Project
title / Development of a sensitive and specific molecular typing method for the epidemiological study of Salmonella
/ DEFRA
project code / OZ0312

Department for Environment, Food and Rural Affairs CSG 15

Research and Development

Final Project Report

(Not to be used for LINK projects)

Two hard copies of this form should be returned to:
Research Policy and International Division, Final Reports Unit
DEFRA, Area 301
Cromwell House, Dean Stanley Street, London, SW1P 3JH.
An electronic version should be e-mailed to
Project title / Development of a sensitive and specific molecular typing method for the epidemiological study of Salmonella
DEFRA project code / OZ0312
Contractor organisation and location / LGC Ltd
Queens Road
Teddington
Middlesex. TW11 0LY
Total DEFRA project costs / £ £228,699.00
Project start date / 03/01/00 / Project end date / 31/01/03
Executive summary (maximum 2 sides A4)
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CSG 15 (9/01) 2

Project
title / Development of a sensitive and specific molecular typing method for the epidemiological study of Salmonella
/ DEFRA
project code / OZ0312

Bacterial pathogens such as Salmonella and Campylobacter are a major cause of infection in the UK, and present a significant public health problem world-wide. Human infection can occur through the consumption of contaminated food or water, by direct person-to-person transmission or direct contact with animals carrying the organisms, or from environmental contamination from faeces of infected animals or people. Identifying the source of infection is central to control the spread of disease, but can be hampered by the inability to discriminate between isolates within the most common Salmonella phage and serotypes using conventional typing methods. The ability to identify food borne pathogens more accurately and rapidly would enable more effective outbreak tracing and facilitate international surveillance of food borne pathogens such as Salmonella. Initiatives such as the Enter-net1 and Global Salm-Surv (GSS)2 surveillance networks have developed frameworks for international data sharing and standardisation of analytical methods, but work on establishing universal characterisation techniques is ongoing. The aim of this project has been to evaluate the use of a molecular typing method, fluorescent AFLP profiling (AFLP)3, as a tool for highly discriminatory characterisation of food borne pathogens, using Salmonella as a model organism.

Until recently, conventional bacteriological methods such as serotyping and phage typing have been used for the characterisation of Salmonella. However, the application of molecular methods, such as plasmid analysis and macro-restriction fragment polymorphism typing by pulsed-field gel electrophoresis (PFGE) have been demonstrated to provide improved discrimination4. The AFLP technique has the potential to further increase sensitivity; allowing multiple regions of the genome to be simultaneously interrogated at the sequence level by restriction endonuclease digestion, with approximately 0.1% of the genome probed by each analysis. The AFLP method can be used to reproducibly generate a detailed genomic profile consisting of a number of accurately sized DNA fragments, by selective amplification of a total digest of total bacterial DNA. The profiles produced from bacterial isolates can then be compared by computer analysis to assess the similarity of strains. The genomic profiles provide a good indication of the relatedness of isolates, and mathematical methods of evaluating evolutionary relationships from AFLP profiling data have been developed5.

To test the suitability of the AFLP method for sensitive characterisation of Salmonella species, over 400 isolates were selected for investigation, from the collection of the Scottish Salmonella Reference Laboratory at Stobhill Hospital in Glasgow. A catalogue of representative strains was produced, including 243 isolates of human origin, 83 animal isolates, 37 from a variety of foods and 26 strains acquired from environmental sources. The isolates had already been characterised by classical typing methods by the Scottish Reference Laboratory. In addition, the majority of the strains had been typed using PFGE, and their antibiotic resistance profile determined. DNA extracts from the resuscitated Salmonella strains were provided by the Scottish Salmonella Reference Laboratory for AFLP analysis at LGC.

