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Journal of Clinical Microbiology, September 2005, p. 4649-4653, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4649-4653.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Increased Genetic Diversity of Neisseria meningitidis Isolates after the Introduction of Meningococcal Serogroup C Polysaccharide Conjugate Vaccines

Mathew A. Diggle¹ and Stuart C. Clarke^1,2*

Scottish Meningococcus and Pneumococcus Reference Laboratory, Stobhill Hospital, Glasgow G21 3UW, United Kingdom,¹ Division of Infection and Immunity, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, United Kingdom²

Received 14 January 2005/ Returned for modification 7 March 2005/ Accepted 4 May 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
During the 1990s, the incidence of meningococcal disease was high in the United Kingdom. This was due primarily to an increase in serogroup C disease, particularly that within the ET-37/ST-11 genetic lineage. Serogroup C meningococcal polysaccharide conjugate vaccines were introduced in the United Kingdom in 1999, but the sequence types of meningococci causing disease since that time have not yet been reported. We have used serogrouping and multilocus sequence typing to characterize meningococci from patients with invasive disease over a 4-year period and show that there is a significant increase in genetic diversity but no genetic evidence of capsule switching.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Polysaccharide conjugate vaccines are providing essential and highly effective immunization against some of the major human pathogens (1, 13, 19, 20, 24). One such pathogen, Neisseria meningitidis, remains an important cause of meningitis and septicemia worldwide (23). Serogroup C meningococcal disease became a particular problem in the United Kingdom during the 1990s and was associated with a high prevalence of the ET37/ST11 clone (6, 16). Prospective enhanced surveillance of those meningococci associated with throat carriage and invasive disease was initiated in the United Kingdom before the implementation of the meningococcal serogroup C conjugate (MenC) vaccines towards the end of 1999 (2, 5, 13). An overall vaccine uptake of 85% was achieved in the United Kingdom for the whole population under 20 years of age (14), and the implementation of MenC vaccines has led to a reduced level of serogroup C meningococcal throat carriage and also a reduced level of invasive disease (13, 14, 17, 18). However, since the population dynamics of meningococcal disease are very fluid, with serogroup B and serogroup C historically undergoing a complementary cyclic pattern (7, 16), it is essential to understand the intra- and interlineage population dynamics of this disease. We used multilocus sequence typing (MLST) to characterize a collection of 432 meningococci, isolated from cases of invasive meningococcal disease before (1999) and during (2000, 2001, and 2002) the introduction of MenC vaccines (years 1 to 4) in Scotland.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Four hundred thirty-two isolates of N. meningitidis from patients with invasive meningococcal disease in Scotland were analyzed between 1999 and 2002 inclusive by MLST, a method which sequences internal fragments of seven housekeeping genes (6, 12). These isolates were isolated from normally sterile sites (blood and cerebrospinal fluid) from patients with meningococcal disease from all 15 National Health Service boards via their representative diagnostic hospital laboratories. Phenotypic and genotypic characterization followed by assignment of nucleotide sequence data to sequence types (STs) and complexes was performed as previously described (4, 6-8). Briefly, internal fragments of seven housekeeping genes—abcZ, adk, aroE, fumC, gdh, pdhC, and pgm—were sequenced using an automated protocol with a Roboseq 4200 (MWG Biotech UK Ltd., Milton Keynes, United Kingdom) and MegaBACE 1000 DNA sequencer (Amersham Biosciences, Little Chalfont, United Kingdom). Alleles were assigned by comparing nucleotide sequence data at that locus to all known alleles at that locus. STs were assigned according to the combination of the seven alleles with reference to the N. meningitidis MLST database (www.mlst.net). Intra- and interlineage comparisons were made using the Sequence Type Analysis and Recombinational Tests (START) suite of programs available at http://pubmlst.org/software/analysis/start (10). Mutation and/or recombination events were calculated by determining the number of substitutions per nonsynonymous (d_N) and synonymous (d_S) site within a particular nucleotide sequence from the allele sequence using the method of Nei and Gojobori (15), which is included in the START software suite. A summary of the single multilocus measure of linkage disequilibrium was achieved by the index of association (I_A) (3, 21) using the START program.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sequence typing data from the 428 meningococci used in this study show that the meningococcal population was highly diverse and that an increasing trend of diversity was noted in individual years, from years 1 to 4 (Fig. 1). The absolute numbers of meningococci isolated in each year were 130 in 1999, 130 in 2000, 80 in 2001, and 88 in 2002. The incidence of serogroup C meningococci decreased during the study, from 58 (45%) in year 1 to 10 (11%) in year 4. The isolation of serogroup B meningococci also changed, resulting in a slight increase from 65 (50%) in year 1 to 69 (78%) in year 4. In year 1, serogroup C meningococci were assigned to only five different STs; 95% of these STs were contained within two complexes (ST-11 complex [93%] and ST-269 complex [2%]) (Table 1). Serogroup B meningococci were assigned to 31 different STs; 68% of these STs were contained within five complexes (ST-44 complex [34%], ST-269 complex [18%], ST-18 complex [8%], ST-35 complex [5%], and ST-32 complex [3%]) (Table 1). However, the serogroups associated with different STs changed between years 1 and 4, such that, in year 4, serogroup C meningococci were assigned to only two different STs; all of these STs were contained within two complexes (ST-11 complex [91%] and ST-269 complex [9%]). Serogroup B meningococci were assigned to 40 different STs; 68% of these STs were contained within five complexes (ST-269 complex [26%], ST-44 complex [19%], ST-32 complex [16%], ST-18 complex [4%], and ST-254 [3%]). Importantly, over the study period, the serogroup C ET-37/ST-11 genetic lineage declined substantially and there was no corresponding increase in serogroup B of the ET-37/ST-11 genetic lineage. Moreover, there was no development of capsule switch (i.e., change of serogroup within the same ST) as determined by serogroup and ST association.

