation , CTS17:204
Available from: 2018-12-20 Created: 2018-12-20 Last updated: 2019-01-07Bibliographically approved
Huseby, D. L. & Hughes, D. (2018). Methods to determine mutational trajectories after experimental evolution of antibiotic resistance.. Methods in Molecular Biology, 1736, 95-103
Open this publication in new window or tab >>Methods to determine mutational trajectories after experimental evolution of antibiotic resistance.
2018 (English)In: Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029, Vol. 1736, p. 95-103Article in journal (Refereed) Published
Abstract [en]

The evolution of bacterial resistance to antibiotics by mutation within the genome (as distinct from horizontal gene transfer of new material into a genome) could occur in a single step but is usually a multistep process. Resistance evolution can be studied in laboratory environments by serial passage of bacteria in liquid culture or on agar, with selection at constant, or varying, concentrations of drug. Whole genome sequencing can be used to make an initial analysis of the evolved mutants. The trajectory of evolution can be determined by sequence analysis of strains from intermediate steps in the evolution, complemented by phenotypic analysis of genetically reconstructed isogenic strains that recapitulate the intermediate steps in the evolution.

Place, publisher, year, edition, pages
New York: Humana Press, 2018
National Category
Microbiology in the medical area
Identifiers
urn:nbn:se:uu:diva-371259 (URN)10.1007/978-1-4939-7638-6_9 (DOI)
Available from: 2018-12-20 Created: 2018-12-20 Last updated: 2019-04-26Bibliographically approved
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