Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles

Abstract

Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5–2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

Introduction

Neisseria gonorrhoeae is a principal pathogen for patients with male urethritis and pharyngitis.^1–4 Currently, the high prevalence of multidrug resistance of this pathogen, including resistance to penicillins, fluoroquinolones, tetracyclines, and most cephalosporins, is of urgent clinical concern worldwide.^5,6 One of the third-generation cephalosporins, cefixime, has been approved for gonococcal infections; however, an increase of resistance to this drug and clinical cases of treatment failure with it have been reported.^7,8 Therefore, the sole, effective, first-line antimicrobial agent, ceftriaxone, in combination with/without azithromycin is recommended for gonococcal urethritis and pharyngitis according to the European STI guidelines, guidelines of the Centers for Disease Control and Prevention, and World Health Organization.^2–4 However, the emergence of ceftriaxone- and azithromycin-resistant N. gonorrhoeae was reported in 2010 and 2012, respectively, and failures of these treatments also have been reported.^9–12 Therefore, the need for alternative antimicrobials for use against multidrug-resistant N. gonorrhoeae infection is pressing.

Carbapenems are broad-spectrum antimicrobials widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria.¹³ Biapenem and doripenem are clinically approved carbapenems in Japan, as are meropenem and imipenem. In addition, an oral penem, faropenem, which is a synthetic β-lactam agent, is also approved for clinical use in Japan.^14,15 Carbapenems and faropenem display high antimicrobial activity against various Gram-positive and -negative bacteria, including multidrug-resistant isolates.^16–18 However, their potencies against cephalosporin-resistant N. gonorrhoeae have not been well elucidated.

The mechanisms of resistance against β-lactam antimicrobials are attributed to amino acid changes caused by nucleotide mutations in penA, mtrR, porB1b (penB), and ponA in N. gonorrhoeae.^19–23 In particular, mosaic mutations in penA, which encodes penicillin-binding protein 2 (PBP2), are the main contributors to penicillin G and cephalosporin resistance. However, genetic associations between mutations in β-lactam resistance-associated genes and decreased susceptibility to carbapenems or faropenem are not well understood.

In this study, we evaluated the susceptibilities of cephalosporin-resistant N. gonorrhoeae isolates to carbapenems and faropenem and their mechanisms of resistance (detecting mutations in penA, mtrR, porB, and ponA) were examined by using whole-genome sequencing, which is a powerful tool to detect gene mutations in multiple loci. In addition, we evaluated the clonality of cephalosporin-resistant N. gonorrhoeae isolates by multilocus sequence types (MLST), N. gonorrhoeae multiantigen sequence types (NG-MAST), and the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) because these typings are suitable methods for provision of valuable information to understand the spread of cephalosporin-resistant N. gonorrhoeae isolates in Japan and across other counties.

Materials and Methods

Bacterial isolates

We used cephalosporin-susceptible (n = 11) and cephalosporin-resistant (n = 12) N. gonorrhoeae clinical isolates in this study. Each isolate was randomly selected from the bacterial collections of the Three Academic Societies Joint Antimicrobial Susceptibility Surveillance Program of N. gonorrhoeae in Japan in 2009 and 2012.^24,25 All isolates were from male patients with urethritis. Isolates that exhibited a minimum inhibitory concentration (MIC) of cefixime of more than 0.25 mg/L were defined as resistant according to the breakpoint of the European Committee of Antimicrobial Susceptibility Testing (EUCAST).²⁶ MICs of cefixime, cefditoren, and ceftriaxone were determined previously.^24,25 All cephalosporin-resistant isolates were susceptible to ceftriaxone and resistant to cefixime. The WHO isolates (WHO F and K) were used as reference strains.²⁷

Whole-genome sequencing and identification of gene mutations associated with β-lactam resistance

Genomic DNA was extracted from cells using a DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany). TruSeq Nano DNA Library Prep (Illumina, San Diego, CA) was used for creating the libraries. Whole-genome sequencing of the 300-bp paired-end libraries was performed using the Illumina MiSeq platform (Illumina). The draft genome was assembled with the CLC Genomics Workbench (Qiagen). Annotation was performed using the DDBJ Fast Annotation and Submission Tool, beta version.²⁸

