Use of a multilocus variable-number tandem repeat analysis method for molecular subtyping and phylogenetic analysis of Neisseria meningitidis isolates

Background The multilocus variable-number tandem repeat (VNTR) analysis (MLVA) technique has been developed for fine typing of many bacterial species. The genomic sequences of Neisseria meningitidis strains Z2491, MC58 and FAM18 have been available for searching potential VNTR loci by computer software. In this study, we developed and evaluated a MLVA method for molecular subtyping and phylogenetic analysis of N. meningitidis strains. Results A total of 12 VNTR loci were identified for subtyping and phylogenetic analysis of 100 N. meningitidis isolates, which had previously been characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing. The number of alleles ranges from 3 to 40 for the 12 VNTR loci; theoretically, the numbers of alleles can generate more than 5 × 1011 MLVA types. In total, 93 MLVA types were identified in the 100 isolates, indicating that MLVA is powerful in discriminating N. meningitidis strains. In phylogenetic analysis with the minimal spanning tree method, clonal relationships, established with MLVA types, agreed well with those built with ST types. Conclusion Our study indicates that the MLVA method has a higher degree of resolution than PFGE in discriminating N. meningitidis isolates and may be a useful tool for phylogenetic studies of strains evolving over different time scales.


Background
Neisseria meningitidis is one of the major causative agents of bacterial meningitis and septicemia in children and young adults [1]. Periodically, it causes large epidemics in Africa, especially in the sub-Saharan meningitis belt, and in Asia [1]; however, it is still a serious problem in many industrialized countries [2,3]. Occasionally, a meningococcal pandemic occurs after large population movements, such as pilgrimages [4,5].
Epidemiological studies of N. meningitidis, using various subtyping methods, allow the identification of a disease out-break and investigation of the disseminating meningococcal strains. With the advent of molecular biology, a number of molecular methods have been developed for epidemiological studies of N. meningitidis. Among the methods, pulsedfield gel electrophoresis (PFGE) and multilocus sequence typing (MLST) are the most frequently used subtyping techniques [6,7]. PFGE usually exhibits high discrimination for bacterial isolates, but it generates fingerprint image data that makes a comparison between laboratories difficult. In contrast, MLST is based on sequence data from seven conserved housekeeping genes; sequences that differ at even a single nucleotide are assigned to different alleles. The combination of alleles at the seven housekeeping genes is designated the sequence type (ST) of the isolate; numerous STs can be obtained. A Neisseria MLST database has been established that allows STs to be compared electronically via the Internet. STs are grouped into clonal complexes by their similarity to a central allelic profile (genotype). These central genotypes are identified by a number of heuristic means, including BURST and split decomposition, along with feedback from public health laboratories and epidemiologists. Once a central genotype has been identified, clonal complexes are defined as including any ST that matches the central genotype at four or more loci unless it more closely matches another central genotype [8]. The accumulation of nucleotide changes in housekeeping genes is a relatively slow process, and the allelic profile of a meningococcal strain is stable over time. Therefore, MLST is a powerful tool for study of global epidemiology of meningococci [6]. However, MLST provides lower discrimination than PFGE for fine typing of some clonal groups of N. meningitidis [9].
In recent years, the multilocus variable-number tandem repeat (VNTR) analysis (MLVA) technique has been developed for fine typing of many bacterial species [10][11][12][13][14][15][16][17][18][19]]. In addition, Yazdankhah et al. [20] have recently developed a MLVA method with four VNTR loci for genotyping of N. meningitidis isolates and successfully differentiated the serogroup W135 isolates from sporadic cases and outbreaks. In this study, we successfully developed a MLVA method with 12 VNTR loci to analyze a panel of N. meningitidis isolates, which had previously been characterized by PFGE and MLST.   [9]. b Number in parentheses indicates the second copy of the locus. The second allele indicated in the parentheses was ignored in the MST analysis. Of the 12 loci, at least 9 were located in coding region of annotated genes ( Table 1). The VNTRDB program used each of the three genomic sequences in turn as a "parent" sequence to search repeat loci and, then, located each of the loci at the other two genomes, so that a locus, for example NMTR9a with only one repeat unit in strains MC58 but with 2 repeat units in strain Z2491 and 3 repeat units in strain FAM18, could be found (Table 1).

