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Epidemiology and Surveillance

Azithromycin Resistance Is Coevolving with Reduced Susceptibility to Cephalosporins in Neisseria gonorrhoeae in Ontario, Canada

Vanessa G. Allen, Christine Seah, Irene Martin, Roberto G. Melano
Vanessa G. Allen
aPublic Health Ontario Laboratories, Toronto, Ontario, Canada
bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
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Christine Seah
aPublic Health Ontario Laboratories, Toronto, Ontario, Canada
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Irene Martin
cNational Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada
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Roberto G. Melano
aPublic Health Ontario Laboratories, Toronto, Ontario, Canada
bDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada
dMount Sinai Hospital, Toronto, Ontario, Canada
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DOI: 10.1128/AAC.02608-13
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ABSTRACT

Azithromycin (AZM) is routinely recommended as a component of dual therapy for gonorrhea in combination with third-generation cephalosporins (3GC). In this study, we examined the prevalence of AZM-resistant (AZMr) Neisseria gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under the current treatment recommendations, and analyzed the clonal distribution of AZMr isolates in Ontario, Canada. Nineteen AZMr clinical isolates (one per patient; MIC, ≥2 μg/ml) were included in the study. Susceptibility profiles of these isolates to 11 antibiotics, molecular typing, characterization of macrolide resistance mechanisms, and penicillin-binding protein 2 (PBP2) patterns were determined for all the isolates. Two groups were defined based on AZMr level; group A isolates displayed high-level resistance (MIC, ≥2,048 μg/ml) due to mutations (A2143G) in the four copies of the 23S rRNA rrl gene, and group B isolates had moderate resistance to AZM (MICs, 2 to 8 μg/ml, C2599T mutation in the rrl gene), with a subgroup belonging to sequence type 3158 (ST3158) (n = 8), which also showed reduced susceptibility to 3GC (MICs, 0.12 to 0.25 μg/ml, PBP2 pattern XXXIV). This AZMr phenotype was not observed in previous provincial surveillance in 2008 (the ST3158 clone was found, with AZM MICs of 0.25 to 0.5 μg/ml associated with mtrR mutations). We hypothesized that the AZM mutant prevention concentration (MPC) in the ST3158 subpopulation we found in 2008 was higher than the MPC in wild-type isolates (AZM MIC, ≤0.031 μg/ml), increasing the chances of additional selection of AZMr mutations. Full AZM resistance is now emerging in this clone together with reduced susceptibility to 3GC, threatening the future efficacy of these antibiotics as therapeutic options for treatment of gonorrhea.

INTRODUCTION

Resistance of Neisseria gonorrhoeae to the third-generation cephalosporins (3GC) cefixime and ceftriaxone (1–3) has raised alarms regarding the imminent loss of the last reliable class of antimicrobials for the treatment of gonorrhea. In an effort to improve treatment efficacy and to delay the further selection of cephalosporin-resistant N. gonorrhoeae, most treatment guidelines now recommend the use of adjunctive azithromycin (AZM) or doxycycline to be administered in conjunction with cephalosporin therapy (4–6). While not recommended as monotherapy except in the case of severe cephalosporin allergy, AZM is often preferred to adjuvant therapy because it offers the convenience of single-dose therapy and demonstrates a lower prevalence of resistance than doxycycline, and preliminary data suggest that AZM may be more effective as synergistic therapy with 3GC, particularly in the treatment of pharyngeal gonorrhea (7–9).

Case reports of AZM-resistant N. gonorrhoeae have been described in many countries, including Canada and the United States (10–18), but only rarely has AZM-resistant N. gonorrhoeae been reported to be associated with concurrent reduced susceptibility to 3GC (16, 17). In Canada, AZM-resistant N. gonorrhoeae was first identified in 1997, and the first AZM-resistant isolates were identified in Ontario in 1998 (10). All isolates in that initial study had AZM MICs of 2 to 4 μg/ml, and to date, no high-level AZM-resistant isolates of N. gonorrhoeae have been reported in the country. More recently, we conducted a surveillance of antimicrobial resistance in gonococci from Ontario and showed a high prevalence of isolates displaying nonsusceptibility (resistant plus intermediate categories) to penicillin (89.2%), tetracycline (72.3%), and ciprofloxacin (29%) and reduced susceptibility to 3GC (18). In addition, 34 isolates (22.8%) displayed only reduced susceptibility to erythromycin (MICs, ≥2 μg/ml) and AZM (MICs, 0.25 to 0.5 μg/ml). Some of these isolates were associated with the N. gonorrhoeae multiantigen sequence typing (NG-MAST) sequence type 3158 (ST3158) and ST1407 (18). ST3158 and ST1407 are closely related (they share the same tbpB allele 110 and have just 2 point mutations of difference between their por alleles) and were previously linked to reduced susceptibility (18–21) or resistance to 3GC (2, 3). In this study, we examined the prevalence of AZM-resistant N. gonorrhoeae from July 2010 to February 2013, assessed the rate of concurrent cephalosporin resistance under current treatment recommendations, and analyzed the clonal distribution of AZM-resistant isolates in Ontario, Canada.