Identification of mutations in genes associated with β-lactam resistance was performed with ResFinder²⁹ at the Center for Genomic Epidemiology using the obtained FASTA files. The allelic number of penA was assigned according to previous studies.^9,30 MLST [the Center for Genomic Epidemiology (https://cge.cbs.dtu.dk/services/MLST/)], NG-MAST [NG-MAST.net (www.ng-mast.net)], and NG-STAR [NG-STAR.net (https://ngstar.canada.ca)] were performed according to previous reports.^9,31,32

Susceptibilities to carbapenems and faropenem

Susceptibilities to meropenem, imipenem, biapenem, doripenem, and faropenem were determined by the agar dilution method using GC agar (Thermo Fisher Scientific, Waltham, MA) supplemented with 0.03 g of guanine HCl, 3 mg of thiamine HCl, 13 mg of p-aminobenzoic acid, 0.01 g of vitamin B₁₂, 0.1 g of cocarboxylase, 0.25 g of nicotinamide adenine dinucleotide, 1 g of adenine, 10 g of l-glutamine, 100 g of glucose, and 0.02 g of ferric nitrate per one liter, as described in the Clinical and Laboratory Standards Institute documentation.³³

Results

Susceptibilities to carbapenems and faropenem

MICs of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem are shown in Table 1. MICs of faropenem and all carbapenems, except meropenem, for cephalosporin-resistant isolates were 8-fold or more higher than those for cephalosporin-susceptible isolates. MICs of meropenem for cephalosporin-susceptible and -resistant isolates were markedly lower than those of other carbapenems and faropenem and they did not differ significantly (by more than one double dilution) for cephalosporin-susceptible isolates.

Table 1.

Genetics and β-Lactam Susceptibilities of Cephalosporin-Susceptible and -Resistant Neisseria gonorrhoeae Clinical Isolates

Strain	MLST	NG-MAST	NG-STAR	Genotypes/mutations				MIC (mg/L)
Strain	MLST	NG-MAST	NG-STAR	penA (allele^a)	mtrR	porB	ponA	CFM^b	CDN^b	CRO^b	MEM	IPM	DOR	BPM	FPM
Cephalosporin-susceptible isolates
WHO-F	10934	3303	2	XV (15.001)	—	porB1A ^c	—	≤0.06	≤0.06	≤0.06	≤0.06	0.125	≤0.06	≤0.06	≤0.06
CSGN1	1588	436	1231	XIX (19.001)	Deletion of Ab^d, G45D	porB1A	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN2	1901	247	127	V (5.002)	Deletion of A	G120K, A121D	L421P	≤0.06	≤0.06	≤0.06	≤0.06	0.125	≤0.06	≤0.06	≤0.06
CSGN3	1588	211	1241	XIX (19.001)	A39T	porB1A	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN4	1901	247	127	V (5.002)	Deletion of A	G120K, A121D	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN5	1901	3356	203	XVIII (18.001)	Deletion of A	G120K, A121D	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN6	7827	6773	1240	V + D101E (106.001)	—	—	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN7	7822	1866	73	II (2.002)	Deletion of A, G45D	G120K, A121D	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN8	7359	6771	231	II (2.002)	A39T	—	—	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN9	1588	5613	1230	XIX (19.001)	—	—	L421P	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
CSGN10	1901	NA	127	V (5.002)	Deletion of A	G120K, A121D	—	≤0.06	≤0.06	≤0.06	≤0.06	0.125	≤0.06	≤0.06	≤0.06
CSGN11	1583	NA	1237	II (2.001)	A39T	—	—	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06	≤0.06
Cephalosporin-resistant isolates
WHO-K	7363	1424	4	Mosaic X (10.001)	Deletion of A, G45D	G120K, A121D	—	1	1	≤0.06	≤0.06	2	1	1	1
CRGN1	1588	4012	1242	Mosaic XXV (108.001)	A39T	porB1A	L421P	0.25	0.5	≤0.06	0.125	1	1	1	1
CRGN2	1901	15533	1243	Mosaic XXVII (27.002)	Deletion of A	G120K, A121N	L421P	0.25	1	0.125	≤0.06	1	0.5	1	1
CRGN3	1588	4012	1242	Mosaic XXV (108.001)	A39T	porB1A	L421P	0.25	0.5	≤0.06	0.125	2	1	1	1
CRGN4	1901	4183	22	Mosaic X (10.001)	Deletion of A	G120K, A121D	L421P	0.5	1	0.125	≤0.06	1	1	1	1
CRGN5	1901	7393	319	Mosaic X (10.001)	Deletion of A	G120K, A121N	L421P	0.25	0.5	0.125	≤0.06	1	0.5	1	1
CRGN6	7363	6778	1229	Mosaic X (10.001)	G45D	G120K, A121D	L421P	0.5	1	0.125	≤0.06	2	0.5	1	1
CRGN7	7363	6778	1229	Mosaic X (10.001)	G45D	G120K, A121D	L421P	0.5	1	≤0.06	≤0.06	2	0.5	1	1
CRGN8	7363	5308	348	Mosaic X (10.001)	—	G120K, A121D	L421P	0.5	1	≤0.06	0.125	2	1	2	1
CRGN9	1901	2958	22	Mosaic X (10.001)	Deletion of A	G120K, A121D	L421P	0.5	1	0.125	0.125	1	0.5	1	1
CRGN10	7363	9551	1236	Mosaic X (10.001)	—	—	L421P	0.25	0.5	≤0.06	0.125	2	1	2	1
CRGN11	1579	6798	22	Mosaic X (10.001)	Deletion of A	G120K, A121D	L421P	0.25	0.5	≤0.06	0.125	1	0.5	1	1
CRGN12	7363	5308	348	Mosaic X (10.001)	—	G120K, A121D	L421P	0.25	1	≤0.06	≤0.06	2	1	1	1

