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Journal of Clinical Microbiology, January 2007, p. 159-167, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01322-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

IS1301 Fingerprint Analysis of Neisseria meningitidis Strains Belonging to the ET-15 Clone{triangledown}

Johannes Elias* and Ulrich Vogel

Institute for Hygiene and Microbiology, University of Würzburg, Würzburg, Germany

Received 27 June 2006/ Returned for modification 16 August 2006/ Accepted 30 October 2006


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Meningococci of the ET-15 clone frequently cause clusters of invasive meningococcal disease (IMD) and are associated with a high case-fatality ratio. Timely typing of strains from outbreaks of IMD caused by this clone is hampered by the low variability of its surface antigens. We present a new Southern blot-based typing method for ET-15 meningococci based on the insertion element IS1301, which was present in all 70 ET-15 strains tested. Fingerprints were stable in vitro over a period of 100 days of cultivation on agar plates. The discriminatory power of IS1301 fingerprinting exceeded that of typing by serogrouping and antigen sequencing of the outer membrane proteins PorA and FetA, as determined by the analysis of 52 epidemiologically unrelated strains. In addition, the method provided conclusive results with regard to the comparison of strains from clusters of IMD. The investigation of insertion sites of IS1301 revealed several new intragenic insertions, among others, into open reading frames homologous to mafB and tspB. A previously described insertion in nadA was present in more than two-thirds of the strains analyzed, suggesting that NadA is probably an unreliable vaccine candidate for the prevention of ET-15 disease.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neisseria meningitidis is a highly diverse facultative bacterial pathogen. In most instances, it colonizes its human host without causing disease. Its occasional invasion, however, can entail devastating diseases like septicemia or meningitis, which are referred to as invasive meningococcal disease (IMD). N. meningitidis continues to feature as a leading cause for bacterial meningitis worldwide. Virtually all disease-causing isolates express a polysaccharide capsule determining their serogroup, the most common of which are A, B, C, Y, and W-135 (39). The majority of invasive strains belong to a limited array of so-called hypervirulent clonal complexes (ST-11, ST-32, ST-8, ST-41/44, ST-1, ST-4, and ST-5), which can be defined by multilocus sequence typing (MLST) (33). These are often coupled to the expression of certain capsular polysaccharides, e.g., strains of the ST-11 complex mostly belong to serogroups C and W-135. ET-15 meningococci represent a subset within the ST-11 complex associated with a higher case-fatality ratio and a higher proportion of sequelae than other clones (14, 30, 51). They were first described in the 1980s in Canada (2) and have since spread throughout the world, bringing about rises in IMD incidence in several countries (29-31, 43). Their identification is facilitated by their rare allele for the enzyme fumarase, as determined by multilocus enzyme electrophoresis (MLEE). The coding gene fumC harbors a point mutation at position 640, which lies outside the sequence used for allele assignment in MLST (49). Preliminary evidence suggests, moreover, that ET-15 meningococci consistently harbor the insertion sequence IS1301, which is rarely observed in the remainder of ST-11 complex strains (22, 28).

Finetyping of meningococci is a prerequisite for the refined analysis of the epidemiology of IMD. At the Reference Centre for Meningococci in Germany (NRZM), a combination of serogrouping and DNA sequence-based antigen typing of the variable regions 1 (VR1) and 2 (VR2) of PorA and the variable region of FetA are routinely employed for this purpose. A recent retrospective cluster analysis performed at the NRZM verified its high discriminatory power (13). Nevertheless, the antigenic diversity of ST-11 meningococci is limited (46). A highly discriminatory tool for the distinction of strains belonging to the ET-15 clone of hypervirulent meningococci is therefore urgently needed to analyze strains isolated from outbreaks of disease.

Typing of bacterial strains based on the distribution of insertion elements within their genomes has been used for a variety of bacterial species. Specifically, IS6110 restriction fragment length polymorphism (RFLP) remains one of the most widely used methods for strain differentiation of the Mycobacterium tuberculosis complex (47). In addition, similar methods have been implemented for the distinction of Mycobacterium avium (17, 48), Yersinia pestis (23), Streptococcus pneumoniae (38), and Streptococcus agalactiae (41).

