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Research Article
30 December 2015

Comparison of Phenotypic and Genotypic Approaches to Capsule Typing of Neisseria meningitidis by Use of Invasive and Carriage Isolate Collections


Neisseria meningitidis serogroup B (MnB) is a leading cause of bacterial meningitis; however, MnB is most commonly associated with asymptomatic carriage in the nasopharyngeal cavity, as opposed to the disease state. Two vaccines are now licensed for the prevention of MnB disease; a possible additional benefit of these vaccines could be to protect against disease indirectly by disrupting nasopharyngeal carriage (e.g., herd protection). To investigate this possibility, accurate diagnostic approaches to characterize MnB carriage isolates are required. In contrast to invasive meningococcal disease (IMD) isolates, which can be readily serogrouped, carriage isolates often lack capsule expression, making standard phenotypic assays unsuitable for strain characterization. Several antibody-based methods were evaluated for their abilities to serogroup isolates and were compared with two genotyping methods (real-time PCR [rt-PCR] and whole-genome sequencing [WGS]) to identify which approach would most accurately ascertain the polysaccharide groups associated with carriage isolates. WGS and rt-PCR were in agreement for 99% of IMD isolates, including those with coding sequences for MnB, MnC, MnW, and MnY, and the phenotypic methods correctly identified serogroups for 69 to 98% of IMD isolates. In contrast, only 47% of carriage isolates were groupable by genotypic methods, due to mutations within the capsule operon; of the isolates identified by genotypic methods, ≤43% were serogroupable with any of the phenotypic methods tested. These observations highlight the difficulties in the serogrouping and capsular genogrouping of meningococcal carriage isolates. Based on our findings, WGS is the most suitable approach for the characterization of meningococcal carriage isolates.


Neisseria meningitidis, a Gram-negative bacterium that causes both epidemic and endemic life-threatening disease, is also an obligate human commensal organism that colonizes the nasopharyngeal mucosa with no or minimal harm to the host, a phenomenon known as carriage (1). Asymptomatic pharyngeal colonization with N. meningitidis in young adults is relatively common, and these asymptomatic carriers represent a potential reservoir for the transmission of pathogenic isolates in the community (2, 3). The rates of asymptomatic carriage in the United States and Europe are highest among adolescents and young adults, peaking at the age of 19 years, and are estimated to be 10% to 35% (46). Rates of transmission and carriage are higher in closed and semiclosed populations, such as university students living in dormitories and military recruits housed in barracks (7). Higher rates of carriage are also found among people in close contact with patients with active meningococcal infections (8). For the majority of people, carriage is an immunizing process that results in systemic, serogroup-specific, protective antibody responses (9, 10). Invasive meningococcal disease (IMD) usually occurs shortly after the onset of colonization of a susceptible host, when the bacteria penetrate the mucosal membranes, overcome host defenses, and invade the bloodstream or meninges, leading to septicemia or meningitis, respectively (11).
IMD is cyclical and varies by age group, being more common among children, especially those <5 years of age. A second peak in disease occurs in adolescence, with rates declining during early adulthood and subsequently decreasing for older age groups (12, 13). Asymptomatic carriers represent a potential source of virulent isolates that cause disease; however, the factors that lead to a change in the carriage state of the organism, resulting in invasive disease, are still poorly understood. Approximately 450 cases of meningococcal disease occurred in the Unites States in 2012, yielding a rate of 0.15 cases/100,000 population (14). The overall incidence rates in Canada (2011 data) and Europe (2012 data) were 0.55 cases/100,000 population and 0.68 cases/100,000 population, respectively (15, 16). In outbreak and epidemic settings, the case rates are considerably higher, i.e., 100 cases/100,000 population to 1,000 cases/100,000 population (17, 18).
The polysaccharide capsule is an important meningococcal virulence factor that protects the bacterium from complement-mediated bactericidal activity of human serum, as well as opsonophagocytic killing in the bacteremic stages of meningococcal disease (1921). N. meningitidis strains are classified into serogroups based on structural differences in their capsular polysaccharides (7, 22, 23). Twelve serogroups have been described, with six (i.e., N. meningitidis serogroup A [MnA], MnB, MnC, MnW, MnX, and MnY) being implicated in the majority of IMD (2427).
Capsule antigens (polysaccharide and polysaccharide conjugate) are potent and efficacious in preventing IMD when formulated as vaccines (28, 29). However, the common sialic acid-containing epitope of the MnB capsule is also present on human neural tissue, which may explain the poor immunogenicity of the MnB capsule; therefore, out of an abundance of caution, the MnB polysaccharide has not been considered a viable vaccine candidate (30). As a result, protein antigens have been employed in the development of MnB vaccines (31). Two vaccines have recently been licensed for the prevention of MnB disease, namely, Trumenba (bivalent rLP2086) (32) and Bexsero (4CMenB) (33).
In areas in which MnC conjugate vaccines were introduced and had widespread implementation, including receipt of the vaccines by very large proportions of adolescents, disease incidence in nonvaccinated populations was dramatically affected due to indirect prevention of disease through reductions in carriage rates, e.g., herd protection/immunity (3437). This effect was not observed with the use of nonconjugated polysaccharide vaccines. It will be important to study whether MnB vaccines have this additional benefit (38). A recent study demonstrated similar levels of herd protection for protein-based (4CMenB) and non-protein-based (MenACWY-CRM) meningococcal vaccines (39). Meningococcal carriage studies can be challenging, due to a combination of reduced capsule expression by carriage strains and a lack of standardized approaches to evaluate carriage isolates in order to identify their associated capsule polysaccharide groups.
Invasive disease isolates are traditionally serogrouped by slide agglutination serogrouping (SASG) assays (23, 40), which use commercial polyclonal antibody reagents raised against prototypical N. meningitidis isolates representing the important disease-associated serogroups. The reagents and laboratory methods used for SASG assays are not standardized across laboratories, especially because the endpoint of the assay is read subjectively with the unaided eye (23, 40). Other serogrouping approaches have employed capsule-specific monoclonal antibodies (MAbs) applied to different platforms, such as dot blotting (41), whole-cell enzyme-linked immunosorbent assays (ELISAs) (42), and flow cytometry (43). Large proportions (70 to 80%) of invasive isolates can be successfully serogrouped using expression-based phenotypic assays (40). In contrast, high percentages of isolates recovered from healthy carriers are reported as nonserogroupable in phenotypic assays; the general explanation for this finding has been the low levels or complete absence of expression of the capsule genes during carriage (4447), which can be explained by several mechanisms, including phase variation of capsule expression genes (44), insertion of genetic mobile elements (48), or a complete absence of the genes required for capsule production, leading to a capsule-null (cnl) phenotype (49, 50). The cnl mutation was reported for ∼16% of nasopharyngeal meningococci collected from healthy children and young adults in two independent carriage studies (45, 50). A recent report by Loh et al. (51) described a complex regulatory mechanism linking growth temperature to capsule gene expression. Molecular mechanisms underlying capsule biosynthesis have been elucidated; polysaccharide synthesis and translocation genes are clustered at a single chromosomal locus called cps. The cps locus consists of six different regions, i.e., A to D, D′, and E (52). Of particular interest for the present study are genes in region A, which encode the enzymes responsible for polysaccharide synthesis (53, 54), and genes in region C, which encode proteins that facilitate polysaccharide transport across bacterial membranes (55, 56).
Capsular genogrouping of carriage isolates can be achieved using rapid sensitive DNA-based methods (40). A number of PCR approaches have been developed to detect targets within the ctrA (capsule transport) gene (52, 57), as well as genes required for serogroup-specific capsule biosynthesis (8, 40, 5861). For serogroup prediction (capsular genogroup assignment), PCR using primers designed for serogroup-specific genes can be applied (6266). Fluorescence-based real-time PCR (rt-PCR) assays were recently developed to confirm the capsule typing of meningococcal isolates (59, 67, 68). Whole-genome sequencing (WGS) data also are being used increasingly for epidemiological typing of bacterial pathogens (6971). In this study, we compared different capsular serogrouping methodologies for the assignment of invasive and carriage isolates to capsular polysaccharide serogroups, in order to determine a methodology for the characterization of carriage isolates that is broadly applicable for different laboratory settings and has the capacity to be standardized.


