Molecular characterization of Neisseria meningitidis isolates recovered from 11-19-year-old meningococcal carriers in Salvador, Brazil

Characterization of meningococci isolated from the pharynx is essential towards understanding the dynamics of meningococcal carriage and disease. Meningococcal isolates, collected from adolescents resident in Salvador, Brazil during 2014, were characterized by multilocus sequence typing, genotyping or whole-genome sequencing. Most were nongroupable (61.0%), followed by genogroups B (11.9%) and Y (8.5%). We identified 34 different sequence types (STs), eight were new STs, distributed among 14 clonal complexes (cc), cc1136 represented 20.3% of the nongroupable isolates. The porA and fetA genotypes included P1.18,25–37 (11.9%), P1.18–1,3 (10.2%); F5-5 (23.7%), F4-66 (16.9%) and F1-7 (13.6%). The porB class 3 protein and the fHbp subfamily A (variants 2 and 3) genotypes were found in 93.0 and 71.0% of the isolates, respectively. NHBA was present in all isolates, and while most lacked NadA (94.9%), we detected the hyperinvasive lineages B:P1.19,15:F5-1:ST-639 (cc32); C:P1.22,14–6:F3-9:ST-3780 (cc103) and W:P1.5,2:F1-1:ST-11 (cc11). This is the first report on the genetic diversity and vaccine antigen prevalence among N. meningitidis carriage isolates in the Northeast of Brazil. This study highlights the need for ongoing characterization of meningococcal isolates following the introduction of vaccines and for determining public health intervention strategies.


Introduction
Neisseria meningitidis is a human commensal bacterium that commonly colonizes the oropharyngeal mucosa, occasionally causing life-threatening disease, such as meningitis or septicemia [1] Meningococcal populations possess a diverse and dynamic structure [2,3] invasive meningococcal cases are caused by a limited number of clonal complexes (cc), known as hyperinvasive lineages, which persistently exist over time despite high rates of recombination [3,4]. The population structure of meningococcal carriage strains is less well defined [5] and some are associated with hypervirulent lineages [6]. Most carriage meningococci lack a capsule and are thus nongroupable (NG). However, commensal strains may play an important role as a reservoir of virulence genes, with implications for meningococcal diversity due to the high frequency of recombination [3]. Multilocus sequence typing (MLST) is used for studying population biology and the evolution of microorganisms [4] and the PubMLST database allows the comparison of global meningococcal strains [7]. While MLST has a low discriminatory power, this has been overcome by characterizing the genes encoding several outer membrane proteins, including: porins A (PorA) and B (PorB) and iron-regulated enterobactin (FetA) [8]. Typing of factor Hbinding protein (FHbp), Neisserial adhesion A (NadA) and Neisserial heparin binding antigen (NHBA) can also improve meningococcal typing and provide information on strain coverage conferred by the serogroup B meningococcal (MenB) vaccines [9]. These antigens were used in the development of two MenB vaccines, the MenB-4C multi-component recombinant vaccine and the MenB-FHbp bivalent vaccine [9,10].
In Brazil, meningococcal disease is endemic with an annual incidence of 1.5-2.0 cases per 100,000 inhabitants [11]. Serogroup C has been responsible for most cases and is historically associated with ST-11 during the 1970s and ST-103 after 2000 [12]. However, there is only limited data describing meningococcal carriage in Brazil [13,14].
Characterization of meningococci isolated from the pharynx is essential towards understanding the dynamics of meningococcal carriage and disease and to determine the potential impact of disease control programs, such as vaccination, on the transmission of meningococci. In 2014, we conducted a cross-sectional study to assess the meningococcal carriage status of 11-19-year-old student's resident in Salvador [15]. In the current work, the meningococcal carriage isolates were characterized by capsular group, ST, and the presence and sequence variability of the porA, porB, fetA, fHbp, nhba, and nadA genes.

Ethics statement
This study was approved by the Ethics Committee at the Gonçalo Moniz Institute, FIO-CRUZ-BA (CAEE # 16099713.1.0000.0040). Written informed consent from all participants (or guardians) in the study were obtained before sample and data collection.

Capsular typing
Capsular groups were characterized by real-time PCR (qPCR), the primers and probes for the ctrA and sodC genes and for serogroups A, B, C, W, Y and X were used as described previously [17,18],. The capsule null locus (cnl) was detected by PCR amplification and sequencing as described previously [19].