The insertion element IS1301 has been classified to the IS5 family, group IS427 (32). Whereas in one study only 13 to 20% of serogroup A, B, and C strains harbored this insertion element, 46 to 89% of serogroup W-135, 29E, and Y strains were positive for IS1301 (22). It has been described to reversibly insert into the siaA gene of serogroup B meningococci, thereby causing phase variation of capsule expression (18). Dolan-Livengood et al. reported insertions into the 5' end of ctrA of the capsule transport operon (ctr) in carrier strains (12). Furthermore, Claus et al. recently detected the inactivation of oatWY (a capsular O-acetyl-transferase) by IS1301 in meningococci of the serogroups W-135 and Y (6). Similarly, disruption of the porA gene has been reported, notably also in ET-15 meningococci (28, 35). Finally, its presence in the nadA gene, which encodes an adhesin promoting invasion into human epithelial cells, was described as a rare feature among meningococcal strains analyzed (10, 11, 28). Nevertheless, the rate of interruption of nadA by IS1301 in ET-15 meningococci has been unknown so far.

We report here the establishment of an IS1301-RFLP typing method for the differentiation of ET-15 meningococci and exemplify its application in different epidemiological settings. In addition, we characterize ten genomic insertion sites in detail, thus providing insight into the genetic variability introduced by IS1301 and allowing the future implementation of PCR-based typing techniques.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial strains and media. All strains were grown on GC agar prepared from Difco-GC medium base (Becton Dickinson GmbH, Heidelberg, Germany) supplemented with PolyViteX mixture (bioMérieux Deutschland GmbH, Nürtingen, Germany). The Neisseria meningitidis serogroup C control strain FAM18 (ST-11) was made available by Mark Achtman (Max Planck Institute for Infection Biology, Berlin, Germany). In addition, 70 meningococcal strains of the electrophoretic type 15, collected between 1991 and 2005, were analyzed. Seven strains from Austria, Canada, Finland, The Netherlands, Norway, Israel, and Iceland were kindly provided by Dominique Caugant (Norwegian Institute of Public Health, Oslo, Norway) and were classified by MLEE (27). Three strains from the Czech Republic, analyzed with MLEE, were donated by Paula Kriz (National Institute of Public Health, Prague, Czech Republic). Three strains from Germany were already characterized by MLST in a carrier study (7) and were additionally subjected to DNA sequencing of the fumC gene, revealing the characteristic single nucleotide polymorphism at position 640 (49). Finally, 57 strains were selected from the strain collection of the Reference Centre for Meningococci in Germany. Of these, five were previously typed by MLEE (19). For the remaining 50 serogroup C and 2 serogroup B strains, MLST confirmed ST-11. Of note, their affiliation to the ST-11 complex was uniformly predicted by the presence of the restriction modification system nmeDIR, as shown by a dot blot approach (9), together with the insertion of IS1655 into the putative restriction modification system 37/1-7 (8). MLST was followed by DNA sequencing of the fumC gene, confirming the presence of allele fumC-3. All ST-11 complex strains were then shown to harbor the specific single nucleotide polymorphism at position 640 of the fumC gene, thus certifying that they belonged to the ET-15 clone (49).

Southern blotting. Genomic DNA from the bacterial strains was isolated using the kit QIAGEN Genomic-tip 100/G (QIAGEN, Hilden, Germany) and digested to completion with the restriction enzyme HincII (New England Biolabs GmbH, Frankfurt am Main, Germany). Fragments were separated by electrophoresis on 0.8% agarose gel and transferred to a nylon membrane according to standard protocols.

Cloning and inverse PCR. DNA fragments of the ET-15 strain DE9246 hybridizing to the IS1301 probe were obtained through isolation from agarose gel after digestion with the restriction endonucleases HincII, BstUI, BsaBI, or MspAI1a (New England Biolabs) using a QIAquick gel extraction kit (QIAGEN). Fragments were either cloned into the vector pBluescript SK+ (Stratagene, Heidelberg, Germany) or subjected to an inverse PCR as follows: ligation with T4 ligase (New England Biolabs) followed by PCR with the outbound IS1301 primers RH1 and RH2 (21). DNA sequencing of the regions flanking the insertion sites was performed with the primers RH1 and RH2 or JE1 (5'-CGATGCTTTACTTGGCTTGCT-3') and JE2 (5'-CTCGCCATTGTTTGTGTTTGC-3').

