Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene

Abstract

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface-exposed amino acid loops of the mature PorB protein) ranged from 5 to 11 and from 8 to 39, respectively. Among porB1a isolates (n = 22) and porB1b isolates (n = 65), 22 and 64 unique genovars, respectively, were identified. All isolates were typeable. The present results provide evidence of a high discriminatory ability, practically the same as that for sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.

FIG. 1.

Sequence variants and polymorphic nucleotide sites in each of the highly polymorphic segments of the porB1a alleles, encoding amino acid loops of the mature PorB1a protein (29), of N. gonorrhoeae isolates (n = 22). The nucleotide present at each polymorphic site among all of the sequence variants is shown for sequence variant 1 in each loop segment. For the other sequence variants, those sites that differ are shown. Dots indicate identity with sequence variant 1, and underlining indicates synonymous mutations. Nucleotide sites that are conserved in all sequence variants are excluded. The length of each loop segment is included, and the sites are numbered above in vertical format based on the nucleotide numbering of a porB1a gene (GenBank accession no. J03029).

FIG. 2.

Sequence variants and polymorphic nucleotide sites in each of the highly polymorphic segments of the porB1b alleles, encoding amino acid loops of the mature PorB1b protein (29), of N. gonorrhoeae isolates (n = 65). The nucleotide present at each polymorphic site among all of the sequence variants is shown for sequence variant 1 in each loop segment. For the other sequence variants, those sites that differ are shown. Dots indicate identity with sequence variant 1, dashes represent alignment gaps (due to indels in some of the variants), and underlining indicates synonymous mutations. Nucleotide sites that are conserved in all sequence variants are excluded. The length of each loop segment, according to the multiple-sequence alignment, is included, and the sites are numbered above in vertical format based on the nucleotide numbering of a porB1b gene (GenBank accession no. J03017).

FIG. 3.

Pyrogram of the sequence analysis of the loop 8 segment of the porB1b allele (sequence variant 1, reverse strand), encoding surface-exposed amino acid loop 8 of the mature PorB1b protein (29), in N. gonorrhoeae. The nucleotide sequences of the entire loop 8 segment, as interpreted by the PSQ SQA (version 2.0) software and after manual editing are shown above the pyrogram. The discrepancy is indicated by underlining in the manually edited sequence and by an arrow in the pyrogram. The cyclic dispensation order of the deoxynucleoside triphosphates is indicated below the pyrogram.