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Genotypic subtyping of multiresistant Salmonella typhimurium DT 104 from food animals and humans
Project Code: B01013
Health Protection Agency
Threlfall, E ;
Veterinary Laboratories Agency, New Haw
The overall aim of the project was, in collaboration with the Veterinary Laboratories Agency (VLA), to standardise and apply recently-developed molecular methods to sub-divide multiple drug resistant (MR) strains of Salmonella enterica serotype Typhimurium definitive phage type (DT) 104 (= MR DT 104) and related phage types for epidemiological purposes.
Previous studies have demonstrated that >80% of MR DT 104 isolates with resistance to ampicillin, chloramphenicol, streptomycin / spectinomycin, sulphonamides and tetracyclines (R-type ACSSpSuT) constitute a single pulsed-field gel electrophoresis (PFGE) profile type, which has been designated Xtm 1. Similarly most isolates of DT 104 of R-type ACSSpSuT 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 we have:
- Investigated further PFGE profile types within MR DT 104 and their distribution
- 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 assay (GAMA), amplified fragment length polymorphism (AFLP), both single-enzyme (SAFLP) and fluorescent (FAFLP).
- 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 VLA for joint outbreak investigations.
In the course of the project:
- PFGE analysis of 853 isolates of MR DT 104 resulted in the identification of 41 distinct pulsed field gel electrophoresis profiles within the phage type. The majority of PFGE profiles were profile type Xtm 1. Eighteen 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 DTs 104a, 104b, 104c, and phage types (PTs) U302 and U309 has provided evidence of the evolutionary divergence of a clonal line of MR DT 104.
- Coupled with PFGE, plasmid profile typing has been shown to be still useful for investigations of outbreaks of MD DT 104.
- GAMA may be useful in outbreaks where isolates exhibit low-level to ciprofloxacin (~15% of MR DT104 isolates).
- LPSA has distinguished two types (A and B), accounting for 80% and 20% of MR DT 104 strains respectively. The method might be a useful adjunct to existing methodologies such as PFGE.
- FAFLP has been demonstrated to have considerable potential for subtyping MR DT104 strains and was able to subtype strains within different PGFE and PP types.
- RAPD, IF and SAFLP methodologies were unable to provide further resolution within the MR DT104 isolates tested. The methods may prove to have application amongst other Salmonella serotypes / phage types.
- The standardisation of methodologies, coupled with the ongoing exchange of molecular subtypes and data between the HPA and VLA has been useful for outbreak investigations.
- Results from the project have been combined with work done under the auspices of B10001 in several investigations of outbreaks of infection
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