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FIG. 1. Phylogenetic relationships of meningococci isolated between 1999 and 2002 (years 1 to 4 [A to D, respectively]) and their association with ST complex and serogroup. Unweighted pair group method with mathematic averages trees were constructed using the START program, and ST complexes were assigned using BURST. Serogroup B is shown in black, serogroup C in white, serogroup Y with hatching, serogroup W135 with waves, and serogoup X with a chessboard design.

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TABLE 1. Complex assignment, association, and intracomplex variationa

Analysis of allele frequency showed that the number of alleles present at each locus for each of the 4 years ranged between eight for adk (adenylate kinase) in year 1 and 20 for aroE (shikimate dehydrogenase) in year 4. The number of polymorphic sites present at each locus for each year ranged between 14 in year 1 (3% of sites for adk) and 128 in year 4 (26% of sites for aroE). The number of alleles as a function of the number of isolates indicated a clear correlation between the increase in the number of alleles identified with an increase in the number of isolates examined. This provides further evidence of meningococcal genetic diversity within the 4 years of our study.

In year 1, disregarding serogroup, meningococci were assigned to 41 different STs within six distinct lineages. These lineages occurred between 1 (0.8%) and 55 (42%) times during year 1. In addition, there were 15 singleton types (11.5%) (i.e., no complex association). Isolates of the ST-11 complex accounted for 55 (42%) isolates and were assigned to two STs (ST-11 [n = 53] and ST-655 [n = 2]), the latter of which is a gdh (glucose-6-phosphate dehydrogenase) locus variant of ST-11. The ST-44 complex accounted for 22 isolates (17%), and these were divided into eight different STs; 14 were ST-41 (64%), 2 were ST-1097 (9%), and ST-44, -46, -146, -170, -1194 and -1255 were each represented by a single isolate (4.5%). In year 2, meningococci were assigned to 47 different STs, an increase of 13% from the previous year. They were further differentiated into nine distinct lineages, an increase of 33% from the previous year, with a 27% decrease in singleton representation. These lineages occurred between 1 (0.8%) and 49 (38%) times during year 2. This was statistically similar to the previous year (P = 0.714). Meningococci of the ST-11 complex continued to account for the majority (38%) of isolates. In year 3, meningococci were assigned to 47 different STs and were differentiated into eight different lineages, a decrease of 11% from the previous year, demonstrating an increase in ST diversity. These lineages occurred between 1 (1%) and 18 (23%) times during year 3. This range variation was significantly smaller (P = 0.0443). This highlights a significant decrease in the representation of multiple isolates and the subsequent increase in single represented isolates. Meningococci of the ST-11 complex continued to account for the majority (25%) of isolates. In year 4, meningococci were assigned 49 different STs, a 4% increase from the previous year and were differentiated into nine different lineages, an increase of 11% from year 3. These lineages occurred between 1 (1%) and 10 (11%) times, and the range of variation was therefore again smaller but not significantly so (P = 0.2621), resulting in a greater proliferation of different STs and thus a greater representation of single isolates. The ST-269 complex accounted for 17 isolates (19%), an increase of 12% from year 3. These were divided into 10 different STs which remained consistent. ST-269 and -275 accounted for four isolates (24%), ST-2205 accounted for two isolates (12%), with ST-13, -283, -467, -1095, -2157, -2166, -2205, and -2319 represented once (6%). Both ST-269 and -275 increased by 25% above year 3, and of the remaining STs, none had been represented in previous years. Interlineage variation accounted for 24 single-locus variations, a 140% increase, combined with 26 double-locus variations, an 8% increase, and 40 satellites, a decrease of 29% from year 3. The singletons accounted for 29 isolates (32%), an increase of 28% from the previous year. These were differentiated into 13 different STs, ST-11 (35%), ST-213 (24%), ST-1345 (7%), and ST-103, -136, -866, -1167, -1466, -2161, -2204, -2239, -231,7 and -2318 were represented once each (3.5%). ST-11 decreased by a further 44% from year 3.

