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Loss of Genomic Diversity in a Neisseria meningitidis Clone Through a Colonization Bottleneck

Abstract Neisseria meningitidis is the leading cause of epidemic meningitis in the “meningitis belt” of Africa, where clonal waves of colonization and disease are observed. Point mutations and horizontal gene exchange lead to constant diversification of meningococcal populations during clonal spread. Maintaining a high genomic diversity may be an evolutionary strategy of meningococci that increases chances of fixing occasionally new highly successful “fit genotypes”. We have performed a longitudinal study of meningococcal carriage and disease in northern Ghana by analyzing cerebrospinal fluid samples from all suspected meningitis cases and monitoring carriage of meningococci by twice yearly colonization surveys. In the framework of this study, we observed complete replacement of an A: sequence types (ST)-2859 clone by a W: ST-2881 clone. However, after a gap of 1 year, A: ST-2859 meningococci re-emerged both as colonizer and meningitis causing agent. Our whole genome sequencing analyses compared the A population isolated prior to the W colonization and disease wave with the re-emerging A meningococci. This analysis revealed expansion of one clone differing in only one nonsynonymous SNP from several isolates already present in the original A: ST-2859 population. The colonization bottleneck caused by the competing W meningococci thus resulted in a profound reduction in genomic diversity of the A meningococcal population.


Introduction
Neisseria meningitidis, a Gram-negative human-specific bacterium, is the major cause of epidemic meningitis in children and adults, particularly across the socalled "African meningitis belt". The epidemiology of N. meningitidis in Africa is characterized by a succession of epidemics, which occur during the dry season (December-May), stop abruptly at the onset of rain and may flare up again in the next dry season. In recent years, the periodicity of epidemic cycles has varied considerably from country to country and their dynamics are still not well understood.
The human nasopharynx is the only habitat of N. meningitidis, but although this bacterium inhabits such a restricted ecological niche, it shows considerable genetic diversity. In Europe complex and temporally relatively stable colonizing meningococcal populations have been observed, which primarily consist of isolates belonging to lineages that have a low virulence (Yazdankhah et al. 2004;Caugant et al. 2007). In contrast, in the African meningitis belt meningococcal colonization is unstable and clonal waves of colonization and disease have been observed (Leimkugel et al. 2007). When the dominating colonizing population belongs to a hypervirulent lineage, these waves are accompanied by outbreaks of invasive meningococcal disease. Lack of a temporally stable and genetically diverse pharyngeal microbiota of meningococci may contribute to the susceptibility of the population of the African meningitis belt to meningococcal disease epidemics (Leimkugel et al. 2007;Chow et al. 2016). Point mutations and horizontal gene exchange lead to constant diversification of meningococcal lineages during clonal spread (Lamelas et al. 2014). In particular the potential for wide variability in cell surface structures allows N. meningitidis lineages to evade immune surveillance.
Meningococci are classified into 12 serogroups based on the composition of their polysaccharide capsule, but only 6 (A, B, C, W, X, and Y) produce frequently invasive disease. Although serogroup A meningococci have been responsible for most epidemics in the meningitis belt in the last 100 years, other serogroups such as C (Chow et al. 2016), W (Taha et al. 2000;Lamelas et al. 2017), and X (Boisier et al. 2006) have also caused outbreaks and are dominating after the introduction of the monovalent serogroup A capsule polysaccharide conjugate vaccine MenAfriVac (Tiffay et al. 2015) in the countries of the meningitis belt (Trotter et al. 2017).
In the framework of a longitudinal meningococcal carriage and disease study in the Kassena-Nankana District (KND) of northern Ghana and an analysis of meningococcal isolates by multilocus sequence typing (MLST), we have observed between 1998 and 2009 three sequential waves of colonization with serogroup A meningococci with the sequence types (ST) 5, 7, and 2859 (Leimkugel et al. 2007). These three closely related lineages have caused meningitis outbreaks in many countries of the meningitis belt (Nicolas, Norheim, et al. 2005). A comparative whole genome sequencing study has revealed that the A: ST-2859 meningococci share a common ancestor with the ST-7 bacteria and differ in multiple homologous recombination events involving incorporation of DNA sequences from meningococci and other Neisseria species (Lamelas et al. 2014). Analysis of the hot spots of recombination indicated that the complex changes in bacterial cell surface structures associated with the recombination events has enabled the A: ST-2859 clone to multiply in host populations that have developed immunity against the A: ST-7 bacteria. A large proportion of individuals belonging to local populations in the meningitis belt may become transiently colonized by dominating clones (Leimkugel et al. 2007) and develop mucosal immunity. When a large enough percentage of the population has become immune to colonization, individuals who are not immune are indirectly protected (herd immunity), leading to the local disappearance of meningococcal clones after a colonization and disease wave (Leimkugel et al. 2007;Lamelas et al. 2014).
Owing to the natural competence of meningococci for transformation and homologous recombination (Rotman and Seifert 2014) the genomic diversity of epidemic N. meningitidis lineages is extensive (Lamelas et al. 2017). Occasionally, individual immune escape variants may expand from these clonal complexes (CCs), as exemplified by the emergence of the A: ST-2859 clone after multiple recombination events from the genomically diversified A: ST-7 population. Here, we demonstrate that also a colonization bottleneck associated with competition for mucosal colonization can cause a massive decrease in genomic diversity in a meningococcal population by expansion of one of the preexisting haplotypes.

