Abstract
Forty-two Neisseria meningitidis isolates were obtained from patients with meningococcal disease in the Norwegian county of Telemark (January 1987 to March 1995), and all were compared by PCR amplicon restriction endonuclease analysis (PCR-AREA) of the dhps gene, chromosomal DNA fingerprinting, and serological analysis. PCR-AREA divided the isolates into 11 classes, of which 4, comprising 15, 8, 6, and 2 isolates, were clonal while the remaining 8 classes were genetically heterogeneous or contained only 1 isolate. Three of the four clonal classes could be tentatively equated with recognized epidemic clones (ET5, ET37, and cluster A4) on the basis of their phenotypic characteristics, while the remaining clone appears to be new. There were significant differences in the geographical distribution of clones, with class 1 (ET5-like) isolates significantly overrepresented in rural parts of Telemark. Class 1 (ET5-like) isolates occurred throughout the study period and were dominant in 1987. Class 2 (ET37-like) isolates occurred from 1988 to 1992, and class 3 isolates (with no recognizable ET affinities) were found only in 1991 and 1992.
Meningococcal disease occurs in the form of sporadic cases, local outbreaks, and epidemics and has a high mortality. Much attention has been paid to methods for typing meningococcal strains in order to trace and control outbreaks and elucidate the global epidemiology of the bacterium, and this has generated a considerable variety of typing methods. The “gold standard” method is isoenzyme electrophoresis (ET typing) (16). This method, which compares electrophoretic polymorphisms in multiple enzymes, defines epidemic clones of Neisseria meningitidis, provides an estimate of genetic similarity, and has provided the basis for inference of the genetic population structure of N. meningitidis. In the clinical setting, a typing system based on serogrouping (capsular polysaccharides), serotyping and subtyping (class 1, 2, and 3 outer membrane proteins), and sulfonamide resistance is more frequently used (6). Although this method may be a useful guide to the clonal affinities of meningococcal strains when applied locally, on a global basis serogroup and, to a lesser extent, serotype are poorly correlated with genetic relatedness (3). More recently, genetic methods, such as whole-genome DNA fingerprinting (12, 18), random amplification of polymorphic DNA (21), repetitive element-based PCR (20), and restriction fragment length polymorphism (RFLP) analysis of PCR products (7, 10), have been successfully applied in epidemiological studies of N. meningitidis. Whole-genome DNA fingerprinting, though useful for tracing outbreak strains, is unsuitable for classification because the restriction pattern is too complex, but other DNA-based methods that generate simpler patterns are more promising.
We have previously described a method (PCR amplicon restriction endonuclease analysis [PCR-AREA]) for PCR-based RFLP analysis of the highly polymorphic chromosomal dhps gene, which determines resistance or sensitivity to sulfonamides in N. meningitidis (10). To evaluate PCR-AREA as a classification method, and in order to study the clonal distribution of meningococcal strains in the Norwegian county of Telemark, we have used PCR-AREA, in combination with DNA fingerprinting and serogroup, serotype and subtype, and sulfonamide resistance determinations, to compare 42 strains isolated from patients with meningococcal disease. This represents all primary cases of meningococcal disease in Telemark from January 1987 to March 1995.
MATERIALS AND METHODS
Samples.
Meningococci were isolated from cerebrospinal fluid (CSF) (16 cases), blood cultures (23 cases), or throat and nasal swabs (3 cases) of 42 patients with invasive meningococcal disease. For the three patients where meningococci were isolated from throat samples, the following criteria led to the diagnosis of invasive meningococcal disease. Patient 1 had petechial bleeding on the arms, legs, back, and face, fever, a high sedimentation rate and C-reactive protein level, normal blood pressure and peripheral circulation, no neck stiffness, and a diagnosis of benign meningococcemia. Patient 5 was admitted to the hospital with symptoms of fulminant meningococcal septicemia (unconsciousness, petechiae, and low blood pressure) and was diagnosed with fulminant meningococcal septicemia. Patient 25 had fever, petechial bleeding, vomiting, poor peripheral circulation, a stiff neck, high C-reactive protein, 6800 cells/ml in the CSF, and a diagnosis of meningococcal meningitis and septicemia.
Throat samples were spread on GC selective medium (9); blood cultures and CSF were spread on chocolate agar. Suspect colonies appearing after 16 h of incubation at 37°C in a 10% CO2 atmosphere were subcultivated on two chocolate agar plates. The first plate was spread to single colonies and used for control of purity; for confirmation of the isolate as N. meningitidis by the oxidase test and the test for the fermentation pattern of glucose, lactose, maltose, and sucrose; for serogrouping; and as a source of innoculum for storage (−70°C). The second plate was spread to confluence and used for extraction of DNA.
Isolates 3 to 42 were collected in the course of the Telemark Meningococcal Project (9, 11). Isolates 1 and 2 represent the two primary cases immediately preceding the start of the project. All of the strains were from isolated primary cases, with the exception of isolate 2, which was the first of two associated cases, and isolates 35-1 and 35-2, which were coprimary cases occurring in the same family. The isolates are numbered consecutively by outbreak number.
