The adaptability of bacterial resistance to antibiotics contributes to its high efficiency during evolution. Tetracycline (TC) is a broad-spectrum antimicrobial agent. Chromatographic analyses and mass spectrometry were used to study the effects of the light illumination of a 462 nm light-emitting diode (LED) on the conformational changes of TC in a phosphate buffer solution (PBS, pH 7.8). Especially, the inactivation of superoxide anion radicals (O
2•
−) and
Escherichia coli (
E. coli), including that of a multidrug-resistant
E. coli (
MDR E. coli), were investigated during the photolysis of TC. A photolysis product of TC (PPT) was generated in an alkaline solution after the illumination of a blue light. The mass spectra of PPT had characteristic ion signals in m/z 459, 445, and 249.1 Da. The PPT has the molecular formula of C
22H
22N
2O
9, and the exact mass is 458.44 g/mol. The inactivation of
MDR E. coli is not significant with TC treatment. The drug-resistant ability of
MDR E. coli has a less significant effect on PPT, and the changed conformation of TC retained the inactivation ability of
MDR E. coli upon blue light photoreaction. With TC, illuminated by a blue light in a pH 7.8 PBS, O
2•
− was generated from TC photolysis, which enhanced the inactivation of
E. coli and
MDR E. coli. A 96.6% inactivation rate of
MDR E. coli was reached with TC under 2.0 mW/cm
2 blue light illumination at 25 ± 3 °C for 120 min, and the effects of the TC-treated photoreaction on
MDR E. coli viability repressed the growth of
MDR E. coli by 4 to 5 logs. The present study of the blue light photoreaction of TC offers a new approach to the inactivation of
MDR E. coli.
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