Meningococcal isolates.

Invasive isolates (n = 97) representing serogroups MnA, MnB, MnC, MnW, and MnY were selected from the Pfizer Invasive Disease collection, which contains isolates from Europe (Czech Republic, France, Germany, Netherlands, and United Kingdom) and the United States (64% of isolates were from the United States) that were collected between 2000 and 2008 (Table 1) (72). Although these isolates had been previously characterized by SASG assays for serogroup determination, they were subjected to blinded operator evaluations in this study. Meningococcal carriage isolates (n = 93) were obtained from pharyngeal swabs from healthy ninth grade students and college/university students living in dormitories and participating in a 1-year-long epidemiological study in Quebec City (in 2010 to 2012) (Table 2) (73).
TABLE 1 Invasive disease isolates
IsolateCapsular genogroup by WGSSequence typeaClonal complexb
PFE-I-0001MnW174ST174 complex
PFE-I-0008MnW1158ST22 complex
PFE-I-0009MnY11ST11 complex/ET-37 complex
PFE-I-0011MnB136ST41/44 complex/lineage 3
PFE-I-0012MnB1260ST103 complex
PFE-I-0002MnB4701ST32 complex/ET-5 complex
PFE-I-0003MnB162ST162 complex
PFE-I-0005MnB162ST162 complex
PFE-I-0098MnY11ST11 complex/ET-37 complex
PFE-I-0006MnW11ST11 complex/ET-37 complex
PFE-I-0013MnA1ST1 complex/subgroup I/II
PFE-I-0015MnY23ST23 complex/cluster A3
PFE-I-0016MnY23ST23 complex/cluster A3
PFE-I-0017MnY23ST23 complex/cluster A3
PFE-I-0018MnY23ST23 complex/cluster A3
PFE-I-0019MnA5ST5 complex/subgroup III
PFE-I-0020MnC11ST11 complex/ET-37 complex
PFE-I-0021MnC1011ST231 complex
PFE-I-0023MnW185ST174 complex
PFE-I-0024MnB269ST269 complex
PFE-I-0025MnB40ST41/44 complex/lineage 3
PFE-I-0027MnB213ST213 complex
PFE-I-0029MnB3279ST213 complex
PFE-I-0030MnB33ST32 complex/ET-5 complex
PFE-I-0031MnB457ST35 complex
PFE-I-0032MnB213ST213 complex
PFE-I-0033MnB457ST35 complex
PFE-I-0034MnB41ST41/44 complex/lineage 3
PFE-I-0010MnB213ST213 complex
PFE-I-0035MnB136ST41/44 complex/lineage 3
PFE-I-0037MnB803ST32 complex/ET-5 complex
PFE-I-0038MnB11ST11 complex/ET-37 complex
PFE-I-0039MnB162ST162 complex
PFE-I-0040MnB32ST32 complex/ET-5 complex
PFE-I-0041MnB2976ST269 complex
PFE-I-0042MnB136ST41/44 complex/lineage 3
PFE-I-0044MnC11ST11 complex/ET-37 complex
PFE-I-0045MnC11ST11 complex/ET-37 complex
PFE-I-0046MnC11ST11 complex/ET-37 complex
PFE-I-0047MnC11ST11 complex/ET-37 complex
PFE-I-0048MnC11ST11 complex/ET-37 complex
PFE-I-0050MnC11ST11 complex/ET-37 complex
PFE-I-0052MnC11ST11 complex/ET-37 complex
PFE-I-0053MnC11ST11 complex/ET-37 complex
PFE-I-0054MnC11ST11 complex/ET-37 complex
PFE-I-0055MnC11ST11 complex/ET-37 complex
PFE-I-0056MnC11ST11 complex/ET-37 complex
PFE-I-0057MnC11ST11 complex/ET-37 complex
PFE-I-0058MnC11ST11 complex/ET-37 complex
PFE-I-0059MnC11ST11 complex/ET-37 complex
PFE-I-0060MnC11ST11 complex/ET-37 complex
PFE-I-0061MnC2006ST103 complex
PFE-I-0062MnC11ST11 complex/ET-37 complex
PFE-I-0063MnC11ST11 complex/ET-37 complex
PFE-I-0064MnY23ST23 complex/cluster A3
PFE-I-0065MnY23ST23 complex/cluster A3
PFE-I-0066MnY23ST23 complex/cluster A3
PFE-I-0067MnY23ST23 complex/cluster A3
PFE-I-0068MnY23ST23 complex/cluster A3
PFE-I-0069MnY23ST23 complex/cluster A3
PFE-I-0070MnY23ST23 complex/cluster A3
PFE-I-0071MnY23ST23 complex/cluster A3
PFE-I-0072MnY23ST23 complex/cluster A3
PFE-I-0073MnY23ST23 complex/cluster A3
PFE-I-0074MnY1625ST23 complex/cluster A3
PFE-I-0075MnY1624ST167 complex
PFE-I-0076MnB32ST32 complex/ET-5 complex
PFE-I-0077MnB32ST32 complex/ET-5 complex