Extraction of DNA for PCR and chromosomal DNA fingerprinting.
DNA was purified by lysozyme–EDTA–Triton X-100 lysis and phenol-chloroform extraction as previously described (12).
PCR.
A 634-bp segment of the chromosomal dhps gene was amplified with the primers NM6 (CGC CAT CAA TTC GGG CAA ATG; nucleotides 711 to 734) and NM7 (TTG GCA GGC AGG GTT TGA; nucleotides 1324 to 1344) (4). The 100-μl PCR mixtures contained 200 ng of purified N. meningitidis DNA, 500 ng of each primer, 1.5 U of Taq polymerase (Promega, Madison, Wis.), 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, 0.2 mM deoxynucleoside triphosphates, and 1.5 mM MgCl2. Reactions were run on an Omnigene thermal cycler (Hybaid, Teddington, United Kingdom) with the following program: 94°C, 60 s; 55°C, 60 s; 72°C, 60 s; 40 cycles.
PCR-AREA.
PCR product (20 μl) was digested with 30 U of CfoI (Promega, Madison, Wis.) in a volume of 25 μl for 1 h at 37°C. The restriction fragments were separated by electrophoresis for 5 h at 400 V on an 8% polyacrylamide gel and visualized by ethidium bromide staining and UV transillumination (10).
DNA fingerprinting.
Purified N. meningitidis DNA (25 μg) was digested with HindIII, and the fragments were separated on 4% polyacrylamide gels and visualized by ethidium bromide staining and UV transillumination as previously described (12).
Serotyping.
The serotype and subtype were determined by dot blot analysis as described by Wedege et al. (19), except that signal detection was done with 3-amino-9-ethyl carbazole and hydrogen peroxide and the strips were photocopied after development.
Serogrouping.
Serogrouping was performed by slide agglutination with N. meningitidis agglutination sera for serogroups A, B, C, Y, and W135 from Murex Diagnostics (Dartford, United Kingdom) according to the manufacturer’s specifications.
Sulfonamide resistance determination.
The MIC of sulfonamide was determined by an agar dilution method. A suspension of one or two freshly grown N. meningitidis colonies was adjusted to a turbidity of 0.5 McFarland units by visual reference to a standard. Twenty-five-microliter samples of the suspension were innoculated onto agar plates containing 2× serial dilutions of sulfathiazole from 2,000 to 1 mg/liter in NM medium. The plates were incubated for 24 h at 37°C in a 10% CO2 atmosphere. NM medium is an enriched variant of HTM medium (8) prepared as follows: 38 g of Mueller-Hinton medium and 5 g of yeast extract (Mast, Merseyside, United Kingdom) were dissolved in 1 liter of distilled water and autoclaved at 121°C. The medium was cooled to 48°C, and 10 ml of 27% glucose, 15 ml of 0.1% hematin, and 1 ml of solution E4 [0.2% cocarboxylase (Sigma catalog no., C-8754), 0.4% NAD, 10% l-glutamine, 26% l-cysteine, and 0.5% Fe(NO3)3 · 9H2O] were added. After the addition of sulfathiazole, the medium was dispensed into petri dishes in 25-ml aliquots. Isolates were classified as resistant, intermediate, or sensitive according to the following system: isolates for which the MICs were ≤16 mg/liter were considered sensitive; isolates for which the MICs were ≥31 but ≤62 mg/liter were considered intermediate; and isolates for which the MICs were ≥250 mg/liter were considered resistant.
Statistical methods.
Statistical analyses were performed with the chi-square test or with Fisher’s exact test where small numbers precluded use of the chi-square test.
RESULTS
Clonal analysis by PCR-AREA and DNA fingerprinting.
PCR-AREA of the 42 isolates revealed 11 different CfoI restriction patterns (classes 1 to 11) for the 634-bp NM6-NM7 PCR product (Fig. 1). Within gels, pattern comparison could be achieved quickly and unambiguously (Fig. 2). A tentative class assignment could be achieved by comparing gels, but final assignments were always made by comparison of lanes on a single gel. Fifteen isolates belonged to class 1, eight isolates belonged to class 2, six isolates belonged to class 3, three isolates belonged to class 6, and two isolates belonged to each of classes 4, 5, and 8. Classes 7, 9, 10, and 11 contained only one isolate each.
FIG. 1.
PCR-AREA of N. meningitidis isolates from Telemark showing the 11 different CfoI digestion patterns. Lane numbers correspond to the class number. The size standards (Std) are U.S. Biochemicals PCR molecular size markers, with fragment sizes of 1,000, 700, 525, 500, 400, 300, 200, 100, and 50 bp.
FIG. 2.
