Research
Clonal Groupings in
Serogroup X Neisseria meningitidis
Sébastien Gagneux,*† Thierry Wirth,‡ Abraham Hodgson,† Ingrid
Ehrhard,§ Giovanna Morelli,‡ Paula Kriz,¶ Blaise Genton,* Tom Smith,*
Fred Binka,† Gerd Pluschke,* and Mark Achtman‡
*Swiss Tropical Institute, Basel, Switzerland; †Navrongo Health
Research Centre, Ministry of Health, Navrongo, Ghana; ‡Max-Planck-Institut
für Infektionsbiologie, Berlin, Germany; §University of Heidelberg,
Heidelberg, Germany; and ¶National Institute of Public Health, Prague,
Czech Republic
The genetic
diversity of 134 serogroup X Neisseria meningitis isolates
from Africa, Europe, and North America was analyzed by multilocus
sequence typing and pulsed-field gel electrophoresis. Although
most European and American isolates were highly diverse, one clonal
grouping was identified in sporadic disease and carrier strains
isolated over the last 2 decades in the United Kingdom, the Netherlands,
Germany, and the United States. In contrast to the diversity in
the European and American isolates, most carrier and disease isolates
recovered during the last 30 years in countries in the African
meningitis belt belonged to a second clonal grouping. During the
last decade, these bacteria have caused meningitis outbreaks in
Niger and Ghana. These results support the development of a comprehensive
conjugate vaccine that would include serogroup X polysaccharide.
Bacterial meningitis due to Neisseria meningitidis (meningococcus)
causes epidemics in Africa usually associated with serogroup A meningococci.
Sporadic cases, outbreaks, and hyperendemic disease in Europe and
the United States are usually caused by serogroups B and C (1).
Occasionally, however, endemic disease and outbreaks are caused
by bacteria belonging to other serogroups, including W135, Y, and
X. Serogroup X N. meningitidis was described in the 1960s
(2,3), and serogroup X meningitis has been observed
in North America (4), Europe (5,6),
Australia (7), and Africa (8,9).
Serogroup X outbreaks have been reported in Niger (10,11) and
Ghana (12). In some cases, serogroup X disease
has been associated with a deficiency of particular complement components
(13,14) or with AIDS (15).
Asymptomatic nasopharyngeal carriage of N. meningitidis
is common, and in only a small percentage of colonized persons do
the bacteria invade the bloodstream and cerebrospinal fluid to cause
disease. Meningococcal populations are highly diverse, and lineages
of meningococci with increased capacity to cause invasive disease
are thought to arise periodically and spread, sometimes globally
(16). Relatively few of these hyperinvasive lineages
or clonal groupings are responsible for most meningococcal disease
worldwide (17). These clonal groupings diversify
during spread (18,19) , primarily as a result
of frequent horizontal genetic exchange (19-21)
. However, many variants are isolated only rarely or from a single
country and are not transmitted further because of bottlenecks associated
with geographic spread and competition (19,22)
. The population structure of N. meningitidis is effectively
panmictic as a result of frequent horizontal genetic exchange (23),
but that of some groupings, such as epidemic serogroup A meningococci,
is largely clonal (24). The population structure
of serogroup X meningococci has not yet been investigated in detail.
After an epidemic of serogroup A disease in 1997-1998 in northern
Ghana (9), we conducted a longitudinal carriage
study to investigate the dynamics of meningococcal carriage during
an interepidemic period (12). We observed a sharp
increase in nasopharyngeal carriage of serogroup X meningococci
by healthy persons, accompanied by several cases of serogroup X
meningitis. To investigate the phylogenetic relationships of these
bacteria, we compared the isolates from Ghana with other serogroup
X meningococci isolated during recent decades in Africa, Europe,
and North America.
