Journal of General Microbiology (1986), 132, 641-652.
Printed in Great Britain
641
Mnltilocus Genotypes Determined by Enzyme Electrophoresis of
Neisseria meningitidis Isolated from Patients with Systemic Disease
and from Healthy Carriers
By D O M I N I Q U E A. C A U G A N T , l * ? K J E L L B 0 V R E , 2 P E T E R G A U S T A D , 2
KLAUS BRYN,' E R I K H O L T E N , 3 E. A R N E H01BY4 A N D
L. ODDVAR F R 0 H O L M l
Department of Methodology and 4Department of Bacteriology, National Institute of Public
Health, Geitmyrsveien 75, Oslo 4, Norway
Kaptein W . Wilhelmsen og Frues Bakteriologiske Institutt, University of Oslo, Rikshospitalet,
Oslo, Norway
Department of Microbiology, Akershus Central Hospital, Nordbyhagen, Norway
(Received 14 June I985 ;revised 9 October 1985)
Variation in nine enzymes in 152 isolates of Neisseria meningitidis from Norway (1 18 from blood
or cerebrospinal fluid of patients with systemic disease and 34 from the pharynx of healthy
carriers) was analysed by starch-gelelectrophoresis.All nine enzymes were polymorphic and the
number of allozymes (electromorphs) identified per locus ranged from 3 to 12, with a mean of
6.1. Among the 152 isolates, 55 unique combinations of electromorphs (electrophoretic types,
ETs) were distinguished. Twenty ETs were represented among the carrier isolates and 37 among
the systemic isolates; hence, only two ETs were found in both groups of isolates. ET-5 was
identified 67 times among the 118 systemic isolates (58%), indicating an association of this ET
with invasiveness; ET-5 was also the most common type among the carrier isolates (18%).
Genetic similarity between ETs was analysed by pairwise comparison of all 55 ETs with respect
to the number of electromorphs by which they differed. No evidence of a general genetic
difference between carrier and case isolates was found. Two well-defined clusters of ETs were
observed, each including one of the two most common ETs identified among the systemic
isolates (ET-5 and ET-37), together with isolates differing from them only at one or two loci. All
isolates of ET-5 and ET-37, as well as their closely related variants defined by the similarity
matrix, were resistant to sulphonamide, independent of their antigenic characteristics and
isolation site. The extensive allozyme variation among isolates of the same serogroup
demonstrated the limited value of serogroupingas an epidemiological tool. All but one isolate of
serotype 15:P 1.16were electrophoreticallysimilar, as were all the 2a :P 1.2 isolates. The 15:P1.15
isolates, however, were genetically heterogeneous. The distribution of alleles in genotypes
identified among the systemic isolates indicated that genetic recombination may occur in
natural populations of N. meningitidis.
INTRODUCTION
Strains of Neisseria meningitidis are characterized by the immunological specificities of their
capsular polysaccharides, i.e. serogroups (Branham, 1953; Slaterus, 1961; Evans et al., 1968);
they are further assigned to serotypes on the basis of antigenic differences among their major
t Present address: Department of Biology, University of Rochester, Rochester, New York 14627, USA.
Abbreviations : ET, electrophoretic type ; NG, non-groupable ; NT, non-typable ; OMP, outer-membrane
protein.
0001-2752 0 1986 SGM
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642
D . A. CAUGANT A N D OTHERS
outer-membrane proteins (Frasch & Chapman, 1972a, b) and to lipopolysaccharide
immunotypes (Mandrel1 & Zollinger, 1977; Apicella, 1979). However, the serogroups and
serotypes actually characterized do not provide a complete description of the antigenic
differences in N . meningitidis. Many isolates, especially those from carriers, do not react with any
of the presently available antisera (Poolman et al., 19SOb; Barvre et al., 1983). Most isolates from
patients are serogroupable, but a large proportion of sporadic-case isolates are non-typable (NT)
(Broud et al., 1979); and epidemic strains are often of a single antigenic type.
Since 1974, the incidence of meningococcal disease has reached an epidemic level in Norway,
with an average of 7.4 cases per 100000 in 1984. At the beginning of the epidemic, serogroups A,
B and C were isolated in about equal frequencies. Since 1979, however, more than 80% of
isolates have been of serogroup B, serogroup A having virtually disappeared (Holten, 1980;
Bavre 8z Gedde-Dahl, 1980; Bavre et al., 1983). In 1984,67% of the B systemic isolates were of
serotype 15, subtype P1.16 (Frarholm et d.,1985).
