B01013: Genotypic subtyping of multiresistant Salmonella typhimurium DT 104 from food animals and humans

Study Duration: June 1999 to March 2002

Contractor: Health Protection Agency

In the tracing of human infections back to their source e.g. from the manufacturing environment, farm etc., it is necessary to be able to separate bacterial species or strains into a sufficiently large number of subtypes that the offending organism can be identified with a reasonable degree of certainty. At the beginning of this project the methods in use to sub-divide salmonella phage types when applied to S. typhimurium DT104 placed more than 95% of the isolates in a single group.

This project aimed to compare a number of molecular techniques e.g. plasmid profiling, pulsed field gel electrophoresis, fluorescent amplified fragment length polymorphisms and sequence polymorphisms in a gene conferring resistance to the antibiotic ciprofloxacin to investigate whether a combination of the methods could subdivide S. typhimurium DT104 strains more effectively.

Established DNA fingerprinting methods such as Pulsed Field Gel Electrophoresis (PFGE) and Plasmid Profiling (PP) have been used successfully to sub-divide Salmonella phage types for epidemiological purposes. However, it is thought that more than 80% of MR DT 104 isolates of R-type ACSSuT constitute a single PFGE type, which has been designated Xtm 1. Similarly most isolates of DT 104 of R-type ACSSuT are characterised by a single plasmid of 60 megadaltons (MDa). The resultant plasmid profile (PP), common to most isolates, has been designated PP A.

Using these established DNA fingerprinting techniques as standard the project has:

a) Investigated further PFGE profile types within MR DT 104 and their distribution.

b) Investigated the applicability of other novel DNA-based techniques for the subdivision of MR DT 104. These techniques have included random Amplified Polymorphic DNA (RAPD) analysis, integron fingerprinting (IF), GyrA mutation analysis (GAMA), amplified fragment length polymorphism (AFLP) both single-enzyme (SAFLP) and fluorescent (FAFLP).

c) Investigated the possibility of lipopolysaccharides analysis (LPSA) for strain differentiation within MR DT 104.

The applicability of the above molecular methods for real-time epidemiological investigations have been has been assessed in collaboration with the Communicable Diseases Surveillance Centre. Data, methods and standard control strains have been exchanged with collaborators at the Verterinary Laboratories Agency (VLA) for joint outbreak investigations.

Outcome/Key results obtained

PFGE analysis of 853 isolates of MR DT 104 resulted in the identification of 41 distinct pulsed field profiles within the phage type. As expected 89% of the isolates were profile Xtm 1. Eighteen of the remaining profiles were represented by a single isolate, while the second most common profile accounted for only 2% of the total. The increase of PFGE types with time, and also of the related phage subtypes 104a, 104b, 104c, U302 and U309 have provided further evidence of the evolutionary divergence of a clonal line of MR DT 104 with time.

The standardisation of methodologies for PFGE and GAMA, coupled with the ongoing exchange of molecular subtypes and data between the Health Protection Agency (HPA) and VLA has been very useful for outbreak investigations.

Plasmid profile typing, again based on a standardised methodology, may still be still useful for outbreak investigations (e.g. for the real-time investigation of the national outbreak of MR DT 104 in the summer of 2000, centred on the West Midlands).

GAMA proved useful in distinguishing several subtypes, but is only applicable in cases where low-level ciprofloxacin resistance is present (~15% of MR DT104 isolates). It should be realised that the method relies on spontaneous chromosomal mutation and therefore does not reflect the clonality seen in other subtyping methods.

LPSA distinguished only two types (A and B), accounting for 80% and 20% of MR DT 104 strains respectively. The observation of variation in LPS between strains of MR DT 104 is in itself of scientific interest. Whilst of limited use alone, LPSA might be a useful adjunct to existing methodologies such as PFGE. For example MR DT 104 isolates of PFGE type Xtm 9 from cases associated with travel to Turkey were distinguished from other cases of the same PFGE type by LPSA.

FAFLP has perhaps the greatest potential for subtyping MR DT104 strains and was able to subtype strains within different PGFE and PP types. However the majority of strains conformed to a single predominant type, whichever combination of restriction enzymes and primers were used. Although the technique is susceptible to variations in DNA extraction, restriction/ligation and PCR reproducibility is possible, particularly when an automated method of DNA extraction is used The method potentially produces many more bands (data points) than other methodologies. When this is combined with possible variations in selection of enzyme and primers, data analysis is complex and time consuming. If the method is to be adapted within and between countries, then further work is required for the standardisation of methodology, data processing and database interrogation.

RAPD, IF and SAFLP - these methodologies provided no further resolution within the MR DT104 isolates tested. However, they may prove to have application amongst other Salmonella serotypes / phage types.

Although the ACSSuT gene cassette was identified amongst other phage types (DT 12 and DT 120), this was due to the evolution of DT104 rather than horizontal gene transfer.

The final report is available from the FSA Library and Information centre.
To obtain a copy, please contact the Enquiry Desk, Dr Elsie Widdowson Library and Information Services, Food Standards Agency (tel: 020 7276 8181/8182 or email: library&info@foodstandards.gsi.gov.uk)

Walker RA, Lawson AJ, Lindsay EA, Ward LR, Wright PA, Bolton FJ, Wareing DR, Corkish JD, Davies RH, Threlfall EJ. (2000). Decreased susceptibility to ciprofloxacin in outbreak-associated multiresistant Salmonella typhimurium DT104. The Vetinary Record 147(14): 395-6

Contact: For any enquiries concerning this research project, please contact the relevant Programme contact or email science@foodstandards.gsi.gov.uk