Cleavase Fragment Length Polymorphism Analysis of Neisseria meningitidis Basic Metabolic Genes

Abstract

Cleavase fragment length polymorphism (CFLP) is a subtyping system based on the property of the enzyme cleavase to recognize junctions between single- and double-stranded regions of DNA formed after denaturation and cooling. To assess the capacity of CFLP for discriminating Neisseria meningitidis serogroup B strains belonging to the electrophoretic type (ET) 5 (ET-5) complex from other serogroup B strains, 30 serogroup B N. meningitidis isolates were subtyped by CFLP with internal fragments of five housekeeping genes, adk, aspC, carA, dhp, and glnA. Two genes (glnA and carA) which demonstrated a high degree of diversity for the serogroup B isolates were then used to further evaluate the suitability of CFLP for screening 50 serogroup C N. meningitidis outbreak-associated and sporadic-case isolates with a single metabolic gene. The results were compared to those from multilocus enzyme electrophoresis (MEE), the current standard subtyping method. CFLP was able to distinguish the ET-5 complex isolates from other serogroup B isolates as efficiently as MEE. Furthermore, CFLP analysis of a single gene was sufficient to identify and cluster the serogroup C isolates belonging to the ET-37 complex from other, unrelated serogroup C isolates but was not capable of differentiating between the isolates of the major individual ETs of this complex (ET-17 and ET-24) causing most serogroup C meningococcal disease outbreaks in the United States. CFLP based on a single gene with a high degree of diversity but not under selective pressure can be applied to the rapid screening of a large number of isolates related to the recognized epidemic complex ET-5 or ET-37. Additionally, CFLP can be used as an initial screening tool to survey the amount of diversity in genes that might be used to develop a DNA sequence-based subtyping system.

Meningococcal disease remains an important public health problem in the United States and worldwide. Multilocus enzyme electrophoresis (MEE) is the “gold standard” method for Neisseria meningitidis subtyping, allowing the identification of transcontinental clonal complexes that have an increased propensity to cause epidemic disease (1, 3, 4, 12, 14, 15, 20). Although it has been successfully applied in many epidemiologic investigations, MEE is time-consuming, expensive, and subject to difficulties in data analysis and interlaboratory comparison. As a result, a system that uses multilocus sequence typing (MLST) of housekeeping genes has recently been developed for the characterization of N. meningitidis (13). One of the biggest advantages of MLST over MEE is the electronic portability of the nucleotide sequence data, enabling rapid global exchange of molecular typing information for epidemiologic comparisons. While the technology for nucleotide sequencing has substantially improved in the last few years, sequencing of multiple housekeeping genes remains time-consuming, even when carried out with an automated sequencer. Cleavase fragment length polymorphism (CFLP) is a subtyping system based on the single-stranded DNA patterns resulting from digestion with the enzyme cleavase, a structure-specific, thermostable nuclease (2). This enzyme recognizes and cleaves secondary structures that consist of double-stranded hairpin regions interspersed with single-stranded regions of DNA and that are formed after denaturation and cooling to an intermediate temperature, in a pattern unique to the nucleotide sequence. The objectives of this study were to evaluate CFLP as a means for rapidly identifying N. meningitidis isolates of two major epidemic-associated electrophoretic type (ET) complexes and to assess its possible usefulness as an initial screening survey for genes that might be used for DNA sequence-based subtyping.

MATERIALS AND METHODS

N. meningitidis strains.

Thirty N. meningitidis serogroup B and 50 serogroup C isolates were assayed by CFLP and MEE. The serogroup B isolates were 19 serotyping and serosubtyping reference strains (9, 10) and 11 sporadic-case isolates obtained through a population-based surveillance system for N. meningitidis that is part of a multistate population-based surveillance project coordinated by the Centers for Disease Control and Prevention (CDC) as part of the Active Bacterial Core Surveillance/Emerging Infections Program Network; 4 of these 11 isolates belonged to the ET-5 complex, and the remaining 7 were only distantly related to this complex. Seventeen of the serogroup C isolates were chosen to represent four epidemiologically defined outbreaks that occurred in California in 1993 (11, 18), New Mexico in 1994 Arizona in 1994 (11), and Georgia in 1998. Strains isolated from a series of cases that occurred in Massachusetts in 1998 were also analyzed. Four or five sporadic-case isolates were selected as controls for each outbreak by use of the available isolate with the closest temporal and geographic proximity. A total of six serogroup C isolates of six ETs closely related to the ETs of the outbreak-associated isolates and therefore within the ET-37 complex (4, 19) and five isolates of ETs only distantly related to this complex were also included. All strains were isolated from normally sterile body sites, such as blood or cerebrospinal fluid, except for the reference strain for serosubtyping, for which information was not available. Serogroup B and serogroup C strains and their selection criteria are listed in Tables 1 and 2, respectively.

