ksgA mutations confer resistance to kasugamycin in Neisseria gonorrhoeae

Abstract

The aminoglycoside antibiotic kasugamycin (KSG) inhibits translation initiation and thus the growth of many bacteria. In this study, we tested the susceptibilities to KSG of 22 low-passage clinical isolates and 2 laboratory strains of Neisseria gonorrhoeae. Although the range of KSG minimum inhibitory concentrations (MICs) was narrow (seven-fold), clinical isolates and laboratory strains fell into three distinct classes of KSG sensitivity, susceptible, somewhat sensitive and resistant, with MICs of 30, 60–100 and 200 μg/mL, respectively. Two genes have previously been shown to be involved in bacterial KSG resistance: rpsI, which encodes the 30S ribosomal subunit S9 protein; and ksgA, which encodes a predicted dimethyltransferase. Although sequencing of rpsI and ksgA from clinical isolates revealed polymorphisms, none correlated with the MICs of KSG. Ten spontaneous KSG-resistant (KSG^R) mutants were isolated from laboratory strain FA1090 at a frequency of <4.4 × 10⁻⁶ resistant colony-forming units (CFU)/total CFU. All isolated KSG^R variants had mutations in ksgA, whilst no mutations were observed in rpsI. ksgA mutations conferring KSG resistance included four point mutations, two in-frame and one out-of-frame deletions, one in-frame duplication and two frame-shift insertions. These data show a narrow range of susceptibilities for the clinical isolates and laboratory strains examined; moreover, the differences in MICs do not correlate with nucleotide polymorphisms in rpsI or ksgA. Additionally, spontaneous KSG^R mutants arise by a variety of ksgA mutations.

Introduction

The aminoglycoside antibiotic kasugamycin (KSG), first isolated from Streptomyces kasugaensis, has been used to prevent rice blast disease caused by the fungus Pyricularia oryzae[1], [2], [3]. KSG inhibits the growth of a wide variety of microorganisms, with reported low toxicity against plants, humans and other animals [2], [4], [5]. However, as an aminoglycoside, some degree of nephrotoxicity and ototoxicity is expected. Several Gram-negative bacteria, including Pseudomonas spp. and Escherichia coli strains, as well as the Gram-positive Bacillus spp. are sensitive to KSG [6]. Although clinical use of KSG has been explored as treatment for Pseudomonas aeruginosa infection in the bladder [4], it is currently only used agriculturally. KSG inhibits translation initiation by blocking transfer RNA (tRNA) binding to the 30S ribosomal subunit; it can be bacteriostatic or bactericidal depending on the concentration used [7]. Recent structural analysis of KSG translation inhibition has provided a detailed mechanism for KSG binding and inhibition [8], [9]. KSG is thought to mimic messenger RNA (mRNA) codon nucleotides and to occupy the peptidyl (P) and exit (E) sites of the ribosome, causing distortion of the mRNA–tRNA codon–anticodon interaction and blocking translation initiation in susceptible organisms [8], [9].

Several KSG resistance mutations have been identified and characterised in E. coli[10], [11], [12], [13] and Bacillus subtilis[14]. The E. coli gene product of ksgA, an adenosine dimethyltransferase KsgA, is responsible for methylation of 16S ribosomal RNA (rRNA) adenosines at positions 1518 and 1519 [15], [16], [17]. Interestingly, this rRNA modification by KsgA appears to be conserved in all species of bacteria, archaea and eukarya studied to date [18], [19]. The exact biological function of this rRNA modification is unknown, and in many bacteria loss of KsgA-dependent methylation is not lethal [20], [21], [22]. Mutations that disrupt KsgA-dependent rRNA methylation are the most common mechanism of KSG resistance in bacteria [11]. Other mutations known to confer KSG resistance include mutation of the target nucleotides A1518 and A1519 in the 16S rRNA [12] and amino acid substitutions in the 30S protein subunit S9, the gene product of rpsI[10]. These mutations may stabilise the mRNA–tRNA interaction perturbed by KSG leading to KSG resistance. Interestingly, rpsI mutations can result in both resistance to and dependence on KSG [10], [23]. For example, one KSG-resistant (KSG^R) rpsI mutant, E. coli strain MV101, required KSG to depress the rate of protein synthesis, otherwise the enhanced ribosomal activity caused by the rpsI mutation was lethal [10]. Both 16S rRNA and rpsI mutations occur less frequently than mutations in ksgA[11], [12].

Neisseria gonorrhoeae is an obligate human pathogen and is the only causative agent of the sexually transmitted infection gonorrhoea. In the USA, gonorrhoea is the second most frequently reported communicable disease, with 339 593 reported cases in 2005 and as many as 700 000 total cases yearly [24]. Many infected individuals may be asymptomatic [25]; however, serious symptomatic infections can occur both in men and women. Notably, N. gonorrhoeae can cause pelvic inflammatory disease (PID) in women [26], epididymitis in men and arthritis both in men and women [27]. Additionally, the pharynx, rectum and conjunctiva are sites often infected by N. gonorrhoeae, thus antibiotics must be effective at controlling the bacteria at multiple mucosal sites. Although antibiotic treatment has historically been effective in control of the disease, the increased prevalence of antibiotic-resistant N. gonorrhoeae has severely limited available treatment options [28]. Accordingly, the US Centers for Disease Control and Prevention (CDC) has classified N. gonorrhoeae as a ‘superbug’, and only cephalosporin drugs are recommended for treatment of gonorrhoea in the USA [29]. With ever-increasing resistance to antibiotics and with disease incidence on the rise in many countries, novel therapies are needed to control the spread of disease. Here we examined the susceptibilities of 22 clinical isolates and 2 laboratory strains of N. gonorrhoeae to KSG and isolated 10 spontaneous KSG^R mutants.