Materials
and Methods
Bacterial Strains
We analyzed 134 N. meningitidis isolates of serogroup X
by pulsed-field gel electrophoresis (PFGE) (130 isolates) or multilocus
sequence typing (MLST) (41 isolates). Of these bacteria, 102 were
isolated in Africa from 1970 to 2000: from meningitis patients (9
isolates) and healthy carriers (70 isolates) in Ghana, 1998-2000;
from healthy carriers in Mali in 1970 (9 isolates) and 1990-91 (4
isolates); and from patients in Chad (1995, 1 isolate), Niger (1997-1998,
4 isolates), and Burkina Faso (1996-1998, 5 isolates). Six isolates
were not tested serologically; the other 96 were NT:P1.5.
Molecular Typing of
Bacteria
PFGE was done by digesting chromosomal DNA prepared in agarose
blocks with NheI and SpeI as described (22),
and MLST by sequencing gene fragments of abcZ, adk,
aroE, fumC, gdh, pdhC, and pgm,
also as described (16; http://www/mlst.net).
The detailed MLST results and sources of isolates have been deposited
in a public database (http://www.mlst.net). Additional MLST data for
31 isolates in 30 sequence types were obtained with permission from
http://www.mlst.net.
Data Analysis
A neighbor-joining tree was constructed by using the numbers of
MLST allele differences with Bionumerics 2.0 (25).
Results
PFGE with two discriminatory rare-cutting enzymes (NheI
and SpeI) was used to identify groups of closely related
strains in 134 isolates of serogroup X N. meningitidis from
countries in Africa, Europe, and North America. All but 3 of 102
isolates from Africa had similar PFGE patterns (Figure
1, clonal grouping X-I). In contrast, 19 of 32 isolates from
Europe and North America had distinct PFGE patterns (Figure
2) that differed from those of the African isolates. However,
among the latter 32 strains, similar PFGE patterns were observed
for 13 isolates from the United Kingdom, Germany, the Netherlands,
and the United States (Figure 2, clonal grouping
X-II).
Forty-one isolates, each representing a distinct PFGE pattern,
were analyzed by MLST. For bacteria from which multiple isolates
with a similar PFGE pattern had been detected, we tested at least
one representative from each year and country of isolation. Together
with other data in the MLST WEB site (http://www.mlst.net),
39 distinct sequence types (STs) have been found in 50 serogroup
X meningococci. The general structure of a neighbor-joining tree
of allelic differences resembles a bush, with little phylogenetic
structure (Figure 3). However, isolates with
similar PFGE patterns were assigned to closely related STs. All
29 clonal grouping X-I isolates analyzed by MLST were in STs ST181,
ST182, or ST751 (Figure 3), which differ
by one to three of the seven gene fragments (Table).
Similarly, all five clonal grouping X-II isolates were in STs 24
and 750, which differ by one of the seven gene fragments (Table).
The three unusual African isolates (strain designations D87, D91,
and D93) were in ST188, which is very distinct from STs of clonal
grouping X-I (Figure 3). These results show
that numerous serogroup X isolates from Africa and nearly half the
serogroup X isolates from Europe and North America belong to two
clonal groupings, while other serogroup X isolates from Europe or
North America are quite diverse.
Serologic Results
African isolates of clonal grouping X-I were NT:P1.5. The 11 North
American and European isolates of clonal grouping X-II for which
serologic data were available were 21:P1.16. Diverse serotype and
serosubtype patterns were found for the other isolates from North
America and Europe
The PFGE patterns distinguished two finer groups (Ia and Ib) in
clonal grouping X-I, which differ consistently in four NheI
and three SpeI fragments (Figure 1).
All 14 group Ia strains tested were either ST181 or ST182, which
differ at one of the seven gene fragments (Table).
All 15 group Ib strains tested were ST751, which differs from ST181
and ST182 at two to three loci (Table). Group
Ia included 10 isolates from Mali (1970-1990), 4 isolates from Niger
(1997-1998), and the sole isolate from Chad (1995), as well as one
of 79 isolates from Ghana (2000). All five isolates from Burkina
Faso (1996-1998) and 78 of 79 isolates from Ghana (1998-2000) were
in group Ib.