TABLE 1.

N. meningitidis serogroup B isolates assayed by CFLP and MEE

Strain (other identification)	Geographic origin or referencesa	Date of collectionb	CFLP pattern					MEE assignment		Serotyped
			adk	aspC	carA	dhp	glnA	ETc	ET-5 complex
Reference
M1080	US	Not known	A	A	C	D	G	1045	No	B:1:P1.7,1
B16B6	US	Not known	B	B	D	B	L	1044	No	B:2a:P1.5,2
2996	The Netherlands	1974	B	D	G	A	J	1046	No	B:2b:P1.5,2
2396	The Netherlands	Not known	C	C	D	A	B	516	No	B:2c:P1.5,2
M981	US	Not known	C	D	H	J	M	1049	No	B:4,7:nt
385	Cuba	1983	C	D	A	F	B	304	Yes	B:4,7:P1.15
M992	US	Not known	C	D	D	D	J	1050	No	B:5:P1.7,1
M136	9	Not known	C	D	D	Nulle	J	1051	No	B:11:nt
S3442	9	Not known	A	E	C	Null	F	1047	No	B:14:P1.23,14
S3446	US	1973	A	E	C	Null	A	1052	No	B:14:P1.14
H44/76	Norway	1976	C	D	A	F	B	304	Yes	B:15:P1.7,16
H355	9	Not known	C	D	A	F	I	1041	Yes	B:15:P1.19,15
BB393	9	Not known	C	D	A	I	I	411	Yes	B:15:P1.3
6557	10	Not known	A	E	C	Null	G	1052	No	B:17,7:P1.23,14
M990	US	Not known	C	D	I	C	E	1048	No	B:NT:P1.6
882066 (Z4008)	The Netherlands	Not known	A	E	C	Null	H	1039	No	B:4,7:P1.4
M982	US	1960	C	D	I	C	E	1053	No	B:NT:P1.9
870227 (H276)	The Netherlands	Not known	C	D	A	F	G	615	Yes	B:4,7:P1.10
S3032	US	1972	C	D	D	E	K	1054	No	B:7:P1.12,16
Surveillance
M3782	Georgia	1992	C	D	D	C	B	1026	No	B:NT:P1.7,13
M1006	Oklahoma	1993	A	E	D	G	B	422	No	B:15:P1.5
M574	Maryland	1/12/94	A	E	E	D	A	403	No	B:4:P1.7,1
M736	Oregon	2/27/94	C	D	A	F	B	301	Yes	B:15:P1.7,16
M1010	Oklahoma	1/6/94	A	E	B	A	A	426	No	B:10:nt
M1019	California	6/20/94	C	D	A	F	B	448	Yes	B:4:P1.7,16
M1072	Oregon	1994	C	F	F	A	D	409	No	B:NT:P1.23,14
M1254	Washington	1994	C	D	A	F	B	301	Yes	ND
M1417	California	2/3/95	C	D	A	F	B	304	Yes	B:15:P1.7,16
M1985	Missouri	8/26/95	A	D	C	H	C	566	No	B:4,7:P1.7,13
M1987	Missouri	8/25/95	A	D	C	H	C	566	No	B:4,7:P1.7,13

^aUS, United States. 

^bMonth/day/year, unless otherwise indicated. 

^cET number. The specific enzyme profiles (reactions) for each ET are available upon request. 

^dSerogroup:serotype:serosubtype, when known, are included for comparison. NT, nonserotypeable; nt, nonserosubtypeable. ND, not determined. 

^eNull, no PCR product. 

TABLE 2.

