ABSTRACTEscherichia coliisolates from inflammatory bowel disease (IBD) patients are often multidrug resistant, including to streptomycin. Streptomycin resistance (StrR) mutations can alter bacterial behavior, which may influence intestinal disease. We generated a spontaneous StrRstrain of the intestinal adherent-invasiveE. coli(AIEC) strain NC101. Whole-genome sequencing revealed a single missense mutation inrpsLthat commonly confers StrR,rpsL-K43N. StrRNC101 exhibited a striking loss of aggregation and significantly increased motility, behaviors that can impact host-microbe interactions. Behavioral changes were associated with reduced transcription ofcsgA, encoding the biofilm component curli, and increased transcription offliC, encoding flagellin. Scanning electron microscopy (SEM) detailed morphologic changes consistent with the observed alterations in multicellular behavior. Because intestinalE. coliisolates exhibit remarkable strain-specific differences, we generated spontaneous StrRmutants of 10 clinicalE. coliphylotype B2 strains from patients with IBD, colorectal cancer, and urinary tract infection. Out of these 10 StrRclinical strains, two had altered colony morphology on Congo red agar (suggesting changes in extracellular products), and three had significant changes in motility. These changes were not associated with a particularrpsLmutation nor with the presence of virulence genes encoding the inflammation-associatedE. colimetabolites yersiniabactin or colibactin. We conclude that common mutations inrpsL, which confer StrR, can differentially alter disease-associated phenotypes across intestinalE. colistrains. These findings highlight the heterogeneity among seemingly similar intestinalE. colistrains and reveal the need to carefully study the strain-specific effects of antibiotic resistance mutations, particularly when using these mutations during strain selection studies.IMPORTANCEWe demonstrate that StrR, commonly acquired through a single point mutation inrpsL(a gene encoding part of the 30S bacterial ribosome), strikingly alters the morphology and behavior of a key intestinal AIEC strain, NC101. These changes include remarkably diminished aggregation and significantly increased motility, traits that are linked to AIEC-defining features and disease development. Phenotypic changes were heterogeneous among other StrRclinicalE. colistrains, underscoring the need to evaluate the strain-specific effects of commonly acquired antibiotic resistance mutations. This is important, as the results of studies using mutant StrREnterobacteriaceaestrains (e.g., for cloning orin vivoselection) may be confounded beyond our demonstrated effects. Long term, these findings can help researchers better distinguish the contribution of specificE. colitraits to functional changes in the microbiota. Evaluating these strain-level differences could provide insight into the diversity of IBD symptoms and lead to improved therapies for microbiota-driven intestinal disorders.