Study Area
The study was conducted in the KND of the Upper-East Region of Ghana, located within the guinea Savannah woodland. The area has two major seasons; a short wet season from June to October and a long dry season for the rest of the year. The district-population is about 140,000, most of them rural, except for the 20,000 inhabitants of Navrongo town. In the KND, people live in compounds with an average of 10 inhabitants (http://www.indepth-network.org/dss_site_ profiles/navrongo.pdf). Between 2007 and 2011, vaccination campaigns with meningococcal serogroup A/C polysaccharide vaccine targeted the whole district population. Ethical clearance for this study was obtained from the responsible institutional and national review boards.

Colonization Isolates
Thirty-seven residential compounds were randomly selected from a complete listing of the district population using the Navrongo Demographic Surveillance System and throat swabs were taken from about 300 individuals per survey. Starting in March 1998 throat swabs were taken twice per year (in April and November) from all inhabitants of the 37 compounds present at the time of the visit who agreed to participate. Study participants in the consecutive carriage surveys thus were at least in part, the same individuals. Here, we present the data of surveys 19 (April 2007) to 27 (April 2011); in each of them between 280 and 364 study participants have been swabbed (table 1).
Throat swabs taken from the study participants were directly inoculated on Thayer-Martin agar plates. Colonies with Neisseria morphology were subcultured. Neisseria meningitidis colonies were identified by standard bacteriological methods as previously described (Leimkugel et al. 2007).

Disease Isolates
Suspected meningitis patients presenting at the War Memorial Hospital (WMH), Navrongo, or one of the four Health Centers of the KND were recruited throughout the study period. A suspected meningitis patient was defined by sudden onset of fever and stiff neck, or fever and stiff neck and altered mental status, in accordance with WHO guidelines. A lumbar puncture was performed before treatment from all suspected meningitis patients, and the cerebrospinal fluid specimen was analyzed by latex agglutination and culture (Leimkugel et al. 2007).

