Development of evolution drugs - antibacterial compounds that block pathways to resistance

AbstractAntibiotic resistance is a worldwide challenge. A potential approach to block resistance is to simultaneously inhibit WT and known escape variants of the target bacterial protein. Here we applied an integrated computational and experimental approach to discover compounds that inhibit both WT and trimethoprim (TMP) resistant mutants of E. coli dihydrofolate reductase (DHFR). We identified a novel compound (CD15-3) that inhibits WT DHFR and its TMP resistant variants L28R, P21L and A26T with IC50 50-75 μM against WT and TMP-resistant strains. Resistance to CD15-3 was dramatically delayed compared to TMP in in vitro evolution. Whole genome sequencing of CD15-3 resistant strains showed no mutations in the target folA locus. Rather, gene duplication of several efflux pumps gave rise to weak (about twofold increase in IC50) resistance against CD15-3. Altogether, our results demonstrate the promise of strategy to develop evolution drugs - compounds which block evolutionary escape routes in pathogens.

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Development of antibacterial compounds that constrain evolutionary pathways to resistance

eLife ◽

10.7554/elife.64518 ◽

2021 ◽

Vol 10 ◽

Author(s):

Yanmin Zhang ◽

Sourav Chowdhury ◽

João V Rodrigues ◽

Eugene I Shakhnovich

Keyword(s):

Dihydrofolate Reductase ◽

Experimental Approach ◽

Whole Genome ◽

Bacterial Protein ◽

Antibacterial Compounds ◽

E Coli ◽

Resistant Strains ◽

Evolutionary Pathways ◽

Resistant Mutants

Antibiotic resistance is a worldwide challenge. A potential approach to block resistance is to simultaneously inhibit WT and known escape variants of the target bacterial protein. Here we applied an integrated computational and experimental approach to discover compounds that inhibit both WT and trimethoprim (TMP) resistant mutants of E. coli dihydrofolate reductase (DHFR). We identified a novel compound (CD15-3) that inhibits WT DHFR and its TMP resistant variants L28R, P21L and A26T with IC50 50-75 µM against WT and TMP-resistant strains. Resistance to CD15-3 was dramatically delayed compared to TMP in in vitro evolution. Whole genome sequencing of CD15-3 resistant strains showed no mutations in the target folA locus. Rather, gene duplication of several efflux pumps gave rise to weak (about twofold increase in IC50) resistance against CD15-3. Altogether, our results demonstrate the promise of strategy to develop evolution drugs - compounds which constrain evolutionary escape routes in pathogens.

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Precise prediction of antibiotic resistance in Escherichia coli from full genome sequences

10.1101/338194 ◽

2018 ◽

Cited By ~ 2

Author(s):

Danesh Moradigaravand ◽

Martin Palm ◽

Anne Farewell ◽

Ville Mustonen ◽

Jonas Warringer ◽

...

Keyword(s):

Machine Learning ◽

Antibiotic Resistance ◽

Population Structure ◽

Genome Sequencing ◽

Diagnostic Tools ◽

Clinical Settings ◽

Whole Genome ◽

Genome Sequences ◽

E Coli ◽

Resistant Strains

AbstractThe emergence of microbial antibiotic resistance is a global health threat. In clinical settings, the key to controlling spread of resistant strains is accurate and rapid detection. As traditional culture-based methods are time consuming, genetic approaches have recently been developed for this task. The diagnosis is typically made by measuring a few known determinants previously identified from whole genome sequencing, and thus is restricted to existing information on biological mechanisms. To overcome this limitation, we employed machine learning models to predict resistance to 11 compounds across four classes of antibiotics from existing and novel whole genome sequences of 1936 E. coli strains. We considered a range of methods, and examined population structure, isolation year, gene content, and polymorphism information as predictors. Gradient boosted decision trees consistently outperformed alternative models with an average F1 score of 0.88 on held-out data (range 0.66-0.96). While the best models most frequently employed all inputs, an average F1 score of 0.73 could be obtained using population structure information alone. Single nucleotide variation data were less useful, and failed to improve prediction for ten out of 11 antibiotics. These results demonstrate that antibiotic resistance in E. coli can be accurately predicted from whole genome sequences without a priori knowledge of mechanisms, and that both genomic and epidemiological data are informative. This paves way to integrating machine learning approaches into diagnostic tools in the clinic.SummaryOne of the major health threats of 21st century is emergence of antibiotic resistance. To manage its economic impact, efforts are made to develop novel diagnostic tools that rapidly detect resistant strains in clinical settings. In our study, we employed a range machine learning tools to predict antibiotic resistance from whole genome sequencing data for E. coli. We used the presence or absence of genes, population structure and isolation year of isolates as predictors, and could attain average precision of 0.93 and recall of 0.83, without prior knowledge about the causal mechanisms. These results demonstrate the potential application of machine learning methods as a diagnostic tool in healthcare settings.

