DNA Methods in Food Safety (eBook)

Molecular Typing of Foodborne and Waterborne Bacterial Pathogens

Omar A. Oyarzabal, Sophia Kathariou (Herausgeber)

eBook Download: EPUB

2014
John Wiley & Sons (Verlag)
978-1-118-27865-9 (ISBN)

Infos

Molecular typing of foodborne pathogens has become an indispensable tool in epidemiological studies. Thanks to these techniques, we now have a better understanding of the distribution and appearance of bacterial foodborne diseases and have a deeper knowledge of the type of food products associated with the major foodborne pathogens. Within the molecular techniques, DNA-based techniques have prospered for more than 40 years and have been incorporated in the first surveillance systems to monitor bacterial foodborne pathogens in the United States and other countries. However, DNA techniques vary widely and many microbiology laboratory personnel working with food and/or water face the dilemma of which method to incorporate.

DNA Methods in Food Safety: Molecular Typing of Foodborne and Waterborne Bacterial Pathogens succinctly reviews more than 25 years of data on a variety of DNA typing techniques, summarizing the different mathematical models for analysis and interpretation of results, and detailing their efficacy in typing different foodborne and waterborne bacterial pathogens, such as Campylobacter, Clostridium perfringens, Listeria, Salmonella, among others. Section I describes the different DNA techniques used in the typing of bacterial foodborne pathogens, whilst Section II deals with the application of these techniques to type the most important bacterial foodborne pathogens. In Section II the emphasis is placed on the pathogen, and each chapter describes some of the most appropriate techniques for typing each bacterial pathogen.

The techniques presented in this book are the most significant in the study of the molecular epidemiology of bacterial foodborne pathogens to date. It therefore provides a unique reference for students and professionals in the field of microbiology, food and water safety and epidemiology and molecular epidemiology.

Dr Omar A. Oyarzabal is Vice President of Technical Services at IEH Laboratories and Consulting Group, Seattle, WA

Dr Sophia Kathariou is Professor of Bioprocessing and Nutrition Sciences at the Department of Food, North Carolina State University, Raleigh, USA

Inhalt

List of Contributors vii

Preface xiii

Section I Typing Method, Analysis, and Applications 1

1 Polymerase Chain Reaction-Based Subtyping Methods 3

Yi Chen and Insook Son

2 Pulsed-Field Gel Electrophoresis and the Molecular
Epidemiology of Foodborne Pathogens 27

Mohana Ray and David C. Schwartz

3 Multilocus Sequence Typing: An Adaptable Tool for
Understanding the Global Epidemiology of Bacterial Pathogens
47

Stephen J. Knabel

4 High-Throughput Sequencing 65

Xiangyu Deng, Lee S. Katz, Patricia I. Fields, and Wei
Zhang

5 Analysis of Typing Results 85

João André Carriço and Mário Ramirez

6 Databases and Internet Applications 113

G. Gopinath, K. Hari, R. Jain, M. H. Kothary, K. G. Jarvis, A.
A. Franco, C. J. Grim, V. Sathyamoorthy, M. K. Mammel, A. R. Datta,
B. A. McCardell, M. D. Solomotis, and Ben D. Tall

7 The Transformation of Disease Surveillance, Outbreak
Detection, and Regulatory Response by Molecular Epidemiology
133

David A. Sweat

Section II Pathogens 163

8 The Genus Bacillus 165

Monika Ehling-Schulz and Ute Messelhäusser

9 Molecular Typing of Campylobacter jejuni 185

Catherine D. Carrillo and Omar A. Oyarzabal

10 DNA Typing Methods for Members of the Cronobacter Genus
205

Susan Joseph and Stephen Forsythe

11 Molecular Subtyping Approaches for Pathogenic Clostridium
spp. Isolated from Foods 249

Brian H. Raphael, Deborah F. Talkington, Carolina Lúquez,
and Susan E. Maslanka

12 Molecular Characterization of Shiga Toxin-Producing
Escherichia coli 275

Pallavi Singh and Shannon D. Manning

13 Molecular Subtyping Methods for Listeria monocytogenes: Tools
for Tracking and Control 303

Sara Lomonaco and Daniele Nucera

14 Salmonella 337

Aaron M. Lynne, Jing Han, and Steven L. Foley

15 Vibrio cholerae 359

Dong Wook Kim

Index 381

Textauszug

1
Polymerase Chain Reaction-Based Subtyping Methods

Yi Chen and Insook Son

Center for Food Safety and Applied Nutrition, Food and Drug Administration, College Park, MD, USA

Polymerase chain reaction (PCR)-based molecular subtyping methods have been developed and applied to study the population genetics and molecular epidemiology of foodborne pathogens for more than two decades. These methods are based on PCR reaction and subsequent analysis of the banding pattern in gel electrophoretic. Some methods involve restriction digestion and ligation. The principles and performance (discriminatory power, epidemiological concordance, ease of use, reproducibility, typeability, etc.) of some typical PCR-based molecular subtyping methods are discussed in the following text.