PFE-I-0078MnB32ST32 complex/ET-5 complex
PFE-I-0079MnB162ST162 complex
PFE-I-0080MnB162ST162 complex
PFE-I-0081MnB162ST162 complex
PFE-I-0082MnA1ST1 complex/subgroup I/II
PFE-I-0083MnA4ST4 complex/subgroup IV
PFE-I-0085MnC917ST37 complex
PFE-I-0086MnC11ST11 complex/ET-37 complex
PFE-I-0087MnC11ST11 complex/ET-37 complex
PFE-I-0088MnC11ST11 complex/ET-37 complex
PFE-I-0089MnW11ST11 complex/ET-37 complex
PFE-I-0091MnY11ST11 complex/ET-37 complex
PFE-I-0092MnY23ST23 complex/cluster A3
PFE-I-0093MnY23ST23 complex/cluster A3
PFE-I-0094MnY103ST103 complex
PFE-I-0095MnY11ST11 complex/ET-37 complex
PFE-I-0096MnW1262ST174 complex
NST, no sequence type, i.e., sequence type not assigned to gene alleles determined by multilocus sequence typing.
ET, electrophoretic type.

Isolate collection and preparation.

Carriage isolates were cultured on modified Thayer-Martin plates (Remel/Thermo Fisher Scientific, Lenexa, KS) for up to 48 h at 37°C in 5% CO2. Suspect colonies were subcultured on trypticase soy agar (TSA) II blood agar (Remel) overnight at 37°C in a 5% CO2 incubator. All isolates were identified as N. meningitidis by Gram staining (BD BBL, Sparks, MD) and oxidase testing (DrySlide; BD BBL, Cockeysville, MD) and were identified to the species level with API NH tests (bioMérieux, Hazelwood, MO). IMD isolates were selected from the Pfizer global IMD collection and cultured on TSA II blood agar as described above.

Genotypic methods. (i) Whole-genome sequencing analysis.

Bacterial isolates were grown overnight on chocolate agar. A 10-μl bacterial loop was used to harvest the growth from one-eighth of a plate into 100 μl of GCK medium (GC medium base [Difco] with 0.042% NaHCO3 and Kellogg's supplements) (74), and the mixture was incubated at 95°C for 5 min to prepare cell suspensions. Genomic DNA (gDNA) was extracted from cell suspensions using magnetic bead technology, as described (Agencourt Genfind V2 system; Beckman Coulter, Brea, CA). The final elution of gDNA was in 100 μl of 10 mM Tris-1 mM EDTA buffer (pH 8). DNA concentrations were measured using the Qubit double-stranded DNA (dsDNA) high-sensitivity assay kit (Life Technologies, Carlsbad, CA) and adjusted to a final value of 0.2 ng/μl with distilled water. The NexteraXT DNA sample preparation protocol (Illumina, San Diego, CA) was followed to prepare DNA libraries for WGS. Library quality was evaluated using the Agilent high-sensitivity DNA kit (product no. G2938-90321; Agilent Technologies, Santa Clara, CA). The libraries were then normalized according to the manufacturer's instructions (Illumina), and the normalized products were evaluated using the Power SYBR Green quantitative PCR assay (Life Technologies, Norwalk, CT). The material was then loaded into the MiSeq instrument (Illumina) for WGS analysis, and genomic data were obtained using the Illumina sequencing platform followed by de novo assembly (CLC Bio, Aarhus, Denmark) to generate contiguous sequences (contigs). Assembled contigs were deposited in the Bacterial Isolate Genome Sequencing Database (BIGSdb) ( with the isolate sources and names and other demographic information, after which contigs were scanned for genes of interest and alleles and capsule groups were assigned (75). WGS data were inspected for the presence of all cps genes for polysaccharide synthesis and transport for a given genogroup, in order to define the genogroup; the absence of a complete coding sequence for any required gene resulted in a nongroupable (NG) designation.

