scholarly journals Sponge-Like: A New Protocol for Preparing Bacterial Ghosts

2013 ◽  
Vol 2013 ◽  
pp. 1-7 ◽  
Author(s):  
Amro A. Amara ◽  
Mounir M. Salem-Bekhit ◽  
Fars K. Alanazi
Keyword(s):  
Gel Electrophoresis ◽  
Chemical Compounds ◽  
Agarose Gel Electrophoresis ◽  
Biotechnological Applications ◽  
E Coli ◽  
Viable Cells ◽  
Inhibition Concentration ◽  
Active Chemical ◽  
Bacterial Ghosts ◽  
First Time

Bacterial Ghosts (BGs) received an increasing interest in the recent years for their promising medicinal and pharmaceutical applications. In this study, for the first time we introduce a new protocol for BGs production.E. coliBL21 (DE3) pLysS (Promega) was used as a model to establish a general protocol for BGs preparation. The protocol is based on using active chemical compounds in concentrations less than the Minimum Inhibition Concentration (MIC). Those chemical compounds are SDS, NaOH, and H2O2. Plackett-Burman experimental design was used to map the best conditions for BGs production. Normal and electronic microscopes were used to evaluate the BGs quality (BGQ). Spectrophotometer was used to evaluate the amount of the released protein and DNA. Agarose gel electrophoresis was used to determine the existence of any residue of DNA after each BGs preparation. Viable cells, which existed after running this protocol, were subjected to lysis by inducing the lysozyme gene carried on pLysS plasmid. This protocol is able to produce BGs that can be used in different biotechnological applications.

scholarly journals Outbreak of KPC-3-producing ST15 and ST348Klebsiella pneumoniaein a Portuguese hospital

2016 ◽  
Vol 145 (3) ◽  
pp. 595-599 ◽  
Author(s):  
D. VUBIL ◽  
R. FIGUEIREDO ◽  
T. REIS ◽  
C. CANHA ◽  
L. BOAVENTURA ◽  
...  
Keyword(s):  
Gel Electrophoresis ◽  
Tertiary Care Hospital ◽  
Tertiary Care ◽  
Pulsed Field Gel Electrophoresis ◽  
Care Hospital ◽  
E Coli ◽  
Carbapenemase Gene ◽  
Sporadic Cases ◽  
First Time ◽  
Sequence Types

SUMMARYTo date, only a few sporadic cases of infections due toKlebsiella pneumoniaecarbapenemase (KPC) producers have been reported in Portugal. Here, we report for the first time an outbreak ofK. pneumoniaeKPC-3 producers in a tertiary-care hospital during 2013. Twenty-seven ertapenem-resistantK. pneumoniaewere identified in patients at a tertiary-care hospital during 2013 isolated predominantly from urine (48·1%) and blood (25·9%) cultures. All isolates were highly resistant toβ-lactam antibiotics and most showed intermediate resistance to imipenem. The more frequentβ-lactamases were TEM- (77·7%), CTX-M- (70·3%) and KPC-type (66·6%). KPC-3 was identified by sequencing. TheblaKPC−3gene was associated with an IncF plasmid, and efficiently transferred toE. coliJ53. Pulsed-field gel electrophoresis typing revealed three clusters of isolates which were further characterized by multi-locus sequence typing as ST11, ST15 and ST348. Ertapenem-resistant ST15 was already in circulation in the hospital, related to expression of OmpK36 modified porin, but the other two sequence types had not been previously found in the hospital. We conclude that the IncF plasmid mediated transfer of KPC-3 in the outbreak and that implementation of carbapenemase gene screening in isolates from patients on admission to hospital is advisable in order to control dissemination of these antimicrobial resistance elements.

scholarly journals Fate of the DNA in plasmid-containing Escherichia coli minicells ingested by human neutrophils

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1394-1400 ◽  
Author(s):  
HB Fox ◽  
P De Togni ◽  
G McMahon ◽  
SB Levy ◽  
JS Robinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Antibiotic Resistance Genes ◽  
Acid Precipitation ◽  
Dna Degradation ◽  
Human Neutrophils ◽  
Agarose Gel Electrophoresis ◽  
Limited Effect ◽  
E Coli

Abstract Escherichia coli minicells containing the plasmid pSC101 (approximately 10 kb) or pBR322 (approximately 4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli: they ingested the minicells, discharged their granule contents into the minicell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.

scholarly journals Temporal Shedding Patterns and Virulence Factors of Escherichia coli Serogroups O26, O103, O111, O145, and O157 in a Cohort of Beef Calves and Their Dams

