Exploring PFGE for Detecting Large Plasmids in Campylobacter jejuni and Campylobacter coli Isolated from Various Retail Meats

Abstract

Campylobacter spp. is one of the most prevalent bacterial pathogens in retail meat, particularly poultry, and is a leading cause of diarrhea in humans. Studies related to Campylobacter large plasmids are limited in the literature possibly due to difficulty in isolating them using available alkaline lysis methods. The objectives of this study were to determine the prevalence of plasmids, particularly large ones, in Campylobacter spp. isolated from various Oklahoma retail meats, and to explore PFGE (Pulsed Field Gel Electrophoresis) as a tool in facilitating the detection of these plasmids. One hundred and eighty nine strains (94 Campylobacter jejuni and 95 Campylobacter coli) were screened for the presence of plasmids using both alkaline lysis and PFGE. Plasmids were detected in 119/189 (63%) using both methods. Most of the plasmids detected by alkaline lysis were smaller than 90 kb and only three were larger than 90 kb. Plasmids over 70 kb in size were detected in 33 more strains by PFGE of which 11 strains contained larger than 90 kb plasmids. Plasmids were more prevalent in Campylobacter coli (73.5%) than in Campylobacter jejuni (52%). BglII restriction analysis of plasmids isolated from 102 isolates revealed 42 different restriction patterns. In conclusion, PFGE was able to detect large plasmids up to 180 Kb in Campylobacter spp. which might have been missed if the alkaline lysis method was solely used. Campylobacter spp. isolated from retail meats harbor a diverse population of plasmids with variable sizes. To our knowledge, this is the first study to use PFGE to detect large plasmids in Campylobacter.

Why Close a Bacterial Genome? The Plasmid of Alteromonas Macleodii HOT1A3 is a Vector for Inter-Specific Transfer of a Flexible Genomic Island

“…PFGE of the extracted DNA was performed using CHEF DRIII device with 1% agarose gels and 0.5X TBE buffer at 14 o C for 11.5 h. Lambda ladder PFGE marker, low range ladder PFGE marker and 1 KB DNA ladder marker were used as molecular size markers. The gel was stained using GelRed stain (Biotum, Hayward, CA 94545) and photographed over an UV transilluminator ( López-Pérez et al, 2012 ; Marasini and Fakhr, 2014 ; Ozdemir and Acar, 2014 ).…”

Molecular Characterization of Staphylococcus aureus Plasmids Associated With Strains Isolated From Various Retail Meats

“…Large plasmids (>60 kb) were screened by PFGE using protocols supplied by the CDC Pulse Net (McDougal et al, 2003;Marasini and Fakhr, 2014). Strains were grown in Typtic Soy Agar (TSA) (Himedia, Mumbai, India) at 37 • C for 16-18 h, harvested by centrifugation, and resuspended in cell suspension buffer (CSB) (100 mM Tris, 100 mM EDTA, PH 8.0) to an OD of 0.9-1.1 at 610 nm.…”

“…After a 4-h incubation at 37 • C, the EC lysis buffer was decanted and TE buffer (4 ml) was added; tubes were then agitated for 30 min at room temperature. The buffer was removed, and the washing was repeated three more times; after the final wash, 4 ml of TE buffer was added and samples were stored at 4 • C. A small section was excised from the plugs and linearized with S1 nuclease as described previously (Marasini and Fakhr, 2014). The plugs were inserted into 1% agarose wells in TBE buffer and electrophoresed with XbaI Salmonella serovar Braenderup H9812 for 14 h in 0.5 X TBE as described (Marasini and Fakhr, 2014).…”

Versatile multi-transgene expression using improved BAC TG-EMBED toolkit, novel BAC episomes, and BAC-MAGIC

“…This supercoiling generates torsional stress which is relieved by local formation of single-stranded regions. Thus, S1 nuclease has been used to cut these single-stranded regions and linearize circular DNA episomes (99–101). After S1 digestion, DNA from the cell clones carrying BAC episomes now showed DNA smears with peak intensities of ∼900 kb and 1 Mb for the DHFR-UG-s3 and HBB-UG-100d3 cell lines, respectively (Figure 6b and Supplementary Figure S3a-b).…”