Tetracycline Rapidly Reaches All the Constituent Cells of Uropathogenic Escherichia coli Biofilms

Abstract

We have developed a method for visualizing Escherichia coli cells that are exposed to tetracycline in a biofilm, based on a previous report that liposomes containing the E. coli TetR(B) protein fluoresce when exposed to this antibiotic. By our method, cells devoid of TetR(B) also exhibited tetracycline-dependent fluorescence. At 50 g of tetracycline ml؊1 , planktonic cells of a uropathogenic E. coli (UPEC) strain developed maximal fluorescence after 7.5 to 10 min of exposure. A similar behavior was exhibited by cells in a 24-or 48-h UPEC biofilm, as examined by confocal laser microscopy, regardless of whether they lined empty spaces or occupied densely packed regions. Further, a comparison of phase-contrast and fluorescent images of corresponding biofilm zones showed that all the cells fluoresced. Thus, all the biofilm cells were exposed to tetracycline and there were no pockets within the biofilm where the antibiotic failed to reach. It also appeared unlikely that niches of reduced exposure to the antibiotic existed within the biofilms.Diseases involving bacterial biofilms are generally chronic and difficult to treat, because bacteria in biofilms are more resistant to antimicrobial agents than their planktonic (freeliving) counterparts. Biofilms contribute to several serious diseases, such as endocarditis, cystitis, cystic fibrosis, and infections of prosthetic devices (4-6a).It is not known why biofilms possess greater resistance. The possibility that this might be due to a lack of penetration of antibiotics has received considerable attention. Although several studies suggest that biofilms permit antibiotic penetration (1,6,8,9,10,12,14), it was not ascertained in these studies if this involved exposure of all constituent cells to the antibiotic.We have developed a method to directly visualize cells coming in contact with tetracycline, based on the report that in the presence of the TetR(B) protein in liposomes (see http:// biosafety.ihe.be/AR/Tetracycline/Menu_Tet for tetracycline resistance determinant nomenclature), this antibiotic fluoresces when activated by UV light (13). TetR(B) is constitutively synthesized in bacteria containing Tn10 and regulates the expression of the tetA(B) gene that encodes the tetracycline efflux pump (2). We report here that the biofilms of uropathogenic Escherichia coli (UPEC) and K-12 strains of E. coli (11) are readily permeable to tetracycline and permit exposure of all the constituent cells to this antibiotic. MATERIALS AND METHODSBacterial strains and plasmids. A clinical isolate of uropathogenic E. coli (AMG1), obtained from the Microbiology Section of the Infectious Diseases Department of the Stanford Medical Center, and our laboratory E. coli strain, AMS6 (11), were employed. A tetracycline-resistant strain of AMG1, referred to as AMG2, containing the TetR(B) protein was generated using the Tn10 transposon, and the " hop" technique; the latter employs a temperature-sensitive phage (561) as the Tn10 vehicle. Transposition was achieved as described previously (15), using...

Biofilm-Related Infections: Bridging the Gap between Clinical Management and Fundamental Aspects of Recalcitrance toward Antibiotics

“…The same observation was made concerning K. pneumoniae biofilms and ciprofloxacin (22). Studies using fluorescent tetracycline demonstrated that 2-day biofilms were less susceptible than planktonic bacteria, whereas tetracycline-mediated fluorescence was present throughout the biofilm within 10 min (47). On the other hand, delayed antibiotic penetration may have important phenotypic consequences.…”

Density-dependent adaptive resistance allows swimming bacteria to colonize an antibiotic gradient

“…The unaltered MIC indicates that the kanamycin resistance of the invading populations is caused by an adaptive mechanism conferring phenotypic resistance, instead of being genetically encoded. Most previously described antibiotic-tolerant phenotypes are based on dormancy (Balaban et al, 2004;Lewis, 2010;Fridman et al, 2014) or reported the transient resistance of matured biofilms that were formed in the absence of antibiotics and exposed only after maturation (Spoering and Lewis, 2001;Anderl et al, 2000;Stone et al, 2002;Amini et al, 2011;Nguyen et al, 2011;Kirby et al, 2012). In contrast, the density-dependent adaptive resistance observed here enables planktonic E. coli to found a population in an antibiotic landscape, and permits swimming motility and population expansion in the presence of a lethal concentration of the antibiotic.…”

Role of biofilm in catheter-associated urinary tract infection☆

“…1,2 For example, in a rabbit model of CAUTI, 400 mg/kg of amdinocillin was required to eliminate Escherichia coli from the surface of the urinary catheter, although the minimum inhibitory concentration of amdinocillin against this organism in the planktonic state was 0.5 μg/mL. 13 Because several studies show that antibiotics can penetrate mature biofilms thoroughly, [14][15][16] the slow growth rates of organisms in biofilms is probably the major factor in conferring resistance. In addition, the juxtaposition of microorganisms of 1 or more species within a biofilm facilitates the transfer of antimicrobial resistance genes.…”