Determining Differential Inhibition of N. gonorrhoeae Penicillin-Binding Proteins by Extended-Spectrum Cephalosporins

<em>Neisseria gonorrhoeae </em>infection is treated with cephalosporins, antibiotics that target outer-membrane penicillin-binding proteins (PBPs), such as PBP1 and PBP2.&nbsp; Mutations in PBPs have led to the rapid development of antibiotic resistance, rendering many cephalosporin treatments ineffective.&nbsp; Despite being a major resistance mechanism, the nature of mutated PBP and drug interactions has yet to be characterized in a conclusive or reproducible manner with whole membrane or pathogen models.&nbsp; Therefore, we sought to identify binding relationships between antibiotic-resistant PBP variants and cephalosporins.&nbsp; We incubated cephalosporins, cefoxitin, ceftriaxone, and cefdinir, with purified PBPs and fluorescent Bocillin, allowing us to visualize the degree of cephalosporins binding using gel electrophoresis and fluorescent imaging.&nbsp; Cefoxitin inhibits PBP1 only, with no effect on PBP2, while ceftriaxone displayed inhibition of both PBP1 and PBP2, with greater influence on PBP1.&nbsp; Cefdinir only displayed inhibition of PBP1 and PBP2 at high concentrations.&nbsp; Based on these results, the ability of ceftriaxone and cefoxitin to synergistically inhibit growth was assessed using Minimum Inhibitory Concentration protocols.&nbsp; Cefoxitin did not significantly change the ability of ceftriaxone to inhibit growth.&nbsp; These results validated the use of purified PBP models to understand the mechanism of individual drugs against antibiotic-resistant strains, but are limited in ability to describe pathogen behavior, especially in an environment with multiple antibiotics.