Novel pena mutations identified in Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone isolated between 2000 and 2014 in Japan

J Antimicrob Chemother 2016; 71: 2466 2470 doi:10.1093/jac/dkw161 Advance Access publication 13 May 2016 Novel pena mutations identified in Neisseria gonorrhoeae with decreased susceptibility to ceftriaxone isolated between 2000 and 2014 in Japan Kensaku Seike 1, Mitsuru Yasuda 1, Kyoko Hatazaki 1, Kosuke Mizutani 1, Kazuya Yuhara 2, Yasuhisa Ito 3, Yoshinori Fujimoto 4, Shin Ito 5, Tomohiro Tsuchiya 1, Shigeaki Yokoi 1, Masahiro Nakano 1 and Takashi Deguchi 1 * 1 Department of Urology, Graduate School of Medicine, Gifu University, Gifu, Japan; 2 Department of Urology, Japanese Red Cross Takayama Hospital, Takayama, Japan; 3 Department of Urology, Ibi Kosei Hospital, Ibi, Japan; 4 Department of Urology, Ogaki Municipal Hospital, Ogaki, Japan; 5 iclinic, Sendai, Japan Introduction *Corresponding author. Tel: +81-58-230-6338; Fax: +81-58-230-6339; E-mail: deguchit@gifu-u.ac.jp K. Seike and M. Yasuda contributed equally to this article. Received 25 January 2016; returned 2 March 2016; revised 6 April 2016; accepted 7 April 2016 Objectives: We examined four clinical strains of Neisseria gonorrhoeae (GU030113, GU110095, GU110332 and GU110362) isolated between 2000 and 2014 in Japan, exhibiting ceftriaxone MICs of 0.5 mg/l, for mutations of the genes associated with penicillin resistance. Methods: The pena, mtrr, porb1b (penb), pona and pilq genes of the strains were sequenced. PBP2s of the strains were aligned to the PBP2s associated with decreased susceptibility to oral cephalosporins, and PBP2s of previously reported strains with decreased susceptibility to ceftriaxone. Results: GU030113 had PBP2 pattern X with an additional substitution of A502T. GU110095 had PBP2 pattern XXVII. GU110332 had PBP2 pattern XXXIV with an additional substitution of P552S. GU110362 had PBP2 composed of pattern X (amino acid positions 1 291) and pattern V (amino acid positions 292 576). GU030113, GU110095 and GU110332 had, G120K and A121D or A121N in PorB1b and L421P in PBP1. GU110362 had A40D in the repressor of MtrR and L421P in PBP1. The strains did not have mutations of pilq1 and pilq2. Conclusions: Addition of A502T to PBP2 pattern X in GU030113 and of P552S to PBP2 pattern XXXIV in GU110332 would possibly contribute to decreased susceptibility to ceftriaxone. In GU110095 and GU110362, it was suggested that, in addition to their altered PBP2s, the enhanced efflux pump, reduced permeability in the outer membrane, another altered target of b-lactams and/or other mechanisms not identified in the present study might contribute to decreased susceptibility. Surveillance of the antimicrobial susceptibility of Neisseria gonorrhoeae in Japan has shown that clinical isolates with decreased susceptibility to oral third-generation cephalosporins, including cefixime, have emerged and spread since the early 2000s. 1 Clinical isolates with cefixime MICs of 0.25 mg/l and 0.5 mg/l accounted for 40.8% and 10.7% of the isolates collected in Japan from 2012 to 2013, respectively. 2 However, most clinical isolates of N. gonorrhoeae with decreased susceptibility to cefixime still exhibit ceftriaxone MICs of 0.25 mg/l or less. 1 3 In 2009, a clinical strain (H041) of N. gonorrhoeae with a ceftriaxone MIC of 2 mg/l was isolated from the pharynx of a female sex worker in Japan, 4 while in 2010, another strain of F89 with ceftriaxone MICs of 1 2 mg/l was isolated in France and Spain. 5,6 Afurther five strains with ceftriaxone MICs of 0.5 mg/l have also been reported. 7 11 Chromosomally mediated penicillin resistance in N. gonorrhoeae is conferred by multifactorial genetic alterations, including those of the pena, mtrr, porb1b (penb), pona and pilq genes. 