scholarly journals A Luminescent Escherichia coli Biosensor for the High Throughput Detection of β-Lactams

2002 ◽  
Vol 7 (2) ◽  
pp. 127-134 ◽  
Author(s):  
Satu J. Valtonen ◽  
Jussi S. Kurittu ◽  
Matti T. Karp
Keyword(s):  
Escherichia Coli ◽  
Model Organism ◽  
Coli Strain ◽  
Bacterial Cells ◽  
Response Curves ◽  
Light Production ◽  
High Throughput Detection ◽  
High Concentrations ◽  
Luciferase Enzyme ◽  
Responsive Element

A group-specific bioluminescent Escherichia coli strain for studying the action of β-lactam antibiotics is described. The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescent are inserted under the control of the β-lactam-responsive element ampR/ampC from Citrobacter freundii. In the presence of β-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction. This biosensor for β-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer. Basic parameters affecting the light production and induction in the gram-negative model organism E. coli SNO301/pBlaLux1 by various β-lactams were studied. The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of β-lactams. Various β-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 μg/ml; piperacillin, 0.0025-25 μg/ml; imipenem, 0.0025-0.25 μg/ml; cephapirin, 0.025-2.5 μg/ml; cefoxitin, 0.0025-1.5 μg/ml; and oxacillin, 25-500 μg/ml. Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay. Different non-β-lactam antibiotics did not cause induction. Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of β-lactam action.

scholarly journals Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

AMB Express ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Josué Daniel Mora-Garduño ◽  
Jessica Tamayo-Nuñez ◽  
Felipe Padilla-Vaca ◽  
Fátima Berenice Ramírez-Montiel ◽  
Ángeles Rangel-Serrano ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Water Samples ◽  
Membrane Integrity ◽  
Analytical Techniques ◽  
Bacterial Cells ◽  
Reporter Strain ◽  
Dna Damaging Agents ◽  
Reporter Strains ◽  
High Concentrations

AbstractThe presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

scholarly journals THE EFFECT ON THE GENETIC APPARATUS OF THE MICROBIAL CELL OF COMPOUNDS BASED ON SUBSTITUTED 1H-INDOL-4-, 5-, 6-, 7-YLAMINES

2019 ◽  
Author(s):  
A. Maseykina ◽  
I. Stepanenko ◽  
S. Yamashkin ◽  
E. Slastnikov
Keyword(s):  
Escherichia Coli ◽  
Alkaline Phosphatase ◽  
Microbial Cell ◽  
Transcriptional Analysis ◽  
Antimicrobial Compounds ◽  
Bacterial Cells ◽  
Response Curves ◽  
Sos Induction ◽  
Sos Chromotest ◽  
Wide Range

