Synthesis and in Silico Modelling of the Potential Dual Mechanistic Activity of Small Cationic Peptides Potentiating the Antibiotic Novobiocin against Susceptible and Multi-Drug Resistant Escherichia coli

Cationic antimicrobial peptides have attracted interest, both as antimicrobial agents and for their ability to increase cell permeability to potentiate other antibiotics. However, toxicity to mammalian cells and complexity have hindered development for clinical use. We present the design and synthesis of very short cationic peptides (3–9 residues) with potential dual bacterial membrane permeation and efflux pump inhibition functionality. Peptides were designed based upon in silico similarity to known active peptides and efflux pump inhibitors. A number of these peptides potentiate the activity of the antibiotic novobiocin against susceptible Escherichia coli and restore antibiotic activity against a multi-drug resistant E. coli strain, despite having minimal or no intrinsic antimicrobial activity. Molecular modelling studies, via docking studies and short molecular dynamics simulations, indicate two potential mechanisms of potentiating activity; increasing antibiotic cell permeation via complexation with novobiocin to enable self-promoted uptake, and binding the E. coli RND efflux pump. These peptides demonstrate potential for restoring the activity of hydrophobic drugs.

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Whole genome sequences of multi-drug resistant Escherichia coli isolated in a Pastoralist Community of Western Uganda: Phylogenomic changes, virulence and resistant genes

10.1101/2020.04.03.023507 ◽

2020 ◽

Author(s):

Jacob Stanley Iramiot ◽

Henry Kajumbula ◽

Joel Bazira ◽

Etienne P. de Villiers ◽

Benon B. Asiimwe

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Whole Genome ◽

Beta Lactamase ◽

Drug Resistant ◽

Genome Sequences ◽

E Coli ◽

Resistant Genes ◽

Pastoralist Community ◽

Western Uganda

AbstractBackgroundThe crisis of antimicrobial resistance is already here with us, affecting both humans and animals alike and very soon, small cuts and surgeries will become life threatening. This study aimed at determine the whole genome sequences of multi-drug resistant Escherichia coli isolated in a Pastoralist Community of Western Uganda: phylogenomic changes, virulence and resistant genes.MethodsThis was a laboratory based cross sectional study. Bacterial isolates analyzed in this study were 42 multidrug resistant E. coli isolated from stool samples from both humans and cattle in pastoralist communities collected between January 2018-March 2019. Most of the isolates (41/42) were resistant to three or more antibiotics (multi-drug resistant) and 21/42 isolates were ESBL producers; 13/42 from human and 8/42 from cattle. Whole Genome Sequencing (WGS) was carried out at the facilities of Kenya Medical Research Institute-Wellcome trust, Kilifi, to determine the phylogenomic changes, virulence and resistant genes.ResultsThe genomes of the human E. coli generally clustered together and away from those of cattle origin. The E. coli isolates were assigned to eight different phylogroups: A, B1, B2, Cladel, D, E, F and G, with a majority being assigned to phylogroup A; while most of the animal isolates were assigned to phylogroup B1. The carriage of multiple AMR genes was higher from the E. coli population from humans than those from cattle. Among these were Beta-lactamase; blaOXA-1: Class D beta-lactamases; blaTEM-1, blaTEM-235: Beta-lactamase; catA1: chloramphenicol acetyl transferase; cmlA1: chloramphenicol efflux transporter; dfrA1, dfrA12, dfrA14, dfrA15, dfrA17, dfrA5, dfrA7, dfrA8: macrolide phosphotransferase; oqxB11: RND efflux pump conferring resistance to fluoroquinolone; qacL, qacEdelta1: quinolone efflux pump; qnrS1: quinolone resistance gene; sul1, sul2, sul3: sulfonamide resistant; tet(A), tet(B): tetracycline efflux pump.A high variation of virulence genes was registered among the E. coli genomes from humans than those of cattle origin.ConclusionThe E. coli of human and cattle origin are largely independent with different ancestral origins. Limited sharing of strains and resistance genes presents a challenge to the hypothesis that AMR in humans is as a result of antibiotic misuse on the farm.

