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Genetic Determinants of Salmonella Resistance to the Biofilm-Inhibitory Effects of a Synthetic 4-Oxazolidinone Analog

Biofilms are resistant to killing by disinfectants and antimicrobials. S. enterica biofilms facilitate long-term host colonization and persistence in food processing environments. Synthetic analogs of 4-oxazolidinone natural products show promise as antibiofilm agents. Here, we show that a synthetic 4-oxazolidinone analog inhibits Salmonella biofilm through effects on both motility and biofilm matrix gene expression. Furthermore, we identify three genes that promote Salmonella resistance to the antibiofilm effects of the compound. This work provides insight into the mechanism of antibiofilm effects of a synthetic 4-oxazolidinone analog in Gram-negative bacteria and demonstrates new mechanisms of intrinsic antimicrobial resistance in Salmonella biofilms. ABSTRACT Biofilms formed by Salmonella enterica are a frequent source of food supply contamination. Since biofilms are inherently resistant to disinfection, new agents capable of preventing biofilm formation are needed. Synthetic analogs of 4-oxazolidinone containing natural products have shown promise as antibiofilm compounds against Gram-positive bacteria. The purpose of our study was 2-fold: to establish the antibiofilm effects and mechanism of action of a synthetic 4-oxazolidinone analog (JJM-ox-3-70) and to establish mechanisms of resistance to this compound in Salmonella enterica serovar Typhimurium (S. Typhimurium). JJM-ox-3-70 inhibited biofilm formation but had no effect on cell growth. The antibiofilm effects were linked to disruption of curli fimbriae and flagellar gene expression and alteration in swimming motility, suggesting an effect on multiple cellular processes. Using a 2-step screening approach of defined multigene and single-gene deletion mutant libraries, we identified 3 mutants that produced less biofilm in the presence of JJM-ox-3-70 than the isogenic WT, with phenotypes reversed by complementation in trans. Genes responsible for S. Typhimurium resistance to the compound included acrB, a component of the major drug efflux pump AcrAB-TolC, and two genes of unknown function (STM0437 and STM1292). The results of this study suggest that JJM-ox-3-70 inhibits biofilm formation by indirect inhibition of extracellular matrix production that may be linked to disruption of flagellar motility. Further work is needed to establish the role of the newly characterized genes as potential mechanisms of biofilm intrinsic antimicrobial resistance. IMPORTANCE Biofilms are resistant to killing by disinfectants and antimicrobials. S. enterica biofilms facilitate long-term host colonization and persistence in food processing environments. Synthetic analogs of 4-oxazolidinone natural products show promise as antibiofilm agents. Here, we show that a synthetic 4-oxazolidinone analog inhibits Salmonella biofilm through effects on both motility and biofilm matrix gene expression. Furthermore, we identify three genes that promote Salmonella resistance to the antibiofilm effects of the compound. This work provides insight into the mechanism of antibiofilm effects of a synthetic 4-oxazolidinone analog in Gram-negative bacteria and demonstrates new mechanisms of intrinsic antimicrobial resistance in Salmonella biofilms.