The study of new antimicrobial compounds includes determining the mechanism of their effect on the microbial cell. As a rule, the effect of most modern synthetic antimicrobials is associated either with the suppression of DNA synthesis, or with the suppression of bacterial protein synthesis at the level of translation or transcription.There are sensitive and simple methods for screening and monitoring the potential genotoxic activity of a wide range of natural and synthetic compounds. To date, the Ames test has been widely used, based on the sensitivity of Salmonella strains to carcinogenic chemicals, although some compounds that cause Ames negative reactions could actually be carcinogenic to animals.Similarly, the SOS chromotest is an SOS transcriptional analysis that can evaluate DNA damage caused by chemical and physical mutagens. It measures the expression of a reporter gene (β-galactosidase). The β-galactosidase enzyme processes ortho-nitrophenyl galactopyranoside to form a yellow compound detected at 420 nm. Then, the induction of β-galactosidase normalizes the activity of alkaline phosphatase, an enzyme expressed constitutively by Escherichia coli. SOS chromotest is also widely used for genotoxicological studies. The answer is quick (several hours) and does not require the survival of the test strain. Dose response curves for various chemicals include a linear region. The slope of this area is taken as a measure of SOS induction.Therefore, an SOS chromotest was selected for the study, which allows one to identify the DNA-mediated effect of the studied compounds.The aim of the work was to evaluate the SOS-inducing activity of antimicrobial compounds based on substituted 1H-indol-4-, 5-, 6-, 7-ylamines.The strain Escherichia coli PQ 37 with the genotype F-thr leu his-4 pyrD thi galE lacΔU169 srl300 :: Th10 rpoB rpsL uvrA rfa trp :: Mis + sfi A :: Mud (Ar, lac) cts, Due to the presence of sfi A genes :: lac Z, lacZ β-galactosidase gene expression in strain PQ 37 is controlled by the promoter of the sfiA gene, one of the components of the E. coli SOS regulon. The indicator of the SOS-inducing activity of the studied compounds in the SOS chromotest is the activity of β-galactosidase, which evaluates the activity of active microorganisms - alkaline phosphatase, which also allows you to control the toxic effect of the studied compounds on bacterial cells.The results showed that 4,4,4-trifluoro-N-(6-methoxy-1,2,3-trimethyl-1H-indol-5-yl)-3-oxobutanamide (1), 4,4,4-trifluoro-N-(6-methyl-2-phenyl-1H-indol-5-yl)-3-oxobutanamide (2) and N-(1,5-dimethyl-2-phenyl-1H-indol-6-yl)-4,4,4-trifluoro-3-oxobutanamide (3) does not possess SOS-inducing activity in the studied concentrations. 4-Hydroxy-8-phenyl-4-(trifluoromethyl)-1,3,4,7-tetrahydro-2H-pyrrolo [2,3-h] -quinolin-2-one (4), 9-hydroxy-5-methyl-2-phenyl-9-(trifluoromethyl)-1,6,8,9-tetrahydro-7Н-pyrrolo-[2,3-f]quinolin-7-one (5), 6-hydroxy-2,3-dimethyl-6-(trifluoromethyl)-1,6,7,9-tetrahydro-8H-pyrrolo[3,2-h]quinolin-8-one (6) and 1,2,3,9-tetramethyl-6-(trifluoromethyl)-1,9-dihydro-8H-pyrrolo [3,2-h]quinolin-8-one (7) showed dose-dependent SOS-inducing activity in bactericidal concentrations. The obtained research results allowed us to identify compounds 4, 5, 6, 7, the mechanism of action of which includes exposure to DNA of a microbial cell.

scholarly journals Persister cells mediate tolerance to metal oxyanions in Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3181-3195 ◽  
Author(s):  
Joe J. Harrison ◽  
Howard Ceri ◽  
Nicole J. Roper ◽  
Erin A. Badry ◽  
Kimberley M. Sproule ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Viable Cell ◽  
Small Population ◽  
Water Soluble ◽  
Bacterial Cells ◽  
Persister Cells ◽  
Term Survival ◽  
E Coli ◽  
Cell Counts ◽  
High Concentrations

Bacterial cultures produce subpopulations of cells termed ‘persisters’, reputedly known for high tolerance to killing by antibiotics. Ecologically, antibiotics produced by competing microflora are only one potential stress encountered by bacteria. Another pressure in the environment is toxic metals that are distributed ubiquitously by human pollution, volcanic activity and the weathering of minerals. This study evaluated the time- and concentration-dependent killing of Escherichia coli planktonic and biofilm cultures by the water-soluble metal(loid) oxyanions chromate (), arsenate (), arsenite (), selenite (), tellurate () and tellurite (). Correlative to previous reports in the literature, control antibiotic assays indicated that a small proportion of E. coli biofilm populations remained recalcitrant to killing by antibiotics (even with 24 h exposure). In contrast, metal oxyanions presented a slow, bactericidal action that eradicated biofilms. When exposed for 2 h, biofilms were up to 310 times more tolerant to killing by metal oxyanions than corresponding planktonic cultures. However, by 24 h, planktonic cells and biofilms were eradicated at approximately the same concentration in all instances. Coloured complexes of metals and chelators could not be generated in biofilms exposed to or , suggesting that the extracellular polymeric matrix of E. coli may have a low binding affinity for metal oxyanions. Viable cell counts at 2 and 24 h exposure revealed that, at high concentrations, all of the metal oxyanions had killed 99 % (or a greater proportion) of the bacterial cells in biofilm populations. It is suggested here that the short-term survival of <1 % of the bacterial population corresponds well with the hypothesis that a small population of persister cells may be responsible for the time-dependent tolerance of E. coli biofilms to high concentrations of metal oxyanions.

