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Journal of Clinical Microbiology, September 2005, p. 4865-4867, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4865-4867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Variable-Number Tandem Repeat Analysis of Meningococcal Isolates Belonging to the Sequence Type 162 Complex

Siamak P. Yazdankhah,1* Konstantinos Kesanopoulos,2 Georgina Tzanakaki,2 Jenny Kremastinou,2 and Dominique A. Caugant1,3

Department of Airborne Infections, Division of Infectious Disease Control, Norwegian Institute of Public Health, Oslo, Norway,1 Meningococcal Reference Laboratory, National School of Public Health, Athens, Greece,2 Institute of Oral Biology, University of Oslo, Oslo, Norway3

Received 7 February 2005/ Returned for modification 20 April 2005/ Accepted 17 June 2005


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ABSTRACT
 
Thirty-one meningococcal isolates from carriers and disease cases belonging to the sequence type (ST) 162 complex, isolated in Greece in 1999 and 2000, were studied by the use of variable-number tandem repeat analysis. Our study demonstrated that the isolates belonging to the ST-162 clonal complex were a heterogeneous group. Based on this heterogeneity, it is unlikely that the disease-associated isolates represent an outbreak.


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TEXT
 
Neisseria meningitidis is an obligate human pathogen of worldwide significance, whose effects range from asymptomatic carriage to lethal systemic infection (4, 13). During nonepidemic periods, approximately 10% of individuals in the general population at any time harbor N. meningitidis in the upper respiratory tract (3).

Most clinical cases of meningococcal disease are caused by strains belonging to serogroups A, B, and C and, to a lesser extent, to serogroups W135 and Y (11). A number of methods have been developed to further characterize meningococcal strains (4). Among these, multilocus sequence typing (MLST), which is based on nucleotide sequence variation in seven housekeeping genes, is considered the "gold standard" for epidemiological studies. The allele profiles of the strains are designated the sequence types (STs), and related STs define clone complexes (9). By the use of this technique, the majority of epidemic and endemic cases of meningococcal disease are found to be caused by a limited number of clonal groups, which have been assigned to the ST-11 complex, the ST-32 complex, the ST-41/44 complex, the ST-5 complex, and the ST-8 complex (7). Studies have shown that, in contrast, meningococcal strains isolated from carriers are highly diverse and comprise many different genotypes (5, 7).

We have previously studied populations of meningococcal isolates recovered from carriers and patients in the Czech Republic, Greece, and Norway (14). In that study, we found that in Greece, ST-162 and closely related STs were predominant both among disease-associated isolates and among carrier isolates. The frequent presence of ST-162 isolates in carriers in Greece indicates the capability of this ST complex to establish a commensal relationship with the host. The prevalence of the ST-162 complex among disease-associated isolates in Greece shows that we face a yet little-known clonal complex with significant invasive ability. Strains of the ST-162 complex have also been reported occasionally from other European countries and from the United States and Asia (http://pubmlst.org/).

With the aim of establishing a variable-number tandem repeat (VNTR) method for molecular typing of N. meningitidis, we previously analyzed a total of 146 meningococci isolated from carriage, endemic, and epidemic cases (15). We concluded that, like pulsed-field gel electrophoresis (PFGE), VNTR analysis has high discriminatory power and may differentiate isolates identified as similar by multilocus enzyme electrophoresis or MLST (2, 6, 12). VNTR is a simple, reliable, and low-cost method compared to the PFGE method. Thus, VNTR analysis might be used for fine typing of meningococcal isolates belonging to the same clonal complex. Furthermore, we observed that VNTR typing might be a useful differential method for short-term epidemiology of meningococcal isolates in relation to outbreaks.

The purposes of this study were to determine the genetic polymorphism of N. meningitidis belonging to the ST-162 complex by using VNTR analysis, to compare VNTR types of carrier and disease-associated isolates, and to determine whether the disease-associated isolates represented an outbreak. The results were compared with MLST and phenotypic (serogrouping, serotyping, and serosubtyping) data.

A total of 31 isolates recovered from carriers and patients in Greece and belonging to the ST-162 complex were included in this study. The 16 (51.6%) isolates from carriers were collected in 1999 from four regions of northern Greece (Ioannina, Serres, Florina, and Evros), where there has been an increasing number of immigrants from neighboring countries (Albania, Bulgaria, and Turkey) (8). The 15 (48.4%) disease-associated isolates were selected among those submitted to the Meningococcal Reference Laboratory in Athens in 1999 and 2000. The disease-associated isolates were from regions different from those where the carrier isolates were obtained. This observation indicates that isolates belonging to the ST-162 complex are widely spread in the country.