Discussion
The general population structure of N. meningitidis is panmictic
as a result of the frequent import of alleles from unrelated Neisseriae
(20,23). Furthermore, several
MLST studies have demonstrated that meningococci from healthy carriers
are highly diverse (16,26) .
Phylogenetic trees of different housekeeping genes from N. meningitidis
are no more congruent with each other than with random trees (27).
Our results for sequence typing of housekeeping genes of serogroup
X meningococci also fit this pattern. Phylogenetic analysis of allele
differences resulted in a bushlike tree that does not seem to contain
any deep phylogenetic information. However, two clonal groupings
were found in this otherwise panmictic group of bacteria. The same
isolates were assigned to both clonal groupings by two independent
methods, MLST and PFGE, indicating that these assignments reflect
real genetic relationships and do not depend on the methods used.
Similar concordant genetic relationships were discerned in epidemic
serogroup A N. meningitidis by multilocus enzyme electrophoresis
(MLEE), random amplification of polymorphic DNA (RAPD), and MLST;
population genetic analyses confirmed that the population structure
of these bacteria is clonal (28). Concordant groupings
were also discerned by MLEE and MLST among the so-called hypervirulent
serogroup B and C isolates of the ET-5 complex, ET-37 complex, lineage
III, and cluster A4 (16). Although their apparent
clonality may reflect an epidemic population structure (23),
this possibility has been excluded for epidemic serogroup A meningococci
(28). Therefore, multiple clonal groupings exist
in N. meningitidis, even though the population structure
of most of the species is panmictic.
The population structure of subgroup III serogroup A meningococci
seems to represent continual, sequential replacement of fit genotypes
by related variants during periods of several years to decades (19).
In subgroup III, nine genoclouds, each consisting of a frequent
genotype plus its rarer, less fit variants, have been identified
during 3 decades of pandemic spread. Our PFGE data for clonal grouping
I of serogroup X suggest that clonal grouping X-I also has a genocloud
structure. Two sets of PFGE variants (group Ia and Ib), which might
each represent a genocloud, were detected in different countries
(Mali, Chad, and Niger; and Burkina Faso and Ghana, respectively).
Additional analyses of polymorphic genes are necessary to clarify
the uniformity of these groups and to test the similarity of their
population structure compared with that of subgroup III.
Both serogroup X clonal groupings described here were isolated
over decades, on multiple occasions, and from diverse locations.
Clonal grouping X-I (1970-2000) was isolated from different countries
in West Africa, and clonal grouping X-II (1986-1999) was isolated
from Europe and North America. For clonal grouping X-I in Ghana,
the disease rate in healthy carriers was estimated to be 3/10,000
(12). Clonal grouping X-1 is thus of considerably
lower virulence than serogroup B ET-5 complex bacteria (disease/carrier
rate of 2,100/10,000 [29]) or serogroup A subgroup
III bacteria during a postepidemic period in a vaccinated population
(100/10,000 [9]).
The relationship between bacterial fitness and clonality has not
yet been investigated extensively in natural isolates. Variation
in virulence between bacterial genotypes leads to more uniformity
in disease isolates than in carriage organism in Streptococcus
pneumoniae (30) and Staphylococcus aureus
(31). However, our data suggest that the clonal
structure of certain meningococcal genotypes need not reflect virulence
but rather is associated with genotypes that are particularly fit
at colonizing the nasopharynx and spreading from person to person.
Although clonal grouping X-I bacteria are less virulent than serogroup
A and B meningococci, they are still pathogenic. Most strains described
here were isolated from asymptomatic carriers or patients with rare
endemic cases. However, group Ia caused a meningitis outbreak with
>60 cases in 1997 in Niger (11). Group Ib caused
a smaller outbreak in 2000 in Ghana (12). These
results suggest that X-I meningococci may even be capable of causing
epidemics. Meningococci are naturally transformable, and horizontal
DNA transfer is frequent in these bacteria (20-22)
. Meningococcal carriage is usually low in interepidemic periods
in Africa (1,12,32,33)
, offering less opportunity for horizontal genetic exchange, which
could account for the low genetic variability in serogroup X meningococci
in Africa.
