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International Journal of Medical Microbiology 294 (2004) 67–73
www.elsevier.de/ijmm
REVIEW
Population structure of pathogenic bacteria revisited
Mark Achtman
Department of Molecular Biology, Max-Planck Institut für Infektionsbiologie, Schumannstrasse 21/22, D-10117 Berlin, Germany
Abstract
This minireview summarizes the historical development of bacterial population genetic concepts since the early
1980s. Initially multilocus enzyme electrophoresis was used to determine population structures but this technique is
poorly portable between laboratories and was replaced in 1998 by multilocus sequence typing. Diverse population
structures exist in different bacterial species. Two distinctive structures are described in greater detail. ‘‘Young’’
organisms, such as Yersinia pestis, have evolved or undergone a severe bottleneck in recent millennia and have not yet
accumulated much sequence diversity. ‘‘genoclouds’’ in subgroup III Neisseria meningitidis arise because of the
accumulation of diversity due to herd immunity, which is then purified during subsequent epidemic spread.
r 2004 Elsevier GmbH. All rights reserved.
Keywords: Bacterial population genetics; Yersinia pestis; Neisseria meningitides
Content
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Y. pestis, a ‘‘Young’’ species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
N. meningitidis subgroup III: clonal expansion and genoclouds. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Introduction
Population genetics as a discipline has developed over
many decades, but only for eukaryotic organisms.
Corresponding author. Tel.: +49-30-2846-0751; fax: +49-30-28460-750
E-mail address: [email protected] (M. Achtman).
1438-4221/$ - see front matter r 2004 Elsevier GmbH. All rights reserved.
doi:10.1016/j.ijmm.2004.06.028
Bacterial population genetics first began when Bob
Selander used the standard eukaryotic technique of
multilocus enzyme electrophoresis (MLEE) on Escherichia coli (Selander and Levin, 1980) to show that
particular combinations of alleles (electrophoretic types,
ETs) were found significantly more often than would be
expected for recombining organisms. From a eukaryotic
viewpoint, the apparent lack of recombination (resulting
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in clones) was novel because mating eukaryotes shuffle
chromosomes and recombine every generation. But
bacteriologists had already previously recognized that
certain uniform groups of pathogens in diverse species
arose by clonal descent from a common ancestor (Old
and Duguid, 1979; Ørskov et al., 1976) and the clonal
concept for bacteria soon received strong support from
a variety of sources of data (Achtman and Pluschke,
1986; Achtman et al., 1983; Ørskov and Ørskov, 1983).
Selander and his disciples (Jim Musser, Tom Whittam, Howard Ochman and Dominique Caugant)
proceeded to use MLEE to map the diversity of one
pathogenic species after another (Selander et al., 1986).
For each species, they identified ETs and clusters of
related ETs, called clonal complexes. Unfortunately,
these schemes have been only of limited use to the
scientific community because extending them depends
on being able to compare a potentially new variant to all
previously defined variants and it is difficult to
reproduce all electrophoretic differences in multiple
laboratories. As a result, each publication used novel
designations, and due to their lack of a uniform system
of nomenclature, sequential analyses are difficult to
relate to each other. Much of the diversity discovered in
those numerous publications is not available for modern
research. For example, thousands of E. coli isolates were
examined by MLEE, but the only currently available
reference E. coli collection is the EcoR collection of 72
isolates that was chosen in 1984 to represent the
diversity known at that time (Ochman and Selander,
1984).