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Research of Associated Resistance to Antimicrobial Agents of Extended Spectrum Beta-Lactamases Escherichia Coli and Klebsiella pneumoniae Strains Isolated in Senegal

Austin Journal of Microbiology ◽

10.26420/austinjmicrobiol.2021.1029 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Ngom B ◽

◽

Wade SF ◽

Diop TA ◽

Diagne R ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Antimicrobial Agents ◽

Diffusion Method ◽

Drug Resistant ◽

Beta Lactamases ◽

E Coli ◽

Sick People ◽

Extended Spectrum ◽

Extended Spectrum Beta Lactamases

Introduction: Some strains of Escherichia coli and Klebsiella pneumoniae produce Extended Spectrum Beta-Lactamases (ESBL) may be responsible for various infections such as urinary infections. These Sick people are treated in the very serious cases by association antibiotics to class to betalactamins, aminosids and quinolons. But proliferation of multi-drug resistant strains involves decreasing therapeutic success. That’s why epidemiological study must be done in all laboratories of bacteriology. Purpose: The aim of the study was to research the resistance phenotypes of our E. coli and K. pneumoniae ESBL strains compared to others families of antibiotics. Material and methods: Thirty two (32) Extended Spectrum betalactamases E. coli and K. pneumoniae strains isolated from either hospitalized patients or sick people who came for consultation were studied. Susceptibility to antimicrobial agents was determined using an antibiotic disk (Bio-Rad) diffusion method on Mueller-Hinton agar (Bio-Rad). The results were interpreted according to the Standards of the French Antibiogram Committee (CA-SFM). Results: The study showed that most of these strains were multi-drug resistant. They were resistant to many beta-lactamines antibiotics. E. coli strains were also resistant at 70,34% to aminosids, at 96,72% to quinolons, at 58,3% to cotrimoxazol, at 26,1% to chloramphénicol and at 21,4% to colistin ; about K. pneumoniae, they were resistant at 72,6% to aminosids, at 88,95% to quinolons, at 86,7% to cotrimoxazol, at 44,4% to chloramphénicol and at 25% to colistin. But all these strains were sensitive at 100% to l’imipenem.

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611. Fosfomycin Resistance of Multidrug-Resistant Escherichia coli and Mechanisms of Fosfomycin Resistance

Open Forum Infectious Diseases ◽

10.1093/ofid/ofz360.680 ◽

2019 ◽

Vol 6 (Supplement_2) ◽

pp. S285-S285

Author(s):

Hyeri Seok ◽

Ji Hoon Jeon ◽

Hee Kyoung Choi ◽

Won Suk Choi ◽

Dae Won Park ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Multidrug Resistant ◽

Resistance Rate ◽

Coli Strain ◽

Resistant Bacteria ◽

E Coli ◽

Broth Microdilution Method ◽

Fosfomycin Resistance

Abstract Background Fosfomycin is one of the antibiotics that may be a candidate for the next-generation antimicrobial agents againt multidrug-resistant bacteria. To date, it is known that the resistance rate is not high for Escherichia coli. However, it is necessary to update the fosfomycin resistance rates in E. coli according to the studies that extended spectrum β-lactamase (ESBL) producing E. coli strains are highly resistance to fosfomycin. We evaluated the resistance rate of fosfomycin, the resistant mechanism of fosfomycin in E. coli, and the activity of fosfomycin against susceptible and resistant strains of E. coli. Methods A total of 283 clinical isolates was collected from patients with Escherichia coli species during the period of January 2018 to June 2018, in three tertiary hospitals of Republic of Korea. In vitro antimicrobial susceptibility tests were performed in all E. coli isolates using the broth microdilution method according to the Clinical and Laboratory Standard Institute (CLSI). Multilocus sequence typing (MLST) of the Oxford scheme was conducted to determine the genotypes of E. coli isolated. Fosfomycin genes were investigated for all fosfomycin-resistant E. coli strains. Results The overall resistance rate to fosfomycin was 10.2%, compared with 53.4%, 46.3%, 41.3%, 31.1%, 10.6%, 2.5%, and 2.1% for ciprofloxacin, cefixime, cefepime, piperacillin/tazobactam, colistin, ertapenem, and amikacin, respectively. The 29 fosfomycin-resistant isolates did not show a clonal pattern on the phylogenetic tree. MurA and glp genes were identified in all strains. FosA3 were identified in two strains and uhp gene were identified in 4 strains. In time-kill curve studies, fosfomycin was more bactericidal than cefixime against all sensitive E. coli strain. Morever, fosfomycin was more bactericidal than piperacillin/tazobactam against ESBL-producing E. coli strain. Conclusion The resistant rate of fosfomycin to E. coli is still low. Fosfomycin was active against E. coli including ESBL producing strains. Disclosures All authors: No reported disclosures.