For more than a decade, many countries in the African meningitis
belt have vaccinated extensively with A/C polysaccharide vaccines
(34). Recently, mass vaccination with conjugated
serogroup C vaccines has been implemented in the United Kingdom,
and strong initial protection has been reported (35).
However, if effective, these vaccines may well select for the spread
of bacteria for which they are not protective (36),
including unusual causes of disease such as serogroups Y, W135,
and X. Capsule switching due to DNA transformation has been documented
(37,38), and effective vaccination against serogroups
A and C may select for capsule switch variants of fit genotypes
expressing a capsular polysaccharide not included in the vaccination
program. The recent outbreaks after the 2000 Hajj pilgrimage, caused
by W135 ET-37 complex meningococci (39,40), may
reflect exactly such selection. These findings support the development
of comprehensive conjugate vaccines that include capsular polysaccharides
from formerly rare causes of disease such as serogroup X.
Acknowledgments
We gratefully acknowledge the helpful comments and support of Alex
Nazzar and Daniel Falush and the receipt of isolates from Dominique
Caugant, Tanja Popovic, Ed Kaczmarski, and Mohamed-Kheir Taha. This
publication made use of the Neisseria MultiLocus Sequence
Typing website (http://neisseria.mlst.net) developed by Man-Suen
Chan, funded by the Wellcome Trust, and located at the University
of Oxford. We thank Keith Jolly for allowing the use of deposited
data. Technical assistance by Santama Abdulai, Titus Teï, Susanne
Faber, Marion Moebes, and Barica Kusecek is greatly appreciated.
This work was funded by the Stanley Thomas Johnson Foundation.
Part of the work (P.K.) was supported by the research grant NI/6882-3
of the Internal Grant Agency of the Ministry of Health of the Czech
Republic.
Dr. Gagneux is a postdoctoral research fellow at the Swiss Tropical
Institute. His research interests focus on the epidemiologic and
genetic characterization of Neisseria meningitidis and Mycobacterium
tuberculosis.
Address for correspondence: Gerd Pluschke, Swiss Tropical Institute,
Socinstrasse 57, 4002 Basel, Switzerland; fax: +41 61-271 8654;
e-mail: gerd.pluschke@unibas.ch
References
- Achtman M. Global epidemiology of meningococcal
disease. In: Cartwright KA, editor. Meningococcal disease. Chichester,
UK: John Wiley, 1995:159-75.
- Bories S, Slaterus KW, Faucon R, Audiffren P, Vandekerkove M.
Peut-on individualiser deux nouveaux groupes sérologiques de Neisseria
meningitidis? Med Trop (Mars) 1966;26:603-16.
- Evans JR, Artenstein MS, Hunter DH. Prevalence
of meningococcal serogroups and description of three new groups.
Am J Epidemiol 1968;87:643-6.
- Ryan NJ, Hogan GR. Severe
meningococcal disease caused by serogroups X and Z. Am J Dis
Child 1980;134:1173.
- Pastor JM, Fe A, Gomis M, Gil D. [Meningococcal
meningitis caused by Neisseria meningitidis of the X serogroup].
Med Clin (Barc) 1985;85:208-9.
- Grahlow WD, Ocklitz HW, Mochmann H. Meningococcal
infections in the German Democratic Republic 1971-1984. Infection
1986;14:286-8.
- Hansman D. Meningococcal
disease in South Australia: incidence and serogroup distribution
1971-1980. J Hyg (Lond) 1983;90:49-54.
- Riou JY, Djibo S, Sangare L, Lombart JP, Fagot P, Chippaux JP,
et al. A
predictable comeback: the second pandemic of infections caused
by Neisseria meningitidis serogroup A subgroup III in Africa,
1995. Bull World Health Organ 1996;74:181-7.
- Gagneux S, Hodgson A, Ehrhard I, Morelli G, Genton B, Smith
T, et al. Microheterogeneity
of serogroup A (subgroup III) Neisseria meningitidis during
an outbreak in northern Ghana. Trop Med Int Health 2000;5:280-7.
