Irish strains of Neisseria meningitidis: characterisation using multilocus sequence typing

British Journal of Biomedical Science,  2003  by Murphy, K M,  O'Donnell, K A,  Higgins, A B,  O'Neill, C,  Cafferkey, M T

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ABSTRACT

A total of 56 Neisseria meningitidis strains are analysed using multilocus sequence typing (MLST). Twenty-nine distinct sequence types (STs) were identified, eight of which were new. Four known hypervirulent clones - ST-11 (electrophoretic type [ET]-37) complex, ST-44 complex (lineage 3), ST-32 (ET-5) complex and ST-8 complex (cluster A4) - were identified by MLST in 35 disease-associated and four carrier strains. Two other clones (ST-22 complex and ST-269 complex) were identified in nine disease-associated and one carrier strain. The remaining strains were heterogeneous. Additional sequencing within the FumC gene further distinguished the ET-15 clone within the ST-11 (ET-37) clonal complex. This resolution of isolates into genetic clones by MLST enhances the more traditional techniques of serotyping and serosubtyping. The data obtained established that hyperendemic meningococcal disease in Ireland could be attributed to strains belonging to four major hypervirulent clones, all of which account for elevated levels of disease worldwide. The extra information provided by MLST will be used to study the population structure and epidemiology of N. meningitidis and will allow a comparison of Irish strains with those circulating globally.

KEY WORDS: Bacterial typing techniques.

Meningococcal infection.

Molecular sequence data.

Neisseria meningitidis.

Sequence analysis.

Introduction

Neisseria meningitidis is a significant global pathogen that causes invasive infection. Invasive meningococcal disease (IMD) is hyperendemic in Ireland, with a laboratory-confirmed invasive infection rate of 13/100,00 population in the epidemiological year 1999/2000.1 This represents one of the highest rates of meningococcal disease in western Europe, where the disease is usually associated with a small number of hypervirulent lineages of serogroup B or C.

Analysis by multilocus enzyme electrophoresis (MLEE) has been used to subdivide meningococcal isolates of all groups into electrophoretic types (ETs).2,3 In particular, most cases of serogroup C disease are caused by ET-37 complex meningococci and derivatives of the cluster A4, whereas serogroup B disease is most frequently caused by meningococci of the lineage 3 or ET-5 complex.4

IMD has many epidemiological manifestations, each of which can be associated with different meningococcal clones. Epidemics and outbreaks are usually associated with organisms from uniform clonal groups, whereas sporadic disease is more likely to be associated with a more diverse group of organisms. Detailed characterisation of N. meningitidis isolates into their correct clonal groups is essential in order to investigate outbreaks, to identify virulent strains, and to trace the genetic links between isolates.

The typing methods currently in use, such as serological typing, MLEE and pulsed-field gel electrophoresis (PFGE), have proved unsuitable for rapid and sensitive typing of meningococci, highlighting the need for improved molecular typing methods. Multilocus sequence typing (MLST), an adaptation of MLEE, has become a widely used method that unambiguously defines relatedness among disease and carriage isolates.5-10 In addition, MLST has identified differences between strains that other molecular methods have failed to detect.11

MLST is based on DNA sequence variation in specific regions of seven housekeeping genes that range from 433-501 bp in length. For each gene fragment, different sequences are assigned as distinct alleles, and each isolate is defined by the combination of alleles at each of seven housekeeping loci. This is known as the allelic profile or sequence type (ST). The STs were assigned to lineages using the BURST programme (http://neisseria.mlst.net).

Within a clonal complex, additional sequencing may be required to further distinguish between clones. One group C clone of the ST-11 (ET-37) complex, referred to as ET-15, is a particularly virulent clone that shows high attack and fatality rates.12,13 MLEE identifies ET-15 meningococci by detecting a point mutation at position 640 within the FumC gene.14 The original FumC gene fragment commonly sequenced by MLST fails to include this mutation; therefore, further sequencing is required to detect the fumC^sub 640^ A substitution.

This study aims to utilise MLST to identify and differentiate between the major clones of N. meningitidis circulating in Ireland. In the case of the ET-37 complex, additional sequencing to detect the fumC^sub 640^ A substitution is performed. This is the first comprehensive, standardised molecular characterisation of Irish meningococcal isolates.

Materials and methods

Bacterial strains

In total, 56 N. meningitidis isolates were selected for inclusion in this study. All had been received at the Irish Meningococcal and Meningitis Reference Laboratory (IMMRL) between 1997 and 2000. The study included 50 isolates from cases of epidemiologically unrelated IMD and six carrier isolates.

The disease-associated strains comprised 22 patient isolates of group C, 23 group B, three group W135, and one each of group X and group Y. The carrier isolates were four group B, one group C and one non-groupable. Details of these isolates are shown in Table 1.