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The evolution of resistance in  Salmonella  to fluoroquinolones (FQs) under a broad range of sub-inhibitory concentrations (sub-MICs) has not been systematically studied. This study investigated the mechanism of resistance development in  Salmonella enterica  serovar Enteritidis ( S . Enteritidis) under sub-MICs of 1/128×MIC to 1/2×MIC of enrofloxacin (ENR), a widely used veterinary FQ. It was shown that the resistance rate and resistance level of  S . Enteritidis varied with the increase in ENR concentration and duration of selection. qRT-PCR results demonstrated that the expression of outer membrane porin (OMP) genes,  omp  C ,  omp  D  and  omp  F , were down-regulated first to rapidly adapt and develop the resistance of 4×MIC, and as the resistance level increased (≥8×MIC), the up-regulated expression of efflux pump genes,  acrB ,  emrB  amd  mdfA , along with mutations in quinolone resistance-determining region (QRDR) gradually played a decisive role. Cytohubba analysis based on transcriptomic profiles demonstrated that  purB ,  purC ,  purD ,  purF ,  purH ,  purK ,  purL ,  purM ,  purN  and  purT  were the hub genes for the FQs resistance. The ‘de novo’ IMP biosynthetic process, purine ribonucleoside monophosphate biosynthetic process and purine ribonucleotide biosynthetic process were the top three biological processes screened by MCODE. This study first described the dynamics of FQ resistance evolution in  Salmonella  under a long-term selection of sub-MICs of ENR in vitro. In addition, this work offers greater insight into the transcriptome changes of  S . Enteritidis under the selection of ENR and provides a framework for FQs resistance of  Salmonella  for further studies.

The Evolution of Fluoroquinolone Resistance in Salmonella under Exposure to Sub-Inhibitory Concentration of Enrofloxacin