scholarly journals Potentiating antibiotic efficacy via perturbation of non-essential gene expression

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Peter B. Otoupal ◽  
Kristen A. Eller ◽  
Keesha E. Erickson ◽  
Jocelyn Campos ◽  
Thomas R. Aunins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Essential Gene ◽  
Gene Knockout ◽  
Multidrug Resistant ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Synergistic Interactions ◽  
Neutral Gene ◽  
Transcriptomic Level ◽  
Antibiotic Efficacy

AbstractProliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

scholarly journals Potentiating antibiotic treatment using fitness-neutral gene expression perturbations

2019 ◽  
Author(s):  
Peter B. Otoupal ◽  
Keesha E. Erickson ◽  
Kristen A. Eller ◽  
Jocelyn Campos ◽  
Thomas R. Aunins ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Treatment ◽  
Bacterial Infections ◽  
Gene Knockout ◽  
Multidrug Resistant ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Neutral Gene ◽  
Targeted Gene Expression ◽  
Intracellular Infections

AbstractThe rapid proliferation of multidrug-resistant (MDR) bacteria poses a critical threat to human health, for which new antimicrobial strategies are desperately needed. Here we outline a strategy for combating bacterial infections by administering fitness neutral gene expression perturbations as co-therapies to potentiate antibiotic lethality. We systematically explored the fitness of 270 gene knockout-drug combinations in Escherichia coli, identifying 114 synergistic interactions. Genes revealed in this screen were subsequently perturbed at the transcriptome level via multiplexed CRISPR-dCas9 interference to induce antibiotic synergy. These perturbations successfully sensitized E. coli to antibiotic treatment without imposing a separate fitness cost. We next administered these fitness neutral gene perturbations as co-therapies to potentiate antibiotic killing of Salmonella enterica in intracellular infections of HeLa epithelial cells, demonstrating therapeutic applicability. Finally, we utilized these results to design peptide nucleic acid (PNA) co-therapies for targeted gene expression reduction in four MDR, clinically isolated bacteria. Two isolates of Klebsiella pneumoniae and E. coli were each exposed to PNAs targeting homologs of the genes csgD, fnr, recA and acrA in the presence of sub-minimal inhibitory concentrations of trimethoprim. We successfully increased each strain’s susceptibility to trimethoprim treatment and identified eight cases in which re-sensitization occurred without a direct fitness impact of the PNA on MDR strains. Our results highlight a promising approach for combating MDR bacteria which could extend the utility of our current antibiotic arsenal.

scholarly journals In Vitro Activities and Inoculum Effects of Ceftazidime-Avibactam and Aztreonam-Avibactam against Carbapenem-Resistant Enterobacterales Isolates from South Korea

Antibiotics ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 912
Author(s):  
Taeeun Kim ◽  
Seung Cheol Lee ◽  
Moonsuk Bae ◽  
Heungsup Sung ◽  
Mi-Na Kim ◽  
...  
Keyword(s):  
Resistance Mechanism ◽  
Fold Increase ◽  
Multidrug Resistant ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Carbapenem Resistant ◽  
High Inoculum ◽  
Inoculum Effect

Ceftazidime-avibactam (CAZ-AVI) and aztreonam-avibactam (AZT-AVI) are novel antibiotic combinations active against multidrug-resistant Gram-negative pathogens. This study aimed to evaluate their in vitro activities and inoculum effects in carbapenem-resistant Enterobacterales (CRE), including carbapenemase-producing (CP)-CRE and non-CP-CRE. A total of 81 independent clinical isolates of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae were collected. CAZ-AVI and AZT-AVI minimal inhibitory concentrations (MICs) were evaluated by broth microdilution using standard and high inocula. The inoculum effect was defined as an ≥8-fold increase in MIC with high inoculum. Phenotypic determination of β-lactam resistance mechanism and PCR for carbapenemase genes were performed. Of the 81 CRE isolates, 35 (43%) were CP-CRE. Overall, 73% of the isolates were susceptible to CAZ-AVI, and 95% had low AZT-AVI MICs (≤8 µg/mL). The MIC50/MIC90s of CAZ-AVI and AZT-AVI were 4/≥512 µg/mL and 0.5/4 µg/mL, respectively. CAZ-AVI was more active against non-CP-CRE than against CP-CRE (susceptibility 80% vs. 63%, p = 0.08; MIC50/MIC90, 2/16 μg/mL vs. 4/≥512 μg/mL), whereas AZT-AVI was more active against CP-CRE (MIC50/MIC90, 0.25/1 μg/mL vs. 0.5/8 μg/mL). All four isolates with high AZT-AVI MIC (≥16 μg/mL) were resistant to CAZ-AVI, but only 18% (4/22) of CAZ-AVI-resistant isolates had high AZT-AVI MIC. The rates of the inoculum effect for CAZ-AVI and AZT-AVI were 18% and 47%, respectively (p < 0.001). Interestingly, the frequency of the AZT-AVI inoculum effect was higher in K. pneumoniae than E. coli (64% vs. 8%, p < 0.001). AZT-AVI is more active against CRE than CAZ-AVI, even in CP-CRE and CAZ-AVI-resistant isolates. The presence of a substantial inoculum effect may contribute to clinical failure in high-inoculum infections treated with AZT-AVI.