Characterization of Bacterial Isolates
Meningococcal isolates were serogrouped by Multiplex PCR (Taha et al. 2000), and selected isolates were analyzed by MLST. DNA extraction was done using a Promega DNA isolation kit and PCR was performed in accordance to standard protocols available on the MLST homepage (http://pubmlst. org/neisseria/). PCR products were sequenced at Macrogen Inc., Europe (Amsterdam, The Netherlands). Allelic profiles were analyzed using applications available on the MLST homepage.
Preparation and Sequencing of Genomic DNA N. meningitidis isolates (supplementary table S1, Supplementary Material online) were grown in liquid Brain Heart Infusion (BactoTM) medium and chromosomal DNA was prepared as described (Lamelas et al. 2014). Briefly, bacterial pellets were resuspended in 0.5 ml of TES buffer (50 mM Tris, 20 mM EDTA, 50 mM NaCl [pH 8]). Two microliters of RNase A (Qiagen 100 mg/ml) and 20% SDS were added to a final concentration of 1% and cells were lysed for 5 min at 42 C. After two phenol:chloroform:isoamyl alcohol (25:24:1, Sigma) extractions and one chloroform:isoamyl alcohol (24:1, Sigma) extraction, DNA was precipitated in two volumes of isopropanol followed by suspension in TE buffer (10 mM Tris, 1 mM EDTA [pH 8]). Ammonium acetate was added to a final concentration of 2.5 M, and the DNA was precipitated in two volumes of ethanol. After two 70% ethanol wash steps and subsequent drying, the DNA was diluted in TE buffer (10 mM Tris, 0.1 mM EDTA [pH 8]).
Multiplexed genomic DNA libraries were prepared with an insert size of 200 using 24 unique index tags. Libraries were combined into pools of 24 and sequenced on an Illumina HiSeq for 75 cycles from each end to produce paired-end reads plus an 8-base index sequence read. Downstream analysis used the index tags to assign reads to individual samples.

Read Alignment and SNP Detection
Variation in the form of single nucleotide polymorphisms (SNPs) was detected using a mapping approach essentially as described (Harris et al. 2010). The paired-end Illumina reads were mapped against the N. meningitidis genome of the A: ST-5 isolate WU2594 (accession numbers FR774048) as reference with an insert size between 50 and 400 bp using SMALT v0.7.4 (http://www.sanger.ac.uk/resources/software/ smalt/), giving, on average, a depth of coverage of 177x. SNPs were identified using SAMtools v1.1. The minimum base call quality to call an SNP was 50; the minimum root mean square of the mapping quality was 30 and the minimum number of reads matching SNP was 4 and per strand 2. SNPs were filtered to remove those at sites with an SNP quality score below 50. SNPs called in phage sequences and repetitive regions of the N. meningitidis reference genome were excluded. Repetitive regions were defined as exact repetitive sequences of 50 bp in length, identified using the repeatmatch function of the MUMmer package (Kurtz et al. 2004).  (3) NG¼nongroupable.
SNPs at sites with heterogeneous mappings (indetermination) were filtered out if the SNP was present in <90% of the reads at that site.

Identification of Recombination Events
To identify recombination events based on high SNP density, we applied a moving window approach using the Gubbins software as described (Croucher et al. 2015).

Phylogenetic Analyses
Maximum-likelihood phylogenetic trees were performed by RAxML v7.0.4 (Stamatakis 2006) using a general timereversible substitution model with c correction for amongsite rate variation. Support for nodes on the trees was assessed using 100 bootstrap replicates.

SNP Pairwise Distance Analysis
To compute the pairwise distance based on the genome wide SNP count between the isolates, we used MEGA6 (Tamura et al. 2013). Two-tailed Mann-Whitney tests were performed using Stata (version 12). The pairwise SNPs distances within the linages were plotted using software package R (http:// www.r-project.org/).  1A). This may be related to repeated vaccination campaigns in the KND with a serogroup A carbohydrate vaccine, which has no effect on colonization, but protects from invasive disease. In general, it has been found that A: ST-2859 meningococci are hypervirulent and can cause major outbreaks, such as the A: ST-5 and A: ST-7 lineages (Si e et al. 2008).