The validity of these interpretations of universal
clonality was questioned in 1993 by a seminal publication (Maynard Smith et al., 1993). John Maynard Smith
and his colleagues pointed out that the evidence for
clonality was not overwhelming and that recombination
is so frequent in some species that apparent clonal
complexes might exist only temporarily and were
doomed to dissipate once sufficient recombination had
occurred. They also introduced the concept that
epidemic spread might result in apparent clonality,
because it temporarily results in numerous isolates that
are very similar, particularly when the focus is on
bacteria that are isolated from invasive disease rather
than from healthy carriers. Numerous examples of
recombination have been described, even among bacteria that are thought to be highly clonal, such as
Salmonella enterica (Brown et al., 2003). Recombination
is so frequent that it is impossible or difficult to
reconstruct the phylogenetic framework of evolution
of many pathogenic species (Feil et al., 2001) and the
genomic content of many organisms consists of a mosaic
of genes that have been imported over millions of years
(Lawrence and Ochman, 1997). The seminal publication
by Maynard Smith and colleagues was so influential that
many scientists began to doubt whether clonal group-
ings could even exist within bacterial species where
recombination was known to occur.
It was not readily possible to test models of
population genetic structure in the early 1990s due to
lack of sequence data. This situation has changed
drastically since 1998, when multilocus sequence typing
(MLST) was introduced (Maiden et al., 1998). MLST
operates on the same principles as MLEE, i.e. fragments
of multiple housekeeping genes (typically seven) scattered around the genome are sequenced from each
isolate and combinations of alleles are referred to as a
sequence type (ST) (Enright and Spratt, 1999). Unlike
MLEE, MLST data are readily comparable between
laboratories, because they are sequence-based, and a
single, publicly available database is established for all
isolates within a species (http://www.mlst.net/). Some of
these databases now include data for previously inconceivably large numbers of isolates. For example, at the
beginning of February, 2004, the database for Neisseria
meningitidis contained information on almost 5000
isolates that were assigned to almost 3500 STs and the
Campylobacter database contained 2215 isolates in 870
STs. Databases for sixteen microbial species are
currently accessible via http://www.mlst.net/, including
unpublished data from my laboratory for E. coli, S.
enterica and Moraxella catarrhalis (http://web.mpiibberlin.mpg.de/mlst).
We no longer lack data. Instead, the major shortage is
tools to classify the diversity and concepts to understand
it (Feil et al., 2004). Furthermore, it has also become
clear that the distinction between clonality and frequent
recombination is too simplistic to reflect the multitude
of population structures that are found in diverse
bacterial species. In this review, I will concentrate on
two particular population structures, exemplified by
Yersinia pestis and subgroup III of serogroup A N.
meningitidis. Other species that I have investigated
possess still different structures but are not described
here because those data have not yet been accepted for
publication.
Y. pestis, a ‘‘Young’’ species
Y. pestis is indistinguishable from Y. pseudotuberculosis by the gold standard of taxonomy, DNA–DNA
hybridization (Bercovier et al., 1980), and should hence
be included within that species by taxonomic criteria.
Sequence comparisons of rRNA genes confirmed this
conclusion: 16S rRNA is identical between the two
species and 23S rRNA is almost identical (Trebesius et
al., 1998). However, it was first after an attempt was
made to apply MLST to Y. pestis (Achtman et al., 1999),
that it became apparent that Y. pestis is simply a clone
of Y. pseudotuberculosis that has evolved in the last few
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M. Achtman / International Journal of Medical Microbiology 294 (2004) 67–73
millennia. Fragments of five housekeeping genes plus a
sixth gene involved in lipopolysaccharide (LPS) biosynthesis were sequenced from 36 isolates that had been
isolated from humans, rodents and fleas from diverse
global sources. The same gene fragments were sequenced from 12 isolates of Y. pseudotuberculosis. Only
one allele was found for each of the gene fragments in Y.
pestis, i.e. all strains possessed identical sequences. In
contrast, 2–5 alleles were found for each gene fragment
in Y. pseudotuberculosis. Furthermore, each of the Y.
pestis alleles was either identical, or almost identical, to
an allele within Y. pseudotuberculosis. Age calculations
showed that Y. pestis has evolved from Y. pseudotuberculosis in the last 1000–20,000 years (Achtman et al.,
1999), which has since been revised to about
10,000–40,000 years (Achtman, 2004).
