The Chromosomal Toxin Gene yafQ Is a Determinant of Multidrug Tolerance for Escherichia coli Growing in a Biofilm

Escherichia coli is refractory to elevated doses of antibiotics when it is growing in a biofilm, and this is potentially due to high numbers of multidrug-tolerant persister cells in the surface-adherent population. Previously, the chromosomal toxin-antitoxin locihipBA andrelBE have been linked to the frequency at which persister cells occur inE. coli populations. In the present study, we focused on thedinJ -yafQ -encoded toxin-antitoxin system and hypothesized that deletion of the toxin geneyafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a ΔyafQ mutant produced biofilms with a structure and a cell density equivalent to those of the parental strain. In-depth susceptibility testing identified that relative to wild-typeE. coli , the ΔyafQ strain had up to a ∼2,400-fold decrease in cell survival after the biofilms were exposed to bactericidal concentrations of cefazolin or tobramycin. Corresponding to these data, controlled overexpression ofyafQ from a high-copy-number plasmid resulted in up to a ∼10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression ofyafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion ofyafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest thatyafQ mediates the tolerance ofE. coli biofilms to multiple but specific antibiotics; moreover, our data imply that this cellular pathway for persistence is likely different from that of multidrug-tolerant cells in stationary-phase planktonic cell cultures.