Transcriptomic Profiling of Ceftriaxone Tolerant- Phenotypes of <i>Neisseria gonorrhoeae</i> Reveals Downregulation of Ribosomal Genes

Background Antibiotic tolerance is associated with failure of antibiotic treatment and accelerates the development of antimicrobial resistance. The molecular mechanisms underlying antimicrobial tolerance remain, poorly understood. In some instances, tolerant bacteria can slow metabolism by extending the lag phase without altering antimicrobial susceptibility. We recently induced ceftriaxone (CRO) tolerance in the N. gonorrhoeae reference strain WHO P. In the current study, we characterized the transcriptomic profiles of these CRO-tolerant phenotypes. Methods To induce tolerance, WHO P strains were grown under three-hour intermittent CRO exposure (10× the MIC), followed by overnight growth in GC broth for seven consecutive days, with cultures maintained in sextuplicate. Two control cultures were maintained without CRO exposure. The tolerance and CRO susceptibility of the isolates were assessed using a modified Tolerance Disc (TD) test. Total RNA was isolated from tolerant isolates (n=12) and control (n = 3) strains, followed by Ribo depletion. Library preparation was carried out using the Stranded TruSeq RNA library preparation kit. Sequencing was performed on a NextSeq6000, v2. 1 × 150 bp (Illumina Inc., San Diego, CA, USA), and data analysis was performed using the CLC Genomics Workbench v22 (clcbio, Denmark). Results Transcriptomic analysis revealed no differentially expressed genes at the first time point that was assessed (after 1 day of CRO exposure). However, after 3 days of CRO exposure, 13 genes were found to be significantly downregulated, including tRNA-Ser (C7S06_RS03100) and tRNA-Leu (C7S06_RS04945) and ribosomal RNA genes (16S and 23S rRNA). Following 7 days of exposure, 51 genes were differentially expressed with most downregulated, such as SecB (Protein-export chaperone SecB) and tRNA-Ser (C7S06_RS01850) and the 16S and 23S ribosomal RNA genes. Conclusions The development of CRO-tolerance in N. gonorrhoeae was associated with the downregulation of various ribosomal genes and associated genes, reflecting a potential mechanism for bacterial survival under antibiotic stress.