Recently, the technique of multilocus enzyme electrophoresis has been applied to the study of
genetic variation in natural populations of Escherichia coli (Selander & Levin, 1980; Caugant et
al., 1981, 1983; Ochman et al., 1983; Ochman & Selander, 1984) and to other bacteria (Levin et
al., 1984; Schill et al., 1984; Musser et al., 1985; Olyhoek et al., 1985). The method consists in
measuring allelic variation at structural gene loci by screening randomly selected cytoplasmic
enzymes for genetically controlled variants (electromorphs or allozymes). The electrophoretic
type (ET) of each strain is determined by its electromorph profile over all loci assayed. The main
advantages of the technique are that all strains can be characterized and, by assaying a relatively
large number of loci, the overall genetic distance between strains and groups of strains can be
estimated.
Here we report the application of the multilocus enzyme electrophoresis technique to isolates
of N . meningitidis from patients with systemic disease and from healthy carriers in Norway.
Polymorphism in enzymes encoded by nine gene loci is analysed for association with
immunological and other characteristics of isolates.
METHODS
Bacrerial strains. The N . meningitidis strains studied were : (i) 34 pharyngeal isolates obtained from healthy
military recruits, between September 1981 and April 1982, in connection with a vaccine trial (Bervre et al., 1983;
Frlaholm er al., 1983); (ii) 35 systemic (blood or cerebrospinal fluid) isolates from cases of meningococcal disease in
the winter of 1981-1982, during a case-control study (Bervre et al., 1983); (iii) 28 systemic isolates from military
personnel (17 cases) and civilians (1 1 cases) in various parts of Norway (1976-1983); and (iv) 55 systemic isolates
from patients in 1984 (Frlaholm et al., 1985). The strains of categories (ii) and (iii) were selected from a large sample
in such a way that, in addition to including a high proportion of B : 15:P1.16 isolates, all serological variants
available were included. In contrast, for the 1984 sample (category iv), all available isolates were studied.
Serogroup and serotype ident$cations. The serogroups were determined by slide agglutination with serogroupspecific sera for A, B, C, W 135,X,Y and Z, with the exception of the category (iii) isolates, which were not tested
against W135 antiserum. Serogrouping of the carrier isolates of category (i) was complemented by gaschromatographic screening for capsular polysaccharides (Bryn et al., 1983) and by agglutination with 29E
antiserum.
Antigen was prepared and serotypes were tested as previously described (Frsholm et al., 1985),with monoclonal
antisera against antigens 1, 2a, 2b, 2c, 5 , 6, 9, 14, 15, P1.2, P1.15 and P1.16.
The serogroup, serotype and subtype (P1 antigen) distributions of the 152 isolates analysed for enzyme variation
are shown in Table 1.
Electrophoresisof enzymes. To prepare protein extracts, isolates were grown overnight at 37 "C in 100 ml tryptic
soy broth (Difco). The bacteria were harvested b j centrifugation, and the bacterial pellets were resuspended by
vortex mixing with 1 ml buffer (0.01 M-Tris/HCI and 0.001 M-EDTA,pH 6.8).The cells were disrupted by freezing
at - 25°C for 48-72 h and then thawing (modified from Braude et a f . , 1983). The bacterial suspensions were then
centrifuged at 27000g for 30 min at 4 "C. The supernatants were sterilized by filtration and stored at - 70 "C until
used for electrophoresis.