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Antibacterial activities of aqueous and methanol extracts of Nigella sativa on some multiple drug resistant diarrheic bacterial agents

Journal of Medicinal Botany ◽

10.25081/jmb.2020.v4.6245 ◽

2020 ◽

pp. 14-19

Author(s):

Adedayo Emmanuel Ogunware ◽

Hassan Zainab Adewunmi

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Nigella Sativa ◽

University Teaching ◽

Multiple Drug ◽

Significant Activity ◽

Drug Resistant ◽

E Coli ◽

Methanolic Extracts ◽

Multiple Drug Resistant

Combinations of various antimicrobial agents have been introduced as an extra successful strategy to combat multiple drug resistant infections. This study was undertaken to evaluate the effects of Nigella sativa seeds on several multi-drug resistant diarrheic bacterial agents. 30 Stool samples were collected from Lagos State University Teaching Hospital (LASUTH), in Nigeria and standard biochemical tests were performed to confirm the diarrheic isolates. Then, antimicrobial susceptibility testing was done on the organisms, followed by screening the effectiveness of Nigella sativa seed extracts on the bacterial agents obtained from the samples. 16 samples tested positive for diarrheic agents Escherichia coli, Escherichia coli 0157H:7 and Klebsiella pneumoniae. The methanolic extracts of Nigella sativa showed the highest zone of inhibition of 12mm for K. pneumoniae at a concentration of 300mg/dl ,10mm for E. coli 0157H:7 at a concentration of 200mg/dl, and 10mm for E. coli at a concentration of 200mg/dl, respectively. The methanolic extracts showed much stronger activity than the aqueous extracts of Nigella sativa which did not show significant activity towards the diarrheic agents isolated.

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Identified Seaweed Compound Diphenylmethane Serves as an Efflux Pump Inhibitor in Drug-Resistant Escherichia coli

Antibiotics ◽

10.3390/antibiotics10111378 ◽

2021 ◽

Vol 10 (11) ◽

pp. 1378

Author(s):

Wen-Jung Lu ◽

Pang-Hung Hsu ◽

Chun-Ju Chang ◽

Cheng-Kuan Su ◽

Yan-Jyun Huang ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Major Elements ◽

Resistant Bacteria ◽

Drug Resistant ◽

Cell Toxicity ◽

Efflux Pump Inhibitor ◽

E Coli ◽

Drug Efflux Pumps ◽

Time Kill

Drug efflux pumps are one of the major elements used by antibiotic-resistant bacteria. Efflux pump inhibitors (EPIs) are potential therapeutic agents for adjunctive therapy, which can restore the activity of antibiotics that are no longer effective against pathogens. This study evaluated the seaweed compound diphenylmethane (DPM) for its EPI activity. The IC50 and modulation results showed that DPM has no antibacterial activity but can potentiate the activity of antibiotics against drug-resistant E. coli. Time-kill studies reported that a combination of DPM and erythromycin exhibited greater inhibitory activity against drug-resistant Escherichia coli. Dye accumulation and dye efflux studies using Hoechst 33342 and ethidium bromide showed that the addition of DPM significantly increased dye accumulation and reduced dye efflux in drug-resistant E. coli, suggesting its interference with dye translocation by an efflux pump. Using MALDI-TOF, it was observed that the addition of DPM could continuously reduce antibiotic efflux in drug-resistant E. coli. Additionally, DPM did not seem to damage the E. coli membranes, and the cell toxicity test showed that it features mild human-cell toxicity. In conclusion, these findings showed that DPM could serve as a potential EPI for drug-resistant E. coli.

