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J. Med. Microbiol. Ð Vol. 50 (2001), 393±395 # 2001 The Pathological Society of Great Britain and Ireland ISSN 0022-2615 EDITORIAL FAFLP: last word in microbial genotyping? Within the forest of medical acronyms there is a dense thicket relating to methods for typing microbes. In recent years this thicket had become almost impenetrable, until a technique of whole genome fragment analysis, ¯uorescent ampli®ed fragment length polymorphism (FAFLP) was described, which some have suggested is the method of choice for bacterial typing. Increasingly, laboratory investigators and the wider medical microbiology and public health community are turning to FAFLP to genotype bacteria [1] and analyse outbreaks. It is, therefore, important to understand the principles that underlie the method and its applicability in clinical practice. FAFLP is a simple procedure that uses a single highly speci®c PCR that selects pre-adapted fragments of DNA and ampli®es them to concentrations easily detectable and accurately sizeable by the laser reader of an automated sequencer. Although in theory FAFLP can be used to analyse the genome of any species from microbe to man, the DNA must ®rst be obtained in a pure state. This excludes specimens that cannot readily be separated from extraneous DNA sources and it may prevent the application of FAFLP to viruses whose nucleic acid can be hard to rid of host or cellular DNA. In any case, most viruses have genomes short enough for partial sequencing to remain the molecular typing method of choice. In contrast, FAFLP is readily applicable to organisms whose DNA can be easily isolated and extracted from a colony or a cell. It is ideal for bacteria and fungi that grow as discrete colonies on inert media, as their DNA is easily extracted in pure form, and their genomes are of the right order of size for FAFLP analysis. The DNA extracted from a single colony or sweep of colonies from a pure plating of a bacterial isolate is cut with two restriction enzymes and double-stranded adapter oligonucleotides that will bind the primers of the PCR are then ligated to the fragments. Various pairs of restriction enzymes have been used to generate the DNA fragments, but for many pathogenic bacterial species the same combination of EcoRI and Mse I has proved satisfactory. When lengths of bacterial DNA of upwards of a million base pairs are restricted in this way many hundreds of fragments result, some with the same restriction site, but others with a different site at each end. Adaptor oligonucleotides are ligated to the sticky ends and the fragments with different restriction sites at each end then take part in the PCR reaction. `Selective' nucleotides can be added to limit the number of fragments that the primer will bind to in a predictable way. Once the fragments have been adapted and primed so that a set will be ampli®ed whose sizes can be resolved accurately by the laser, the PCR can proceed. For most species there will still be suf®cient fragments ampli®ed to produce enough polymorphisms for analysis. These ampli®ed fragments will have been selected in an unweighted fashion from throughout the genome and therefore be representative of the whole genome. This is a considerable advantage over other microbial typing methods. The ampli®ed fragments are separated in the slab or capillary polyacrylamide gel of an automated sequencer. A recent comparison of laser-based measurements of fragment length with the lengths predicted from the known whole genome sequence of Escherichia coli K12 has shown that, on the ABI 377 sequencing apparatus, sizing is accurate to within one base pair [2]. Analysis of DNA from isolates of a bacterial pathogen from a suspected outbreak is based on pair-wise comparisons between the numbers of fragments ampli®ed from the DNA of each isolate, and their sizes. For isolates from the same bacterial species some of these fragments will be of the same size and by implication derived from the same conserved genome sequence. Other fragments will be polymorphic. If no polymorphic fragments are found in a comparison of a group of isolates the implication is that, on the basis of those particular FAFLP conditions, the group constitutes a single strain (clone). This is an important conclusion when, for instance, an outbreak is being investigated. While ultimate proof of the clonality of a group