scholarly journals Compensatory mutations modulate the competitiveness and dynamics of plasmid-mediated colistin resistance in Escherichia coli clones

2020 ◽  
Vol 14 (3) ◽  
pp. 861-865 ◽  
Author(s):  
Qiu E. Yang ◽  
Craig MacLean ◽  
Andrei Papkou ◽  
Manon Pritchard ◽  
Lydia Powell ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Fitness Landscape ◽  
Fitness Cost ◽  
Colistin Resistance ◽  
Bacterial Populations ◽  
Compensatory Evolution ◽  
Neutral Mutations ◽  
The Cost ◽  
Negative Epistasis

AbstractThe emergence of mobile colistin resistance (mcr) threatens to undermine the clinical efficacy of the last antibiotic that can be used to treat serious infections caused by Gram-negative pathogens. Here we measure the fitness cost of a newly discovered MCR-3 using in vitro growth and competition assays. mcr-3 expression confers a lower fitness cost than mcr-1, as determined by competitive ability and cell viability. Consistent with these findings, plasmids carrying mcr-3 have higher stability than mcr-1 plasmids across a range of Escherichia coli strains. Crucially, mcr-3 plasmids can stably persist, even in the absence of colistin. Recent compensatory evolution has helped to offset the cost of mcr-3 expression, as demonstrated by the high fitness of mcr-3.5 as opposed to mcr-3.1. Reconstructing all of the possible evolutionary trajectories from mcr-3.1 to mcr-3.5 reveals a complex fitness landscape shaped by negative epistasis between compensatory and neutral mutations. Our findings highlight the importance of fitness costs and compensatory evolution in driving the dynamics and stability of mobile colistin resistance in bacterial populations, and they highlight the need to understand how processes (other than colistin use) impact mcr dynamics.

scholarly journals Resistance/fitness trade-off is a barrier to the evolution of MarR inactivation mutants in Escherichia coli

2020 ◽  
Vol 76 (1) ◽  
pp. 77-83 ◽  
Author(s):  
Lisa Praski Alzrigat ◽  
Douglas L Huseby ◽  
Gerrit Brandis ◽  
Diarmaid Hughes
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Efflux Pump ◽  
Drug Susceptibility ◽  
Fitness Cost ◽  
Competitive Growth ◽  
Contributing Factors ◽  
Trade Off ◽  
Compensatory Evolution ◽  
The Cost

Abstract Background Mutations that inactivate MarR reduce susceptibility to ciprofloxacin and competitive growth fitness in Escherichia coli. Both phenotypes are caused by overexpression of the MarA regulon, which includes the AcrAB-TolC drug efflux pump. Objectives We asked whether compensatory evolution could reduce the fitness cost of MarR-inactivating mutations without affecting resistance to ciprofloxacin. Methods The cost of overexpressing the AcrAB-TolC efflux pump was measured independently of MarA overexpression. Experimental evolution of MarR-inactive strains was used to select mutants with increased fitness. The acquired mutations were identified and their effects on drug susceptibility were measured. Results Overexpression of the AcrAB-TolC efflux pump was found not to contribute to the fitness cost of MarA regulon overexpression. Fitness-compensatory mutations were selected in marA and lon. The mutations reduced the level of MarA protein thus reducing expression of the MarA regulon. They restored growth fitness but also reduced resistance to ciprofloxacin. Conclusions The fitness cost caused by overexpression of the MarA regulon has multiple contributing factors. Experimental evolution did not identify any single pump-independent cost factor. Instead, efficient fitness compensation occurred only by mechanisms that reduce MarA concentration, which simultaneously reduce the drug resistance phenotype. This resistance/fitness trade-off is a barrier to the successful spread of MarR inactivation mutations in clinical isolates where growth fitness is essential.

scholarly journals Alleviation of C⋅C Mismatches in DNA by the Escherichia coli Fpg Protein

2021 ◽  
Vol 12 ◽  
Author(s):  
Almaz Nigatu Tesfahun ◽  
Marina Alexeeva ◽  
Miglė Tomkuvienė ◽  
Aysha Arshad ◽  
Prashanna Guragain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Excision Repair ◽  
Dna Lesions ◽  
Base Pairs ◽  
E Coli ◽  
Thymine Glycol ◽  
Base Excision ◽  
The Cost ◽  
Primary Substrate

DNA polymerase III mis-insertion may, where not corrected by its 3′→ 5′ exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The kcat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The KM values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

scholarly journals In Vivo Bioluminescence Imaging for the Study of Intestinal Colonization by Escherichia coli in Mice

