INTRODUCTION

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Bacterial meningitis due to Neisseria meningitidis (the meningococcus) continues to be of global importance for public health authorities. While pandemics affecting China and Africa are usually caused by meningococci of the A capsular serogroup, sporadic meningitis, outbreaks, and hyperendemic disease in Central Europe and the United States are usually caused by serogroups B and C (2). For sporadic meningococcal meningitis, public health efforts often include bacteriological analysis of throat swabs obtained from close contacts of the patient and the treatment of healthy nasopharyngeal carriers with prophylactic antibiotics and/or vaccines (5).

Meningococci isolated from the healthy contacts of a diseased patient (carrier isolates) are not necessarily related to the strain causing disease (case isolates), even if all these isolates express the same capsular serogroup. Various subtyping methods have been used to test relationships among the strains from a cohort, including serotyping and serosubtyping with monoclonal antibodies (MAbs) (11), pulsed-field gel electrophoresis (PFGE) (7), multilocus enzyme electrophoresis (MLEE) (2), ribotyping (22), the randomly amplified polymorphic DNA method (25), and PCR-restriction fragment length polymorphism analysis (16). Recently, a novel portable approach, multilocus sequence typing (MLST), which is based on the DNA sequences of six housekeeping gene fragments, has been developed (18). MLST allows assignment of meningococci to clonal groups within a globally accessible, continuously expanding central database.

In outbreak situations, so many bacteria may be isolated that only some of the carrier strains, usually selected on the basis of their capsular serogroup by latex agglutination, are evaluated in detail. However, the expression of capsular polysaccharide by serogroup B meningococci can undergo phase variation, resulting in the isolation of strains from carriers which are not obviously related to the index strain because they are capsule negative and nonserogroupable (6, 14, 15). The siaD gene encodes a polysialyltransferase which is needed for the synthesis of capsular polysialic acid chains. siaD can be amplified from serogroup B and C meningococci by PCRs (6), enabling the determination of the potential serogroups of bacteria which have become phenotypically nonserogroupable. Here we describe a two-step PCR, an improved version of the siaD PCR (6), which distinguishes between serogroup B, C, W135, and Y meningococci.

Antigenic variation through horizontal genetic exchange can also lead to capsule switching among highly related bacteria (23). Such capsule-switching variants would be classified as unrelated to the parent strain by classical serogrouping. Similarly, antigenic variation can also lead to the switching of other antigens (13), including those used for serological subtyping, such as PorA (21). Thus, serological methods cannot reliably recognize the relatedness of meningococci and epidemiological analyses should rely primarily on molecular techniques, particularly those based on multiple loci scattered around the chromosome (3).

We report here on the molecular investigation of meningococci isolated from individuals who had been in contact with a patient with severe serogroup C meningococcal disease.

	MATERIALS AND METHODS

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Meningococcal isolates, culture conditions, and identification. Blood cultures were performed with the BactT/Alert System from Organon Teknika (Durham, N.C.). Cerebrospinal fluid was cultured on Columbia blood agar (Difco, Augsburg, Germany) and chocolate agar (Difco), and enrichment cultures were performed with brain heart infusion medium (Difco) supplemented with factors V and X (Difco). Nasopharyngeal swabs from 60 individuals with contact with the index patient were cultured on chocolate agar plates. After overnight growth at 37°C in a 5% CO₂ atmosphere, colonies containing oxidase-positive, gram-negative diplococci were tested with the RAPID NH System (LD Labor Diagnostika, Leiden, Germany) for the biotyping of Neisseria and Haemophilus. Serogrouping was performed by latex agglutination with the Directigen Meningitis Combo Test (Becton Dickinson, Meylan, France) and by an enzyme-linked immunosorbent assay (ELISA) with the following MAbs: MAb 1087 (specific for the serogroup A capsule), MAb 735 (serogroup B), MAbs 924 and 1125 (serogroup C), MAb 1508 (serogroup W135), and MAb 1938 (serogroup Y). For the ELISA, meningococci were grown overnight on chocolate agar, and 20 µl of a bacterial suspension (optical density at 600 nm = 0.15) was added to each well of a microtiter plate (Greiner, Solingen, Germany) which had been coated with poly-D-lysine (Sigma). After drying, the bacteria were fixed with phosphate-buffered saline (PBS)-0.05% glutaraldehyde for 10 min at room temperature. Thereafter, nonspecific binding sites were blocked by incubation with PBS-1% bovine serum albumin for 1 h at 37°C. Binding of the anticapsule antibodies and of a secondary peroxidase-conjugated anti-mouse immunoglobulin antibody (Dianova, Hamburg, Germany) was performed for 1 h in PBS-1% bovine serum albumin. Intermediate wash steps were performed with PBS. ET-37 reference strains were a kind gift of Dominique A. Caugant (World Health Organization Collaborating Centre for Reference and Research on Meningococci, National Institute of Public Health, Oslo, Norway). Serogroup B isolate B1940 has been described elsewhere (9), and the reference serogroup W135 and Y strains were ATCC 750020 and ATCC 55989, respectively.

