Jack H Lambert, Rayven Todd, Thomas W Bagwell, Damani Andre, Raybun Spelts, Fantasia Gorham, Jamie Woods, Ayomide H Adeyemi, Shondia Evans, Rafael Ponce-Terashima, Kenneth I Onyedibe
Background/Objectives: Antimicrobial resistance threatens public health globally, and critical access hospitals (CAHs) serving rural communities face structural barriers to implementing antibiotic stewardship programs (ASPs). This study evaluated whether a regional educational intervention could improve antimicrobial stewardship knowledge and implementation among rural healthcare professionals. Methods: A quantitative, quasi-experimental repeated cross-sectional evaluation was conducted following Antibiotic Stewardship conferences in 2023 and 2024. Participants from 38 rural and critical access hospitals completed pre-conference, post-conference, 6-month follow-up, and implementation surveys. Quantitative data were analyzed using descriptive statistics and unpaired chi-square tests, with Fisher's exact test substituted where expected cell counts were below 5, with significance set at p < 0.05. Results: Seventy healthcare professionals participated across both years. Correct identification of the use of an antibiogram to guide empiric (rather than definitive) therapy improved from 27% pre-conference to 58% post-conference (p = 0.029). Confidence in antibiogram interpretation increased from 67% to 100% post-intervention (p = 0.004, Fisher's exact) but declined to 50% (5 of 10) at 6-month follow-up. Knowledge of the minimum isolate threshold required to construct an antibiogram improved from 23% (7 of 30) pre-conference to 64% (7 of 11) at 6-month follow-up (p = 0.026, Fisher's exact). Despite this, prescribing intent for conditions where antibiotics are typically unnecessary showed minimal improvement: recommendations for antibiotic use in middle ear infections decreased from 50% to 36.84%, and for mpox from 20% to 15.79%. All participants reported intent to modify clinical practice; however, time constraints, technology limitations, and lack of resources were consistently cited as primary barriers for implementation across both survey years. Conclusions: A regional educational intervention improved short-term antimicrobial stewardship knowledge and confidence among rural healthcare professionals, but knowledge retention declined over time and structural barriers persisted. Education alone is insufficient; sustained reinforcement and rural-adapted ASP models incorporating protected ASP time, decision-support tools and external partnerships are needed to translate knowledge gains into consistent stewardship practice.