227

Epidemiol. Infect. (1992), 109, 227-239 Printed in Great Britain

Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes H. NI', A. I. KNIGHT', K. A. V. CARTWRIGHT2 AND J. J. McFADDEN'* 'Molecular Microbiology Group, School of Biological Sciences, University of Surrey, Guildford, Surrey, GU2 5XH, UK 2 Public Health Laboratory, Gloucester Royal Hospital, Great Western Rd, Gloucester, GL1 3NN

(Accepted 28 April 1992) SUMMARY

The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorphism (RFLP) analysis using a number of random DNA probes and a probe containing a truncated copy of the meningococcal insertion sequence 11106. The data were used to estimate genetic distance between all pairs of strains and to construct phylogenetic trees for meningococcal strains. B15:P1.16R strains isolated from cases of systemic meningococcal disease in two health districts with a high incidence of disease were clonal in contrast to similar strains from cases occurring in other parts of the UK. Strains from these areas, which contain a similar genomic deletion. were found to be derived from two distinct lineages within the B15: P1. 16R phylogenetic group. RFLP data demonstrated that present serological typing systems for the meningoccus do not necessarily reflect true genetic relationships. INTRODUCTION

Variations in the capsular polysaccharide permit the division of Neisseria meningitidis into a number of serogroups [1]. Strains may be further divided into serotypes on the basis of variation in the class 2 or 3 outer membrane protein (OMP) [2], and may be serosubtyped according to variations in the class 1 OMP [3]. Sulphonamide resistance is also utilized as an epidemiological marker. More recently multilocus enzyme electrophoretic typing (ET-typing) has been used to allocate strains to a number of ET types [4]. Strains of serogroup B have predominated in the UK and in Europe over the last decade; During the 1980s there was an increase in disease caused by group B type 15 subtype 16 (B15: P1.16), mainly sulphonamide-resistant (R) strains in the UK [5] and North Western Europe [6]. Although B15:P1.16R disease has occurred throughout the UK certain health districts including Gloucester [7] and Plymouth [8] have shown a prolonged increased incidence of endemic disease due to this strain. Current vaccines based on meningococcal polysaccharides are unsatisfactory; those consisting of meningococcal polysaccharides A and C give only short lived *

Author for correspondence.

H. NI AND OTHERS 228 immunity and group B polysaccharide is a poor immunogen. Considerable eforts are therefore being directed into the development of vaccines based on more immunogenic surface components [9]. The success of such vaccines will depend on detailed knowledge of the epidemiology of disease caused by N. meningitidis and on the relationship between serogroups, serotypes and subtypes. Using a DNA probe pLUS210 homologous to a repetitive element in the meningococcal genome, we were able to divide strains into a number of RFLP types. B15 :P1.16R strains from two localities of increased endemic disease (Gloucester and Plymouth) gave identical banding patterns (termed 'outbreaktype') suggesting a single clone, possibly of high virulence, may have been responsible for the elevated rate of disease in these districts [10]. The outbreaktype banding pattern was shown to be due to an insertion or deletion event (for simplicity subsequently referred to as a deletion) downstream from the gene (porA) coding for the class 1 OMP. This deletion removed one copy of the repetitive element contained within pUS210; this element was found to be a truncated form of a novel neisserial insertion sequence 181106 [11]. The same deletion was found in strains from the two districts with a high incidence of disease. However, Fox and colleagues [12]. using a random DNA probe, typed a selection of UK meningococcal strains and found that B15: PI. 16R strains were of two distinct RFLP types, with one type including Gloucester and the second type including Plymouth strains. In addition. McGuinness and colleagues [13] identified a point mutation in the N. mreningitidis porA gene coding for one of two variable regions (VR2) determining sero-subtype; this mutation distinguished between B 15: P1. 16R strains which had predominated in Gloucester and Plymouth Health Districts. However the use of any single character for typing strains does not provide reliable phylogenetic data that may be used for examining the genetic relationship between strains. WVe therefore examined a selection of UK meningococcal strains with five further random probes in addition to the repetitive probe pUS210 and used this data to determine the phylogenetic relationships between strains in the UK. MATERIALS AND METHODS

Strains N. mneningitidis G3 (NCIMB 40248) is an outbreak-type strain isolated from the Gloucester Health District. Additional meningococcal strains (Table 1) were supplied by Dr D. Jones, Meningoeoccal Reference Laboratory (MRL), Manchester and Dr R. G. Fallon, Ruchill Hospital, Glasgow. These strains were clinical isolates from a number of locations in the UK and were collected between 1980 and 1991. The strains used for diversity analysis were isolated between 1984 and 1990. DNA manipulations DNA extractions and Southern blotting ofN. as previously described [10].