MATERIALS AND METHODS

Gonococcal isolates and susceptibility testing.Public Health Ontario Laboratories (PHOL) is the microbiological reference service for the province of Ontario, Canada. PHOL provides confirmation and susceptibility testing for culture isolates of N. gonorrhoeae to all private and hospital laboratories in the province (population in 2013, 13.5 million [see http://www.statcan.gc.ca/tables-tableaux/sum-som/l01/cst01/demo02a-eng.htm]). Analysis of the PHOL gonococcal susceptibility database (from July 2010 to February 2013) allowed us to identify 57 strains with AZM MICs of ≥1 μg/ml (defined as resistant by the European Committee on Antimicrobial Susceptibility Testing [EUCAST] guidelines [available at http://www.eucast.org/]). In concordance with the Gonococcal Isolate Surveillance Project (GISP) criteria for isolates with alert value MICs (protocol available at http://www.cdc.gov/std/gisp/GISP-Protocol07-15-2010.pdf), each isolate (one per patient) with an AZM MIC of ≥2 μg/ml was included in this study. Isolates with AZM MICs of 2 to 8 μg/ml were considered moderately resistant, and those with MICs of ≥256 μg/ml were considered highly resistant (13). Reduced susceptibility to cefixime was defined as an MIC of ≥0.12 μg/ml (22). Identification of all N. gonorrhoeae isolates was performed as described previously (18), and the isolates were stored at −86°C. All of the isolates were subcultured twice on GC agar containing 1% defined growth supplement (23) at 37°C in 5% CO2 for 20 to 24 h before antimicrobial testing and DNA extraction were performed. The MICs of penicillin, ceftriaxone, cefixime, ertapenem, ciprofloxacin, tetracycline, erythromycin, spectinomycin, gentamicin, and solithromycin (CEM-101, a novel fluoroketolide currently in clinical development) were determined by the Etest (bioMérieux, Marcy l'Etoile, France) or the agar dilution method, and Clinical and Laboratory Standards Institute (CLSI) guidelines (23) were used to interpret the results, except in the cases of AZM (EUCAST guidelines) and ertapenem, gentamicin, and solithromycin (for which there are no breakpoints available in the CLSI and EUCAST guidelines). N. gonorrhoeae ATCC 49226 and strains F, G, K, L, N, O, and P of the 2008 WHO N. gonorrhoeae reference strain panel were used as quality control strains (24).

Molecular studies.DNA extracts of each isolate were prepared as described and stored at −20°C (18). All primers used in this study were previously published (18). Mutations in the mtrR gene/promoter, the L4 and L22 riboprotein genes, and the 4 copies of 23S rRNA genes were identified by PCR and sequencing. The presence of acquired enzymatic mechanisms and efflux pumps involved in macrolide resistance was screened by PCR. PBP2 patterns were also determined in all strains, as described previously (18). The presence of blaTEM and tetM genes was investigated in one isolate that was highly resistant to penicillin and tetracycline. The nucleotide and deduced amino acid sequences were analyzed with the Vector NTI version 10.3.0 analysis software package (Life Technologies). Searches of the sequences were performed with the BLAST program (available at the National Center for Biotechnology Information website [http://www.ncbi.nlm.nih.gov/]). Multiple-sequence alignments were performed with the ClustalW2 program (available at the European Bioinformatics Institute website [http://www.ebi.ac.uk/Tools/msa/clustalw2/]). N. gonorrhoeae isolates were typed using the NG-MAST method (25). The edited and trimmed sequences were uploaded into a publicly accessible database on the NG-MAST website (http://www.ng-mast.net) to obtain the allele and ST designations. A phylogenetic tree showing clonal relatedness of NG-MAST was created using the MEGA5 program (26).