scholarly journals Rapid Accumulation of Motility-Activating Mutations in Resting Liquid Culture ofEscherichia coli

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Darren J. Parker ◽  
Pınar Demetci ◽  
Gene-Wei Li
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Liquid Medium ◽  
Regulatory Networks ◽  
Gene Knockout ◽  
Single Gene ◽  
Point Mutations ◽  
Content Type ◽  
E Coli ◽  
Keio Collection

ABSTRACTExpression of motility genes is a potentially beneficial but costly process in bacteria. Interestingly, many isolate strains ofEscherichia colipossess motility genes but have lost the ability to activate them under conditions in which motility is advantageous, raising the question of how they respond to these situations. Through transcriptome profiling of strains in theE. colisingle-gene knockout Keio collection, we noticed drastic upregulation of motility genes in many of the deletion strains compared to levels in their weakly motile parent strain (BW25113). We show that this switch to a motile phenotype is not a direct consequence of the genes deleted but is instead due to a variety of secondary mutations that increase the expression of the major motility regulator, FlhDC. Importantly, we find that this switch can be reproduced by growing poorly motileE. colistrains in nonshaking liquid medium overnight but not in shaking liquid medium. Individual isolates after the nonshaking overnight incubations acquired distinct mutations upstream of theflhDCoperon, including different insertion sequence (IS) elements and, to a lesser extent, point mutations. The rapidity with which genetic changes sweep through the populations grown without shaking shows that poorly motile strains can quickly adapt to a motile lifestyle by genetic rewiring.IMPORTANCEThe ability to tune gene expression in times of need outside preordained regulatory networks is an essential evolutionary process that allows organisms to survive and compete. Here, we show that upon overnight incubation in liquid medium without shaking, populations of largely nonmotileEscherichia colibacteria can rapidly accumulate mutants that have constitutive motility. This effect contributes to widespread secondary mutations in the single-gene knockout library, the Keio collection. As a result, 49/71 (69%) of the Keio strains tested exhibited various degrees of motility, whereas their parental strain is poorly motile. These observations highlight the plasticity of gene expression even in the absence of preexisting regulatory programs and should raise awareness of procedures for handling laboratory strains ofE. coli.

scholarly journals Predicting growth rate from gene expression

2018 ◽  
Vol 116 (2) ◽  
pp. 367-372 ◽  
Author(s):  
Thomas P. Wytock ◽  
Adilson E. Motter
Keyword(s):  
Gene Expression ◽  
Growth Rate ◽  
Population Level ◽  
Phenotypic Traits ◽  
Phenotypic Characteristics ◽  
K Nearest Neighbors ◽  
E Coli ◽  
Learning Technique ◽  
Genetic Level ◽  
Antibiotic Efficacy

Growth rate is one of the most important and most complex phenotypic characteristics of unicellular microorganisms, which determines the genetic mutations that dominate at the population level, and ultimately whether the population will survive. Translating changes at the genetic level to their growth-rate consequences remains a subject of intense interest, since such a mapping could rationally direct experiments to optimize antibiotic efficacy or bioreactor productivity. In this work, we directly map transcriptional profiles to growth rates by gathering published gene-expression data from Escherichia coli and Saccharomyces cerevisiae with corresponding growth-rate measurements. Using a machine-learning technique called k-nearest-neighbors regression, we build a model which predicts growth rate from gene expression. By exploiting the correlated nature of gene expression and sparsifying the model, we capture 81% of the variance in growth rate of the E. coli dataset, while reducing the number of features from >4,000 to 9. In S. cerevisiae, we account for 89% of the variance in growth rate, while reducing from >5,500 dimensions to 18. Such a model provides a basis for selecting successful strategies from among the combinatorial number of experimental possibilities when attempting to optimize complex phenotypic traits like growth rate.

scholarly journals Modulated efficacy CRISPRi reveals evolutionary conservation of essential gene expression-fitness relationships in bacteria

2019 ◽  
Author(s):  
John S. Hawkins ◽  
Melanie R. Silvis ◽  
Byoung-Mo Koo ◽  
Jason M. Peters ◽  
Marco Jost ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cellular Networks ◽  
High Throughput ◽  
Essential Gene ◽  
Evolutionary Conservation ◽  
Essential Genes ◽  
E Coli ◽  
Gene Expression Levels

