New Ceftriaxone- and Multidrug-Resistant Neisseria gonorrhoeae Strain with a Novel Mosaic penA Gene Isolated in Japan

Abstract

We have characterized in detail a new ceftriaxone- and multidrug-resistant Neisseria gonorrhoeae strain (FC428) isolated in Japan in 2015. FC428 differed from previous ceftriaxone-resistant strains and contained a novel mosaic penA allele encoding a new mosaic penicillin-binding protein 2 (PBP 2). However, the resistance-determining 3′-terminal region of penA was almost identical to the regions of two previously reported ceftriaxone-resistant strains from Australia and Japan, indicating that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread.

TEXT

Ceftriaxone is the last remaining option for empirical first-line antimicrobial monotherapy of gonorrhea, but the evolving resistance in Neisseria gonorrhoeae threatens its use, i.e., in monotherapy and in the dual-therapy regimens together with azithromycin (1–3). Four different ceftriaxone-resistant N. gonorrhoeae strains previously isolated in Japan (H041 [4] and GU140106 [5]), France (6) and Spain (F89 [7]), and Australia (A8806 [8]) have been characterized in detail. None of these strains have spread widely nationally or internationally. However, once a ceftriaxone-resistant gonococcal strain spreads, gonorrhea control will become exceedingly difficult. Consequently, dual antimicrobial therapy, mainly ceftriaxone and azithromycin, is now recommended in Europe, the United States, Canada, and Australia, which hopefully will mitigate the development of antimicrobial resistance (AMR) and, at a minimum, the spread of AMR strains (1). It is a great concern that frequently occurring intraspecies and interspecies DNA transfer among Neisseria spp. continues to develop new mosaic penA alleles encoding novel penicillin-binding protein 2 (PBP 2), which is the main lethal target for all β-lactam antimicrobials, resulting in ceftriaxone resistance in N. gonorrhoeae (1–3, 9, 10).

We report a new ceftriaxone- and multidrug-resistant N. gonorrhoeae strain (FC428) isolated in January 2015 in Osaka, Japan, from a male urethritis patient, who was successfully treated with 2 g of spectinomycin intramuscularly at his first attendance at a sexually transmitted disease (STD) clinic. The patient was in his twenties. No information regarding the sexual orientation of the patient or sexual contacts were available.

FC428 was cultured on modified Thayer-Martin medium and species verified using Gonochek-II (TCS Biosciences Ltd., Buckingham, United Kingdom) and the HN-20 rapid system identification test (Nissui, Tokyo, Japan). A nitrocefin test (Thermo Scientific Yokohama, Japan) showed that FC428 was a penicillinase-producing N. gonorrhoeae (PPNG) strain. According to Etest (bioMérieux, Marcy l'Etoile, France), FC428 had a ceftriaxone MIC of 0.5 μg/ml, i.e., was resistant according to EUCAST breakpoints (www.eucast.org) and nonsusceptible according to CLSI breakpoints (www.clsi.org), which only state susceptible or nonsusceptible. FC428 was additionally resistant to cefixime (MIC, 1 μg/ml), benzylpenicillin (MIC, >32 μg/ml), and ciprofloxacin (MIC, >32 μg/ml) but susceptible to spectinomycin (MIC, 8 μg/ml) and azithromycin (MIC, 0.25 μg/ml) (www.clsi.org and www.eucast.org) (Table 1).

TABLE 1.

Comparison of characterized ceftriaxone-resistant Neisseria gonorrhoeae strains

Characteristic	FC428	GU140106 (5)	A8806 (8)	F89 (6, 7)	H041 (4)
Location, yr of isolationa	Japan, 2015	Japan, 2014	Australia, 2013	France and Spain, 2010	Japan, 2009
MIC (μg/ml)
Ceftriaxone	0.5	0.5	0.5	1–2	2–4
Azithromycin	0.25	0.5	0.25	1	1
Spectinomycin	8	32	≤64	16	16
MLSTb	1903	7363	7363	1901	7363
NG-MAST	3435	6543	4015	1407	4240
porB	1053	3854	1059	908	2594
tbpB	21	10	10	110	10
Key alternations in PBP2	A311V, T483S	A311V, T483S	A311V, T483S	A501P in mosaic PBP 2 XXXIV	A311V, T316P, T483Sc

^aMLST, multilocus sequence typing; NG-MAST, N. gonorrhoeae multiantigen sequence typing.

^bMLST ST1903 (abcZ126, adk39, aroE67, fumC157, gdh148, pdhC153, and pgm65) differs with two and three loci from ST7363 (abcZ59, adk39, aroE67, fumC78, gdh148, pdhC153, and pgm65) and ST1901 (abcZ109, adk39, aroE170, fumC111, gdh148, pdhC153, and pgm65) (differences are underlined), respectively.

^cKey amino acid alternations causing the ceftriaxone resistance (15).

Multilocus sequence typing (MLST) and N. gonorrhoeae multiantigen sequence typing (NG-MAST) were performed as previously described (11–13). FC428 belonged to MLST sequence type 1903 (ST1903) and NG-MAST ST3435, which differ from the previously described ceftriaxone-resistant gonococcal strains (4–8). For example, MLST ST1903 differs by two and three loci from ST7363 and ST1901, respectively (Table 1). In our laboratory, among all Japanese gonococcal isolates examined using MLST (n = 1,327) and NG-MAST (n = 1,476), no ST1903 or ST3435 (porB1053 or tbpB21) strain, respectively, has been found. However, MLST ST1903 strains have been identified, for example, in 2005 to 2007 in Thailand among PPNG strains (14). Notably, FC428 is also a PPNG with bla_TEM-135. Regarding NG-MAST, porB1053 has not been found in our collection of Japanese gonococcal isolates, although tbpB21 is the fifth most prevalent allele (111 [7.5%] of 1,476 isolates).

