Mutations causing in vitro resistance to azithromycin in Neisseria gonorrhoeae

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Abstract

In 1999, a cluster of gonococcal isolates exhibiting high Minimal Inhibitory Concentrations (MICs), to azithromycin (2.0–4.0 mg/l) were identified in Kansas City, MO. Isolates were characterized by auxotype/serovar class, lipoprotein (Lip) subtyping and sequencing of the mtrR gene, which has been implicated in decreased azithromycin susceptibility in the gonococcus. Isolates were Pro/IB-3 and contained the 17c Lip subtype. Molecular characterization of the mtrR gene revealed a 153 base pair insertion sequence located between the mtrR/mtrC promoter and the mtrC gene. Some isolates also contained a frame shift within the mtrR gene. Transformation of these mutations into an azithromycin-sensitive recipient strain resulted in transformants with MICs as high as 2.0 mg/l and inactivation of the mtrD gene reduced azithromycin MICs 270-fold. These results demonstrated that the mtr mutations were responsible for the increased MICs in these isolates.

Introduction

Azithromycin and erythromycin belong to the macrolide family of antibiotics. While neither antimicrobial is currently recommended by Centers for Disease Control and Prevention (CDC) for the treatment of gonorrhoea, a 2 g dose of azithromycin is FDA approved for the treatment of uncomplicated gonorrhoea [1]. Unfortunately, the 2 g dose of azithromycin causes a high level of gastrointestinal distress. A 1 g dose of azithromycin is well tolerated but is insufficiently effective with a cure rate of only 93% [1].

One of the mechanisms of in vitro resistance to azithromycin and erythromycin in Neisseria gonorrhoeae is the multiple transferable resistance (mtr) efflux system that confers resistance to structurally diverse hydrophobic agents such as detergents, dyes, and macrolide antibiotics [2], [3], [4], [5], [6], [7]. Recent studies have demonstrated that mutations in the promotor regions of the mtrR and mtrC genes, which are part of the mtr efflux operon, produce increased minimal inhibitory concentrations (MICs) of azithromycin ranging from 0.25 to 0.5 mg/l of azithromycin [8], [9].

In this report, we characterized the mtr operon of 12 isolates collected from individual males in Kansas City, MO as part of the Gonococcal Isolate Surveillance Project (GISP) from March to October 1999 [10], [11]. The MICs of these isolates were considerably higher than those previously described having MICs of 1.0–4.0 mg/l of azithromycin. All patients had been treated with cefixime so there was no report of treatment failure with azithromycin [10].

Section snippets

Bacterial strains

All 12 pre-treatment isolates characterized were sent to the CDC, frozen in trypticase soy broth containing 20% glycerol and were stored at −70 °C. Additional strains or sequences used or examined included strains 28Bl, F-28, FA19 and 4 isolates from Kansas City with MICs≤0.125 mg/l of azithromycin (Table 1).

Strain characterization and culture

Isolates were characterized by auxotype and serovar (A/S) as described previously [12], [13]. Agar dilution susceptibilities of isolates to penicillin G, tetracycline, spectinomycin,

Relatedness of isolates

As shown in Table 1, all 12 of the Kansas City Azi/Ery isolates were Pro/IB-3 A/S class and the 17c Lip subtype. The MIC ranges for antibiotics other than Azi/Ery were as follows: penicillin G, 0.5–1.0 mg/l; tetracycline, 1.0–2.0 mg/l; ceftriaxone, 0.008–0.015 mg/l; cefixime, 0.015–0.03 mg/l, and ciprofloxacin, 0.002–0.008 mg/l.

Identification of mtrR mutations in clinical isolates

The region coding for the mtrR protein of both Azi/Ery-resistant and -sensitive isolates of N. gonorrhoeae was sequenced from approximately 324 nucleotides upstream of

Discussion

One mechanism conferring reduced susceptibility of N. gonorrhoeae to macrolide antibiotics, specifically azithromycin and erythromycin has been demonstrated to be a function of the expression of an efflux system regulated by the mtrR product [2], [5]. While these earlier isolates exhibited only slightly increased MICs of azithromycin, isolates obtained in Kansas City over a 8-month period demonstrated significantly elevated values that ranged from 1.0 to 4.0 mg/l of azithromycin. This

Acknowledgements

The authors wish to thank Dr Joyce Joyce and Dr Gerald Hoff of the Kansas City Health Department and Dr Catherine McLean of the Division of STD Prevention, CDC, for their contributions to this study. The authors also wish to thank Laura Doyle of the Cleveland Regional GISP laboratory for initial antimicrobial resistance determination and Dr William Shafer of Emory University for providing the mtrD::kan construct.

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