Species status of Neisseria gonorrhoeae: evolutionary and epidemiological inferences from multilocus sequence typing

Abstract

Background: Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species.

Results: A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters.

Conclusion: The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present.

Figure 1

Polymorphic sites in concatenated gonococcal housekeeping gene sequences. The polymorphic sites are shown for each concatenated sequence of seven housekeeping gene fragments. The positions of the polymorphic sites, and the genes in which they occur are indicated along the top.

Figure 2

The temporal and geographic distribution of 66 STs described for N. gonorrhoeae. A neighbour-joining tree was constructed from the concatenated MLST sequences from 66 STs obtained from 149 gonococcal isolates. Each circle denotes a particular ST and the countries and regions of isolation, if known are shown. Green circles indicate STs from isolates collected in Liverpool, UK between 1981 and 1991, pink circles indicate STs from isolates collected in Liverpool, UK between 2000 and 2001 and blue circles indicate STs from isolates collected elsewhere, including geographically and epidemiologically unlinked isolates. The two closely related STs obtained from the AHU- isolates are shown.

Figure 3

MLST data resolves N. gonorrhoeae, N. meningitidis and N. lactamica into three clusters. (a) A neighbour-joining tree was constructed from the concatenated MLST sequences for each species. (b) A splits graph was constructed from the concatenated MLST sequences for each species. Bootstrap values are indicated for the main branches (2000 replications).

Figure 4

Alleles from a single locus (gdh) resolved species specific clusters. A neighbour-joining tree was constructed from the gdh allele sequences for each species. Bootstrap values are indicated for the main branches (2000 replications).