This study examined the microbicidal activity of 222-nm UV radiation (UV₂₂₂), which is potentially a safer alternative to the 254-nm UV radiation (UV₂₅₄) that is often used for surface decontamination. Spores and/or growing and stationary-phase cells of Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis, Staphylococcus aureus, and Clostridioides difficile and a herpesvirus were all killed or inactivated by UV₂₂₂ and at lower fluences than with UV₂₅₄B. subtilis spores and cells lacking the major DNA repair protein RecA were more sensitive to UV₂₂₂, as were spores lacking their DNA-protective proteins, the α/β-type small, acid-soluble spore proteins. The spore cores' large amount of Ca²⁺-dipicolinic acid (∼25% of the core dry weight) also protected B. subtilis and C. difficile spores against UV₂₂₂, while spores' proteinaceous coat may have given some slight protection against UV₂₂₂ Survivors among B. subtilis spores treated with UV₂₂₂ acquired a large number of mutations, and this radiation generated known mutagenic photoproducts in spore and cell DNA, primarily cyclobutane-type pyrimidine dimers in growing cells and an α-thyminyl-thymine adduct termed the spore photoproduct (SP) in spores. Notably, the loss of a key SP repair protein markedly decreased spore UV₂₂₂ resistance. UV₂₂₂-treated B. subtilis spores germinated relatively normally, and the generation of colonies from these germinated spores was not salt sensitive. The latter two findings suggest that UV₂₂₂ does not kill spores by general protein damage, and thus, the new results are consistent with the notion that DNA damage is responsible for the killing of spores and cells by UV₂₂₂IMPORTANCE Spores of a variety of bacteria are resistant to common decontamination agents, and many of them are major causes of food spoilage and some serious human diseases, including anthrax caused by spores of Bacillus anthracis Consequently, there is an ongoing need for efficient methods for spore eradication, in particular methods that have minimal deleterious effects on people or the environment. UV radiation at 254 nm (UV₂₅₄) is sporicidal and commonly used for surface decontamination but can cause deleterious effects in humans. Recent work, however, suggests that 222-nm UV (UV₂₂₂) may be less harmful to people than UV₂₅₄ yet may still kill bacteria and at lower fluences than UV₂₅₄ The present work has identified the damage by UV₂₂₂ that leads to the killing of growing cells and spores of some bacteria, many of which are human pathogens, and UV₂₂₂ also inactivates a herpesvirus.

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