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Sub-lethal Concentrations of Phytochemicals (Carvacrol and Oregano) Select for Reduced Susceptibility Mutants of Escherichia coli O23:H52

Polish Journal of Microbiology ◽

10.33073/pjm-2020-003 ◽

2020 ◽

Vol 69 (1) ◽

pp. 121-125

Author(s):

AFNAN A. AL-MNASER ◽

MARTIN J. WOODWARD

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Coli Strain ◽

Whole Genome ◽

Avian Pathogenic Escherichia Coli ◽

Antibiotic Resistant ◽

E Coli ◽

Pathogenic Escherichia Coli ◽

Survival Mechanisms

In vitro studies aimed at studying the mechanism of action of carvacrol and oregano as natural anti-bacterial agents to control multiple antibiotic-resistant avian pathogenic Escherichia coli (APEC) strain O23:H52 isolated from chicken were performed. Derivatives with increased minimum inhibitory concentrations (MIC) to the phytochemicals were selected after growing Escherichia coli (E. coli) strain O23:H52 at sub-lethal concentrations of carvacrol and oregano for a period of 60 days. Whole-genome sequencing (WGS) of two derivatives revealed a missense mutation in cadC and marR: the genes responsible for survival mechanisms and antibiotic resistance by efflux, respectively.

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Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02665-16 ◽

2017 ◽

Vol 61 (5) ◽

Cited By ~ 11

Author(s):

Jacynthe L. Toulouse ◽

Thaddeus J. Edens ◽

Lorea Alejaldre ◽

Amee R. Manges ◽

Joelle N. Pelletier

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Dihydrofolate Reductase ◽

Multiple Resistance ◽

Whole Genome ◽

Type Ii ◽

Content Type ◽

E Coli ◽

Reductase Gene ◽

Multiple Resistance Genes

ABSTRACT Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli. Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.

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Switching an active site helix in dihydrofolate reductase reveals limits to sub-domain modularity

10.1101/2021.06.18.448971 ◽

2021 ◽

Author(s):

Victor Y. Zhao ◽

João V. Rodrigues ◽

Elena R. Lozovsky ◽

Daniel L. Hartl ◽

Eugene I. Shakhnovich

Keyword(s):

Catalytic Activity ◽

Dihydrofolate Reductase ◽

Strongly Correlated ◽

Thermal Stabilities ◽

E Coli ◽

In Vitro Measurements ◽

Flux Model ◽

Α Helix ◽

Dynamics Simulations

To what degree are individual structural elements within proteins modular such that similar structures from unrelated proteins can be interchanged? We study sub-domain modularity by creating 20 chimeras of an enzyme, E. coli dihydrofolate reductase (DHFR), in which a catalytically important, 10-residue α-helical sequence is replaced by α-helical sequences from a diverse set of proteins. The chimeras stably fold but have a range of diminished thermal stabilities and catalytic activities. Evolutionary coupling analysis indicates that the residues of this α-helix are under selection pressure to maintain catalytic activity in DHFR. We performed molecular dynamics simulations using replica exchange with solute-tempering. Chimeras with low catalytic activity exhibit non-helical conformations that block the binding site and disrupt the positioning of the catalytically essential residue D27. Simulation observables and in vitro measurements of thermal stability and substrate binding affinity are strongly correlated. Several E. coli strains with chromosomally integrated chimeric DHFRs can grow, with growth rates that follow predictions from a kinetic flux model that depends on the intracellular abundance and catalytic activity of DHFR. Our findings show that although α-helices are not universally substitutable, the molecular and fitness effects of modular segments can be predicted by the biophysical compatibility of the replacement segment.