Typical PCR-AREA gel showing classes 1, 2, 4, and 5. The lanes are numbered according to the class. The standard (St) is a mixture of PCR products and restriction fragments. Fragment sizes are 645, 217, 100, 83, 74, and 62 bp.
Isolates within each PCR-AREA class were compared by chromosomal DNA fingerprinting. The results of DNA fingerprinting were classified according to the following system: unrelated patterns (differing by at least 7 bands but more typically by >20 bands) were each given a letter code (a, b, c, etc.), while groups of related patterns (differing by no more than 5 bands) were given a single common letter code subdivided by a number code (a1, a2, a3, etc.). Within a subdivision (e.g., a1) all of the strains displayed identical DNA fingerprints. The results are presented in Table 1.
TABLE 1.
Properties of isolates in the 11 PCR-AREA classes
| Class | Isolate no. | Serogroup | Serotype | Sulfonamide resistance (MIC)a | Yr of isolation | DNA fingerprint |
|---|---|---|---|---|---|---|
| 1 | 1 | B | 15:P1.7,16 | R (1,000) | 1987 | a1 |
| 2 | B | 15:P1.7,16 | R (1,000) | 1987 | a2 | |
| 3 | B | 15:P1.7,16 | R (1,000) | 1987 | a3 | |
| 4 | B | 15:P1.7,16 | R (1,000) | 1987 | a4 | |
| 5b | B | Not tested | R | 1987 | a5 | |
| 8 | B | 15:P1.7,16 | R (1,000) | 1988 | a1 | |
| 10 | B | 15:P1.7,16 | R (1,000) | 1988 | a1 | |
| 12 | B | 15:P1.7,16 | R (1,000) | 1988 | a6 | |
| 26 | B | 15:P1.7,16 | R (1,000) | 1991 | a7 | |
| 30 | B | 15:P1.2,5 | R (500) | 1992 | a8 | |
| 32 | B | 1,19:P1.7,16 | R (1,000) | 1992 | a9 | |
| 34 | B | 1,19:P1.7,16 | R (1,000) | 1992 | a9 | |
| 36 | B | 15:P1.7,16 | R (1,000) | 1993 | a10 | |
| 37 | B | 15:P1.12,13,13a | R (1,000) | 1993 | a11 | |
| 40 | B | 15:P1.7,16 | R (1,000) | 1995 | a12 | |
| 2 | 7 | C | 2a:P1.2,5 | R (1,000) | 1988 | b1 |
| 9 | C | 2a:P1.2,5 | R (1,000) | 1988 | b1 | |
| 11 | C | 2a:P1.2,5 | R (1,000) | 1988 | b2 | |
| 15 | C | 2a:P1.2,5 | R (1,000) | 1989 | b3 | |
| 16 | C | 2a:P1.2,5 | R (1,000) | 1990 | b4 | |
| 17 | C | 2a:P1.2,5 | R (1,000) | 1990 | b5 | |
| 27 | C | 2a:P1.2,5 | R (1,000) | 1991 | b6 | |
| 33 | C | 2a:P1.2,5 | R (1,000) | 1992 | b6 | |
| 3 | 22 | B | 16:- | S (8) | 1991 | c1 |
| 24 | B | 16:P1.2,5 | S (4) | 1991 | c2 | |
| 25 | B | 16:P1.2,5 | S (8) | 1991 | c1 | |
| 28 | B | 16:- | S (4) | 1991 | c3 | |
| 35-1 | B | 16:P1.2,5 | S (8) | 1992 | c2 | |
| 35-2 | B | 16:P1.2,5 | S (8) | 1992 | c2 | |
| 4 | 13 | B | 4:P1.9 | I (31) | 1988 | d |
| 39 | B | NT:P1,16 | I (31) | 1994 | e | |
| 5 | 14 | Y | 14,19:P1.16 | S (8) | 1989 | f |
| 38 | Y | 14,19:P1.2,5,6 | I (31) | 1994 | g | |
| 6 | 20 | W135 | 16:P1.7 | I (31) | 1990 | h |
| 23 | C | 21:P1.5 | S (16) | 1991 | i | |
| 31 | B | NT:P1.13,13a | S (8) | 1992 | j | |
| 7 | 6 | B | 8,19:P1.1,7 | I (31) | 1988 | |
| 8 | 18 | C | 2b:P1.2,5 | R (250) | 1990 | k1 |
| 19 | C | 2b:P1.2,5 | R (250) | 1990 | k2 | |
| 9 | 21 | B | 4:P1,16 | I (31) | 1990 | |
| 10 | 29 | B | 4:P1.14,15 | I (62) | 1992 | |
| 11 | 41 | B | 4:- | S (8) | 1995 |
R, resistant; I, intermediate; S, sensitive. MICs are in milligrams per liter.
Isolate not viable. The serogroup and sulfonamide resistance were based on laboratory results obtained at the time of isolation. The DNA data were based on a stored DNA sample.
Phenotypic analyses.