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Phenotypic and Genotypic Characterization of ESBL-, AmpC-, and Carbapenemase-Producing Klebsiella pneumoniae and Escherichia coli Isolates

Medical Principles and Practice ◽

10.1159/000500311 ◽

2019 ◽

Vol 28 (6) ◽

pp. 547-551 ◽

Cited By ~ 9

Author(s):

Hossein Kazemian ◽

Hamid Heidari ◽

Roya Ghanavati ◽

Sobhan Ghafourian ◽

Fateme Yazdani ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Antimicrobial Agents ◽

Drug Resistant ◽

E Coli ◽

Molecular Tests ◽

Pcr Method ◽

Hospital Acquired ◽

Genotypic Characteristics

Objectives: Drug resistance among gram-negative bacteria is a worldwide challenge. Due to the importance of drug-resistant Klebsiella pneumoniae and Escherichia coli strains in hospital-acquired infections, we aimed to determine the phenotypic and genotypic characteristics of ESBL-, AmpC-, and carbapenemase-producing isolates obtained from hospitalized patients in Tehran and Ilam (Iran). Materials and Methods: In total, 90 K. pneumoniae isolates and 65 E. coli isolates were collected from various infections. Phenotypic identification of bacterial isolates was performed using standard methods. Phenotypic screening of ESBL, AmpC, and carbapenemase enzymes was carried out. Detection of ESBL, AmpC, and carbapenemase genes was also performed by the PCR method. Results: Phenotypic detection tests showed that 36 (40%) K. pneumoniae and 23 (35.4%) E. coli isolates were ESBL producers. Moreover, 18 (20%) and 6 (9.2%) K. pneumoniae and E. coli isolates were AmpC producers, respectively. Modified Hodge test results indicated that 39 (43.3%) K. pneumoniae and 18 (27.7%) E. coli isolates produced carbapenemase. Molecular tests showed that 40% of K. pneumoniae and 36.9% of E. coli isolates were ESBL positive. AmpC was detected in 24.4 and 13.8% of K. pneumoniae and E. coli isolates. Carbapenemase was detected in 34 (37.8%) K. pneumoniae and 13 (20%) E. coli isolates. Conclusion: In this study, 3 K. pneumoniae isolates simultaneously carried ESBL, AmpC, and carbapenemase genes. Up-to-date strategies such as combination therapy or utilization of new antimicrobial agents might help to combat such drug-resistant organisms.

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Novel mechanisms of TolC-independent decreased bile-salt susceptibility in Escherichia coli

FEMS Microbiology Letters ◽

10.1093/femsle/fnaa083 ◽

2020 ◽

Vol 367 (10) ◽

Author(s):

Vuong Van Hung Le ◽

Patrick J Biggs ◽

David Wheeler ◽

Ieuan G Davies ◽

Jasna Rakonjac

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Sodium Deoxycholate ◽

Coli Strain ◽

Receptor Protein ◽

Phosphotransferase System ◽

Multidrug Efflux Pump ◽

Knock Out ◽

E Coli ◽

Camp Receptor

ABSTRACT Bile salts, including sodium deoxycholate (DOC), are secreted into the intestine to aid fat digestion and contribute to antimicrobial protection. Gram-negative pathogens such as Escherichia coli, however, are highly resistant to DOC, using multiple mechanisms of which the multidrug efflux pump AcrAB-TolC is the dominant one. Given that TolC-mediated efflux masks the interaction of DOC with potential targets, we sought to identify those targets by identifying genes whose mutations cause an increase in the MIC to DOC relative to the ∆tolC parental strain, that lacks TolC-associated functional efflux pumps. Using a mutant screen, we isolated twenty independent spontaneous mutants that had a higher MICDOC than the E. coli parental ∆tolC strain. Whole genome sequencing of these mutants mapped most mutations to the ptsI or cyaA gene. Analysis of knock-out mutants and complementation showed that elimination of PtsI, a component of the carbohydrate phosphotransferase system, or one of the two key proteins involved in cAMP synthesis and signaling, adenylate cyclase (CyaA) or cAMP receptor protein (Crp) causes low-level increased resistance of a ∆tolC E. coli strain to DOC.

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New indicator Escherichia coli strain for rapid and accurate detection of supF mutations

Genes and Environment ◽

10.1186/s41021-020-00167-x ◽

2020 ◽

Vol 42 (1) ◽

Author(s):

Ruriko Fukushima ◽

Tetsuya Suzuki ◽

Hiroyuki Kamiya

Keyword(s):

Escherichia Coli ◽

Mammalian Cells ◽

Indicator Strain ◽

Coli Strain ◽

Chromosomal Dna ◽

Amber Mutation ◽

E Coli ◽

Accurate Detection ◽

Mutation Spectra ◽

Forward Mutation

Abstract Background The supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed. Results The gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01. Conclusion RF01 is a new and useful indicator E. coli strain for analyzing supF mutations.