Allele numbers were determined by NG-STAR.³²

These MICs were cited from previous articles.^24,25

The strains have porB1A, and other strains have porB1B.

Deletion of adenine residue in the 13-bp inverted repeat region of the mtrR promoter.

CFM, cefixime; CDN, cefditoren; CRO, ceftriaxone; MPM, meropenem; IPM, imipenem; DOR, doripenem; BPM, biapenem; FPM, faropenem; MIC, minimum inhibitory concentration; MLST, multilocus sequence types; NG-STAR, N. gonorrhoeae sequence typing for antimicrobial resistance; NG-MAST, N. gonorrhoeae multiantigen sequence types; NA, not available.

MLST, NG-MAST, and NG-STAR

By whole-genome sequencing, we identified eight MLST types, 17 NG-MAST types, and 17 NG-STAR types among the isolates (Table 1). In cephalosporin-resistant isolates, MLST ST7363 was the most common (five isolates), and these were divided into three NG-MAST types (ST5308, ST6778, and ST9551) and three NG-STAR types (ST348, ST1229, and ST1236). MLST ST1901 was shared in cephalosporin-susceptible and -resistant isolates (four isolates each) and consisted of different NG-MAST and NG-STAR types. MLST ST1588 was found in three susceptible and two resistant isolates, and the NG-MAST types of susceptible strains were different. The NG-STAR types were varied in cephalosporin-susceptible isolates, while some common NG-STARs (NG-STAR ST22, 348, 1229, and 1242) were found in cephalosporin-resistant isolates (Table 1). NG-STAR ST1229, ST1231, ST1236, ST1240, ST1242, and ST1243 were submitted as new types in this study.

Identification of mutations in penA, mtrR, porB, and ponA

By whole-genome sequencing of cephalosporin-susceptible and -resistant N. gonorrhoeae clinical isolates, we detected several gene mutations attributed to amino acid substitutions and deletions in penA, mtrR, porB, and ponA (Table 1).

The allele types of penA were assigned according to previous reports.^9,30 Alleles II, XIX, and V (three isolates each), V with D101E, and XVIII (one isolate each) were identified in cephalosporin-susceptible isolates. There were three types of mosaic alleles, X (nine isolates), XXV (two isolates), and XXVII (one isolate), in cephalosporin-resistant isolates. In NG-STAR penA allelic types, 106.001, 108.001, and 27.002 were newly identified in this study, thus we submitted them as new penA allelic types.

