The Use of Pulsed Field Gel Electrophoresis in Listeria monocytogenes Sub-Typing - Harmonization at the European Union Level

Félix, Benjamin; Dao, Trinh Tam; Lombard, Bertrand; Brisabois, Adrien Assere Anne; Roussel, Sophie

doi:10.5772/38578

Cited by 13 publications

(14 citation statements)

References 21 publications

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“…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.…”

Section: Discussionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

“…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].…”

Section: Introductionmentioning

confidence: 99%

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

López

Suárez

Niubó

et al. 2015

J Infect Dev Ctries

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Introduction: Group A streptococci (GAS) is responsible of several human diseases ranging from mild infection to severe invasive toxinmediated disease and post-infectious sequelae. Accordingly, a GAS surveillance program based on molecular techniques is advisable for its epidemiological control. Pulsed-field gel electrophoresis (PFGE) is the gold standard for GAS molecular subtyping, but a major disadvantage is the length of the procedure, which takes 1-3days of work, minimum. The aim of this study was to develop a rapid and cost-effective procedure for PFGE subtyping of GAS isolates. Methodology: Different incubation times of GAS, immobilized in agarose miniplugs, in solutions containing lysozyme and/or mutanolysine followed by solutions with urea instead of proteinase K, were assayed. DNA was restricted with SmaI and the fingerprints were obtained in clamped homogeneous electric field (CHEF) chambers and minichambers. The modified procedure was used to subtype 22 GAS isolates. Results: Intact DNA molecules of GAS immobilized in agarose miniplugs were prepared incubating the cells, in situ, with a solution containing lysozyme for 4hours, followed by the incubation in a non-enzymatic solution with urea for 2hours. SmaIDNA macrorestriction fragments were well resolved in 5hours and 14minutes by electrophoresis in a CHEF minichamber at 10V/cm. This procedure for GAS DNA preparation was useful for fingerprinting GAS strains in the format of CHEFMapper (BioRad). Conclusions: The procedure took 13 hours for GAS strains subtyping. Both sample preparation and electrophoresis in CHEF minichamber represent an economic alternative for performing massive epidemiological studies of this human pathogen.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

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

López

Suárez

Niubó

et al. 2015

J Infect Dev Ctries

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“…Apa I/ AscI -PFGE is routinely used at the EURL for the surveillance of food, animals and environmental isolates at the national and European level [14,15]. One of the main EURL activities is to develop or/and evaluate and keep up to date with new molecular subtyping methods and deploy them through the European NRL network.…”

Section: Introductionmentioning

confidence: 99%

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping

Roussel

Félix

Grant³

et al. 2013

BMC Microbiology

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BackgroundListeriosis is a severe infection which mainly affects pregnant women, neonates and immuno-compromised adults. ANSES’s Laboratory for Food safety has been the European Union Reference Laboratory (EURL) for L. monocytogenes in the food chain since 2006. Pulsed Field Gel Electrophoresis (PFGE) is routinely used in the EURL for the surveillance of L. monocytogenes isolated from foods, animals and the environment. One of the main EURL activities is to evaluate alternative molecular subtyping methods to PFGE, and integrate their use within the National Reference Laboratories (NRL) network. Since 2008, the United Kingdom (UK)-NRL for L. monocytogenes at the Health Protection Agency (HPA), London, has used fluorescent Amplified Fragment Length Polymorphism (fAFLP) for the routine surveillance of L. monocytogenes isolated from human clinical cases, food and food processing environments in the UK. This study compares fAFLP with PFGE for subtyping L. monocytogenes.ResultsA panel of 109 L. monocytogenes isolates from either human cases of listeriosis, foods, food processing environments and animals were used for the comparative evaluation. Among these, 2 strains were tested from duplicate culture by both methods. The panel also included field isolates, isolates associated with outbreaks or sporadic cases and reference strains. The two strains tested in duplicate displayed the same fAFLP and PFGE types. Strains known to be epidemiologically associated with one another were found to have unique PFGE and fAFLP types. FAFLP and PFGE divided the strains into 76 and 82 distinct profiles, or types, respectively. The discriminatory index calculated was 0.993 and 0.996 for fAFLP and PFGE, respectively.ConclusionsThe discriminatory ability of fAFLP was similar to that of PFGE for the subtyping of L. monocytogenes isolates. As a less labour intensive technique fAFLP may be a better method to use than PFGE in investigating outbreaks of human listeriosis and tracking the source of contamination in food processing facilities in real time.

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“…The "gold standard" for molecular subtyping for foodborne pathogens (e.g., Salmonella , Shigella , pathogenic E. coli , Campylobacter , Yersinia , Vibrio , and Listeria monocytogenes ) used by the federal government and internationally is PFGE (Felix et al 2012 ;Fields et al 2011 ;Peters 2009 ), despite the relative abundance of other approaches, many of which have been evaluated, utilized by CDC, and shown to have superior discriminatory power over PFGE. For example, MLVA was used along with PFGE in an outbreak investigation associated with peanut butter ( http://www.cdc.gov/salmonella/typhimurium/strains_table.html ).…”

Section: Approaches Used By Government Agenciesmentioning

confidence: 99%

Processing Plant Investigations: Practical Approaches to Determining Sources of Persistent Bacterial Strains in the Industrial Food Processing Environment

Kornacki¹

2014

The Microbiological Safety of Low Water Activity Foods and Spices

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Persistent pathogenic bacterial strains may result from many sources, such as repetitively contaminated ingredients brought into the plant environment, unrepaired roof leaks, and workers routinely entering the plant from agricultural or other contaminated areas, as well as from a microbial population that has established itself in the plant environment, perhaps originating from one or more of these venues. Pathogenic bacteria may also adapt to dry conditions and survive for years under these conditions, including within the production environment and in dry raw ingredients, a phenomena documented to occur with strains of Salmonella . These can be very diffi cult situations to effectively investigate and resolve, made even worse by certain false paradigms employed by investigators, Molecular subtyping approaches to identify the occurrence of persistent strains are usually required.

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The Use of Pulsed Field Gel Electrophoresis in Listeria monocytogenes Sub-Typing - Harmonization at the European Union Level

Cited by 13 publications

References 21 publications

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

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

Fluorescence amplified fragment length polymorphism compared to pulsed field gel electrophoresis for Listeria monocytogenes subtyping

Processing Plant Investigations: Practical Approaches to Determining Sources of Persistent Bacterial Strains in the Industrial Food Processing Environment

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