(ii) TaqMan rt-PCR.

All N. meningitidis isolates were tested using an rt-PCR assay targeting the porA (encoding the class 1 or PorA protein) and ctrA genes (76, 77). All isolates (regardless of porA and ctrA results) were evaluated further in rt-PCR assays specific for the eight capsule serogroups, as described by Rojas et al. (68). Isolates that were ctrA negative or did not give positive results in any of the serogroup-specific assays were characterized as nongroupable (NG).

Phenotypic methods. (i) Slide agglutination serogrouping.

N. meningitidis isolates were serogrouped by SASG assays using commercial antisera (Difco, Sparks, MD), as described previously (40). For the carriage isolates, SASG1 and SASG2 assays were performed at two different laboratories, using independently supplied commercial reagents. In the case of the IMD isolates, SASG1 and SASG2 assays were performed in the same laboratory with two different lots of commercial reagents. Briefly, for each isolate, a few colonies were picked from an overnight culture plate to make a suspension in 0.1 to 0.2 ml of 1× phosphate-buffered saline (PBS). On a sectioned glass microscope slide, 100 μl of bacterial suspension was mixed with 20 to 100 μl of test serum; the slide was rocked gently for 1 min. The slide was observed for evidence of clumping, indicating a positive agglutination reaction. A saline control was used to detect nonspecific autoagglutination.

(ii) Dot blotting.

Dot blot assays were conducted as described previously (78). Briefly, one loopful of each overnight culture (at 37°C in 5% CO2) was harvested from chocolate agar (Remel), transferred into 200 μl saline, and incubated at 56°C for 30 min. Two-microliter cell suspensions were dotted on nitrocellulose membranes (0.45-μm pore size; Bio-Rad Laboratories, Hercules, CA). After 30 min of drying at room temperature, membranes were blocked with 1% bovine serum albumin (BSA) in 1× PBS. MAbs specific for MnA, MnB, MnC, MnW, and MnY were added directly into the blocking buffer, at various dilutions of 1:200 to 1:1,000. Blots were incubated for 1 h at room temperature with gentle rocking. Membranes were washed separately four times with 20 mM Tris (pH 7.5)-180 mM NaCl plus 0.05% Tween 20 (vol/vol) and then were incubated for 1 h with goat anti-mouse IgG conjugated to alkaline phosphatase (1:1,000 dilution; Sigma-Aldrich, St. Louis, MO). Each membrane included type isolate controls. Monoclonal antibody reagents for serogroups MnA, MnC, MnW, and MnY were described previously (78), and anti-MnB was provided by the National Institute of Biological Standards and Control (NIBSC) (South Mimms, United Kingdom).

(iii) Flow cytometry.

Serogrouping by fluorescence-activated cell sorting (FACS) analysis was conducted as described previously (43). The MAbs utilized were as described for the dot blot assay.

(iv) Live-cell phenotypic assay.

Monoclonal antibodies (MAbs) specific for MnA, MnB, MnC, MnW, and MnY capsular antigens were adsorbed batchwise onto protein G magnetic beads (Magne; Promega, Madison, WI), to a concentration of 0.16 μg IgG per μl, and were rinsed in Dulbecco's phosphate-buffered saline (DPBS) to remove unbound antibody. Beads (3 μl) were distributed in duplicate into separate wells of a low-protein-binding, white, 96-well plate (Greiner, Monroe, NC). Control wells received beads adsorbed with normal mouse IgG or no beads, to monitor specificity and nonspecific binding, respectively. N. meningitidis test isolates were prepared by scraping a 2-cm area from a freshly grown plate (chocolate agar; Remel) with a 10-μl loop and suspending the cells in 1 ml of GC medium base supplemented with Isovitalex (BD), in the wells of a 96-deep-well block. Two 10-fold serial dilutions of the cells were prepared in the same medium, and 100 μl of the 1:100 dilution was transferred to the reaction plate containing antibody beads. The suspension was incubated for 45 min at 37°C in 5% CO2 with shaking (300 rpm). A Tecan HydroSpeed plate washer equipped with a magnetic base station was used to remove unbound cells (six washes). Cells remaining bound to the antibody beads were detected using the luciferase-based BacTiter-Glo lysis reagent (Promega, Madison, WI), which measures ATP released from live bacteria via its associated luminescence. After incubation with the reagent at 37°C for 10 min at room temperature, the plate was sealed with transparent tape and results were read on a luminometer (Spectramax M5e; Molecular Devices, Sunnyvale, CA). Results were corrected for nonspecific binding by dividing the average of replicate reads observed with antigen-specific antibodies by the average of reads obtained for mouse IgG controls. Values ≥2-fold greater than the mouse IgG baseline value were reported as positive results.

Statistical analysis.

Concordance between methods was determined using a concordance rate for matching assay results for each isolate; the denominator was the total number of samples tested for the paired methods. The 95% confidence intervals (CIs) for the concordance rates were calculated using the Clopper-Pearson exact method (79).


Genotypic characterization by WGS.

