scholarly journals Calculating number of Co-Migrated DNA Fragments across Pulsed Field gel electrophoresis (PFGE) profiles: Image analysis algorithm v2 (protocols.io.bthtnj6n)

protocols.io ◽  
2021 ◽  
Author(s):  
Ibrahim El-khalil
Keyword(s):  
Image Analysis ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Dna Fragments ◽  
Analysis Algorithm ◽  
Pulsed Field ◽  
Image Analysis Algorithm ◽  
Pfge Profiles

scholarly journals Novel algorithms for PFGE bacterial typing: Number of co-migrated DNA fragments, linking PFGE to WGS results and computer simulations for evaluation of PulseNet international typing protocols

2020 ◽  
Author(s):  
Ibrahim-Elkhalil M. Adam ◽  
Isam Abdokashif ◽  
Asia Elrashid ◽  
Hiba Bayoumi ◽  
Ahmed Musa ◽  
...  
Keyword(s):  
Image Analysis ◽  
In Silico ◽  
Pathogenic Bacteria ◽  
Restriction Enzymes ◽  
Dna Fragments ◽  
Evaluation Tool ◽  
Bacterial Typing ◽  
Analysis Algorithm ◽  
Image Analysis Algorithm ◽  
Pfge Profiles

AbstractBackgroundStandard protocols for Pulsed-field gel electrophoresis (PFGE) were adopted and being used in a global scale for surveillance of many bacterial food-borne diseases. Matched PFGE bands are considered regardless of co-migration of different DNA fragments. Molecular epidemiology is turning toward whole genome sequencing (WGS). Although, WGS results can be digested In-silico, PFGE and WGS data are being compared separately. We describe a new image analysis algorithm that enables identification of how many DNA fragments co-migrate during PFGE. We built a database that compare described PFGE results to in-silico obtained digestion models (from WGS). Reliability of the method was assessed in-silico using novel computer simulation approach. From WGS, 1,816 digestion model (DMs) were obtained as recommended by PulseNet international. Simulation codes were designed to predict PFGE profiles when DMs are separated at 5% PFGE resolution in addition to expected co-migration levels.ResultsPFGE simulation has shown that about 35% of DNA fragments co-migrate at 5% PFGE resolution. Similar result was obtained when wet-lab PFGE profiles were analyzed using image analysis algorithm mentioned earlier. When image analysis results were compared to DMs, results returned by geltowgs.uofk.edu database revealed reasonable relatedness to DMs. In terms of number of PFGE typable DNA fragments, 45,517 were typable (representing 46.54% out of 97,801). Previously mentioned typable fragments (in terms of typable sizes) comprised 91.24% of the sum of nucleotides of all chromosomes tested (7.24 billion bp). However, significant variations were shown within and between different digestion protocols.ConclusionsIdentification of co-migration levels will reveal the third dimension of PFGE profiles. This will provide a better way for evaluating isolate relationships. Linking old PFGE results to WGS by means of simulation demonstrated here will provide a chance to link millions of PFGE epidemiological data accumulated during the last 24 years to the new WGS era. Evaluation of population dynamics of pathogenic bacteria will be deeper through space and time. Selection of restriction enzymes for PFGE typing will have a powerful in-silico evaluation tool.

Isolation and Pulsed-Field Gel Electrophoresis Typing of Listeria monocytogenes from Modified Atmosphere Packaged Fresh-Cut Vegetables Collected in Ireland

2006 ◽  
Vol 69 (10) ◽  
pp. 2524-2528 ◽  
Author(s):  
GILLIAN A. FRANCIS ◽  
DAVID O'BEIRNE
Keyword(s):  
Listeria Monocytogenes ◽  
Gel Electrophoresis ◽  
Fruits And Vegetables ◽  
Pulsed Field Gel Electrophoresis ◽  
Leafy Vegetables ◽  
Modified Atmosphere ◽  
Pulsed Field ◽  
Fruit Samples ◽  
Fresh Cut ◽  
Pfge Profiles

