Commensal Neisseria species are reservoirs of antimicrobial resistance for pathogenic Neisseria. Azithromycin resistance occurs at both low and high levels; however, the mechanisms by which this resistance is transferred between species are not fully understood. It remains unclear how acquired macrolide resistance genes, such as msrD, and intrinsic chromosomal mechanisms, such as 23S rRNA and mtrR mutations, contribute to azithromycin resistance.
To address this gap, in vitro transformation experiments were performed using the commensal species Neisseria subflava as a donor. Two donor strains exhibited low azithromycin MICs (4 and 8 µg/mL), while two additional donor strains showed high‑level resistance (MICs >256 µg/mL); both high‑level donors carried the msrD gene. Two Neisseria meningitidis and two Neisseria gonorrhoeae strains were used as recipients, all of which were initially susceptible to azithromycin, with baseline MICs ranging from 0.125 to 1.5 µg/mL. Following transformation, azithromycin MICs were determined for all transformants, and acquisition of msrD was assessed by PCR.
Transformation with low‑level azithromycin‑resistant N. subflava donors resulted in clear increases in azithromycin MICs in both recipient species. N. meningitidis transformants exhibited MICs of 12 to 16 µg/mL, while N. gonorrhoeae transformants showed smaller increases to 2 to 4 µg/mL indicating species‑specific differences in resistance expression and/or concurrent expression of one or more additional intrinsic resistance mechanisms.
In contrast, transformation using DNA from N. subflava which are highly azithromycin‑resistant resulted in N. meningitidis strains with MICs ranging from 48 to 128 µg/mL. Transformation of N. gonorrhoeae with DNA from high‑level resistant donors was unsuccessful. Notably, the msrD was not detected in any of the transformants, suggesting that acquisition of msrD was not required for high‑level resistance and indicating that one or more chromosomal alterations acquired during transformation contributed to the increased azithromycin resistance, compared with the wild‑type recipient strain.
Future work will focus on genomic and phenotypic characterisation of the transformed strains to identify genetic changes associated with increased resistance. Such analyses will help clarify the mechanisms contributing to enhanced azithromycin resistance in these species and may inform understanding of evolution to resistance in closely related bacterial pathogens.