meningitidis DNA were performed

DNA probes Probes were obtained from N. meningitidis EcoR I genomic libraries constructed

DNA probe analysis of N. meningitidis

229

Table 1. Strain information Straians

Serotyp)e*

13.-: Pl 16 11 1315:PI.16R A( 73 13 r): 1)P1.1(6 1337(6 131 5: P 1. I (i ('242 13 15: P 1. 1I ('24(2 B 1 5: P 1. 1 6 ('457 131: PlP.1 6 ('504 B115: P1.16 (225 134:PI.15 sI 13 15R)1 III1

Isolation Pl ymIlouthi (IIoucester lincoln

Date 04/84 06/84 08/80

I'eterborough 08/81 Leicester

08/82

08/82 08/82 11/82 08/82 1984 1984 1985 1985

(G 7

Cnt

(422

('21)

(424 (G55

Cnt

lBelfast Southenid Shrewsburv Leicester Scotland(l Scotland(l Scotland Scotland Scotland Scotlaind Scotland Scotland Scotland Birminlgham Southampton (loucester (Gloucester (Gloucester (Cloucester (Gloucester

Cnt

Gloucester

06/86

(457

(n

tR NG'15

(Gloucester

04/87

S3 S4

1315R 1B115: Pl.1 1R

85

I14:1~1.151i

134: P1. 114 S6 87 NG4: P1. 15R S8 B34:Pl.15R S9 134: PI. I5R1 El781 lin3t: E1 1. 1514 F971 13 15R (1.S

('4

(467

G'96F (I21)S (41J((i h:5), (

(C497 (49)8

('2:)h

(499

C2a5

('2a,-

(G12: (2a: 1' 1 1. ('I126 B2 hS (I

27

Bh

5

S

(G131 G 132 (G17:3 (4136

B2bS B2bS (C2a: P'l.12R B4:Pl.7S (4148 (C7

(4133,

B1

5.:P11.161(

(4169 B315:P1'.1 6R :1'1.16R G(112 1315 (46I 57 B( P1. 16R GI.71 315-: 1'1.2161 (449 1315:P1 161R (,' 648 BLl5: 1'1.161R 131 PI 1 6R GI 1() N4:

(G108 13 15: E'1. I 6R GPP45 2 B15:P'1.16R B15:L1316R (412 P2

BIS31:P1.16R

B15: PI. 16R

P8

P1) P12

1 5: P1. 16R B1I5:P1 .16R B1 5:P1.16R B15:Pl. 16R B

1988 1988 1988 08.84 08/85 12/8.5 12/85 04/86 06/86 06/86

(Glouc ester

11/87

Gloucester (Gloucester (Gloucester (Gloucester (4loucester (Gloucester

01/89

( Ilotcester

12/89

(Glouecstcr (I4loucester (Gloucester (Gloucester Cloucester (dloucester

12/89 12/89 04/91 04/91 04/91

01 /89

(1/89 01/89 12/89 1 2/89

(louicester

04/91 04/92

Gloucester (Gloucester

04/91 04/91

(loucester

04/91

Gloucester Gloucester GTloucester Gloucester Gloucester (GlouLcester Gloucester (Gloucester Gloucester

04/91 12/89 12/85 01/89 07/86 10/86 10/86 06/89 12/89 01/85 12/85 07/86 11/86 03/87 10/87

Plvmouth Plmouth

P4

1987 1988

Plymouth Plymouth Plymouth Plymouth

Strainis

Serotvpe*

P14 B15: P1.16R P16 B15:P1.16R P18 B15:P1.16R L,299 B15:P1.16R L376 B15: P1.16R J2007 B15:P1.16R J705 B15:PI.16R L744 B115: PI.16R E74 B15:PI.16R K992 B15:P1.16R F921 B115: P1.16R (G1506 B15:P1.16R K1475 B15: P1. 16R G79 B15:P1.16R F133 B15:P1.16R H1793 B15:P1.16R H2347 B15: P1. 16R L274 B15:P1.16R 1,61 B15:P1.16R L273 B15:PI.16R 1,1571 B15:P1.16R 1J334 B15:P1.16R (G190 315PI .16R G 177 132b:Pl.108 (G33 N(G (_34 N(S G35 NG G38 Bnt:P1.12R (484 NGntS (4 107 B4: P1. 15$ G( 110 B2bS (4 119 Bnt: P1.5 (47 1 1315: P1.6 (4106 1348 (4175 B15:P1.16R G37 BnitR

