The Evolution of Heteroresistance via Small Colony Variants in Escherichia coli Following Long Term Exposure to Bacteriostatic Antibiotics.

Traditionally, bacteriostatic antibiotics are agents able to arrest bacterial growth. Despite being unable to kill bacterial cells, when they are used clinically the outcome of these drugs is frequently as effective as when a bactericidal drug is used. We explore the dynamics of Escherichia coli after exposure to two ribosome-targeting bacteriostatic antibiotics, chloramphenicol and azithromycin, for thirty days. The results of our experiments provide evidence that bacteria exposed to these drugs replicate, evolve, and generate a sub-population of small colony variants (SCVs) which are resistant to multiple drugs. These SCVs contribute to the evolution of heteroresistance and rapidly revert to a susceptible state once the antibiotic is removed. Stated another way, exposure to bacteriostatic drugs selects for the evolution of heteroresistance in populations previously lacking this trait. More generally, our results question the definition of bacteriostasis as populations exposed to bacteriostatic drugs are replicating despite the lack of net growth.

The book of Lambda does not tell us that naturally occurring lysogens of Escherichia coli are likely to be resistant as well as immune.

The most significant difference between bacteriophages functionally and ecologically is whether they are purely lytic (virulent) or temperate. Virulent phages can only be transmitted horizontally by infection, most commonly with the death of their hosts. Temperate phages can also be transmitted horizontally, but upon infection of susceptible bacteria, their genomes can be incorporated into that of their host's as a prophage and be transmitted vertically in the course of cell division by their lysogenic hosts. From what we know from studies with the temperate phage Lambda and other temperate phages, in laboratory culture, lysogenic bacteria are protected from killing by the phage coded for by their prophage by immunity; where upon infecting lysogens, the free temperate phage coded by their prophage is lost. Why are lysogens not only resistant but also immune to the phage coded by their prophage since immunity does not confer protection against virulent phages? To address this question, we used a mathematical model and performed experiments with temperate and virulent mutants of the phage Lambda in laboratory culture. Our models predict and experiments confirm that selection would favor the evolution of resistant and immune lysogens, particularly if the environment includes virulent phage that shares the same receptors as the temperate. To explore the validity and generality of this prediction, we examined 10 lysogenic Escherichia coli from natural populations. All 10 were capable of forming immune lysogens, but their original hosts were resistant to the phage coded by their prophage.

Evaluating the potential efficacy and limitations of a phage for joint antibiotic and phage therapy of Staphylococcus aureus infections.

In response to increasing frequencies of antibiotic-resistant pathogens, there has been a resurrection of interest in the use of bacteriophage to treat bacterial infections: phage therapy. Here we explore the potential of a seemingly ideal phage, PYOSa, for combination phage and antibiotic treatment of Staphylococcus aureus infections. This K-like phage has a broad host range; all 83 tested clinical isolates of S.aureus tested were susceptible to PYOSa Because of the mode of action of PYOSa, S. aureus is unlikely to generate classical receptor-site mutants resistant to PYOSa; none were observed in the 13 clinical isolates tested. PYOSa kills S. aureus at high rates. On the downside, the results of our experiments and tests of the joint action of PYOSa and antibiotics raise issues that must be addressed before PYOSa is employed clinically. Despite the maintenance of the phage, PYOSa does not clear populations of S. aureus Due to the ascent of a phenotyically diverse array of small-colony variants following an initial demise, the bacterial populations return to densities similar to that of phage-free controls. Using a combination of mathematical modeling and in vitro experiments, we postulate and present evidence for a mechanism to account for the demise-resurrection dynamics of PYOSa and S. aureus Critically for phage therapy, our experimental results suggest that treatment with PYOSa followed by bactericidal antibiotics can clear populations of S. aureus more effectively than the antibiotics alone.

Antibiotic Killing of Diversely Generated Populations of Nonreplicating Bacteria.

Nonreplicating bacteria are known to be (or at least commonly thought to be) refractory to antibiotics to which they are genetically susceptible. Here, we explore the sensitivity to killing by bactericidal antibiotics of three classes of nonreplicating populations of planktonic bacteria: (i) stationary phase, when the concentration of resources and/or nutrients are too low to allow for population growth; (ii) persisters, minority subpopulations of susceptible bacteria surviving exposure to bactericidal antibiotics; and (iii) antibiotic-static cells, bacteria exposed to antibiotics that prevent their replication but kill them slowly if at all, the so-called bacteriostatic drugs. Using experimental populations of Staphylococcus aureus Newman and Escherichia coli K-12 (MG1655) and, respectively, nine and seven different bactericidal antibiotics, we estimated the rates at which these drugs kill these different types of nonreplicating bacteria. In contrast to the common belief that bacteria that are nonreplicating are refractory to antibiotic-mediated killing, all three types of nonreplicating populations of these Gram-positive and Gram-negative bacteria are consistently killed by aminoglycosides and the peptide antibiotics daptomycin and colistin, respectively. This result indicates that nonreplicating cells, irrespectively of why they do not replicate, have an almost identical response to bactericidal antibiotics. We discuss the implications of these results to our understanding of the mechanisms of action of antibiotics and the possibility of adding a short-course of aminoglycosides or peptide antibiotics to conventional therapy of bacterial infections.

Leaky resistance and the conditions for the existence of lytic bacteriophage.

