Heterogeneity and Subtyping

GUIDELINES FOR GENOTYPING

MATCHING AIMS WITH METHODS: Genomic diversity within is now well established and a plethora of molecular typing techniques is available, many of which have been applied successfully to distinguish between isolates. However, no single molecular method has yet emerged as an internationally accepted basis for typing or subtyping purposes. Although consensus guidelines for appropriate use and evaluation of microbial epidemiologic typing systems have been proposed (129), they are aimed mainly at phenotypic methods applicable in the characterization of isolates implicated in community or hospital outbreaks. For , the situation is perhaps unique as no reliable phenotyping methods are available for comparison, and outbreaks of infection, which usually provide an important basis for validating typing methods, are not a feature of the epidemiology of the species. The earliest typing methods used for , such as restriction digest analysis (including PFGE), ribotyping, and other forms of blot hybridization, have been now largely superceded by PCR-based methods such as RAPD, REP, and RFLP analysis. This trend was highlighted in a recent analysis of performance criteria for typing methods, in which the superiority of the PCR methods was demonstrated, including better typeability, higher discrimination power, and better general ease of use (17). While there are a number of important reasons to characterize individual strains of , the typing method employed is largely dictated by the specific aim of the investigation, and there is no single method that can satisfy all requirements. Some guidelines are provided in Table 1 for various typing methods and the purposes for which they might be most appropriate. They are grouped in three general categories, i.e., markers for potentially enhanced pathogenicity, precise identification of strain differences, and global population structure analysis. Some comments on the relative advantages and disadvantages of the different methods are provided.

FUTURE NEEDS

The enormous expansion in detailed information about the fine genetic structure of , promoted by the availability of two complete genome sequences, continues to focus interest on strain-specific attributes, and hence a wider recognition of the importance of precise methods for strain identification. The present evidence is that strain genotypes appear to be relatively stable, at least under in vitro conditions. However, in view of the in vivo plasticity of the genome, it will be essential to monitor how typing markers are affected by genetic drift. Aspects that could merit development in the future include direct typing from clinical specimens without the need for culture, development of global standardized data sets of genomic fingerprints for different PCR-based methods, and development of sequence-based typing using standard sets of loci in housekeeping as well as pathogenicity marker genes.