Preparation of probes. An IS1301 probe was constructed using the primers SH42 (5'-TTGAGCTAGGGTCATGG-3') and SH46 (5'-AAATCAGGGTTAGGTTTCTT-3'). The PCR product was purified employing a QIAquick PCR purification kit (QIAGEN) and labeled with digoxigenin-11-dUTP using a random primed DNA labeling kit as described by the manufacturer (Roche Diagnostics, Penzberg, Germany). Probes for the IS1301 insertion sites were generated by PCR using chromosomal DNA of the IS1301-negative strain FAM18 as a template. Oligonucleotide primers are listed in Table 1.


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  		TABLE 1. Primer pairs used to generate probes for insertion sites of strain DE9246

 
Screening for the presence of IS1301 in nadA. The presence of an insertion in nadA (NMC1970, according to the unpublished annotation of the genome sequence of strain FAM18) was screened by using a PCR with the primer pair HC306 and HC307, specific for sequences flanking insertion site no. 10 (Table 1). A PCR product of approximately 1.2 kb suggested an insertion of IS1301, whereas a product of approximately 0.4 kb was interpreted as a lack thereof. A NMC1970 probe was generated with the same primers and genomic DNA of strain FAM18 as a template. It was used to identify restriction fragments representing IS1301 insertions within nadA in Southern blots.

Fingerprint analysis. IS1301 fingerprint patterns were analyzed with Gelcompar II software (Windows XP, version 4.0; Applied Maths, Sint-Martens-Latem, Belgium). Developed films were digitized using a scanner with an optical resolution of 200 dpi (Hewlett-Packard ScanJet 7450C). The banding patterns were normalized using the digoxigenin-labeled DNA molecular weight marker III as an external reference (Roche Diagnostics; 14 fragments ranging from 125 bp to 21 kb). At least every fifth lane was a reference lane. In general, bands were automatically assigned by the computer and corrected manually after visual inspection of the original films. Similarities of the banding patterns were measured using the Dice binary coefficient. The position tolerance and optimization of band matching were each set to 1.0%. The unweighted-pair group method using arithmetic averages was used for dendrogram display.

Finetyping. All 70 strains were finetyped as follows: serogrouping of meningococcal isolates was accomplished by slide agglutination using monoclonal antibodies NmA 932, NmB 735, NmW135 1509, and NmY 1938 (produced on request by Chiron-Behring, Marburg, Germany) and the Neisseria meningitidis group C agglutinating sera (Remel, Lenexa, KS). Further typing was accomplished by amplification and DNA sequencing of VR1 and -2 of the porA gene encoding porin A and of VR1 of the fetA gene encoding the FetA protein (40, 42).

DNA sequence analysis and statistics Blast analyses were carried out using the web interfaces provided by NCBI (http://www.ncbi.nlm.nih.gov/BLAST/), TIGR (http://tigrblast.tigr.org/cmr-blast/) and The Wellcome Trust Sanger Institute (http://www.sanger.ac.uk/cgi-bin/blast/submitblast/n_meningitidis). The calculation of the discriminatory indices and their confidence intervals for finetyping and IS1301-RFLP typing was performed as described previously (16, 24).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Presence of IS1301 and stability of the banding patterns in vitro. IS1301 was present in all 70 ET-15 isolates analyzed (100%). The visibly discernible number of insertion elements ranged from six to twelve per strain (Fig. 1). IS1301-RFLP patterns of two strains (DE9246 and DE9260) remained stable after daily passaging on GC agar for a period of 100 days (data not shown).


Figure 1
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  		FIG. 1. IS1301 DNA fingerprint patterns of 70 meningococcal strains of the ET-15 clone and the corresponding dendrogram. The position of each IS1301 band was normalized using an external reference to facilitate mutual comparability. The Dice coefficient was used to calculate similarities and the unweighted-pair group method using arithmetic averages was used for dendrogram display (scale represents percent similarity). Shaded boxes labeled with letters a to e highlight strains from clusters of meningococcal disease. (a, County of Unna; b, Greater Karlsruhe; c, Greater Bremen; d, County of Rottal/Inn; e, County of Oberallgäu and adjacent counties; see the text). The IS1301 insertion in nadA is marked by an arrow. The asterisks denote strains where fetA was not typeable due to deletion.