Mutation and/or recombination events for the 432 isolates in this study, calculated using d_N/d_S ratios, showed that nonsynonymous nucleotide sequence changes were selected out of the population (Table 2). These data were compared to invasive meningococci contained within the global data set (http://neisseria.mlst.net). The data were comparable to the global data set, with only a few differences between the ratio values, except for aroE, suggesting that the proteins the genes encode are well conserved. The d_N/d_S ratio value was >1 for aroE, indicating that the proteins that these genes encode are not so well conserved and nonsynonymous changes were corrected faster than their occurrence by mutation. The index of association (I_A) for the meningococcal population in years 1 to 4 was high (between 2.564 and 4.469), indicating a clonal population. However, the clonality reduced year by year in reflection of the reduction in serogroup C ST-11 strains.

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TABLE 2. Natural selection and recombination in meningococci between 1999 and 2002 as determined by dN/dS ratios and IA valuesa

Top Abstract Introduction Materials and Methods Results Discussion References

 
A number of studies have been performed with collections of meningococci that cause disease (6, 9, 11, 12, 22). This is reflected by the large number of STs now present within the MLST database. These studies, with which these STs are related, have centered upon carriage and disease, and have been performed mostly during time periods where the selective pressures are normal (i.e., outside of vaccine programs). Our study has taken all available invasive isolates over a 4-year period, starting prior to the implementation of MenC vaccines in the United Kingdom and then continuing through the following 3-year period after vaccine implementation, to determine the genetic diversity of the disease-causing meningococcal population. We have shown that the genetic diversity of disease-causing meningococci has significantly increased after the introduction of MenC vaccines and that this increase has reflected a significant decrease in serogroup C ET-37/ST-11 meningococci which has not been accompanied by an increase in serogroup B meningococci of the same genetic lineage. The diversity of the current disease-causing meningococcal population is due, however, to the presence of new combinations of alleles arising from recombination, thus pertaining to new ST profiles rather than allele mutation. The increase in recombination between years 1 to 4 may therefore be due to natural selection and the increased ability of non-ST-11 meningococci to fill the niche left by this clone. Interestingly, this niche has been filled by meningococci of numerous STs and from a number of clonal complexes. Currently, therefore, there is no hyperendemic clone present in the United Kingdom that has directly replaced the ST-11 clone. However, some clones, such as the ST-269 clone, must be monitored closely, as they have increased year by year since 1999. Our findings will have an important impact on conjugate vaccine strategy for the control of meningococcal disease, as well as other bacterial pathogens, because the medium to long-term effects of vaccines directed against a single serogroup clearly have an effect on the meningococcal population biology. Although the introduction of MenC vaccines has not led directly to serogroup switching (to serogroup B), a definite increase in numbers of clones of serogroup B has been observed, although much of this may be due to the typical cyclical pattern of serogroup B/C incidence. Further retrospective and prospective studies must therefore be performed in order to gain further insights into the whole meningococcal population, not just that causing invasive disease, including the emergence of important genetic lineages. Although, in the present study, we included meningococci from almost 1 full year prior to the introduction of the MenC vaccine, analysis of disease-causing isolates in additional years prior to vaccine implementation would be useful. Our laboratory is currently analyzing such data from a 30-year period prior to implementation of the MenC vaccine. Our data will also be merged with meningococcal carriage study data so that the relationship between carriage and disease can be better understood.

 
We thank all members of the SMPRL for their general support, as well as Tim Mitchell, Glasgow University, Martin Maiden, Oxford University, and Brian Spratt, Imperial College, for useful comments. This publication made use of the Neisseria Multi Locus Sequence Typing and SMPRL MLST websites (http://neisseria.mlst.net and http://phoenix.medawar.ox.ac.uk/smprl) developed by Man-Suen Chan and Keith Jolley and sited at the University of Oxford. The development of this site has been funded by the Wellcome Trust and European Union.

Our laboratory is funded by the Meningitis Association (Scotland), Chief Scientist's Office of the Scottish Executive and National Services Division of the Scottish Executive.

 
* Corresponding author. Present address: Dept. of Improving Health and Quality, Portsmouth City PCT, Finchdean House, Milton Road, Portsmouth PO3 6DP, United Kingdom. Phone: 44 23 9283 5020. Fax: 44 23 9723 3292. E-mail: stuartcclarke{at}hotmail.com.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, September 2005, p. 4649-4653, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4649-4653.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Diggle, M. A. Articles by Clarke, S. C. Search for Related Content PubMed PubMed Citation Articles by Diggle, M. A. Articles by Clarke, S. C.