G39

BJ12R

M2 M3 M4 M5 M6 M7 M8 M9 M1O Mlt M12 M13 M14 M15 M16 M17 G74 G87 G28 G83 G85 G86 G S105

B15: PI. 15R B15:P1.16R B15:P1.15R B15 P1.15R B15: P1.15R B15: P1.12R B15:P1.15R B15:P1.15R B15:P1.15R B15:P1.16R B15:P1.16R B15:P1.16R B15:P1.16R B15:P1.16R B15:Pl.16R B15:P1.16R Bnt Bnt: P1.28

29ES B4: P.15S Bnt: PI.5S MAt: '1. 2S B44

Isolation

Plymouth Plymouth Plymouth London London Birmin1gham Birmingham Birmingham Norwich Norwich Westcliffe Westcliffe Liverpool Liverpool Liverpool Liverpool Liverpool Liverpool Liverpool Liverpool Liverpool London Gloucester Gloucester (loucester (loucester Gloucester Gloucester Gloucester Gloucester (floucester Gloucester Gloucester Gloucester

Gloucester Gloucester Gloucester Suffolk Kent Essex Cheshire Somerset London Warwick Dorset Lancashire Cheshire Lancashire Suffolk Sussex Yorkshire LaIncashire London Gloucester Gloucester Gloucester Gloucester Gloucester (loucester Gloucester

D)ate 05/88 11/88 02/89 01/90 01/90 10/88 03/88 08/90 01/84 04/89 10/85 05/86 05/89 01/86 02/85 07/87 11/87 01/90 01/90 01/90 05/90 01/90 04/91 04/91 08/86 08/86 08/86 10/86 09/88 05/89 07/89 12/89 01/87 05/89 04/91 08/89

08/89 10/84 11/84 12/84 01/85 02/85 02/85 03/85 03/85 03/85 04/85 01/86 01/86 01/86 01/86 02/86 02/86 03/86 09/88 08/86 09/88 09/88 (9/88 05/89

H. NI

230

AND OTHERS

Table 1. (cont.) Serotvl) e* Strainis G43 BntS G54 Cnt G53 NGS G42 NG15 G51 NG15S G63 NG15R G64 NGR G32 NGnt:P1.12S.12 G45 B15:P1.16R GIO B15:P1.15R G46 B15:P1.16R G114 B15:P1.16R G26 B15:P1.16R G50 B15:P1.16R G121 B15:P1.16R G123 B15:P1.16R P3 B15:P1.16R P5 B15:P1.16R P7 B15:P1.16R P9 B15:P1.16R Pll B15:P1.16R P13 B15:P1.16R * R.

Isolation

Date

Gloucester 10/86 Gloucester 12/86 Gloucester 12/86 Gloucester 10/86 Gloucester 12/86 Gloucester 07/87 Gloucester 07/87 Gloucester 08/86 Gloucester 04/84 Gloucester 12/85 Gloucester 10/86 Gloucester 10/89 Gloucester 10/86 Gloucester 12/86 Gloucester 12/89 Gloucester 12/89 Plymouth 07/85 Plymouth 07/86 Plymouth 09/86 Plymouth 03/87 Plymouth 04/87 Plymouth 01/88 sulphonamide resistance;

Strainis

Serotvpe* B15:P1.16R

P15 P17 B15:P1.16R P19 B15:P1.16R L107 B15:P1.16R L163 B15:P1.16R J2263 B15:P1.16R F104 B15:P1.16R G2369 B15:P1.16R F242 B15:P1.16R H6 B15:P1.16R Ft 113 1B15:P1.16R K869 B15:P1.16R G919 B15:PI.16R CG54 131-5: PI. 16R4 F485 B135: P1.16R H1525 B15:P1.16R J12063 131;5:PI.16R L443 B15:PI.16R 1,73 1315: Pl .16R 1,1715B1 15: P1. 16R L578 B315:P1.16R 8,

IsolatioIn

Date

Plymouth Plymouth Plymouth

06/88 11/88 03/89

London London Birmingham Birmingham Birmingham Norwich Norwich Westeliffe Westcliffe

01/90 01/90 11/88 01/85 06/86 03/85 01/87 12/85 03/89 02/86 01/86

Liverpool ,iverpool Liverpool Liverpool Liverpool

Liverpool Liverpool Liverpool Londoin

05/85 05/87 08/88 01/90 01/90 01/90 02/90

sulphoiiamide sensitive.

in either pUC18 or pBR322. Randomly selected probes (with insert size) were: pUS250 (4 2 kb), pUS251 (3-2 kb), pUS252 (2 5 kb), pUS253 (6 2 kb) in pUC18 and pUS254 (12 5 kb) in pBR322. Probes were selected for this work on the basis of containing large DNA inserts and in giving polymorphic banding patterns when hybridized to digests of meningococcal DNA in Southern blots. The repetitive probe pUS210 [10] contained a truncated copy of IS1106 [11].