 
Discriminatory power of IS1301 RFLP in comparison to finetyping. Finetyping of meningococcal strains, as performed on a routine basis at the NRZM, comprises serogrouping and sequencing of VR1 and -2 of porA and the variable region of fetA (42). In two strains analyzed herein, FetA typing was negative due to deletion of the target sequence (DE9260, DE9301; unpublished data). In total, 13 different finetypes, i.e., unique combinations of the variables above, could be distinguished. IS1301-RFLP typing, however, revealed 37 unique banding patterns as determined by computer-assisted analysis using Gelcompar. In order to compare the discriminatory powers of both methods, the discriminatory indices (DI) were calculated using 52 epidemiologically unrelated strains (16, 24), i.e., including only one pattern per recognized cluster of disease. In total, five spatiotemporal clusters of disease were considered that had caused public awareness and public health activities. The results show a typing resolution of IS1301-RFLP typing that was significantly higher than that of routinely used finetyping for ET-15 meningococci (DI for IS1301-RFLP, 0.979 [95% confidence interval, 0.962 to 0.996]; DI for serogroup-PorA-FetA typing, 0.814 [95% confidence interval, 0.749 to 0.879]).

Stability of IS1301 patterns in clusters of meningococcal disease. Several strains from outbreaks of ET-15 meningococcal disease were retrospectively analyzed by IS1301-RFLP (Fig. 2). Twenty-six of all the strains (37.1%) were associated to perceived spatiotemporal disease clusters, which were grouped into the following entities.


Figure 2
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  		FIG. 2. Geographic expansion, finetypes, and IS1301 DNA fingerprint patterns of clusters caused by ET-15 meningococci. Finetypes and IS1301-RFLP patterns: {blacklozenge}, figures in brackets designate the number of strains belonging to this type or pattern (only given where more than one type or pattern per cluster was detected); {clubsuit}, letters in parentheses refer to highlighted fingerprint patterns in Fig. 1; {spadesuit}, only cases analyzed at the reference centre are provided; the asterisk marks clusters where epidemiologically, not directly, related regional occurrences of the same IS1301-RFLP pattern were coupled to cases with a reported epidemiological link.

 
(i) Short-term, spatially confined clusters displaying a single unique banding pattern. This group comprised a community outbreak in the area of Bremen between February and March 2003, an institutional cluster in the county of Unna in March 2003, and a regional outbreak in the county of Oberallgäu between February and April 2004, which was followed by an immunization campaign (20). Further analysis of ET-15 strains in the region of Unna revealed an additional strain with the same fingerprint pattern isolated in May 2003. Similarly, three further strains with IS1301-RFLP patterns indistinguishable from those in Oberallgäu were found in close spatial proximity: one from the same county (Oberallgäu) isolated in April 2003, and two further strains from counties approximately 40 and 90 km further north sampled in May 2003 and March 2004, respectively (Fig. 2 depicts the geographic expansion of all cases).

(ii) A short-term, spatially confined cluster with two banding patterns. This group is represented by a single cluster in the county of Rottal/Inn, which emerged in February 1998 after the carnival season (19). Here, IS1301-RFLP revealed two distinct patterns. This, however, concurred with the finetyping results, which revealed two different finetypes, suggesting that the outbreak was caused by two distinguishable ET-15 strains.

(iii) A protracted hyperendemic situation with similar banding patterns. This situation occurred in the region of Greater Karlsruhe over a period of 23 months from December 1999 to November 2001, prompting the implementation of a vaccination campaign (1). While all cases were assigned the same finetype (C:P1.5-1,10-8:F4-1), IS1301-RFLP showed three similar, yet distinct patterns, whereby the predominant comprised five of seven strains. The diversification could have been the result of microevolution taking place over a period of almost two years. In conclusion, however, each cluster of IMD showed unique banding patterns not identified among the remainder of the strains analyzed.