AbstractEssential genes are the central hubs of cellular networks. Despite their importance, the lack of high-throughput methods for titrating their expression has limited our understanding of the fitness landscapes against which essential gene expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate specific levels of CRISPRi activity and demonstrate its broad applicability in bacteria. Using libraries of mismatched sgRNAs, we characterized the expression-fitness relationships of essential genes in Escherichia coli and Bacillus subtilis. Remarkably, these relationships co-vary by pathway and are predominantly conserved between E. coli and B. subtilis despite ~ 2 billion years of evolutionary separation, suggesting that deeply conserved tradeoffs underlie bacterial homeostasis.One Sentence SummaryBacterial essential genes have varying responses to CRISPRi knockdown that are largely conserved across ~2 billion years of evolution.

scholarly journals Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions

Genes ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 53
Author(s):  
Francesca Bushell ◽  
John M. J. Herbert ◽  
Thippeswamy H. Sannasiddappa ◽  
Daniel Warren ◽  
A. Keith Turner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Essential Gene ◽  
Acid Stress ◽  
Short Chain Fatty Acids ◽  
Coli Strain ◽  
E Coli ◽  
Kegg Pathways ◽  
Relative Contribution ◽  
External Stresses ◽  
Genome Level

Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism’s ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

scholarly journals Amotosalen is a bacterial multidrug efflux pump substrate potentially affecting its pathogen inactivation activity

2021 ◽  
Author(s):  
Alex B. Green ◽  
Katelyn E. Zulauf ◽  
Katherine A. Truelson ◽  
Lucius Chiaraviglio ◽  
Meng Cui ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Efflux Pumps ◽  
Multidrug Resistant ◽  
Pathogen Inactivation ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Rnd Efflux Pumps

AbstractPathogen inactivation is a strategy to improve the safety of transfusion products. The Cerus Intercept technology makes use of a psoralen compound called amotosalen in combination with UVA light to inactivate bacteria, viruses and protozoa. Psoralens have structural similarity to bacterial multidrug-efflux pump substrates. As these efflux pumps are often overexpressed in multidrug-resistant pathogens and with recent reported outbreaks of transfusion-associated sepsis with Acinetobacter, we tested whether contemporary drug-resistant pathogens might show resistance to amotosalen and other psoralens based on multidrug efflux mechanisms through microbiological, biophysical and molecular modeling analysis. The main efflux systems in Enterobacterales and Acinetobacter baumannii, tripartite RND (resistance-nodulation-cell division) systems which span the inner and outer membranes of Gram-negative pathogens and expel antibiotics from the bacterial cytoplasm into the extracellular space, were specifically examined. We found that amotosalen was an efflux substrate for the TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump from Acinetobacter baumannii, and that minimal inhibitory concentrations for contemporary bacterial isolates in vitro approached and exceeded the concentration of amotosalen used in the approved platelet and plasma inactivation procedures. These findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.ImportancePathogen inactivation is a strategy to enhance the safety of transfused blood products. We identify the compound, amotosalen, widely used for pathogen inactivation, as a bacterial multidrug efflux substrate. Specifically, experiments suggest that amotosalen is pumped out of bacteria by the major TolC-dependent RND efflux pumps in E. coli and the AdeABC efflux pump in Acinetobacter baumannii. Such efflux pumps are often overexpressed in multidrug-resistant pathogens. Importantly, the minimal inhibitory concentrations for contemporary multidrug-resistant Enterobacterales, Acinetobacter baumannii, Pseudomonas aeruginosa, Burkholderia spp., and Stenotrophomonas maltophilia isolates approached or exceeded the amotosalen concentration used in approved platelet and plasma inactivation procedures, potentially as a result of efflux pump activity. Although there are important differences in methodology between our experiments and blood product pathogen inactivation, these findings suggest that otherwise safe and effective inactivation methods should be further studied to exclude possible gaps in their ability to inactivate contemporary, multidrug-resistant bacterial pathogens.

scholarly journals The Pheno- and Genotypic Characterization of Porcine Escherichia coli Isolates

2021 ◽  
Vol 9 (8) ◽  
pp. 1676
Author(s):  
Tanja Bernreiter-Hofer ◽  
Lukas Schwarz ◽  
Elke Müller ◽  
Adriana Cabal-Rosel ◽  
Maciej Korus ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Resistance Genes ◽  
Multidrug Resistant ◽  
Whole Genome Sequence ◽  
Health Concern ◽  
Multi Drug Resistance ◽  
Virulence Determinants ◽  
Minimal Inhibitory Concentrations ◽  
E Coli ◽  
Antimicrobial Resistance Genes

