scholarly journals Control of pH during plasmid preparation by alkaline lysis of Escherichia coli

2008 ◽  
Vol 378 (2) ◽  
pp. 224-225 ◽  
Author(s):  
Cheri Cloninger ◽  
Marilyn Felton ◽  
Bonnie Paul ◽  
Yasuko Hirakawa ◽  
Stan Metzenberg
Keyword(s):  
Escherichia Coli ◽  
Alkaline Lysis ◽  
Plasmid Preparation

scholarly journals Horizontal Transfer of Antimicrobial Resistance by Extended-Spectrum β Lactamase-Producing Enterobacteriaceae

2011 ◽  
Vol 3 (01) ◽  
pp. 037-042 ◽  
Author(s):  
Varsha K Vaidya
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Resistance Genes ◽  
Environmental Conditions ◽  
Horizontal Transfer ◽  
Hospital Setting ◽  
Alkaline Lysis ◽  
Antibiotic Resistant ◽  
Extended Spectrum ◽  
Synergy Test

ABSTRACT Background: The purpose of this work was to study the acquisition of new antibiotic-resistant genes carried by extended spectrum β-lactamase (ESBL)-producing Enterobacteriaceae via horizontal transfer to understand their rampant spread in the hospitals and in the community. Materials and Methods: A retrospective analysis of 120 ESBL screen-positive isolates of Escherichia coli and Klebsiella pneumoniae, which were subjected to antimicrobial susceptibility testing, was carried out. The Double Disc Synergy Test (DDST) and Inhibitor-Potentiation Disc Diffusion Test (IPDD) were employed for confirmation of ESBL activity. The transferability of the associated antibiotic resistance for amoxicillin, amikacin, gentamicin, cefotaxime and ceftriaxone was elucidated by intra- and intergenus conjugation in Escherichia coli under laboratory as well as under simulated environmental conditions. Transformation experiments using plasmids isolated by alkaline lysis method were performed to study the transferability of resistance genes in Klebsiella pneumoniae isolates. Results : ESBL production was indicated in 20% each of the Escherichia coli and Klebsiella pneumoniae isolates. All the ESBL isolates showed co- resistance to various other groups of antibiotics, including 3GC antibiotics, though all the isolates were sensitive to both the carbapenems tested. Conjugation-mediated transfer of resistance under laboratory as well as environmental conditions at a frequency of 3-4 x 10-5 , and transformation-mediated dissemination of cefotaxime and gentamicin resistance shed light on the propensity of ESBL producers for horizontal transfer. Conclusions: The transfer of resistant markers indicated availability of a large pool of resistance genes in the hospital setting as well as in the environment, facilitating long-term persistence of organisms.

scholarly journals PREVALENCE OF EXTENDED SPECTRUM BETA-LACTAMASES (ESBLS) PRODUCING ESCHERICHIA COLI AND KLEBSIELLA PNEUMONIAE AMONG HOSPITALIZED PATIENTS FROM NIGERIA

2021 ◽  
Vol 5 (2) ◽  
pp. 584-595
Author(s):  
Olivia Sochi Egbule ◽  
Bernard O. Ejechi
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
General Hospital ◽  
Hospitalized Patients ◽  
Diffusion Method ◽  
Alkaline Lysis ◽  
Beta Lactamases ◽  
Resistance Patterns ◽  
Extended Spectrum Beta Lactamases ◽  
Synergy Test

The aim of this study was to determine the resistance patterns and ESBLs production among clinical isolates of Escherichia coli and Klebsiella pneumoniae in two government hospitals of Delta State, Nigeria. Urine, blood and wound samples were aseptically collected from hospitalized patients, bacteriologically processed and isolates identified using standard protocols. Antimicrobial susceptibility testing was determined by disc diffusion method. The plasmid DNA of Multidrug resistance (MDR) isolates were extracted by alkaline lysis method. Phenotypic ESBL production of the MDR isolates was done by Double Disc Synergy Test (DDST) while PCR was used to detect blaCTX-M, blaSHV and blaTEM among isolates. A total of 217 isolates were obtained, of which 161(74.2%) and 56(25.8%) were Escherichia coli and Klebsiella pneumoniae respectively. The antimicrobial resistance varied from one location to another. All isolates obtained from blood of general hospital Warri (GHW) were 100% resistant to amoxicillin clavulanic acid and the cephalosporins (ceftazidime, cefotaxime, and cefuroxime). Isolates from General hospital Agbor (GHA) showed high resistance of 75.0% to cefotaxime, 93.8% to each of ceftazidime and cefuroxime. Overall low resistance to nitrofurantoin was observed in E. coli isolates obtained from urine of GHW (27.5%) and GHA (20.8%). Out of 217 isolates, 75.1% (163/217) were MDR, of which 36.8% and 39.3% produced ESBL by DDST and PCR respectively. The most common ESBL gene was blaCTX-M expressed by 28(17.2%) of the isolates. The high prevalence of MDR and ESBL underscores the need for a continuous local monitoring of antibiotic resistance.    