Analysis of RFLP data Banding patterns were coded for each probe using the program ELBAMAP [14] in order to construct a binary code database dependent on the presence or absence of a specific band. This was then used to construct a similarity matrix [15]. The matrix was analyzed using the KITSCH program from the PHYLIP package of phylogeny inference programs using the SEQNET node at the SERC Daresbury Laboratory, to produce a phylogenetic tree. The KITSCH program uses the Fitch-Margoliash method [16, 17] to find the tree which minimizes: Sum of squares

EE (obs-exp)2 j

obs2

where obs is the observed distance between species i and j and exp is the expected distance, computed as the sum of the lengths (amounts of evolution) of the segments of the tree from species i to species j. The mean isolation date for strains isolated from each health district was calculated as follows: X [Y+N(D/365)]

DNA probe analysis of N. meningitidis

231

kb 23-1 94

66 4.4

23 2-0

1 2 3 4 5 6 7 8 91011121314151617181920 Fig. 1. Representative Southern blot demonstrating banding patterns obtained after probing Dra I digested N. meningitidis DNA with pUS254. Lanes: t, AHind III size markers; 2, G3 (B15:P1.16); 3, Pt (1315:PI.16); 4, G74 (Bnt); 5, G83 (B4:PI.1.5): 6. G85 (Bnt:PI.5); 7, G86 (Bnt:P1.12); 8. G105 (B34); 9, 043 (Bnt); 10. 028 (29E); 1, GO54 (Cnt); 12, G32 (NCGnt: Pl.12); 13, G53 (NG); 14, G42 (NG15); 15, G51 (NG15), 16, G63 (NC' 15): 17. G64 (NG): 18, G67 (NG 15) ; 19, G 120 (NG4: P 1.16) ; 20, APst I size markers.

where Y is the year in which the strain was isolated. D is the number of days elapsed in that year before the strain was isolated, and N is number of strains studied in each health district. The differences in the means were analysed by t test. Strain diversity (h) was estimated using the equation.

n(1- X2)

(n-i) Standard errors due to sampling were calculated by the equation [18]: Sd =

(2n(2

-

) {2(2n -2) [X3-(Xi)2] ± [X2-

where X is the proportion of each RFLP type in an area and n is the number of strains studied in each area. RESULTS

Preliminary data obtained from DNA probes demonstrated that some probes gave identical banding patterns for closely related strains irrespective of the digest

232

H. NI

AND OTHERS

M3,M13.MI6,M17,A273,B376(B15:PI.I6)

S5,S6,S8(B4:P1,1S),S7(NG4:PI.15) M7(BI5:PI.12),SI, S2 (B15) P1 (BI5:PI.16)

A

_C346 (BI5:PI.16) G190*(B15:PI.16)

{C225 (B4:PI.15) Mr, M2, M4, M6, M9 (BIS:PI.15) MIO(BIS:PI.I5) M5(BI5:PI.15)

Ml1I,M14-(BI5:PI.I6) EC-457,C242,S4*(B15:PI.16)

c

S3(B15) * G45*,GI55*,GI57',GI75

(BIS:PI.16)

_G7'(C)MI5,S4(Bi5:PI.16)

G3*,G133 (Bi5:P1.16)G8*(C) G63-(NGI5) G64 (NG)

G38(BntPI.12) M121(B15:P1.16) F971 (B15) G84(NGnt)

UG37(Bnt)

G33(NG) G74(Bnt) E781 (Bnt:Pi,15) G83(B4:PI,15) G43(Bnt)

G87(Bnt:Pi.2)

S9(B4:PI.15) G21(29E)15) ~~~~~~~~~~~~~~~~~~G42(NG G53(NG)

a

a

G54(CO;)

G71(BI5:PI.6) G96(C2b)

G97(C2b:Pl.2) G55(Cni) G IO(B2b) G119(Bnt) G132(B2b) G126(B2b) G127(B2b) G34(NG)

d

G9i.G99,GI23(C2a) GS7(Cnt) G2(C2b) G177(B2b:PI.10) G171 (CMaPI.2)