Two isolate collections (invasive, n = 97; carriage, n = 93) were subjected to whole-genome sequencing (WGS) to determine whether the isolates had complete cps pathways, to serve as positive controls for the capsular genogrouping and serogrouping assays under evaluation (Tables 1 and 2). Using WGS, the isolates were then placed into one of several categories, i.e., (i) complete cps operon, (ii) incomplete cps operon missing essential synthesis or transport genes, with or without an insertion sequence (IS) element, (iii) incomplete cps operon missing one or more of the accessory genes (e.g., O-acetyltransferase), with or without an IS element, or (iv) cps operon deletion (capsule null [cnl]).
TABLE 2 Carriage isolates
IsolateCapsular genogroup by WGSSequence typeaClonal complexb
PFE-C-0001NG-ctrAneg53ST53 complex
PFE-C-0002NG-ctrApos269ST269 complex
PFE-C-0003NG-ctrAneg53ST53 complex
PFE-C-0004NG-ctrAneg198ST198 complex
PFE-C-0005MnW184ST22 complex
PFE-C-0006NG-ctrAneg823ST198 complex
PFE-C-0008MnW184ST22 complex
PFE-C-0009NG-ctrAneg53ST53 complex
PFE-C-0010NG-ctrAneg43ST41/44 complex/lineage 3
PFE-C-0011MnY23ST23 complex/cluster A3
PFE-C-0012NG-ctrAneg53ST53 complex
PFE-C-0013NG-ctrAneg53ST53 complex
PFE-C-0014NG-ctrAneg823ST198 complex
PFE-C-0015NG-ctrAneg43ST41/44 complex/lineage 3
PFE-C-0017MnY23ST23 complex/cluster A3
PFE-C-0018MnBpartial269ST269 complex
PFE-C-0019MnY23ST23 complex/cluster A3
PFE-C-0020MnY23ST23 complex/cluster A3
PFE-C-0021NG-ctrAneg53ST53 complex
PFE-C-0022MnY23ST23 complex/cluster A3
PFE-C-0023NG-ctrAneg53ST53 complex
PFE-C-0024NG-ctrAneg823ST198 complex
PFE-C-0025MnBpartial6473ST41/44 complex/lineage 3
PFE-C-0026NG-ctrAneg1136ST1136 complex
PFE-C-0027MnC944ST41/44 complex/lineage 3
PFE-C-0028NG-ctrAneg43ST41/44 complex/lineage 3
PFE-C-0029NG-ctrAneg198ST198 complex
PFE-C-0030NG-ctrAneg53ST53 complex
PFE-C-0031NG-ctrAneg53ST53 complex
PFE-C-0032MnY23ST23 complex/cluster A3
PFE-C-0033MnY23ST23 complex/cluster A3
PFE-C-0034MnB6473ST41/44 complex/lineage 3
PFE-C-0035MnB43ST41/44 complex/lineage 3
PFE-C-0036MnC944ST41/44 complex/lineage 3
PFE-C-0037NG-ctrAneg1117ST1117 complex
PFE-C-0039MnY23ST23 complex/cluster A3
PFE-C-0040NG-ctrAneg823ST198 complex
PFE-C-0041MnY23ST23 complex/cluster A3
PFE-C-0042MnW184ST22 complex
PFE-C-0043MnB2726ST32 complex/ET-5 complex
PFE-C-0044MnB2726ST32 complex/ET-5 complex
PFE-C-0045NG-ctrApos23ST23 complex/cluster A3
PFE-C-0046NG-ctrAneg1136ST1136 complex
PFE-C-0047MnY23ST23 complex/cluster A3
PFE-C-0048MnB269ST269 complex
PFE-C-0049MnY23ST23 complex/cluster A3
PFE-C-0050NG-ctrAneg823ST198 complex
PFE-C-0051NG-ctrAneg1117ST1117 complex
PFE-C-0052MnB6473ST41/44 complex/lineage 3
PFE-C-0053NG-ctrAneg823ST198 complex
PFE-C-0054NG-ctrAneg198ST198 complex
PFE-C-0055NG-ctrAneg53ST53 complex
PFE-C-0056NG-ctrApos269ST269 complex
PFE-C-0057MnB2726ST32 complex/ET-5 complex
PFE-C-0058NG-ctrAneg53ST53 complex
PFE-C-0059NG-ctrAneg198ST198 complex
PFE-C-0060MnB2726ST32 complex/ET-5 complex
PFE-C-0061MnY23ST23 complex/cluster A3
PFE-C-0062NG-ctrAneg198ST198 complex
PFE-C-0063NG-ctrAneg1117ST1117 complex
PFE-C-0064MnW5389ST22 complex
PFE-C-0065MnW5389ST22 complex
PFE-C-0066MnB6473ST41/44 complex/lineage 3
PFE-C-0067MnY23ST23 complex/cluster A3
PFE-C-0068NG-ctrAneg53ST53 complex
PFE-C-0069MnY23ST23 complex/cluster A3
PFE-C-0070MnB43ST41/44 complex/lineage 3
PFE-C-0071MnW184ST22 complex
PFE-C-0072NG-ctrAneg823ST198 complex
PFE-C-0073MnW184ST22 complex
PFE-C-0074MnB2726ST32 complex/ET-5 complex
PFE-C-0075NG-ctrAneg53ST53 complex
PFE-C-0076NG-ctrAneg823ST198 complex
PFE-C-0077NG-ctrAneg823ST198 complex
PFE-C-0079MnB6473ST41/44 complex/lineage 3
PFE-C-0080MnC944ST41/44 complex/lineage 3
PFE-C-0081MnW184ST22 complex
PFE-C-0082MnY23ST23 complex/cluster A3
PFE-C-0083MnB6473ST41/44 complex/lineage 3
PFE-C-0086MnY23ST23 complex/cluster A3
PFE-C-0087MnW184ST22 complex
PFE-C-0088MnC278ST35 complex
PFE-C-0089MnY23ST23 complex/cluster A3
PFE-C-0090NG-ctrAneg1117ST1117 complex
PFE-C-0091NG-ctrAneg823ST198 complex
PFE-C-0092NG-ctrAneg823ST198 complex
PFE-C-0093NG-ctrAneg1136ST1136 complex
NST, no sequence type, i.e., sequence type not assigned to gene alleles determined by multilocus sequence typing.
ET, electrophoretic type.
The invasive isolates were initially scanned for the presence of porA, ctrA, and sodC as N. meningitidis species-specific markers; two invasive isolates had porA gene deletions, and one isolate had a partial porA deletion. All 97 invasive isolates were positive for both ctrA and sodC and were assigned to capsular polysaccharide groups by WGS based on the cps locus. Nine isolates had an incomplete cps locus with genetic lesions (insertions/deletions) in accessory genes. Four MnW isolates had truncated cssF (formerly oatWY) genes, two of which had insertions within the cssF gene, identified as IS1016 and IS1301 (one each) (8082). One MnY isolate had a truncation in cssF with an IS1016 insertion. Three MnC isolates had IS1301 insertions in the cssE gene (formerly oatC), with an additional MnC isolate having a truncation of the cssE gene (see Table S1 in the supplemental material). These results indicated that all 97 invasive isolates should be groupable by the rt-PCR assay and serogroupable by the phenotypic assays, because the accessory genes (cssE and cssF) are not essential for capsule expression (81).
Similarly, WGS was conducted on the carriage isolates (n = 93), and isolates were screened for the presence of the porA, ctrA, and sodC genes; one isolate had a partial porA deletion, and three isolates were negative for sodC. In contrast to the invasive isolates, only 35 carriage isolates (38%) had a complete cps locus by WGS (capsular serogroup MnB [18%], MnC [4%], or MnY [15%]). The remainder of the isolates (n = 58) had an incomplete cps locus, with 40% of the isolates missing ctrA. Thirty-nine (67%) of the 58 isolates were ctrA negative; of those, 32 isolates lacked capsule biosynthesis and capsule transport genes and thus were classified as capsule operon-null (cnl) isolates (83, 84). Fifteen isolates had insertions bearing coding sequences from IS1301 (8082), including nine in cssF (MnW) and three each in cssA (MnY) and csb (MnB). An additional isolate had an IS30-family insertion sequence associated with truncation of the cssA gene (MnY) (85) (see Table S1 in the supplemental material). These findings suggest that 44 carriage isolates should be groupable by the rt-PCR assay and serogroupable by the phenotypic assays. With this baseline genotypic information established, the accuracy of five diagnostic assays was assessed to determine which technique (genotypic or phenotypic) was best able to determine the capsular group (genogroup or serogroup).