In experimental cultures, when bacteria are mixed with lytic (virulent) bacteriophage, bacterial cells resistant to the phage commonly emerge and become the dominant population of bacteria. Following the ascent of resistant mutants, the densities of bacteria in these simple communities become limited by resources rather than the phage. Despite the evolution of resistant hosts, upon which the phage cannot replicate, the lytic phage population is most commonly maintained in an apparently stable state with the resistant bacteria. Several mechanisms have been put forward to account for this result. Here we report the results of population dynamic/evolution experiments with a virulent mutant of phage Lambda, λVIR, and Escherichia coli in serial transfer cultures. We show that, following the ascent of λVIR-resistant bacteria, λVIR is maintained in the majority of cases in maltose-limited minimal media and in all cases in nutrient-rich broth. Using mathematical models and experiments, we show that the dominant mechanism responsible for maintenance of λVIR in these resource-limited populations dominated by resistant E. coli is a high rate of either phenotypic or genetic transition from resistance to susceptibility-a hitherto undemonstrated mechanism we term "leaky resistance." We discuss the implications of leaky resistance to our understanding of the conditions for the maintenance of phage in populations of bacteria-their "existence conditions."

Phagocytes, Antibiotics, and Self-Limiting Bacterial Infections.

Most antibiotic use in humans is to reduce the magnitude and term of morbidity of acute, community-acquired infections in immune competent patients, rather than to save lives. Thanks to phagocytic leucocytes and other host defenses, the vast majority of these infections are self-limiting. Nevertheless, there has been a negligible amount of consideration of the contribution of phagocytosis and other host defenses in the research for, and the design of, antibiotic treatment regimens, which hyper-emphasizes antibiotics as if they were the sole mechanism responsible for the clearance of infections. Here, we critically review this approach and its limitations. With the aid of a heuristic mathematical model, we postulate that if the rate of phagocytosis is great enough, for acute, normally self-limiting infections, then (i) antibiotics with different pharmacodynamic properties would be similarly effective, (ii) low doses of antibiotics can be as effective as high doses, and (iii) neither phenotypic nor inherited antibiotic resistance generated during therapy are likely to lead to treatment failure.

A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death.

We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding "bacteriostatic" antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures. IMPORTANCE: Chemotherapeutic agents, including antibiotics, have been used for more than a century; nevertheless, there are still major gaps in our understanding of how these drugs operate which limit future advances in antibacterial chemotherapy. Although the molecular mechanisms by which antibiotics bind to their target structures are largely known, fundamental questions about how these drugs actually kill and/or inhibit the replication of bacteria remain unanswered and subjects of controversy. We postulate that for the broad class of ribosome-binding bacteriostatic antibiotics, their reducing the number of active (functional) ribosomes per cell provides a sufficient explanation for the abatement of replication and the low rate of decline in densities of viable cells of bacteria exposed to these drugs. Using E. coli K-12 constructs with deletions of from one to six of the seven ribosome-RNA operons and the ribosome-binding bacteriostatic antibiotics tetracycline, chloramphenicol, and azithromycin, we tested this hypothesis. The results of our experiments are consistent with this "numbers game" hypothesis.

Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

Microtubules regulate disassembly of epithelial apical junctions.

BACKGROUND: Epithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion. RESULTS: Calcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex. CONCLUSION: Our data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins.

Neutrophil migration across tight junctions is mediated by adhesive interactions between epithelial coxsackie and adenovirus receptor and a junctional adhesion molecule-like protein on neutrophils.

Neutrophil (polymorphonuclear leukocytes [PMN]) transepithelial migration during inflammatory episodes involves a complex series of adhesive interactions and signaling events. Previous studies have shown that key adhesive interactions between leukocyte CD11b/CD18 and basally expressed fucosylated glycoproteins followed by binding to desmosomal-associated JAM-C are key elements of the transmigration response. Here we provide the first evidence that PMN-expressed junctional adhesion molecule-like protein (JAML) regulates transmigration via binding interactions with epithelial coxsackie and adenovirus receptor (CAR). Experiments with a JAML fusion protein revealed specific binding of JAML to epithelial CAR expressed at tight junctions in T84 cell monolayers and normal human colonic mucosa. Furthermore, JAML-CAR binding is mediated via the membrane distal immunoglobulin (Ig) loop of CAR and the membrane proximal Ig loop of JAML. PMN bound to immobilized CAR but not JAML in a divalent cation-independent manner. Lastly, in assays of PMN transepithelial migration, JAML/CAR fusion proteins and their antibodies significantly inhibited transmigration in a specific manner. Taken together, these results indicate that JAML and CAR are a novel pair of adhesion molecules that play an important role in modulating PMN migration cross epithelial tight junctions. These findings add a new element to a multistep model of PMN transepithelial migration and may provide new targets for anti-inflammatory therapies.

Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex.

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.

Involvement of the junctional adhesion molecule-1 (JAM1) homodimer interface in regulation of epithelial barrier function.

Junctional adhesion molecule-1 (JAM1) is a tight junction-associated immunoglobulin superfamily protein implicated in the regulation of tight junctions and leukocyte transmigration. The structural basis for the function of JAM1 has yet to be determined. Here we provide evidence that JAM1 homodimer formation is important for its function in epithelial cells. Experiments were conducted to determine the effects of a panel of JAM1 monoclonal antibodies on epithelial barrier recovery after transient disruption by calcium switch. Two monoclonal antibodies were observed to inhibit barrier recovery in contrast to another monoclonal antibody that had no effect. Epitope mapping by phage display revealed that both inhibitory antibodies bind to a region of JAM1 located within the N-terminal Ig-like loop (residues 111-123). Competition experiments with synthetic peptides and site-directed mutagenesis confirmed the location of this epitope. Analysis of the crystal structure of JAM1 revealed that this epitope includes residues within the putative homodimer interface, and one of the two inhibitory antibodies was then shown to block JAM1 homodimer formation in vitro. Finally, mutations within the homodimer interface were shown to prevent enrichment of JAM1 at points of cell contact, presumably by interference with homophilic interactions. These findings suggest that homodimer formation may be important for localization of JAM1 at tight junctions and for regulation of epithelial barrier function.