Antibiotics have been one of the most successful forms of therapy in medicine. However, the efficiency of antibiotics is compromised by the emergence of antibiotic-resistant pathogens. To reduce antibiotic resistance, complete understanding of bacterial tactics to defend themselves against antibiotics is necessary. Small-noncoding RNAs (sRNAs) modulate gene expression by base-pairing with multiple target mRNAs. Cellular levels of Hfq-dependent sRNAs influence antibiotic resistance by modulating expression of specific target genes; therefore, such sRNAs could be a good tool to identify target mRNAs that modulate antibiotic susceptibility and may themselves be used as druggable molecules. Here, we report the identification of genes and pathways associated with OxyS RNA-mediated cephalothin resistance using phenotypic and expression analyses of OxyS-regulated genes identified by RNA-seq, literature mining, or predictions. From our studies we found that the differential expression of 27 OxyS-regulated genes was involved in cephalothin susceptibility. Among them, 17 gene knockouts showed resistance to the drug and nine from them is associated with cAMP receptor protein (CRP), a transcriptional dual regulator in E. coli. Moreover, levels of OxyS and OxyS-modulated genes (cycA and cysH) were also altered in multidrug-resistant (MDR) E. coli strains. Together, our data suggest that OxyS extensively modulates gene expression in multiple pathways to develop cephalothin resistance. In addition, OxyS and its regulated target genes, either individually or in combination, could be used as molecular markers and targets for the identification and eradication of cephalothin-resistant strains. (C) 2018 Elsevier Inc. All rights reserved.