Whole-genome sequencing
The N. meningitidis isolates that were not fully characterized by molecular typing were analyzed by whole-genome sequencing. Genomic DNA was extracted [24] and sequenced using MiSeq v2 chemistry (Illumina, San Diego, CA, USA). Genome assembly was carried out using CLC Genomics Workbench, ver 9.0.0 (CLC bio, Aaarhus, Denmark) with read trimming and mapping of reads back to contigs. The MLST alleles, STs and cc were identified by comparison of the assembled genomes with PubMLST [7] alleles using a BLAST search (https://blast.ncbi. nlm.nih.gov/Blast.cgi). Sequences of PorA, PorB, FetA, NadA, NHBA and FHbp were identified as described previously [24].

Phylogenetic analysis
Single nucleotide polymorphisms (SNPs) were identified using kSNP version 3 software [25] with a kmer length of 25. A maximum likelihood phylogenetic tree was constructed from the core SNPs and the Tamura-Nei model, using MEGA7 [26] and 500 bootstraps iterations.

Genomic diversity of the N. meningitidis isolates
The genetic relatedness of the 45 N. meningitidis isolates that could not be fully characterized by molecular typing was assessed using whole-genome sequencing. The phylogenetic analysis revealed that isolates from the same cc clustered together (Fig 1). A total of 7131 core SNPs were identified with a difference of 0-3847 between all isolates analyzed.

Discussion
In the present study, we evaluated the molecular characteristics of meningococcal carriage isolates recovered from 11-19-year-old students, resident in Salvador, Brazil. Most of the N. meningitidis isolates were NG, which is consistent with other carriage studies [19,28,29]. Although the capsule is not required for person-to-person transmission [19] there is evidence that loss of the capsule enhances the capacity of meningococci to colonize the human nasopharynx and to avoid human defense systems [30]. Furthermore, in some instances, capsuledeficient strains have caused invasive disease [31].
Among the groupable carriage isolates, the most common included genogroups MenB and MenY, in agreement with previous reports [28,32,33]. In addition, we found a low prevalence of MenC carriage among the students, which may be related to the mass vaccination campaign with a MenC conjugate vaccine that was conducted in Salvador in 2010 [34]. Although the vaccination status of the participants was not available, the MenC vaccination campaign for 10-24-year-olds may have had some effect on the low MenC colonization rates seen in this study. As seen in studies from the United Kingdom, the introduction of a MenC conjugate vaccine to the adolescent and young adult population was responsible for a 67% reduction in MenC colonization rates compared to non-vaccinated individuals [35]. However, we were unable to evaluate the impact of MenC conjugate vaccine on meningococcal carriage due to the lack of baseline carriage data prior to the vaccination campaign.
Molecular typing revealed that the N. meningitidis isolates were highly diverse, as expected for a carrier population [2]. We characterized 34 STs belonging to 14 cc and found an association with some of the capsular groups, as previously reported [29,36]. The cc1136 and cc198 were most common and, as observed in our study, these cc can be found among carriage and cnl-positive isolates [36]. Indeed, the genetic relatedness of the 45 isolates analyzed by wholegenome sequencing found that isolates belonging to the same cc were more closely related and formed distinct phylogenetic clusters (Fig 1).
In agreement with previous reports of carriage and invasive isolates, we found an association between genogroup B and cc41/44, cc32 and cc4821 [6,28,33]. Interestingly, one of the NG cc32 isolates clustered with a genogroup B cc32 isolate and had the same genotype profile except for the NHBA protein variant (Table 1). This NG cc32 isolate lacked the csb gene, which is required for capsule synthesis.
Genogroup Y was associated with cc23 and cc175 and isolates belonging to cc23 have been reported to be involved with invasive disease in the USA, South America, Europe and South Africa [37]. Furthermore, cc175 was responsible for over 17% of MenY invasive cases in Brazil, during 2007Brazil, during -2011. These results demonstrate the continuing circulation of pathogenic isolates among carriers.
Previous studies found that a small proportion of carriage isolates belonged to hyperinvasive lineages [6,29]. Furthermore, it is known that these lineages can persist over many decades and spread around the world, despite high rates of recombination [3]. In this study, we identified three hyperinvasive isolates associated with meningococcal disease cases in Brazil. Of note, the strain C:P1.22,14-6:F3-9:ST-3780 (cc103), differing only in the PorA VR1 subtype, is responsible for most meningococcal disease cases in Brazil. It was also identified as the causa- There are few reports describing the distribution of the vaccine antigen alleles among N. meningitidis carriage isolates [28,45]. Overall, the PorA, PorB and FetA variants identified in this study were highly variable within the same genogroup and cc, as well as the presence of the same antigenic allele in different cc.
In conclusion, this study presents an overview of the molecular diversity and vaccine antigen content of N. meningitidis carriage isolates in Salvador, Brazil. Continuous monitoring of antigen variability, including carriage isolates from other age groups, as well as isolates from meningococcal cases, will be needed to monitor the impact of the anti-meningococcal vaccination strategies on the carriage population, as well as to contribute to future public health decisions on vaccine usage.