scholarly journals Exploring the Contribution of the AcrB Homolog MdtF to Drug Resistance and Dye Efflux in a Multidrug Resistant E. coli Isolate

Antibiotics ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 503
Author(s):  
Sabine Schuster ◽  
Martina Vavra ◽  
Ludwig Greim ◽  
Winfried V. Kern
Keyword(s):  
Fluorescent Dyes ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Coli Strain ◽  
Double Knockout ◽  
E Coli ◽  
Knockout Mutants ◽  
Antibiotic Agents ◽  
Single Knockout

In Escherichia coli, the role of RND-type drug transporters other than the major efflux pump AcrB has largely remained undeciphered (particularly in multidrug resistant pathogens), because genetic engineering in such isolates is challenging. The present study aimed to explore the capability of the AcrB homolog MdtF to contribute to the extrusion of noxious compounds and to multidrug resistance in an E. coli clinical isolate with demonstrated expression of this efflux pump. An mdtF/acrB double-knockout was engineered, and susceptibility changes with drugs from various classes were determined in comparison to the parental strain and its acrB and tolC single-knockout mutants. The potential of MdtF to participate in the export of agents with different physicochemical properties was additionally assessed using accumulation and real-time efflux assays with several fluorescent dyes. The results show that there was limited impact to the multidrug resistant phenotype in the tested E. coli strain, while the RND-type transporter remarkably contributes to the efflux of all tested dyes. This should be considered when evaluating the efflux phenotype of clinical isolates via dye accumulation assays. Furthermore, the promiscuity of MdtF should be taken into account when developing new antibiotic agents.

Urban wastewater disinfection for agricultural reuse: effect of solar driven AOPs in the inactivation of a multidrug resistant E. coli strain

2015 ◽  
Vol 178 ◽  
pp. 65-73 ◽  
Author(s):  
Giovanna Ferro ◽  
Antonino Fiorentino ◽  
María Castro Alferez ◽  
M. Inmaculada Polo-López ◽  
Luigi Rizzo ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Coli Strain ◽  
Urban Wastewater ◽  
E Coli ◽  
Wastewater Disinfection

scholarly journals Molecular survey of mcr1 and mcr2 plasmid mediated colistin resistance genes in Escherichia coli isolates of animal origin in Iran

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Kayhan Ilbeigi ◽  
Mahdi Askari Badouei ◽  
Hossein Vaezi ◽  
Hassan Zaheri ◽  
Sina Aghasharif ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Companion Animals ◽  
Multidrug Resistant ◽  
Animal Origin ◽  
Colistin Resistance ◽  
E Coli ◽  
High Level ◽  
Farming Industry

Abstract Objectives The emergence of colistin-resistant Enterobacteriaceae from human and animal sources is one of the major public health concerns as colistin is the last-resort antibiotic for treating infections caused by multidrug-resistant Gram-negative bacteria. We aimed to determine the prevalence of the prototype widespread colistin resistance genes (mcr-1 and mcr-2) among commensal and pathogenic Escherichia coli strains isolated from food-producing and companion animals in Iran. Results A total of 607 E. coli isolates which were previously collected from different animal sources between 2008 and 2016 used to uncover the possible presence of plasmid-mediated colistin resistance genes (mcr-1 and mcr-2) by PCR. Overall, our results could not confirm the presence of any mcr-1 or mcr-2 positive E. coli among the studied isolates. It is concluded that despite the important role of food-producing animals in transferring the antibiotic resistance, they were not the main source for carriage of mcr-1 and mcr-2 in Iran until 2016. This study suggests that the other mcr variants (mcr-3 to mcr-9) might be responsible for conferring colistin resistance in animal isolates in Iran. The possible linkage between pig farming industry and high level of mcr carriage in some countries needs to be clarified in future prospective studies.

scholarly journals CK1α ablation in keratinocytes induces p53-dependent, sunburn-protective skin hyperpigmentation

2017 ◽  
Vol 114 (38) ◽  
pp. E8035-E8044 ◽  
Author(s):  
Chung-Hsing Chang ◽  
Che-Jung Kuo ◽  
Takamichi Ito ◽  
Yu-Ya Su ◽  
Si-Tse Jiang ◽  
...  
Keyword(s):  
Target Genes ◽  
Neutralizing Antibody ◽  
Double Knockout ◽  
Melanocyte Stimulating Hormone ◽  
Kit Ligand ◽  
Skin Physiology ◽  
P53 Activation ◽  
P53 Target Genes ◽  
Single Knockout