12 14 For decreased susceptibility to cefixime and ceftriaxone, alterations of PBP2 encoded by the pena gene, particularly mosaic structures of PBP2 composed of fragments analogous to PBP2s in commensal 3 11,13 17 Neisseria species, play a principal role. Our surveillance of antimicrobial susceptibility of N. gonorrhoeae in Japan since 2000 has identified five isolates with ceftriaxone MICs of 0.5 mg/l among 2126 isolates tested. The mechanism of resistance of one strain (GU140106) has been reported previously. 11 In the present study, we examined the four other strains for mutations in the genes associated with b-lactam resistance # The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com 2466

N. gonorrhoeae with decreased susceptibility to ceftriaxone JAC as well as their relatedness to other strains previously reported to have decreased susceptibility to ceftriaxone. Methods The four isolates with ceftriaxone MICs of 0.5 mg/l (GU030113, GU110095, GU110332 and GU110362) were successfully recovered from storage at 2708C. MICs of penicillin G (Wako Pure Chemical Industries, Ltd, Osaka, Japan), cefixime (Astellas Pharma, Tokyo, Japan), ceftriaxone (Chugai Pharmaceutical Co., Ltd, Tokyo, Japan), tetracycline (Wako Pure Chemical Industries, Ltd), azithromycin (Sigma Aldrich Corporation, St Louis, MO, USA), spectinomycin (LKT Laboratories, Inc., St Paul, MN, USA) and levofloxacin (Daiichi Sankyo Company, Ltd, Tokyo, Japan) were determined again by the agar dilution method. 18 N. gonorrhoeae ATCC49226 was used as the quality control strain. The pena, mtrr, porb1b (penb), pona and pilq genes of the strains were sequenced as previously described. 3,13,14 The pena genes of GU030113, GU110095, GU110332 and GU110362 were deposited in GenBank under accession nos LC113985, LC114149, LC114150 and LC113986, respectively. The PBP2s derived from the pena genes were aligned to PBP2 of penicillin-susceptible strain LM306 (GenBank accession no. M32091), PBP2 pattern V associated with penicillin resistance, 3 PBP2s (patterns V, X and XXXIV) associated with decreased susceptibility to cefixime, 3,15 PBP2s of two strains with ceftriaxone MICs of 1 2 mg/l (H041 and F89) and PBP2s of five strains with ceftriaxone MICs of 0.5 mg/l isolated in Japan in 2000 or 2001 (GP853), China in 2007, Austria in 2011, Australia in 2013 (A8806) and Japan in 2014 (GU140106). 4 11 In the following text and Table 1, the amino acid positions in PBP2 are identified according to numbering of the NCBI reference sequence of N. gonorrhoeae PBP2 pattern V (GenBank accession no. WP_012503873.1). Molecular epidemiological characterization of the strains was performed by means of MLST and N. gonorrhoeae multiantigen sequence typing (NG-MAST) as previously reported. 4,5 This study was approved by the Institutional Review Board of the Graduate School of Medicine, Gifu University, Gifu, Japan (reference no. 26-341). Results and discussion Table 1. Characteristics of the four clinical strains of N. gonorrhoeae with decreased susceptibility to ceftriaxone Characteristic GU030113, GU110095 and GU110332 were isolated from the urethra of men with gonococcal urethritis (Table 1). GU110362 Strain GU030113 GU110095 GU110332 GU110362 Isolation of the strains year 2003 2011 2011 2011 specimen source urethral swab urethral swab urethral swab pharyngeal swab Demographics of patients sex male male male male age (years) 22 35 28 44 sexual partner casual woman female sex worker girlfriend female sex worker symptoms dysuria and urethral dysuria and urethral dysuria and urethral asymptomatic treatment 100 mg of cefcapene pivoxil three times/day for 7 days test-of-cure for N. gonorrhoeae not performed not performed