Transposon mutagenesis identifies genes which control antimicrobial drug tolerance in stationary-phase Escherichia coli

• Published in: FEMS microbiology letters Vol. 243; no. 1; pp. 117 - 124
• Main Authors: Hu, Yanmin, Coates, Anthony R.M.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Elsevier B.V 01.02.2005; Blackwell Publishing Ltd; Blackwell; Oxford University Press
• Subjects: Animals; Anti-Bacterial Agents - pharmacology; Anti-Bacterial Agents - therapeutic use; Antibiotic tolerance; Antibiotics; Antiinfectives and antibacterials; Antimicrobial agents; Bacteria; Bacteriology; Biological and medical sciences; Chemotherapy; Ciprofloxacin; DNA Transposable Elements; DNA, Intergenic - genetics; Drug Tolerance; E coli; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Escherichia coli - growth & development; Escherichia coli Infections - drug therapy; Escherichia coli Infections - microbiology; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Female; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Bacterial; Genes; Genetics; Genomes; Gentamicin; Growth rate; Humans; Kanamycin; Kanamycin - pharmacology; Kanamycin - therapeutic use; Mice; Mice, Inbred ICR; Microbiology; Mutagenesis; Mutagenesis, Insertional; Mutants; Open reading frames; Polymerase Chain Reaction; Rifampin; Stationary phase; Thigh; Thigh - microbiology; Transposon; Transposon mutagenesis; Transposons; Mutagenesis; Drug tolerance; Transposon; Escherichia coli; Polymerase chain reaction; Resistance; Gene; Bacteria; Identification; Transposable element; Antimicrobial agent; Enterobacteriaceae
• ISSN: 0378-1097; 1574-6968
• DOI: 10.1016/j.femsle.2004.11.049

Tolerance to antimicrobial agents is a universal phenomenon in bacteria which are no longer multiplying or whose growth rate slows. Since slowly multiplying bacteria occur in clinical infections, extended periods of antimicrobial chemotherapy are needed to eradicate these organisms and to achieve cure. In this study, the molecular basis of antibiotic tolerance was investigated using transposon mutagenesis. We screened 5000 Escherichia coli Tn 10Cam mutants for reduction of kanamycin tolerance in late stationary phase and found that 4935 mutants were able to grow to late stationary phase. Reduced tolerance was observed in nine mutants which became sensitive to killing by kanamycin. The mutant KS639 was the most sensitive one to kanamycin, and its genome was disrupted in an intergenic region which lies between aldB and yiaW open reading frames. This mutant showed increased sensitivity not only to kanamycin but also to gentamicin, ciprofloxacin and rifampicin. Reduced tolerance of KS639 to kanamycin was also observed in a murine thigh infection model. P1 transduction to the wild type strains confirmed that the intergenic region was responsible for the tolerance of the bacterium to antibiotics. Using PCR-directed one-step gene replacement, we inactivated the genes aldB, yiaW and yiaV. We also deleted the intergenic region. There was no difference in kanamycin tolerance between each mutant (Δ aldB, Δ yiaW and Δ yiaV) and the parental strain. But the mutant lacking the intergenic region showed reduced tolerance to kanamycin. These data suggest that the intergenic region between aldB and yiaW genes may be involved in tolerance to antimicrobial agents in E. coli. Furthermore, they show that it is important in murine infection during antibiotic treatment and lead to a faster kill of the mutant bacteria.