Isolates were further classified by serogroup, serotype, and sulfonamide resistance. Twenty-eight isolates belonged to serogroup B, 11 isolates belonged to serogroup C, 2 isolates belonged to serogroup Y, and 1 isolate belonged to serogroup W135. The serogroups, serotypes, and subtypes are summarized in Table 2. Twenty-six isolates were sulfonamide resistant, nine isolates were sulfonamide sensitive, and seven isolates had an intermediate level of resistance. There were several clear correlations of phenotypic characteristics. All serogroup B isolates that were either serotype 15 or subtype P1.7,16 (14 isolates) were also sulfonamide resistant. All six B:16 isolates were sulfonamide sensitive. Ten of 11 serogroup C isolates were serotype 2a or 2b, subtype P1.2,5, and sulfonamide resistant.
TABLE 2.
Serogroups, serotypes, and subtypes of N. meningitidis strains from Telemark
| Serogroup | Serotype (no. of isolates) | Subtype (no. of isolates) |
|---|---|---|
| B (n = 28) | 4 (4) | P1.9 (1) |
| P1.16 (1) | ||
| P1.14,15 (1) | ||
| NSTc (1) | ||
| 15 (12) | P1.7,16 (10) | |
| P1.2,5 (1) | ||
| P1.12,13,13a (1) | ||
| 16 (6) | NST (2) | |
| P1.2,5 (4) | ||
| 1,19 (2) | P1.7,16 (2) | |
| 8,19 (1) | P1.7,16 (1) | |
| NTa (2) | P1.13,13a (1) | |
| P1.16 (1) | ||
| NDb (1) | ND (1) | |
| C (n = 11) | 21 (1) | P1.5 (1) |
| 2a (8) | P1.2,5 (8) | |
| 2b (2) | P1.2,5 (2) | |
| Y (n = 2) | 14 (2) | P1.6 (1) |
| P1.2,5,6 (1) | ||
| W135 (n = 1) | 16 (1) | P1.7 (1) |
NT, nontypeable.
ND, not determined.
NST, nonsubtypeable.
The distribution of phenotypes by PCR-AREA class is shown in Table 3. PCR-AREA classes 1, 2, 3, and 8 were highly homogeneous with respect to their phenotypic characteristics and DNA fingerprint patterns. The 15 class 1 isolates were all in serogroup B, had high-level sulfonamide resistance, and were dominated by strains of serotype 15 (12 isolates) and of subtype P1.7,16 (12 isolates). DNA fingerprinting of class 1 isolates revealed 12 related DNA fingerprinting patterns (a1 to a12). Isolates 1, 8, and 10 and isolates 34 and 36 were identical by all criteria (Table 4). The eight class 2 isolates were all in serogroup C, serotype 2a:P1.2,5, and were resistant to sulfonamide. Six related fingerprint patterns (b1 to b6) could be identified. Two pairs of isolates identical by all criteria (Table 4) were found. The six class 3 isolates were all in serogroup B, serotype 16, and were sensitive to sulfonamide. Three related fingerprint patterns were found (c1 to c3), and three isolates were identical by all criteria (Table 4). The two isolates of class 8 were both C:2b:P1.2,5. They were resistant to sulfonamide, but the MIC for them was lower than those for class 1 and 2 isolates. They had related, but not identical, DNA fingerprints. We conclude that strains of these four classes are clonally related.
TABLE 3.
Distribution of phenotypes by PCR-AREA classa
| Class (no. of isolates) | Phenotypeb | No. of isolates |
|---|---|---|
| 1 (15) | B:15:P1.7,16:R | 10 |
| B:1,19:P1.7,16:R | 2 | |
| B:15:P1.2,5:R | 1 | |
| B:15:P1.12,13,13a:R | 1 | |
| Not tested | 1 | |
| 2 (8) | C:2a:P1.2,5:R | 8 |
| 3 (6) | B:16:-:S | 2 |
| B:16:P1.2,5:S | 4 | |
| 4 (2) | B:4:P1.9:I | 1 |
| B:NT:P1.16:I | 1 | |
| 5 (2) | Y:14,19:P1.16:S | 1 |
| Y:14,19:P1.2,5,6:I | 1 | |
| 6 (3) | W135:16:P1.1,7:I | 1 |
| C:21:P1.5:S | 1 | |
| B:NT:P1.13,13a:S | 1 | |
| 8 (2) | C:2b:P1.2,5:R | 2 |
PCR-AREA classes 7, 9, 10, and 11 contain single isolates and are not included in this table.
Serogroup:serotype:subtype:sulfonamide susceptibility (R, resistant; I, intermediate; S, sensitive).
TABLE 4.