N. meningitidis serogroup C isolates assayed by CFLP and MEE

Strain	Geographic origin	Date of collectiona	Epidemiologic description and propertiesb	CFLP patternc		MEE assignment		Serotypee
				glnA	carA	ETd	ET-37 complex
M140	California	2/14/93	CA outbreak related	24	24	24	Yes	C:2a:P1.14
M141	California	2/18/93	CA outbreak related	24	24	24	Yes	C:2a:P1.14
M145	California	1/1/93	CA outbreak related	24	24	24	Yes	C:2a:P1.14
M146	California	2/8/93	CA outbreak related	24	24	24	Yes	C:2a:P1.14
M149	California	2/20/93	CA outbreak related	24	24	24	Yes	C:2a:P1.14
M62	California	7/13/93	Sporadic case	24	24	13	Yes	C:2a:P1.5,2
M201	California	9/14/93	Sporadic case	24	24	24	Yes	C:2a:nt
M239	California	9/22/93	Sporadic case	195	195	195	Yes	C:NT:P1.5,2
M746	California	4/8/94	Sporadic case	24	24	17	Yes	C:2a:P1.5
M941	California	6/1/94	Sporadic case	24	24	24	Yes	C:2a:P1.14
M630	Arizona	2/24/94	AZ outbreak related	24	24	17	Yes	C:2a:P1.5,2
M631	Arizona	2/22/94	AZ outbreak related	24	24	138	Yes	C:2a:P1.5,2
M632	Arizona	2/19/94	AZ outbreak related	24	24	17	Yes	C:2a:P1.5,2
M633	Arizona	2/24/94	AZ outbreak related	24	24	17	Yes	C:2a:P1.5,2
M634	Arizona	2/22/94	AZ outbreak related	24	24	226	Yes	C:2a:P1.5,2
M160	Arizona	3/26/94	Sporadic case	24	24	24	Yes	C:2a:nt
M433	Arizona	1994	Sporadic case	24	24	226	Yes	C:2a:P1.5,2
M615	Georgia	2/25/94	Sporadic case	24	24	24	Yes	C:2a:nt
M626	New York	2/20/94	Sporadic case	24	24	24	Yes	C:2a:P1.5,2
M4619	Georgia	2/28/98	GA outbreak related	24	24	203	Yes	C:2a:nt
M4642	Georgia	4/2/98	GA outbreak related	24	24	203	Yes	C:2a:P1.2
M1532	New Mexico	4/29/94	NM outbreak related	147	147	147	No	C:NT:nt
M1533	New Mexico	12/29/94	NM outbreak related	24	24	17	Yes	C:2a:P1.5,2
M1534	New Mexico	3/8/95	NM outbreak related	24	24	17	Yes	C:2a:P1.5,2
M1535	New Mexico	3/7/95	NM outbreak related	24	24	17	Yes	C:2a:P1.5,2
M1536	New Mexico	3/17/95	NM outbreak related	24	24	17	Yes	C:2a:P1.5,2
M393	Utah	12/19/93	Sporadic case	50	50	50	Yes	C:2a:P1.5,2
M993	Oklahoma	12/3/93	Sporadic case	24	24	24	Yes	C:2a:P1.5,2
M1177	California	11/13/94	Sporadic case	24	24	196	Yes	C:2a:P1.5,2
M1252	Illinois	11/22/94	Sporadic case	24	24	17	Yes	C:2a:P1.5,2
M1291	Colorado	12/22/94	Sporadic case	24	24	141	Yes	C:2a:P1.5,2
M5120	Massachusetts	5/4/98	MA cluster	24	24	233	Yes	C:2a:P1.5,2
M5119	Massachusetts	5/6/98	MA cluster	24	24	233	Yes	C:2a:P1.5,2
M5118	Massachusetts	6/26/98	MA cluster	24	24	233	Yes	C:2a:nt
M2654	Massachusetts	2/96	Sporadic case	24	24	24	Yes	C:2a:nt
M2655	Massachusetts	2/96	Sporadic case	24	24	24	Yes	C:2a:nt
M4594	Massachusetts	12/12/97	Sporadic case	203	24	203	Yes	C:2a:P1.23,14
M4695	Massachusetts	4/1/98	Sporadic case	24	24	24	Yes	C:2a:P1.5,2
M4698	Massachusetts	1/30/98	Sporadic case	24	24	24	Yes	C:2a:P1.5,2
M80	Georgia	10/30/89	ET-37 complex	24	24	33	Yes	C:2a:P1.5,2
M3761	Tennessee	1992	ET-37 complex	24	24	78	Yes	C:2a:P1.2
M3733	California	1992	ET-37 complex	18	24	18	Yes	C:2a:P1.5,2
M1352	New York	1/5/95	ET-37 complex	24	24	132	Yes	C:2a:P1.5,2
M2752	California	5/14/96	ET-37 complex	24	24	163	Yes	C:2a:P1.5,2
M2964	California	8/3/96	ET-37 complex	201	24	201	Yes	C:2a:P1.14
M210	Tennessee	8/16/93	Not ET-37 complex	55	55	55	No	C:4,7:nt
M1096	Tennessee	6/9/94	Not ET-37 complex	56	56	56	No	C:NT:nt
M420	Georgia	1/4/94	Not ET-37 complex	56	56	127	No	C:NT:P1.7,13
M1560	Georgia	3/4/95	Not ET-37 complex	197	197	197	No	C:21:P1.5,2
M1569	Georgia	3/26/95	Not ET-37 complex	698	698	698	No	C:2a:P1.5,2

^aMonth/day/year unless otherwise indicated. 