Diagnostic methodologies.

Figure 1 shows the distributions of the various serogroups and capsular genogroups in the invasive and carriage isolate collections, as determined by each of the methodologies. Tables 3 and 4 show the agreement between the five methods for the invasive and carriage isolate collections, respectively, using WGS as the standard method. Table 5 shows the agreement between the methods for the isolates in the two isolate sets that harbored a capsule operon sufficient to express capsule.
FIG 1 Serogroup distributions among invasive isolates (n = 97) (A) and carriage isolates (n = 93) (B) determined by different diagnostic methods. Isolates underwent serogrouping by SASG1 and SASG2 assays, LCPA, dot blotting, and FACS and capsular genogrouping by rt-PCR and WGS. Proportions of isolates in each serogroup, as identified by the indicated assays, are presented.
TABLE 3 Agreement between genotypic and phenotypic methods used for grouping invasive isolates (n = 97)
WGS capsular genogroup assignmentrt-PCR capsular genogroup agreement (%)Phenotypic method agreement (%)a
MnA (n = 4)1007510010010075
MnB (n = 33)974812949794
MnC (n = 29)1009397100100100
MnW (n = 7)10010010010086100
MnY (n = 24)100100100969696
Pairwise concordance rates (Clopper-Pearson exact method) for WGS, rt-PCR, FACS, LCPA, and dot blotting were ≥96.9% (95% CI, 91.2 to 100%).
TABLE 4 Agreement between genotypic and phenotypic methods used for grouping carriage isolates (n = 93)
WGS capsular genogroup assignmentrt-PCR capsular genogroup agreement (%)Phenotypic method agreement (%)a
MnB (n = 17)1002424243535
MnC (n = 4)10000000
MnW (n = 9)10000112222
MnY (n = 14)1007971867979
NG, ctrA negative (n = 39)72727910010092
NG, ctrA positive (n = 10)1060100100100100
Pairwise concordance rates (Clopper-Pearson exact method) for serogroup assignment by FACS, dot blotting, or LCPA versus WGS were ≤20.4% (95% CI, 11.0 to 30.1%) and that for rt-PCR versus WGS was 47.3% (95% CI, 36.9 to 57.9%).
Four samples were not tested by FACS.
TABLE 5 Agreement in serogroup assignments for cps-positive isolates
Type of classification and assayAgreement for groupable isolatesa (%)
Invasive isolates (n = 97)Carriage isolates (n = 44)
    Dot blot9739
Capsular genogroup  
Isolates determined by WGS to have complete capsule operons.

Characterization of invasive disease isolates.

