The chromosomal toxin gene yafQ is a determinant of multidrug tolerance for Escherichia coli growing in a biofilm

Abstract

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 loci hipBA and relBE have been linked to the frequency at which persister cells occur in E. coli populations. In the present study, we focused on the dinJ-yafQ-encoded toxin-antitoxin system and hypothesized that deletion of the toxin gene yafQ might influence cell survival in antibiotic-exposed biofilms. By using confocal laser scanning microscopy and viable cell counting, it was determined that a Delta yafQ 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-type E. coli, the Delta yafQ strain had up to a approximately 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 of yafQ from a high-copy-number plasmid resulted in up to a approximately 10,000-fold increase in the number of biofilm cells surviving exposure to these bactericidal drugs. In contrast, neither the inactivation nor the overexpression of yafQ affected the tolerance of biofilms to doxycycline or rifampin (rifampicin). Furthermore, deletion of yafQ did not affect the tolerance of stationary-phase planktonic cells to any of the antibacterials tested. These results suggest that yafQ mediates the tolerance of E. 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.

FIG. 1.

Biofilm formation by wild-type E. coli K-12 BW25113 is similar to that of its isogenic ΔyafQ mutant. (a) Biofilm mean viable cell counts and standard deviations were comparable for the two strains, and this assessment was based on the indicated number of control measurements (n). When their structures were examined by CLSM, the wild-type (b) and mutant (c) strains also had similar structures at the air-liquid-surface interface of polystyrene pegs. Each microscopy panel is a representative of four independent replicates and captures an area of 238 by 238 μm.

FIG. 2.

Biofilm populations of the ΔyafQ strain have decreased numbers of cells surviving exposure to cefazolin and tobramycin compared to the numbers of parental E. coli K-12 BW25113 cells. Mean log killing was calculated after the biofilms had been exposed to cefazolin (a) and tobramycin (b) for 24 h. Each datum point is based on 4 to 16 independent replicates. Asterisks, significant difference in log killing of the mutant population compared to that of the wild-type population by using the criteria indicated in Materials and Methods. The mean viable cell counts for the biofilms not exposed to antibiotics were 6.19 ± 0.31 and 5.99 ± 0.19 log₁₀ CFU/peg for the wild-type and ΔyafQ strains, respectively.

FIG. 3.

Biofilm populations of the ΔyafQ strain and isogenic parental strain E. coli K-12 BW25113 had similar numbers of cells surviving exposure to doxycycline and rifampin. Mean log killing was calculated from the viable cell counts after the biofilms had been exposed to doxycycline (a) and rifampin (b) for 24 h. Each datum point is based on 4 to 20 independent replicates. The mean viable cell counts for the biofilms not exposed to antibiotics were 5.84 ± 0.58 and 5.85 ± 0.29 log₁₀ CFU/peg for the wild-type and ΔyafQ strains, respectively.

FIG. 4.

Overexpression of yafQ from a high-copy-number plasmid increased the number of cells in E. coli K-12 BW25113 biofilm populations surviving exposure to bactericidal concentrations of cefazolin and tobramycin. Mean log killing was calculated from the viable cell counts after the biofilms had been exposed to cefazolin (a) and tobramycin (b) for 24 h. The biofilms were incubated with 5 μM IPTG for 4 h before antibiotic exposure. All assays were conducted with 30 μg/ml chloramphenicol in the growth medium to select for the pCA24N expression vector. Each datum point is based on four to eight independent replicates. Asterisks, significant difference in log killing of the mutant population compared to that of the wild-type population by using the criteria indicated in Materials and Methods. The mean viable cell counts for the biofilms unexposed to antibiotics were 5.34 ± 0.80 and 5.44 ± 0.15 log₁₀ CFU/peg for the wild-type and ΔyafQ strains, respectively.

FIG. 5.

Overexpression of yafQ from a high-copy-number plasmid had no effect on the number of cells in E. coli K-12 BW25113 biofilm populations surviving exposure to doxycycline or rifampin. Mean log killing was calculated from the viable cell counts after the biofilms had been exposed to doxycycline (a) and rifampin (b) for 24 h. The biofilms were incubated with 5 μM IPTG for 4 h before antibiotic exposure. All assays were conducted with 30 μg/ml chloramphenicol in the growth medium to select for the pCA24N expression vector. Each datum point is based on four to eight independent replicates. The mean viable cell counts for the biofilms not exposed to antibiotics were 5.38 ± 0.09 and 5.65 ± 0.22 log₁₀ CFU/peg for the wild-type E. coli K-12 BW25113 strain bearing pCA24N_ptac and pCA24N_ptac yafQ, respectively.

FIG. 6.

Stationary-phase planktonic cell populations of ΔyafQ and isogenic parental strain E. coli K-12 BW25113 had similar numbers of cells surviving exposure to cefazolin and tobramycin. Mean log killing was calculated after the biofilms had been exposed to cefazolin (a) and tobramycin (b) for 24 h. Each datum point is based on four to eight independent replicates. The mean viable cell counts for the stationary-phase planktonic populations not exposed to antibiotics were 8.74 ± 0.35 and 8.99 ± 0.06 log₁₀ CFU/ml for the wild-type and ΔyafQ strains, respectively.