Associations between indistinguishablea isolates
| Groupb | Isolate no. | Datec | Connection |
|---|---|---|---|
| 1-a1 | 1 | Jan 1987 | None known. Geographically separate. |
| 8 | Jan 1988 | ||
| 10 | Oct 1988 | ||
| 1-a9 | 32 | May 1992 | None known. Geographically separate. |
| 34 | Nov 1992 | ||
| 2-b1 | 7 | Mar 1988 | Temporally associated. Both from Grenland urban district. |
| 9 | Jun 1988 | ||
| 2-b6 | 27 | Nov 1991 | Temporally associated. Both from Grenland urban district. |
| 33 | May 1992 | ||
| 3-c2 | 24 | Jun 1991 | Same town. 35-1 and 35-2 coprimary in same family; no connection with 24 known. |
| 35-1 | Dec 1992 | ||
| 35-2 | Dec 1992 |
Identical in PCR-AREA class, DNA fingerprint, serogroup, serotype (except isolate 24), and sulfonamide resistance.
Group name is a combination of PCR-AREA class and DNA fingerprint pattern.
Jan, January; Oct, October; Nov, November; Mar, March; Jun, June; Dec, December.
The two isolates of class 4 were both in serogroup B and had an intermediate level of sulfonamide resistance, but their serotypes and DNA fingerprint patterns differed. The two isolates of class 5 were both in serogroup Y, serotype 14,19, but their subtypes, sulfonamide resistances, and DNA fingerprints differed. Isolates of class 6 had different serogroups, serotypes, sulfonamide resistances, and DNA fingerprints. Classes 7, 9, 10, and 11 contained only one member each, all serogroup B isolates with low-to-intermediate levels of sulfonamide resistance. Three of these four isolates were serotype 4, but all had different subtypes. We conclude that the presence of similar PCR-AREA patterns is not indicative of clonal relations in these strains.
Association between indistinguishable isolates.
Five groups of two to three indistinguishable isolates could be identified (Table 4). They are designated by their PCR-AREA classes and DNA fingerprint patterns. These isolates had identical serogroups, serotypes, serosubtypes, and sulfonamide resistances, with the exception of isolate 22, which differed from other isolates in group 3-c1 in being nonsubtypeable (Table 1). There was a maximum of 21 months between cases of disease caused by indistinguishable isolates. We are not aware of any social connection between these patients, except in the case of isolates 35-1 and 35-2 (3-c2), which were from siblings who both fell sick in the space of 24 h. However, the cases of disease caused by strains of group 2-b1 and 2-b6 were sufficiently close in time and space that a connection is to be suspected.
Correlation of PCR-AREA class with sulfonamide MIC.
Sulfonamide resistance in N. meningitidis is caused by alterations of the chromosomal dhps gene (15), which is the target for PCR-AREA. The PCR-AREA pattern is therefore expected to correlate with sulfonamide resistance. There is a nearly exact correlation between the PCR-AREA patterns and the sulfonamide MICs for the 31 isolates of classes 1, 2, 3, and 8; these strains are closely related, with a generally high degree of phenotypic homogeneity. When the genetically heterogeneous classes 4, 5, and 6 are examined, we see that only class 4 is homogeneous with respect to the MIC of sulfonamide. Thus, similar PCR-AREA patterns in unrelated strains do not predict sulfonamide MICs with any degree of exactitude (Table 1).
Temporal distribution of clones.
Figure 3 shows the occurrence of isolates belonging to the three major PCR-AREA classes during the 8 years of the study. Class 1 was dominant in 1987 and since then has made a moderate but persistent contribution to cases of meningococcal disease in Telemark. Class 2 was first observed in 1988 and persisted until 1992, after which it disappeared (a single class 2 case has since been observed, in January 1997 [data not shown]). Class 3 was the most temporally confined clone, with all cases occurring in 1991 and 1992.
FIG. 3.
Temporal distribution of PCR-AREA classes from 1987 to 1995.
Geographical distribution of clones.
The population of Telemark (163,000) is concentrated in the coastal urban-industrial area of Grenland (population, 89,000). The remainder of the county is predominantly rural and thinly populated, with a population of 74,000. Thirty-one of the 42 cases in this study were associated with the Grenland area (the incidence for the study period was 35 per 100,000), while 13 cases were associated with the rural part of the county (the incidence for the study period was 17.5 per 100,000). Two cases had affinities to both rural and industrial parts of the county.
When the geographical location of a case is considered in relation to the clonal classes found, a clear difference between class 1 and the other classes emerges (Table 5). Class 2 and class 3 are confined to cases occurring within the Grenland industrial area (P = 0.003; Fisher’s exact test), and there was also a strong tendency for strains of classes 4 to 11 to occur in the Grenland area. The contrary is found for class 1, which is twice as frequent outside the Grenland area (χ2 = 12.16; P < 0.005).
TABLE 5.
Geographical distribution of N. meningitidis clones in Telemark
| Class | No. of isolates
|
χ2 | P value | ||
|---|---|---|---|---|---|
| Grenlanda | Telemarkb | Total | |||
| 2c | 7 | 0 | 7 | 0.003f | |
| 3d | 5 | 0 | 5 | 0.003f | |
| 4 to 11c | 9 | 3 | 12 | ||
| Subtotale | 21 | 3 | 24 | ||
| 1 | 5 | 10 | 15 | 12.16 | <0.005 |
| Total | 26 | 13 | 39 | ||
Grenland industrial district (population, 89,000).