Deletion of adenine residue in the 13 bp inverted repeat region of the mtrR promoter was identified in six and five cephalosporin-susceptible and -resistant isolates, respectively. In MtrR, G45D was detected in two cephalosporin-susceptible and -resistant isolates each, and A39T in three and two, respectively. In PorB, porB1A was identified in two cephalosporin-susceptible and -resistant isolates each. G120K was found in four and nine, respectively. Among them, four susceptible and seven resistant isolates shared A121D, and two cephalosporin-resistant isolates possessed A121N. In PonA, all of the N. gonorrhoeae clinical isolates except three cephalosporin-susceptible isolates possessed L421P.

Discussion

In the present study, all cephalosporin-resistant isolates (resistant to cefixime and cefditoren, but susceptible to ceftriaxone) possessed mosaic allele X, XXV, or XXVII of penA (Table 1). Allele X, which was the most predominant among the cephalosporin-resistant isolates in this study, is also the most prevalent in cephalosporin-resistant N. gonorrhoeae clinical isolates in Japan.³¹ On the other hand, we identified a new allele, V with D101E, of penA in cephalosporin-susceptible isolates.

MLST ST7363 and ST1901 are known as major lineages of cephalosporin-resistant N. gonorrhoeae that spread in the clinical setting throughout the world, especially in Japan. These frequently possess the mosaic allele X, in addition to XXXIV and XXXVIII in penA.^9,31,34 In this study, these were dominant in cephalosporin-resistant isolates and most of them possessed the mosaic allele X. These observations suggest that the trend of spreading cephalosporin-resistant N. gonorrhoeae has not changed in Japan. Whereas MLST ST1588 and ST1901 were identified in both cephalosporin-susceptible and -resistant isolates, their NG-MAST types properly discriminate cephalosporin susceptibility.

Cephalosporin-resistant N. gonorrhoeae has been increasing in Japan. NG-MAST ST1407 with mosaic allele XXXIV in penA is known to be a major cephalosporin-resistant N. gonorrhoeae sequence type worldwide, including Japan.³¹ Another previous study reported genotypes of high-level cefixime- and ceftriaxone-resistant N. gonorrhoeae strains isolated in Japan. The HO41 strain belonged to MLST ST7363 and NG-MAST ST2594 with penA-37.001, and FC428 and FC160 strains were MLST ST1903 and NG-MAST ST3435 with penA-60.001.³⁵ These NG-MAST sequence types and mosaic allele types of penA were not identified in the study.

NG-STAR was developed by the National Microbiology Laboratory, the Public Health Agency of Canada, in 2017.³² Whereas varied NG-STAR types were identified in cephalosporin-susceptible isolates, some common NG-STAR types were observed in cephalosporin-resistant isolates. We could not evaluate the association between the strains in this study and those in previous studies because NG-STAR types have not been determined in most of the previous studies. In a recent report, NG-SATR types, ST233 and ST226, were found to be ceftriaxone-resistant isolates in Japan.³⁵ NG-STAR should be a useful tool for a detailed understanding of the spread of antimicrobial-resistant N. gonorrhoeae in Japan in future epidemiological studies.

It has been reported that ertapenem and meropenem possess lower MICs for cephalosporin-resistant N. gonorrhoeae than cephalosporins and penicillins.^30,36,37 However, other carbapenems have not been evaluated. We investigated the susceptibilities of cephalosporin-resistant N. gonorrhoeae isolates with mosaic alleles in penA to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and an oral penem, faropenem. We found that imipenem, doripenem, biapenem, and faropenem showed little antimicrobial activity against cephalosporin-resistant N. gonorrhoeae. In contrast, the MIC of meropenem for cephalosporin-resistant isolates did not increase in comparison with other carbapenems and faropenem. This result was similar to a previous study, which found that meropenem still retains its efficacy against cephalosporin-resistant N. gonorrhoeae, but imipenem does not.³⁰ Although meropenem has good antimicrobial activity in vitro, further investigations on its pharmacokinetics and tissue distribution in the pharynx and urethra will be required for clinical application to the treatment of N. gonorrhoeae infection.