Techniques of starch-gel electrophoresis were similar to those described by Selander et al. (1971). Nine enzymes
were stained: malic enzyme, NADP-linked (ME; EC 1.1.1.40); glucose-6-phosphate dehydrogenase (G6PDH;
EC 1.1. I .49); phenylalanyl-leucine peptidase (PLP; EC 3.4.11 .-); isocitrate dehydrogenase (IDH;
EC 1.1.1.42); 6-phosphogluconate dehydrogenase (6PGDH; EC 1.1.1.44); aconitase (ACO; EC 4.2.1.3);
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Enzyme genotypes of’Neisseria meningitidis
643
Table 1. Serogroup and serotype distribution in I18 case isolates and 34 carrier isolates
(indicated by the numbers in parentheses) o j N . meningitidis analysed for enzyme variation
Serogroup . . . NG
A
B
C
29E
Y
W135
Seroty pe
NT :N T :P1.2
N T :P1.15
N T : P1.16
2a:2a : P1.2
2a : P1.15
2b :P1.15
14:P1.16
15 :15:P1.2
15:P1.15
15:P1.16
1
glutamate dehydrogenase, NADP-linked (GDH-1; EC 1 . 4 . 1 .4); glutamate dehydrogenase, NAD-linked (GDH2; EC 1.4.1.2); and alcohol dehydrogenase (ADH; EC I . 1.1.1). Tris/citrate buffer (pH 8.0) was used for the
electrophoresis of the first six enzymes listed above and Poulik buffer (pH 8.7) was used for the GDHs and ADH
(see Selander et al., 1971).
The electrophoretic mobilities of the enzymes were recorded in a first-approach by comparison with two
reference strains with very different electromorph combinations (ET-5 and ET-18; see Table 2). All isolates that
differed from the reference strains were next compared to each other, side by side on a gel, for the nine enzymes
assayed. (About 10% of the isolates analysed were grown several times and cultures lysed by freezing and thawing
or by sonication for I min. Mobilities of the enzymes from protein extracts of different cultures of the same strain
were always identical.) Electromorphs were numbered in order of decreasing anodal mobility, and the
combination of electromorphs at the nine enzyme loci was determined for all isolates. Although the genes coding
for these enzymes have not been mapped on the chromosome of N . meningitidis, it is assumed that the enzymes are
encoded by chromosomal genes and so electromorphs were equated with alleles at each locus.
Sulphonamide suscepribility testing. Minimum inhibitory concentrations of sulphadiazine were determined as
described by Bsvre et a f . (1983). Isolates with minimum inhibitory concentrations of S mg I-’ or lower were
considered to be sulphonamide-sensitive; those with values of 10 to 50mg 1-’ were considered to be of
intermediate susceptibility; those with values of 100 mg I-’ or higher were considered resistant.
Sodium dodecyl sulpltate-polyacrylumide gel electrophoresis of outer-membrane proteins ( O M P s ) . OM Ps were
prepared according to the procedure of Mocca & Frasch (1982). The Laemmli (1970) system with 1So; (w/v)
acrylamide gels was used for electrophoresis.
RESULTS
Electrophoretic variation
All nine enzymes assayed were polymorphic. The number of electromorphs (alleles) identified
at each locus ranged from three for GDH-1 to 12 for IDH, with a mean of 6.1. No enzymic
activity was detected for G6PDH in one B : N T : - isolate, which was also atypical for a
meningococcus in that it did not ferment glucose, maltose or saccharose. Twenty-four isolates
had either little or no activity for ADH; all of these were considered as having a ‘null’ allele.
Among the 152 isolates there were 55 distinctive combinations of alleles (ETs) (Table 2). There
were 20 ETs among the 34 carrier isolates (ETs 1 to 20) and 37 ETs among the 118 isolates from
cases (ETs 5, 7 and 21 to 55). Only two ETs (ET-5 and ET-7) were represented among isolates
recovered from both carriers and patients.
Genic diversity per locus, expressed as the probability that two randomly chosen ETs have
different electromorphs at a given locus, is shown in Table 3 for the 20 ETs of carrier isolates, the
37 ETs of patient isolates and all El’s pooled. Mean genic diversity was higher among ETs
isolated from patients than from carriers, primarily because of variation at the G6PDH locus,
for which six electromorphs were identified among the patient isolates but only two among those
from carriers.