^bGroups of strains are arranged as a particular outbreak and the controls for that outbreak. Outbreak related as defined in reference 6. ET-37 complex, strains of ETs representing the ET-37 complex. Not ET-37 complex, strains of ETs different from the ET-37 complex. 

^cCFLP patterns for the glnA and carA genes are designated the same as the ET. 

^dET number. The specific enzyme profiles (reactions) for each ET are available upon request. 

^eSerogroup:serotype:serosubtype are included for comparison. NT, nonserotypeable; nt, nonserosubtypeable. 

CFLP analysis.

The bacteria were incubated on blood agar plates with 5% sheep blood (BBL Microbiology Systems, Cockeysville, Md.) overnight at 35°C in a 5% CO₂ atmosphere. Whole-cell suspensions in 0.01 M Tris buffer (pH 8.0) were boiled for 10 min and used as a template for PCR amplification. PCR products were derived from five target genes: adenylate kinase (adk), aspartate transaminase (aspC), carbamoylphosphate synthetase (carA), dihydropteroate synthase (dhp), and glutamine synthetase (glnA). The criteria for gene selection included enzymes that are used in MEE and that have a high degree of diversity and availability of sequences in GenBank. The strands were labeled with 6-carboxyfluorescein (FAM) or 4,7,2′,7′-tetrachloro-6-carboxyfluorescein (TET) fluorescence dye (Glen Research, Sterling, Va.) and purified with a Qiaquick 8 PCR Purification Kit (Qiagen Inc., Valencia, Calif.). Approximately 100 fmol of the labeled DNA substrate was denatured at 95°C for 2 min, cooled to a suitable collapse temperature, and digested with 25 U of the structure-specific cleavase I enzyme provided in the CFLP Evaluation Kit (Third Wave Inc., Madison, Wis.). The manufacturer’s instructions were followed, except for the use of 95% formamide instead of the stop solution to terminate the reaction because of the background caused by the dye present in that solution. The cleavase reaction optimization of time and temperature was performed at temperatures ranging from 50 to 65°C and times ranging from 2 to 6 min as previously described (2). The labeled primer sequences, annealing temperatures for PCR amplification, DNA product sizes, and CFLP digestion conditions are described in Table 3.

TABLE 3.

Primer sequences and CFLP conditions for doubly fluorescence-labeled PCR products from target genes adk, aspC, carA, dhp, and glnA

Gene	Fluorescence-labeled primers (sequences)a	PCR annealing temp (°C)	PCR product (bp)	CFLP digestion conditions (°C/min)
adk	TET (AC TTC GGT TTG CTC GTG GTA)	68	479	58/2
	FAM (GG CAA AGG CAC TCA GGC G)
aspC	TET (AA CCC WAC CGG TAT CGA CCC TAC GC)	58	470	55/2
	FAM (GT CAA GCC GCT GAA AGA GAA CAT GCC)
carA	TET (GA GTG GAT GCC CGC CTG TTC)	65	919	62/4
	FAM (AT TTG GAT TAC GCC GCC CTA C)
dhp	TET (TG CTA CTT CAC CGA CAA CAT)	65	400	65/4
	FAM (TC TCA CGC CCG ATT CTT TKT)
glnA	TET (CG TAC AGG TTT TTA TCG GCA GG)	58	1,104	60/1
	FAM (AA GGC AAG CAG CAC CAC TTT AC)

^aW, equal mixture of A and T; K, equal mixture of G and T. 

