High-Level Azithromycin Resistance Occurs in Neisseria gonorrhoeae as a Result of a Single Point Mutation in the 23S rRNA Genes

Abstract

High-level azithromycin resistance (AZM-HR), defined as a MIC of >256 mg/liter, emerged in Neisseria gonorrhoeae in the United Kingdom in 2004. To determine the mechanism of this novel phenotype, isolates from the United Kingdom that were AZM-HR (n, 19), moderately AZM resistant (MICs, 2 to 8 mg/liter) (n, 26), or sensitive (MICs, 0.12 to 0.25 mg/liter) (n, 4) were screened for methylase (erm) genes and for mutations in the mtrR promoter region, associated with efflux pump upregulation. All AZM-resistant isolates and 12 sensitive isolates were screened for mutations in domain V of each 23S rRNA allele. All AZM-HR isolates contained the A2059G mutation (Escherichia coli numbering) in three (3 isolates) or four (16 isolates) 23S rRNA alleles. Most (22/26) moderately AZM resistant isolates contained the C2611T mutation in at least 3/4 alleles. The remainder contained four wild-type alleles, as did 8/12 sensitive isolates, while one allele was mutated in the remaining four sensitive isolates. Serial passage of AZM-sensitive colonies on an erythromycin-containing medium selected AZM-HR if the parent strain already contained mutation A2059G in one 23S rRNA allele. The resultant AZM-HR strains contained four mutated alleles. Eight isolates (five moderately AZM resistant and three AZM-HR) contained mutations in the mtrR promoter. No methylase genes were detected. This is the first evidence that AZM-HR in gonococci may result from a single point mutation (A2059G) in the peptidyltransferase loop in domain V of the 23S rRNA gene. Mutation of a single allele is insufficient to confer AZM-HR, but AZM-HR can develop under selection pressure. The description of a novel resistance mechanism will aid in screening for the AZM-HR phenotype.

Molecular mechanism of macrolide–lincosamide resistance in Moraxella catarrhalis

“…On the other hand, in a Gram-negative diplococcus, Neisseria gonorrhoeae, which is similar to M. catarrhalis in that it also contains four copies of the rrn operon (Chisholm et al, 2010), a single mutation at position 2059 in at least three of the 23S rRNA alleles confers high-level resistance to azithromycin (MIC.256 mg l 21 ) (Chisholm et al, 2010). However, it has not been investigated whether one or more mutated 23S rRNA alleles can confer high-level macrolidelincosamide resistance in M. catarrhalis.…”

“…As a result, transformants 49143TF25 and 19TF24 with the single A2058T mutation in three or four of the 23S rRNA alleles demonstrated a high level of resistance to 14-, 15-and 16-membered macrolides and lincosamide (Table 2). Furthermore, a previous report has demonstrated that clinical isolates of N. gonorrhoeae with a single mutated 23S rRNA allele (A2059G) showed extremely low azithromycin MICs (0.06-0.125 mg l 21 ) (Chisholm et al, 2010). Moreover, since other transformants, viz.…”

“…Transformation experiments were performed as described by Chisholm et al (2010), with some modifications. Briefly, M. catarrhalis ATCC 49143 and Mc19 were suspended in 1 ml TE buffer (pH 8.0) to the equivalent of a McFarland standard of 3.0; these were then inoculated on BHI agar plates and incubated for 1 h at 37 uC in an atmosphere of 95 % air and 5 % CO 2 .…”

“…In general, mechanisms underlying resistance against these antimicrobials are known to involve active efflux of the antibiotics and modification of the ribosomal target by enzymes such as rRNA methylase, enzymic inactivation or mutations in the 23S rRNA gene, as described previously in various organisms by Chisholm et al (2010), Li et al (2011) and Versalovic et al (1996). Another mechanism, involving point mutations in genes encoding the ribosomal proteins L4 (rplD) and L22 (rplV), has been reported in some clinical isolates (Matsuoka et al, 2004;Tait-Kamradt et al, 2000).…”

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…This mechanism of resistance is therefore of special relevance in microorganisms with only one or two rrn loci (Leclercq, 2002;Vester & Douthwaite, 2001) also being described in microorganisms with three or four rrn loci (Chisholm et al, 2010;Saito et al, 2012). Enterobacteriaceae such as E. coli, Salmonella spp.…”