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644
D . A . CAUGANT AND OTHERS
Table 2. Electromorph composition at nine enzyme loci of’ 55 electrophoretic types (ETs)
identiJied among 152 Norwegian isolates of N . meningitidis
No. of isolates from:
&
Patients
ET Healthy (systemic I
no. carriers
ME
disease)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
1
I
5
1
6
2
1
I
1
1
1
1
1
1
4
2
1
1
1
1
67
1
3
3
1
2
1
3
3
3
3
2
5
1
2
3
3
3
1
1
1
5
1
I
1
1
2
1
1
1
1
1
1
3
1
7
1
1
1
1
2
1
1
1
I
2
1
1
1
1
1
1
1
1
3
5
I
1
1
1
2
3
3
1
3
1
3
3
3
3
1
4
3
4
1
I
3
1
1
1
1
4
3
3
3
1
3
1
4
3
3
3
Reldtive electrophoretic mobility of allozymes
h
G6PDH
3
3
3
3
1
3
1
3
3
3
3
3
1
3
3
3
3
3
3
3
5
1
1
3
3
3
4
3
4
4
3
3
1
4
3
3
3
4
6
3
1
4
5
4
3
2
3
3
1
3
5
3
3
3
3
PLP
3
3
:j
7
ti
7
4
5
)
1
)
8
4
4
4
2
5
4
i
?
.
-
4
I
3
/
6
9
4
/
4
c
z
5
4
7
1
4
2
4
5
4
4
7
6
2
6
7
4
5
1
7
2
4
4
2
4
5
\
IDH
6PGDH
ACO
GDH-I
GDH-2
ADH
7
6
2
2
8
7
7
7
5
7
3
1
4
12
7
9
5
7
7
11
6
8
5
3
7
7
8
7
7
7
7
12
8
7
5
12
5
10
3
5
8
6
3
6
5
5
5
11
6
7
6
5
12
9
7
3
4
1
1
1
4
4
4
4
4
4
4
4
1
2
4
4
4
4
4
4
4
4
3
4
4
2
4
4
4
4
4
4
4
4
4
2
2
4
2
4
4
2
4
4
3
3
3
2
3
3
1
3
3
3
3
3
3
2
2
2
3
3
3
3
3
3
3
3
3
2
3
3
3
3
3
3
3
3
2
3
4
3
3
4
3
3
3
3
3
3
3
3
3
3
2
4
3
3
3
2
2
2
2
2
2
2
2
3
3
4
1
2
2
1
2
1
2
2
1
2
2
2
2
2
3
2
3
2
2
2
3
2
2
3
4
3
3
2
3
2
2
4
3
3
3
4
3
2
4
2
3
3
2
3
1
1
1
5
3
1
1
1
6
6
1
5
1
6
3
3
6
7
1
1
3
3
3
1
3
3
5
4
3
1
1
5
1
5
1
1
5
2
6
1
6
5
5
1
1
1
6
1
5
2
5
3
4
4
4
2
2
4
5
4
4
4
2
5
2
4
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1
1
3
2
2
1
2
1
2
2
2
1
1
1
1
2
2
1
2
1
3
2
1
2
2
2
1
2
1
2
1
2
1
1
1
1
1
1
1
2
1
1
1
1
1
1
1
2
1
1
2
1
1
2
Enzyme genotypes of' Neisseria meningitidis
-
645
Table 3. Genic diversity at nine enzyme loci in N . meningitidis
Isolates from*
Carriers
(n = 20)
Enzyme
ADH
ME
IDH
6PGDH
G6PDH
GDH-2
GDH-1
PLP
ACO
Mean
b
Cases
(n = 37)
e
H
4t
4
11
4
2
3
3
7
3
4.6
0.553
0.689
0.842
0-695
0.268
0.416
0.574
0.832
0.195
0.562
r
e
H
3t
4
9
7
6t
4
3
7
4
5.2
0.597
0.630
0.85 1
0.772
0.703
0.338
0.486
0.809
0,494
0.63 1
Total
(n = 5 5 )
&
e
H
4t
5
12
7
6t
4
3
9
5
6.1
0.615
0.657
0.85 1
0.747
0.564
0.347
0.516
0.8 17
0.417
0.615
* e, Number of electromorphs; H , genic diversity calculated as --n
1 - Ex;,where x, is the frequency of the
n-1
electromorph among the electrophoretic types, and n is the number of ETs (Nei, 1978).
t Includes a 'null' electromorph.