not performed negative culture result of pharyngeal swab MIC (mg/l) of antimicrobial agents penicillin G 4 16 8 2 cefixime 1 1 0.5 1 ceftriaxone 0.5 0.5 0.5 0.5 tetracycline 4 8 4 2 azithromycin 1 1 0.5 0.25 spectinomycin 32 32 32 32 levofloxacin 16 16 8 8 Analysis of the pena, mtrr, porb1b, pona and pilq genes PBP2 X+A502T XXVII XXXIV+P552S X (1-291)+V (292-576) mtrr/mtrr A40D in the repressor of MtrR PorB1b G120K, A121D G120K, A121N G120K, A121N PBP1 L421P L421P L421P L421P PilQ type VII type VII type VII type VI Genotyping MLST 1901 9751 1901 7363 NG-MAST 668 3935 13194 5687 2467

Seike et al. was isolated from the pharynx of an asymptomatic man. The three men with gonococcal urethritis were treated with ceftriaxone or cefcapene, but the test-of-cure for N. gonorrhoeae was not performed. The asymptomatic man with the pharyngeal infection was treated with a single dose of 1 g of ceftriaxone. Culture of the pharyngeal swab was negative for N. gonorrhoeae 17 days after treatment. The decreased susceptibility of all four isolates to ceftriaxone (MIC 0.5 mg/l) was confirmed on re-testing after retrieval from frozen storage. In addition, all four isolates were resistant to penicillin G, tetracycline and levofloxacin based on the breakpoints of ofloxacin (Table 1). 19 They showed decreased susceptibility to cefixime and ceftriaxone. The MICs of azithromycin ranged from 0.25 to 1 mg/l. In Japan, a single-dose regimen of extended-release azithromycin (2 g) is available to treat gonorrhoea. Our previous study on the microbiological efficacy of this regimen on gonococcal urethritis suggested that GU030113 and GU110095 with azithromycin MICs of 1 mg/l might be resistant to this regimen. 20 For spectinomycin, all of the strains were susceptible. Although all the strains had altered PBP2 (Table 1), their PBP2s did not match any of the previously reported strains with decreased susceptibility to ceftriaxone (Figure 1). 3 11 GU030113 had an additional substitution of A502T in PBP2 pattern X. In F89 with ceftriaxone MICs of 1 2 mg/l, A502P was found in its mosaic PBP2. 5 In the Chinese strain with a ceftriaxone MIC of 0.5 mg/l, A502V was found in its non-mosaic PBP2. 8 The substitutions at amino acid position 502 in PBP2s, including A502T, were reported to contribute to decreased susceptibility to ceftriaxone. 16 For GU030113, A502T could possibly contribute to decreased susceptibility to ceftriaxone, although transformation studies are needed to confirm its association with decreased susceptibility to ceftriaxone. GU110332 had mosaic PBP2 pattern XXXIV with P552S. F89 had mosaic PBP2 pattern XXXIV with A502P and the Austrian strain with a ceftriaxone MIC of 0.5 mg/l had mosaic PBP2 pattern XXXIV with T535A. 5,9 Strains of N. gonorrhoeae harbouring mosaic PBP2s, including pattern XXXIV, commonly decrease their susceptibility to cefixime, but do not always decrease ceftriaxone susceptibility. 3,4,7,8,15 17 SM-1, SM-2 and SM-3 strains having PBP2 pattern XXXIV, which were collected in 2008 in San Francisco, USA, exhibited cefixime MICs of 0.125 0.25 mg/l, but ceftriaxone MICs of 0.03 0.06 mg/l. 15 A previous transformation study reported that the introduction of PBP2 pattern XXXIV with an additional substitution at amino acid position 502 or 552 increased ceftriaxone MICs in a ceftriaxonesusceptible strain. 17 Based on this finding, the decreased susceptibility to ceftriaxone of strain GU110332, which had PBP2 belonging to pattern XXXIV, might reflect an additional substitution at amino acid position 552. For GU110095 and GU110362, it is uncertain whether their respective PBP2 pattern XXVII and novel PBP2 could contribute to their decreased susceptibility to ceftriaxone. GU030113, GU110095 and GU110332 had a single nucleotide deletion in the mtrr, G120K and A121D or A121N in PorB1b and L421P in PBP1 (Table 1). GU110362 had A40D in the repressor of MtrR and L421P in PBP1. The A40D in the repressor of MtrR detected in GU110362 was different from A39T or G45D reported to confer increased resistance to hydrophobic antimicrobials. 