Genotypic characterization and genome comparison reveal insights into potential vaccine coverage and genealogy of Neisseria meningitidis in military camps in Vietnam

Bacterial isolation and typing

All invasive N. meningitidis strains were isolated from CSF and all carrier strains were isolated from mouthwash samples at the Laboratory of Microbiology, Military Institute of Preventive Medicine, Hanoi as described previously (Tran et al., 2019). Two isolates, NMB_VN2013 and NMB_VN2015, were from the CSF of two confirmed meningitis cases discovered in 2013 and 2015 at Military Unit 2, a camp in the geographical closeness to Military Unit 1, the camp where the previously reported DuyDNT isolate was identified. Both cases were treated at the military hospital and subsequently recovered. Serogroup identification and multi-locus sequence typing (MLST) were either done according to previously described standard methods (World Health Organization, 2011) or by manually extracting the corresponding sequences from WGS data.

DuyDNT isolate was renamed NMB_VN2014 here for convenience and consistency. Available laboratory records of the Laboratory of Microbiology, Military Institute of Preventive Medicine, Hanoi were reviewed and a suitable collection of 25 isolates were chosen based on reported year (before and after 2013–2015), location (Military Unit 1, 2, and two nearby units here named Unit 3 and Unit 4) and availability of molecular characterization data (Table 1). Serogroup data, MLST data, fHbp and porA allelic variants were obtained from laboratory records. Case-related metadata, including year, clinical status, and location were obtained where available.

Antibiotic susceptibility testing

Susceptibility of isolates to seven antibiotics, namely ampicillin, ciprofloxacin, cefotaxime, ceftriaxone, rifampicin, meropenem and chloramphenicol, was examined using E-test strip (bioMerieux, France) following the manufacturer guideline and MIC values were determined. Susceptibility was interpreted according to CLSI 2018 breakpoints (CLSI, 2018).

Genome sequencing and analysis

Genomic DNA was extracted using GeneJET Genomic DNA Purification Kit (Thermofisher Scientific) in accordance with the manufacturer’s instruction. Samples’ quality was checked before sequenced using the Illumina HiSeq 4000 system (Macrogen). Genome assembly and annotation were performed as previously described (Tran et al., 2019). Annotated amino acid sequences were used to identify genes involved in antibiotic resistance (Tran et al., 2019). Additionally, allelic profile of relevant antibiotic resistant genes from PubMLST was extracted from WGS data of each genome, and where applied, PSI-BLAST was used to find homologous sequence. Antigenic profile, antibody cross reactivity prediction and allelic variants of virulent factors including capsular genes, Maf-toxin island, and outer membrane vesicle (OMV) genes were analyzed with gMATS (Muzzi et al., 2019) and the PubMLST server using default parameters.

Genome comparison and phylogenetic analysis

Assembled genomes were submitted to the PubMLST website and allelic variants were automatically assigned for each locus. Genomes were compared at the seven loci of MLST scheme, the 53 loci of ribosomal MLST scheme, and the 1605 loci of the core genome cgMLST scheme for N. meningitidis. Allelic variants were further processed in Excel and where necessary, manually removed from comparisons. Genomes were aligned using the progressive Mauve software (Darling, Mau & Perna, 2010), and the phylogenetic distance between strains was determined at the whole genome level. Neighbor-Net networks were constructed from various comparisons implemented on the PubMLST and visualized by SplitsTree (Huson, 1998).

Raw sequence data of 18 Southeast Asian N. meningitidis was obtained from the European Nucleotide Archive (project PRJEB30968) (Batty et al., 2019), assembled using Spades V3.11.1 (Bankevich et al., 2012), and assembled contigs were used for phylogenetic network analysis by SplitsTree and other sequence comparisons.

Characterization of NMB_VN2013 and NMB_VN2015 isolates

Inference of the Vietnamese isolates’ genealogy

Neighbor-Net analysis was used to construct the phylogenetic networks of three Vietnamese isolates and the well-studied 108-isolate global N. meningitidis collection (Bratcher et al., 2014) based on the seven-locus (MLST), 53-locus (rMLST) and 1605-locus (cgMLST) comparisons. This collection represents the major hyper-invasive lineages/sub-lineages recorded worldwide in the last 70 years. All three methods grouped the Vietnamese isolates into a well-defined, separate clade from the rest of the network. While similar relationships for the Vietnamese isolates were maintained in all analyses, the 1605-locus cgMLST analysis was able to place the Vietnamese branch at the stem of the previously defined Lineage 3 (ST-41/44 clonal complex) (Fig. 1). Additionally, only cgMLST analysis could separate the Vietnamese isolates into three unique strains; while MLST comparison grouped NMB_VN2013 and NMB_VN2015 into one ST, and rMLST identified NMB_VN2013 and NMB_VN2014 as a single strain; showing the close relationship among these strains.

A more refined cluster was observed when the 18 Southeast Asian invasive N. meningitidis were added to the genealogical analysis. Eleven chloramphenicol-resistant isolates, including three Vietnamese isolates, four Thai isolates, and two each from Laos and Cambodia, formed a distinct group, diverged from the rest of Southeast Asian isolates. This lineage seemed to have rapidly expanded clonally in recent years, though this could partially be due to better case report and laboratory detection, since data from this region was scarce up until recently although still remains limited. Although NeighborNet analysis placed this lineage as a divergent branch from other chloramphenicol susceptible isolates in the region, due to the limited number of samples, it remains a possibility for their origin.

