A Model System for Sensitive Detection of Viable E. coli Bacteria Combining Direct Viability PCR and a Novel Microarray-Based Detection Approach

We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.

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Nested PCR protocol for the rapid detection of Escherichia coli in potable water

Canadian Journal of Microbiology ◽

10.1139/m96-110 ◽

1996 ◽

Vol 42 (8) ◽

pp. 862-866 ◽

Cited By ~ 32

Author(s):

David Juck ◽

Jordan Ingram ◽

Michèle Prévost ◽

Josée Coallier ◽

Charles Greer

Keyword(s):

Escherichia Coli ◽

Nested Pcr ◽

Potable Water ◽

Test Organism ◽

Bacterial Cells ◽

Nested Polymerase Chain Reaction ◽

Fecal Indicator ◽

E Coli ◽

Pcr Products ◽

Polymerase Chain

A rapid and sensitive method for the detection of low levels of bacteria in potable water was developed. The fecal indicator bacterium Escherichia coli was used as the test organism in a filtration concentration–nested polymerase chain reaction (PCR) protocol, combined with ethidium bromide visualization of PCR products. Two sets of primers were designed from the E. coli specific β-glucuronidase gene (uidA), the primary pair producing a 486-bp fragment that was used as template for the nested primer pair delineating a 186-bp fragment. This protocol can detect 1–10 bacterial cells/50 mL water sample within 6–8 h, in contrast to traditional culturing or Southern hybridization methods which require 2–3 days for results.Key words: nested PCR, sensitive, detection, potable water.

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RNA Polymerases from Bacillus subtilisand Escherichia coli Differ in Recognition of Regulatory Signals In Vitro

Journal of Bacteriology ◽

10.1128/jb.182.21.6027-6035.2000 ◽

2000 ◽

Vol 182 (21) ◽

pp. 6027-6035 ◽

Cited By ~ 77

Author(s):

Irina Artsimovitch ◽

Vladimir Svetlov ◽

Larry Anthony ◽

Richard R. Burgess ◽

Robert Landick

Keyword(s):

Escherichia Coli ◽

Bacterial Species ◽

In Vitro Transcription ◽

Rna Polymerases ◽

Bacterial Cells ◽

Signal Recognition ◽

E Coli ◽

Promoter Recognition ◽

Species Specific

ABSTRACT Adaptation of bacterial cells to diverse habitats relies on the ability of RNA polymerase to respond to various regulatory signals. Some of these signals are conserved throughout evolution, whereas others are species specific. In this study we present a comprehensive comparative analysis of RNA polymerases from two distantly related bacterial species, Escherichia coli and Bacillus subtilis, using a panel of in vitro transcription assays. We found substantial species-specific differences in the ability of these enzymes to escape from the promoter and to recognize certain types of elongation signals. Both enzymes responded similarly to other pause and termination signals and to the general E. coli elongation factors NusA and GreA. We also demonstrate that, although promoter recognition depends largely on the ς subunit, promoter discrimination exhibited in species-specific fashion by both RNA polymerases resides in the core enzyme. We hypothesize that differences in signal recognition are due to the changes in contacts made between the β and β′ subunits and the downstream DNA duplex.

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Analysis of the monitoring study of the antibiotic-resistance of the agents of purulent-inflammatory processes of soft tissue

Reports of Vinnytsia National Medical University ◽

10.31393/reports-vnmedical-2018-22(2)-10 ◽

2018 ◽

Vol 22 (2) ◽

pp. 285-288

Author(s):

A.P. Prevar ◽

A.V. Kryzshanovskaya ◽

V.A. Radionov ◽

V.M. Mrug

Keyword(s):