I = I
ilh
E T distribution among isolates from healthy carriers and systemic disease cases
Of the ETs identified among carriers 75 % were recovered from only one individual, whereas
the comparable proportion was 81 % among cases (Table 2). However, the mean number of
individuals from whom an ET was recovered was higher among the cases (3-2hosts per ET) than
among the carriers (1.7 hosts per ET). This results from the high representation of ET-5, which
was identified 67 times among the 118 isolates from patients (58%). ET-5 was also the most
frequently encountered ET among the carriers (18%). Excluding .ET-5, the proportion of
individual hosts per ET was the same among carriers and cases (1.4).
Temporal stability of the ETs
Among the few ETs that were identified several times in the sample of isolates analysed, some
were recovered over a period of years. ET-5 was found among the oldest isolates analysed (1976)
until the most recent samples; ET-7 was isolated from a carrier in the winter of 1981-1982 and
also from the spinal fluid of a patient in 1984; and ET-37, the second most common ET found in
the meningococcal epidemic, was recovered during the period 1977 to 1984.
Relationship among ETs
The genetic distance between ETs was estimated by counting the number of electromorph
differences between all pairs of ETs. In Fig. 1, the ETs are ordered by degree of similarity
according to the method of Sneath (1957) and pairs of ETs that differed by one to four
electromorphs are represented in the matrix of similarity. Several ETs (1 1, 13, 24 and 34) were
not related to any other ET identified; they differed by as many as four electromorphs from the
most closely related ETs. Fig. 1 shows that there are three well-defined clusters among the
isolates analysed. One cluster comprised ETs 12, 17 and 20 (identified among the carrier
isolates), and two clusters of ETs were related to the two most frequently recovered case strains :
ETs 5, 22, 23, 27, 33, 41 and 49 as one group, and ETs 35, 37, 40, 45, 46 and 52 as the other.
However, those two common ETs among the case isolates (ET-5 and ET-37) differed from each
other at all nine enzyme loci. Most of the remaining ETs fell into two related clusters (left side of
Fig. l), connected by ETs 2, 3, 9 and 47.
Relationship between ET and capsular antigen
The number of ETs, the maximum number of electromorph differences between pairs of ETs
and estimates of genic diversity among isolates of the same capsular antigen are presented in
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D . A . CAIJGANT A N D OTHERS
646
NG:15:-
11
I .
24
13
20
17
12
53
43
44
42
46
45
52
40
37
35
21
54
51
15
48
A:NT:B:NT:-
C : 2a : P 1.21-. B: 2a:
B.
n:21
B:NT:-
x
x
B I N G :N T
7
X
X
B PI.
B:14:P
NG: IS/NT: PI. 16. B : 15: PI. l6/15/-. B : N T : PI. I6/-, c : 15 : P 1.
NC:N T -
X
X
B N T r x x x x
X
x
X
x
X
x
49
41
33
27
23
22
5
10
39
38
4
16
Y:Za.-.
B:2a
x x x x
x
x x x x
x x x
x
x x x x
x
*
x
x
x
x x
x x
x
x
x
x
x
NG
14
50
36
47
9
3
2
29
26
30
32
28
19
31
25
18
55
8
1;
B:
1 6 8 55182531192832302629 2 3 9 4 7 3 6 5 0 1 4 1 6 4382910 5 222327334] 49748155154213537405245464244435312172013243411 ETs
Fig. 1 . Pairwise comparison of the 55 ETs of N . menrngitidis with respect to the number of
electromorphs by which they differed: m, I ; El, 2 ; 0,
3 ; x , 4.
Table 4. All serogroups included isolates that were genetically distant. In particular, both
serogroup B and serogroup C isolates comprised ET-5 and ET-37, which differ at all loci. The
average diversity within serogroups was 96% of the total genic diversity. Different capsular
antigens were also associated with a single ET. For example, ET-5 included isolates of serogroups
B and C and non-groupable (NG) isolates; ET-37 included six isolates of serogroup C and one
isolate of serogroup B; ET-47 included one serogroup B and one serogroup Y isolate. In addition,
an NG carrier isolate (ET-9) differed from ET-47 only in having a ‘null’ at ADH. The most
extensive variation was found for ET-15, which was represented by four carrier isolates with
four different serogroups (B, C, W I35 and NG).