MLVA genotyping
The MLVA genotyping was performed on 100 N. meningitidis isolates, which were collected between 1996 and 2002, and their PFGE patterns and ST types were characterized previously [9]. The results showed that the majority of the isolates carried only one copy of each of the 12 loci; however, five isolates carried extra copy of NMTR1, NMTR7, NMTR9 or NMTR18 locus, two isolates did not carry the NMTR1 locus, and three isolates did not carry the NMTR12 locus ( Table 2). The number of alleles at each of the 12 loci ranged from 3 to 40 alleles counted on the 100 isolates analyzed (Table 3). Six loci (NMTR1, NMTR2, NMTR7, NMTR9, NMTR10 and NMTR12) had more than 10 alleles and four loci (NMTR1, NMTR2, NMTR7 and NMTR9) had a high allelic polymorphism index (≥ 0.9) ( Table 3). Based on the allele number for each of the 12 loci determined in this study, at least 5 × 10 11 MLVA allelic profiles (MLVA types) are expected.
A total of 93 MLVA types were identified for the 100 isolates ( Table 2). The majority of MLVA types represented only one isolate; however, each TW4, TW5, TW51, TW52, and TW62 types represented two isolates and TW3 represented three isolates. TW62 was identified in two serogroup B isolates (NM255 and NM256), which were obtained from two cases in a meningococcal disease outbreak in a family. TW52 was identified in two serogroup C isolates (NM377 and NM378) with a close epidemiological relationship. TW3, TW4, and TW5 were identified in serogroup Y isolates collected from sporadic cases; the isolates were derived from a newly imported clone [9]. The two serogroup W135 isolates with TW51 type were collected in cases at a 2-year interval. Phylogenetic tree built with MLVA profiles As shown in the previous study [9], PFGE exhibited a higher degree of discrimination than MLST for the isolates analyzed. However, the results of this study showed that MLVA exhibited much higher resolution than PFGE on the same panel of isolates. MLVA discriminated all of the serogroup B isolates and 29 of 31 serogroup W135 isolates, which were collected from sporadic cases ( Table 2). In contrast, only two ST type and four PFGE patterns were identified in the 31 serogroup W135 isolates ( Table 2). Only one ST type and two PFGE patterns were identified in the 11 serogroup Y isolates (Table 2). However, these isolates were further discriminated into seven MLVA genotypes.

Phylogenetic analysis
The clonal relationships among the 100 isolates were constructed with the MLVA types by the minimal spanning tree (MST) method. In the analysis with 12 loci, MLVA types matching at eight or more loci were regarded as clonally related. Consequently, eight distinct MLVA groups were established and the grouping feature established with the MLVA types had good agreement with that built with ST types (Figure 1). The two serogroup A isolates were characterized as different MLVA types (TW48 and TW59), differing in three loci, both carried ST-7 type within the ST-5 complex (  TW1, TW2, TW27, TW55 and TW63) were separated from the T4 group. However, they had a closer genetic relationship with the genotypes within the T4 group.
All the MLVA types, except TW65 and TW88, representing the serogroup W135 isolates, were clustered in T7 group. The two MLVA types (TW25 and TW52), identified in three serogroup C isolates, had a closer clonal relationship with the W135 isolates than other serogroup isolates, although they differed at five loci with the closest MLVA types within the T7 group. A total of 32 MLVA types were identified in the 31 serogroup W135 and three serogroup C isolates; in contrast, only two ST types (ST-11 and its single locus variant, ST-3016) were found in the isolates ( Table 2). The isolates with TW25 and TW52 types emerged in 2001 and 2002, respectively. Since TW25 and TW52 differed in as many as seven loci, the two MLVA strains should not be derived from a common imported strain.
The serogroup Y isolates shared a close clonal relationship as the seven MLVA types, forming a compact cluster. Six MLVA types differed in only one or two loci with the founder type, TW3, which was identified in the earliest collected isolates in Taiwan.

MLVA allelic profiles of isolates from patient-contact episodes
Five isolates, collected from healthy contacts of four patients were characterized by MLVA. The MLVA profiles were identical for isolates from three episodes. Two isolates from the fourth episode differed in a single locus, NMTR-7 (Table 4).

Discussion
Our data demonstrate that the MLVA method is powerful for subtyping and useful for phylogenetic investigation of N. meningitidis isolates. The MLVA exhibited a much higher discriminatory power than PFGE for the isolates tested and the resulting data agreed well with the epidemiological observations. Of the 100 N. meningitidis isolates Our study showed that the clonal relationships between the isolates, established with MLVA types, was in good agreement with those built with ST types. As shown on Figure 1, strains within a ST complex or ST group shared more common VNTR loci. Among the 12 loci, four (NMTR1, NMTR2, NMTR7 and NMTR 12) were highly polymorphic; they could have higher variation rates. The remaining loci could have moderate and low variation rate. Thus, different sets of VNTR loci may be useful for phylogenetic investigation of isolates evolving over different time scales. Phylogenetic investigations of spreading of N. meningitidis strains over a long time scale will best be carried out using loci with a low or moderate variation rate. Forensics and outbreak investigations may use loci with a higher variation rate. In our study, the MST grouping features built with 10 or 11 loci, which excluded one or two highly polymorphic loci, such as NMTR1, NMTR2 or both from 12 loci, remained similar but tighter to that with 12 loci (data not shown). Therefore, use of more VNTR loci with a lower variation rate will increase the power of MLVA in phylogenetic studies of N. meningitidis strains evolving over a long time scale.
The allelic profiles of the 11 serogroup Y isolates demonstrated the level of stability for the 12 VNTR loci. The comparison of the allelic profiles indicated that VNTR2 had the highest variation rate; five additional alleles at NMTR2, but only one at NMTR1, NMTR7 and NMTR9, evolved in the serogroup Y isolates over a 2-year time span. The stability of the VNTR loci was also demonstrated by the comparison of the allelic profiles of isolates from four patient-contact episodes. Although a single locus variant was observed in isolates from a patient-contact episode (Table 4), this MLVA method should be sta-ble enough for forensic and outbreak investigations. Since variation normally occurs in only a small portion of isolates from an outbreak [15], such variation is usually not a problem for interpretation of MLVA data.

