QUANTITATIVE BIOFILM ASSAY USING A MICROTITER PLATE TO SCREEN FOR ENTEROAGGREGATIVE ESCHERICHIA COLI

2004 ◽  
Vol 71 (5) ◽  
pp. 687-690 ◽  
Author(s):  
NAOKO WAKIMOTO ◽  
JALALUDDIN SHEIKH ◽  
JAV SARANTUYA ◽  
JUNICHIRO NISHI ◽  
JAMES P. NATARO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Microtiter Plate

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

scholarly journals Evaluating the Five Escherichia coli Derivative Strains as Platform for Arginine Deiminase Overproduction

2020 ◽  
Author(s):  
Sara Abdollahi ◽  
Mohammad Hossein Morowvat ◽  
Amir Savardashtaki ◽  
Cambyz Irajie ◽  
Sohrab Najafipour ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Recombinant Protein ◽  
Microtiter Plate ◽  
Arginine Deiminase ◽  
Expression Vectors ◽  
Efficacy Evaluation ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Bacterial Enzyme ◽  
Host Strains

Abstract Escherichia coli is one of the most preferred host microorganisms for the production of recombinant proteins due to its well-characterized genome, availability of various expression vectors and host strains. Choosing a proper host strain for the overproduction of a desired recombinant protein is very important because of the diversity of genetically modified expression strains. This study attempted to evaluate the five host strains including BL21 (DE3), Rosetta (DE3), DH5α, XL1-BLUE and SHuffle in terms of arginine deiminase (ADI) production and enzyme activity. Arginine deiminase (ADI) was chosen a bacterial enzyme which degrades L-arginine. It is effective in treatment of some types of human cancers like melanoma and hepatocellular carcinoma (HCC) which are arginine-auxotrophic. Five mentioned E. coli strains were cultivated. The pET-3a was used as the expression vector. The competent E. coli cells were obtained through CaCl 2 method. It was then transformed with the construct of pET3a-ADI using heat shock strategy. The ADI production levels were examined by 10% SDS-PAGE analysis. The ability of host strains for expression of the requested recombinant protein was compared. The enzymatic activity of the obtained recombinant ADI from each studied strain was assessed by a colorimetric 96-well microtiter plate assay. All the five strains exhibited a significant band at 46 kDa. BL21 (DE3) produced the highest amount of ADI protein followed by Rosetta (DE3). The following activity assay showed that ADI from BL21 (DE3) and Rosetta (DE3) had the most activity. There are some genetic and metabolic differences among the various E. coli strains, leading to differences in the amount of recombinant protein production. The results of this study can be used for the efficacy evaluation of the five studied strains for the production of similar pharmaceutical enzymes. The strains also could be analyzed in terms of proteomics.

scholarly journals Genome-Wide Transposon Mutagenesis Reveals a Role for pO157 Genes in Biofilm Development in Escherichia coli O157:H7 EDL933

2010 ◽  
Vol 78 (6) ◽  
pp. 2377-2384 ◽  
Author(s):  
Supraja Puttamreddy ◽  
Nancy A. Cornick ◽  
F. Chris Minion
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Virulence Plasmid ◽  
Microtiter Plate Assay ◽  
Severe Diarrhea ◽  
Wide Range ◽  
Intergenic Regions ◽  
Biofilm Development

ABSTRACT Enterohemorrhagic Escherichia coli O157:H7, a world-wide human food-borne pathogen, causes mild to severe diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. The ability of this pathogen to persist in the environment contributes to its dissemination to a wide range of foods and food processing surfaces. Biofilms are thought to be involved in persistence, but the process of biofilm formation is complex and poorly understood in E. coli O157:H7. To better understand the genetics of this process, a mini-Tn5 transposon insertion library was constructed in strain EDL933 and screened for biofilm-negative mutants using a microtiter plate assay. Ninety-five of 11,000 independent insertions (0.86%) were biofilm negative, and transposon insertions were located in 51 distinct genes/intergenic regions that must be involved either directly or indirectly in biofilm formation. All of the 51 biofilm-negative mutants showed reduced biofilm formation on both hydrophilic and hydrophobic surfaces. Thirty-six genes were unique to this study, including genes on the virulence plasmid pO157. The type V secreted autotransporter serine protease EspP and the enterohemolysin translocator EhxD were found to be directly involved in biofilm formation. In addition, EhxD and EspP were also important for adherence to T84 intestinal epithelial cells, suggesting a role for these genes in tissue interactions in vivo.

scholarly journals Beta-Glucuronidase (GusA) reporter enzyme assay for Escherichia coli

2019 ◽  
Author(s):  
Madeleine Huber ◽  
Jörg Soppa
Keyword(s):  
Escherichia Coli ◽  
Translational Regulation ◽  
Enzyme Assay ◽  
Microtiter Plate ◽  
E Coli ◽  
Reporter Enzyme ◽  
Keio Collection ◽  
Beta Galactosidase ◽  
Transcriptional Fusions ◽  
Microtiter Plate Format