Excluding Grenland industrial district (population, 74,000).
Two isolates associated with both the Grenland industrial district and the rest of Telemark were excluded from this analysis.
Isolates 35-1 and 35-2, which occurred within 1 day in the same family, are regarded as a single case.
Total of classes 2 to 11.
Fisher’s exact test.
DISCUSSION
Usefulness of PCR-AREA.
PCR-AREA was developed in order to address the limitations of DNA fingerprinting (12) in classification. The complex band pattern generated by DNA fingerprinting allows precise identification of the strain present in a patient among strains isolated from contacts, but exhaustive cross-comparison of patterns in more than a handful of strains is unachievable. The PCR-AREA restriction pattern of 8 to 15 CfoI (HhaI) fragments is simpler and should allow exhaustive cross-comparison of strains. However, the reduced information density of PCR-AREA patterns is expected to reduce its precision relative to DNA fingerprinting and to give rise to more “false calls” of strain identity.
We found that PCR-AREA fulfilled our expectations, allowing exhaustive cross-comparison of 42 meningococcal strains in a way that correlates meaningfully with other criteria of strain similarity. Four PCR-AREA classes, containing 31 of a total of 42 isolates, consisted of strains that were clonally related, as evidenced by the results of phenotypic characterization and DNA fingerprinting. Three classes, containing seven isolates, contained strains that were phenotypically and genotypically heterogeneous. The remaining four classes contained only one isolate each. No phenotypically identical isolates with different PCR-AREA patterns were encountered. This indicates that PCR-AREA is a sensitive detector of strain similarity but that it occasionally groups unrelated strains. This may be because the same restriction pattern can represent different sequences, or the same dhps sequence may be present in unrelated strains due to the effects of transformation. In epidemiological studies, PCR-AREA will be useful as a first-step analysis method where there is a need to detect groups of closely similar, epidemiologically related strains in large strain collections prior to the application of more high-resolution methods, such as DNA fingerprinting or ET typing.
In the clinical context, PCR-AREA should also be of use for quickly identifying the contacts of patients with meningococcal disease who carry the disease-causing strain and should be given prophylactic treatment. Although in this study PCR-AREA was performed on purified DNA, the assay can also be performed on crude lysates of primary colonies, and a result can be achieved within 8 hours of the isolation of a primary colony. Rapid detection will prevent secondary cases and transmission of the strain to new carriers.
Other authors have recognized the potential of PCR-based analyses for rapid epidemiological analysis of meningococcal strains. Guibourdenche et al. (7) performed RFLP analysis of the pilA-pilB locus and found good correlation with the results of ET typing. Newcombe et al. (14) performed genetic serosubtyping by single-stranded conformational polymorphism analysis of the amplified porA VR1 region after direct amplification from clinical samples, allowing recognition of the similarity of outbreak strains. Woods et al. (20) have pioneered a multilocus PCR approach based on the distribution of repetitive elements in the meningococcal genome which gives results that are in good accordance with those of ET typing but which has a slightly lower discriminatory power.
PCR-AREA pattern and sulfonamide resistance.
PCR-AREA targets the meningococcal chromosomal dhps gene, which determines sulfonamide resistance in N. meningitidis. In the strains studied here there is a high degree of correlation between the PCR-AREA pattern and the level of sulfonamide resistance. This is, however, primarily due to the fact that the collection is dominated by clonal isolates with high degrees of phenotypic and genotypic similarity. In the nonclonal classes 4, 5, and 6, the sulfonamide MICs correlate poorly with the PCR-AREA patterns. This may be because a single PCR-AREA pattern can encompass both resistant and sensitive dhps alleles or because other genetic loci may play a role in determining the MIC of sulfonamide.
Clonal structure of strains from Telemark.
One of the most striking, and at first sight puzzling, aspects of our results is that while homogeneous (clonal) PCR-AREA classes (classes 1, 2, 3, and 8) are typically large (15, 8, 6, and 2 members, respectively), heterogeneous (nonclonal) classes (classes 4, 5, and 6) are small (2, 2, and 3 isolates, respectively). This observation may be accounted for by the proposed genetic structure of meningococcal populations (13, 17). Meningococcal populations have a basically panmictic structure, where free exchange of genetic material between strains results in a very low degree of linkage disequilibrium—that is to say, genetic markers associate randomly. Upon this panmictic structure is imposed an element of clonality caused by the spread of epidemic strains. The spread of such strains exceeds the rate of genetic recombination, so that the strains are genetically homogeneous. Our results conform well to this model. Members of classes 1, 2, 3, and 8 are epidemic strains, belonging to the clonal part of the meningococcal population, and are genetically homogeneous and numerous. Members of classes 4, 5, and 6 are nonepidemic strains belonging to the panmictic population; they have similarity in the dhps gene, due presumably to transformational spread of dhps alleles through the meningococcal population, but are otherwise dissimilar. The small sizes of these classes reflect the great variety of dhps sequences present in the meningococcal population (4).
