A variety of phenotypic methods have been used both independently and in combination for subdivision within serovars. Those currently in use include bacteriophage typing (phage typing) and resistance (antibiogram) typing.
Bacteriophage Typing
The underlying principle of phage typing is the host specificity of bacteriophages and on this basis several phage-typing schemes have been developed for serovars of clinical or epidemiological importance. (For extensive review of the strengths and weaknesses of these schemes, see White, 1926; Threlfall, 2005a.) The most important schemes internationally are those for S. Typhi, S. Paratyphi A and B, S. Enteritidis, S. Typhimurium, and S. Virchow.
Salmonella Typhi
The first phage typing scheme was based on the principle of phage adaptation and was developed for the differentiation of Typhi; in this scheme, progressive adaptations were made of Vi phage II – specific for Vi (capsular) antigen of Typhi – which is highly adaptable and shows a high degree of specificity for the last strain on which it has been propagated. The extraordinary adaptation of Vi phage II is due in part to the selection of spontaneously occurring host-range phage mutants by the bacterium and in part to a nonmutational phenotypic modification of phage by the host strain. The method of Vi phage typing was standardized in 1947 and with further adaptations of Vi phage II, a further 95 types have been defined and internationally recognized, bringing the total number of Vi PTs to 106.
Other Serovars
In contrast to the phage typing scheme for Typhi, phage typing schemes for other serovars depend on, to a limited extent, phage adaptability and, for the most part, are based on patterns of lysis produced by serologically distinct phages isolated from a variety of sources. More than 70 PTs are now recognized in the Enteritidis scheme, the value of which was realized on an international scale following the global pandemic of Enteritidis from the late 1980s, extending to 2006. This scheme was instrumental in identifying the global pandemic PT 4, and more recently, in detecting and monitoring the emergence of non-PT 4s associated with eggs from poultry flocks in several different European countries. A major achievement has been the standardization of phage typing for S. Enteritidis throughout Europe and one scheme, that of Ward and colleagues (1987), is now in use in reference laboratories for human salmonellosis in 22 European countries as well as in Australia, Japan, and Canada (Fisher and Threlfall, 2005).
For S. Typhimurium, almost 300 PTs have now been recognized and designated using the scheme of Anderson and colleagues (1977). This scheme is the most commonly used worldwide, although local schemes have been developed for individual countries. The importance of this scheme is well illustrated by the universal recognition of the multiple drug-resistant (MDR) epidemic clone of S. Typhimurium DT 104. However, of note is that PTs cannot always be regarded as indicative of clonality because PT conversions may result from the acquisition of both plasmids and bacteriophages. A further problem with phage typing is that because of the necessity to propagate and maintain bacteriophage stocks and to implement strict quality control procedures, the procedure is best performed by highly trained staff in reference laboratories and not by workers in individual laboratories on an ad hoc basis.
Resistance (Antibiogram) Typing
The pattern of susceptibility/resistance to selected antimicrobial drugs can be a very useful screen for epidemiological investigations. Because of mutation and/or plasmid acquisition such patterns cannot be regarded as definitive. Nevertheless, patterns such as ACSSuT for S. Typhimurium DT 104 and for the identification of Salmonella Genomic Island 1 (SGI-1), and ACSSuTTm (Tm, trimethoprim) for S. Typhi have become very useful markers and have been used on a global basis to assist in the identification of epidemic clones or drug resistance islands.
Of particular importance in the use of susceptibility/ resistance patterns (R-types) as epidemiological markers, and also in the international surveillance of antimicrobial drug resistance in salmonella is the standardization/harmonization of methodologies coupled with the interpretation of results. For human salmonella isolates within Europe this has been achieved, in the first instance, by the harmonization of methods of susceptibility testing in all reference laboratories with responsibility for human referrals. Following international agreement on the definitions of resistance and susceptibility based on various methodologies, it has been possible to combine and analyze the antimicrobial drug resistance data originating from all countries within Europe who participate in the Enter-net surveillance network.