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Mutations Conferring Aminoglycoside and Spectinomycin Resistance in Borrelia burgdorferi

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.50.2.445-452.2006 ◽

2006 ◽

Vol 50 (2) ◽

pp. 445-452 ◽

Cited By ~ 39

Author(s):

Daniel Criswell ◽

Virginia L. Tobiason ◽

J. Stephen Lodmell ◽

D. Scott Samuels

Keyword(s):

16S Rrna ◽

Borrelia Burgdorferi ◽

Type Strain ◽

Wild Type Strain ◽

Small Subunit ◽

Wild Type ◽

Spectinomycin Resistance ◽

E Coli ◽

Resistant Mutants

ABSTRACT We have isolated and characterized in vitro mutants of the Lyme disease agent Borrelia burgdorferi that are resistant to spectinomycin, kanamycin, gentamicin, or streptomycin, antibiotics that target the small subunit of the ribosome. 16S rRNA mutations A1185G and C1186U, homologous to Escherichia coli nucleotides A1191 and C1192, conferred >2,200-fold and 1,300-fold resistance to spectinomycin, respectively. A 16S rRNA A1402G mutation, homologous to E. coli A1408, conferred >90-fold resistance to kanamycin and >240-fold resistance to gentamicin. Two mutations were identified in the gene for ribosomal protein S12, at a site homologous to E. coli residue Lys-87, in mutants selected in streptomycin. Substitutions at codon 88, K88R and K88E, conferred 7-fold resistance and 10-fold resistance, respectively, to streptomycin on B. burgdorferi. The 16S rRNA A1185G and C1186U mutations, associated with spectinomycin resistance, appeared in a population of B. burgdorferi parental strain B31 at a high frequency of 6 × 10−6. These spectinomycin-resistant mutants successfully competed with the wild-type strain during 100 generations of coculture in vitro. The aminoglycoside-resistant mutants appeared at a frequency of 3 × 10−9 to 1 ×10−7 in a population and were unable to compete with wild-type strain B31 after 100 generations. This is the first description of mutations in the B. burgdorferi ribosome that confer resistance to antibiotics. These results have implications for the evolution of antibiotic resistance, because the 16S rRNA mutations conferring spectinomycin resistance have no significant fitness cost in vitro, and for the development of new selectable markers.

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Antibacterial activities of epiroprim, a new dihydrofolate reductase inhibitor, alone and in combination with dapsone.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.6.1376 ◽

1996 ◽

Vol 40 (6) ◽

pp. 1376-1381 ◽

Cited By ~ 35

Author(s):

H H Locher ◽

H Schlunegger ◽

P G Hartman ◽

P Angehrn ◽

R L Then

Keyword(s):

Dihydrofolate Reductase ◽

Biological Fluids ◽

Pneumocystis Carinii ◽

Antibacterial Activities ◽

Gram Negative Bacteria ◽

Highly Active ◽

Excellent Activity ◽

Resistant Strains ◽

Better Than

Epiroprim (EPM; Ro 11-8958) is a new selective inhibitor of microbial dihydrofolate reductase. EPM displayed excellent activity against staphylococci, enterococci, pneumococci, and streptococci which was considerably better than that of trimethoprim (TMP). EPM was also active against TMP-resistant strains, although the MICs were still relatively high. Its combination with dapsone (DDS) was synergistic and showed as in vitro activity superior to that of the TMP combination with sulfamethoxazole (SMZ). The EPM-DDS (ratio, 1:19) combination inhibited more than 90% of all important gram-positive pathogens at a concentration of 2 + 38 micrograms/ml. Only a few highly TMP-resistant staphylococci and enterococci were not inhibited. EPM was also more active than TMP against Moraxella catarrhalis, Neisseria meningitidis, and Bacteroides spp., but it was less active than TMP against all other gram-negative bacteria tested. Atypical mycobacteria were poorly susceptible to EPM, but the combination with DDS was synergistic and active at concentrations most probably achievable in biological fluids (MICs from 0.25 +/- 4.75 to 4 + 76 micrograms/ml). EPM and the EPM-DDS combination were also highly active against experimental staphylococcal infections in a mouse septicemia model. The combination EPM-DDS has previously been shown to exhibit activity in Pneumocystis carinii and Toxoplasma models and, as shown in the present study, also shows good activity against a broad range of bacteria including many strains resistant to TMP and TMP-SMZ.

