Escherichia coli Harboring a Natural IncF Conjugative F Plasmid Develops Complex Mature Biofilms by Stimulating Synthesis of Colanic Acid and Curli▿ †

It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F+ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump.

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a well-known inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket

Aminoacyl β-naphthylamides as substrates and modulators of AcrB multidrug efflux pump

Efflux pumps of the resistance-nodulation division superfamily, such as AcrB, make a major contribution to multidrug resistance in Gram-negative bacteria. Inhibitors of such pumps would improve the efficacy of antibiotics, and ameliorate the crisis in health care caused by the prevalence of multidrug resistant Gram-negative pathogens. Phenylalanyl-arginine β-naphthylamide (PAβN), is a wellknown inhibitor of AcrB and its homologs. However, its mechanism of inhibition is not clear. Because the hydrolysis of PAβN in Escherichia coli was nearly entirely dependent on an aminopeptidase, PepN, expression of PepN in periplasm allowed us to carry out a quantitative determination of PAβN efflux kinetics through the determination of its periplasmic concentrations by quantitation of the first hydrolysis product, phenylalanine, after a short period of treatment. We found that PAβN is efficiently pumped out by AcrB, with a sigmoidal kinetics. We also examined the behavior of PAβN homologs, Ala β-naphthylamide, Arg β-naphthylamide, and Phe β-naphthylamide, as substrates of AcrB and as modulators of nitrocefin efflux through AcrB. Furthermore, molecular dynamics simulations indicated that the mode of binding of these compounds to AcrB affects the modulatory activity on the efflux of other substrates. These results, and the finding that PAβN changes the nitrocefin kinetics into a sigmoidal one, suggested that PAβN inhibited the efflux of other drugs by binding to the bottom of the distal binding pocket, the so-called hydrophobic trap, and also by interfering with the binding of other drug substrates to the upper part of the binding pocket

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms▿ †

Biofilms are considered to be highly resistant to antimicrobial agents. Several mechanisms have been proposed to explain this high resistance of biofilms, including restricted penetration of antimicrobial agents into biofilms, slow growth owing to nutrient limitation, expression of genes involved in the general stress response, and emergence of a biofilm-specific phenotype. However, since combinations of these factors are involved in most biofilm studies, it is still difficult to fully understand the mechanisms of biofilm resistance to antibiotics. In this study, the antibiotic susceptibility of Escherichia coli cells in biofilms was investigated with exclusion of the effects of the restricted penetration of antimicrobial agents into biofilms and the slow growth owing to nutrient limitation. Three different antibiotics, ampicillin (100 μg/ml), kanamycin (25 μg/ml), and ofloxacin (10 μg/ml), were applied directly to cells in the deeper layers of mature biofilms that developed in flow cells after removal of the surface layers of the biofilms. The results of the antibiotic treatment analyses revealed that ofloxacin and kanamycin were effective against biofilm cells, whereas ampicillin did not kill the cells, resulting in regrowth of the biofilm after the ampicillin treatment was discontinued. LIVE/DEAD staining revealed that a small fraction of resistant cells emerged in the deeper layers of the mature biofilms and that these cells were still alive even after 24 h of ampicillin treatment. Furthermore, to determine which genes in the biofilm cells are induced, allowing increased resistance to ampicillin, global gene expression was analyzed at different stages of biofilm formation, the attachment, colony formation, and maturation stages. The results showed that significant changes in gene expression occurred during biofilm formation, which were partly induced by rpoS expression. Based on the experimental data, it is likely that the observed resistance of biofilms can be attributed to formation of ampicillin-resistant subpopulations in the deeper layers of mature biofilms but not in young colony biofilms and that the production and resistance of the subpopulations were aided by biofilm-specific phenotypes, like slow growth and induction of rpoS-mediated stress responses

Exposure of conjugative plasmid carrying Escherichia coli biofilms to male-specific bacteriophages

Escherichia coli carrying a natural conjugative F-plasmid generates F-pili mating pairs, which is important for early biofilm formation. In this study, we investigated the effect of male-specific filamentous single stranded DNA bacteriophage (f1) and RNA bacteriophage (MS2) on the formation of biofilms by E. coli carrying a natural conjugative F-plasmid. We showed that the early biofilm formation was completely inhibited by addition of the f1 phage, but not the MS2 phage. This suggests that the tip of F-pili is the specific attachment site for mating pairs formation and the side of F-pili has a non-obligatory role during biofilm formation. The inhibitory effect of the f1 phage was dependent on the time of addition during the biofilm formation. No inhibitory effect was observed when the f1 phages were added to the mature biofilms. This resistant mechanism of the mature biofilms could be attributed to the biofilm-specific phenotypes representing that the F-pili mating pairs were already formed and then the curli production commenced during the biofilm maturation. The pre-formed mating pairs seemed to resist the f1 phages. Altogether, our results indicate a close relationship between the presence of conjugative plasmid and male-specific bacteriophages within sessile biofilm communities, as well as the possibility of using the male-specific bacteriophages to control biofilm formation