2009 ◽  
Vol 76 (1) ◽  
pp. 264-274 ◽  
Author(s):  
M.-L. Foucault ◽  
L. Thomas ◽  
S. Goussard ◽  
B. R. Branchini ◽  
C. Grillot-Courvalin
Keyword(s):  
Escherichia Coli ◽  
Bioluminescence Imaging ◽  
Light Emission ◽  
Direct Method ◽  
Firefly Luciferase ◽  
Intestinal Colonization ◽  
Bacterial Populations ◽  
Bioluminescence Signal

ABSTRACT Bioluminescence imaging (BLI) is emerging as a powerful tool for real-time monitoring of infections in living animals. However, since luciferases are oxygenases, it has been suggested that the requirement for oxygen may limit the use of BLI in anaerobic environments, such as the lumen of the gut. Strains of Escherichia coli harboring the genes for either the bacterial luciferase from Photorhabdus luminescens or the PpyRE-TS and PpyGR-TS firefly luciferase mutants of Photinus pyralis (red and green thermostable P. pyralis luciferase mutants, respectively) have been engineered and used to monitor intestinal colonization in the streptomycin-treated mouse model. There was excellent correlation between the bioluminescence signal measured in the feces (R 2 = 0.98) or transcutaneously in the abdominal region of whole animals (R 2 = 0.99) and the CFU counts in the feces of bacteria harboring the luxABCDE operon. Stability in vivo of the bioluminescence signal was achieved by constructing plasmid pAT881(pGB2ΩPamiluxABCDE), which allowed long-term monitoring of intestinal colonization without the need for antibiotic selection for plasmid maintenance. Levels of intestinal colonization by various strains of E. coli could be compared directly by simple recording of the bioluminescence signal in living animals. The difference in spectra of light emission of the PpyRE-TS and PpyGR-TS firefly luciferase mutants and dual bioluminescence detection allowed direct in vitro and in vivo quantification of two bacterial populations by measurement of red and green emitted signals and thus monitoring of the two populations simultaneously. This system offers a simple and direct method to study in vitro and in vivo competition between mutants and the parental strain. BLI is a useful tool to study intestinal colonization.

scholarly journals Advantage of the F2:A1:B- IncF Pandemic Plasmid over IncC Plasmids in In Vitro Acquisition and Evolution of blaCTX-M Gene-Bearing Plasmids in Escherichia coli

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Anne-Claire Mahérault ◽  
Harry Kemble ◽  
Mélanie Magnan ◽  
Benoit Gachet ◽  
David Roche ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Experimental Evolution ◽  
Negative Impact ◽  
Fitness Cost ◽  
M Gene ◽  
Content Type ◽  
E Coli ◽  
Conjugative Plasmids ◽  
K 12

ABSTRACT Despite a fitness cost imposed on bacterial hosts, large conjugative plasmids play a key role in the diffusion of resistance determinants, such as CTX-M extended-spectrum β-lactamases. Among the large conjugative plasmids, IncF plasmids are the most predominant group, and an F2:A1:B- IncF-type plasmid encoding a CTX-M-15 variant was recently described as being strongly associated with the emerging worldwide Escherichia coli sequence type 131 (ST131)-O25b:H4 H30Rx/C2 sublineage. In this context, we investigated the fitness cost of narrow-range F-type plasmids, including the F2:A1:B- IncF-type CTX-M-15 plasmid, and of broad-range C-type plasmids in the K-12-like J53-2 E. coli strain. Although all plasmids imposed a significant fitness cost to the bacterial host immediately after conjugation, we show, using an experimental-evolution approach, that a negative impact on the fitness of the host strain was maintained throughout 1,120 generations with the IncC-IncR plasmid, regardless of the presence or absence of cefotaxime, in contrast to the F2:A1:B- IncF plasmid, whose cost was alleviated. Many chromosomal and plasmid rearrangements were detected after conjugation in transconjugants carrying the IncC plasmids but not in transconjugants carrying the F2:A1:B- IncF plasmid, except for insertion sequence (IS) mobilization from the fliM gene leading to the restoration of motility of the recipient strains. Only a few mutations occurred on the chromosome of each transconjugant throughout the experimental-evolution assay. Our findings indicate that the F2:A1:B- IncF CTX-M-15 plasmid is well adapted to the E. coli strain studied, contrary to the IncC-IncR CTX-M-15 plasmid, and that such plasmid-host adaptation could participate in the evolutionary success of the CTX-M-15-producing pandemic E. coli ST131-O25b:H4 lineage.