Abstract The beta-Glucuronidase (GusA) is a long-known reporter enzyme for many different species [1]. The E. coli gusA gene is often used in plant research because plants lack an endogenous gusA gene. In E. coli, the transcript of the gusA gene is more stable than that of the highly used reporter gene beta-galactosidase (lacZ) [2]. The GusA activity can be determined using the artificial substrate p-nitrophenyl-β-D-glucopyranosid (pNPG). pNPG is converted to glucoronic acid and para-nitrophenol (pNP), which can be quantified spectrometrically at 405 nm. To avoid background, it is best to use an E. coli strain with a deletion of the chromosomal gusA gene, which is available e.g. at the Keio collection [3]. The gusA gene can be used for transcriptional fusions, e.g. to characterize promoters, and also for translational fusions, e.g. to study translational regulation. The assay was adapted to the microtiter plate format to enable the parallel handling of a large number of samples. The “procedure” (see below) describes an application with the gusA gene in a translational fusion with the gene of interest cloned under the control of the inducible arabinose promoter PBAD.

scholarly journals The Escherichia coli Efflux Pump TolC Promotes Aggregation of Enteroaggregative E. coli 042

2007 ◽  
Vol 76 (3) ◽  
pp. 1247-1256 ◽  
Author(s):  
Naoko Imuta ◽  
Junichiro Nishi ◽  
Koichi Tokuda ◽  
Rika Fujiyama ◽  
Kunihiro Manago ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Efflux Pump ◽  
Microtiter Plate ◽  
Virulence Plasmid ◽  
Wild Type ◽  
Planktonic Cells ◽  
Microtiter Plate Assay ◽  
E Coli ◽  
Aggregative Adherence

ABSTRACT Enteroaggregative Escherichia coli (EAEC) is an emerging enteric pathogen in both developing and industrialized countries. EAEC is defined as a diarrheal pathogen based on its characteristic aggregative adherence to HEp-2 cells in culture and its biofilm formation on the intestinal mucosa. We have reported that the novel protein AatA, which is encoded on the EAEC virulence plasmid pAA2, localizes to the outer membrane and facilitates export of the dispersin Aap across the outer membrane. Because AatA is an E. coli efflux pump TolC homolog, we investigated the role of TolC in the virulence of EAEC. No difference in Aap secretion was observed between the wild type and its tolC mutant (042tolC). However, characteristic aggregation in high-glucose Dulbecco's minimal essential medium for the wild type was diminished for 042tolC. In a microtiter plate assay, there were significantly more planktonic cells for 042tolC than for the wild type, while there were significantly fewer spontaneously precipitated cells on the substratum for 042tolC than for the wild type. In a HEp-2 cell adherence test, 042tolC showed less aggregative adherence than did the wild type. The strong aggregation and aggregative adherence were restored in the complement strain with tolC. In a transwell assay, planktonic cells of 042tolC decreased when cocultured with the wild type or the complement, while precipitated cells of 042tolC increased when cocultured with them. These results suggest that TolC promotes the aggregation and adhesion of EAEC 042 by secreting an assumed humoral factor.

scholarly journals Chemical Composition and Antibacterial Activity of Angelica archangelica Root Essential Oil

2017 ◽  
Vol 12 (2) ◽  
pp. 1934578X1701200 ◽  
Author(s):  
Milica G. Aćimović ◽  
Snežana Đ. Pavlović ◽  
Ana O. Varga ◽  
Vladimir M. Filipović ◽  
Mirjana T. Cvetković ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Antibacterial Activity ◽  
Essential Oil ◽  
Inhibitory Concentration ◽  
Microtiter Plate ◽  
Microtiter Plate Assay ◽  
Natural Preservative ◽  
E Coli ◽  
Angelica Archangelica ◽  
Root Essential Oil

Roots of wild growing Angelica archangelica L. from Mt. Ozren (Serbia) were subjected to hydrodistillation and GC-MS analysis. The roots contained 0.10% of essential oil with α-pinene (29.7%), δ-3-carene (14.2%), and a mixture of β-phellandrene and limonene (13.2%) as main compounds. The modified resazurin microtiter-plate assay was used to evaluate the antibacterial activity of the essential oil against Staphylococcus aureus and Escherichia coli. The minimum inhibitory concentration (MIC) values were 14.2 μL/mL for S. aureus and 28.4 μL/mL for E. coli, while the minimum bactericidal concentrations (MBC) were 56.8 μL/mL and 113.6 μL/mL, respectively. According to the obtained results, the angelica root essential oil can be applied as a natural preservative in food and as a natural antibiotic for the treatment of several infectious diseases caused by these two bacteria.

scholarly journals Pengaruh Ekstrak Alpinia galanga L Terhadap Produksi Biofilm pada Escherichia coli

2021 ◽  
Vol 10 (1) ◽  
pp. 24-30
Author(s):  
Didik Wahyudi ◽  
Syahran Wael
Keyword(s):  
Escherichia Coli ◽  
Optical Density ◽  
Crystal Violet ◽  
Microtiter Plate ◽  
Alpinia Galanga ◽  
One Way Anova

