VCOM Research Day Program Book 2023

Graduate Student Research Biomedical 14 Creation and Infectious Dose Determination of Streptomycin-Resistant Mutants of Neisseria Gonorrhoeae Strains: a Mouse Model Study

Babatomiwa Kikiowo, Aloka B. Bandara, Nader S. Abutaleb and Mohamed N. Seleem Corresponding author: Kbabatomiwa@vt.edu

Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg

Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrhoeae, a human pathogen that can colonize male and female genital mucosal surfaces. Gonococcal infections pose a significant global public health concern; the Centers for Disease Control and Prevention reported 677,769 cases of gonorrhea in 2020. The rapid occurrence of N. gonorrhoeae resistance to all classes of antibiotics could lead to untreatable gonorrhea. Thus, novel and effective anti- N. gonorrhoeae drugs are urgently needed. N. gonorrhoeae strain FA1090 is the only strain used in in vivo mouse models because of its natural resistance to streptomycin, a necessary antibiotic utilized in the models to inhibit the commensal flora in the lower genital tract of mice to enhance N. gonorrhoeae colonization. However, this strain is susceptible to all antibiotics used to treat Neisseria , such as ceftriaxone and azithromycin, and therefore, not suitable for use in drug discovery studies. To test the efficacy of new

drugs against clinical isolates, such as ceftriaxone resistance and azithromycin-resistance strains in vivo , the streptomycin resistance is a required phenotype. Hence, our aim in this study is to construct streptomycin-resistant mutants of two clinically important N. gonorrhoeae strains exhibiting resistance to the frontline antibiotics, ceftriaxone and azithromycin, used in the treatment of gonorrhea that could be utilized in vivo. The rpsL gene encoding for resistance to streptomycin was isolated from N. gonorrhoeae FA1090 and cloned into the pMR32 plasmid. The resultant plasmid was introduced into wild-type N. gonorrhoeae WHO-X (ceftriaxone resistant) and CDC-181 (azithromycin-resistant) strains. The resulting mutant strains were confirmed to be resistant to streptomycin (minimum inhibitory concentrations, MICs >1024 µg/mL). A pilot study to determine the infectious dose of the newly developed mutant strains was performed using a murine model of N. gonorrhoeae genital tract infection. Groups (n=5)

of ovariectomized female 8-week-old BALB/c mice were inoculated intravaginally with 20 µL of different doses of N. gonorrhoeae . An antibiotic cocktail was administered throughout the experiment to increase the susceptibility to N. gonorrhoeae and minimize vaginal flora. Vaginal swabs were collected every other day and N. gonorrhoeae CFU were counted. The constructed streptomycin-resistant mutants, like the N. gonorrhoeae FA1090 strain, survived in mice for seven days. Based on this study, the newly constructed streptomycin-resistant strains have the potential to be used in mouse models to investigate the efficacy of drugs against azithromycin-resistant and ceftriaxone-resistant N. gonorrhoeae strains.

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