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Mechanisms Accounting for Fluoroquinolone Resistance in Escherichia coli Clinical Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00665-08 ◽

2008 ◽

Vol 53 (1) ◽

pp. 235-241 ◽

Cited By ~ 95

Author(s):

Sonia K. Morgan-Linnell ◽

Lauren Becnel Boyd ◽

David Steffen ◽

Lynn Zechiedrich

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Efflux Pump ◽

Resistance Mechanisms ◽

Clinical Isolates ◽

Fluoroquinolone Resistance ◽

Multidrug Efflux Pump ◽

Topoisomerase Iv ◽

E Coli ◽

Genes Encoding

ABSTRACT Fluoroquinolone MICs are increased through the acquisition of chromosomal mutations in the genes encoding gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE), increased levels of the multidrug efflux pump AcrAB, and the plasmid-borne genes aac(6′)-Ib-cr and the qnr variants in Escherichia coli. In the accompanying report, we found that ciprofloxacin, gatifloxacin, levofloxacin, and norfloxacin MICs for fluoroquinolone-resistant E. coli clinical isolates were very high and widely varied (L. Becnel Boyd, M. J. Maynard, S. K. Morgan-Linnell, L. B. Horton, R. Sucgang, R. J. Hamill, J. Rojo Jimenez, J. Versalovic, D. Steffen, and L. Zechiedrich, Antimicrob. Agents Chemother. 53:229-234, 2009). Here, we sequenced gyrA, gyrB, parC, and parE; screened for aac(6′)-Ib-cr and qnrA; and quantified AcrA levels in E. coli isolates for which patient sex, age, location, and site of infection were known. We found that (i) all fluoroquinolone-resistant isolates had gyrA mutations; (ii) ∼85% of gyrA mutants also had parC mutations; (iii) the ciprofloxacin and norfloxacin MICs for isolates harboring aac(6′)-Ib-cr (∼23%) were significantly higher, but the gatifloxacin and levofloxacin MICs were not; (iv) no isolate had qnrA; and (v) ∼33% of the fluoroquinolone-resistant isolates had increased AcrA levels. Increased AcrA correlated with nonsusceptibility to the fluoroquinolones but did not correlate with nonsusceptibility to any other antimicrobial agents reported from hospital antibiograms. Known mechanisms accounted for the fluoroquinolone MICs of 50 to 70% of the isolates; the remaining included isolates for which the MICs were up to 1,500-fold higher than expected. Thus, additional, unknown fluoroquinolone resistance mechanisms must be present in some clinical isolates.

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UDP-N-Acetylmuramic Acid l-Alanine Ligase (MurC) Inhibition in atolCMutant Escherichia coli Strain Leads to Cell Death

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02890-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 6165-6171 ◽

Cited By ~ 6

Author(s):

Vaishali Humnabadkar ◽

K. R. Prabhakar ◽

Ashwini Narayan ◽

Sreevalli Sharma ◽

Supreeth Guptha ◽

...

Keyword(s):

Escherichia Coli ◽

Efflux Pump ◽

Coli Strain ◽

Cellular Activity ◽

Wild Type ◽

Compound A ◽

Content Type ◽

E Coli ◽

In The Wild ◽

High Concentrations

ABSTRACTThe Mur ligases play an essential role in the biosynthesis of bacterial peptidoglycan and hence are attractive antibacterial targets. A screen of the AstraZeneca compound library led to the identification of compound A, a pyrazolopyrimidine, as a potent inhibitor ofEscherichia coliandPseudomonas aeruginosaMurC. However, cellular activity againstE. coliorP. aeruginosawas not observed. Compound A was active against efflux pump mutants of both strains. Experiments using anE. colitolCmutant revealed accumulation of the MurC substrate and a decrease in the level of product upon treatment with compound A,indicating inhibition of MurC enzyme in these cells. Such a modulation was not observed in theE. coliwild-type cells. Further, overexpression of MurC in theE. colitolCmutant led to an increase in the compound A MIC by ≥16-fold, establishing a correlation between MurC inhibition and cellular activity. In addition, estimation of the intracellular compound A level showed an accumulation of the compound over time in thetolCmutant strain. A significant compound A level was not detected in the wild-typeE. colistrain even upon treatment with high concentrations of the compound. Therefore, the lack of MIC and absence of MurC inhibition in wild-typeE. coliwere possibly due to suboptimal compound concentration as a consequence of a high efflux level and/or poor permeativity of compound A.

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