- Etienne J, Sperber G, Adamou A, Picq JJ. [Epidemiological
notes: meningococcal meningitis of serogroup X in Niamey (Niger)].
Med Trop (Mars) 1990;50:227-9.
- Campagne G, Schuchat A, Djibo S, Ousseini A,
Cisse L, Chippaux JP. Epidemiology
of bacterial meningitis in Niamey, Niger, 1981-96. Bull World
Health Organ 1999;77:499-508.
- Gagneux S, Hodgson A, Smith T, Wirth T, Ehrhard I, Morelli G,
et al. Prospective study of a serogroup X Neisseria meningitidis
outbreak in Northern Ghana. J Infect Dis 2002;185:618-26.
- Swart AG, Fijen CA, te Bulte MT, Daha MR, Dankert J, Kuijper
EJ. [Complement
deficiencies and meningococcal disease in The Netherlands].
Ned Tijdschr Geneeskd 1993;137:1147-52.
- Fijen CA, Kuijper EJ, Te BM, van de Heuvel MM, Holdrinet AC,
Sim RB, et al. Heterozygous
and homozygous factor H deficiency states in a Dutch family.
Clin Exp Immunol 1996;105:511-6.
- Morla N, Guibourdenche M, Riou JY. Neisseria
spp.
and AIDS. J Clin Microbiol 1992;30:2290-4.
- Maiden MC, Bygraves JA, Feil E, Morelli G, Russell JE, Urwin
R, et al. Multilocus
sequence typing: a portable approach to the identification of
clones within populations of pathogenic microorganisms. Proc
Natl Acad Sci U S A 1998;95:3140-5.
- Caugant DA. Population
genetics and molecular epidemiology of Neisseria meningitidis.
APMIS 1998;106:505-25.
- Caugant DA, Froholm LO, Bovre K, Holten E, Frasch CE, Mocca
LF, et al. Intercontinental
spread of a genetically distinctive complex of clones of Neisseria
meningitidis causing epidemic disease. Proc Natl Acad
Sci U S A 1986;83:4927-31.
- Zhu P, van der Ende A, Falush D, Brieske N, Morelli G, Linz
B, et al. Fit
genotypes and escape variants of subgroup III Neisseria meningitidis
during three pandemics of epidemic meningitis. Proc Natl Acad
Sci U S A 2001;98:5234-9.
- Linz B, Schenker M, Zhu P, Achtman M. Frequent
interspecific genetic exchange between commensal Neisseriae
and Neisseria meningitidis. Mol Microbiol 2000;36:1049-58.
- Kriz P, Giorgini D, Musilek M, Larribe M, Taha
MK. Microevolution
through DNA exchange among strains of Neisseria meningitidis
isolated during an outbreak in the Czech Republic. Res Microbiol
1999;150:273-80.
- Morelli G, Malorny B, Muller K, Seiler A, Wang JF, del Valle
J, et al. Clonal
descent and microevolution of Neisseria meningitidis during
30 years of epidemic spread. Mol Microbiol 1997;25:1047-64.
- Smith JM, Smith NH, O'Rourke M, Spratt BG. How
clonal are bacteria? Proc Natl Acad Sci U S A 1993;90:4384-8.
- Achtman M, van der Ende A, Zhu P, Koroleva IS, Kusecek B, Morelli
G, et al. Molecular
epidemiology of serogroup A meningitis in Moscow, 1969 to 1997.
Emerg Infect Dis 2001;7:420-7.
- Bionumerics 2.0. Sint-Martens-Latem, Belgium: Applied Maths;
2000.
- Jolley KA, Kalmusova J, Feil EJ, Gupta S, Musilek M, Kriz P,
et al. Carried
meningococci in the Czech Republic: a diverse recombining population.
J Clin Microbiol 2000;38:4492-8.
