OBJECTIVE:Comparison of studies evaluating patient-to-patient transmission of organisms is difficult, given the lack of standardized criteria. We used fluoroquinolone-resistant Escherichia coli (FQREC) as a model to characterize variability in definitions of relatedness across studies and to evaluate the resultant impact on study conclusions. DESIGN:Narrative review and cohort study. METHODS:The narrative review compared relatedness criteria across studies of FQREC. Additionally, an existing database was used to compare relatedness of isolates on the basis of molecular criteria alone versus molecular plus clinical criteria with different temporal cutoffs (hospitalization overlap of ≥1 day or allowance for nonoverlap of hospitalization dates of ≤7 days or ≤30 days). RESULTS:Forty-six articles met narrative review inclusion criteria. Sixteen studies exclusively utilized molecular criteria to define relatedness. Thirty studies included molecular and clinical criteria. Of these, 6 included temporal data (ie, time period of isolate identification), 10 included patient location, and 14 included proximity and temporal criteria. For the database analysis, 353 patients were colonized with FQREC. There were 2 main clusters containing 48 and 17 related isolates within 49 pulsed-field gel electrophoresis types. Among the clusters, 18.4% of isolates were related by molecular criteria. Incorporating clinical criteria, fewer isolates were considered related: 5.7% of isolates using 30-day criteria, 3.1% using 7-day criteria, and 1.4% using 1-day overlap. CONCLUSIONS:There is considerable variability in definitions of relatedness of FQREC. Utilizing molecular criteria alone to define relatedness overestimates transmission compared with definitions including clinical criteria. Standard definitions of relatedness in studies of antimicrobial-resistant organisms are needed.