Recycling antibiotics into GUMBOS: a new combination strategy to combat multi-drug-resistant bacteria.

The emergence of multi-drug-resistant bacteria, coupled with the lack of new antibiotics in development, is fast evolving into a global crisis. New strategies utilizing existing antibacterial agents are urgently needed. We propose one such strategy in which four outmoded ß-lactam antibiotics (ampicillin, carbenicillin, cephalothin and oxacillin) and a well-known antiseptic (chlorhexidine di-acetate) were fashioned into a group of uniform materials based on organic salts (GUMBOS) as an alternative to conventional combination drug dosing strategies. The antibacterial activity of precursor ions (e.g., chlorhexidine diacetate and ß-lactam antibiotics), GUMBOS and their unreacted mixtures were studied with 25 clinical isolates with varying antibiotic resistance using a micro-broth dilution method. Acute cytotoxicity and therapeutic indices were determined using fibroblasts, endothelial and cervical cell lines. Intestinal permeability was predicted using a parallel artificial membrane permeability assay. GUMBOS formed from ineffective ß-lactam antibiotics and cytotoxic chlorhexidine diacetate exhibited unique pharmacological properties and profound antibacterial activity at lower concentrations than the unreacted mixture of precursor ions at equivalent stoichiometry. Reduced cytotoxicity to invasive cell types commonly found in superficial and chronic wounds was also observed using GUMBOS. GUMBOS show promise as an alternative combination drug strategy for treating wound infections caused by drug-resistant bacteria.

Minimizing human infection from Escherichia coli O157:H7 using GUMBOS.

OBJECTIVES: Reduction in faecal shedding of Shiga toxin-producing enterohaemorrhagic Escherichia coli (EHEC) in food-producing animals is a viable strategy to minimize human disease initiated by exposure to these microorganisms. To this end, an intervention strategy involving the electrostatic hybridization of two commonly used anti-infective agents for veterinary practice (i.e. chlorhexidine and ampicillin) was evaluated to curtail EHEC-transmitted disease from ruminant sources. Chlorhexidine di-ampicillin is a novel group of uniform material based on organic salts (GUMBOS) with inherent in vitro antibacterial activity that comes from its parent antimicrobial ions, chlorhexidine and ampicillin. METHODS: Antibacterial activities for chlorhexidine diacetate, sodium ampicillin, chlorhexidine di-ampicillin and stoichiometrically equivalent 1 : 2 chlorhexidine diacetate : sodium ampicillin were assessed using the serial 2-fold dilution method and time-kill studies against seven isolates of E. coli O157:H7 and one non-pathogenic E. coli 25922. Further studies to investigate synergistic interactions of reacted and stoichiometrically equivalent unreacted antimicrobial agents at MICs and possible mechanisms were also investigated. RESULTS: Synergism and in vitro antibacterial activities against EHEC were observed in this study, which suggests chlorhexidine di-ampicillin could be a useful reagent in reducing EHEC transmission and minimizing EHEC-associated infections. Likewise, chlorhexidine di-ampicillin reduced HeLa cell toxicity as compared with chlorhexidine diacetate or the stoichiometric combination of antimicrobial agents. Further results suggest that the mechanisms of action of chlorhexidine di-ampicillin and chlorhexidine diacetate against E. coli O157:H7 are similar. CONCLUSIONS: Reacting antimicrobial GUMBOS as indicated in this study may enhance the approach to current combination drug therapeutic strategies for EHEC disease control and prevention.

In vitro synergism of trifluorothymidine and ganciclovir against HSV-1.

PURPOSE: To determine whether trifluorothymidine (TFT) and ganciclovir (GCV) are synergistic against herpes simplex virus type 1 (HSV-1). METHODS: TFT and GCV activity against 12 strains of HSV-1 (including an acyclovir-resistant strain) was measured by plaque-forming unit (PFU) inhibition. Cellular toxicity was assessed with an MTT dye reduction assay. Synergism was determined by calculating fractional inhibitory concentration (FIC indices) based on PFU reduction. RESULTS: Concentrations of TFT resulting in 50% inhibition of PFUs (IC(50)) of acyclovir-susceptible HSV-1 strains ranged from 3.07 ± 0.36 to 12.52 ± 0.61 µM. GCV IC(50) values ranged from 0.40 ± 0.02 to 1.59 ± 0.14 µM. IC(50) values of TFT and GCV against the acyclovir-resistant strain were 15.40 ± 3.17 and 93.00 ± 9.64 µM, respectively. Concentrations of TFT or GCV resulting in 50% cell cytotoxicity (CC(50)) were 0.99 ± 0.01 and 92.91 ± 8.92 µM, respectively. TFT and GCV combined (10:1) were 10 times more potent against all acyclovir-susceptible HSV-1 strains. For 8 of 12 HSV-1 strains, the IC(50) of TFT and GCV combined was lower than the CC(50) of either drug. For acyclovir-susceptible HSV-1 strains, TFT and GCV combined generated a FIC index of <0.5, suggesting strong synergism between the two drugs. The FIC value for TFT and GCV combined against the acyclovir-resistant HSV-1 strain was 0.84, indicating nonantagonism. CONCLUSIONS: TFT and GCV are synergistic against acyclovir-susceptible HSV-1 at concentrations significantly less toxic than if each antiviral were used as a sole agent.