scholarly journals The Hek Outer Membrane Protein of Escherichia coli Strain RS218 Binds to Proteoglycan and Utilizes a Single Extracellular Loop for Adherence, Invasion, and Autoaggregation

2007 ◽  
Vol 76 (3) ◽  
pp. 1135-1142 ◽  
Author(s):  
Robert P. Fagan ◽  
Matthew A. Lambert ◽  
Stephen G. J. Smith
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Epithelial Cells ◽  
Membrane Protein ◽  
Molecular Level ◽  
Coli Strain ◽  
Bacterial Cells ◽  
Extracellular Loop ◽  
Gram Negative ◽  
E Coli

ABSTRACT Escherichia coli is the principal gram-negative causative agent of sepsis and meningitis in neonates. The pathogenesis of meningitis due to E. coli K1 involves mucosal colonization, transcytosis of epithelial cells, survival in the bloodstream, and eventually invasion of the meninges. The last two aspects have been well characterized at a molecular level. Less is known about the early stages of pathogenesis, i.e., adhesion to and invasion of epithelial cells. We have previously reported that the Hek protein causes autoaggregation and can mediate adherence to and invasion of epithelial cells. Here, we report that Hek-mediated adherence is dependent on binding to glycosoaminoglycan, in particular, heparin. The ability to hemagglutinate, autoaggregate, adhere, and invade is contingent on a putative 25-amino-acid loop that is exposed to the outside of the bacterial cells.

Efficacy of Washing with a Commercial Flatbed Brush Washer, Using Conventional and Experimental Washing Agents, in Reducing Populations of Escherichia coli on Artificially Inoculated Apples†

2001 ◽  
Vol 64 (2) ◽  
pp. 159-163 ◽  
Author(s):  
BASSAM A. ANNOUS ◽  
GERALD M. SAPERS ◽  
ANGELA M. MATTRAZZO ◽  
DENISE C. R. RIORDAN
Keyword(s):  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Coli Strain ◽  
Trisodium Phosphate ◽  
Hammer Mill ◽  
Bacterial Cells ◽  
Cross Contamination ◽  
E Coli ◽  
Commercial Practice ◽  
Press Cloth

Conventional and experimental washing formulations were applied with a commercial flatbed brush washer under conditions representative of commercial practice to determine their efficacy in decontaminating apples inoculated with a nonpathogenic Escherichia coli strain. Golden Delicious apples (18 kg) inoculated with E. coli were mixed with approximately 109 kg of uninoculated Fuji apples (distinctly different in appearance) in a wet dump tank containing 1,325 liters of water at 20°C for 15 min. The combined apples were washed in a flatbed brush washer with the following washing solutions: water at 20°C, water at 50°C, 200 ppm of chlorine (pH 6.4) at 20°C, 8% trisodium phosphate at 20°C, 8% trisodium phosphate at 50°C, 5% hydrogen peroxide at 20°C, 5% hydrogen peroxide at 50°C, 1% APL Kleen 245 at 50°C, and two-stage washing treatments using the combination of 1% APL Kleen 245 at 20 or 50°C followed by 5% hydrogen peroxide at 35 or 50°C. None of the washing treatments tested under the conditions of this experiment significantly reduced the E. coli populations on the inoculated apples or in cider made from these apples, probably as a consequence of the inability of this washing system to inactivate or remove the bacterial cells in inaccessible calyx and stem areas of apples. These results are important because they demonstrate the need for new fruit washing technology that can overcome this limitation. Also, there was no significant cross-contamination of the Fuji apples in the dump tank. Significant cross-contamination of cider, made with uninoculated apples, occurred in the hammer mill and/or the press cloth when these units were not sanitized following a trial with inoculated apples.