Conclusion
MLVA exhibits a higher degree of resolution than PFGE for fine typing of N. meningitidis isolates and produces portable data that can easily be used for comparisons between laboratories via the Internet. MLVA data can also be used to investigate phylogenetic relationship between N. meningitidis strains. Therefore, MLVA can be adopted as an epidemiological tool for forensics and disease outbreak investigations, and for investigating clonal relationship among meningococcal strains. However, the mutation rate for each VNTR loci is still unknown. To fully exploit the value of MLVA, more VNTR loci need to be explored and more N. meningitidis isolates, of known epidemiological history, need to be characterized. , searches tandem repeat loci from one of the three genomic sequences and then locates the positions of each of the loci at the other two compared genomes. The three genomic sequences are used in turn as the "parent" sequence, so that a locus with only one repeat unit at a genome, but with two or more repeat units at other genomes, will not be missed. Searches found more than 300 repeat loci that were common to all the three strains and had variable repeat units between the three strains. Twenty-three repeat loci that had short repeat unit length (≤ 30 bp), more than 85% repeat sequence identity, and no indels were selected for further evaluation with 10 genetic distinct strains. Twelve loci, which were detected in all of the 10 testing isolates and amplified with only one amplicon, were chosen for genotyping of N. meningitidis isolates ( Table 1).

Preparation of crude bacterial DNA
Meningococcal isolates, stored at -70°C, were plated onto trytic soy agar with 5% sheep blood and incubated overnight at 37°C under a 5% CO 2 atmosphere. A loopful (10 µl) of bacterial growth was removed from the plate, suspended in 100 µl of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0) in an Eppendorf tube, and boiled for 10 min. After centrifugation at 3700 g for 10 min, the supernatant was transferred to a new tube and used for PCR amplification.

PCR amplification and analysis of VNTR regions
The primer sets specific to the 12 VNTR regions are listed on Before size analysis the fluorescent amplicons were diluted in water, usually at a 1:100 or 1:200 ratio, then separated by capillary electrophoresis on an ABI Prism 3130 Genetic Analyzer with GeneScan 500 LIZ Size Standard (cat # 4322682; Applied BioSystems). Data were collected and lengths of amplicons were determined with GeneScan Data Analysis Software, ver 3.7 (Applied Bio-Systems). All amplicons with different lengths from each locus were subjected to nucleotide sequence determination to verify the repeat sequence and the numbers of repeat units in the amplicons. The primers (without dye label) used for nucleotide sequence determination were the same as the primer sets used for PCR amplification. DNA sequencing was performed using the ABI Prism Big Dye Terminator cycle sequencing ready reaction kit and an ABI Prism 3130 Genetic Analyzer. The numbers of repeat units for the 12 VNTR loci (Table 1) and the predicted sizes of amplicons (Table 5) for the N. meningitidis strains Z2491, MC58 and FAM18 were taken as the standards to infer the number of repeat unit of each locus for the isolates tested.

Data analysis
The numbers of repeat units for each locus were saved as "Character Type" data in BioNumerics software (version 3.5; Applied Maths, Kortrijk, Belgium) and then subjected to cluster analysis using the Minimum Spanning Tree method. The polymorphism information index or Nei's diversity index (DI) was calculated for evaluating allele diversity as 1-Σ (allele frequency) 2 .

Bacterial strains
A total of 105 N. meningitidis isolates, collected from meningitis patients and healthy contacts, were included in this study. The collection from patients comprised 2 serogroup A isolates, 52 serogroup B isolates, 3 serogroup C isolates, 31 serogroup W135 isolates, 11 serogroup Y and 1 non-groupable isolate ( Table 2). They were collected from sporadic cases between 1996 and 2002 in Taiwan, except two pairs of isolates (NM255 and NM256; NM377 and NM378), which were, respectively, isolated from a meningococcal disease outbreak in a family and from two cases with a close temporal and spatial connection. All the 100 isolates from patients have been characterized by PFGE and MLST in a previous study by Chiou et al. [9]. Five isolates from healthy contacts were collected from four independent patient-contact episodes ( Table 4).

Authors' contributions
JC Liao designed all the primers and MLVA analyzed all the isolates. CC Li was in charge of searching potential VNTR loci by computer software and MST clustering analyses. CS Chiou initiated and managed the project, analyzed data, and wrote the report. All authors read and approved the final manuscript.