On an international basis, meningococcal clones are defined by their ET type, determined by multilocus enzyme electrophoresis (3, 16, 18). Although there is considerable phenotypic variation within clones, it is nonetheless possible to identify certain typical phenotypes which allow tentative assignment of meningococci to ET types on the basis of phenotype. Three of the clones that we have identified here, classes 1, 2, and 8, have striking phenotypic affinities with recognized meningococcal clones. Class 1 has the phenotypic characteristics of strains of the ET5 complex (B:15:R), and its PCR-AREA pattern is identical to those of isolates from our laboratory known to belong to ET5. ET5 strains caused a prolonged period of hyperendemic meningococcal disease in Norway in the period 1975 to 1979 (1), and our results confirm that this clone continues to be abundant in Norway. Class 2 has the phenotypic characteristics of ET37 strains (C:2a:P1.2:R). This clone has been present in Norway since the beginning of the 1970s (5). Class 8 has the phenotypic characteristics of cluster A4 (B,C:2b:P1.2), which has been present worldwide for many years (2, 5). Class 3 does not appear to resemble any known clone and may therefore represent a local miniepidemic, as is reflected by its geographical restriction to the Grenland industrial area and temporal restriction to 1991 and 1992.
Geographical distribution of clones.
The geographical distributions of classes 1 (B:15; ET5-like), 2 (C: 2a; ET37-like), and 3 (B:16) differ significantly. Classes 2 and 3 are confined to cases associated with the Grenland urban district. Class 3 appears to have arisen in this district and to have subsequently died out without spreading to other parts of the county. Class 2 (ET37-like) has been endemic in Norway for more than 10 years, so its confinement to the Grenland district is difficult to explain. Class 1 resembles ET5, which has been present in Norway since the 1970s and should thus have had ample time to achieve a homogeneous distribution, so its predilection for the rural areas of Telemark is similarly difficult to account for. Environmental factors or patchy distribution and a tendency for the social networks that facilitate transmission to be exclusively rural or urban are possible explanations.
Indistinguishable isolates.
Five small groups of two to three indistinguishable isolates were identified in this study. Such isolates are probably epidemiologically related. Although cases of disease caused by indistinguishable isolates occurred within 2 years of each other, we found no noticeable geographical clustering, nor are we aware of any links between cases, with the single exception of two coprimary cases occurring in the same family. Although it is likely that a thorough investigation would reveal chains of common contacts between patients with disease caused by identical isolates, such an investigation is undesirable because of the burden of guilt which might be placed on these contacts. Cases caused by indistinguishable isolates represent failures of the objective of the Telemark Meningococcal Project (9, 11), which is to prevent secondary cases by directed chemoprophylaxis given to carriers of the disease-causing strain among a patient’s contacts identified by DNA fingerprinting. Such failures probably arise because of difficulties in identifying and contacting all relevant contacts.
ACKNOWLEDGMENTS
We acknowledge the able technical help of Berit Nyland and Anne-Gry Allum. We also wish to express our gratitude to Dominique Caugant for her help in identifying the clonal affinities of our strains.
This study was conducted by Randi Kersten Aakre in partial fulfillment of the requirements for the degree of Cand. Scient. R.K.A. wishes to acknowledge the support of her supervisor, Berit Johansen.