scholarly journals Automated Filtration-Based High-Throughput Plasmid Preparation System

1999 ◽  
Vol 9 (5) ◽  
pp. 463-470 ◽  
Author(s):  
Masayoshi Itoh ◽  
Tokuji Kitsunai ◽  
Junichi Akiyama ◽  
Kazuhiro Shibata ◽  
Masaki Izawa ◽  
...  
Keyword(s):  
High Throughput ◽  
Large Scale ◽  
Microtiter Plate ◽  
Linear Process ◽  
Alkaline Lysis ◽  
Filtration Method ◽  
Cell Harvesting ◽  
Automated Processing ◽  
Plasmid Preparation ◽  
Plasmid Purification

Current methods of plasmid preparation do not allow for large capacity automated processing. We have developed an automated high-throughput system that prepares plasmid DNA for large-scale sequencing. This system is based on our previously reported filtration method. In this method, cell harvesting, alkaline lysis, and plasmid purification occur in a single 96-well microtiter plate from which sequence-ready DNA samples are collected. The plates are designed to allow all reagents to be injected from above the wells and the spent reagents to be aspirated from below. This design has enabled us to build a linear process plasmid preparation system consisting of an automated filter plate stacker and a 21-stage automated plasmid preparator. The 96-well plates used are outfitted with glass-filters that trap Escherichia coli before the plates are stacked in the automated stacker. The plates move from the stacker to each of the 21 stages of the preparator. At specific stages, various reagents or chemicals are injected into the wells from above. Finally, the plates are collected in the second stacker. The optimal throughput of the preparator is 40,000 samples in 17.5 hr. Here, we describe a pilot experiment preparing 15,360 templates in 160 specially designed 96-well glass-filter plates. The prepared plasmids were subjected to restriction digestion, DNA sequencing, and transcriptional sequencing.

Comparison of Alkaline Lysis with Electroextraction and Optimization of Electric Pulses to Extract Plasmid DNA from Escherichia coli

2013 ◽  
Vol 246 (11) ◽  
pp. 861-867 ◽  
Author(s):  
Saša Haberl ◽  
Marko Jarc ◽  
Aleš Štrancar ◽  
Matjaž Peterka ◽  
Duša Hodžić ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasmid Dna ◽  
Alkaline Lysis ◽  
Electric Pulses

scholarly journals Antibiotic Sensitivity Pattern and Plasmid Profile of Bacteria Isolated from Diabetic Ulcers in Mbano Metropolis, Imo State, Southeastern Nigeria

2021 ◽  
Vol 6 (1) ◽  
pp. 38-46
Author(s):  
E.O Nwankwo ◽  
◽  
E. E. Nwagbara ◽  
K.N. Onusiriuka
Keyword(s):  
Escherichia Coli ◽  
Plasmid Profile ◽  
Klebsiella Pneumonia ◽  
Diabetic Patients ◽  
Southeastern Nigeria ◽  
Alkaline Lysis ◽  
Diabetic Ulcers ◽  
E Coli ◽  
Microbiological Methods ◽  
Sensitivity Pattern