12 It is uncertain whether A40D could contribute to enhanced efflux pump activity in GU110362. Other alterations detected in the Figure 1. Sequences of only the altered amino acids in PBP2 of N. gonorrhoeae strains GU030113, GU110095, GU110332 and GU110362, which are aligned to the WT PBP2 of the pena gene of penicillin-susceptible strain LM306, PBP2 pattern V associated with penicillin resistance, PBP2 patterns X and XXXIV associated with decreased susceptibility to oral cephalosporins, PBP2s of H041 and F89 with high-level resistance to ceftriaxone and PBP2s of GP853, the Chinese strain isolated in 2007, A8806, the Austrian strainisolatedin2011andgu140106withdecreasedsusceptibilityto ceftriaxone. The amino acid positions in PBP2 were identified according to numbering of the NCBI reference sequence of N. gonorrhoeae PBP2 pattern V (GenBank accession no. WP_012503873.1). 2468

N. gonorrhoeae with decreased susceptibility to ceftriaxone JAC mtrr, PorB1b and PBP1 were associated with penicillin resistance in N. gonorrhoeae. The strains had a PilQ amino acid ST of VI or VII, but did not have mutations of pilq1 (F595L) and pilq2 (E666K) associated with penicillin susceptibility. 14 GP853 with a ceftriaxone MIC of 0.5 mg/l did not have substitutions associated with decreased susceptibility to ceftriaxone such as those at amino acid position 311, 316, 474, 502 or 552 in its mosaic PBP2. 7 However, GP853 had penicillin resistance-associated genetic mutations in the mtrr, porb1b (penb) and/or pona genes, which were identical or analogous to those observed in our strains. In such strains as GU110095, GU110362 and GP853, it was suggested that in addition to alterations of PBP2s, enhanced efflux pump activity and reduced permeability in the outer membrane, another altered target of b-lactams and/or other mechanisms not identified in this study might contribute to the decreased susceptibility to ceftriaxone. The strains were assigned to the following MLSTs: GU030113 and GU110332 as ST1901, GU110095 as ST9751 and GU110362 as ST7363 (Table 1). NG-MAST assigned all four strains to different types. H041, A8806 and GU140106, which had substitutions at amino acid positions 311, 316 and/or 484, had the MLST of ST7363. 4,10,11 One way such strains evolve into ceftriaxone-resistant ones might be by acquiring substitutions at amino acid position 311, 316 or 484. F89, the Austrian strain and GU110332 harbouring PBP2 pattern XXXIV with an additional substitution had the MLST of ST1901. 5,9 Another way such strains evolve into ceftriaxoneresistant ones might be by acquiring additional substitutions in PBP2 pattern XXXIV. The prevalence of clinical isolates of N. gonorrhoeae exhibiting ceftriaxone MICs of 0.5 mg/l is still low. However, the emergence and spread of such isolates might be the prelude to the wider emergence of strains that are fully resistant to ceftriaxone, which would be a major public health crisis were it to happen. Acknowledgements We thank the patients of the Japanese Red Cross Takayama Hospital (Takayama, Japan), Ibi Kosei Hospital (Ibi, Japan), Ogaki Municipal Hospital (Ogaki, Japan) and iclinic (Sendai, Japan). Funding This work was supported in part by the Japan Society for the Promotion of Science (JSPS), Japan [Grant-in-Aid for Scientific Research (C) 25462509 and (C) 26462442]. Transparency declarations None to declare. 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