- Holmes EC, Urwin R, Maiden MC. The
influence of recombination on the population structure and evolution
of the human pathogen Neisseria meningitidis. Mol Biol
Evol 1999;16:741-9.
- Bart A, Barnabé C, Achtman M, Dankert J, van der Ende A, Tibayrenc
M. Strong linkage disequilibrium between different genetic markers
challenges the epidemic clonality model in Neisseria meningitidis
serogroup A isolates. Infection, Genetics and Evolution 2001;1:117-22.
- Cartwright KA, Stuart JM, Jones DM, Noah ND.
The Stonehouse survey: nasopharyngeal carriage of meningococci
and Neisseria lactamica. Epidemiol Infect 1987;99:591-601.
- Smith T, Lehmann D, Montgomery J, Gratten M, Riley ID, Alpers
MP. Acquisition
and invasiveness of different serotypes of Streptococcus pneumoniae
in young children. Epidemiol Infect 1993;111:27-39.
- Day NPJ, Moore CE, Enright MC, Berendt AR,
Maynard Smith J, Murphy MD, et al. A link between virulence and
ecological abundance in natural populations of Staphylococcus
aureus. Science 2001;292:114-6.
- Blakebrough IS, Greenwood BM, Whittle HC, Bradley AK, Gilles
HM. The
epidemiology of infections due to Neisseria meningitidis
and Neisseria lactamica in a northern Nigerian community.
J Infect Dis 1982;146:626-37.
- Hassan-King MK, Wall RA, Greenwood BM. Meningococcal
carriage, meningococcal disease and vaccination. J Infect
1988;16:55-9.
- Tikhomirov E, Santamaria M, Esteves K. Meningococcal
disease: public health burden and control. World Health Stat
Q 1997;50:170-7.
- Ramsay ME, Andrews N, Kaczmarski EB, Miller E. Efficacy
of meningococcal serogroup C conjugate vaccine in teenagers and
toddlers in England. Lancet 2001;357:195-6.
- Maiden MC, Spratt BG. Meningococcal
conjugate vaccines: new opportunities and new challenges.
Lancet 1999;354:615-6.
- Swartley JS, Marfin AA, Edupuganti S, Liu LJ, Cieslak P, Perkins
B, et al. Capsule
switching of Neisseria meningitidis. Proc Natl Acad
Sci U S A 1997;94:271-6.
- Vogel U, Claus H, Frosch M. Rapid
serogroup switching in Neisseria meningitidis. N Engl
J Med 2000;342:219-20.
- Taha MK, Achtman M, Alonso JM, Greenwood B, Ramsay M, Fox A,
et al.
Serogroup W135 meningococcal disease in Hajj pilgrims. Lancet
2000;356:2159.
- Popovic T, Sacchi CT, Reeves MW, Whitney AM, Mayer LW, Noble
CA, et al. Neisseria
meningitidis
serogroup W135 isolates associated with the ET-37 complex.
Emerg Infect Dis 2000;6:428-9.
Table.
Multilocus sequence typing results of two serogroup X Neisseria
meningitidis clonal groupings |
|
ST |
Allele numbers
|
Country
(no. of isolates) |
Year |
|
AbcZ
|
Adk
|
aroE
|
FumC
|
gdh
|
pdhC
|
Pgm
|
|
24
|
2
|
5
|
2
|
7
|
15
|
20
|
5
|
Netherlands (1), United States (1)
|
1986, 1993
|
750
|
2
|
5
|
2
|
9
|
15
|
20
|
5
|
United Kingdom (2), Germany (1)
|
1998-1999
|
181
|
10
|
3
|
15
|
7
|
5
|
41
|
31
|
Mali (6), Chad (1), Niger (2), Ghana (1)
|
1970-2000
|
182
|
10
|
3
|
15
|
26
|
5
|
41
|
31
|
Mali (4)
|
1970
|
751
|
10
|
3
|
15
|
7
|
8
|
41
|
6
|
Burkina Faso (3), Ghana (12)
|
1996-2000
|
|
|
|