The first stage of the project focussed on development of a robust protocol for the generation of AFLP profiles. To facilitate inter-laboratory comparison it was decided to utilise commercially available AFLP reagents provided as a Microbial Fingerprinting Kit. However, in the last year of the project, the highly variable quality of commercially available reagents necessitated a change to a “home-brew” method, based on the original method of Vos et al 3. The effect of a variety of parameters on the robustness and quality of AFLP profiles was assessed, and an optimised protocol based on the method provided with the AFLP kit was developed. To promote reproducible data interpretation and comparability of results between analysts and laboratories, and to ensure uniformity of profile peaks between runs, methods for normalisation of data and criteria for profile acceptance were developed. However, the inherent variability in profile intensity was a significant challenge to the reproducibility of the method, with subjective calling of signals on the borderline of automated detection. The variation in fragment intensity is one of the main problems with robust isolate characterisation by AFLP analysis.

An extensive range of commercially available PCR primer and restriction enzyme combinations were evaluated for sensitivity against a set of closely related isolates. In addition, a number of novel primer and enzyme combinations, designed to target hyper-variable regions of the Salmonella genome, have been developed to increase the discriminatory power of the technique. The most discriminatory combination identified, using EcoRI-T and TaqI-0 selective primers, was used to profile the extensive catalogue of isolates. The AFLP profiles produced were then compared with known phenotypic characteristics of the isolates to identify markers correlating with particular traits. To facilitate identification of unique markers for particular characteristics, a searchable database was developed at LGC using Microsoft Access. The database allows both storage of isolate profiles and comparison of molecular and phenotypic characteristics of strains, and also facilitates matching of AFLP profiles of unknown samples with those already entered into the database. The matching function to templates stored within the database is designed to be of use in potential outbreak tracing. The capability of the system to identify unknown isolates was tested by a blind trial. It was possible to correctly assign the serotype of thirty seven of forty unknown isolates by AFLP profile matching, and identification to the individual isolates was possible for nine of the samples, facilitated in part by small numbers of templates of certain rarer serotypes within the AFLP database. However, in contrast to published studies where smaller numbers of isolates were examined6,18, robust correlation between molecular markers and phage type or other phenotypic characteristics could not be established.

In conclusion, the AFLP typing approach offers several advantages over currently used typing methods, including the accuracy of the technique, and the accessibility of the information to database storage. However, there are a number of barriers to widespread uptake. Specifically, the method is not robust, and the complex, multi-step nature of the profiling method can lead to high failure rates through variation in reaction efficiency. In turn, this lack of consistent efficiency of the AFLP process causes significant variation in signal intensity. Consequent subjective data interpretation leads to poor reproducibility of profiles produced, despite the highly accurate fragment sizing capability of the process. In addition, the cost of both the capital equipment and high cost per unit test reduces the accessibility of the method, precluding its use as a universal typing tool. The relatively high costs of reagents are unavoidable as the method is patented with a sole licensed supplier of reagents for commercial use of the technique. Overall, the discriminatory power of the technique was not found to be sufficiently high to warrant extensive further effort in overcoming the very real drawbacks of the method. However, the advent of new techniques such a mcicroarray based analysis, with the ability to interrogate a larger number of potentially informative loci simultaneously, could provide an alternative route to informative bacterial typing. Indeed, results of the application of microarray analysis to Salmonella typing have recently been published20.

CSG 15 (9/01) 2

Project
title / Development of a sensitive and specific molecular typing method for the epidemiological study of Salmonella
/ DEFRA
project code / OZ0312
Scientific report (maximum 20 sides A4)
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CSG 15 (9/01) 2

Project
title / Development of a sensitive and specific molecular typing method for the epidemiological study of Salmonella
/ MAFF
project code / OZ0312

