Identification of a mutation associated with erythromycin resistance in Bordetella pertussis: implications for surveillance of antimicrobial resistance

Abstract

Erythromycin treatment failures and in vitro resistance of Bordetella pertussis have been reported on several occasions in the past few years, but the mechanism of resistance has not been described. One potential mechanism, genetic modification of the erythromycin-binding site on the 23S rRNA of the 50S ribosomal subunit, has been observed in other bacteria. To explore this possibility, we amplified the portion of the 23S rRNA gene encoding the central loop of domain V. DNA sequencing and restriction fragment length polymorphism of the PCR products showed that each of the four erythromycin-resistant B. pertussis strains tested contained an A-to-G transition mutation at position 2058 (Escherichia coli numbering) of the 23S rRNA gene. The mutation was not found in seven erythromycin-susceptible isolates tested. Two of the resistant isolates were heterozygous, containing at least one mutant copy and one wild-type copy of the 23S rRNA gene. These results indicate that erythromycin resistance in these strains is likely due to a mutation of the erythromycin-binding site in the 23S rRNA gene. Identification of the resistance mechanism will facilitate development of molecular susceptibility testing methods that can be used directly on clinical specimens in the absence of an isolate.

FIG. 1.

Screening for A2058G and A2059G mutations in B. pertussis by PCR-RFLP analysis. (A). BsaI (lanes 1 to 5) or BbsI (lanes 6 to 10) digestion of a 521-bp fragment of the 23S rDNA gene of erythromycin-resistant B. pertussis clinical isolates (A228, C353, and MN2531), heterogeneous strain C352, and erythromycin-susceptible strain MN2726. The 521-bp fragment was generated by PCR amplification using primers 1907U and 2408L as described in Materials and Methods. Lanes: M, 100-bp ladder (Life Technologies); 1 and 6, B. pertussis A228; 2 and 7, B. pertussis C352; 3 and 8, B. pertussis C353; 4 and 9, B. pertussis MN2531; 5 and 10, B. pertussis MN2726. (B). BbsI digestion of the 521-bp fragment of additional isolates of B. pertussis. Lanes: M, 100-bp ladder (Life Technologies); 1 to 7, erythromycin-susceptible clinical isolates B. pertussis MN277, MN973, MN1286, MN1699, MN1773, MN1893, and MN2726; 8, erythromycin-resistant B. pertussis isolate MN253.

FIG. 2.

Sequence chromatograms for wild-type (MN2726), homogeneously erythromycin-resistant (A228 and C353), and heterogeneous (C352) strains of B. pertussis. The nucleotide at position 2047 is surrounded by a box. Note the presence and relative amounts of the residual A peak at position 2047 in the chromatograms of heterozygous strains A228 and C352.