RESULTS

Nineteen isolates of N. gonorrhoeae with AZM MICs of ≥2 μg/ml were identified in Ontario from July 2010 to February 2013 from a total of 2,800 nonduplicated N. gonorrhoeae isolates received at PHOL during that period of time (0.7%). These 19 isolates were recovered from patients between 20 and 72 years old, most of them male (16/19), who were treated in 8 different hospitals/clinics in the greater Toronto area. Two isolates displayed high-level AZM resistance (MIC, ≥2,048 μg/ml) (group A), and 17 isolates showed moderate resistance to AZM (MIC, 2 to 8 μg/ml) (group B) (Table 1).

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TABLE 1

Susceptibility profiles of azithromycin-resistant N. gonorrhoeae isolates (n = 19)

The two isolates in group A were resistant to ciprofloxacin and had high erythromycin MICs. One of them (STI133/2010) was also highly resistant to tetracycline and penicillin (the tetM and blaTEM-135 genes were identified in this isolate). Both isolates were susceptible to 3GC and spectinomycin and had low ertapenem MICs (Table 1). Interestingly, these isolates were 2- to 10-fold more resistant to gentamicin than the isolates in group B. The solithromycin MICs were ≤32 μg/ml, lower than those observed for erythromycin and AZM. Both isolates displayed a single adenine deletion within a 13-bp inverted repeat sequence located in the mtrR promoter and the mutation A2143G (N. gonorrhoeae numbering; A2059 in Escherichia coli numbering) in the four copies of the 23S rRNA rrl gene (Fig. 1, Table 2). One isolate also had the mutation G45D in MtrR (STI133/10 was designated GN640 in reference 27). No other mechanism of macrolide resistance was found. The two isolates belonged to different clones, ST5343 and ST4980, unrelated to the STs found in group B (Fig. 2, Table 2).

FIG 1
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FIG 1

Secondary structure of the peptidyl transferase center, domain V, of the 23S rRNA (E. coli numbering). Point mutations found in this study that were associated with macrolide resistance are shown in circles (N. gonorrhoeae numbering).

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TABLE 2

Molecular results of azithromycin-resistant N. gonorrhoeae isolates (n = 19)

FIG 2
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FIG 2

Phylogenetic tree of concatenated sequences of por and tbpB alleles for all NG-MAST sequence types (ST) identified in this study. MEGA5 was used to construct the phylogenetic tree using the neighbor-joining method (26). The tree topology is based on distance estimates using the “number of nucleotide differences” model. (A) Isolates highly resistant to azithromycin (MICs, ≥2,048 μg/ml); (B) isolates displaying reduced susceptibilities to ceftriaxone and cefixime (isolate STI137, only to cefixime) harboring the mosaic PBP2 allele type XXXIV.

The isolates from group B displayed high erythromycin MICs, most of them were resistant to ciprofloxacin, tetracycline, and penicillin, and all of them were susceptible to 3GC and spectinomycin (Table 1). However, 8 of them had reduced susceptibilities to cefixime (MIC, ≥0.12 μg/ml) and ceftriaxone (MICs, 0.06 to 0.12 μg/ml). Low MIC values were obtained for gentamicin (0.8 to 3 μg/ml), solithromycin (0.25 to 0.5 μg/ml), and ertapenem (0.003 to 0.032 μg/ml). The ertapenem MICs were slightly higher in the cluster of isolates with reduced susceptibilities to cefixime (ertapenem MICs, 0.023 to 0.032 μg/ml versus ≤0.012 μg/ml in the 3GC-susceptible strains). The mutation C2599T (C2611 in E. coli numbering) was found in the four copies of the 23S rRNA rrl gene of all the isolates of group B (Fig. 1, Table 2). Despite the fact that mutations in the mtrR gene or its promoter and/or in the riboprotein L4 gene were also observed in some isolates (Table 2), the mutation C2599T appears to be the main mechanism of AZM resistance found in this group, since some strains did not have mutations in other target genes related to macrolide resistance. No acquired methylase, efflux pump, or esterase genes or mutations in the riboprotein L22 gene were detected in any of the isolates. The most prevalent clone found in this group belonged to ST3158 (n = 8) or was closely related to it (n = 1, ST6915, tbpB 110; por 4133, 2 point mutations difference with por 1914 in ST3158) (Table 2). These 9 isolates harbored the mosaic PBP2 allele type XXXIV (Fig. 2, Table 2) and were isolated in 8 clinics/hospitals.