Consecutive Meningococcal Colonization and Disease
A first three W: ST-2881(cc175) meningitis cases were identified in spring of 2009 ( fig. 1B). At that time the W colonization rate was still below our detection limit defined by pharyngeal sampling of about 300 individuals per survey. In the following four colonization surveys from fall 2009 to spring 2011, W ST-2881 meningococcal colonization rates between 6.6% and 16.7% were observed (table 1 and  The genomes from the 27 isolates belonging to the second A: ST-2859 colonization and disease wave were unusually homogenous with no new recombinations found. Only four isolates differed from the overall population in either two SNPs (isolates 3338 and 3319) or one SNP (isolates 3340 and 3322) that represent most likely point mutations. The genomes of 23/27 isolates, including all seven disease isolates from 2010 to 2011, were thus identical, providing evidence for clonal expansion after the colonization bottleneck in 2009. In contrast, the genomic diversity of the 16 sequenced isolates from 2007 to 2009 was significantly higher (P-value <0.01) than that of the 27 isolates from 2010 to 2011 (fig. 2B). The genome data are indicative for the expansion of one clone that differs in only a single SNP at position 1655554 from five isolates (2808, 2809, 2810, 2811, and 2934) belonging to the original A: ST-2859 population ( fig. 2A). This SNP produces an R to G amino acid substitution in the conserved hypothetical integral membrane protein NMAA_141. All isolates belonging to the second A: ST-2859 wave shared the recombination blocks previously identified in the common ancestor of all A: ST-2859 isolates (Lamelas et al. 2014) and no additional recombination event was identified. Overall, results thus show that after the colonization bottleneck the second A: ST-2859 colonization and disease wave was caused by the spread of one haplotype from the more heterogeneous population causing the first A: ST-2859 colonization and disease wave.

Discussion
Maintaining a high genomic diversity may be an evolutionary strategy of meningococci that increases chances of fixing occasionally new highly successful "fit genotypes" (Zhu et al. 2001) or immune escape variants out of an immensely diverse repertoire of genomic variants present in spreading clones. In particular, the high competence for transformation and homologous recombination of meningococci (Rotman and Seifert 2014) would normally foster the maintenance of this diversity, because it will allow beneficial mutations to be transmitted through the population without the fitter genotype replacing the entire population of genotypes. However, recombination may also enable the combination of multiple beneficial variants creating a highly fit haplotype which could rapidly sweep to fixation. Based on these observations, an epidemic clonality model has been proposed, which postulates that meningococcal populations undergo occasional rounds of clonal propagation in an otherwise recombining population structure (Smith et al. 1993). More recently it has been argued that a predominant clonal evolution model fits best to the population genetics of N. meningitidis (Tibayrenc and Ayala 2015).
Here, we present results of the second phase (2007-2011) of the first longitudinal meningococcal colonization and disease study in the meningitis belt of sub-Saharan Africa. Our sampling strategy allowed us to describe the dynamics of meningococcal carriage and disease in the KND over a 13 year study period that included both meningococcal outbreaks and interepidemic periods.  Nicolas, Norheim, et al. 2005;Boisier et al. 2006). In 2003, W: ST-2881 meningitis cases have also been reported from Benin, Nigeria  and Burkina Faso (Mueller et al. 2007) and outbreaks have been reported from Cameroon (Massenet et al. 2013) and Ghana (Lamelas et al. 2017).
Interestingly, the A: ST-2859 meningococci re-emerged in the KND after a gap of 2 years, when their colonization rate was for most of the time below our detection limit of about 0.3%. Our previous comparative whole genome sequencing analyses have indicated that the A: ST-2859 clone represents a new adaptive genotype that has emerged from A: ST-7 meningococci by multiple recombination events. The A: ST-2859 bacteria apparently evaded A: ST-7 lineage-specific mucosal immunity through multiple changes in noncapsular cell surface components (Lamelas et al. 2014). Based on these findings, we had hypothesized that the re-emerging A: ST-2859 bacteria studied here would belong to a new "fit genotype" or immune escape variant that has accumulated additional recombination blocks. However, we observed a completely different scenario: there is no evidence, that the A: ST-2859 colonization and disease isolates re-emerging in 2011 contain a recombination block not present in the A: ST-2859 isolates from 2007 to 2009. Also at the level of point mutations no accumulation of genomic changes was observed and 23 of the 27 "new" isolates differed in only one SNP from five of the "old" isolates. The genomes of the four other "new" isolates differed from the other 23 in only 1 or 2 additional SNPs. The second A: ST-2859 colonization wave was thus unusually homogenous, providing a striking example on how genomic diversity of an epidemic clone can collapse through the spread of a particular haplotype. In the scenario described here, the elimination of genomic variation appears to be related to a colonization bottleneck caused by the W: ST-2881 meningococci, but that does not exclude that the clone that emerged in 2010 is a particular fit haplotype.

Funding
This work was supported by the Swiss National Science Foundation (grant number 310030-152840) and by the Volkswagen Foundation.