2004 ◽  
Vol 70 (3) ◽  
pp. 1708-1716 ◽  
Author(s):  
M. C. Pearce ◽  
C. Jenkins ◽  
L. Vali ◽  
A. W. Smith ◽  
H. I. Knight ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Hybridization ◽  
Gel Electrophoresis ◽  
Temporal Pattern ◽  
Immunomagnetic Separation ◽  
Pulsed Field Gel Electrophoresis ◽  
Beef Calves ◽  
E Coli ◽  
First Time ◽  
The Relationship

ABSTRACT This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx 1, eae, and ehl genes, 6.5% carried vtx 1 and vtx 2, and one isolate carried vtx 2 only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx 2, and none carried vtx 1. Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx 1 or vtx 2. All but one serogroup O157 isolate carried vtx 2, eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.

scholarly journals Fate of the DNA in plasmid-containing Escherichia coli minicells ingested by human neutrophils

Blood ◽  
1987 ◽  
Vol 69 (5) ◽  
pp. 1394-1400
Author(s):  
HB Fox ◽  
P De Togni ◽  
G McMahon ◽  
SB Levy ◽  
JS Robinson ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Antibiotic Resistance Genes ◽  
Acid Precipitation ◽  
Dna Degradation ◽  
Human Neutrophils ◽  
Agarose Gel Electrophoresis ◽  
Limited Effect ◽  
E Coli

Escherichia coli minicells containing the plasmid pSC101 (approximately 10 kb) or pBR322 (approximately 4 kb) were opsonized and incubated with human neutrophils. The neutrophils responded to the minicells as they would to native E coli: they ingested the minicells, discharged their granule contents into the minicell-containing phagosomes, and expressed a respiratory burst. After one hour of incubation, the fate of the ingested plasmid DNA was examined. No DNA degradation was detected by trichloroacetic acid precipitation or agarose gel electrophoresis. Moreover, when pBR322 recovered from ingested minicells was transformed into E coli, no mutations in either of the antibiotic resistance genes carried by the plasmid were detected out of many thousand transformants screened. These findings confirm the surprisingly limited effect of neutrophils on ingested DNA.

Characterization and transformation of plasmid pAA-1 found in an antarctic cryptoendolithic bacterium

1996 ◽  
Vol 42 (6) ◽  
pp. 571-576 ◽  
Author(s):  
Christian G. Gliesche ◽  
Marina Jendrach ◽  
Klaus Peissl ◽  
Jörg Siebert ◽  
Peter Hirsch
Keyword(s):  
Plasmid Dna ◽  
Gel Electrophoresis ◽  
Natural Transformation ◽  
Restriction Map ◽  
Agarose Gel ◽  
Dry Valleys ◽  
Agarose Gel Electrophoresis ◽  
Bacterial Isolates ◽  
E Coli ◽  
Natural Genetic Transformation

Sixty-three bacterial isolates from antarctic sandstone samples (Linnaeus Terrace, Asgard Range, McMurdo Dry Valleys) were screened for the presence of plasmid DNA. Twenty-seven percent of all the isolates (mainly Gram-positives) harbored one or more plasmids of low molecular mass (1.1–16.3 kb). Strain AA-341 contained plasmid pAA-1 (2.9 kb), as demonstrated by agarose gel electrophoresis and restriction endonuclease digests. This plasmid conferred resistance to chromium and ampicillin. It was not conjugative, but it could be transferred by electroporation to chromium- and ampicillin-sensitive strains AA-330, AA-338, and E. coli HB101. A restriction map of pAA-1 was constructed with HindIII, SalI, ScaI, AvaII, EcoRI, PvuII, BamHI, and DraI. Electrotransfer of this plasmid from E. coli HB101(pAA-1) to strain AA-330 was demonstrated. By natural genetic transformation, plasmid pAA-1 could be transferred into the sensitive strain AA-334, which thereby became resistant to chromium and ampicillin. The importance of such processes for the colonization of stressed environments is discussed.Key words: Antarctica, cryptoendolithic bacteria, plasmids, resistance to chromium, natural transformation.

scholarly journals Curing of Escherichia coli Antibiotic Resistance Plasmids by Aspirin

2014 ◽  
Vol 8 (1) ◽  
pp. 30-35
Author(s):  
Ibtesam H. Al Musawi ◽  
Ali A. Al Zaag
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Large Plasmid ◽  
Agarose Gel Electrophoresis ◽  
E Coli ◽  
Resistance Plasmids ◽  
Minimum Number

