Analysis of phenotypic variants of the serogroup C ET-15 clone of Neisseria meningitidis by pulsed-field gel electrophoresis.

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1 JCM Accepts, published online ahead of print on 9 May 2007 J. Clin. Microbiol. doi: /jcm Copyright 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Analysis of phenotypic variants of the serogroup C ET-15 clone of Neisseria meningitidis by pulsed-field gel electrophoresis. In Canada, two waves of hyperendemic meningococcal diseases were documented in the last 20 years. Both were due to a unique clone of serogroup C N. meningitidis designated by MLEE as ET-15 (1). When this clone first appeared, it was characterized by the antigenic formula C:2a:P1.5,2 (1). In 2001, antigenic variants of this ET-15 clone characterized as C:2a:P1.7,1 (2) or C:2a:P1.5 (3) emerged to cause outbreaks (4). In this study, we examined whether PFGE can be used as a discriminatory tool to differentiate between the antigenic variants C:2a:P1.7,1, C:2a:P1.5, and C:2a:P1.5,2 of the ET-15 clone of N. meningitidis. Eleven serogroup C ET-15 N. meningitidis isolates from invasive meningococcal disease (IMD) cases were selected for this study. Ten of the eleven serogroup C isolates were identified as serotype 2a (1). DNA sequencing of the porb gene of the nontypeable isolate identified it as a serotype 2a mutant containing a previously described mutational hotspot (10). There were four different combinations of serosubtype antigens observed for these eleven isolates: five with the P1.5 antigen, three with the P1.7,1 antigens, two with the P1.5,2 antigens, and one with the P1.2 antigen. The pora genes of these strains were sequenced and their PorA VRs were summarized in Figure 1. MLST was performed according to the established method by Maiden et al. (12) and isolates were assigned sequence types (STs) according to the Neisseria MLST 1

2 website ( An additional region in the fumc gene was amplified to determine the presence of a G to A point mutation at position 360, characteristic of ET-15 strains (11).All eleven isolates contained this particular point mutation and were therefore classified as ET-15 strains. PFGE analysis of the eleven isolates that represented the three antigenic variants, C:2a:P1.2,5, C:2a:P1.7,1 and C:2a:P1.5, was performed, as described by Tyler and Tsang (23). Restriction enzyme-digestion of genomic DNA with Nhe1 (data not shown) and Spe1 indicated that serogroup C ET-15 variants had overall similarity and were difficult to distinguish based on the banding patterns they exhibited (Figure 1). Using Spe1, PFGE pattern I was unique to isolates with the serosubtype P1.5 antigen. However, pattern II was common to all three ET-15 antigenic variants, while pattern III was common to C:2a:P1.2,5 and C:2a:P1.7,1 isolates, and pattern IV was common to C:2a:P1.2,5 and C:2a:P1.5 isolates. Despite the widespread acceptance of the PFGE method (13, 24), the data presented in this study, showing an apparent lack of correlation between isolates DNA fingerprints and their antigenic profiles, serves to illustrate a potential limitation of PFGE in the analysis of N. meningitidis strains for molecular epidemiology studies of IMD. Nevertheless, for localized outbreak analysis, PFGE is still a very useful tool to identify strains linked to a common source (15,20). In summary, a number of typing tools, including both phenotypic and genotypic methods, should be used in combination with 2

3 carefully documented epidemiological information for surveillance and analysis of meningococcal disease. (480 words) We would like to thank the Directors and staff of Provincial Public Health Laboratories for providing strains for this study. We thank Averil Henderson and Jan Stoltz for the serotyping and PFGE data and the DNA core facility at the Public Health Agency of Canada s National Microbiology Laboratory for DNA sequencing work. This publication made use of the Neisseria Multi Locus Sequence Typing website ( neisseria/) developed by Keith Jolley and Man-Suen Chan and sited at the University of Oxford. The development of this site has been funded by the Wellcome Trust and European Union. Marissa L. Cameron and Raymond S. W. Tsang * Vaccine Preventable Bacterial Diseases, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada R3E 3R2. * Phone: (204) Fax: (204) Raymond_Tsang@phac-aspc.gc.ca 3