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

2014 ◽  
Vol 58 (10) ◽  
pp. 6165-6171 ◽  
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.

scholarly journals Quantitative Determination of the Biodegradable Polymer Poly(β-hydroxybutyrate) in a Recombinant Escherichia coli Strain by Use of Mid-Infrared Spectroscopy and Multivariative Statistics

2000 ◽  
Vol 66 (8) ◽  
pp. 3415-3420 ◽  
Author(s):  
Mustafa Kansiz ◽  
Helen Billman-Jacobe ◽  
Don McNaughton
Keyword(s):  
Escherichia Coli ◽  
Ftir Spectroscopy ◽  
Statistical Technique ◽  
Coli Strain ◽  
Pls Regression ◽  
Bacterial Cells ◽  
Optimal Model ◽  
Mid Infrared ◽  
Predicted Values

ABSTRACT Fourier transform infrared (FTIR) spectroscopy in combination with the partial least squares (PLS) multivariative statistical technique was used for quantitative analysis of the poly(β-hydroxybutyrate) (PHB) contents of bacterial cells. A total of 237 replicate spectra from 34 samples were obtained together with gas chromatography-determined reference PHB contents. Using the PLS regression, we were able to relate the infrared spectra to the reference PHB contents, and the correlation coefficient between the measured and predicted values for the optimal model with a standard error of prediction of 1.49% PHB was 0.988. With this technique, there are no solvent requirements, sample preparation is minimal and simple, and analysis time is greatly reduced; our results demonstrate the potential of FTIR spectroscopy as an alternative to the conventional methods used for analysis of PHB in bacterial cells.

scholarly journals Disruption of Membrane by Colistin Kills Uropathogenic Escherichia coli Persisters and Enhances Killing of Other Antibiotics

2016 ◽  
Vol 60 (11) ◽  
pp. 6867-6871 ◽  
Author(s):  
Peng Cui ◽  
Hongxia Niu ◽  
Wanliang Shi ◽  
Shuo Zhang ◽  
Hao Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Infections ◽  
Enhancement Effect ◽  
Bacterial Cells ◽  
Sulfa Drugs ◽  
Content Type ◽  
Bacterial Membranes ◽  
Highly Active ◽  
High Concentrations

ABSTRACTPersisters are small populations of quiescent bacterial cells that survive exposure to bactericidal antibiotics and are responsible for many persistent infections and posttreatment relapses. However, little is known about how to effectively kill persister bacteria. In the work presented here, we found that colistin, a membrane-active antibiotic, was highly active againstEscherichia colipersisters at high concentrations (25 or 50 μg/ml). At a clinically relevant lower concentration (10 μg/ml), colistin alone had no apparent effect onE. colipersisters. In combination with other drugs, this concentration of colistin enhanced the antipersister activity of gentamicin and ofloxacin but not that of ampicillin, nitrofurans, and sulfa drugsin vitro. The colistin enhancement effect was most likely due to increased uptake of the other antibiotics, as demonstrated by increased accumulation of fluorescence-labeled gentamicin. Interestingly, colistin significantly enhanced the activity of ofloxacin and nitrofurantoin but not that of gentamicin or sulfa drugs in the murine model of urinary tract infection. Our findings suggest that targeting bacterial membranes is a valuable approach to eradicating persisters and should have implications for more effective treatment of persistent bacterial infections.

Mechanisms involved in effects of antimicrobial peptides, bactenectins ChBac3.4, ChBac5, and mini-ChBac7.5Nb, on bacterial cells

2017 ◽  
pp. 43-50
Author(s):  
О.В. Шамова ◽  
М.С. Жаркова ◽  
П.М. Копейкин ◽  
Д.С. Орлов ◽  
Е.А. Корнева
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Activity ◽  
Innate Immunity ◽  
Infectious Diseases ◽  
Metabolic Activity ◽  
Capra Hircus ◽  
Bacterial Cells ◽  
Antibiotic Resistant ◽  
Resistant Strains

