Thithiwat May

Increased Antibiotic Resistance of Escherichia coli in Mature Biofilms

ABSTRACT 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.

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.

Induction of Multidrug Resistance Mechanism in Escherichia coli Biofilms by Interplay between Tetracycline and Ampicillin Resistance Genes

ABSTRACT Biofilms gain resistance to various antimicrobial agents, and the presence of antibiotic resistance genes is thought to contribute to a biofilm-mediated antibiotic resistance. Here we showed the interplay between the tetracycline resistance efflux pump TetA(C) and the ampicillin resistance gene (blaTEM-1) in biofilms of Escherichia coli harboring pBR322 in the presence of the mixture of ampicillin and tetracycline. E. coli in the biofilms could obtain the high-level resistance to ampicillin, tetracycline, penicillin, erythromycin, and chloramphenicol during biofilm development and maturation as a result of the interplay between the marker genes on the plasmids, the increase of plasmid copy number, and consequently the induction of the efflux systems on the bacterial chromosome, especially the EmrY/K and EvgA/S pumps. In addition, we characterized the overexpression of the TetA(C) pump that contributed to osmotic stress response and was involved in the induction of capsular colanic acid production, promoting formation of mature biofilms. However, this investigated phenomenon was highly dependent on the addition of the subinhibitory concentrations of antibiotic mixture, and the biofilm resistance behavior was limited to aminoglycoside antibiotics. Thus, marker genes on plasmids played an important role in both resistance of biofilm cells to antibiotics and in formation of mature biofilms, as they could trigger specific chromosomal resistance mechanisms to confer a high-level resistance during biofilm formation.

RelE-Mediated Dormancy Is Enhanced at High Cell Density in Escherichia coli

Bacteria show remarkable adaptability under several stressful conditions by shifting themselves into a dormant state. Less is known, however, about the mechanism underlying the cell transition to dormancy. Here, we report that the transition to dormant states is mediated by one of the major toxin-antitoxin systems, RelEB, in a cell density-dependent manner in Escherichia coli K-12 MG1655. We constructed a strain, IKA121, which expresses the toxin RelE in the presence of rhamnose and lacks chromosomal relBE and rhaBAD. With this strain, we demonstrated that RelE-mediated dormancy is enhanced at high cell densities compared to that at low cell densities. The initiation of expression of the antitoxin RelB from a plasmid, pCA24N, reversed RelE-mediated dormancy in bacterial cultures. The activation of RelE increased the appearance of persister cells against β-lactams, quinolones, and aminoglycosides, and more persister cells appeared at high cell densities than at low cell densities. Further analysis indicated that amino acid starvation and an uncharacterized extracellular heat-labile substance promote RelE-mediated dormancy. This is a first report on the induction of RelE-mediated dormancy by high cell density. This work establishes a population-based dormancy mechanism to help explain E. coli survival in stressful environments.

Localized Expression Profiles of rpoS in Escherichia coli Biofilms

Although importance of the rpoS gene on biofilm formation by Escherichia coli has been suggested, there has not been any report showing where the rpoS is expressed during biofilm formation process. Since physiological state of the cells in the biofilms is considerably heterogeneous, the expression of the rpoS gene must be heterogeneous. In this study, in situ spatial expression of the rpoS gene during biofilm formation was investigated with an rpoS‐gfp transcriptional fusion mutant strain. A ribosomal binding site and a gene encoding a green fluorescent protein were introduced into the downstream of the rpoS gene, which enabled us to observe the in situ spatial expression of the rpoS gene during biofilm formation processes without any disturbance of the rpoS expression. In the early stages of the biofilm formation process, the rpoS gene was expressed in the most of the cells. On the other hand, the rpoS expression was observed only at the outside of the biofilms during the late stages of the biofilm formation process. The in situ spatial expression of the rpoS gene in the biofilm was verified by quantifying the expression levels of the rpoS at the outside and the inside of the biofilms with the real time RT‐PCR. In addition, global gene expression analysis was performed with DNA microarray to investigate physiological difference between the outside and the inside of the biofilms. This heterogeneous rpoS expression profile suggested that the cells at the outside of the biofilm need to express the rpoS to shift the physiological state to the stationary growth mode such as induction of various stress responses and suppression of the motility. Biotechnol. Bioeng. 2009;103: 975–983.

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.

Enterobactin is required for biofilm development in reduced‐genome Escherichia coli

A variety of bacterial cell surface structures and quorum signalling molecules play a role in biofilm development in Escherichia coli. However, here we show that an engineered reduced-genome E. coli mutant that lacks 17.6% of the parental E. coli genome, including the genes involved in the synthesis of various cell surface structures, such as type 1 fimbriae, curli, exopolysaccharide polymers and the autoinducer-2 signalling molecule, is able to develop mature biofilms. Using temporal gene expression profiling, we investigated phenotypic changes in reduced-genome biofilms in relation with the genes encoding the synthesis of different amino acids that were differentially expressed during biofilm formation. We identified and characterized entB, marR, dosC, mcbR and yahK genes, as involved in biofilm formation by the reduced-genome E. coli. Of these, for a first time, we demonstrated that overproduction of entB and yahK, which encode an enterobactin for iron transport and a hypothetical oxidoreductase protein, respectively, promoted biofilm development and maturation. Our results indicate that specific types of genes contribute to phenotypic changes in reduced-genome E. coli biofilms. In addition, this work demonstrates that the functions of biofilm-specific genes could be analysed through experiments using the reduced-genome E. coli.

Impacts of hydrophilic colanic acid on bacterial attachment to microfiltration membranes and subsequent membrane biofouling

In order to examine the interactions between physicochemical properties of specific extracellular polymeric substances (EPS) and membrane biofouling, we investigated the impacts of hydrophilic colanic acid, as a model extracellular polysaccharide component, on initial bacterial attachment to different microfiltration (MF) membranes and membrane biofouling by using Escherichia coli strains producing different amounts of colanic acid. In a newly designed microtiter plate assay, the bacterial attachment by an E. coli strain RcsF(+), which produces massive amounts of colanic acid, decreased only to a hydrophobic membrane because the colanic acid made cell surfaces more hydrophilic, resulting in low cell attachment to hydrophobic membranes. The bench-scale cross-flow filtration tests followed by filtration resistance measurement revealed that RcsF(+) caused severe irreversible membrane fouling (i.e., pore-clogging), whereas less extracellular polysaccharide-producing strains caused moderate but reversible fouling to all membranes used in this study. Further cross-flow filtration tests indicated that colanic acid liberated in the bulk phase could rapidly penetrate pre-accumulated biomass layers (i.e., biofilms) and then directly clogged membrane pores. These results indicate that colanic acid, a hydrophilic extracellular polysaccharide, and possible polysaccharides with similar characteristics with colanic acid are considered as a major cause of severe irreversible membrane fouling (i.e., pore-clogging) regardless of biofilm formation (dynamic membrane).