Serotyping. The serotypes and serosubtypes of the meningococcal isolates were determined by a previously described ELISA (1) with a set of serotype-specific (2a, 2b, 4, 14, 15, and 21) and serosubtype-specific (P1.1 through P1.15) antibodies, which were provided by J. Suker and I. Feavers (National Institute for Biological Standards and Control, Hertfordshire, United Kingdom).

cps PCR. The siaA, siaB, and siaC genes of region A of the cps locus (9, 23) yielded a 2-kb product after amplification by colony PCR with primers SH39 and UE16 (Table 1). The PCR conditions were initial denaturation (94°C, 5 min), followed by 36 cycles of annealing (45°C, 1 min), extension (72°C, 90 s), and denaturation (94°C, 1 min) and then one cycle of annealing (45°C, 1 min) and a final extension (72°C, 10 min). The PCR conditions for the amplification of the siaD genes (9, 10, 23) were the same, except that the following annealing temperatures (product sizes) were used: 52°C for primers UE12-UE13 (1.8 kb), 40°C for primers HC2-HC4 (2.1 kb), and 50°C for primers HC39-HC50 (extension time, 2 min; 2.4 kb) and primers HC44-HC50 (extension time, 2 min; 3.2 kb). The recently described siaD (B/C) PCR with primers B/CsiaD1 and B/CsiaD2, which exclusively amplify serogroup B and C genes (6), was also performed as a control.

PFGE. PFGE was performed as described previously (19) after digestion of the DNA blocks with the restriction endonucleases SpeI and NheI.

MLST. The sequences of fragments from the abcZ, adk, aroE, gdh, pdhC, and pgm genes were determined as described previously (18) and were compared to the sequences of formerly known alleles. Novel allelic variants were assigned the allelic designations adk13 (ST54), aroE20 (ST54), and gdh18 (ST55). New combinations of the six alleles were assigned the designations ST54 through ST56 (see http://mlst.zoo.ox.ac.uk).

	RESULTS

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Epidemiological description and properties of strains. A German male (age, 17 years) developed severe meningococcal septicemia and skin hemorrhaging on his return to Germany from Prague, Czech Republic, where he had attended an international youth gathering with a group of German scouts for 3 days. Serogroup C N. meningitidis was isolated from cultures of both blood (strain 2120) and nasopharyngeal swabs (strain 2121) from the patient; no bacteria could be cultivated from the cerebrospinal fluid. Strains 2120 and 2121 were nonserotypeable and were of the P1.5,2 serosubtype. Disease outbreaks in the Czech Republic since 1993 have been caused by serogroup C meningococci of the ET-37 complex, usually serotype 2a and serosubtype P1.5,2 (17). Such Czech strains belong to a particular MLEE electrophoretic type (ET) called ET-15 (17), which has also caused hyperendemic disease in Canada (4). The patient recovered without complications after treatment with penicillin.

Serogroup-specific PCR. In order to determine the capsular genotypes of the nongroupable isolates, PCRs which can amplify the siaA to siaC and siaD genes, respectively, of region A from serogroup B, C, W135, or Y meningococci were developed. Region A of the cps locus contains the genes required for the synthesis of activated sialic acid (siaA to siaC) and for the polymerization of sialic acid (siaD). The siaA to siaC genes are identical in serogroups B, C, W135, and Y (9, 10, 23). Thus, the siaA to siaC PCR allows the differentiation of serogroups B, C, W135, and Y from the other serogroups of meningococci. In contrast, the siaD genes of serogroup B and C meningococci are 64% identical and differ considerably from the siaD genes of serogroups W135 and Y, which are 98% identical (9, 10, 23). Primers which bind to unique upstream and downstream regions flanking siaD were developed to distinguish serogroups B and C. The homology between the siaD genes of serogroup W135 and Y strains is so high that a common reverse primer was used together with serogroup-specific forward primers which bind within the siaD gene (Table 1). The siaD PCR yielded a larger DNA fragment with serogroup Y strains than with serogroup W135 strains due to a 630-bp insertion in the region downstream of siaD which is present among all serogroup Y strains analyzed to date (11a). Thus, these serogroups can be distinguished among reference strains by siaD PCRs.

View larger version (81K): [in this window] [in a new window]   FIG. 1.   Determination of the capsular genotype of nonserogroupable carrier strains by PCR amplification of sia genes. The strains tested are listed at the top, and the molecular sizes of the standards are given on the left. Lanes: , negative control without template DNA; B, serogroup B strain B1940 (9); C, index strain 2120 (serogroup C, this study); W, serogroup W135 strain (ATCC 750020); Y, serogroup Y strain (ATCC 55989), other numbers at the top are nonserogroupable carrier isolates.

MLST. The MLST procedure, which consists of the sequencing of six independent gene fragments (18), was applied to the two strains isolated from the patient, seven of the carrier isolates, and two representative ET-15 strains of the ET-37 complex (strain 259/43 from the Czech Republic and strain 88/048 from Canada). Both case isolates and both representative ET-15 strains were ST11, the sequence type (ST) most commonly associated with strains of the ET-37 complex.