The incidence of Listeria monocytogenes in modified atmosphere packaged fresh-cut fruits and vegetables from chill cabinets of a supermarket in Ireland was investigated over a 2-year period. Overall, 9.58% of fresh-cut produce was contaminated with Listeria spp. Various species of Listeria were isolated from samples, including L. monocytogenes, L. seeligeri, L. innocua, L. welshimeri, and L. ivanovii. No fruit samples contained detectable L. monocytogenes. Overall, a total of 21 L. monocytogenes isolates (2.9% of samples) were recovered from a range of products, including dry coleslaw mix (80% shredded cabbage and 20% shredded carrot), bean sprouts, and leafy vegetables such iceberg, romaine, and radicchio lettuce and mixed salad leaves (curly endive, escarole, and radicchio leaves). Dry coleslaw mix appeared to have the highest incidence of Listeria contamination (20%) compared with other products. Listeria contamination was more frequent (P < 0.05) during the summer and autumn months than during the winter and spring months. The 21 L. monocytogenes isolates were subsequently subtyped by genomic macrorestriction techniques using ApaI with pulsed-field gel electrophoresis (PFGE). PFGE of digested DNA produced bands of 79 to 518 kb. Four PFGE profiles were identified, and approximately 50% of the isolates were associated with profile 1. This study indicates that fresh-cut vegetables packaged under a modified atmosphere can support growth of numerous species of Listeria, including L. monocytogenes.

scholarly journals Molecular typing of Escherichia coli O157[ratio ]H7 (H−) isolates from cattle in Japan

1999 ◽  
Vol 122 (2) ◽  
pp. 337-341 ◽  
Author(s):  
M. AKIBA ◽  
T. MASUDA ◽  
T. SAMESHIMA ◽  
K. KATSUDA ◽  
M. NAKAZAWA
Keyword(s):  
Escherichia Coli ◽  
Molecular Typing ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Escherichia Coli O157 ◽  
Outbreak Strain ◽  
Pulsed Field ◽  
E Coli ◽  
Biological Methods ◽  
Pfge Profiles

A total of 77 Escherichia coli O157[ratio ]H7 (H−) isolates from cattle in Japan were investigated by molecular biological methods. Most of these isolates (43 isolates) possessed the stx2 gene, but not stx1. Fifteen bacteriophage types and 50 pulsed-field gel electrophoresis (PFGE) profiles were observed. One isolate was indistinguishable from the human outbreak strain by these methods. This indicates that cattle must be considered as a possible source of human E. coli O157[ratio ]H7 infection in Japan.

scholarly journals Intermediately virulent Rhodococcus equi isolates from pigs in Slovenia: discovery of new plasmid types and assessment of genetic diversity by pulsed-field gel electrophoresis

2009 ◽  
Vol 54 (No. 3) ◽  
pp. 111-117 ◽  
Author(s):  
M. Pate ◽  
M. Ocepek ◽  
I. Zdovc ◽  
C. Minato ◽  
Y. Ohtsu ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Gel Electrophoresis ◽  
Detailed Comparison ◽  
Rhodococcus Equi ◽  
Pulsed Field Gel Electrophoresis ◽  
High Genetic Diversity ◽  
Pulsed Field ◽  
Large Plasmids ◽  
Multiple Strains ◽  
Pfge Profiles

The presence of large plasmids in 30 <I>Rhodococcus equi</I> strains isolated from pig lymph nodes with granulomatous changes was investigated. Plasmid DNAs were isolated and digested with the restriction endonucleases <I>Bam</I>HI, <I>Eco</I>RI, <I>Eco</I>T22I and <I>Hind</I>III for detailed comparison and estimation of plasmid sizes. A total of nine isolates were identified as intermediately virulent (VapB-positive), harbouring large plasmids of type 5 (<I>n</I> = 5) and four new variants that we tentatively designated as type 19 (<I>n</I> = 1), 20 (<I>n</I> = 1), 21 (<I>n</I> = 1) and 24 (<I>n</I> = 1). All isolates were subjected to genotyping with pulsed-field gel electrophoresis (PFGE). High genetic diversity was observed: 21 distinct genotypes were detected; five were found in multiple isolates and the others were unique. Isolates of the same plasmid type exhibited different PFGE profiles and vice versa. In a few cases, multiple strains from certain farms were analysed, the majority of which exhibited diverse PFGE profiles. Our findings demonstrate the presence of a wide variety of <I>R. equi</I> strains even in small confined environments such as farms. This is the first molecular epidemiology study of intermediately virulent <I>R. equi</I> isolates from Slovenian pigs.