1 Introduction

The aim of the project has been to evaluate the AFLP technique as a method for identification and comparison of Salmonella isolates. The use of the AFLP molecular profiling method has been proposed, to improve inter-laboratory comparability and data-sharing due to the potential to very accurately size the characteristic fragments produced. This should enable replacement of the less discriminatory serotyping and phage typing methods that have historically been routinely used in reference laboratories. Methods to allow effective comparison of strains isolated from biological, food and environmental sources could improve epidemiological monitoring, and facilitate identification of the source of infection in outbreak situations. We have investigated the ability of AFLP to discriminate effectively between and within Salmonella serotypes, using a set of 400 isolates chosen by the Scottish Salmonella Reference Laboratory from the extensive collection of strains received by the Reference Laboratory for testing. The samples were from a variety of sources, and had already been typed using conventional methods and tested for resistance to a range of antibiotics. In addition, we have developed a database system to store the AFLP fingerprints of the Salmonella isolates together with known phenotypic characteristics. The database provides a facility to match fingerprints of unknown strains to the characterised isolates in the database, facilitatating isolate identification. The ability to identify isolates from the AFLP profiles has been tested in a blind trial, and the serotypes of thirty-seven of the forty strains tested were correctly identified, with several of the isolates being identified exactly. Despite these promising results, problems have been encountered both in ensuring that profiles are analysed using a coherent and uniform approach and in generating consistent AFLP profiles. The AFLP method is not yet sufficiently automated to permit completely objective data analysis, and thus cannot yet be adopted routinely by Reference laboratories. However, the potential for highly discriminatory analysis should ensure that the technique forms part of the range of molecular epidemiological tools that are applied to pathogen identification in the future.

The annual report on the project submitted in February 2001 contains very detailed information on the early work to validate and optimise the AFLP technique for analysis of Salmonella isolates. The early work will be presented in this Final report in summary, together with more detailed results and analysis of AFLP profiling of the extensive range of isolates from the Scottish Salmonella Reference Laboratory. The AFLP protocols used, the Salmonella Strain Catalogue, geographical origin of the strains, and the details of the searchable Microsoft Access database are included in Appendices.

2 Investigation of the utility of AFLP for Salmonella typing

2.1 Basis of the AFLP technique

Figure 1: Schematic diagram of the AFLP process

The AFLP method is based on selectively amplifying restriction fragments from a total digest of genomic DNA. Bacterial cells are lysed to release the genomic DNA, which can then be purified using any of a variety of methods prior to AFLP analysis. The first step of the fingerprinting process involves digesting DNA with two restriction enzymes to create fragments. Linkers (short synthetic pieces of DNA) are then ligated to the ends of the fragments. Two rounds of PCR are then performed; the first amplifies all fragments flanked with linkers; the second is a selective reaction that reduces the complexity of the mixture. The selective amplification employs two primers, one of which is fluorescently labelled to enable fragments to be resolved and detected by polyacrylamide gel electrophoresis using an ABI PRISM 377. A schematic diagram of the process is shown in Figure 1, and the details of the method we have employed are provided in Appendix A. The protocol is based on the AFLP™ Microbial Fingerprinting Protocol supplied by PE Applied Biosystems, which is based on the original method of Vos et al 3

Figure 2: AFLP profiles fractionated on a sequencing gel and electropherogram of a gel region

An example of a sequencing gel with AFLP profiles is shown in Figure 2. On this gel the profiles are generated with the EcoRI-0/MseI-C selective primer combination (blue fragments), and the internal size standard in each lane is visualised as red fragments. The fluorescent signals are read as the gel is run, and the data is then imported into GeneScan and Genotyper analysis software. The electropherogram in Figure 2 shows signal peaks in a region of the sequencing gel.

2.2  Optimisation of the AFLP methodology

The various stages of the AFLP process were optimised to ensure maximal reproducibility of the method, from culture of the strains for test, to DNA extraction, profile production and data analysis. The results of investigating the impact of the various parts of the process on the overall method are presented here.

2.2.1 Effect of Salmonella growth conditions on AFLP profiling

In order to determine whether growth conditions had any effect on DNA yield and AFLP profile quality, a test isolate was chosen from the catalogue, S. enteritidis 971669, and grown under aerated and static conditions (shaking at 200rpm). In addition three test strains were grown in rich media (Brain Heart Infusion) and minimal medium, and at three different temperatures (30oC, 37oC and 42oC). DNA was then extracted and profiled to assess any effects of the different growth conditions on AFLP profiles. AFLP profiles were not significantly different under the growth conditions examined. Although a few additional peaks were present in several of the samples these were not reproducibly observed and were attributable to differences in overall profile intensity that resulted in very weak peaks becoming detected as part of the profile. In conclusion, the AFLP profiles are independent of the growth conditions investigated.