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Disrupting folate metabolism reduces the capacity of bacteria in exponential growth to develop persisters to antibioticsBacteria can survive high doses of antibiotics through stochastic phenotypic diversification. We present initial evidence that folate metabolism could be involved with the formation of persisters. The aberrant expression of the folate enzyme gene fau seems to reduce the incidence of persisters to antibiotics. Folate-impaired bacteria had a lower generation rate for persisters to the antibiotics ampicillin and ofloxacin. Persister bacteria were detectable from the outset of the exponential growth phase in the complex media. Gene expression analyses tentatively showed distinctive profiles in exponential growth at times when bacteria persisters were observed. Levels of persisters were assessed in bacteria with altered, genetically and pharmacologically, folate metabolism. This work shows that by disrupting folate biosynthesis and usage, bacterial tolerance to antibiotics seems to be diminished. Based on these findings there is a possibility that bacteriostatic antibiotics such as anti-folates could have a role to play in clinical settings where the incidence of antibiotic persisters seems to drive recalcitrant infections.