Randomly amplified polymorphic DNA

Randomly amplified polymorphic DNA (RAPD) technique is a PCR technique widely used for subtyping various bacterial pathogens. It was first described by Williams et al. (1990) and unlike conventional PCR arbitrary PCR primers are used and the target PCR products of RAPD are unknown. The primers are usually 9–10 bp long and are arbitrarily chosen by the researcher or can be randomly generated by computers. The arbitrary primer can simultaneously anneal to multiple sites in the whole genome under low stringent conditions. When the two primers anneal within a few kilobases of each other in the proper direction, a fragment is amplified. These products can then be separated by gel electrophoresis and the banding patterns of different isolates compared. The genomic locations where these primers anneal are usually specific to a genotype and thus RAPD patterns are used for subtyping purposes. The annealing of arbitrary primers can be affected by only a few nucleotide differences. Because of its marked sensitivity, RAPD PCR has proven useful for differentiating both Gram-positive and Gram-negative bacteria, especially for closely related species or epidemiologically related strains (Hadrys, Balick, and Schierwater, 1992; Power, 1996; Milch, 1998) (Figure 1.1).

Figure 1.1 Randomly amplified polymorphic DNA analysis using arbitrary primers. Arbitrarily designed short primers (8–12 nucleotides) anneal to a large template of genomic DNA. When two primers anneal in the opposite direction to two genomic locations that are reasonably distant from each other, a fragment is amplified. These randomly amplified fragments are then analyzed by gel electrophoresis, resulting in a different pattern of amplified DNA fragments on the gel. To enhance priming with short primers, many primers are designed with a GC content between 10 and 70% and low annealing temperatures are used.

RAPD has been widely used to subtype various foodborne pathogens such as Listeria monocytogenes, Salmonella, and Escherichia coli O157:H7. Nilsson et al. (1998) developed and optimized a RAPD subtyping method for Bacillus cereus that showed excellent reproducibility. Mazurier et al. (1992) investigated the epidemiologic relevance of RAPD using well-characterized outbreak isolates of L. monocytogenes and found that RAPD correctly classified 92 out of 102 isolates into corresponding epidemic groups. Aguado, Vitas, and García-Jaloń (2001) performed RAPD and serotyping analysis to study the cross-contamination of L. monocytogenes in processed food products. Using RAPD, the authors illustrated that the strains isolated from different meat type and brand on the same date had identical subtypings, suggesting cross-contamination. The authors also found that RAPD and serotyping results were concordant, but RAPD demonstrated higher discriminatory power. The authors finally concluded that RAPD was an easy method that could be used to identify cross-contamination in post-processing environment. Vogel et al. (2001) analyzed 148 L. monocytogenes strains from vacuum-packed cold-smoked salmon produced in 10 different smokehouses using RAPD with 4 different primers separately. The authors demonstrated that RAPD provided higher discriminatory power than ribotyping and serotyping for epidemiologic typing of L. monocytogenes; however, the discriminatory power of RAPD was not as good as pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP). The authors obtained 16 reproducible RAPD profiles and the clustering of isolates using the 4 primers was identical. They identified dominant RAPD types in products from each smokehouse but also found identical RAPD types in different smokehouses, and concluded that these were persistent strains in the smokehouse environment. This study was reported in 2001. It would be interesting to reanalyze those strains with identical RAPD types from different smokehouses using other discriminatory methods that were developed in the last decade. In an earlier study conducted in Japan (Yoshida et al., 1999), researchers analyzed 20 epidemiologically unrelated L. monocytogenes strains isolated from different animals and locations and on different dates, and identified 18 types by RAPD using 4 primers. They also analyzed seven epidemiologically related L. monocytogenes strains isolated from raw milk and a bulk tank on a dairy farm and showed that those strains had the same RAPD type. The results demonstrated that RAPD was epidemiologically concordant. O'Donoghue et al. (1995) used RAPD to study the diversity of L. monocytogenes of different serotypes and the authors reported that serogroup 1/2 of L. monocytogenes strains are genetically more diverse than serogroup 4, a finding that was confirmed by many other subtyping methods. Kim et al. (2005) studied a set of E. coli O157:H7 strains using RAPD and discovered that RAPD could not differentiate O157 strains that varied in the degree of virulence. Another study of E. coli O157:H7 (Vidovic, Germida, and Korber, 2007) demonstrated that RAPD yielded excellent discriminatory power for differentiating E. coli O157:H7 from animal sources.