G162(C2a:P1.2)

I l I _

G173(C2a:Pi.12) (NG 15) ~~~~~~~GSI Gl31 (B2b) .~~~~~~~~~~~~~~ ~~~~~~~~~~G86(B4:PIS)

~~~~~~~~~~~~~~~~~~~~G24(Cnt) G136(B4:PI.7) G169(Bntpl.16)

r b

r

1 I I I

~~~~~~~~~~~~~~~G12D(NG4:PI.16)

II e

~~~~~~~~~~~~~~~G67(NG15) G85 (BntPI.5) GIO7(B4:PI.15) ~~~~~~~~~~~~~~G39(BI2Z ~~~~~~~~~~~~~~~~G35SNG) ~~~~~~~~~~~~~~~~~~GI48(C7)

lIG17(Cut)

G32(NGntP1.12)

04

03

02

0.1

0

Genetic distance Fig. 2. (A) Phylogenetic relationship between N. meningitidis strains derived using randomly selected probes. Strains designation and serotypes are shown. Strain G3 was from the Gloucester Health District; strain P1 was from the Plymouth Health District. G3 is indistinguishable to 16 additional strains from the Gloucester District and one strain from the Plymouth District. Strain Pl was identical to 17 strains from the Plymouth District and one strain from the Gloucester District. Strains designated outbreak-type (with pUS210/EcoRI) are indicated *.

I~,l ~ G162(Ca:9P8I,.)G123(Ca

DNA probe analysis of N. meningitidis

233

S7(NG4:PI1j5),S6,S8(B4:P1,15) MM17A273(B15:P1.16)

S3(B15)

M3(Bl5:PI,16)

B

_ ,{

M12

15TPI,16)

~~~C242WBl5:P.16) M7(BI5:P1.12)

M14*(BIS:PI.16)

MIl-(Bl5:PI.16)

Pt*(BlS:P.16),S2'(B15)

S4-(B15:PI.16) SI (B15)

G155-, G45*(Bl1:PI,16)G71(C),G64 '(NG) G31,G1331(BI5:PI.16),G8'(C) G190(BOT:PI.16) C457(B15:PI.16)

C

G63'(NG15)

G1751(BI5:PI.16)

G157*(BI5:PI.16)

C504(B15:P1.16) M16(B15:P1.16) M13(B15:P1.16)

M15*(B15:PI.16) B376(B15PI.16)

M9(B15:PI.15)

M8(B15:PI.15)

M5(B5:P1.15)

M8 (B15:P1.15) S5(B4:PI.15)

(M246(B15:PI.16) M6(B1S:PI.15)

I846 (B1TP1.16)

C725(B4:PI.15)

M10O(BIS:Tl.15

G37(Bnt) P971 (B15P1) G38(BntPl. 12) E781 (BntP1.15)

~~~~~~~~~~~~~~~~~G43(Bot) G83(B4:PI.15) G74(BPt)

2|

S9(B4:P.15)

G87(BntPl.2) G84(NGnt) G71(BIS:PI.6) G32(NGn:P1.12) G148(CT) G17(Cnt)

_

a

G28('9E) G35 (NG) G33(NG)

~~~~~~~~~~~~~~~G120(NG4:Pl.16) G67(NG15) ~~~~~~~~~~~~~~~~~~G85(BntPl.15) ___ |G 51 (NGI5) ~~~~~~~~~~~~~~~~G169(BntLPI.16)

&

d

E

I l

G86(BW:PI.15) G24 (Cnt)

,

e

~~~~~~~~~~~~~~~~~G39(B12) 'G106(B4) (B4) ~~~~~~~~~~~~~~~~~G105

I

4

GI131 (B2b)

I

~~~~~~~~~~~~~~~~~~~~~~~G107(B4:PI.15) ~~~~~~~~~~~~~~~~~~~~~~G136(B4:PI.7)

I

I

G54(Cnt)

l

(CMafll.) ~~~~~~~~~~~~~~~~~~~~G171

~~~~~~~~~~G173(C2a:Pl.12) ~~~~~~~~~~~~G57(Cat) ~~~~~~~~~~~~~~~~~~~GII9(Bnt),GII0(B2b) ~~~~~~~~~~~~~~~~G96(C2b),97(C2b:P.2) G~~~~~~~~~~~~~~~~~~132 GlZ7(Bb))

I

*

I e l §

Phylogenetic and epidemiological analysis of Neisseria meningitidis using DNA probes.

The genetic relationships between various serotypes and serogroups of meningococcal strains were investigated by restriction fragment-length polymorph...
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