of isolates will only come from sequencing their whole genomes, which is scarcely practical, further proof can be obtained if necessary by examining other sets of genomic fragments generated under different FAFLP conditions, i.e., with other restriction enzymes or modi®ed primers. These changed conditions may identify infrequent polymorphisms in closely similar bacterial strains that were not evident at ®rst. By altering the restriction enzymes and the selectivity of the primers the resolving power of the method can be increased, or decreased, as required. FAFLP is more rapid than pulsed-®eld gel electrophoresis (PFGE). Once the FAFLP procedure has been set up, a group of single species isolates from an outbreak can be compared both with each other and with sporadic unrelated isolates of the same species, Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 23:13:26 394 EDITORIAL serotype or phage type (or both), within 48 h. The comparison is objective because the fragments are laser sized, and the results are not weighted in favour of particular genes such as those that code for an easily detectable phenotype like antibiotic resistance or, as is the case for multilocus sequence typing [3], in favour of `housekeeping' genes each requiring their own ampli®cation procedure. In addition, FAFLP results are reproducible in the same laboratory and, when based on the same sequencer and therefore identical sizing, between laboratories. If standard FAFLP conditions were agreed internationally, these results could be shared between specialist groups, especially if use were made of close interval ¯uorescent size markers as controls and if panels of strains representing the range of strain variation within each species were made available to all collaborators. Although high resolution between similar microbial isolates is the most immediate advantage that FAFLP offers there may be further bene®ts in the future. Computer analysis of the number and sizes of fragments generated by each isolate allows phylogenetic trees to be drawn, and these trees may throw light into dark taxonomic corners where phenotypic tests have failed to clarify intra-species relationships. For example, close concordance has been shown between multilocus enzyme electrophoresis and ¯uorescent FAFLP analyses of the E. coli strain collection, ECOR [4]. FAFLP could equally readily be applied to examining the genomic relationships between E. coli and its related enterobacterial species, an area where numerical taxonomy based on combinations of biochemical and other phenotypic tests, and genotyping by the sequencing of housekeeping genes, are at odds [5]. There are still some technical hurdles that prevent the wider use of FAFLP. Without inter-laboratory agreement on FAFLP conditions for each species, and proof that fragment sizes will be accurately determined, it is uncertain whether FAFLP results can be `portable' between laboratories. Until this can be shown FAFLP will only be applied to relatively small-scale analyses in single laboratories. However, if conditions for transferability can be agreed between laboratories, then valuable surveillance schemes can be set up to monitor the spread of signi®cant strains of the major pathogens. Nevertheless, FAFLP promises to be a rapid, objective and cost-effective genotyping technique that will discriminate between isolates of important human bacterial pathogens and enhance local investigations. The list of pathogens in Table 1 to which FAFLP has been applied is by no means exhaustive. What other limitations does FAFLP have? Because it samples the entire genome it must have an isolate to work upon, and not just a gene fragment directly ampli®ed from a clinical specimen. Thus, if an isolate cannot be obtained, for instance because of prior antibiotic treatment, FAFLP analysis is impossible. Table 1. Major pathogens genotyped by FAFLP Organism Reference no. Campylobacter jejuni E. coli O157 Mycobacterium tuberculosis Neisseria meningitidis Salmonella enterica Staphylococcus aureus Streptococcus pyogenes Enterococcus faecium Chlamydia spp. 7 8 9 10 11 12,13 14,15 16 17 FAFLP also has high start up costs and, for all but the least heterogeneous species (e.g., Mycobacterium tuberculosis), fragment analysis requires software not yet freely available for every sequencer. FAFLP is cheap (c. 20 $US per sample [6]) by the standards of other genotyping