Nonenzymatic protocol for Pseudomonas aeruginosa DNA preparation and rapid subtyping by mini pulsed‐field gel electrophoresis

Abstract

The fastest protocol for Pseudomonas aeruginosa subtyping by contour clamped homogeneous electric field (CHEF) electrophoresis takes around 20 h. It includes enzymatic sample preparation, DNA restriction and fragment separation. Here, P. aeruginosa cells embedded in agarose miniplugs were lysed and deproteinized by incubating the miniplugs for 30 min in a single nonenzymatic solution. DNA molecules were digested for 2 h with 5 U of XbaI, and fragments were separated in 4.96 h by miniCHEF electrophoresis at 10 V/cm. Total time for P. aeruginosa subtyping was 8 h. Control experiments included DNA preparation by enzymatic or nonenzymatic protocols, different times of DNA restriction and comparisons of DNA separations done by miniCHEF or CHEF electrophoresis. Both methods and chambers gave similar results, but the rapid nonenzymatic method and the miniCHEF gave them in less time. Cells grown in broth or on plates were useful for nonenzymatic DNA preparation. Thirteen P. aeruginosa isolates were successfully fingerprinted using the protocol described here.

Cost-effective procedure for Streptococcus pyogenes immobilized DNA preparation and miniPFGE subtyping

“…In our experiments, the SmaI DNA from S. pyogenes resolved in the miniCHEF showed band patterns similar to those previously reported [27,30], but they were obtained 3.8-4.5 times faster due to the fact that the miniCHEF uses greater electric field strength. We were able to reproduce in the miniCHEF the patterns obtained in CHEF conventional chambers by applying equivalent running conditions as had previously been reported [32][33]35]. These results confirm that equations that describe the DNA migration in CHEF [42] were also able to bring the suitable running conditions for rapid DNA fingerprinting of S. pyogenes by miniCHEF electrophoresis.…”

“…DNA suitable for PFGE fingerprinting [32][33]35]. However, an initial step of cell lysis with lytic enzymes (lysozyme or mutanolysin) was necessary for preparing GAS DNA, probably due to the different composition of the cell walls of Gram-positive and Gram-negative bacteria.…”

“…In addition, this procedure yielded patterns with more than 90% of DNA suitable for electrophoresis based on R(%) value calculation (Table 1, Figure 2). This result permitted the selection of the most economical option (lysozyme) in the lysis step and reduced the incubation times, helped by the small thickness of the miniplugs (0.07 cm) [33].…”

“…S. cerevisiae, E. histolytica, P. aeruginosa, V. cholerae, Salmonella, and Aeromonas genomes have been analyzed by CHEF electrophoresis in minichambers, reducing the running time at least twofold compared to the time needed in the CHEF chamber [32][33][35][36][37][38][39]. In this paper, we present a rapid and cost-effective PFGE procedure for subtyping GAS strains in approximately 13 hours after cell harvesting.…”

“…Immobilized DNA of Saccharomyces cerevisiae, Pseudomonas aeruginosa, Vibrio cholerae, Aeromonas, Salmonella, and Streptococcus pneumoniae have been successfully prepared by nonenzymatic methods [31][32][33][34][35]. S. cerevisiae, E. histolytica, P. aeruginosa, V. cholerae, Salmonella, and Aeromonas genomes have been analyzed by CHEF electrophoresis in minichambers, reducing the running time at least twofold compared to the time needed in the CHEF chamber [32][33][35][36][37][38][39].…”