Re-evaluation, Optimization, and Multilaboratory Validation of the PulseNet-Standardized Pulsed-Field Gel Electrophoresis Protocol for Listeria monocytogenes

Abstract

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

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

“…On the other hand, in our previous experience, the rapid enzymatic protocol for S. pyogenes has the same reproducibility problems as the original PulseNet protocol for L. monocytogenes reported by Halpin et al [23]. The modifications developed in 2010 by Halpin et al [23] to the PulseNet protocol for L. monocytogenes, which mainly were increasing the lysis temperature to 56°C, may also improve the reproducibility of S. pyogenes DNA preparation for PFGE.…”

“…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, the resistance to in situ lysis of the cell wall of Gram-positive bacteria is known [23].…”

“…There are other rapid PFGE protocols reported for subtyping Gram-positive bacteria [19,[23][24][25]34,[43][44][45]. Matushek et al reported a rapid procedure for DNA preparation of Gram-positive bacteria for pulsed field, reducing the incubation times to 4 or 5 hours in some cases.…”

“…The time consumed by the original procedure for GAS DNA preparation was up to two to three days [17]. In recent years, optimized PFGE protocols for bacterial subtyping have reduced the bacterial DNA preparation time to 2-4 hours, but they still involve the use of solutions that contain cell-wall-disrupting enzymes and proteases or huge amounts of restriction enzymes, and some of them do not generate reproducible results of good quality, probably due to inefficient bacterial lysis [18][19][20][21][22][23][24]. As well, the separation of DNA macrorestriction fragments takes between 18 and 24 hours in the CHEF chamber [18][19][20][21][22][23][24].…”

“…In recent years, optimized PFGE protocols for bacterial subtyping have reduced the bacterial DNA preparation time to 2-4 hours, but they still involve the use of solutions that contain cell-wall-disrupting enzymes and proteases or huge amounts of restriction enzymes, and some of them do not generate reproducible results of good quality, probably due to inefficient bacterial lysis [18][19][20][21][22][23][24]. As well, the separation of DNA macrorestriction fragments takes between 18 and 24 hours in the CHEF chamber [18][19][20][21][22][23][24]. Procedures for GAS subtyping have been reported based on these optimized protocols, but in general, they have the same mentioned drawbacks in relation to the DNA preparation and the long electrophoresis times [25][26][27][28][29][30].…”