Background: Ceftriaxone-resistant Neisseria gonorrhoeae (NG) is a major global public health concern. Ceftriaxone resistance is mediated by mosaic penA alleles which were acquired from commensal Neisseria species through horizontal gene transfer (HGT). To elucidate this mechanism, we aimed to transform NG into ceftriaxone-resistant by employing a cell-to-cell transformation model using a commensal Neisseria species harboring the mosaic penA60 allele.
Methods: We reviewed the literature to identify the most common mosaic penA alleles associated with ceftriaxone-resistant NG. In the laboratory, we transformed the commensal N. cinerea ATCC14685 into ceftriaxone-resistant (NC-CR) using donor penA60 DNA from ceftriaxone-resistant CDC-1280 NG and used it for cell-to-cell transformation trials. Transformation trials were performed in liquid media using various concentrations of NC-CR and ceftriaxone-susceptible NG. To identify NG transformants and determine minimum inhibitory concentrations (MIC), post-cultured samples were plated on GC agar plates with increasing concentrations of ceftriaxone. Transformation trials with donor penA60 DNA instead of NC-CR were also performed with ceftriaxone-susceptible NG multi-locus sequencing type (MLST) ST1901, a strain type commonly associated with ceftriaxone resistance.
Results: The mosaic penA60 allele was the most common allele type (74.4%, (218/293)) associated with NG with MIC ≥ 0.25 µg/mL. Transformation of N. cinerea and NG with donor penA60 DNA increased the MIC to 1 µg/mL and 0.5 µg/mL, respectively. Despite repeated trials under varied experimental conditions, cell-to-cell transformation of NG into ceftriaxone-resistant was not observed based on identification of transformants using selective ceftriaxone-containing media. Trials using purified NC-CR DNA, also did not result in NG transformation. However, transformation of NG ST1901 was consistently achieved using a 3.5 kb purified PCR product containing the full-length penA60 allele suggesting that smaller DNA fragments are necessary for transformation; the degree of DNA fragmentation during cell-to-cell transformation is unknown.
Conclusions: NG ST1901 can be transformed into ceftriaxone-resistant using donor penA60 DNA, but transformation was not observed using a cell-to-cell co-culturing model. Additional studies employing single-cell analysis, instead of culture-based approaches, for detection of transformants are warranted. The use of a pharyngeal NG controlled human infection model might be useful for characterizing HGT events between commensal Neisseria species and NG under natural conditions.