Escherichia (E.) coli is the main causative pathogen of neonatal and post-weaning diarrhea and edema disease in swine production. There is a significant health concern due to an increasing number of human infections associated with food and/or environmental-borne pathogenic and multidrug-resistant E. coli worldwide. Monitoring the presence of pathogenic and antimicrobial-resistant E. coli isolates is essential for sustainable disease management in livestock and human medicine. A total of 102 E. coli isolates of diseased pigs were characterized by antimicrobial and biocide susceptibility testing. Antimicrobial resistance genes, including mobile colistin resistance genes, were analyzed by PCR and DNA sequencing. The quinolone resistance-determining regions of gyrA and parC in ciprofloxacin-resistant isolates were analyzed. Clonal relatedness was investigated by two-locus sequence typing (CH clonotyping). Phylotyping was performed by the Clermont multiplex PCR method. Virulence determinants were analyzed by customized DNA-based microarray technology developed in this study for fast and economic molecular multiplex typing. Thirty-five isolates were selected for whole-genome sequence-based analysis. Most isolates were resistant to ampicillin and tetracycline. Twenty-one isolates displayed an ESBL phenotype and one isolate an AmpC β-lactamase-producing phenotype. Three isolates had elevated colistin minimal inhibitory concentrations and carried the mcr-1 gene. Thirty-seven isolates displayed a multi-drug resistance phenotype. The most predominant β-lactamase gene classes were blaTEM-1 (56%) and blaCTX-M-1 (13.71%). Mutations in QRDR were observed in 14 ciprofloxacin-resistant isolates. CH clonotyping divided all isolates into 51 CH clonotypes. The majority of isolates belonged to phylogroup A. Sixty-four isolates could be assigned to defined pathotypes wherefrom UPEC was predominant. WGS revealed that the most predominant sequence type was ST100, followed by ST10. ST131 was detected twice in our analysis. This study highlights the importance of monitoring antimicrobial resistance and virulence properties of porcine E. coli isolates. This can be achieved by applying reliable, fast, economic and easy to perform technologies such as DNA-based microarray typing. The presence of high-risk pathogenic multi-drug resistant zoonotic clones, as well as those that are resistant to critically important antibiotics for humans, can pose a risk to public health. Improved protocols may be developed in swine farms for preventing infections, as well as the maintenance and distribution of the causative isolates.

Modulation of the drug resistance by Platonia insignis Mart. extract, ethyl acetate fraction and Morelloflavone/Volkensiflavone (biflavonoids) in Staphylococcus aureus strains overexpressing efflux pump genes

2020 ◽  
Vol 21 ◽  
Author(s):  
Andressa Kelly Ferreira e Silva ◽  
Antonielly Campinho dos Reis ◽  
Emanuelly Elanny Andrade Pinheiroc ◽  
Jonas Nascimento de Sousa ◽  
Felipe Araújo de Alcântara Oliveira ◽  
...  
Keyword(s):  
Natural Products ◽  
Ethyl Acetate ◽  
Native Species ◽  
Efflux Pump ◽  
Public Health Problem ◽  
Ethyl Acetate Fraction ◽  
Multidrug Resistant ◽  
Global Public Health ◽  
Minimal Inhibitory Concentrations ◽  
Subinhibitory Concentrations

Background: Microbial resistance to antibiotics is a global public health problem, which requires urgent attention. Platonia insignis is a native species from the eastern Brazilian Amazon, used in the treatment of burns and wounds. Objectives: To evaluate the antimicrobial activity of the hydroalcoholic extract of P. insignis (PIHA), the ethyl acetate fraction (PIAE), and its subfraction containing a mixture of biflavonoids (BF). Moreover, the effect of these natural products on the antibiotic activity against S. aureus strains overexpressing efflux pump genes was also evaluated. Methods: Minimal inhibitory concentrations were determined against different species of microorganisms. To evaluate the modulatory effect on the Norfloxacin-resistance, the MIC of this antibiotic was determined in the absence and presence of the natural products at subinhibitory concentrations. Inhibition of the EtBr efflux assays were conducted in the absence or presence of the natural products. Results: PIHA showed a microbicidal effect against S. aureus and C. albicans, while PIAE was bacteriosctatic for S. aureus. PIAE and BF at subinhibitory concentrations were able to reduce the MIC of Norfloxacin acting as modulating agents. BF was able to inhibit the efflux of EtBr efflux in S. aureus strains overexpressing specific efflux pump genes. Conclusion: P. inignisis a source of efflux pump inhibitors, including volkensiflavone and morelloflavone which were able to potentiate the Norfloxacin activity by NorA inhibition, being also able to inhibit QacA/B, TetK and MsrA. Volkensiflavone and morelloflavone could be used as adjuvant in the antibiotic therapy of multidrug resistant S. aureus strains overexpressing efflux pumps.

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