All 97 invasive isolates were positive for porA and ctrA by the TaqMan rt-PCR assay. Capsule group assignments by rt-PCR were in agreement with WGS results for 96 of the isolates (Table 3). One isolate (PFE-I-0010) was nongroupable by rt-PCR, as it gave positive results with both the MnA and MnB primer sets. The WGS data showed that a complete MnB locus, including the capsule biosynthesis genes cssA-cssC and csb, was present in this isolate. WGS analysis did not identify any sequences for the MnA operon, indicating a false-positive result by rt-PCR for this isolate. In addition, two isolates that lacked the porA gene by WGS gave false-positive results by rt-PCR.
Using the phenotyping approaches, we were able to serogroup between 69% and 97% of invasive isolates, with the methods using monoclonal antibody reagents (FACS, dot blotting, and live-cell phenotypic assay [LCPA]) being the most sensitive and specific (Tables 3 and 5). As expected, all nine isolates with an incomplete cps locus (four MnW, four MnC, and one MnY) were found to express the capsule by the various serogrouping approaches, as the loss of the functional O-acetyltransferase genes did not affect capsule expression or detection in the serogrouping assays (81).
Table 5 shows the percent agreement in serogroup assignment in comparisons of the five phenotypic assays versus WGS. Dot blotting, LCPA, and FACS demonstrated ≥97% agreement with WGS and also demonstrated that the one isolate (PFE-I-0010) that was called NG (positive for both MnA and MnB) by rt-PCR expressed a MnB capsule. The SASG2 and SASG1 assays showed 69% and 79% overall agreement, respectively, with WGS data. The SASG1 and SASG2 assays had 100% agreement with WGS in assigning serogroups W and Y (Table 3); however, the rates of agreement for serogroup B determination were much lower (48% and 12%, respectively) and differed greatly between the two SASG panels. The SASG1 and SASG2 assays resulted in NG results for ∼20% and ∼30% of the isolates tested, respectively. For the SASG1 panel, 89% of the NG assignments were determined by the other phenotypic assays to be MnB, in agreement with the WGS results. For the SASG2 panel, 100% of the NG assignments were determined to be MnB by the other phenotypic methods and WGS. The incorrect identifications by the phenotypic methods are presented in Table S2 in the supplemental material.

Characterization of N. meningitidis carriage isolates.

All 93 carriage isolates were porA positive, with 65 (70%) of the isolates testing positive for ctrA by rt-PCR. A capsule genogroup was assigned to 56 (60%) of the ctrA-positive isolates by rt-PCR. Of the capsular serogroupable isolates identified by WGS (n = 44), dot blotting, LCPA, and FACS correctly grouped 17 isolates (39%), 18 isolates (41%), and 19 isolates (43%), respectively (Table 5). Specifically for MnB isolates, these methods correctly identified 4 (24%), 6 (35%), and 5 (29%), respectively, of the 17 MnB isolates genogrouped by WGS. Conversely, rt-PCR incorrectly identified 12 isolates (8 MnB and 4 MnY); the cps locus carried by these isolates was incomplete, with partial or interrupted csb and synA genes, respectively. Dot blotting, LCPA, FACS, SASG2 assays, and the genotypic methods were in agreement on NG assignments for all 10 nongroupable ctrA-positive isolates. Similarly, dot blotting, FACS, and the genotypic methods were in agreement on NG assignments for all 39 nongroupable ctrA-negative isolates. SASG1 and SASG2 assays correctly assigned serogroups to 14 (32%) and 13 (29%) of the 44 serogroupable isolates, respectively, as determined by rt-PCR and confirmed by WGS. The incorrect identifications by the phenotypic methods are presented in Table S3 in the supplemental material.