Антимикробные пептиды (АМП) системы врожденного иммунитета - соединения, играющие важную роль в патогенезе инфекционных заболеваний, так как обладают свойством инактивировать широкий спектр патогенных бактерий, обеспечивая противомикробную защиту живых организмов. В настоящее время АМП рассматриваются как потенциальные соединения-корректоры инфекционной патологии, вызываемой антибиотикорезистентными бактериями (АБР). Цель данной работы состояла в изученим механизмов антибактериального действия трех пептидов, принадлежащих к семейству бактенецинов - ChBac3.4, ChBac5 и mini-ChBac7.5Nb. Эти химически синтезированные пептиды являются аналогами природных пролин-богатых АМП, обнаруженных в лейкоцитах домашней козы Capra hircus и проявляющих высокую антимикробную активность, в том числе и в отношении грамотрицательных АБР. Методы. Минимальные ингибирующие и минимальные бактерицидные концентрации пептидов (МИК и МБК) определяли методом серийных разведений в жидкой питательной среде с последующим высевом на плотную питательную среду. Эффекты пептидов на проницаемость цитоплазматической мембраны бактерий для хромогенного маркера исследовали с использованием генетически модифицированного штамма Escherichia coli ML35p. Действие бактенецинов на метаболическую активность бактерий изучали с применением маркера резазурина. Результаты. Показано, что все исследованные пептиды проявляют высокую антимикробную активность в отношении Escherichia coli ML35p и антибиотикоустойчивых штаммов Escherichia coli ESBL и Acinetobacter baumannii in vitro, но их действие на бактериальные клетки разное. Использован комплекс методик, позволяющих наблюдать в режиме реального времени динамику действия бактенецинов в различных концентрациях (включая их МИК и МБК) на барьерную функцию цитоплазматической мембраны и на интенсивность метаболизма бактериальных клеток, что дало возможность выявить различия в характере воздействия бактенецинов, отличающихся по структуре молекулы, на исследуемые микроорганизмы. Установлено, что действие каждого из трех исследованных бактенецинов в бактерицидных концентрациях отличается по эффективности нарушения целостности бактериальных мембран и в скорости подавления метаболизма клеток. Заключение. Полученная информация дополнит существующие фундаментальные представления о механизмах действия пролин-богатых пептидов врожденного иммунитета, а также послужит основой для биотехнологических исследований, направленных на разработку на базе этих соединений новых антибиотических препаратов для коррекции инфекционных заболеваний, вызываемых АБР и являющимися причинами тяжелых внутрибольничных инфекций. Antimicrobial peptides (AMPs) of the innate immunity are compounds that play an important role in pathogenesis of infectious diseases due to their ability to inactivate a broad array of pathogenic bacteria, thereby providing anti-microbial host defense. AMPs are currently considered promising compounds for treatment of infectious diseases caused by antibiotic-resistant bacteria. The aim of this study was to investigate molecular mechanisms of the antibacterial action of three peptides from the bactenecin family, ChBac3.4, ChBac5, and mini-ChBac7.5Nb. These chemically synthesized peptides are analogues of natural proline-rich AMPs previously discovered by the authors of the present study in leukocytes of the domestic goat, Capra hircus. These peptides exhibit a high antimicrobial activity, in particular, against antibiotic-resistant gram-negative bacteria. Methods. Minimum inhibitory and minimum bactericidal concentrations of the peptides (MIC and MBC) were determined using the broth microdilution assay followed by subculturing on agar plates. Effects of the AMPs on bacterial cytoplasmic membrane permeability for a chromogenic marker were explored using a genetically modified strain, Escherichia coli ML35p. The effect of bactenecins on bacterial metabolic activity was studied using a resazurin marker. Results. All the studied peptides showed a high in vitro antimicrobial activity against Escherichia coli ML35p and antibiotic-resistant strains, Escherichia coli ESBL and Acinetobacter baumannii, but differed in features of their action on bacterial cells. The used combination of techniques allowed the real-time monitoring of effects of bactenecin at different concentrations (including their MIC and MBC) on the cell membrane barrier function and metabolic activity of bacteria. The differences in effects of these three structurally different bactenecins on the studied microorganisms implied that these peptides at bactericidal concentrations differed in their capability for disintegrating bacterial cell membranes and rate of inhibiting bacterial metabolism. Conclusion. The obtained information will supplement the existing basic concepts on mechanisms involved in effects of proline-rich peptides of the innate immunity. This information will also stimulate biotechnological research aimed at development of new antibiotics for treatment of infectious diseases, such as severe in-hospital infections, caused by antibiotic-resistant strains.

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