Insertion sites within strain DE9246. Ten insertion sites within strain DE9246 were characterized by DNA sequencing and alignment to the genome of strain FAM18. Blast searches yielded high sequence similarities to the genome of FAM18 in nine of the ten insertion sites analyzed. In five cases, IS1301 appeared to disrupt open reading frames (ORFs). Interestingly, three of these ORFs encode proteins homologous to structures expressed on the cell surface, like the adhesin NadA (10), the putative adhesin MafB (36), and the so-called T-cell- and B-cell-stimulating Neisseria-specific antigen TspB. Probes hybridizing with the different insertion sites identified their position within the HincII-digested genomic DNA of DE9246. The positions relative to the unpublished genome sequence of strain FAM18, produced by the Sanger Centre, the orientation of the insertion element IS1301, and its location relative to identified ORFs are detailed in Fig. 3. For one insertion site (designated 'insertion site no. 4'), Blast searches yielded no similarities to neisserial proteins on the amino acid level but showed similarity to a hypothetical protein from Pseudomonas entomophila (47% identity over a length of 163 amino acids; accession no. CAK16407). Also, the GC content of this DNA sequence was low, with 35.4% compared to 51.6% of the host genome, suggesting a possible nonneisserial origin.


Figure 3
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  		FIG. 3. IS1301 insertion sites within the strain DE9246 characterized in more detail. The external reference digoxigenin-labeled DNA molecular weight marker III (Roche Diagnostics GmbH, Penzberg, Germany) is displayed for comparison and marked ‘Ref.’ (the length of the fragments in kilobase pairs is given on the left side). a, Orientation relative to the unpublished complete sequence of FAM18. b, ORF designation, orientation relative to the sequence of FAM18 (in parentheses), and annotation (in brackets) are provided where available. c, The names of the proteins (in brackets) are given where available; percent identity on the amino acid level and the number of identical amino acids is added in parentheses. Sequence data were produced by the Neisseria meningitidis Serogroup C Strain FAM18 Sequencing Group at the Sanger Institute, downloadable at ftp://ftp.sanger.ac.uk/pub/pathogens/nm.

 
The target consensus sequence 5'-AYTAG-3', with duplication of the central TA after insertion, was identified previously for IS1301 (21). We found corresponding target sequences in four instances (insertion sites ‘4,’ ‘5,’ ‘8,’ and ‘9b'). Weber et al. reported the insertion of IS1301 into 5'-ACTAA-3' in capsule polysaccharide synthesis genes (50), which we also detected for sites '5b’ and ‘10.’ Additionally, we registered the following insertion spots: 5'-ATTAA-3' (site '1'), 5'-GCTAA-3' (site '6'), 5'-TCTAA-3' (site '7'), and 5'-TCTAG-3' (site '9'). The DNA sequences of the insertion sequences themselves were found to be identical for each identified site. In particular, no variability in the length of the poly(T) stretch at position 303 of IS1301 was found. Interestingly, all copies differed from the published sequence of IS1301 (GenBank accession no. Z49092) by the insertion of d(A) at position 45, which is located 37 bp upstream of the start of ORF1 (GenBank accession no. CAA88914).

Disruption of nadA in ET-15 meningococci. NadA is a meningococcal adhesin and a proposed vaccine candidate. We assessed by PCR the proportion of strains in which the ORF encoding NadA (NMC1970) appeared to be disrupted by IS1301 to determine the share of ET-15 meningococci that would be targetable by antibodies directed against NadA. The proportion of ET-15 strains harboring IS1301 in the nadA gene was 68.6%. Four strains out of seventy (5.7%; DE9426, DE9439, DE9481, and DE9792) were negative in both the PCR and hybridization approaches, indicating loss of the target sequence. The IS1301/nadA Southern blot band is shown in Fig. 1. It is likely that hypervirulent strains of the ET-15 clone frequently do not express a functional NadA protein.

Nucleotide sequence accession number. The nucleotide sequence for T- and B-cell-stimulating Neisseria-specific antigen TspB has been submitted to GenBank under accession no. AJ010115.