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In Vitro Activity of a Novel Siderophore-Cephalosporin, GT-1 and Serine-Type β-Lactamase Inhibitor, GT-055, against Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. Panel Strains

Antibiotics ◽

10.3390/antibiotics9050267 ◽

2020 ◽

Vol 9 (5) ◽

pp. 267 ◽

Cited By ~ 3

Author(s):

Le Phuong Nguyen ◽

Naina Adren Pinto ◽

Thao Nguyen Vu ◽

Hyunsook Lee ◽

Young Lag Cho ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Vitro Activity ◽

Intrinsic Activity ◽

In Vitro Activity ◽

E Coli ◽

Acinetobacter Spp ◽

Resistant Strains ◽

Lactamase Inhibitor

This study investigates GT-1 (also known as LCB10-0200), a novel-siderophore cephalosporin, inhibited multidrug-resistant (MDR) Gram-negative pathogen, via a Trojan horse strategy exploiting iron-uptake systems. We investigated GT-1 activity and the role of siderophore uptake systems, and the combination of GT-1 and a non-β-lactam β-lactamase inhibitor (BLI) of diazabicyclooctane, GT-055, (also referred to as LCB18-055) against molecularly characterised resistant Escherichia coli, Klebsiella pneumoniae and Acinetobacter spp. isolates. GT-1 and GT-1/GT-055 were tested in vitro against comparators among three different characterised panel strain sets. Bacterial resistome and siderophore uptake systems were characterised to elucidate the genetic basis for GT-1 minimum inhibitory concentrations (MICs). GT-1 exhibited in vitro activity (≤2 μg/mL MICs) against many MDR isolates, including extended-spectrum β-lactamase (ESBL)- and carbapenemase-producing E. coli and K. pneumoniae and oxacillinase (OXA)-producing Acinetobacter spp. GT-1 also inhibited strains with mutated siderophore transporters and porins. Although BLI GT-055 exhibited intrinsic activity (MIC 2–8 μg/mL) against most E. coli and K. pneumoniae isolates, GT-055 enhanced the activity of GT-1 against many GT-1–resistant strains. Compared with CAZ-AVI, GT-1/GT-055 exhibited lower MICs against E. coli and K. pneumoniae isolates. GT-1 demonstrated potent in vitro activity against clinical panel strains of E. coli, K. pneumoniae and Acinetobacter spp. GT-055 enhanced the in vitro activity of GT-1 against many GT-1–resistant strains.

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In Vitro and In Vivo Antibacterial Activities of SM-216601, a New Broad-Spectrum Parenteral Carbapenem

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.10.4185-4196.2005 ◽

2005 ◽

Vol 49 (10) ◽

pp. 4185-4196 ◽

Cited By ~ 27

Author(s):

Yutaka Ueda ◽

Katsunori Kanazawa ◽

Ken Eguchi ◽

Koji Takemoto ◽

Yoshiro Eriguchi ◽

...

Keyword(s):

Bacterial Infections ◽

Antibacterial Activities ◽

Multiresistant Pathogens ◽

E Coli ◽

Wide Range ◽

Agar Dilution ◽

Resistant Strains ◽

Mrsa Strains

ABSTRACT SM-216601 is a novel parenteral 1β-methylcarbapenem. In agar dilution susceptibility testing, the MIC of SM-216601 for 90% of the methicillin-resistant Staphylococcus aureus (MRSA) strains tested (MIC90) was 2 μg/ml, which was comparable to those of vancomycin and linezolid. SM-216601 was also very potent against Enterococcus faecium, including vancomycin-resistant strains (MIC90 = 8 μg/ml). SM-216601 exhibited potent activity against penicillin-resistant Streptococcus pneumoniae, ampicillin-resistant Haemophilus influenzae, Moraxella catarrhalis, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis, with MIC90s of less than 0.5 μg/ml, and intermediate activity against Citrobacter freundii, Enterobacter cloacae, Serratia marcescens, and Pseudomonas aeruginosa. The therapeutic efficacy of SM-216601 against experimentally induced infections in mice caused by S. aureus, E. faecium, E. coli, and P. aeruginosa reflected its in vitro activity and plasma level. Thus, SM-216601 is a promising candidate for nosocomial bacterial infections caused by a wide range of gram-positive and gram-negative bacteria, including multiresistant pathogens.

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