The rest of isolates also clustered into two groups, one included NM03, NM14, and NM15 that clustered to the ST-41 sub-lineage of ST-41/44 clonal complex, while the remaining isolates clustered together, seemingly formed a group connecting the chloramphenicol-resistant lineage with ST-44 sub-lineage of ST-41/44 clonal complex (Fig. 1).

Relationships among Vietnamese isolates revealed by genome comparisons

Of the 1605 loci compared, Genome Comparator identified 1245 identical loci and 355 variable loci in at least one genome of the three isolates in this study. Five loci were missing in all three isolates, four of those were pseudo genes and one encoded for a phage-related protein. Fifteen loci were paralogous loci presumably resulting from assembly of repetitive sequences and thus were excluded from the final analysis. Resultant variable loci were further examined to exclude all variations from genes/pseudo-genes encoding for hypothetical proteins. Consequently, from the initially identified 355 variable loci, we confirmed 264 loci that contained either point mutations, insertions/deletions, or allelic replacements from the three genomes. Among these, a number of sequence variables were observed in adjacent genes, suggesting frequent recombination events between genomes. Pairwise, the genomes of NMB_VN2013 and NMB_VN2014 showed the highest similarity, and NMB_VN2014 and NMB_VN2015 pair showed the lowest. However, pairwise phylogenetic distance between strains calculated from the progressiveMauve alignment using whole genome content was in the range of 0.0158 to 0.0112, indicating a close relationship among them.

Besides core genome comparison, sequences of the known genetic determinants for the virulence of invasive N. meningitidis, namely the capsular gene cluster, the Maf-toxin island, and genes encoding for the outer membrane vesicle (OMV) peptides and pilin, from the genomes of three isolates were also compared. The resulting differences, after excluding all variables due to assembly or in the coding sequence of hypothetical proteins, are listed in Table 4. Overall, higher sequence variability could be observed among the three genomes at these loci. From 19 loci of the capsular gene cluster being present for comparison, four confirmed variable loci were rfbA, cssA, ctrF, and cnl. Except for two assigned alleles found in two loci (allele 113, cssA locus of NMB_VN2015’s genome and allele 4, ctrF locus of NMB_VN2013’s genome), the rest of the alleles were newly identified and not yet assigned an allelic number. Four out of 45 loci of the Maf-toxin genomic island included in this comparison, namely anmK, mafIo2MGI-2, mafA_MGI-3, and mafB_MGI-3, appeared to be different. Sequences of the anmK locus of NMB_VN2015 and the mafB_MGI-3 locus of NMB_VN2014 genome appeared to be novel, unassigned alleles. Detailed nucleotide changes analysis revealed a higher percentage of changed nucleotides compared to any other single locus in both mafA_MGI-3 and mafB_MGI- 3, which were adjacent to each other, suggesting a recombination event could have happened in this region. Compared to the capsular gene cluster and Maf-toxin island, the CDS of OMV and pilin genes showed higher sequence diversity with variables scattered among loci.

To better understand the probable evolutionary context of these strains, we have further collected and analyzed 25 isolates representing a collection of both historical and later emerged isolates from the same or nearby military units with the three in this study (Table 1). Epidemiological data of these 25 isolates revealed the predominance of serogroup B N. meningitidis (78.5%) and the recurrence of two major lineages centered around ST-1576 and ST-4821 in all camp sites (Table 1). The predominance of serogroup B agrees with the previous report from sporadic cases in the region (Pancharoen et al., 2000). The other serogroup observed was serogroup C (21.5%). Both ST-1576 and ST-4821 were major hyper-virulent STs with a long history and worldwide distribution. Antigenic profiles associated with these major STs show high similarity to strains described before in China during 1978–2013 (Zhu et al., 2015). Besides these, strains representing singleton and novel STs were also frequently observed throughout the years and locations. Available antigenic profiles suggested frequent exchange of genetic material via recombination among strains and reservoirs, with several alleles (P1.20, P1.7-2) associated with the major lineages frequently recurring in different combinations. Clonal expansion was also observed, indicated by the emergence of novel sequence types, and showed no distinct cluster in regard of geological locations, reflecting the close and frequent contact nature of the training units of new military recruits, suggesting close transmission extending beyond individual camps seemed to be the main driving force for N. meningitidis prevalence and expansion within the military environment in Vietnam. From searching through the PubMLST database, many emergent STs identified were limited to these units only, highlighting the niche characteristic of N. meningitidis population of the military camps.

N. meningitidis remains the main cause of sporadic meningitis and sepsis in military recruit camps (Tran et al., 2019; Sim et al., 2013; Keiser, Hamilton & Broderick, 2011). Accurate identification and characterization of the causative strain is crucial for the success of treatment for patients and prophylaxis for contact persons, as well as prevention of outbreaks. Records of Vietnamese and Southeast Asian N. meningitidis isolates are still extremely limited, thus it is not possible to determine the origin of these strains, or how they have arisen. Study at the genomic level of additional historical invasive and carriage strains collected at these and nearby camps, or nearby regions can help identify the phylogenetic routes that led to their emergence. On the other hand, since the military setting is among the highest risk group for meningococcal disease in adults, continual effort is needed to provide the surveillance data essential for effective policy making and preparation for response in case of potential outbreaks in the future.