A number of species have now been recognized that
possess little sequence diversity and are of recent
descent. The time since a last common ancestor for
Mycobacterium tuberculosis is about the same as for Y.
pestis (Sreevatsan et al., 1997; Gutacker et al., 2002).
Bacillus anthracis also shows very little sequence
diversity (Hill et al., 2004; Keim et al., 1999) and is
probably of recent origin. Some other pathogens that do
not have species status are also highly uniform because
of severe bottlenecks in the last 50,000 years, e.g. S.
enterica Typhi (Kidgell et al., 2002) and Plasmodium
falciparum (Rich et al., 1998; Conway et al., 2000;
Volkman et al., 2001; Joy et al., 2003). What is
somewhat unusual about Y. pestis is that it has not
simply undergone a recent bottleneck, but rather has
actually evolved from Y. pseudotuberculosis within this
time frame. Taken together, these various young groups
of pathogens define a novel population structure,
possibly best designated as ‘‘Young’’, which does not
fall into the clonal versus recombination debate.
What has happened to Y. pestis since it evolved?
Initial steps in its evolution may have resulted due to the
acquisition of two virulence plasmids (Achtman et al.,
1999). Subsequently, the genome of Y. pestis has been
disrupted by multiple insertions of IS100 (Achtman et
al., 1999; Motin et al., 2002) and other insertion
elements (Deng et al., 2002; Parkhill et al., 2001),
resulting in deletions (Radnedge et al., 2002) and
genomic rearrangements (Deng et al., 2002). Numerous
genes have been inactivated by these mechanisms as well
as by frameshift mutations (Deng et al., 2002). However,
a comparison of 3237 homologous coding sequences
between two Y. pestis genomes revealed only 38
synonymous polymorphisms in non-repetitive DNA
(Achtman, 2004), confirming that there has been
extremely little accumulation of neutral sequence
diversity during its limited time of existence.
Microbiologists have identified considerable diversity
within Y. pestis by a variety of methods. Y. pestis can be
subdivided into biovars that differ in their nutritional
69
properties (Anisimov et al., 2004), into ribotypes
(Guiyoule et al., 1994), and by the locations of IS100
(Motin et al., 2002), the presence of DNA islands
(Radnedge et al., 2002) or the number of repeats within
repetitive DNA stretches (Adair et al., 2000). Splitting
Y. pestis into subtypes may be useful for epidemiological
purposes, although even this remains to be proven, but
does not negate my primary conclusion that this species
is young and amazingly uniform.
N. meningitidis subgroup III: clonal expansion
and genoclouds
As mentioned above, the MLST database for N.
meningitidis (meningococci) contains almost 3500 STs
for 5000 bacteria isolated from diseased patients and
healthy carriers since 1917 (Fig. 1). Of these, 231
meningococci expressing the serogroup A capsular
polysaccharide have been tested by MLST but only 49
STs were detected. The isolates tested by MLST
represent the most diverse genotypes found among
approximately 1000 isolates from diverse epidemics and
global sources by more sensitive typing methods
(MLEE, RAPD and PCR-RFLP) (Olyhoek et al.,
1987; Wang et al., 1992; Bart et al., 1998; Achtman et
al., 2001; Zhu et al., 2001) and reflect the continued
surveillance of outbreaks and carrier isolates in diverse
countries. 33 of the 49 STs cluster together tightly, with
five STs containing the majority of isolates, and
represent the known global diversity of serogroup A
meningococci from all epidemics and hyperendemic
disease since the 1950s.
This extraordinary uniformity can be partially explained by the hypothesis that serogroup A meningococci represent the clonal expansion since 1805, when
the first meningitis epidemic was reported (Vieusseux,
1806), of an organism that acquired genes encoding the
A capsular polysaccharide (Achtman et al., 2001). The
epidemic model proposed by Maynard Smith et al.