After cleavage, 6 μl of each reaction sample was mixed with 1 μl of the internal lane standard Genescan-2500 TAMRA (Perkin Elmer/Applied Biosystems, Foster City, Calif.), and the mixture was dried under vacuum. The samples were resuspended in 4 μl of deionized formamide and denatured by being heated at 92°C for 2 min. The labeled DNA fragments were resolved on a 6% acrylamide–8 M urea gel in Tris-borate buffer for 7 h at 1,500 V by use of a model 373 automated DNA sequencer and 672 GeneScan software (Perkin Elmer/Applied Biosystems). Fingerprint patterns were determined by visual comparison of the electropherograms generated by the 672 GeneScan software. Slightly different CFLP patterns were observed when PCR products were purified with a PCR Clean Up Kit from Boehringer Mannheim Biochemicals (data not shown); therefore, this kit was not used in this study. For the 30 serogroup B isolates, five genes (adk, aspC, carA, dhp, and glnA) were used to validate the capability of CFLP to discriminate the ET-5 complex strains from other serogroup B strains. The 50 serogroup C isolates were assayed by CFLP with two genes not only to evaluate this method for rapidly discriminating the ET-37 complex strains from other ET strains but also to estimate its potential for differentiating outbreak-associated from non-outbreak-associated strains. The genes glnA and carA were used for this purpose because of their high degree of CFLP diversity for the serogroup B strains.

MEE analysis.

All strains were subtyped by MEE; 24 enzymes were used with methodology described previously (16). In order to compare CFLP to MEE, the genetic relatedness of the serogroup B strains was analyzed by use of the respective dendrograms generated with PHYLIP software (8).

RESULTS

CFLP analysis of five metabolic genes in serogroup B strains.

Among the 30 serogroup B isolates, we found 13 CFLP patterns or types for glnA, 10 for dhp, 9 for carA, 6 for aspC, and 3 for adk. By MEE, 25 distinct ETs were identified. More variability for the adk gene was identified by CFLP than by MEE, since three types were observed by CFLP and no diversity was observed for this locus by MEE. Similarly, nine CFLP types and five MEE types were observed for carA. For aspC, 6 CFLP types and 10 MEE types were observed with all combinations of the two MEE bands. The genetic relatedness of the strains analyzed by CFLP and MEE is illustrated by the dendrograms shown in Fig. 1. The majority of strains were grouped into similar clusters by both CFLP and MEE analyses. The nine ET-5 complex strains showed almost identical clustering patterns in both methods. Relationships of some strains were not the same in CFLP and MEE analyses. For example, the serological reference strains B16B6 and 2996 were found closely related by MEE but were found different by CFLP.

FIG. 1.

Dendrograms generated from CFLP and MEE subtyping of the 30 N. meningitidis serogroup B isolates. The group of nine closely related strains of the ET-5 complex is clustered together by both methods (box). The bar indicates genetic distance for both CFLP and MEE.

CFLP analysis of single metabolic genes in serogroup C strains.

With a single exception (strain M1532), all of the outbreak-related strains from California, New Mexico, Arizona, and Georgia and three strains from a cluster in Massachusetts showed the same glnA and carA CFLP patterns. Strain M1532, which was isolated in New Mexico and which displayed a unique pattern for both genes, was characterized as ET-147 (which differs from ET-24 in 17 of 24 enzymes), while the ETs of all other outbreak-related strains were members of the ET-37 complex (Table 2). Only 1 of 19 sporadic-case isolates (M239) was identified to be of an ET not closely related to the ET-37 complex (Table 2), and this isolate could be differentiated from the outbreak-associated strains by both glnA and carA. Two additional sporadic-case isolates (M393 and M4594) could be differentiated from the outbreak-associated strains by glnA. Isolate M393 also had a distinct carA pattern. Figure 2 shows glnA and carA CFLP patterns of an outbreak-related strain (M140) and a sporadic-case isolate (M239) from Los Angeles County, Calif. Analysis of N. meningitidis serogroup C isolates revealed that the glnA gene was more diverse than the carA gene, as was observed for the serogroup B isolates. No differences were observed for the carA gene among the six isolates of ETs closely related to the ET-37 complex (Table 2). Only two of those isolates (M2964 and M3733) showed minor differences in glnA patterns. In contrast, the five isolates of ETs distantly related to the ET-37 complex (Table 2) exhibited unique patterns for both genes, confirming the screening capacity of CFLP with a single metabolic gene in differentiating ET-37 complex strains from strains of other ETs.

FIG. 2.

Electropherograms generated by the 672 GeneScan software showing the CFLP patterns of serogroup C isolates M140 (an outbreak-associated ET-24 isolate) and M239 (a sporadic-case isolate not related to the ET-37 complex) from California. The two upper electropherograms are cleaved single-stranded PCR products derived from the glnA gene, and the two lower electropherograms were derived from the carA gene. The x axis shows the scan number, and the y axis shows relative fluorescence.