Soft Tissues ◽

High Sensitivity ◽

Biochemical Properties ◽

Antimicrobial Drugs ◽

Strict Adherence ◽

E Coli ◽

Monitoring Study ◽

Treatment Measures ◽

Inflammatory Processes ◽

Main Factor

The main factor in the treatment of suppurative and inflammatory processes is the timely optimization of treatment measures taking into account the nature of the microflora and its susceptibility to antimicrobial drugs. The purpose of the study is to monitor the spectrum of microorganisms – pathogens of purulent-inflammatory processes of soft tissues in surgical patients; study of the sensitivity of isolated strains to antibiotics. The material was collected in accordance with aseptic rules. The identification of a pure culture of bacteria was carried out according to morphological, culture, biochemical properties, and the presence of virulence enzymes. Sensitivity of bacteria to antibiotics was determined by the standard disks method (by Kirby-Bauer’s). 255 patients with purulent-inflammatory processes of soft tissues were examined for the period from 2014 to 2017. 229 strains of isolated bacteria were included to Escherichia coli, Citrobacer freundii, Enterobacter cloacae, E.aerogenes, Proteus vulgaris, Staphylococcus aureus, S.epidermidis, Streptococcus pyogenes, S.viridians, S.agalactiae, Pseudomonas aeruginosa. The main cause of purulent-inflammatory processes of soft tissues is Staphylococci (67,2%). Compared to previous studies, the number of P.aeruginosa isolated cultures increased (7.9%). In monoculture and in association with other microorganisms, E. coli (9.6% of cases), E.cloacae et aerogenes (3.9% of cases), P.vulgaris (3.9% of cases), C.freundi (2.5% of cases), S.agalactiae, S.pyogenes, S.viridans (3.5%). The number of associated sows reaches 12%. Clinical strains of microorganisms remain most sensitive to fluoroquinolones, cephalosporins, and also retains high sensitivity to gentamicin, lincomycin, rifampicin, which is important for empirical antibiotic therapy. To increase the effectiveness of antibacterial therapy, strict adherence to the mode of appointment of antibiotics, justification of indications, a combination of antibiotics of different spectrum of action, mandatory correction after determining the sensitivity of the pathogen.

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Toxic Effect of 2-ethyl (bicyclo[2.2.1] heptane) on Bacterial Cells

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-67-72 ◽

2019 ◽

Vol 35 (6) ◽

pp. 67-72 ◽

Cited By ~ 1

Author(s):

I.V. Manukhov ◽

L.S. Yaguzhinsky ◽

M.V. Bermeshev ◽

M.A. Zisman ◽

V.G. Pevgov ◽

...

Keyword(s):

Toxic Effect ◽

Atmospheric Oxygen ◽

Inducible Promoter ◽

Oxygen Species ◽

Bacterial Cells ◽

Unique Identifier ◽

E Coli ◽

Lux Biosensors ◽

High Level ◽

Ministry Of Higher Education

Toxic effect of 2-ethylnorbornane (2-ethyl(bicyclo[2.2.1]heptane) (EBH)) on bacteria has been studied using the E. coli pRecA-lux and E. coli pKatG- lux cells as lux-biosensors. It was shown that the addition of EBH to the incubation medium leads to death and growth retardation, high level oxidative stress and DNA damage in E. coli cells. It is assumed that the oxidation of EBH with atmospheric oxygen causes the formation of reactive oxygen species in the medium, which makes a major contribution to the toxicity of this substance. biosensor, luciferase, bioluminescence, inducible promoter, PrecA, PkatG The authors are grateful to Stanislav Filippovich Chalkin for the development of interdisciplinary ties in the scientific community. The work was financially supported by the Ministry of Higher Education and Science of Russia (Project Unique Identifier RFMEFI60417X0181, Agreement No. 14.604.21.0181 of 26.09.2017).

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Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6380-6385.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6380-6385 ◽

Cited By ~ 52

Author(s):

R. Temmerman ◽

L. Masco ◽

T. Vanhoutte ◽

G. Huys ◽

J. Swings

Keyword(s):

Nested Pcr ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Temporal Changes ◽

Rrna Gene ◽

Specific Pcr ◽

Gradient Gel Electrophoresis ◽

Species Specific ◽

Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

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SEPSIS MARKERS AND SENSITIVITY OF STAPHYLOCOCCUS AUREUS AND ESCHERICHIA COLI ISOLATES IN A GENERAL HOSPITAL SETTING

Vestnik ◽

10.53065/kaznmu.2021.86.69.013 ◽

2021 ◽

pp. 68-74

Author(s):

М.Е. Рамазанов ◽

В.Н. Сон ◽

М.Р. Рысулы ◽

С.Т. Турсуналиев ◽

Е.Б. Еспенбетов

Keyword(s):