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although ET-15 meningococci represent only a small share of all occurring strains causing IMD in many countries (in Germany, their rate between 2002 and 2005 was 6.1%), their particular epidemiologic profile sets them apart from other hypervirulent lineages. Since their emergence in Canada (2) and worldwide spread therefrom, they have featured regularly in reports describing outbreaks (5, 19, 20, 25, 26, 44) and rises in incidences in several countries (29-31, 43, 51). Also, the generally graver clinical picture of invasive disease caused by this clone (14, 30, 51) further delineates its epidemiologic distinctiveness. Another noteworthy feature of ET-15 meningococci is their predilection to strike teenagers (e.g., 59% of the ET-15 cases between 2002 and 2005 in Germany were over 10 years old), who represent an age group for which different activities and events have been shown to increase the risk of acquiring IMD further than the risk in childhood (45).

For the monitoring of circulating meningococcal strains, we employ a finetyping routine encompassing the determination of the serogroup and sequence-based typing of the variable regions of the outer membrane proteins PorA and FetA. This approach permits a refined surveillance of meningococci with a fairly high discriminatory power. For the typing of ET-15 meningococci, however, the comparatively low variability of surface-bound targets within this clone limits the efficiency of this method.

We report here the establishment of a novel typing approach for ET-15 meningococci that has a high discriminatory power yet shows sufficient stability to enable epidemiological analyses, as shown by the in vitro passaging experiments reported herein and by the satisfying stability of patterns in strains isolated from outbreaks of meningococcal disease. The in vitro stability of patterns seen with strain DE9246 might be due to the possibly nonfunctional state in which IS1301 was found for all insertion sites analyzed in more detail. The length of the poly(T) stretch at position 222 of the transposase gene in all cases resulted in the presence of two open reading frames, which are probably nonfunctional. However, this hypothesis needs confirmation by in vitro experiments assessing phase variation rates of IS1301.

Since IMD caused by the ET-15 clone often occurs in a clustered fashion, the timely typing of the isolated strains is important to guide public health measures. The regional eruptions of IMD around Karlsruhe and Oberallgäu from 1999 to 2001 and 2004 (Fig. 2) were both followed by local vaccination campaigns (1, 20). In both cases, IS1301-RFLP corroborated the results obtained by finetyping, which suggested the regional emergence of a single strain of ET-15 meningococci. In the case of the Karlsruhe cluster, IS1301-RFLP additionally revealed two similar, yet distinct patterns for two cases. Considering the temporal extension of almost two years, this might have been attributable to microevolutionary changes within the original emerging clone. Strains from the smaller clusters in Greater Bremen and Unna were undistinguishable by DNA sequence-based finetyping. Moreover, both clusters appeared within a short time frame, which raised questions as to the existence of an epidemiological link. IS1301-RFLP, however, clearly confirmed that these accumulations represented flare-ups of disease caused by two distinct clones. Finally, the cluster in the county of Rottal/Inn revealed the simultaneous involvement of two distinct strains, already apparent after finetyping alone. The intriguing finding is reminiscent of what has been found by molecular investigation of a university outbreak cluster where cases were caused by two different strains (15). A difference of several independent markers, i.e., PorA, FetA, and IS1301 insertions, during a temporarily confined cluster is unlikely due to diversification occurring during the spread of the outbreak strain within a few days. It can rather be assumed that in the case of the carnival-associated cluster in Rottal/Inn (19), locally prevalent risk factors and risk behavior (45) promoted the independent attack by two different strains.

In addition to representing a useful technique for the monitoring of ET-15 disease, IS1301-RFLP is less time-consuming than pulsed-field gel electrophoresis, because it relies on the simple separation of DNA fragments in standard agarose gels. Results can be obtained as soon as 48 to 72 h. Nevertheless, one has to consider the limitations of IS1301-RFLP: (i) only typing of viable bacterial strains is possible, (ii) interlaboratory portability is limited (although possible if standardization is achieved between facilities), and (iii) it is only applicable to ET-15 strains.

