Molecular Genotyping Methods and Computerized Analysis for the Study of Salmonella enterica

Ana Belén Vivanco, Juan Alvarez, Idoia Laconcha, Nuria López-Molina, Aitor Rementeria, and Javier Garaizar

1. Introduction

Salmonella enterica is widely recognized as a major cause of foodborne diseases in humans and animals and has been isolated from environmental sources in increasing numbers worldwide (1,2). Conventional typing methods such as serotyping and phage typing have been and still are the mainstay in descriptive epidemiology of this microorganism. Nevertheless, limitations on the availability of phage reagents circumscribes the performance of such technique in reference laboratories.

The resolving power of epidemiological typing has been expanded during recent years through the molecular analysis of microbial DNA. The broader availability of the reagents and equipment is accelerating their generalized use in clinical and public health laboratories. Important differences in the performance criteria of the genotyping techniques (typability, reproducibility, stability, and discriminatory power) and the convenience criteria (flexibility, accessibility, and ease of use) exist between them, and there is no ideal typing system for universal use (3,4).

Most of these powerful strain-discriminative techniques are based on comparison of electrophoretic patterns or fingerprints, for which computer-assisted strategies and software packages have been developed to help in construction and analysis of micro-bial databases. Several initiatives, such as PulseNet (http://www.cdc.gov/pulsenet) or Harmony (http://www.phls.org.uk/inter/harmony), have arisen during recent years for international construction of such fingerprinting databases, which will allow the rapid detection of new strains and the spread of pathogenic clones of bacteria through different regions or countries. Nevertheless, complete consensus has not yet been achieved on the techniques to use or the criteria for interpretation of the results, but these goals may be reached soon.

The purpose of this chapter is to describe some of the fingerprinting methods that are currently available to differentiate strains of Salmonella enterica and the computer-assisted strategies that can be used for their analysis. We describe here methods based on DNA restriction such as pulsed-field gel electrophoresis (PFGE) (2,5), methods based on DNA amplification such as polymerase chain reaction (PCR) fingerprinting (6) methods, based on DNA restriction and amplification such as infrequent restriction site PCR (IRS-PCR) (7), and an approach to their computerized analysis and generation of fingerprinting databases. When the purpose of the study is the analysis of a limited number of epidemiologically related isolates, a visual analysis of the gel is faster and more efficient. However, if the objective of the analysis is surveillance of a pathogen over time or requires the analysis of higher numbers of isolates, then computerized analysis is more convenient. We use PCR fingerprinting and IRS-PCR methods when a quick analysis of a limited number of isolates in a gel is required. We use the PFGE method for more permanent epidemiological studies, including those that require interlaboratory comparisons, and we suggest their use for construction of computerized libraries of fingerprints with software packages.

2. Materials

2.1. Pulsed-Field Gel Electrophoresis

1. TES buffer: 10 mM Tris-HCl, pH 8.0, 10 mM Na2EDTA, 1 M NaCl. Sterilize by auto-claving and store at 4°C.

2. Certified Low-Melt agarose (Bio-Rad).

3. Lysis buffer: 1 M NaCl, 10 mM Tris-HCl, pH 8.0, 200 mM Na2EDTA, 0.5% w/v N-lauryl-sarcosine, and 0.2% w/v sodium deoxycholate. Make up 100 mL of solution. Filter sterilize and store at room temperature. Add 1 mg/mL lysozyme, and 2 ^g/mL RNAse prior to use (see Note 1).

4. ESP buffer: 0.5 MNa2EDTA, pH 8.0, 1% w/v N-laurylsarcosine. Filter-sterilize and store at room temperature. Add 1 mg/mL Proteinase K prior to use.

5. TE buffer: 10 mM Tris-HCl, pH 8.0, 10 mM Na2EDTA. Sterilize by autoclaving and store at 4°C.

6. PMSF solution: dissolve 17 mg phenylmethylsulfonyl fluoride (PMSF; Sigma) in 1 mL of isopropanol (see Note 2). Store at -20°C. If necessary, redissolve before using warming up to 56°C.

