Multidrug-resistant Neisseria gonorrhoeae infection in heterosexual men with reduced susceptibility to ceftriaxone, first report in Thailand

Abstract

The global rapid emergence of azithromycin/ceftriaxone resistant Neisseria gonorrhoeae threatens current recommend azithromycin/ceftriaxone dual therapy for gonorrhea to ensure effective treatment. Here, we identified the first two N. gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Among 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, two isolates (NG-083 and NG-091) from urethral swab in male heterosexual patients had reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L). Both were multidrug resistant and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). NG-083 and NG-091 remained susceptible to azithromycin (MIC of 1 mg/L and 0.5 mg/L, respectively). Reduced susceptibility to ceftriaxone was associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region. NG-083 belonged to sequence type (ST) 7235 and NG-091 has new allele number of tbpB with new ST. Molecular docking revealed ceftriaxone weakly occupied the active site of mosaic XXXIV penicillin-binding protein 2 variant in both isolates. Molecular epidemiology results revealed that both isolates display similarities with isolates from UK, USA, and The Netherlands. These first two genetically related gonococcal isolates with decreased ceftriaxone susceptibility heralds the threat of treatment failure in Thailand, and importance of careful surveillance.

Figure 1

The amino acid sequence alignments of different PBP2 patterns of 9 N. gonorrhoeae isolates including nonmosaic PBP2 pattern II, VII, XIV, XIX, XVI, XVIII, XXXXIV, mosaic XXXIV, and new mosaic pattern (GC-013) were compared with the wild type PBP2 of N. gonorrhoeae strain LM306 (GenBank accession no. AAA25463).

Figure 2

The structure of mosaic XXXIV PBP2 variant of N. gonorrhoeae clinical isolate (a) showing the location of important mutations and the active site sequence motifs, enlarge of (b) C terminal domain (c) N terminal domain (d) Site of phosphorylation. Mutations were modeled by PyMOL [DeLano, 2002] (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.).

Figure 3

Molecular docking of ceftriaxone in the active site of a) wild type PBP2 and b) mosaic XXXIV PBP2 variant. The standard macromolecule was extracted from rcsb.org/ (PDB: 3EQU) and the mutations were modeled by PyMOL [DeLano, 2002] (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrödinger, LLC.).

Figure 4

Phylogenetic analyses based on core genome from draft genomes of Neisseria gonorrhoeae NG-083 and NG-091 clinical isolated in Thailand and from varying N. gonorrhoeae WGS investigations conducted elsewhere (available on NCBI database) were determined for the number of single nucleotide polymorphisms (SNPs) by Core-Genome SNP Analysis. C1-C4 represent major clusters.

Figure 5

Time-kill curve of ceftriaxone plus azithromycin against N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone (a) NG-083 and (b) NG-091.

Figure 6

Confocal imaging analysis (3D and cross sectional) of biofilm structure of N. gonorrhoeae isolates (a,b) NG-083 and (c,d) NG-091treated with MIC concentration (0.125 mg/L) of ceftriaxone.

Figure 7

Effects of different concentration of ceftriaxone on N. gonorrhoeae isolates (n = 2). Isolate NG-083; (a) in vitro biomass (b) in vitro Live/Dead cell ratio (c) in vitro bio-volume inhibition and isolate NG-091; (d) in vitro biomass (e) in vitro bio-volume inhibition (f) in vitro Live/Dead cell ratio.