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<OL> <LI> Understand the phenomenon of antimicrobial persistence in the food borne pathogen Salmonella enterica serotype Typhimurium (S. typhimurium) by physiologically and genetically characterizing subpopulations of cells that resist killing by different antimicrobial agents. <LI>Identify and compare commonly used antimicrobial agents, in addition to antibiotics, that can also invoke persistence in S. typhimurium.<LI> Isolate and characterize new S. typhimurium mutants with altered ability to enter into a persistent state. <LI>Determine if persistent S. typhimurium are altered in their pathogenicity using a mouse model.
NON-TECHNICAL SUMMARY: It has long been observed that treatment of bacteria with distinct classes of antibiotics is generally ineffective at completely killing the entire population. A small subset of cells enter into a non-growing state known as persistence. Persistent bacteria are able to survive exposure to a variety of antimicrobial agents therefore reducing the efficacy of the treatment. A better understanding of persistence should lead to new, more effective treatments to eliminate bacterial contamination.
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APPROACH: Susceptibility testing assays will be used to measure the response of S. typhimurium to a variety of antibiotics and other antimicrobial compounds. Enrichment techniques as well as flow cytometer methods will be used to isolate subpopulations of cells that persist through the treatment, but are not resistant to the antimicrobial agent. Persistent cells will also be characterized by microscopy and by exposure to fluorescent dyes to determine the physiological state of the cells. Transposon mutagenesis will also be used to isolate new mutants with either heightened or decreased ability to enter into a persistent state. It will also be determined if persistent cells are altered in pathogenicity by determining the LD50 of infected mice.
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PROGRESS: 2003/09 TO 2007/08 <BR>
OUTPUTS: The goal of this project was to understand the basis of antimicrobial persistence. In contrast to resistance, persistence refers to the ability of bacteria to tolerate antimicrobials that kill the majority of the population. A small subpopulation of cells enter into a non-growing state that can survive treatment with antibiotics and disinfectants, that is lethal for the majority of the culture. Although difficult to study, we have sought to understand persistence, and to assess its potential role in food safety, by isolating mutants of Salmonella enterica Ser Typhimurium that show increased levels of persistence to antibiotics (Fig. 3). Our activities included quantifying the levels of persistence in S. enterica using assays measuring survival to a variety of different antibiotics that target different cellular processes, including cell wall biosynthesis and protein synthesis. We also developed a genetic screen to identify new "high persistence" mutants of S. enterica. These mutants were characterized by the persistence assays. The product resulting from these studies include 8 new S. enterica mutants strains that show elevated persistence to antibiotics. By mapping the genes responsible for this phenotype, we will be able to identify the cellular systems responsible for tolerance of antimicrobials. Upon publication, these mutants will be made available to the scientific community interested in similar studies. <BR> PARTICIPANTS: Gregory Phillips, Ph.D., Principal investigator Ronald Griffith, DVM, Ph.D.,Co-principal investigator Andrew Slattery, M.S., graduate student researcher Alec Victorsen, graduate student researcher
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IMPACT: 2003/09 TO 2007/08 <BR>
The outcomes of these studies include new bacterial strains to study the molecular basis of antimicrobial persistence in Salmonella. In general, the mutants showed an extended lag phase prior to resuming growth at the same rate as wild type. This is not the sole explanation for persistence, however, as mutants defective in other processes (ackA and pta), which also showed an extended lag phase, did not display high persistence. Since we were interested in identifying potential gain-of-function mutations, we chose to isolate the high persistence mutants by chemical mutagenesis. While this strategy will yield a wide range of mutations, they are more difficult to map than simple transposon insertions. We have, however, identified genetically linked markers to 4 of the mutations and are currently mapping their locations. This information will subsequently lead us to identity of the mutations responsible for high persistence. A potential change of condition that could result from these studies is improved methods for treatment of antimicrobial agents, including antibiotics and disinfectants, to avoid inducing a state of high persistence in food-borne pathogens.