Methodological development of VNTR analysis for N. meningitidis has been reported elsewhere (15). In brief, four VNTR areas in the genome of N. meningitidis were analyzed for genetic polymorphism by using the primers listed in Table 1. The annealing temperatures for the four PCRs were 59°C, 57°C, 63°C, and 57°C for VNTR01, -02, -06, and -08, respectively. PCR products (5 µl) resulting from amplifications of the four VNTR loci for each strain were mixed together and electrophoresed on a 2% agarose gel. Gels were stained by ethidium bromide and photographed under UV illumination. When the PCR products of the four VNTR loci were mixed, only three bands were detected in some isolates (Fig. 1), as a result of equal or very similar PCR product sizes in these isolates. The gel photographs were analyzed using BioNumerics software (Applied Maths BVBA, Sint-Martens-Latem, Belgium), and a dendrogram was constructed by using the Dice coefficient of similarity and cluster analysis with the unweighted-pair group method with arithmetic averages. The position tolerance and the optimization were set up at 0.6 and 1.0, respectively.


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  		TABLE 1. PCR primers for VNTR analysis of N. meningitidis



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  		FIG. 1. Genetic relationships among meningococcal isolates belonging to the ST-162 complex, as determined by VNTR analysis. The left side of the gel photo is the top of the gel. nd, not done; C, carrier; P, patient.

All disease-associated isolates belonged to serogroup B, which also predominated among carrier isolates. This was in agreement with data published by Tzanakaki et al. (10), who identified the ST-162 complex among serogroup B meningococcal isolates recovered from patients and their close family contacts in Greece. Phenotype B:4:P1.14 was the most frequent, but various other serotypes (1, 14, 15, and 22) and serosubtypes (P1.2, P1.4, P1.6, and P1.12) were found in association with the ST-162 complex isolates. All disease-associated isolates were from patients under 17 years of age, and most of them (n = 8) were isolated from infants (1 year old or younger).

Figure 1 illustrates the genetic similarity of the meningococcal isolates belonging to the ST-162 complex, based on VNTR analysis. The VNTR assay divided the 31 isolates into 26 VNTR types; the disease-associated and carrier isolates were divided into 13 types each, while MLST assigned the disease and carrier isolates to 4 and 5 STs, respectively. There were five pairs of isolates that were 100% similar by VNTR analysis. Two of the 16 carrier isolates displayed 100% similarity with two disease-associated isolates. Isolates with the same VNTR types differed in their phenotypes. It is known that serological examination of meningococcal isolates for typing has some limitations, which include the inability to subtype all isolates with available reagents, poor expression, or masking of the surface antigens. Furthermore, two isolates (disease-associated isolate GR-33/00 and carrier isolate GRS-88) with different STs showed similar VNTR patterns. It has been shown that other techniques with high discriminatory power, such as PFGE, may sometimes identify isolates with different phenotypes as similar (1, 12).

We have previously demonstrated that VNTR typing is capable of distinguishing between meningococcal isolates belonging to the ST-32 complex and those belonging to the ST-11 complex (15). In these complexes, the same patterns were obtained for epidemiologically related isolates from a relatively confined area and during a limited period of time (15). Therefore, the VNTR diversity demonstrated in the ST-162 complex in Greece suggests that this clonal complex has circulated for some time in the country and that the disease-associated isolates do not represent an outbreak situation. However, the VNTR assay has allowed recognition of an outbreak caused by isolates belonging to the ST-162 complex in a kindergarten in Athens, Greece (Tzanakaki et al., unpublished data).

VNTR analysis of the isolates belonging to the ST-162 complex demonstrated further heterogeneity of this group of meningococcal isolates. The ability of the VNTR analysis to distinguish among the ST-162 complex isolates may be useful to identify subtypes of this clonal complex in an outbreak situation.


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ACKNOWLEDGMENTS
 
This work was supported by the EU-MenNet project of the European Commission, contract QLK2-CT-2001-01436.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Airborne Infections, Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 4404 Nydalen, NO-0403 Oslo, Norway. Phone: 47 22 62 28 09. Fax: 47 21 62 28 01. E-mail: siya{at}fhi.no. Back


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Journal of Clinical Microbiology, September 2005, p. 4865-4867, Vol. 43, No. 9
0095-1137/05/$08.00+0     doi:10.1128/JCM.43.9.4865-4867.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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