Comparison of Danish Isolates of Salmonella entericaSerovar Enteritidis PT9a and PT11 from Hedgehogs (Erinaceus europaeus) and Humans by Plasmid Profiling and Pulsed-Field Gel Electrophoresis

2000 ◽  
Vol 38 (10) ◽  
pp. 3631-3635 ◽  
Author(s):  
B. Nauerby ◽  
K. Pedersen ◽  
H. H. Dietz ◽  
M. Madsen
Keyword(s):  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Phage Type ◽  
Pulsed Field Gel Electrophoresis ◽  
Clonal Lineage ◽  
S1 Nuclease ◽  
Pulsed Field ◽  
Plasmid Profiling ◽  
Susceptibility Patterns ◽  
Pfge Profiles

During the years 1994 to 1998, 10 strains of Salmonella enterica serovar Enteritidis phage type 11 (PT11) and 6 PT9a strains were isolated from Danish hedgehogs, together with 7 strains that did not yield phage susceptibility patterns conforming with any known phage type (routine dilution no conformity [RDNC]). From 1995 to 1998, five Danish patients were reported infected with serovar Enteritidis PT11 and two with PT9a. All serovar Enteritidis PT11, PT9a, and RDNC isolates from hedgehogs and humans were analyzed by pulsed-field gel electrophoresis (PFGE), plasmid profiling, and restriction fragment length polymorphism (RFLP) of plasmids. By use of S1 nuclease and HindIII, the PT11 and PT9a isolates had identical plasmid profiles and RFLP patterns, which differed from the RDNC profiles. The PFGE profiles were identical for all serovar Enteritidis PT11 and PT9a strains from hedgehogs, four of five human strains of serovar Enteritidis PT11, and two human strains of serovar Enteritidis PT9a, irrespective of restriction enzyme, whereas the last human strain deviated slightly when NotI was used but not when XbaI or SpeI was used. The results indicate that serovar Enteritidis PT9a and PT11 are closely related and that PT11 and PT9a from Danish hedgehogs and humans belong to the same clonal lineage.

scholarly journals Pulsed-Field Gel Electrophoresis Profile Changes Resulting from Spontaneous Chromosomal Deletions in Enterohemorrhagic Escherichia coli O157:H7 during Passage in Cattle

2009 ◽  
Vol 75 (17) ◽  
pp. 5719-5726 ◽  
Author(s):  
Noriyo Yoshii ◽  
Yoshitoshi Ogura ◽  
Tetsuya Hayashi ◽  
Takashi Ajiro ◽  
Toshiya Sameshima ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Electrophoresis ◽  
Pulsed Field Gel Electrophoresis ◽  
Enterohemorrhagic Escherichia Coli ◽  
Sequencing Analysis ◽  
Pulsed Field ◽  
Chromosomal Deletions ◽  
Profile Changes ◽  
Dna Sequencing Analysis ◽  
Pfge Profiles

ABSTRACT A total of 905 enterohemorrhagic Escherichia coli (EHEC) O157:H7 isolates that were recovered from experimentally infected cattle, in addition to the inoculated strain, were analyzed by pulsed-field gel electrophoresis (PFGE). Twelve PFGE profiles other than that of the inoculated strain were observed. We successfully identified five distinct chromosomal deletions that affected the PFGE profiles using whole-genome PCR scanning and DNA sequencing analysis. The changes in PFGE profiles of EHEC O157:H7 isolates after passage through the intestinal tract of cattle were partially generated by deletion of chromosomal regions.

scholarly journals Whole-Genome Sequences of Two Listeria monocytogenes Serovar 1/2a Strains Responsible for a Severe Listeriosis Outbreak in Central Italy

2018 ◽  
Vol 6 (24) ◽  
Author(s):  
Massimiliano Orsini ◽  
Alessandra Cornacchia ◽  
Claudio Patavino ◽  
Marina Torresi ◽  
Patrizia Centorame ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Gel Electrophoresis ◽  
Central Italy ◽  
Pulsed Field Gel Electrophoresis ◽  
Single Band ◽  
Whole Genome ◽  
Genome Sequences ◽  
Content Type ◽  
Pulsed Field ◽  
Pfge Profiles

ABSTRACT We report the whole-genome sequences of two Listeria monocytogenes strains responsible for a severe invasive listeriosis outbreak in central Italy that occurred in 2015 and 2016. These two strains differ by a single band in their pulsed-field gel electrophoresis (PFGE) profiles.

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