Clinical and antiviral efficacy of an ophthalmic formulation of dexamethasone povidone-iodine in a rabbit model of adenoviral keratoconjunctivitis.

PURPOSE: To determine the efficacy of a new formulation of topical dexamethasone 0.1%/povidone-iodine 0.4% (FST-100) in reducing clinical symptoms and infectious viral titers in a rabbit model of adenoviral keratoconjunctivitis. METHODS: Rabbit corneas were inoculated bilaterally with 2×10(6) plaque-forming-units (PFU) of adenovirus type 5 (Ad5) after corneal scarification. Animals were randomized 1:1:1:1 (five rabbits per group) to FST-100, 0.5% cidofovir, tobramycin/dexamethasone (Tobradex; Alcon Laboratories, Fort Worth, TX) ophthalmic suspension, and balanced salt solution (BSS; Alcon Laboratories). Treatment began 12 hours after viral inoculation and continued for 7 consecutive days. The eyes were clinically scored daily for scleral inflammation (injection), ocular neovascularization, eyelid inflammation (redness), friability of vasculature, inflammatory discharge (pus), and epiphora (excessive tearing). Eye swabs were collected daily before treatment for the duration of the study. Virus was eluted from the swabs and PFU determined by titration on human A549 cells, according to standard procedures. RESULTS: The FST-100 treatment resulted in significantly lower clinical scores (P<0.05) than did the other treatments. The 0.5% cidofovir exhibited the most ocular toxicity compared with FST-100, tobramycin/dexamethasone, and balanced salt solution treatments. FST-100 and 0.5% cidofovir significantly (P<0.05) reduced viral titers compared with tobramycin/dexamethasone or balanced salt solution. CONCLUSIONS: FST-100 was the most efficacious in minimizing the clinical symptoms of adenovirus infection in rabbit eyes. FST-100 and 0.5% cidofovir were both equally effective in reducing viral titers and decreasing the duration of viral shedding. By providing symptomatic relief in addition to reducing infectious virus titers, FST-100 should be a valuable addition to treatment of epidemic adenoviral keratoconjunctivitis.

Nona-D-arginine amide suppresses corneal cytokines in Pseudomonas aeruginosa keratitis.

PURPOSE: Nona-D-arginine (D9R) amide suppressed interleukin 1ß production during Pseudomonas aeruginosa corneal infection. The purpose of this study was to determine the cellular disposition of D9R and its effect on other inflammatory mediators induced by infection. METHODS: Mouse eyes received 5 µL of either phosphate-buffered saline (PBS, pH 7.4) or 100-µM D9R hourly for 5 hours (total of 6 drops per eye) immediately after corneal wounding and infection with 1 × 10 colony-forming units (CFUs) of P. aeruginosa strain PAO1. At 6, 12, and 24 hours postinfection, eyes were scored on a scale of 0 (normal eye) to +4 (corneal perforation). After scoring, mice were killed and eyes enucleated. Whole eyes were used for determining viable CFUs per eye. Corneas were excised for quantitation of tumor necrosis factor α, interferon γ, interleukin 10, and granulocyte-macrophage colony-stimulating factor. The fate of D9R in cells was determined using a labeled peptide. RESULTS: Eyes treated with D9R had significantly lower disease scores (P ≤ 0.001) and fewer CFUs (P ≤ 0.01) than those in PBS-treated eyes. No corneal cytokines were detected in any D9R-treated eyes. In contrast, beginning at 12 hours postinfection, increasing amounts of tumor necrosis factor alpha, interleukin 10, and granulocyte-macrophage colony-stimulating factor were detectible in corneas of PBS-treated eyes. Within 60 minutes, D9R accumulated in the cell nucleus and nucleolus and remained for over 24 hours. CONCLUSION: D9R reduces the severity of P. aeruginosa ocular infection in part by reducing bacterial burden and in part by controlling a destructive proinflammatory response. D9R might be a useful alternative to steroids in treating other inflammation-mediated pathologies of the eye.

Nona-D-arginine amide for prophylaxis and treatment of experimental Pseudomonas aeruginosa keratitis.

