Whole-Genome Sequencing for Characterization of Capsule Locus and Prediction of Serogroup of Invasive Meningococcal Isolates

Abstract

Invasive meningococcal disease is mainly caused by Neisseria meningitidis serogroups A, B, C, X, W, and Y. The serogroup is typically determined by slide agglutination serogrouping (SASG) and real-time PCR (RT-PCR). We describe a whole-genome sequencing (WGS)-based method to characterize the capsule polysaccharide synthesis (cps) locus, classify N. meningitidis serogroups, and identify mechanisms for nongroupability using 453 isolates from a global strain collection. We identified novel genomic organizations within functional cps loci, consisting of insertion sequence (IS) elements in unique positions that did not disrupt the coding sequence. Genetic mutations (partial gene deletion, missing genes, IS insertion, internal stop, and phase-variable off) that led to nongroupability were identified. The results of WGS and SASG were in 91% to 100% agreement for all serogroups, while the results of WGS and RT-PCR showed 99% to 100% agreement. Among isolates determined to be nongroupable by WGS (31 of 453), the results of all three methods agreed 100% for those without a capsule polymerase gene. However, 61% (WGS versus SASG) and 36% (WGS versus RT-PCR) agreements were observed for the isolates, particularly those with phase variations or internal stops in cps loci, which warrant further characterization by additional tests. Our WGS-based serogrouping method provides comprehensive characterization of the N. meningitidis capsule, which is critical for meningococcal surveillance and outbreak investigations.

Web-Based Genome Analysis of Bacterial Meningitis Pathogens for Public Health Applications Using the Bacterial Meningitis Genomic Analysis Platform (BMGAP)

“…Next, each resulting assembly is assessed by average depth of coverage, as reported by the SPAdes assembler, and the evenness of coverage across contig. Assemblies are analyzed by three core modules: species identification, capsule characterization, and molecular typing (Topaz et al, 2018;Marjuki et al, 2019;Potts et al, 2019). The overall BMGAP workflow including each core module is illustrated in Figure 1.…”

“…It is possible that the expression of NmY capsule in these two isolates could be lower than the visual detectable range for SASG given that all of the genetic elements required for expression of the NmY capsule are present according to WGS and PCR. Another consideration for these discrepancies could be heterogeneous NmY cultures in which reversible mutations such as internal stops could cause polyagglutination or no agglutination in SASG while genotypically being detected as NmY by WGS and PCR (Marjuki et al, 2019). Discrepancy resolution data is summarized in Table 3.…”

“…Biochemical and molecular laboratory tests can be time consuming and labor-intensive, but the recent proliferation of whole genome sequencing [WGS] technology has created an opportunity for streamlining the characterization of bacterial meningitis pathogens. Automated sequence analysis pipelines that identify the bacterial species (Topaz et al, 2018), Nm serogroup (Marjuki et al, 2019), and Hi serotype (Potts et al, 2019) provide a proof of concept for using WGS to elucidate bacterial meningitis pathogens.…”

Molecular insights into meningococcal carriage isolates from Burkina Faso 7 years after introduction of a serogroup A meningococcal conjugate vaccine

“…The resulting paired-end 250 bp sequence reads were trimmed using Cutadapt [21] and underwent de novo assembly using SPAdes 3.7 [22]. The genome assembly was used to confirm the isolate species [23], to obtain multilocus sequence typing (MLST) information from PubMLST [24], and to characterize the capsule locus, as described by Marjuki et al [25]. This capsule analysis includes identifying the capsule genes present, checking each capsule gene for mutations and reporting the capsule genotype.…”

Meningococcal Deduced Vaccine Antigen Reactivity (MenDeVAR) Index: a Rapid and Accessible Tool that Exploits Genomic Data in Public Health and Clinical Microbiology Applications

“…Multilocus sequence typing (MLST), based on housekeeping genes, is the most widely-used approach to characterising bacterial variants, facilitating communication among laboratories internationally and the identification of hyperinvasive meningococci (12). Typing bacterial genetic diversity of medically important features, such as polysaccharide capsules (13,14), antimicrobial resistance genes (15), and vaccine antigens (16) can be achieved through similar geneby-gene approaches (17). For example, the Bexsero ® Antigen Sequence Typing (BAST) scheme was established to characterise and describe vaccine antigen variants, using data derived through WGS or sequencing of individual genes (16).…”