The study was undertaken to evaluate the bacteriology and antibiogram of isolates from diabetic patients with chronic foot ulcers in Nigeria. A total of 150 pus samples were collected and processed according to standard aerobic and anaerobic microbiological methods. Antibiogram was done using Kirby-Bauer method. Biofilm tests, ESBL & AmpC production was conducted using Congo red agar, Double disc synergy test and Cefoxitin disc test respectively. Total number of isolates obtained was 210. The Plasmid profiles of some of the Multi-Drug Resistance (MDR) isolates were carried out using the alkaline lysis method for plasmid extraction and electrophoresis on agarose gel with standard markers. The most frequently isolated aerobic organism in the study was Escherichia coli (32.1%) while the least occurring was Enterobacter spp (1.57%). For the anaerobes, Peptostreptococcus spp (40%) was the highest isolated bacterium. Percentage of Extended Spectrum -lactamase ( ESBL) producers among E. coli isolates was 44%. Percentages of biofilm formation potential among the isolates were: E. coli (36.8%), S. aureus (23.1%) and Proteus vulgaris (4.2%). Escherichia coli and S. aureus showed considerable levels of resistance to some common antibiotics. No methicilin resistant S. aureus was encountered. AmpC producers encountered were Klebsiella pneumonia (10%) and E. coli (8.1%). Post-curring antibiogram tests revealed that nine isolates carried plasmids, suggesting that the mode of resistance may be plasmid mediated. Keywords: Diabetic ulcers, Bacteria, Antibiogram, Plasmid profile

scholarly journals OPTIMASI METODE LISIS ALKALI UNTUK MENINGKATKAN KONSENTRASI PLASMID

2015 ◽  
Vol 2 (2) ◽  
pp. 60
Author(s):  
Dudi Hardianto ◽  
Alfik Indarto ◽  
Nurtjahjo Dwi Sasongko
Keyword(s):  
Escherichia Coli ◽  
Molecular Biology ◽  
Low Cost ◽  
Cesium Chloride ◽  
Alkaline Lysis ◽  
Isolation Methods ◽  
Plasmid Isolation ◽  
Biology Laboratory

Plasmids are extra chromosomal molecules of DNA that replicate autonomously and found in prokaryote and eukaryote cells. There are a number of methods that are used to isolate plasmids, such as alkaline lysis, boiling lysis, using cesium chloride, and chromatography. Amongst the disadvantages in plasmid isolation methods are lengthy time especially when handling a large number of samples, high cost, and low purity. Alkaline lysis is the most popular for plasmid isolation because of its simplicity, relatively low cost, and reproducibility. This method can be accomplished in 50 minutes to one hour. In this research, the alkaline lysis method was developed to obtain suitable plasmid for applications in a molecular biology laboratory. The aim of this research was to reduce contaminants and improve yield of plasmid. The result of isolation of pICZA plasmid in Escherichia coli gave the concentration of 3.3 to 3.8 µg/µL with the purity of 1.99.Keywords: Plasmid isolation, pICZ A, Escherichia coli, rapid, alkaline lysis  ABSTRAKPlasmid merupakan molekul DNA ekstrakromosomal yang bereplikasi secara mandiri dan ditemukan dalam sel prokariot dan eukariot. Banyak metode yang digunakan untuk isolasi plasmid, seperti: lisis alkali, lisis dengan pemanasan, penggunaan sesium klorida, dan kromatografi. Kelemahan beberapa metode isolasi DNA adalah waktu isolasi yang lama terutama saat isolasi plasmid dalam jumlah banyak, mahal dan kemurniannya yang rendah. Metode lisis alkali merupakan metode yang sangat umum untuk isolasi plasmid karena mudah dilakukan, relatif murah, dan reprodusibilitas. Metode ini dapat dilakukan dalam 50 menit sampai 1 jam. Pada penelitian ini dikembangkan metode lisis alkali untuk memperoleh plasmid yang sesuai untuk penggunaan di laboratorium biologi molekuler. Tujuan dari penelitian ini adalah untuk mengurangi jumlah kontaminan dan meningkatkan konsentrasi plasmid. Hasil isolasi plasmid pICZA dalam Escherichia coli mempunyai konsentrasi antara 3,3 sampai 3,8 µg/µL dan kemurniannya 1,99.Kata Kunci: Isolasi plasmid, pICZ A, Escherichia coli, cepat, lisis alkali

Pulsed-field gel electrophoresis typing, antibiotic resistance, and plasmid profiles of Escherichia coli strains isolated from foods