Casein kinase 1α (CK1α), a component of the β-catenin destruction complex, is a critical regulator of Wnt signaling; its ablation induces both Wnt and p53 activation. To characterize the role of CK1α (encoded byCsnk1a1) in skin physiology, we crossed mice harboring floxedCsnk1a1with mice expressing K14–Cre–ERT2to generate mice in which tamoxifen induces the deletion ofCsnk1a1exclusively in keratinocytes [single-knockout (SKO) mice]. As expected, CK1α loss was accompanied by β-catenin and p53 stabilization, with the preferential induction of p53 target genes, but phenotypically most striking was hyperpigmentation of the skin, importantly without tumorigenesis, for at least 9 mo afterCsnk1a1ablation. The number of epidermal melanocytes and eumelanin levels were dramatically increased in SKO mice. To clarify the putative role of p53 in epidermal hyperpigmentation, we established K14–Cre–ERT2CK1α/p53 double-knockout (DKO) mice and found that coablation failed to induce epidermal hyperpigmentation, demonstrating that it was p53-dependent. Transcriptome analysis of the epidermis revealed p53-dependent up-regulation of Kit ligand (KitL). SKO mice treated with ACK2 (a Kit-neutralizing antibody) or imatinib (a Kit inhibitor) abrogated the CK1α ablation-induced hyperpigmentation, demonstrating that it requires the KitL/Kit pathway. Pro-opiomelanocortin (POMC), a precursor of α-melanocyte–stimulating hormone (α-MSH), was not activated in the CK1α ablation-induced hyperpigmentation, which is in contrast to the mechanism of p53-dependent UV tanning. Nevertheless, acute sunburn effects were successfully prevented in the hyperpigmented skin of SKO mice. CK1α inhibition induces skin-protective eumelanin but no carcinogenic pheomelanin and may therefore constitute an effective strategy for safely increasing eumelanin via UV-independent pathways, protecting against acute sunburn.

scholarly journals Contributions of A2A and A2B adenosine receptors in coronary flow responses in relation to the KATP channel using A2B and A2A/2B double-knockout mice

2011 ◽  
Vol 301 (6) ◽  
pp. H2322-H2333 ◽  
Author(s):  
Maryam Sharifi Sanjani ◽  
Bunyen Teng ◽  
Thomas Krahn ◽  
Stephen Tilley ◽  
Catherine Ledent ◽  
...  
Keyword(s):  
Coronary Flow ◽  
Channel Blocker ◽  
Cardiovascular Disorders ◽  
Therapeutic Approaches ◽  
Double Knockout ◽  
Cgs 21680 ◽  
Pathological Conditions ◽  
Sch 58261 ◽  
Single Knockout

Adenosine plays a role in physiological and pathological conditions, and A2 adenosine receptor (AR) expression is modified in many cardiovascular disorders. In this study, we elucidated the role of the A2BAR and its relationship to the A2AAR in coronary flow (CF) changes using A2B single-knockout (KO) and A2A/2B double-KO (DKO) mice in a Langendorff setup. We used two approaches: 1) selective and nonselective AR agonists and antagonists and 2) A2AKO and A2BKO and A2A/2BDKO mice. BAY 60-6583 (a selective A2B agonist) had no effect on CF in A2BKO mice, whereas it significantly increased CF in wild-type (WT) mice (maximum of 23.3 ± 9 ml·min−1·g−1). 5′- N-ethylcarboxamido adenosine (NECA; a nonselective AR agonist) increased CF in A2BKO mice (maximum of 34.6 ± 4.7 ml·min−1·g−1) to a significantly higher degree compared with WT mice (maximum of 23.1 ± 2.1 ml·min−1·g−1). Also, CGS-21680 (a selective A2A agonist) increased CF in A2BKO mice (maximum of 29 ± 1.9 ml·min−1·g−1) to a significantly higher degree compared with WT mice (maximum of 25.1 ± 2.3 ml·min−1·g−1). SCH-58261 (an A2A-selective antagonist) inhibited the NECA-induced increase in CF to a significantly higher degree in A2BKO mice (19.3 ± 1.6 vs. 0.5 ± 0.4 ml·min−1·g−1) compared with WT mice (19 ± 3.5 vs. 3.6 ± 0.5 ml·min−1·g−1). NECA did not induce any increase in CF in A2A/2BDKO mice, whereas a significant increase was observed in WT mice (maximum of 23.1 ± 2.1 ml·min−1·g−1). Furthermore, the mitochondrial ATP-sensitive K+ (KATP) channel blocker 5-hydroxydecanoate had no effect on the NECA-induced increase in CF in WT mice, whereas the NECA-induced increase in CF in WT (17.6 ± 2 ml·min−1·g−1), A2AKO (12.5 ± 2.3 ml·min−1·g−1), and A2BKO (16.2 ± 0.8 ml·min−1·g−1) mice was significantly blunted by the KATP channel blocker glibenclamide (to 0.7 ± 0.7, 2.3 ± 1.1, and 0.9 ± 0.4 ml·min−1·g−1, respectively). Also, the CGS-21680-induced (22 ± 2.3 ml·min−1·g−1) and BAY 60-6583-induced (16.4 ± 1.60 ml·min−1·g−1) increase in CF in WT mice was significantly blunted by glibenclamide (to 1.2 ± 0.4 and 1.8 ± 1.2 ml·min−1·g−1, respectively). In conclusion, this is the first evidence supporting the compensatory upregulation of A2AARs in A2BKO mice and demonstrates that both A2AARs and A2BARs induce CF changes through KATP channels. These results identify AR-mediated CF responses that may lead to better therapeutic approaches for the treatment of cardiovascular disorders.