The meningococcal capsule is a virulence factor, as exemplified by the low frequency of unencapsulated N. meningitidis isolates causing invasive disease (86, 87). The capsule protects the bacterium from bactericidal activity during invasion and dissemination (51, 8890). In this study, we demonstrated that ≥97% of invasive disease isolates expressed capsule in vitro and could be readily characterized by both genotypic and phenotypic methodologies. Serogrouping of carriage isolates phenotypically, as well as by rt-PCR, was challenging; this was particularly true for MnB isolates. This is due to reduced or no capsule expression in the carriage state, which has been reported to be the result of phase variation and/or genetic deletions or insertions in the cps locus (4447). A subset of nongroupable isolates has been described in the literature, with deletion of the complete cps locus yielding capsule-null (cnl) mutants (45, 50, 84). WGS confirmed the loss of the ctr (capsule transport) operon and capsule biosynthesis genes in 32 (34%) of the carriage isolates tested. In agreement with published work, the capsule-null isolates in our collection were restricted to the following clonal complexes: sequence type 53 (ST53) (38%), ST198 (41%), ST1117 (12%), and ST1136 (9%) (91). Additionally, 10% of the carriage isolates were negative by rt-PCR for serogroup-specific genes, although they did carry the ctrA gene. Although carriage isolates may be missing the capsule locus or harbor loci with insertions or deletions, this does not necessarily predispose such isolates to be nonvirulent, as N. meningitidis is naturally genetically competent and has the capacity to obtain the cps pathway genes through horizontal genetic exchange (9294). In the carriage state in the human nasopharynx, cocolonization with other pathogenic and nonpathogenic N. meningitidis strains has the potential to lead to genetic exchange and capsule switching (95, 96). Furthermore, although IMD caused by cnl meningococci is rare, there have been reports of cnl isolates causing disease, mainly in immunocompromised patients (86, 87).
WGS identified 44 carriage isolates (47%) that contained a complete cps operon; of those isolates, less than one-half were phenotypically positive by the various phenotypic diagnostics tests (Table 5). Phenotypic assays were more effective in serogrouping invasive isolates than carriage isolates; ≥95% of invasive isolates were successfully grouped by FACS, dot blotting, and LCPA (Table 5). Although the capsule is an important virulence factor, cps expression is regulated at different stages of growth and infection (47). The recent study by Loh et al. (51) demonstrated that environmental factors (e.g., temperature) play roles in the expression of capsule and surface antigens through a thermosensor. Those authors identified an 8-bp repeat motif whose deletion resulted in dysregulation of the cssA promoter. In the present study, alignment of the intergenic region containing the ctrA and cssA promoters from carriage isolates that failed to express the capsule and capsule-positive control isolates did not reveal any clues, at the sequence level, to explain the lack of capsule expression by the carriage isolates (data not shown). Isolates in our collection were shown to have as many as three of the 8-bp repeat motifs described by Loh et al. (51); additional studies will be required to determine the role of these sequences in capsule expression in vivo.
WGS is rapidly becoming the gold standard for bacterial epidemiological studies (6971). In this study, we have highlighted its potential usefulness for the characterization of meningococcal carriage isolates. Although the rt-PCR assay appears to be highly predictive of capsule genogrouping, especially for invasive N. meningitidis isolates (99%), this approach relies on the detection of a small fragment (<120 bp) of one gene in the cps operon for each capsular genogroup, and assay sensitivity can be affected by the high degree of genetic plasticity, which we confirmed especially when evaluating carriage isolates. Selection of the target sequences is critical and may be different in different geographical locations.
Several targets (porA, ctrA, and sodC) have been employed as N. meningitidis-specific diagnostic targets for rt-PCR assays (97, 98). In this study, we looked at all three targets by WGS in both of our isolate collections. As prior studies have found (50, 84), ctrA is absent in a large percentage of carriage isolates; in our collection, only 54/93 carriage isolates (58%) were positive for ctrA, whereas 100% of invasive isolates were positive for this gene. Both porA (99% and 97%, respectively) and sodC (97% and 100%, respectively) were well represented in the carriage and invasive isolate collections. Our data confirm that either of these target genes could be a reliable N. meningitidis-specific marker for an rt-PCR diagnostic assay.
The carriage and IMD isolate collections were not contemporary or from the same geographical locations. However, data collected over the past 5 years from diverse geographical locations, which showed that the levels of capsule expression in carriage isolates were the same (data not shown), suggested that geographical locations may not affect our final conclusions. Additionally, the IMD isolates were previously characterized by SASG assays; therefore, the isolates tested were not from an unbiased set, although all assays were conducted in a blinded fashion. As reported previously, IMD isolates are efficiently serogrouped by SASG assays; therefore, this was not thought to affect the overall conclusions of the study (40).
With the introduction of vaccines targeting MnB, carriage studies are essential for monitoring the ability of the new vaccines to prevent carriage acquisition and/or to shorten the duration of carriage episodes and for assessing the development of herd protection (12). Identification of the appropriate diagnostic tools to conduct surveillance following vaccination will be critical. The present study demonstrates the utility of using WGS to accurately type bacterial isolates recovered during clinical studies. Based on our field study, rt-PCR diagnostic assays seems unlikely to provide the reliability necessary for carriage surveillance, due to a large number of misclassified isolates with a mutated or deleted cps locus. Our findings suggest that the use of WGS to type carriage isolates accurately, in conjunction with the use of FACS to determine the capsule phenotype, may offer a superior algorithm to characterize carriage isolates for most purposes, compared to single methods. Remaining challenges for the development of WGS as an interpretive tool for disease surveillance, confirmatory diagnosis, and case management include the development of an appropriate infrastructure for data generation, analysis, and storage. We conclude that a combination of genotypic and phenotypic methods will improve N. meningitidis serogroup determination and the understanding of N. meningitidis carriage and epidemiology.


We acknowledge Alita Miller and Robert Donald for helpful conversations and Li Hao for assistance with bioinformatics analyses.
This study was sponsored by Pfizer Vaccine Research.
C.H.J., N.M., E.R., L.A., J.H., J.C.H., L.K.M., Q.J., L.P., J.E., K.U.J., and A.S.A. are current or former employees of Pfizer and may own stock in the company. R.G. and P.D.W. have received research grants and/or reimbursement of travel expenses from Pfizer. L.W.M. and X.W. have no conflicts of interest and do not receive financial support or have other interests in commercial companies.

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Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 54Number 1January 2016
Pages: 25 - 34
Editor: D. J. Diekema
PubMed: 26311858


Received: 29 May 2015
Returned for modification: 11 July 2015
Accepted: 20 August 2015
Published online: 30 December 2015


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C. Hal Jones
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Naglaa Mohamed
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Eduardo Rojas
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Lubomira Andrew
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Johanna Hoyos
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Julio C. Hawkins
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Lisa K. McNeil
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Qin Jiang
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Leonard W. Mayer
Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Xin Wang
Meningitis and Vaccine Preventable Diseases Branch, Division of Bacterial Diseases, National Center for Immunizations and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA
Rodica Gilca
Institut National de Santé Publique du Québec, Quebec City, Canada
Laval University, Quebec City, Canada
CHU de Québec, Quebec City, Canada
Philippe De Wals
Institut National de Santé Publique du Québec, Quebec City, Canada
Laval University, Quebec City, Canada
CHU de Québec, Quebec City, Canada
Louise Pedneault
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Joseph Eiden
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Kathrin U. Jansen
Pfizer Vaccine Research and Development, Pearl River, New York, USA
Annaliesa S. Anderson
Pfizer Vaccine Research and Development, Pearl River, New York, USA


D. J. Diekema


Address correspondence to C. Hal Jones, [email protected].

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