Reproducibility is one of the biggest concerns of RAPD. Certain factors such as DNA quality and concentration, the type of Taq polymerase employed, and PCR reaction conditions can all affect the reproducibility of RAPD PCR. Therefore, it is critical to maintain the greatest consistency in DNA template quality, reagent selection, and experimental design for successful RAPD PCR. In addition, because the arbitrary primers are not specifically designed for certain genomic loci, the hybridization of the primers to the genome can be partial, which confound the PCR reaction. A RAPD protocol used by Nath, Maurya, and Gulati (2010) had only 40% reproducibility when subtyping Salmonella typhi strains isolated from typhoid patients between 1987 and 2006 in India. Penner et al. (1993) conducted an inter-laboratory reproducibility study of RAPD protocols with different primers and found two major variables with RAPD. One variable was that small and large polymorphic fragments were not always reproduced and therefore the size ranges of DNA fragments were different among the laboratories. The other major variable was that reproducible results were obtained with only four of the five primers using the same reaction conditions. These results highlight the importance of protocol optimization and the maintenance of consistent thermal cyclers among different laboratories when performing RAPD. Davin-Regli et al. (1995) demonstrated that variations in the concentration of template DNA could significantly affect the reproducibility of RAPD banding patterns. Bidet et al. (2000) evaluated three RAPD protocols using different single primers each for subtyping Clostridium difficile, and the reproducibility were only 88, 67, and 33% for the three primers. Due to the very low reproducibility of RAPD, the authors cautioned that the discriminatory power might be an overestimation.

Amplified fragment length polymorphism (AFLP)

AFLP is a highly discriminatory subtyping method used for molecular subtyping. With AFLP, genomic DNA is purified and digested with two restriction enzymes and then two different restriction-specific adaptors are ligated to ends of the restriction fragments (Figure 1.2). PCR primers, which are complementary to the adaptors, are designed to selectively amplify a subset of the ligated restriction fragments under stringent PCR conditions. In order to further select a subset of fragments to amplify, PCR primers are usually designed with a specific base or doublet or triplet of bases adjacent to either restriction site, and thus only the subset of genomic fragments that have matching bases adjacent to the restriction sites are amplified. PCR amplicons are then analyzed by gel electrophoresis, and gel patterns (polymorphisms between and within restriction sites) are used to assign subtypes (Savelkoul et al., 1999; Foley et al., 2004; Foley, Zhao, and Walker, 2007; Singh and Mohapatra, 2008).

Figure 1.2 Amplified fragment length polymorphism analysis. A DNA template is first digested with two restriction enzymes, preferably a hexa-cutter and a tetra-cutter; and then the restriction fragments are ligated to the adaptors. Primers are designed to be complementary to the adapter and restriction site sequences, and their 3′ ends were added by a random nucleotide for...

Produkt-Details

• Erscheint lt. Verlag: 21.8.2014
• Sprache: englisch
• Themenwelt: Naturwissenschaften ► Biologie
• Themenwelt: Technik ► Lebensmitteltechnologie
• Themenwelt: Weitere Fachgebiete ► Land- / Forstwirtschaft / Fischerei
• Schlagworte: Analytical Chemistry • Analytische Chemie • Appearance • bacterial • Better • Biomolecules (DNA, RNA, Peptides, etc.) • Biomoleküle (DNA, RNA, Peptide) • Biomoleküle (DNA, RNA, Peptide) • Chemie • Chemistry • deeper • Diseases • Distribution • Epidemiological • First • Foodborne • Food Science & Technology • Incorporated • indispensable • Knowledge • Lebensmittelforschung u. -technologie • Microbiology, Food Safety & Security • Mikrobiologie u. Nahrungsmittelsicherheit • molecular • Pathogens • Studies • techniques • thanks • Tool • Understanding • years
• ISBN-10: 1-118-27865-8 / 1118278658
• ISBN-13: 978-1-118-27865-9 / 9781118278659