2012 ◽  
Vol 58 (11) ◽  
pp. 1278-1287 ◽  
Author(s):  
Ahmet Uysal ◽  
Yusuf Durak
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Gel Electrophoresis ◽  
Bacterial Contamination ◽  
Pulsed Field Gel Electrophoresis ◽  
Diffusion Method ◽  
Alkaline Lysis ◽  
Pulsed Field ◽  
E Coli ◽  
Plasmid Profiles

Bacterial contamination in foods and antimicrobial resistance levels of common pathogenic strains causing food-borne disease are important in human health. Thus, typing technologies are important tools to determine primary sources of bacterial contamination. In this study, 40 Escherichia coli strains isolated from 85 food samples were evaluated in terms of genetic diversity, susceptibility to certain antibiotics, and plasmid profiles. Pulsed-field gel electrophoresis was used to identify the genetic relations of E. coli isolates. It was determined that the 40 E. coli strains revealed 32 different pulsotypes represented by 6 subtypes. Antibiotic susceptibility tests conducted by using a disc diffusion method against 15 antibiotics showed that although the isolates revealed 14 different types of resistance profiles, the strains showed the greatest resistance to ampicillin (77.5%), followed by ticarcillin–clavulanic acid (30%), tetracycline (22.5%), and cephalothin (14.5%). Plasmid isolations studies of the strains conducted by the method of alkaline lysis revealed that 18 (45%) of 40 E. coli strains contain 31 different plasmid bands ranging between 64.4 and 1 kb. The results showed that PFGE was a powerful method in tracking sources of food contamination and that the antibiotic resistance levels of food isolates were high and should be monitored.

scholarly journals Plasmid Profiling and Occurrence of β-lactamase Enzymes in Multi-drug Resistant Uropathogenic Escherichia coli in Kathmandu, Nepal

2019 ◽  
Author(s):  
Sabnum Shrestha ◽  
Nabaraj Adhikari ◽  
Komal Raj Rijal ◽  
Basudha Shrestha ◽  
Bipin Adhikari ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urinary Tract ◽  
Phenylboronic Acid ◽  
Tertiary Hospital ◽  
Urine Samples ◽  
Drug Resistant ◽  
Alkaline Lysis ◽  
E Coli ◽  
Plasmid Profiling ◽  
Tract Infections

Abstract Background Emergence of Extended Spectrum β-Lactamases (ESBL) among gram-negative bacteria, predominantly Escherichia coli in Nepal has been alarming. The main objectives of this study were to determine the prevalence of ESBL, ABL (AmpC type β-Lactamase), MBL (Metallo β-Lactamase) and KPC (Klebseilla pneumoniae carbapenamase) producing multi-drug resistant uropathogenic E. coli and their correlation with plasmid profiling pattern among the patients suspected of or having urinary tract infection in a tertiary hospital in Kathmandu, Nepal. Methods The mid-stream urine samples were collected from patients suspected of or having urinary tract infections (UTI) and were inoculated in Cystine Lactose electrolyte deficient (CLED) agar. The ESBL E. coli were detected by combined disc diffusion technique using cefotaxime with and without clavulanate. The isolates were screened for ABL by inhibitor-based method using cefoxitin alone and/or with cloxacillin. MBL/KPC were detected using combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA) or Ethylene diamine tetra acetic acid (EDTA), or both PBA and EDTA. Plasmids were extracted by alkaline lysis method from isolates and profiled on agarose gel electrophoresis. Results Out of total 2,661 urine samples, E. coli were isolated in 64.5% (507/788), among which 170 (33.5%) were MDR isolates. All MDR isolates were resistant to amoxicillin and third generation cephalosporins but were highly sensitive to imipenem (94.12%, 160/170), amikacin (92.94%, 158/170) and nitrofurantoin (86.47%, 147/170). Among 170 MDR isolates, 78.2% (133/170) were ESBL, 46.3% (50/170) were AmpC, 11.2% (19/170) were MBL and 0.6% (1/170) was KPC producers. Coproduction of β-Lactamases was detected in 24.12% (41/170) of isolates. E. coli isolates showed one plasmid (>33.5 Kb) which was unanimously present in all the isolates. Overall, 44 different plasmid profile groups were identified based on the molecular weight and number of plasmids. β-lactamase producers were relatively resistant to higher number of antibiotics (≤10) than non-producer (≤8) and number of plasmids were higher in β-lactamase producers (≤7) than non-producers (≤5).

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