7. 10X TBE buffer: 890 mM Tris-HCl, 890 mM boric acid, 20 mM Na2EDTA. Sterilize by autoclaving and store at room temperature.

8. Electrophoresis buffer: 75 ^M thiourea (Sigma) in 0.5X TBE buffer (see Note 3). Prepare fresh and discard after use.

9. Pulsed-field Certified Agarose (Bio-Rad).

10. Rare-cutting restriction endonucleases Xbal, Spel, and Blnl and appropriate buffers (Boehringer Mannheim).

11. Ethidium bromide solution: prepare a concentrate stock solution of ethidium bromide (200 ^g/mL solution) using ultrapure water. Ethidium bromide is sensitive to light: use an opaque bottled to store the stock solution. Caution: Ethidium bromide is also carcinogenic. Always wear gloves when handling materials containing ethidium bromide. Follow decontamination instructions using activated charcoal or incineration.

12. Lambda ladder concatamers (Bio-Rad).

13. CHEF-DR II Pulsed-field Electrophoresis System (Bio-Rad).

14. Long-wave UV transilluminator for visualization of agarose gels (UltraLum).

15. MP4 Land camera (Polaroid).

2.2. PCR Fingerprinting

1. Tissue culture grade sterile water (Sigma).

2. 10X PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3 (Applied Biosystems).

3. 25 mM MgCl2 (Applied Biosystems).

4. AmpliTaq Polymerase 5 U/^L (Applied Biosystems).

5. PCR dNTPs solution: 10 mM each of dATP, dCTP, dGTP, and dTTP (Applied Biosystems).

6. PCR primers: ERIC2 (5'-AAG TAA GTG ACT GGG GTG AGC G-3'), M13 (5'-GTA AAA CGA CGG CCA GT-3'), and OPS-19 (5'-GAG TCA GCA G-3') (Applied Biosystems; see Note 4).

7. Thermal cycler for PCR: Robocycler gradient 96 (Stratagene).

8. 1X TBE buffer.

9. Molecular grade agarose (Bio-Rad).

10. Gel loading solution (Sigma).

11. pGEM DNA marker (Promega).

12. 200 ^g/mL Ethidium bromide stock solution.

13. Standard apparatus for the electrophoresis of agarose gels and power supply (Bio-Rad).

14. Long-wave UV transilluminator for visualization of agarose gels (UltraLum).

15. MP4 Land camera (Polaroid).

2.3. Infrequent Restriction Site PCR

1. Lysis solution: mix 5 M guanidine thiocyanate with 0.1 MNa2EDTA in 20 mL of distilled water. Heat to 65°C to dissolve and add 1.7 mL of 30% (v/v) Sarkosyl. When cold, make up to 100 mL with distilled water. Keep at room temperature and light protected.

4. Isopropanol.

6. TE buffer: 10 mM Tris-HCl, pH 8.0, 10 mM Na2EDTA. Sterilize by autoclaving and store at 4°C.

7. Restriction endonucleases XbaI, Hhal, and TaqI and appropriate buffers (Boehringer Mannheim).

8. T4 DNA ligase 10 U/^L and appropriate buffer (Bioline).

9. ATP solution: 10 mM ATP, 50 mM Tris-HCl, pH 7.5 (Bioline).

10. DNA adapters and primers (Applied Biosystems) (Tables 1 and 2).

11. Tissue culture grade sterile water (Sigma).

12. 10X PCR buffer: 500 mM KCl, 100 mM Tris-HCl, pH 8.3 (Applied Biosystems).

13. 25 mM MgCl2 (Applied Biosystems).

14. AmpliTaq Polymerase 5 U/^L (Applied Biosystems).

15. PCR dNTPs solution: 10 mM of either dATP, dCTP, dGTP, or dTTP (Applied Biosystems).

16. 30% (w/v) Acrylamide/bis solution, 37.5:1 (Bio-Rad).

17. 10% (w/v) Ammonium persulfate solution (Bio-Rad).

19. Gel loading solution (Sigma).

20. 1X TBE buffer.

Table 1

Adapters for Ligation in IRS-PCR

Table 1

Adapters for Ligation in IRS-PCR

Restriction enzyme

Adapters

Sequence

Hhal

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