REFERENCES
- 1.Achtman M. Global epidemiology of meningococcal disease. In: Cartwright K, editor. Meningococcal disease. Chichester, United Kingdom: Wiley; 1995. pp. 159–176. [Google Scholar]
- 2.Caugant D A. Epidemiologie moleculaire de Neisseria meningitidis. L’analyse des clones. Ann Inst Pasteur. 1994;5:130–137. [Google Scholar]
- 3.Caugant D A, Mocca L F, Frasch C E, Frøholm L O, Zollinger W D, Selander R K. Genetic structure of Neisseria meningitidis populations in relation to serogroup, serotype, and outer membrane protein pattern. J Bacteriol. 1987;169:2781–2792. doi: 10.1128/jb.169.6.2781-2792.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Fermér C. Horizontal transmission and mutational adaptation of sulfonamide resistance in pathogenic strains of Neisseria meningitidis. Doctoral thesis. Uppsala, Sweden: University of Uppsala; 1996. [Google Scholar]
- 5.Flægstad T, Kaaresen P I, Stokland T, Gutteberg T. Factors associated with fatal outcome in childhood meningococcal disease. Acta Pædiatr. 1995;84:1137–1142. doi: 10.1111/j.1651-2227.1995.tb13513.x. [DOI] [PubMed] [Google Scholar]
- 6.Greenwood B M. Meningococcal infections. In: Weatherall D J, et al., editors. Oxford textbook of medicine. Oxford, United Kingdom: Oxford University Press; 1996. pp. 533–544. [Google Scholar]
- 7.Guibourdenche M, Giorgini D, Guèye A, Larribe M, Riou J-Y, Taha M-K. Genetic analysis of a meningococcal population based on polymorphism of the pilA-pilB locus: a molecular approach for meningococcal epidemiology. J Clin Microbiol. 1997;35:745–750. doi: 10.1128/jcm.35.3.745-750.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Jørgensen J H, Redding J S, Maher L A, Howell A W. Improved medium for antimicrobial susceptibility testing of Haemophilus influenzae. J Clin Microbiol. 1987;25:2105–2113. doi: 10.1128/jcm.25.11.2105-2113.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kristiansen B E, Tveten Y, Ask E, Reiten T, Knapskog A B, Steen-Johnsen J S, Hopen G. Preventing secondary cases of meningococcal disease by identifying and eradicating disease-causing strains in close contacts of patients. Scand J Infect Dis. 1992;24:165–173. doi: 10.3109/00365549209052608. [DOI] [PubMed] [Google Scholar]
- 10.Kristiansen B E, Fermér C, Jenkins A, Ask E, Swedberg G, Sköld O. PCR amplicon restriction endonuclease analysis of the chromosomal dhps gene of Neisseria meningitidis: a method for studying spread of the disease-causing strain in contacts of patients with meningococcal disease. J Clin Microbiol. 1995;33:1174–1179. doi: 10.1128/jcm.33.5.1174-1179.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Kristiansen B E, Knapskog A B, Reiten T, Steen-Johnsen J, Hopen G. Meningokokkprosjekt Telemark. Tidsskr Nor Lægeforen. 1993;113:2933–2937. [PubMed] [Google Scholar]
- 12.Kristiansen B E, Sørensen B, Bjorvatn B, Falk E, Fosse E, Bryn H, Frøholm L O, Gaustad P, Bøvre K. An outbreak of group B meningococcal disease: tracing the causative strain of Neisseria meningitidis by DNA fingerprinting. J Clin Microbiol. 1986;23:764–767. doi: 10.1128/jcm.23.4.764-767.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Maynard Smith J, Smith N H, O’Rourk M, Spratt B. How clonal are bacteria? Proc Natl Acad Sci USA. 1993;90:4384–4388. doi: 10.1073/pnas.90.10.4384. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Newcombe J, Dyer S, Blackwell L, Cartwright K, Palmer W H, McFadden J. PCR–single-stranded confirmational polymorphism analysis for non-culture-based subtyping of meningococcal strains in clinical specimens. J Clin Microbiol. 1997;35:1809–1812. doi: 10.1128/jcm.35.7.1809-1812.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Rådström P, Fermér C, Kristiansen B E, Jenkins A, Sköld O, Swedberg G. Transformational exchanges in the dihydropteroate synthase gene of Neisseria meningitidis: a novel mechanism for acquisition of sulfonamide resistance. J Bacteriol. 1992;174:6386–6393. doi: 10.1128/jb.174.20.6386-6393.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Selander R K, Caugant D A, Ochman H, Musser J M, Gilmour N, Whittam T S. Methods of multilocus enzyme electrophoresis for bacterial population genetics and systematics. Appl Environ Microbiol. 1986;51:873–884. doi: 10.1128/aem.51.5.873-884.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Spratt B G, Smith N H, Zhou J, O’Rourke M, Feil E. The population genetics of the pathogenic Neisseria. In: Baumberg S, et al., editors. Population genetics of bacteria. Cambridge, United Kingdom: Cambridge University Press; 1995. pp. 143–160. [Google Scholar]
- 18.Strathdee C A, Tyler S D, Ryan J A, Johnson W M, Ashton F E. Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada. J Clin Microbiol. 1993;31:2506–2508. doi: 10.1128/jcm.31.9.2506-2508.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Wedege E, Høiby E A, Rosenqvist E, Frøholm L O. Serotyping and subtyping of Neisseria meningitidis isolates by co-agglutination, dot-blotting and ELISA. J Med Microbiol. 1990;31:195–201. doi: 10.1099/00222615-31-3-195. [DOI] [PubMed] [Google Scholar]
- 20.Woods C R, Koeuth T, Estabrook M M, Lupski J R. Rapid determination of outbreak-related strains of Neisseria meningitidis by repetitive element-based polymerase chain reaction. J Infect Dis. 1996;174:760–767. doi: 10.1093/infdis/174.4.760. [DOI] [PubMed] [Google Scholar]
- 21.Woods J P, Kersulyte D, Tolan R W, Berg C M, Berg D E. Use of arbitrarily primed polymerase chain reaction to type disease and carrier strains of Neisseria meningitidis isolated during a university outbreak. J Infect Dis. 1994;169:1384–1389. doi: 10.1093/infdis/169.6.1384. [DOI] [PubMed] [Google Scholar]