scholarly journals Quercetin Rejuvenates Sensitization of Colistin-Resistant Escherichia coli and Klebsiella Pneumoniae Clinical Isolates to Colistin

2021 ◽  
Vol 9 ◽  
Author(s):  
Yishuai Lin ◽  
Ying Zhang ◽  
Shixing Liu ◽  
Dandan Ye ◽  
Liqiong Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Alternative Pathway ◽  
Membrane Damage ◽  
New Drugs ◽  
Colistin Resistance ◽  
Cell Membrane Damage ◽  
E Coli

Colistin is being considered as “the last ditch” treatment in many infections caused by Gram-negative stains. However, colistin is becoming increasingly invalid in treating patients who are infected with colistin-resistant Escherichia coli (E. coli) and Klebsiella Pneumoniae (K. pneumoniae). To cope with the continuous emergence of colistin resistance, the development of new drugs and therapies is highly imminent. Herein, in this work, we surprisingly found that the combination of quercetin with colistin could efficiently and synergistically eradicate the colistin-resistant E. coli and K. pneumoniae, as confirmed by the synergy checkboard and time-kill assay. Mechanismly, the treatment of quercetin combined with colistin could significantly downregulate the expression of mcr-1 and mgrB that are responsible for colistin-resistance, synergistically enhancing the bacterial cell membrane damage efficacy of colistin. The colistin/quercetin combination was notably efficient in eradicating the colistin-resistant E. coli and K. pneumoniae both in vitro and in vivo. Therefore, our results may provide an efficient alternative pathway against colistin-resistant E. coli and K. pneumoniae infections.

scholarly journals PixR, a novel activator of conjugative transfer of IncX4 resistance plasmids, mitigates the fitness cost of mcr-1 carriage in Escherichia coli

2021 ◽  
Author(s):  
Lingxian Yi ◽  
Romain Durand ◽  
Frédéric Grenier ◽  
Jun Yang ◽  
Kaiyang Yu ◽  
...  
Keyword(s):  
Transcriptional Regulator ◽  
Fitness Cost ◽  
Host Bacterium ◽  
Regulator Gene ◽  
Rna Seq ◽  
Colistin Resistance ◽  
Bacterial Populations ◽  
Transfer Genes ◽  
Resistance Plasmids ◽  
Competition Assays

The emergence of the plasmid-borne colistin resistance gene mcr-1 threats public health. IncX4-type plasmids are one of the most epidemiologically successful vehicles for spreading mcr-1 worldwide. Since MCR-1 is known for imposing a fitness cost to its host bacterium, the successful spread of mcr-1-bearing plasmids might be linked to high conjugation frequency, which would enhance the maintenance of the plasmid in the host without antibiotic selection. However, the mechanism of IncX4 plasmids conjugation remains unclear. In this study, we used high-density transposon mutagenesis to identify factors required for IncX4 plasmid transfer and 18 genes were identified, including five with annotations unrelated to conjugation. The Cappable-seq and RNA-seq analysis confirmed that a novel transcriptional regulator gene, pixR, directly regulates the transfer of IncX4 plasmids by binding the promoter of 13 essential transfer genes to increase their transcription. Plasmid invasion and co-culture competition assays revealed that pixR is essential for the spread and persistence of mcr-1>-bearing IncX4 plasmids in bacterial populations, and effective conjugation is crucial for alleviating the fitness cost exerted by mcr-1 carriage. The existence of the IncX4-specific pixR gene increases plasmid transmissibility while promoting the invasion and persistence of mcr-1-bearing plasmids in bacterial populations, which helps explain their global prevalence.

scholarly journals Conjugal Transfer, Whole-Genome Sequencing, and Plasmid Analysis of Four mcr-1-Bearing Isolates from U.S. Patients

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Wenming Zhu ◽  
Adrian Lawsin ◽  
Rebecca L. Lindsey ◽  
Dhwani Batra ◽  
Kristen Knipe ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Resistance ◽  
Genome Sequencing ◽  
Resistance Genes ◽  
Whole Genome ◽  
Colistin Resistance ◽  
Content Type ◽  
Plasmid Analysis ◽  
Antimicrobial Resistance Genes

ABSTRACT Four Enterobacteriaceae clinical isolates bearing mcr-1 gene-harboring plasmids were characterized. All isolates demonstrated the ability to transfer colistin resistance to Escherichia coli; plasmids were stable in conjugants after multiple passages on nonselective media. mcr-1 was located on an IncX4 (n = 3) or IncN (n = 1) plasmid. The IncN plasmid harbored 13 additional antimicrobial resistance genes. Results indicate that the mcr-1-bearing plasmids in this study were highly transferable in vitro and stable in the recipients.

