Short communicationComparison of two AFLP methods and PFGE using strains of Listeria monocytogenes isolated from environmental and food samples obtained from Piedmont, Italy
Highlights
► We typed 103 well characterized L. monocytogenes food and environmental strains. ► We compared two AFLP methods and PFGE in characterizing the same set of samples. ► We evaluated discriminatory power, typeability and concordance of the three methods. ► We evidenced a low typeability for one of the AFLP method considered. ► We observed similar discriminatory power and a good agreement between methods.
Introduction
Listeria monocytogenes is a foodborne pathogen causing listeriosis mainly in certain well-defined high-risk groups. In 2008, the reported incidence of listeriosis in Europe was 0.3/100,000 population. In Italy, it has mostly caused sporadic cases with a reported incidence of 0.1/100,000 in 2007 and similar to incidences reported in 2005 (EFSA, 2007). More specifically, there were 45 notified cases reported in the last 10 years (1998–2008) in the Piedmont region, area where this study was carried out (Ministero del Lavoro, 2006). Because of its high case-fatality rate (20–30%), listeriosis ranks among the most frequent causes of death due to foodborne illness (EFSA, 2010).
L. monocytogenes has been recovered from raw or processed foods including dairy products, meat products, vegetables and seafood and can be found in the environment, mostly in soil and silage.
In order to reduce L. monocytogenes contamination, to confirm outbreaks sources, establish transmission patterns, and eliminate reservoirs of epidemic strains, there is a need for subtyping methods capable of discerning epidemiologically related strains.
Phenotypic methods, such as serotyping and phage typing, often yield low discriminatory power. However, serology is still largely being used to determine the prevalence of specific serotypes of bacterial species.
Since the 1980s, the number of genotypic methods was extended by the development of restriction-fragment-based methods such as pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). PFGE typing was shown to be a very accurate and reproducible method for fine structure comparison and molecular subtyping of L. monocytogenes (Graves and Swaminathan, 2001, Hyytiä-Trees et al., 2007).
AFLP analysis is a DNA fingerprinting technique based on the selective amplification of genomic restriction fragments. AFLP has been reported to yield more complex patterns when compared to other available DNA fingerprinting methods, thus likely increasing strain discrimination (Razin, 2006). The strategy of using two different restriction enzymes and subsequent selective amplification results in the generation on highly discriminatory banding patterns (Aarts et al., 1999, Vogel et al., 2001). In addition, the use of fluorescent labels allows AFLP patterns to be viewed by automated laser fluorescence analysis, permitting inter-laboratory data comparison (Aarts et al., 1999). These features, combined with the possibility for automation and high-throughput analysis, make AFLP an interesting alternative to currently used whole-genome fingerprinting techniques (Aarts et al., 1999, Vogel et al., 2001).
Recently, a number of new AFLP protocols using different restriction enzymes, were shown to have high typeability, discriminatory power and reproducibility (Keto-Timonen et al., 2007, Keto-Timonen et al., 2003, Parisi et al., 2010, Wulff et al., 2006). However, these studies were difficult to compare as different numbers of strains from different sources were analyzed.
Therefore, the purpose of the present study was to compare, in terms of typeability and discriminatory power, two available AFLP methods with PFGE using a set of 103 well-characterized L. monocytogenes strains isolated from environmental and food samples collected in Italy.
Section snippets
Test strains
A total of 103 unrelated and well-characterized L. monocytogenes strains isolated from different environmental and food sources were analyzed (Fig. 1). Food samples were collected from food-production plants, supermarkets and small food markets from Piedmont, Italy. The environmental samples consisted of swabs collected from the environment in food production plants and work surfaces in contact with meat or dairy products. Strains were isolated from: meat and meat processing plants (n = 64),
Identification
All 103 test strains that previously had been biochemically identified as L. monocytogenes were also confirmed as L. monocytogenes by prfA PCR.
PFGE
All 103 test strains were typeable with both AscI and ApaI macrorestriction analysis. The generated patterns divided the strains into 56 types grouped into two major clusters (I and II) diverging at a S.L. of 36.6% (Fig. 1). These clusters reflect the genetic division of L. monocytogenes into Lineage I and II, respectively. Among the 23 strains
Discussion
In this research, two AFLP methods and PFGE were compared in regard to discriminatory power, typeability and concordance. All methods clustered L. monocytogenes strains into two major clusters. These two clusters may reflect the division of L. monocytogenes species into three genetic lineages: Lineage I (serotypes 1/2b, 4b, 3b), Lineage II (1/2a, 1/2c, 3a) and Lineage III (4a, 4c, some 4b) (Wiedmann et al., 1997, Ward et al., 2004, Neves et al., 2008). The clustering of isolates allowed the
Acknowledgements
We would like to thank Prof S. Knabel for his assistance in the revision process.
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