scholarly journals Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome

2020 ◽  
Vol 29 (16) ◽  
pp. 2723-2735 ◽  
Author(s):  
Jesús-José Ferre-Fernández ◽  
Elena A Sorokina ◽  
Samuel Thompson ◽  
Ross F Collery ◽  
Emily Nordquist ◽  
...  
Keyword(s):  
Anterior Segment ◽  
Vertebrate Development ◽  
Double Knockout ◽  
Rieger Syndrome ◽  
Forkhead Box ◽  
Combined Genotypes ◽  
Craniofacial Defects ◽  
Winged Helix Transcription Factor ◽  
Single Knockout

Abstract The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a−/− single homozygous and foxc1−/− double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/−;foxc1b−/−) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.

scholarly journals High Prevalence and Diversity of Cephalosporin-Resistant Enterobacteriaceae Including Extraintestinal Pathogenic E. coli CC648 Lineage in Rural and Urban Dogs in Northwest Spain

Antibiotics ◽  
2020 ◽  
Vol 9 (8) ◽  
pp. 468 ◽  
Author(s):  
Fátima Abreu-Salinas ◽  
Dafne Díaz-Jiménez ◽  
Isidro García-Meniño ◽  
Pilar Lumbreras ◽  
Ana María López-Beceiro ◽  
...  
Keyword(s):  
High Risk ◽  
Antimicrobial Resistance ◽  
One Health ◽  
Potential Role ◽  
Multidrug Resistant ◽  
E Coli ◽  
Rural And Urban ◽  
High Prevalence ◽  
Healthy Dogs

The aim of this work was to assess the prevalence of extended spectrum-β-lactamase (ESBL)- and carbapenemase-producing Enterobacteriaceae in fecal samples recovered from rural and urban healthy dogs in Northwest Spain (Galicia) to identify potential high-risk clones and to molecularly characterize positive isolates regarding the genes coding for ESBL/pAmpC resistance and virulence. Thirty-five (19.6%) out of 179 dogs were positive for cephalosporin-resistant Enterobacteriaceae, including Escherichiacoli and Klebsiella pneumoniae (39 and three isolates, respectively). All the isolates were multidrug resistant, with high rates of resistance to different drugs, including ciprofloxacin (71.4%). A wide diversity of ESBL/pAmpC enzymes, as well as E. coli phylogroups (A, B1, C, D, E, F and clade I) were found. The eight isolates (20.5%) found to conform to the ExPEC status, belonged to clones O1:H45-clade I-ST770 (CH11-552), O18:H11-A-ST93-CC168 (CH11-neg), O23:H16-B1-ST453-CC86 (CH6-31), and O83:H42-F-ST1485-CC648 (CH231-58), with the latter also complying the uropathogenic (UPEC) status. The three K. pneumoniae recovered produced CTX-M-15 and belonged to the ST307, a clone previously reported in human clinical isolates. Our study highlights the potential role of both rural and urban dogs as a reservoir of high-risk Enterobacteriaceae clones, such as the CC648 of E. coli and antimicrobial resistance traits. Within a One-Health approach, their surveillance should be a priority in the fight against antimicrobial resistance.