Relationship hetween ET and serotype
The frequency distributions of ETs within each serotype (serotype plus subtype combination)
identified more than once among the isolates from patients are listed in Table 5, together with
the genic diversity within serotype. Because most carrier isolates were non-typable (NT), they
were not included in this table. The average genic diversity of isolates characterized by the same
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Enzyme genotypes of’ Neisseria meningitidis
647
Table 4. Electrophoretic type ( E T ) diversity within serogroups of N . meningitidis
Serogroup
A
B
C
W135
Y
29E
NG
Total
No. of
isolates
No. of
ETs
3t
4
102
16
2
2
1
3.
t Includes one or more pairs
Genic
diversity*
5
31t
8
2
2
I
16
55
25
152
* See footnote. Table
Maximum no.
of differences
between ETs
0-407
0.598
0.627
0,778
0.556
9
9
7
5
__
-
8
9
0.609
0.615
of ETs differing only by a ‘null’ electromorph.
Table 5. Electrophoretic type ( E T ) diversity within serotypes of’systemic isolates of
N . meningitidis
Serotype
15 :P1.16
15 : P I . IS
2a :PI .2
I5 :NT : PI. I6
NT : PI. IS
2a:N T : PI .2
NT :Total
No. of
isolates
No. of
ETs
ET no. (no. of
i5olates if > I )
54
5
7
6
3
4
4
16
5
4
2
5
2
3
4
3
15
I I0
35
5(50), 23, 33, 34, 41
5(2), 28, 32, 53
35, 37(6)
5(9), 23.1). 36. 48. 49
5(5), 31
21, 29, 30
37, 40, 47. 52
25, 35(2), 45
5 , 7, 24, 26. 27, 28. 38,
39, 42(2). 43, 44, 46,
50. 51. 55
I5
* See footnote. Table
Maximum no.
of differences
between ETs
6
8
Genic
diversity*
7
5
6
7
8
8
0.344
0.556
0.1 1 I
0.51 1
0.556
0.556
0.407
0.630
0.6 14
9
0.6 I 7
1
3
serotype was only 77:i of the total diversity, but the genic diversity varied according to the
serotype considered. The 54 isolates that were serotype 15 :P1.16 included only five ETs, of
which four were genetically related (ETs 5 , 23, 33 and 41). ET-5 alone was represented 50 times
among the 54 1 5 : P 1 .16 isolates. The last 15 : P 1.16 isolate (ET-34) differed from that group by as
many as six of the nine enzymes. Whereas all 53 related isolates were either of serogroup B or of
serogroup C, the unrelated 15 : P 1.16 isolate was of serogroup W 135. The reaction of the W 135
strain with antiserum 15 was weaker than that of the B and C isolates of that serotype. Serotype
2a: P1.2 was also quite homogeneous, with only two closely related ETs represented (ET-35 and
ET-37). In contrast, the 15:P1.15 serotype included isolates with very different ETs and the
genic diversity among the 15 : P1.15 isolates was 90% of the total diversity.
Among systemic isolates for which either serotype or subtype could not be defined, the 15 :-strains corresponded mainly to serological variants of the 15 : P1.16 strains, as nine out of 15
were ET-5 and four others were closely related to this ET. In contrast, the 15:- isolates found in
carriers were not related to ET-5. Of the six NT:P1.16 systemic isolates, five were ET-5. The
sixth (ET-31) was quite different but related to an isolate of the same serotype found among the
carriers (ET-8) (see Fig. 1). Like the 15:P1.15 isolates, the N T : P l . l 5 isolates were
heterogeneous. Three each of the 2a :- and NT: P 1.2 isolates were similar to the 2a :P 1.2 isolates,
but each category included an isolate that differed from the others by seven and eight
electromorphs, respectively.
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D . A . C A U G A N T A N D OTHERS
Among the NT:- isolates, three of the four belonging to serogroup A were similar to each
other (ETs 42 and 43 differed only by a null at ADH); whereas the other one differed at five of
the nine enzyme loci. The 12 B and C NT:- case isolates corresponded to 12 different ETs,
including ET-5, a variant of ET-5 (ET-27), and a variant of ET-37 (ET-46). The genic diversity
of the NT:- isolates was equivalent to the total genic diversity.
