Predominant porB1A and porB1B genotypes and correlation of gene mutations with drug resistance in Neisseria gonorrhoeae isolates in Eastern China

Abstract

Background: Variations of porB1A and porB1B genes and their serotypes exist in Neisseria gonorrhoeae isolates from different geographical areas, and some site mutations in the porB1B gene correlate with drug resistance.

Methods: The β-lactamase production of N. gonorrhoeae isolates was determined by paper acidometric test and nitrocefin discs. The porB1A and porB1B genes of 315 non-penicillinase-producting N. gonorrhoeae (non-PPNG) strains were amplified by PCR for sequencing to determine serotypes and site mutations. A duplex PCR was designed to simultaneously detect both porB1A and porB1B genes. Penicillin and tetracycline resistance was assessed by an in vitro drug sensitivity test.

Results: Of the N. gonorrhoeae isolates, 31.1% tested positive for porB1A and 68.9% for porB1B genes. All the 98 porB1A+ isolates belonging to IA6 serotype with either no mutation at the 120 and 121 sites (88.8%) or a D120G (11.2%) mutation and were no resistance to both penicillin and tetracycline. Among the 217 porB1B+ isolates, 26.7%, 22.6% and 11.5% belonged to IB3, IB3/6 and IB4 serotypes, respectively. Particularly, two novel chimeric serotypes, IB3/6-IB2 and IB2-IB4-IB2, were found in 77 and 8 porB1B+ isolates. Two hundred and twelve (97.7%) of the porB1B+ isolates were presented G120 and/or A121 mutations with 163 (76.9%) at both sites. Interestingly, within the 77 porB1B+ isolates belonging to IB3/6-IB2 serotype, 15 were discovered to possess novel deletions at both A121 and N122 sites. All the replacement mutations at these sites in PorB1B were correlated with resistance and the deletion mutation showed the highest resistance.

Conclusion: N. gonorrhoeae isolates circulating in Eastern China include a sole PorB1A serotype (IA6) and five PorB1B serotypes. Multiple mutations in porB1B genes, including novel A121 and N122 deletions, are correlated with high levels of penicillin and tetracycline resistance.

Figure 1

Amplicons of entire or partial porB1A and porB1B genes. Note: Lane M: DNA marker (BioColor); Lanes 1 and 2: amplicons of entire porB1A gene (981 bp) and porB1B gene (1044 bp), respectively; Lanes 3 and 4: amplicons of partial porB1A gene (520 bp) and partial porB1B gene (583 bp and 201 bp) by duplex PCR, respectively; Lane 5: blank control.

Figure 2

Amino acid sequences from porB1A genes of N. gonorrhoeae isolates. Note: (IA6): reported amino acid sequence of IA6 serotype (GenBank accession No: L19962); (1) and (2): PorB1A sequences from 56 and 23 gonococcal isolates, respectively; (3) to (5): PorB1A sequences from 8 gonococcal isolates; (6) PorB1A sequences from 11 gonococcal isolates. Underlined areas indicate positions of primers.

Figure 3

Amino acid sequences from porB1B genes of N. gonorrhoeae strains. Note: (IB3), (IB3/6), (IB2) and (IB4): reported PorB1B sequences belonging to serotypes IB3 (GenBank accession No: U75639), IB3/6 (GenBank accession No: U75641), IB2 (GenBank No: U75640) and IB4 (GenBank No: AF090797), respectively; (1): PorB1B sequence from 58 gonococcal isolates referring to IB3 serotype; (2): PorB1B sequence from 49 gonococcal isolates referring to IB3/6 serotype (D means the 27 isolates with A121D mutation, the 19 isolates with A121G mutation and the remaining 3 isolates with no mutation at the A121 site); (3): PorB1B sequence from 62 gonococcal isolates referring to IB3/6-IB2 chimeric serotype without A121 and N122 deletion (K means the 53 strains with G120K mutation and the remaining 9 strains with G120N mutation); (4): PorB1B sequence from 15 gonococcal isolates referring to IB3/6-IB2 chimeric serotype with A121 and N122 deletions; (5): PorB1B sequence from 25 gonococcal isolates referring to IB4 serotype; (6): PorB1B sequence from 8 gonococcal isolates referring to IB2-IB4-IB2 chimeric serotype. Underlined areas indicate the positions of primers. The signal "/" means lack of the amino acid residue.