To evaluate the genetic association with decreased susceptibility to carbapenems and faropenem, we compared antimicrobial susceptibilities of N. gonorrhoeae clinical isolates based on the presence of gene mutations in penA, ponA, mtrR, and porB (Table 2). We observed the strongest association between the increase of carbapenem/faropenem MICs and the presence of mosaic alleles in penA. It is reported that mutations in mtrR (which encodes transcriptional repressor of efflux pump operon mtrCDE), porB (which encodes porin), and ponA (which encodes PBP1) are involved in resistance to penicillins and/or cephalosporins,^19–22 and mutations of mtrR and porB are also involved in elevation of carbapenem MICs.³⁷ However, we observed no correlation between the increase of carbapenem/faropenem MICs and mutations in ponA, mtrR, and porB. These results indicated that the decrease of carbapenem/faropenem susceptibility was related to mosaic mutations in penA.

Table 2.

Carbapenem Minimum Inhibitory Concentrations and Mutations in Genes Associated with Resistance of Neisseria gonorrhoeae to Cephalosporins and Penicillins

Antimicrobials	No. of isolates with MIC (mg/L)
Mutations	≤0.06	0.12	0.25	0.5	1	2
Meropenem
penA nonmosaic
ponA	2
mtrR	2
mtrR + porB	1
ponA + mtrR + porB	6
penA mosaic
ponA	2
ponA + porB	1
ponA + mtrR + porB	4	5
Imipenem
penA nonmosaic
ponA	2
mtrR	2
mtrR + porB		1
ponA + mtrR + porB	5	1
penA mosaic
ponA						2
ponA + porB						1
ponA + mtrR + porB					6	3
Doripenem
penA nonmosaic
ponA	2
mtrR	2
mtrR + porB	1
ponA + mtrR + porB	6
penA mosaic
ponA					2
ponA + porB					1
ponA + mtrR + porB				6	3
Biapenem
penA nonmosaic
ponA	2
mtrR	2
mtrR + porB	1
ponA + mtrR + porB	6
penA mosaic
ponA					1	1
ponA + porB						1
ponA + mtrR + porB					9
Faropenem
penA nonmosaic
ponA	2
mtrR	2
mtrR + porB	1
ponA + mtrR + porB	6
penA mosaic
ponA					2
ponA + porB					1
ponA + mtrR + porB					9

The affinity of carbapenems/faropenem to PBP2, which is encoded by penA, has not been evaluated in N. gonorrhoeae. Ochiai et al. reported that the decreased susceptibility of cephalosporins (cefdinir and cefixime) was attributed to decreased affinity of mosaic PBP2 in N. gonorrhoeae.³⁸ In Escherichia coli, it has been reported that strains expressing wild-type and some mutated PBP2s exhibited lower MICs of meropenem compared with those of other carbapenems.³⁹ Meropenem might have higher affinity to the mosaic PBP2 and higher antimicrobial activity against N. gonorrhoeae than other carbapenems and faropenem. Further studies are needed to elucidate this issue.

Recently, ceftriaxone-resistant N. gonorrhoeae having penA-60.001 has been disseminated worldwide.^35,40 Meropenem is shown to exhibit low MIC (less than 0.125 mg/L) in ceftriaxone-resistant isolates,^9,30 although these isolates possessed other mosaic alleles, penA-37.001 and penA-42.001.³⁵ There is some evidence therefore that meropenem may serve as an alternative antimicrobial agent for the treatment of multidrug-resistant N. gonorrhoeae, and penA is a promising target for development of new antimicrobials against N. gonorrhoeae infection.

Footnotes

Acknowledgments

This work was supported by grants from the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID), the Ministry of Education, Culture, Sport, Science and Technology in Japan, the Japan Agency for Medical Research and Development, and MEXT for the Joint Research Program of the Research Center for Zoonosis Control, Hokkaido University. This work was also partly supported by a grant from JSPS KAKENHI (17K15688) and the Yuasa Memorial Foundation. The funding sources did not play any role in the study design; collection, analysis, and interpretation of data; writing of the report; and decision to submit the article for publication.

Disclosure Statement

No competing financial interests exist.

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