An isolate of Escherichia coli (E. coli) was isolated from urine sample due to person infected with urinary tract infection (UTI).The isolate was resistant to the following antibiotics: Ampicillin, Cotrimoxazole, Chloramphenicol and Tetracycline. Agarose gel electrophoresis of its plasmids content has revealed the presence of single large plasmid and two small plasmids bands. The large plasmid was conjugative and contained the resistance genes for four antibiotics. In vitro curing of this plasmid was achieved by treatment with salicylic acid (aspirin) with 150,200,250 and 300 µg/ml as indicated by the elimination of resistance, also by absence of large plasmid band following agarose gel electrophoresis. In vivo curing was conducted using New Zealand rabbits. UTI was induced by bacterial inoculation via urethral catheterization. The E. coli from urine samples of the rabbits, the count of which was proportional to the type of treatment. Minimum number of colonies was associated with group treated with metheprim was aspirin 300mg/kg daily dosage. This result may indicate that the side effect of metheprim was in its maximum with aspirin. Survivor bacteria may indicate incomplete exposure to the drug.

scholarly journals Enhancement of Recombinase-Aided Amplification (Raa) Assay by Betaine and Pullulan

2021 ◽  
Author(s):  
Jinrong Wang ◽  
Guowei Song ◽  
Yue Ming ◽  
Jing Pan ◽  
Ruiqing Zhang ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Gel Electrophoresis ◽  
Nucleic Acid Amplification ◽  
Basic Method ◽  
Clinical Samples ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Fluorescence Methods ◽  
Threshold Time ◽  
First Time

Abstract In this research, nucleic acid amplification enhancers suitable for recombinase-aided amplification (RAA) assay were studied for the first time, and amplification of a long-fragment (509 bp) by the RAA assay was initially explored. Using recombinant plasmids and clinical samples, RAA fluorescence and basic methods were used to evaluate the efficacy. The fluorescence method was evaluated by threshold time (TT) and fluorescence value, and the basic method was interpreted by 2% agarose gel electrophoresis. Taking a previously established RAA assay for HPV18 as an example, we demonstrated that the addition of 0.2, 0.4, and 0.6 M betaine and 10% pullulan could enhance RAA. The new RAA assays with betaine and pullulan were called B-RAA and P-RAA, respectively. In the B-RAA and P-RAA fluorescence methods, TT values could be shortened by 1.72–2.32 and 2.60 minutes, respectively, and fluorescence values could be enhanced by 8847.25–9094.37 and 5250 mv, respectively. In the basic method, sensitivity could be increased by 10-fold. We successfully amplified a long-fragment of 509 bp in a P-RAA assay with a sensitivity of 102 copies/μL (compared with 103 copies/μL in the RAA assay). We thus conclude that betaine and pullulan are effective additives to enhance RAA assays.

scholarly journals Genetic study of Salmonella spp. Producting Betalatimase (ESBLs)

2010 ◽  
Vol 7 (1) ◽  
pp. 254-260
Author(s):  
Baghdad Science Journal
Keyword(s):  
Genetic Study ◽  
Gel Electrophoresis ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Biochemical Tests ◽  
Salmonella Spp ◽  
Small Plasmid ◽  
E Coli ◽  
Extended Spectrum ◽  
Synergy Test

Ten isolates were collected from different clinical sources from laboratory in medicine century . These isolates were belonging to the genus Salmonella depending on morphological and biochemical tests . The antibiotic scussptibility tests against 10 antibiotics were examined , and it was found that the 60% isolates have multiple resistant to antibiotic ,(70%) of isolates were resistant to ampicillin,(50%) were resistant to augmentin ,(40%) were resistant to ceftriaxone ,(20%) were resistant to cefotaxime and (10%) were resistant to ciprofloxacin and tetracycline while all isolates showed sensitivity to piperacillin, imipenem, amikacin and erythromycin .The ability of Salmonela isolates to produce ?-lactamase enzymes were tested using iodometric method , and the results showed that all isolates produced this enzyme.The ability of these isolates to produce Extended-Spectrum ?-lactamase (ESBLs)were also determined by double disc synergy test , only five isolates produced these enzyme. Agarose gel electrophoresis showed that Salmonella isolates ?-lactamase producer have two small plasmid bands . Transformation experiments revealed that these plasmids were capable to transform E. coli MM294, an observation which indicates the ability of these plasmids to show their expression in more than one host.

Luminography – an Alternative Assay for Detection of von Willebrand Factor Multimers

1988 ◽  
Vol 60 (02) ◽  
pp. 133-136 ◽  
Author(s):  
R Schneppenheim ◽  
H Plendl ◽  
U Budde
Keyword(s):  
Von Willebrand Factor ◽  
Gel Electrophoresis ◽  
Detection Methods ◽  
Agarose Gel ◽  
Agarose Gel Electrophoresis ◽  
Luminescence Assay ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryA luminescence assay was adapted for detection of von Willebrand factor multimers subsequent to SDS-agarose gel electrophoresis and electroblotting onto nitrocellulose. The method is as fast as chromogenic detection methods and appears to be as sensitive as autoradiography without the disadvantages of the latter.

Sign in / Sign up

Export Citation Format

Share Document