PFGE. MLST is a highly conservative genotyping method that is insensitive to microevolution and that is therefore highly suitable for determining relationships between bacterial strains and for long-term epidemiology. In contrast, PFGE is very sensitive to microevolution and can reveal minor differences between related strains (19). Bacterial DNA was analyzed by PFGE after digestion with SpeI and NheI to determine if minor differences distinguished any of the related strains (Fig. 2). Several bands differed between the ST11 strains isolated in Canada and the Czech Republic and between either of those bacteria and the ST11 isolates from the throat and blood of the German patient. In contrast, the latter two strains were almost identical, except that after digestion with SpeI, the DNA from the throat isolate yielded a fragment of 170 kb, while the DNA from the blood isolate yielded a fragment of 130 kb. Similarly, after NheI digestion, the throat isolate yielded a 160-kb band while the blood isolate yielded a 120-kb band (Fig. 2). These results suggest that this strain had suffered a 40-kb deletion during invasion from the throat to the bloodstream. No differences were observed between the four ST55 isolates after digestion with NheI, and three of those isolates, including strains 2122 (nongroupable, genotype C) and 2123 (serogroup C) isolated from a carrier (scout 2) were also indistinguishable after digestion with SpeI. The other three carrier isolates (ST32, ST54, and ST56, respectively) yielded unique PFGE patterns, as expected.

View larger version (39K): [in this window] [in a new window]   FIG. 2.   PFGE analysis of case and carrier strains after digestion with SpeI or NheI. The strains tested and their STs are indicated at the top of each panel, and the positions of molecular weight (MW) markers are given on the left. Representative ET-15 strains from Canada (strain 88/048) and the Czech Republic (strain 259/43) are next to the isolates from the blood (strain 2120) and throat (strain 2121) from the index patient. Other strains were isolated from carriers and are described in Table 2. The positions of the bands which differ between strains 2120 and 2121 are indicated by asterisks.

	DISCUSSION

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A serogroup C strain was isolated from a patient who had returned from the Czech Republic where serogroup C ET-37 complex meningococci have recently caused epidemics of meningitis and septicemia (17). Due to the increased risk of disease among the contacts of patients with sporadic cases of meningococcal disease, close contacts were offered prophylactic antibiotic therapy and their carrier status was determined in order to estimate whether the index strain had spread. The data presented here demonstrate that serogrouping of meningococci isolated from healthy carriers is an inadequate criterion for determining spread. The molecular mechanisms of reversible capsule phase variation have recently been elucidated for serogroup B meningococci (14, 15), and the data presented here suggest that capsule phase variation (whose mechanism remains to be elucidated) may also occur among serogroup C meningococci. Two highly related meningococci were isolated from one healthy carrier, and the meningococci were identical by MLST and PFGE analysis but differed in their expression of the C polysaccharide. Two other carrier isolates possessed genes for the expression of the C or the Y polysaccharide, although they were nonserogroupable. If the results for serogroup B meningococci can be extrapolated, these nonserogroupable strains are potentially capable of reversion to encapsulation and are potential sources of disease. These observations indicate that serogrouping of carrier isolates should rely on molecular techniques as well as on classical serological methods. The PCR methods described here allow determination of the B, C, W135, and Y capsular genotypes within 2 days after colony isolation and could readily be implemented in most laboratories for this purpose. Informed decisions on whether to offer prophylactic therapy to unrelated contacts could then be reached after both microbiological and capsular genotyping data were available.

Molecular typing methods. Both short-term and long-term epidemiological studies depend on reliable typing methods which can be used to determine whether bacteria spread between individuals. The results obtained by serological methods are not conclusive because many isolates are not typeable and because the serotype and serosubtype are not uniform even within highly related bacteria such as those of the ET-37 complex (24) due to frequent horizontal genetic exchange (21). Similarly, the ST55 bacteria described here were variably serotype 4 and nontypeable (Table 2). Phylogenetic methods based on multiple loci scattered around the chromosome are necessary for reliable analysis of the relationships between bacteria (3). Of these, the MLST method has been shown to be as reliable as MLEE and even more conservative (18). The MLST analysis showed unambiguously that the strains from the index patient belonged to the ET-37 complex; almost all ET-37 complex strains are ST11, and all ST11 strains belong to the ET-37 complex. MLST also showed that none of the strains isolated from the 60 contacts were of the ET-37 complex. Inefficient spread of ET-37 complex bacteria has also been found in other investigations (1a, 20). Four strains isolated from three carriers within one family all belonged to the novel ST ST55, and the three other carrier strains investigated belonged to three other STs. One of the latter was serogroup B, ET-5 complex, which has been associated with hyperendemic disease in many countries (8).

Relationships among ET-37 bacteria. By the MLEE scheme ET-37 complex strains from patients with hyperendemic disease in Canada differ from other ET-37 complex strains by one enzyme and have therefore been referred to as "ET-15" (4). ET-37 strains from recent outbreaks in the Czech Republic are indistinguishable by MLEE from the Canadian strains (17), suggesting that there might be an epidemiological link between disease in both countries. The index patient described here became sick on the way home from a trip to the Czech Republic and may therefore have been colonized with ET-37 complex bacteria during that trip.