4 References 1. Ashton, F. E., J. A. Ryan, A. Borczyk, D. A., Caugant, L. Mancino, and D. Huang Emergence of a virulent clone of Neisseria meningitidis serotype 2a that is associated with meningococcal group C disease in Canada. J. Clin. Microbiol. 29: Law, D. K., A. M. Henderson, R. S. Tsang DNA Sequence analysis of the PorB protein of nonserotypeable serogroup C ET-15 meningococci suggests a potential mutational hot spot on their serotype antigens. J. Clin. Microbiol. 42: Maiden, M.C.J., J.A. Bygraves, E. Feil, G. Morelli, J.E. Russell, R. Urwin, Q. Zhang, J. Zhou, K. Zurth, D.A. Caugant, I.M. Feavers, M. Achtman, and R.G. Spratt Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci., 95: Patrick, D. M., S. Champagne, S. H. Goh, G. Arsenault, E. Thomas, C. Shaw, T. Rahim, F. Taha, M. Bigham, V. Dubenko, D. Skowronski, and R. C. Brunham Neisseria meningitidis carriage during an outbreak of serogroup C disease. Clin. Infect. Dis. 37: Popovic T., S. Schmink, N.A. Rosenstein, G.W. Ajello, M.W. Reeves, B. Plikaytis, S.B. Hunter, E.M. Ribot, D. Boxrud, M.L. Tondella, C. Kim, C. Noble, E. 4

5 Mothershed, J. Besser, and B.A. Perkins Evaluation of pulsed-field gel electrophoresis in epidemiological investigations of meningococcal disease outbreaks caused by Neisseria meningitidis serogroup C. J. Clin. Microbiol. 39: Tsang, R.S.W., L. Kiefer, D.K.S. Law, J. Stoltz, R. Shahin, S. Brown, and F. Jamieson Outbreak of serogroup C meningococcal disease caused by a variant of Neisseria meningitidis serotype 2a ET-15 in a community of men who have sex with men. J. Clin. Microbiol. 41: Tsang, R. S. W., C. M. Tsai, P. Zhu, L. Ringuette, M. Lorange, and D. K. S. Law Phenotypic and genetic characterization of a unique variant of serogroup C ET-15 meningococci (with the antigenic formula C:2a:P1.7,1) causing invasive meningococcal disease in Quebec, Canada. J. Clin. Microbiol. 42: Tsang, R. S. W., D. K. S. Law, A. M. Henderson, M. L. Blake, and J. Stoltz Increase in serogroup C meningococcal disease in Canada is associated with antigenic changes in the protein antigens of the ET-15 clone of Neisseria meningitidis. J. Infect. Dis. 194: Tyler, S., and R. Tsang Genetic analysis of Canadian isolates of C:2a:P1.2,5 and B:2a:P1.2,5 Neisseria meningitidis strains belonging to the hypervirulent clone of ET-15. Can. J. Microbiol. 50:

6 10. Tyrrell, G. J., L. Chui, M. Johnson, N. Chang, R. P. Rennie, J. A. Talbot, and The Edmonton Meningococcal Study Group Outbreak of Neisseria meningitidis in Edmonton, Alberta, Canada. Emerg. Infect. Dis. 8: Vogel, U., H. Claus, M. Frosch, and D. A. Caugant Molecular basis for differentiation of the ET-15 clone within the ET-37 complex of Neisseria meningitidis. J. Clin. Microbiol. 38:

7 Figure 1. Eleven PFGE profiles of Spe1-digested DNA (using Dice coefficient with a position tolerance 1.5% of and optimization of 2.0% on BioNumerics 3.5 software) representing the three Neisseria meningitidis serogroup C ET-15 antigenic variants C:2a:P1.5,2, C:2a:P1.7,1, and C:2a:P

8 96Year Antigens VR1 VR2 VR3 Pattern 2002 C:2a:P C:2a:P I 2000 C:(2a):P1.(5),2* C:2a:P C:2a:P1.7, II 2004 C:2a:P C:2a:P1.7, C:2a:P1.7, III 2002 C:2a:P1.5, C:2a:P C:2a:P1.5, IV 98 * The phenotype of this strain was C:NT:P1.2; antigens in brackets were deduced by DNA sequencing of the serotype and serosubtype antigen genes. This serotype 2a strain has a single non-synonymous point mutation that led to the non-serotypeable phenotype (2). 100 PorA 15