(1993) can be excluded, because two major clonal
groupings with strong linkage disequilibrium were
indicated by MLEE, RAPD and MLST of epidemic
serogroup A meningococci (Bart et al., 2001). This then
raises the question how clonal expansion can prevail
when recombination is frequent. Why are there so few
STs among epidemic serogroup A and why are these
bacteria so uniform?
One of the characteristics of epidemic meningococcal
disease is that epidemics in any one country only last for
2–3 years, even before the introduction of antibiotics
and vaccines against A capsular polysaccharide.
Furthermore, it is not only disease that disappears but
also carriage of serogroup A meningococci returns to
endemic levels very quickly (Hassan-King et al., 1988;
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Subgroup III
Fig. 1. Minimal spanning tree of the relationships among 3322 STs within N. meningitidis. Data from http://www.pubmlst.org/ as of
early February, 2004. The tree shows one minimal spanning tree of relationships between sequence types and was generated with
Bionumerics V 3.5 (Applied Maths, Belgium). Colored nodes represent clonal complex notations at the PubMLST web site. The
arrow points to STs that belong to subgroup III of serogroup A.
Gagneux et al., 2002b), possibly due to herd immunity.
Towards the end of an epidemic, the frequency of both
mutators and serological variants increases dramatically
(Crowe et al., 1989; Linz et al., 2000; Richardson et al.,
2002), presumably due to immune pressure (Meyers
et al., 2003). Although these variants may temporarily
escape herd immunity, they normally seem to be less fit
than their parents and are lost by bottlenecks during
spread from country to country (Zhu et al., 2001). As a
result, most of the isolates from a pandemic that spans
multiple countries and continents are indistinguishable,
even by very sensitive methods, but multiple, unique
variants can be isolated in each country. The resulting
population type was designated a ‘‘genocloud’’ (Zhu
et al., 2001). With time, fit variants can arise, which are
transmitted as efficiently as their parents and form new
genoclouds. Most of these are also lost with time but
some are transmitted so efficiently that they can spread
pandemically to diverse countries and continents.
During the last 35 years, nine genoclouds have been
detected during three pandemic waves caused by ST5
and ST7 and their minor variants (Zhu et al., 2001).
The purification of variants during epidemic spread of
serogroup A meningococci ensures that these bacteria
remain clonal. At any one time, the diversity is highly
limited and disappears continuously as these bacteria
spread. Variants cannot accumulate in any one area
because herd immunity ensures that no single genocloud
survives in one location for more than a few years. This
mechanism is probably not limited to serogroup A,
because epidemic spread has also been observed for
serogroup X (Gagneux et al., 2002a, b). However,
epidemic spread is rarely observed unless it is accompanied by disease and it is the combination of both
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M. Achtman / International Journal of Medical Microbiology 294 (2004) 67–73
properties, efficient transmission and frequent disease,
that is so unique to serogroup A meningococci.
Concluding remarks
The combination of MLST with high-throughput
sequencing has drastically increased the amount of
information available on the diversity of pathogenic
bacteria. Our knowledge of natural diversity will
continue to increase rapidly in the near future, especially
as other numerical techniques with lower cost and
higher throughput (variable number tandem repeat and
single nucleotide polymorphism analysis) are applied to
large strain collections. The problem for the future will
be obtaining samples from those developing countries
where disease is most frequent and handling the large
datasets that arise. Interdisciplinary population genetic
insights are needed to guide the planning of such
experiments and novel algorithms will be needed to
evaluate the data. So far, the hints given by our current
understanding of the population structure of pathogenic
bacteria indicate that fascinating insights into biological
problems of general interest will be possible.
Acknowledgements
The recent work described here was supported by the
Deutsche Forschungsgemeinschaft priority program
1047 ‘‘Ecology of bacterial pathogens: molecular and
evolutionary aspects’’ (Grant Ac 36/9).
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