DISCUSSION

The emergence of multidrug-resistant gonococci is threatening the few therapeutic options still available for the treatment of gonorrhea. To address the increasing cephalosporin resistance of N. gonorrhoeae worldwide, current guidelines recommend a combination of cephalosporin and AZM as the first line of treatment for gonorrhea (4–6, 28). Cases of AZM resistance in gonococci have been rare and sporadic and only occasionally associated with cephalosporin resistance. In this study, we identified the emergence of two populations of AZM-resistant N. gonorrhoeae in Ontario, Canada, both of them with specific mutations in the macrolide's target, the 23S rRNA, as the main mechanisms of resistance (Tables 1 and 2). One of these groups included two isolates of N. gonorrhoeae displaying high-level AZM resistance. The second population of N. gonorrhoeae isolates moderately resistant to AZM is equally concerning, because it was associated with a worldwide disseminated clonal complex with decreased susceptibility to the cephalosporins, a combination that can threaten the efficacy of current therapeutic recommendations.

The prevalence of AZM resistance in Ontario has been slowly increasing since 2008, at which time no AZM-resistant isolates (MICs, ≥1 μg/ml) were detected. In this study, although only 0.6% (19/2,800) of all isolates collected from July 2010 to February 2013 were resistant to AZM (MICs, ≥2 μg/ml), 16 out of 19 isolates (84%) were recovered from February 2012 to February 2013, mostly from community hospitals and clinics. These results suggest that this phenotype is becoming more common in the province. Nine out of 19 isolates were multidrug resistant and belonged to the clonal group ST3158 or a related sequence type (Table 2, Fig. 2). This was one of the prevalent clones detected in 2008, showing reduced susceptibility to macrolides (MICs, 0.25 to 0.5 μg/ml versus ≤0.031 μg/ml in a wild-type isolate) associated with mutations in the mtrR promoter (18). These results indicate that the most significant mechanism of reduced susceptibility to macrolides in isolates from 2008 involved the overproduction of the MtrCDE efflux pump, an unspecific mechanism of resistance affecting macrolides, β-lactams, and tetracyclines. A theoretical explanation for the changes observed from 2008 (when only reduced susceptibility to AZM was found) to 2012-2013 (when moderate resistance to AZM was detected) is that the AZM mutant prevention concentration (MPC) in the 2008 clonal subpopulation was higher than the MPC in wild-type isolates, which could increase the chances of additional selection of AZM resistance mutations. Zhao and Drlica introduced the concept of MPC (the lowest concentration at which no mutants are selected) and the mutant selection window (a drug concentration range within which mutants are selectively enriched, with the window delimited by the MIC and the MPC) (29–31). Applying this concept, if an organism already has a mutation conferring reduced in vitro susceptibility to an antibiotic, even if the mutation does not confer clinical resistance, then the MPC will be increased, facilitating the selection of higher levels of resistance. In that context, and considering the recommended suboptimal (and potential mutant selector) AZM therapy dose of 1g (4, 6, 28, 32), isolates belonging to ST3158 with reduced susceptibilities to macrolides due to increased expression of the mtrCDE efflux pump gene (i.e., isolates with mtrR promoter mutations from 2008) could theoretically benefit with a higher MPC, increasing the chances of selection of additional resistance mutations during therapy. As observed in the clone ST3158 from 2012-2013, mutation C2599T in the four copies of the 23S rRNA gene affects more specifically the binding of macrolides, increasing the AZM MICs from 0.25 to 0.5 μg/ml in 2008 (18) to 2 to 8 μg/ml. This new level of AZM resistance can also increase the MPC and the mutant selection window of this bacterial subpopulation, giving it advantage over other gonococcal isolates in the presence of suboptimal concentrations of AZM and favoring the selection of additional mutations, like A2143G, not yet described in clone ST3158. Further studies are ongoing to define MPCs and the mutant selection windows of wild-type isolates, isolates with reduced AZM susceptibilities, and isolates moderately resistant to AZM, all belonging to clone ST3158 identified in Ontario. Another Canadian study showed a prevalence of ST3158 (and the related ST1407) in gonococci isolated in 2001 to 2010 that had reduced susceptibilities to 3GC (21). All of them had erythromycin MICs of ≥1 μg/ml and harbored the same promoter deletion affecting mtrCDE expression that was found in isolates from Ontario in 2008, which suggests that that clonal group was disseminated across the country. Besides reduced susceptibility or moderated resistance to AZM, all ST3158 or related types from 2008 (18) and from 2012-2013 (this study) also had reduced susceptibilities to 3GC due to PBP2 pattern XXXIV. Additional studies on national isolates from 2005 to 2012 indicated that, of 119 isolates tested, 50.4% had PBP2 pattern XXXIV, most of them belonging to ST1407 or a related sequence type (e.g., ST3158 [I. Martin, unpublished results]). This clonal group expressing this particular PBP2 pattern was also detected in other countries, all of them displaying reduced susceptibilities to 3GC and in some cases related to cefixime treatment failure (19, 20, 22, 33–35). Also, this same PBP2 pattern with the mutation A501P was present in 3GC-resistant isolates detected in Europe, both of which belonged to ST1407 (2, 3). The presence in Ontario of ST3158 expressing the PBP2 pattern XXXIV is currently increasing the risk of selecting 3GC resistance. In addition, this PBP2 mosaic correlates in our study with increased ertapenem MICs (0.016 to 0.032 μg/ml versus 0.003 to 0.012 μg/ml in strains with another PBP2 pattern). Ertapenem was proposed as a potential therapeutic option for the treatment of gonorrhea (36); if this carbapenem is added to future treatment regimens, the evolution of ertapenem resistance should be monitored.