Escherichia coli merupakan bakteri Gram negatif berbentuk batang, mampu menyebabkan infeksi di beberapa bagian tubuh, dan ditemukan telah resisten terhadap berbagai antibiotic, salah satu factor penyebabnya adalah kemampuannya membentuk biofilm pada jaringan. Alpinia gallanga L memiliki kemampuan menghambat pembentukan biofilm terhadap beberapa bakteri. Tujuan Penelitian ini adalah untuk mengetahui kemampuan ekstrak Alpinia galangal L dalam menghambat produksi biofilm Escherichia coli. Penelitian ini di awali dengan ekstraksi rimpang lengkuas dengan etanol menggunakan metode masersi, kemudian dibuat konsentrasi 10%, 20%, 30%, 40%, dan 50%. Escherichia coli diisolasi dari kasus Infeksi Saluran Kemih (ISK). kemudian di lakukan karakterisasi fisiologisnya dan uji kepekaan terhadap antibiotik. Uji penghambatan biofilm Escherichia coli dilakukan dengan metode microtiter plate culture dengan menggunakan spektrofotometer pada panjang gelombang 595nm, Hasil pengukuran produksi biofilm berupa besarnya nilai Optical Density crystal violet 0,1%, setiap perlakuan menggunakan ulangan 8 kali, data yang didapatkan dianalisis dengan One Way Anova. Hasil Penelitian menunjukan bahwa Ekstrak Alpinia galanga L mampu menghambat produksi biofilm Escherichia coli pada konsentrasi 30%.

scholarly journals Identification phenotypic and genotypic characterization of biofilm formation in Escherichia coli isolated from urinary tract infections and their antibiotics resistance

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Elnaz Davari Abad ◽  
Amin Khameneh ◽  
Leila Vahedi
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Urinary Tract Infections ◽  
Biofilm Formation ◽  
Microtiter Plate ◽  
Antibiotics Resistance ◽  
E Coli ◽  
Microbial Characteristics ◽  
Tract Infections ◽  
Resistance Rates

Abstract Objective Urinary tract infections (UTIs) are the most common infectious diseases, and Escherichia coli is the most common pathogen isolated from patients with UTIs. The products of sfa, afa and foc genes are important for binding of the bacterium to urinary tract epithelium. Our aim was to investigate these genes in E. colis isolated from patients with UTIS. The frequencies of the genes were determined using PCR. Biofilm formation and antibiotic resistance rates were determined using microtiter plate and disk diffusion methods, respectively. The P < 0.05 was considered statistically significant. Results The frequencies of sfa, afa and foc were 75.3%, 17.5% and 22.5%, respectively showing a significantly higher prevalence of the sfa gene. The most effective antibiotics against the E. colis were nitrofurantoin and amikacin. The highest microbial resistance rates were also observed against amoxicillin and ampicillin. Furthermore, 12.7%, 6.3%, 74.7% and 6.3% of the isolates showed strong, moderate, weak capacities and no connections to form biofilms, respectively. The expression of the sfa gene was significantly associated with forming strong biofilms. Regarding the variabilities in the characteristics of E. coli strains associated with UTIs, it seems reasonable to adjust diagnostic and therapeutic methods according to the regional microbial characteristics.

scholarly journals Increase in biofilm formation by Escherichia coli under conditions that mimic the mastitic mammary gland

2014 ◽  
Vol 44 (4) ◽  
pp. 666-671 ◽  
Author(s):  
João Carlos Miguel Costa ◽  
Isis de Freitas Espeschit ◽  
Fábio Alessandro Pieri ◽  
Laércio Anjos Benjamin ◽  
Maria Aparecida Scatamburlo Moreira
Keyword(s):  
Escherichia Coli ◽  
Mammary Gland ◽  
Biofilm Formation ◽  
Culture Media ◽  
Control Strategies ◽  
Bovine Mastitis ◽  
Microtiter Plate ◽  
Spectrophotometric Analysis ◽  
Biofilm Production ◽  
Glucose Availability

Bacterial biofilms are involved in the aggravation and recurrence of clinical mastitis in dairy herds. Several factors such as pH, temperature, concentration of O2 and glucose can affect their induction and growth rates. In this study, biofilm production was demonstrated by 27 Escherichia coli strains isolated from bovine mastitis at different pH values depending on the availability of glucose, mimicking conditions found in mammary glands affected by the disease. Biofilm formation was analyzed by spectrophotometric analysis in microtiter plate with 16 different culture media and by scanning electron microscopy. Biofilm formation was greater in isolates cultured under conditions associated with low glucose availability (0.5% or 1.5%) and with either an acidic (5.5) or alkaline (8.5) pH, compared to conditions associated with high glucose availability (2.5% or 3.5%) and near-neutral pH (6.5 or 7.5). Results indicate possible favoring of biofilm production in the later stages of the infectious process caused by E. coli, when the gland environment is less propitious to bacterial growth due to the stress conditions mentioned above; contrasting with the environment of the healthy mammary gland, in which there is no limitation on nutrients or conditions of particular alkalinity or acidity. Thus, knowledge of the stage in which is the infection and environmental conditions of the mammary gland that cause increased production of biofilms is of paramount importance to guide the most appropriate control strategies to prevent relapse after treatment of bovine mastitis, an economically important disease in dairy cattle worldwide.

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