Escherichia Coli ◽

Staphylococcus Aureus ◽

Hospital Setting ◽

Bloodstream Infections ◽

High Sensitivity ◽

Clinical Isolates ◽

Empiric Antibiotic Therapy ◽

E Coli ◽

Number Of Patients ◽

Empiric Antibiotic

Представлены результаты проспективного обследования 80 больных ГКБ №7 с бактериемией с октября 2019 года по февраль 2021 года из различных отделений госпиталя. Производилась оценки показателей маркеров сепсиса - пресепсина, прокальцитонина и С-реактивного белка (СРБ) в крови больных в динамике эмпирической терапии антимикробными препаратами (АМП). Наибольшее число больных с выявленной бактериемией находилось в отделении ОАРИТ - 39 пациентов, у 25 из них был диагностирован сепсис по шкале СЕПСИС III, вызванный известными патогенами Staphylococcus aureus (46,6%) и Escherichia coli (36,6%). Для эмпирического лечения применялись различные антибиотики: ампенициллин, амикацин, меропенем, цефотаксим, метрид, ципрофлоксацин, ципрокс, цефлокс, цефазолин, цефтриаксон, левофлоксацин. Уровни прокальцитонина составляют для больных с клиническими изолятами E. coli 20,8±3,1нг/мл, а для изолятов St. aureus 15,7±1,8 нг/мл. После терапии АМП наблюдается значительное снижение показателей до 1,43±0,6 и 2,3±0,9 нг/мл., что позволяет признать эффективность эмпирической антибиотикотерапии при инфекциях кровотока. Высокая чувствительность клинических изолятов Escherichia coli отмечена к препаратам группы карбапенемов - имипенему и меропенему (90,9%), низкая к эртапенему (72,7%). 100% чувствительность все изоляты показали по отношению к АМП из группы глицилциклинов - тигециклину, который структурно сходен с тетрациклинами. Высокой резистеностью клинические изоляты Staphylococcus aureus обладают к пенициллину (92,9%), липопептиду природного происхождения даптомицину (85,8%) и препарату из группы линкозамидов - клиндамицину (64,3%). The results of a prospective examination of 80 patients with bacteremia from October 2019 to February 2021 from various departments of the hospital are presented. The largest number of patients with detected bacteremia were in the OARIT department - 39 patients, 25 of them were diagnosed with sepsis according to the SEPSIS III scale, caused by known pathogens Staphylococcus aureus (46.6%) and Escherichia coli (36.6%). For empirical treatment, various antibiotics were used: ampenicillin, amikacin, meropenem, cefotaxime, metrid, ciprofloxacin, ciprox, ceflox, cefazolin, ceftriaxone, levofloxacin. Procalcitonin levels for patients with clinical E. coli isolates are 20.8 ± 3.1 ng / ml, and for St. aureus 15.7 ± 1.8 ng / ml. After AMP therapy, there is a significant decrease in indicators to 1.43 ± 0.6 and 2.3 ± 0.9 ng / ml, which makes it possible to recognize the effectiveness of empiric antibiotic therapy for bloodstream infections. High sensitivity of clinical isolates of Escherichia coli was noted to drugs of the carbapenem group - imipenem and meropenem (90.9%), low to ertapenem (72.7%). All isolates showed 100% sensitivity to AMPs from the glycylcycline group - tigecycline, which is structurally similar to tetracyclines. Clinical isolates of Staphylococcus aureus are highly resistant to penicillin (92.9%), natural lipopeptide daptomycin (85.8%), and a drug from the lincosamide group - clindamycin (64.3%).

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One-Step Preparation of Competent E. coli: Transformation and Storage of Bacterial Cells in the Same Solution

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot101212 ◽

2021 ◽

Vol 2021 (11) ◽

pp. pdb.prot101212 ◽

Cited By ~ 1

Author(s):

Michael R. Green ◽

Joseph Sambrook

Keyword(s):

Escherichia Coli ◽

Convenient Method ◽

Plasmid Dna ◽

Transformation Efficiency ◽

Bacterial Cells ◽

E Coli ◽

One Step ◽

And Storage

This protocol describes a convenient method for the preparation, use, and storage of competent Escherichia coli. The reported transformation efficiency of this method is ∼5 × 107 transformants/µg of plasmid DNA.

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PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation

Microbiology ◽

10.1099/mic.0.001031 ◽

2021 ◽

Vol 167 (3) ◽

Author(s):

Sathi Mallick ◽

Shanti Kiran ◽

Tapas Kumar Maiti ◽

Anindya S. Ghosh

Keyword(s):

Escherichia Coli ◽

Biofilm Formation ◽

Type Species ◽

Pentose Phosphate ◽

Bacterial Cells ◽

Surface Adhesion ◽

Content Type ◽

Penicillin Binding Proteins ◽

E Coli ◽

Link Type

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

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