To the best of our knowledge, these are the first N. gonorrhoeae isolates with high-level AZM resistance (MIC, ≥2,048 μg/ml) in Canada. A point mutation at position A2143G, known as a key position for macrolide interaction with the ribosome (12, 14), was the basis of that phenotype. Mutations in the MtrR protein, the repressor of the mtrCDE operon, also contribute to the high-level macrolide-resistant phenotype, but only for reduced susceptibility or moderate resistance to azithromycin (37). Low solithromycin MICs were observed even in isolates with AZM MICs of ≥2,048 μg/ml, as recently reported (27, 38). In contrast to AZM, which binds only one site on the 23S ribosomal subunit, solithromycin would have three sites of interaction, thereby enhancing its activity due to the higher number of binding sites (39). Our results, in concordance with X-ray structure studies of solithromycin in complex with the E. coli ribosomes (39), indicate that the point mutations found in the 23S rRNA of AZM-resistant gonococci (A2143G and C2599T; A2059 and C2611 in E. coli numbering) are not sufficient to produce high-level solithromycin resistance. Clinical trials (in development) will reveal its efficacy in vivo.

One of the isolates (STI133/2010) displaying high-level AZM resistance harbored the blaTEM-135 gene. TEM-135 has only one amino acid difference from TEM-1 (M182T), which is also found in TEM-type extended-spectrum β-lactamases (ESBLs) as a stabilizer mutation (e.g., TEM-20 contains mutation G238S, which extends the substrate profile of the enzyme to 3GC-like cefotaxime, and the stabilizer M182T mutation) (40, 41). TEM-135 would be a potential direct precursor of an ESBL, which could produce high-level resistance to 3GC.

Clonal coevolution of macrolide resistance and reduced susceptibility to 3GC has been detected in Ontario since 2012. This emergence and the potential threat of cephalosporin resistance due to the point mutation A501P in PBP2 pattern XXXIV and/or ESBL selection in isolates resistant to AZM can lead to discontinuation of macrolides and 3GC as therapeutic options for the treatment of gonorrhea and deserve continuous surveillance.

ACKNOWLEDGMENTS

We thank the microbiology staff of the Sexually Transmitted Infections and Antimicrobial Susceptibility sections at PHOL. We also thank Prabhavathi Fernandes (Cempra Inc.) for supplying solithromycin (CEM-101).

FOOTNOTES

    • Received 29 November 2013.
    • Returned for modification 28 December 2013.
    • Accepted 2 February 2014.
    • Accepted manuscript posted online 10 February 2014.
  • Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Azithromycin Resistance Is Coevolving with Reduced Susceptibility to Cephalosporins in Neisseria gonorrhoeae in Ontario, Canada
Vanessa G. Allen, Christine Seah, Irene Martin, Roberto G. Melano
Antimicrobial Agents and Chemotherapy Apr 2014, 58 (5) 2528-2534; DOI: 10.1128/AAC.02608-13

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Azithromycin Resistance Is Coevolving with Reduced Susceptibility to Cephalosporins in Neisseria gonorrhoeae in Ontario, Canada
Vanessa G. Allen, Christine Seah, Irene Martin, Roberto G. Melano
Antimicrobial Agents and Chemotherapy Apr 2014, 58 (5) 2528-2534; DOI: 10.1128/AAC.02608-13
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