Abstract: 【Abstract】 Objective To investigate the in vitro antibacterial activity of antimicrobial peptides LL37 against Neisseria gonorrhoeae （N. gonorrhoeae）. Methods Seventy-five clinical isolates of N. gonorrhoeae were collected from the STD Clinic, Hospital for Skin Diseases, Chinese Academy of Medical Sciences in 2023. Their antimicrobial susceptibility to antibiotics was determined using the agar dilution method. In the growth inhibition assay, three N. gonorrhoeae strains （two clinically isolated multidrug-resistant strains and one reference strain FA1090） were treated with gradient concentrations of LL37 （7.2, 3.6, 1.8, 0.9, 0.45, 0.225, and 0.112 5 μmol/L） or sterile water for 2 hours. Bacterial suspensions were then prepared and inoculated onto agar plates, and colony-forming units were counted. The minimum inhibitory concentrations （MICs） of LL37 against the 75 clinical isolates and the reference strain FA1090 were determined using the broth microdilution method. The combined bactericidal effect of LL37 and ceftriaxone against 33 multidrug-resistant N. gonorrhoeae strains was evaluated using the checkerboard dilution method. Statistical analysis for paired samples was performed using the Wilcoxon signed-rank test. Results Seventy-five clinical isolates of N. gonorrhoeae were obtained from 73 male patients with urethritis and 2 female patients with cervicitis, including 33 multidrug-resistant strains and 42 non-multidrug-resistant strains. The growth inhibition assay showed that the survival rates of the three N. gonorrhoeae strains decreased with an increase in LL37 concentrations （range, 0.025 - 7.2 μmol/L）, and approached 0 at LL37 concentrations ≥ 3.6 μmol/L. The MICs of LL37 against the 75 N. gonorrhoeae isolates ranged from 1.8 to 7.2 μmol/L, and both the MIC?? and MIC?? were 3.6 μmol/L. The combined antimicrobial susceptibility test of LL37 and ceftriaxone against 33 multidrug-resistant strains showed synergistic effects in 3 strains （9.1%） and indifferent effects in 30 strains （90.9%）, with no antagonistic effect observed. The overall combined effect of LL37 and ceftriaxone was indifferent （geometric mean of the fractional inhibitory concentration index = 0.97）. Among the 28 strains with reduced susceptibility or resistance to ceftriaxone, the MIC of ceftriaxone decreased after combination with LL37 （Z = ?4.21, P < 0.001）. Conclusions In vitro experiments showed that LL37 had antibacterial activity against N. gonorrhoeae. The combined effect of LL37 and ceftriaxone was indifferent for most strains. LL37 may have potential for the treatment of gonorrhea.

Key words: Neisseria gonorrhoeae, Antimicrobial cationic peptides, LL37, Minimum inhibitory concentration, Ceftriaxone, Cefixime, Checkerboard method, In vitro techniques