Relationship between ET and type of disease
For 98 of the 118 isolates that caused systemic disease, the type of infection was known: 57
patients had meningitis, 32 had septicaemia, five had both meningitis and septicaemia, and four
had other types of systemic disease.. Of the 68 isolates that were ET-5 or related types, 38 were
recovered from meningitis cases (56% 'and 24 from septicaemia (35 %). Of the eight isolates that
were ET-37 or related ETs, six were isolated from meningitis cases (75%) and two from
septicaemia (25%). ET-5 and related ETs were involved in the five cases where patients had
both meningitis and septicaemia. Three of the four cases that did not lead to meningitis or
septicaemia were due to strains not related to ET-5 or ET-37.
Sulphonamide susceptibility
Among the 34 carrier isolates, six were sulphonamide-resistant, three were of intermediate
susceptibility and the remainder were sensitive. The six sulphonamide-resistant isolates were
ET-5. Among the systemic isolates, the sulphonamide susceptibility was determined for isolates
of categories (i) and (iv), and nine of the category (iii) isolates (see Methods). All 77 isolates that
were either ET-5 and related ETs or ET-37 and related ETs were sulphonamide-resistant. The
minimum inhibitory concentration of sulphadiazine was, however, generally lower for ET-37
and variants (200 mg 1 - l ) than for the ET-5 family (500 mg 1-1 or more). All isolates that were
not of the ETs mentioned above were of intermediate susceptibility (1) or sensitive (19), with the
exception of two isolates corresponding to ET-28, which were also resistant (100 mg 1-1 and
200 mg 1-l, respectively).
I) I SC U S S I0N
The analysis of electrophoretic variation in nine enzymes revealed extensive allozyme
variation in N . meningitidis isolates from Norway. The mean genic diversity per locus among the
55 ETs of N . meningitidis (0.60) was higher than that obtained for 279 ETs of E. coli (0.52) from
diverse geographic sources analysed at 12 enzyme loci in a study by Ochman et al. (1983). In
considering only the six enzymes common to these two studies (ADH, IDH, 6PGDH, G6PDH,
PLP and ACO), the mean genic diversity was then 0.66 for N . meningitidis and 0-54 for E. coli.
No evidence of a general genetic difference between isolates from healthy carriers and from
patients with systemic disease was provided by this study. Distributions of electromorphs in
carrier and case isolates were quite similar for most loci; several genotypes were closely related
or identical at all nine enzymes in both groups of isolates. The most common ET (ET-5) among
systemic isolates was also the most prevalent among asymptomatic ones. The high frequency of
ET-5 among the systemic isolates, however, indicates an association of this ET with
invasiveness.
As most carrier isolates are NG :NT, serological methods do not detect the extent of crossinfection between healthy hosts. Multilocus enzyme electrophoresis, however, demonstrated
that 25 % of the ETs (five of 20) identified among military recruits at a single camp were shared
by two or more healthy carriers. Because of the prevalence of ET-5 among systemic isolates in
Norway, the occurrence of six carrier isolates of this ET may be the result of multiple infection
external to the military camp. However, the four other ETs that were recovered from two or
more hosts were not identified among the case strains. Although the common occurrence of
those ETs in the general carrier population of Norway cannot be ruled out, those multiple
recoveries can be more readily explained by cross-infection among the military recruits. The
analysis of OMP patterns supported this view; all isolates of a given ET from this military camp
also had the same OMP pattern (Fig. 2). Variation in low-molecular-mass protein was noted for
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Enzyme genot-vpes of Neisseria meningitidis
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D. A . C A U G A N T A N D OTHERS
isolates characterized as ET-3, ET-15 and ET-16, but this was not considered further because of
the high variability of that class of protein (isolates from an individual may vary in lowmolecular-mass OMP ; Poolman et al., 1980a).
Although the four ET-15 isolates each had a different serogroup (NG, B, Y and W 135), all had
a similar OMP pattern. The finding of isolates identical in both enzyme and OMP patterns but
differing in capsular antigens implies that the serogroup evolves rapidly in response to
environmental factors. This was already suggested by the observation that during an outbreak of
meningococcal disease among military recruits caused by serogroup B :serotype 2 strains, about
25 % of the case-contacts who became carriers harboured W 135:2 strains and only 2% carried
B :2 (DeVoe, 1982). The three capsular polysaccharides associated with ET-15 are polymers of
N-acetylneuraminic acid (serogroup B), alternating with glucose (serogroup Y) or galactose
(serogroup W135) (Craven et al., 1978). Probably only a small enzymic change in the bacterium
is necessary to shift from one capsular type to another. Moreover, it has been shown that a single
N . meningitidis strain could elaborate both the Y and the W 135 capsular polysaccharides (Brandt
et al., 1980). The variability of the capsular antigen both among carrier and among case strains
of the same ET provides further evidence of the limitations of serogrouping as an
epidemiological tool (Griffiss et al., 1977).