The penA allele of FC428 (penA_FC428) was sequenced as previously described (13) to investigate the relatedness with the penA alleles of the previously reported ceftriaxone-resistant gonococcal strains (H041, GU140106, F89, and A8806 [4–8]). The deduced amino acid sequence of PBP 2 in FC428 elucidated a shared trait with the ceftriaxone-resistant strains GU140106 (5) and A8806 (8). As in GU140106 (5) and A8806 (8), the mosaic PBP 2 of FC428 possessed two (A311V and T483S) of the three (A311V, A316P, and T483S) critical amino acid substitutions resulting in the ceftriaxone resistance in H041 (4, 15). Transformation of PCR-amplified full-length penA_FC428, performed as previously described, to the ceftriaxone-susceptible N. gonorrhoeae strain NG9807 (4, 13) verified that the penA_FC428 allele caused the ceftriaxone resistance, i.e., the ceftriaxone MIC of the recipient NG9807 increased 32-fold (from 0.016 μg/ml to 0.5 μg/ml). The mtrR and penB resistance determinants, which also increase the MICs of ceftriaxone, were determined in both of these strains, as previously described (4, 13). Both FC428 and the recipient NG9807 contained these resistance determinants, i.e., a single-nucleotide (A) deletion in the promoter region of mtrR and a G120K alteration in PorB1b. However, while FC428 possessed a wild-type sequence of MtrR and an A121D alteration in PorB1b, NG9807 had a G45D alteration in MtrR and an A121N alteration in PorB1b. The penA_FC428 was also further compared to penA of the GU140106 (penA_GU140106) and A8806 (penA_A8806) strains, which had a ceftriaxone MIC (0.5 μg/ml) identical to that of FC428 (Table 1). The 5′-terminal region of penA_FC428 (nucleotide positions 1 to 293) was identical to the corresponding regions of penA_GU140106 and penA_A8806. The 3′-terminal region of penA_FC428 was also nearly identical (99.9%; only one nucleotide mismatch was found at position 1296) to the corresponding regions of penA_GU140106 and penA_A8806 (positions 919 to 1749 and 907 to 1749, respectively). However, the central region of penA_FC428 showed substantially lower nucleotide sequence similarity with both penA_GU140106 and penA_A8806 (83.7% and 93.5%, respectively). On the contrary, the central region of penA_FC428 was identical to the corresponding region of penA of the penicillin- and cephalosporin-susceptible N. gonorrhoeae strain FA1090 (penA_FA1090). In fact, the entire 5′-terminal half of penA_FC428 (nucleotide positions 1 to 904) was identical to the corresponding region of penA_FA1090, while the 3′-terminal half was completely different (Fig. 1). All this indicates that the new mosaic penA_FC428 and the ceftriaxone resistance of FC428 evolved due to acquisition of the identical ceftriaxone resistance-determining 3′-terminal region of penA encoding PBP 2, which caused the ceftriaxone resistance in the recently reported ceftriaxone-resistant strains from Japan (5) and Australia (8). Accordingly, FC428, GU140106 (5), and A8806 (8) represent different N. gonorrhoeae strains (according to MLST, NG-MAST, and complete penA sequencing), but they all harbor the mainly identical ceftriaxone resistance-determining 3′-terminal region of penA. This indicates that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread. Taking advantage of the unique and characteristic construct of penA_FC428, the development of a PCR specific for penA_FC428 and utilization of this PCR for rapid molecular screening of ceftriaxone-resistant strains (among cultured strains and samples for nucleic acid amplification tests) might be valuable. A comprehensive investigation of the origin of this unique penA allele might also elucidate the existence of a genetic source or reservoir of these ceftriaxone resistance-determining PBP 2 regions, which repeatedly donate these and other PBP 2 regions to different lineages of N. gonorrhoeae. The origin of the ceftriaxone resistance-determining PBP 2 region in FC428 has not been possible to identify; however, the genetic source is most likely some commensal Neisseria species.

FIG 1.

Mosaic penA genes of the ceftriaxone-resistant Neisseria gonorrhoeae strains FC428 (present study), GU140106 (5), A8806 (8), and the ceftriaxone-susceptible genome-sequenced gonococcal strain FA1090. Sequence similarities of penA_FC428 (GenBank accession no. LC113953), penA_GU140106 (GenBank accession no. LC056026), penA_A8806 (D. M. Whiley, personal communication), and penA_FA1090 (GenBank accession no. AE004969) are shown by rectangles. The regions conserved among ceftriaxone-resistant strains are shown by black rectangles, and unique regions of each strain are indicated by gray rectangles. The penA_FA1090 and penA regions in the ceftriaxone-resistant strains similar to penA_FA1090 are shown by white rectangles. * indicates one nucleotide difference at position 1296 of penA: G in penA_FC428, and A in penA_GU140106 and penA_A8806.

In conclusion, a new ceftriaxone- and multidrug-resistant N. gonorrhoeae strain (FC428) isolated in Japan in 2015 has now been characterized. Our results indicate that both ceftriaxone-resistant strains and conserved ceftriaxone resistance-determining PBP 2 regions might spread. According to pharmacodynamic analyses (16), using <1 g of ceftriaxone for the treatment of gonorrhea caused by strains, like FC428 (ceftriaxone MIC, 0.5 μg/ml), is unlikely to clear the infection. Accordingly, dual antimicrobial therapy (ceftriaxone plus azithromycin [1]) might be crucial to introduce in additional regions globally. Continuous strengthened AMR surveillance in the Osaka/Kyoto area of Japan and other regions worldwide is essential.

Nucleotide sequence accession number.

The complete nucleotide sequence of the penA gene of FC428 has been deposited in DDBJ under accession no. LC113953.