scholarly journals Activity of Tigecycline or Colistin in Combination with Zidovudine against Escherichia coli Harboring tet(X) and mcr-1

2020 ◽  
Vol 65 (1) ◽  
pp. e01172-20 ◽  
Author(s):  
Yu-Feng Zhou ◽  
Ping Liu ◽  
Shu-He Dai ◽  
Jian Sun ◽  
Ya-Hong Liu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistant ◽  
Therapeutic Options ◽  
Infection Model ◽  
Synergistic Activity ◽  
Colistin Resistance ◽  
Content Type ◽  
E Coli ◽  
Antiretroviral Drug

ABSTRACTAlternative therapeutic options are urgently needed against multidrug-resistant Escherichia coli infections, especially in situations of preexisting tigecycline and colistin resistance. Here, we investigated synergistic activity of the antiretroviral drug zidovudine in combination with tigecycline or colistin against E. coli harboring tet(X) and mcr-1 in vitro and in a murine thigh infection model. Zidovudine and tigecycline/colistin combinations achieved synergistic killing and significantly decreased bacterial burdens by >2.5-log10 CFU/g in thigh tissues compared to each monotherapy.

Utilizing in vitro DNA assembly to engineer a synthetic T7 Nanoluc reporter phage for Escherichia coli detection

2019 ◽  
Vol 11 (3) ◽  
pp. 63-68 ◽  
Author(s):  
Elsi M Pulkkinen ◽  
Troy C Hinkley ◽  
Sam R Nugen
Keyword(s):  
Escherichia Coli ◽  
Liquid Culture ◽  
Detection Methods ◽  
Luciferase Expression ◽  
Dna Assembly ◽  
Bacterial Populations ◽  
Low Concentrations ◽  
Health And Disease ◽  
T7 Phage

Abstract Bacteria have major role in regulating human health and disease, therefore, there is a continuing need to develop new detection methods and therapeutics to combat them. Bacteriophages can be used to infect specific bacteria, which make them good candidates for detecting and editing bacterial populations. However, creating phage-based detection assays is somewhat limited by the difficulties in the engineering of phages. We present here a synthetic biology strategy to engineer phages using a simple in vitro method. We used this method to insert a NanoLuc luciferase expression cassette into the T7 phage, in order to construct the NRGp6 reporter phage. The synthetic NRGp6 phage was used to efficiently detect low concentrations of Escherichia coli from liquid culture. We envision that our approach will benefit synthetic biologists for constructing different kinds of engineered phages, and enable new approaches for phage-based therapeutics and diagnostics.

scholarly journals Fitness Cost and Compensation Mechanism of Sulfonamide Resistance Genes (Sul1, Sul2, and Sul3) in Escherichia Coli

2021 ◽  
Author(s):  
Yuqiao Zhou ◽  
Jiehong Fang ◽  
Zaeim Davood ◽  
Daofeng Qu ◽  
Jianzhong Han
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Fitness Cost ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Compensation Mechanism ◽  
Compensatory Evolution ◽  
Differential Proteins ◽  
Sulfonamide Resistance ◽  
Sulfonamide Resistance Gene

Abstract Background: The fitness cost of antibiotic resistance is a crucial factor to determine the evolutionary success of resistant bacteria. Even if the selection pressure in the environment is eliminated, drug-resistant bacteria can still compensate for drug-resistant genes' fitness cost through some compensation mechanisms. The fitness cost and compensatory evolution of antibiotic resistance are an essential part of bacterial evolution.Result: Engineered bacteria with the same genetic background that carry sulfonamide resistance gene were generated to explore the fitness cost of sulfonamide resistance gene in Escherichia coli. There were significant differences in the protein expression of the two-component system pathway (fliZ, fliA, fliC and lrhA), folate biosynthesis pathway (sul1, sul2 and sul3), ABC transporter system (ugpC, rbsA and gsiA), and outer membrane pore protein OmpD through the comparative analysis of differential proteins compared to sensitive bacteria. Thus, we could speculate the possible fitness compensation mechanism. Finally, qRT-PCR was used to verify the functions of some differential proteins at the transcriptional level.Conclusions: The study of fitness cost assessment and compensatory evolution of bacterial resistance will help understand the development track of antibiotic resistance of bacterial pathogens and provide new ideas for solving antibiotic resistance issues.

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