Some serotype : subtype combinations were strongly associated with the genotype as defined
by allozyme pattern. Only closely related ETs were found in 2a: P1.2 and 15 :P1.16 isolates (with
the exception of a W 135 : 15 :PI. 16 isolate). The heterogeneity of the 15 :P1.15 isolates, however,
cannot be explained.
Competence in genetic transformation in N . meningitidis strains correlates with the presence
of fimbriae (Frsholm et al., 1973). Upon isolation, all strains are fimbriated and competent
(Jyssum & Lie, 1965; DeVoe & Gilchrist. 1975). Because the transformation frequency may
exceed the mutation frequency by a factor of lo4 (Frsholm et al., 1973), it may be assumed that
some of the ETs that are closely similar to ET-5 and ET-37 were generated by genetic
recombination between strains. For example, ET-23, which was identified five times in the
collection of case isolates, was identical to ET-5 at all loci except IDH, where it had a ‘5’
electromorph instead of an ‘8’. The ‘5’ electromorph at IDH was encountered in the sample of
case isolates only in ET-37 and related ETs and in two ET-47 isolates characterized by the
antigenic types B :2a : P 1.15 and Y :2a :-. Because of the prevalence of ET-37 and closely related
variants, the origin of the ‘5’ electromorph in ET-23 may be the result of genetic recombination
between the two dominant ETs occurring in patients. The fact that one of the ET-23 isolates was
characterized by the antigenic type B : 15 : PI .2 supports this hypothesis and suggests that in that
case the recombinational event also included the P1 protein. Another ET-23 isolate was
B :15:P1.16, and the three others were B :15 :-. Although the OMP patterns of the ET-23 isolates
were similar, except for one of the B : 15 :- isolates, the variation in P1 protein suggested that the
different ET-23 isolates were due to several independent recombinational events. The multiple
recoveries of ET-23 may imply that the fitness of the strain was increased by the new
combination of genes.
The distribution of sulphonamide resistance among isolates correlates perfectly with the
enzyme pattern. All the isolates that were ET-5, ET-37 or related ETs were resistant to
sulphonamide, regardless of serotype. For example, the two ET-5 isolates that were atypical in
serotype (having serotype I5 :P 1.15 rather than the common 15 : PI. 16 for this ET) were also
resistant. It may be important to follow the evolution in the Norwegian population of the only
other ET (ET-28) that was resistant to sulphonamide, since it was recovered from two cases of
septicaemia, one of which was fatal.
With the exception of one isolate of N . meningitidis, all Neisseria species are reported to have
G6PDH (Holten, 1974). One of the isolates analysed in this study (ET-46) showed no G6PDH
activity and was also atypical in its sugar fermentation pattern. The taxonomic position of this
isolate was confirmed by its allozyme profile in the eight other enzymes : it was related to ET-37.
Its OMP pattern was also similar to those of other ET-37 isolates, although it was NT : -- rather
than 2a: P1.2 like most representatives of ET-37.
The results of this study confirm the existence of genetic heterogeneity among B:15
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Enzyme genotypes of Neisseria meningitidis
65 1
N . meningitidis isolates in Norway, as earlier detected by DNA restriction endonuclease
fingerprinting (Kristiansen et al., 1984). Methods that analyse a significant part of the genetic
information contained in the strains, such as multilocus enzyme electrophoresis or DNA
fingerprinting, have important epidemiological applications and may be of particular value for
the study of bacteria in which serological characters evolve rapidly, as seems to be the case in the
meningococcus.
We thank Karin Bolstad for preparing the antigens and performing the serotyping, Erik Jantzen for his help
with the computer analyses, Robert K. Selander for his comments on the manuscript, and Patricia E. Pattison for
her assistance in preparing the manuscript.
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