DISCUSSION

In this study, CFLP and MEE were compared to investigate the capacity of CFLP to discriminate meningococci belonging to the ET-5 complex, which have been responsible for many outbreaks in Europe, South Africa, Central America, and South America (3, 4, 15) and, more recently the United States (5, 14, 20), from other serogroup B strains. All strains of the ET-5 complex were clearly distinguishable from other serogroup B strains by both CFLP and MEE. CFLP detected more variation than MEE for some genes, resulting in more alleles per locus, similar to the results obtained by MLST analysis (13). The number of CFLP patterns obtained generally correlated with the relative amount of DNA sequence diversity observed for the genes used. For instance, meningococcal adk gene sequences may differ at ∼1% of nucleotide sites (7); conversely, the glnA gene has been found to be unusually variable, with diversity observed at 4.4% of nucleotide sites (17, 21). The congruence between CFLP and MEE was excellent for most of the strains tested, but some differences were found, as also reported by Maiden et al. (13) when comparing MLST and MEE. The reasons for these differences are not known, nor are the actual relationships of the strains. Because the two methods examine different properties of strains, it is possible that both typing schemes are correct. More importantly, the sensitivity of the CFLP method for differentiating the ET-5 complex strains is equivalent to that of the standard MEE method.

The usefulness of CFLP in rapidly screening serogroup C isolates of the ET-37 complex (1, 4, 11, 12, 18, 19) was also evaluated. CFLP analysis of a single metabolic gene could consistently differentiate strains belonging to the ET-37 complex from serogroup C strains unrelated to this complex. However, it was of particular epidemiologic significance to determine if ET-17 and ET-24 could be differentiated from other ETs of this complex and from each other, as these ETs are the most frequently identified ETs of the ET-37 complex and are also most frequently associated with serogroup C meningococcal disease outbreaks in the United States. Although ET-17 and ET-24 differ from each other for only 3 of 24 enzymes in MEE, the outbreaks that they cause have always been epidemiologically distinct. No outbreaks in which both of these ETs were simultaneously detected have been reported. The CFLP patterns of strains of ET-17 and ET-24 were indistinguishable by use of both glnA and carA genes, suggesting that perhaps the selection and evaluation of another gene(s) may be needed in this particular situation; possible genes are those encoding the peptidase, phosphoenolpyruvate carboxykinase, or fumarase enzymes that differentiate ET-17 and ET-24 in MEE. A weakness of MEE analysis results from the fact that ET-17 and ET-24 are also frequently identified in strains isolated from sporadic cases through population-based epidemiologic surveillance; therefore, it is impossible to distinguish outbreak-associated and sporadic-case isolates of these ETs. This is also generally true for strains of other ETs of this complex, as specific ETs are identified in both outbreak-associated and sporadic-case isolates. It is not completely unexpected for CFLP based on a single gene not to be able to differentiate outbreak-associated from sporadic-case isolates of the ET-37 complex, especially because of the large number of loci examined by MEE (24 in our study). Further CFLP evaluation of genes used in the MEE panel may result in the identification of a gene(s) capable of providing this differentiation.

CFLP has the potential to be widely used for initial screening because, unlike MEE, CFLP can easily and rapidly screen a large number of strains. With the model 377 automated sequencer, 96 strains can be assayed for a single gene within 48 h. Additionally, CFLP analysis is a DNA-based subtyping method with the capacity for direct assignment of alleles based on the nucleotide sequences of genes. CFLP can be applied to the rapid screening of a large number of strains during investigations of outbreaks and/or surveillance systems. CFLP is as efficient as and more rapid than MEE in identifying strains of the ET-5 and ET-37 complexes. Because of the sensitivity in detecting nucleotide changes (2), CFLP based on a single metabolic gene with a high degree of diversity but not under selective pressure may be used to generate an appropriate level of discrimination among isolates. As clearly demonstrated in this study, CFLP not only can rapidly screen for the major epidemic clones of serogroups B and C, e.g., clonal complexes ET-5 and ET-37 but also, more importantly, can provide guidance regarding the choice of genes for the DNA sequence-based approach to the molecular subtyping of N. meningitidis. The development of such a subtyping system with a level of discrimination comparable to or surpassing that achieved by the current gold standard, MEE, will enable rapid global exchange of data and comparison of molecular subtyping information for both research and epidemiologic purposes. The use of CFLP as a screening tool to develop a DNA sequence-based subtyping method will greatly simplify the creation of a reliable and reproducible database for N. meningitidis or other microorganisms.