procedures, but it cannot match the very modest costs of established although lower resolution phenotypic tests such as phage typing. Also, it has yet to be shown that it can accommodate the same large numbers of isolates that a reference laboratory routinely phage types or serotypes. The advantages of FAFLP are clear: it relies on a single PCR, it uses instrumentation that is now commonly available and it can de®ne clones and clone complexes within important pathogenic species with speed and ease. It is much cheaper than other genotyping methods based on sequencing. For these reasons FAFLP is now establishing itself as the method of choice for high resolution microbial typing. PHILIP MORTIMER and CATH ARNOLD Molecular Biology Unit Central Public Health Laboratory London NW9 5HT References 1. Savelkoul PHM, Aarts HJM, DeHaas J et al. Ampli®edfragment length polymorphism analysis: the state of an art. J Clin Microbiol 1999; 37: 3083±3091. 2. Arnold C, Metherell L, Clewley JP, Stanley J. Predictive modelling of ¯uorescent FAFLP: a new approach to the molecular epidemiology of E. coli. Res Microbiol 1999; 150: 33±44. 3. Maiden MCJ, Bygraves JA, Feil E et al. Multilocus sequence typing: a portable approach to the identi®cation of clones within populations of pathogenic microorganisms. Proc Natl Acad Sci USA 1999; 95: 3140±3145. 4. Arnold C, Metherell L, Willshaw G, Maggs A, Stanley J. Predictive ¯uorescent ampli®ed fragment length polymorphism analysis of Escherichia coli: a high resolution typing method with phylogenetic signi®cance. J Clin Microbiol 1999; 37: 1274±1279. 5. Pupo GM, Lan R, Reeves R. Multiple independent origins of Shigella clones of Escherichia coli and convergent evolution of many of their characteristics. Proc Natl Acad Sci USA 2000; 97: 10567±10572. 6. Olive DM, Bean P. Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 1999; 37: 1661±1669. 7. Duim B, Wassenar TM, Rigter A, Wagenaar JA. High Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 23:13:26 EDITORIAL 8. 9. 10. 11. 12. resolution genotyping of Campylobacter strains isolated from poultry and humans with FAFLP ®ngerprinting. Appl Environ Microbiol 1999; 65: 2369±2375. Smith D, Willshaw G, Stanley J, Arnold A. Genotyping of verocytotoxin-producing Escherichia coli O157: comparison of isolates of a prevalent phage type by ¯uorescent ampli®edfragment length polymorphism and pulsed-®eld gel electrophoresis analyses. J Clin Microbiol 2000; 38: 4616±4620. Goulding JN, Stanley J, Saunders N, Arnold C. Genome sequence-based ¯uorescent ampli®ed fragment length polymorphism (FAFLP) analysis of Mycobacterium tuberculosis. J Clin Microbiol 2000; 38: 1121±1126. Goulding J, Hookey JV, Stanley J et al. Fluorescent ampli®ed fragment length polymorphism genotyping of Neisseria meningitidis identi®es clones associated with invasive disease. J Clin Microbiol 2000; 38: 4580±4585. Aarts HJM, van Lith LAJT, Keijer J. High-resolution genotyping of Salmonella strains by FAFLP-®ngerprinting. Lett Appl Microbiol 1998; 26: 131±135. Grady R, Desai M, O'Neill G, Cookson B, Stanley J. Genotyping of epidemic methicillin-resistant Staphylococcus aureus phage-type 15 by ¯uorescent ampli®ed-fragment length 395 polymorphism. J Clin Microbiol 1999; 37: 3198±3203. 13. Grady R, O'Neill G, Cookson B, Stanley J. Fluorescent FAFLP analysis of the MRSA epidemic. FEMS Microbiol Lett 2000; 187: 27±30. 14. Desai M, Tanna A, Wall R, Efstratiou A, George R, Stanley J. Fluorescent ampli®ed-fragment length polymorphism analysis of an outbreak of group A streptococcal invasive disease. J Clin Microbiol 1998; 36: 3133±3137. 15. Desai M, Efstratiou A, George R, Stanley J. High resolution genotyping of Streptococcus pyogenes serotype M1 isolates by ¯uorescent FAFLP analysis. J Clin Microbiol 1999; 37: 1948±1950. 16. Antonishyn NA, McDonald RR, Chan EL et al. Evaluation of ¯uorescence-based ampli®ed fragment length polymorphism analysis for molecular typing in hospital epidemiology: comparison with pulsed-®eld gel electrophoresis for typing strains of vancomycin-resistant Enterococcus faecium. J Clin Microbiol 2000; 38: 4058±4065. 17. Morre SA, Ossewaarde JM, Savelkoul PHM et al. Analysis of genetic heterogeneity in Chlamydia trachomatis clinical isolates of serovars D, E, and F by ampli®ed fragment length polymorphism. J Clin Microbiol 2000; 38: 3463±3466. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Thu, 15 Jun 2017 23:13:26