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 toxinantitoxin 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 ⌬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 ⌬yafQ strain had up to a ϳ2,400-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-copynumber plasmid resulted in up to a ϳ10,000-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.Biofilms, which are assemblages of microorganisms encased in a self-produced matrix of extracellular polymers, are a prevalent mode of microbial life that is thought to be responsible for many chronic, recalcitrant infections (8,13,30,31). The bacteria in biofilms have decreased sensitivities to antibiotics and other antimicrobial agents, and this decreased sensitivity occurs independently of the presence of resistance determinants on mobile genetic elements (7). Recent findings suggest that growth in a biofilm might be a population-based strategy for bacterial survival, as multidrug resistance and tolerance can be linked to an ongoing process of phenotypic diversification among the surface-adherent cells (16,39). One type of phenotypic variant produced by single-species bacterial populations is the persister cell, a metabolically quiescent cell that neither grows nor dies when it is exposed to bactericidal concentrations of antibiotics (2,3,19,36,38). Although subpopulations of these physiological variants represent 0.1% or less of logarithmically growing planktonic cell cultures (29, 36), the proportion of persister cells in biofilms might be as high as 10% (36). Therefore, it is reasonable to hypothesize that persister cells contribute to the multidrug tolera...

RETRACTED: Bacterial persistence by RNA endonucleases

“…Cells were grown in LB containing chloramphenicol (50 μg/mL) at 37°C. cases, toxin overproduction strongly increased the persister fraction with the two different antibiotics, thus supporting that the initial observations (32)(33)(34)(35) are generally valid (Fig. S3).…”

“…Simultaneously, our observations allow us to propose a long sought after common function to TA loci. The combined observations that overproduction of mRNases induced a persister-like state (32,35) and that mRNase-induced stasis was reversible (30) prompted us to investigate the suggested connection between persistence and TA loci. We found strong evidence that TA loci encoding mRNases and the persistence phenomenon are intimately connected: Activation of endogenously encoded RelE and ectopic production of five mRNases induced antibiotic tolerance (Fig.…”

“…Cells overproducing TA-encoded mRNases were, similar to persister cells, tolerant to antibiotics (32)(33)(34)(35). Moreover, cell populations enriched for persisters had increased levels of TA mRNAs (33,34), and deletion of yafQ (encoding a RelE homolog) resulted in reduced persistence of cells in a biofilm but not in planktonic cells (32).…”

Regulation of phenotypic variability by a threshold-based mechanism underlies bacterial persistence

“…Threshold regulation of phenotypic variability may be a general property of those motifs, whose function would be to differentiate bacterial populations into distinct subpopulations to cope with stress (21). Recent evidence that implicates TA modules in Myxococcus development (43) and in biofilm formation (44,45) support that view. Finally, through linking a genetic regulatory motif and its phenotypic effect, we now have a basic understanding of the connections between multiple elements of bacterial persistence: genotype, phenotype, and fitness.…”

The diguanylate cyclase YddV controls production of the exopolysaccharide poly-N-acetylglucosamine (PNAG) through regulation of the PNAG biosynthetic pgaABCD operon

“…Biofilm and planktonic cells differ significantly in their physiology, in their gene expression pattern and even in their morphology. In particular, biofilm cells are characterized by production of adhesion factors and extracellular polysaccharides (EPS), resistance to environmental stresses, and lower sensitivity to antibiotics compared with planktonic cells (Costerton et al, 1995;Anderl et al, 2000;Harrison et al, 2007Harrison et al, , 2009). …”