Tigecycline efflux in Acinetobacter baumannii is mediated by TetA in synergy with RND-type efflux transporters

2020 ◽  
Vol 75 (5) ◽  
pp. 1135-1139 ◽  
Author(s):  
Wuen Ee Foong ◽  
Jochen Wilhelm ◽  
Heng-Keat Tam ◽  
Klaas M Pos
Keyword(s):  
Acinetobacter Baumannii ◽  
Efflux Pump ◽  
Gene Knockout ◽  
Drug Susceptibility ◽  
Major Facilitator Superfamily ◽  
Rt Pcr ◽  
E Coli ◽  
Rnd Efflux Pump ◽  
Major Facilitator

Abstract Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.

scholarly journals Cloning and Characterization of SmeDEF, a Novel Multidrug Efflux Pump from Stenotrophomonas maltophilia

2000 ◽  
Vol 44 (11) ◽  
pp. 3079-3086 ◽  
Author(s):  
Ana Alonso ◽  
José L. Martínez
Keyword(s):  
Stenotrophomonas Maltophilia ◽  
Efflux Pump ◽  
Bacterial Pathogen ◽  
Drug Susceptibility ◽  
Multidrug Resistant ◽  
Transcriptional Unit ◽  
Chromosomal Dna ◽  
Multidrug Efflux Pump ◽  
E Coli ◽  
Wide Range

ABSTRACT Stenotrophomonas maltophilia is a nosocomial bacterial pathogen intrinsically resistant to several antibiotics. The mechanisms involved in this intrinsic multiresistance phenotype are poorly understood. A library of chromosomal DNA from a spontaneous multidrug-resistant S. maltophilia D457R mutant (A. Alonso and J. L. Martinez, Antimicrob. Agents Chemother. 41:1140–1142, 1997) was screened for complementation of erythromycin susceptibility on an antibiotic-hypersusceptible Escherichia coli ΔacrABstrain. Cloning and further analysis revealed that a 6-kbp region constituting a transcriptional unit was capable of complementing the antibiotic-susceptible phenotype of an E. coli ΔacrABstrain. We identified three open reading frames, smeD,smeE and smeF, which code for members of the membrane fusion protein, resistance nodulation division, and outer membrane factor families, respectively. Drug susceptibility assays indicated that the SmeDEF system cloned in E. coli mediates resistance to a wide range of antibiotics. Ethidium bromide and norfloxacin accumulation experiments in the presence and in the absence of carbonyl cyanide m-chlorophenylhydrazone showed that this system constitutes a drug efflux pump dependent on the membrane proton motive force. The presence of high levels of smeDEFmRNA in the multiresistant D457R mutant was consistent with the high levels of SmeF (formerly Omp54) observed in the same strain. In contrast, transcription levels of smeDEF in the D457 strain were tiny, which correlates with the low levels of SmeF observed for this strain. Also, for both the D457 and D457R strains, we observed growth phase-dependent regulation in which the highest level of transcription corresponded to early exponential phase, with transcription decreasing throughout the growth curve to undetectable levels at 24 h.

Increasing of the Aminoglicosyde Antibiotic Activity Against a Multidrug-Resistant E. coli by Turnera ulmifolia L. and Chlorpromazine

2009 ◽  
Vol 11 (4) ◽  
pp. 332-335 ◽  
Author(s):  
Henrique D. M. Coutinho ◽  
José G. M. Costa ◽  
Edeltrudes O. Lima ◽  
Vivyanne S. Falcão-Silva ◽  
José P. Siqueira-Júnior
Keyword(s):  
Bacterial Resistance ◽  
Inhibitory Concentration ◽  
Ethanol Extract ◽  
Multidrug Resistant ◽  
Coli Strain ◽  
Minimal Bactericidal Concentration ◽  
Efflux System ◽  
E Coli ◽  
Turnera Ulmifolia ◽  
Conventional Antibiotics

In this study, an ethanol extract of Turnera ulmifolia L. (EETU) and chlorpromazine (CPZ) were tested for their antimicrobial activity alone or in combination with conventional antibiotics against two strains of Escherichia coli (E. coli). The growth of neither E. coli strain was inhibited by the extract. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration values were ≥1 mg/ml for both the strains of E. coli. However, the extract did increase the antimicrobial effects of amikacin, neomycin, and tobramycin. A similar effect of CPZ on amikacin, kanamycin, and tobramycin indicated the involvement of an efflux system in the resistance to these aminoglycosides. Results suggest that extracts from T. ulmifolia could be used as a plant-derived natural product with resistance-modifying activity, constituting a new weapon against bacterial resistance to antibiotics.

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