Several insertion sites for the insertion sequence IS1301 have been described so far. A reversible insertional inactivation of siaA was described by Hammerschmidt et al. (18). They found an in vitro reexpression frequency of the polysaccharide capsule of 10–4 and hypothesized involvement of IS1301-mediated phase variation in the process of invasion of meningococci. Genetic analyses of carrier strains led to the discovery of a different insertion in siaA and two further occurrences in the 5' end of ctrA (12). In addition, Weber et al. described several hitherto unknown inserts of IS1301 into the following genes of region A of the capsule gene complex (cps): siaA, siaC, siaDW, and siaDB (50). Two distinct insertions were also recently shown in oatWY encoding a capsular O-acetyltransferase (6). PorA represents yet another surface-expressed molecule that was shown to be inactivated by IS1301 (35). Such PorA-negative strains were also recovered from patients with IMD, and serosubtypability of the affected strains was abolished (28). Finally, IS1301 was described in nadA (10, 11), which encodes a neisserial adhesin and invasin (4). Since NadA was shown to induce bactericidal antibodies even after mucosal vaccination (3), it has emerged as a promising possible vaccine component. It was reported to be present in close to 100% of the hypervirulent lineages ST-32, ST-11, and ST-8, whereas it was shown to be absent in ST-41/44 strains (10).

Surprisingly, the rate of insertion into nadA in ET-15 meningococci amounts to 68.6%. As for the previously mentioned genes, it is conceivable that this insertion is reversible. Nevertheless, while phase-variable expression for NadA has been shown to be mediated by changes in the number of microsatellite repeats upstream of its promoter (34), the modulation of its expression by mobile genetic elements has not been demonstrated. Ultimately, however, our analyses might have implications for vaccine coverage attainable by vaccines including NadA.

Hilse et al. detected an insertion of IS1301 in the serogroup B strain B1940 upstream of frpC, which codes for a repeat-in-toxin exoprotein (21). We found a different insertion site 48 bp upstream of NMC1806, which is highly homologous to FrpD (GenBank accession no. L06299) (Fig. 3). Prochazkova et al. predict that frpD and frpC form a single operon termed frpDC, which is controlled by a putative iron-regulated promoter, presumably located ~300 bp upstream of frpD (37).

Further intragenic insertions were found in ORFs homologous to the adhesin MafB (36) and to the T-cell- and B-cell-stimulating Neisseria-specific antigen TspB (GenBank accession no. AJ010115). Once again, these examples reveal the tendency of IS1301 to inactivate genes coding for molecules exposed to the immune system of the host.

In conclusion, our work presents a highly discriminatory technique with moderate hands-on time for the timely typing of ET-15 clusters. In the future, our approach might be simplified by a multiplex PCR method addressing only variable insertion sites. Moreover, we provide a detailed compilation of new IS1301 insertion sites, highlighting the role, possibly more prominent than previously supposed, of this mobile genetic element for the establishment of immune escape variants in N. meningitidis. Further research comparing the nature of hypervirulent lineages might elucidate to what extent this feature contributes to the epidemiological distinctiveness of ET-15 meningococci.


   ACKNOWLEDGMENTS
 
We are indebted to Matthias Frosch for continuous support. Heike Claus is acknowledged for helpful discussions and critical reading of the manuscript. We thank Christine Meinhardt for expert technical assistance and Stefan Niemann for invaluable hints and tips.

This study was supported by DFG grant VO 718/3 to Ulrich Vogel and Matthias Frosch and by the German Ministry of Health as part of the funding for the National Reference Centre for Meningococci (NRZM).

We extend our thanks to the Sanger Centre for providing genome sequence data of strain FAM18 prior to publication. These sequence data were produced by the Neisseria meningitidis Serogroup C Strain FAM18 Sequencing Group at the Sanger Institute, downloadable at ftp://ftp.sanger.ac.uk/pub/pathogens/nm. Finally, we gratefully acknowledge the donation of strains by Dominique Caugant (Oslo), Paula Kriz (Prague), and Mark Achtman (Berlin).


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Hygiene and Microbiology, University of Würzburg, Josef-Schneider-Str. 2, Building E1, 97080 Würzburg, Germany. Phone: 49 931 20146905. Fax: 49 931 20146445. E-mail: jelias{at}hygiene.uni-wuerzburg.de. Back

{triangledown} Published ahead of print on 8 November 2006. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Anonymous. 2002. Erkrankungen durch Meningokokken der Serogruppe C: Örtliche Häufung führte zu einer öffentlichen Impfempfehlung. Epidemiologisches Bulletin (Robert-Koch-Institute) 2002:175-177.
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Journal of Clinical Microbiology, January 2007, p. 159-167, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01322-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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