Download FAFLP: last word in microbial genotyping?

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,
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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
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