PURPOSE: Nona-D-arginine amide (D9R) affected a cure of Pseudomonas aeruginosa corneal disease when combined with ciprofloxacin. In this study, we show that D9R alone and prophylactic treatment with D9R significantly reduced corneal pathology and bacterial burden associated with P. aeruginosa infection. MATERIALS AND METHODS: Right eyes of Swiss Black mice received a 5 microl drop of either phosphate buffered saline (PBS, pH 7.4) or100 microM D9R every half-hour or hourly for 5 hr (total of 6-12 drops/eye) immediately after corneal wounding and subsequent infection with 1 x 10(6) colony forming units (CFU) of a cytotoxic strain of P. aeruginosa (ATCC 19660). For prophylactic treatment, eyes were treated hourly for 5 hr (total of 6 drops/eye) before infection with an invasive strain of P. aeruginosa (PAO1). At 24 hr post infection, all eyes were evaluated for pathology and scored on a scale of 0 (normal eye) to +4 (corneal perforation). Mice were then sacrificed and eyes were harvested for CFU determination (N = 15) or histopathology (N = 5). RESULTS: P. aeruginosa-infected eyes treated with prophylactic D9R or with D9R after infection had significantly less ocular pathology (P < or = 0.001) and bacterial burden (P < or = 0.01) than eyes treated with PBS. CONCLUSIONS: D9R (100 microM) would be a beneficial addition to current antimicrobial therapy for P. aeruginosa corneal disease. Furthermore, D9R can be incorporated into ophthalmic solutions as a prophylactic agent with the potential of reducing the incidence and severity of P. aeruginosa ocular infection.

Nona-D-arginine therapy for Pseudomonas aeruginosa keratitis.

PURPOSE: Nona-D-arginine amide (D9R) was evaluated as treatment for Pseudomonas aeruginosa keratitis when administered alone and with ciprofloxacin. METHODS: Mouse corneas were infected with P. aeruginosa. Immediately after infection and hourly for 5 hours, eyes received 5 microL of either Dulbecco phosphate-buffered saline (D-PBS), 10 microM D9R, or 100 microM D9R. At 16 hours postinfection (PI) and then hourly for 5 hours, eyes treated with D9R or D-PBS then received 5 microL ciprofloxacin (0.08%) or deionized water. On days 1, 7, and 14 PI, eyes were scored on a scale of 0 (normal eye) to +4 (corneal perforation). On day 1 PI, mice were euthanatized and eyes were harvested for histopathology or colony-forming unit (CFU) determination. At 6, 12, and 24 hours PI, corneas treated with 100 microM D9R or D-PBS alone were harvested for the determination of IL-1beta cytokine concentrations. RESULTS: Eyes treated with 10 or 100 microM D9R and ciprofloxacin had significantly less disease than eyes treated with D-PBS and ciprofloxacin. Fewer than 30 CFUs were recovered from any eye treated with ciprofloxacin. Eyes treated with D9R alone had significantly less disease and lower IL-1beta cytokine concentrations than D-PBS-treated eyes; however, there were no significant differences in CFU (> or = 4 log(10)) between these groups. CONCLUSIONS: Administration of 10 or 100 microM D9R effectively reduced the abnormality associated with P. aeruginosa keratitis. Treatment with D9R and ciprofloxacin was superior to treatment with antibiotic alone by reducing ocular disease through suppression of the proinflammatory cytokine IL-1beta and eradicating viable bacteria from the eye.

Polyphosphate kinase 1 and the ocular virulence of Pseudomonas aeruginosa.

PURPOSE: To determine the role of polyphosphate kinase 1 (PPK1) in the ocular virulence of Pseudomonas aeruginosa. METHODS: Using a mouse model of infection, P. aeruginosa strains PAO1, PAOM5 (an isogenic mutant of PAO1 deficient in PPK1), and PAOM5+PPK1 (the mutant complemented with PPK1 on plasmid pHEPAK11) were compared for ocular virulence. These strains were also characterized with respect to traits associated with survival and pathogenicity in an ocular environment. RESULTS: The PPK1-deficient strain PAOM5 was significantly less virulent than either wild-type PAO1 or the complemented mutant (P <0.016). Loss of virulence was not associated with serum sensitivity or diminished adherence to the cornea. However, PAOM5 has an increased susceptibility to oxidative stress and was cleared from corneal tissue significantly better (P <0.006) than either the wild-type or restored strain. Furthermore, the PPK1-deficient mutant produced significantly less (P <0.022) pyocyanin. CONCLUSIONS: PPK1 is essential for a successful ocular infection by P. aeruginosa. The loss of ocular virulence is probably due to the dysregulation of multiple genes, including those responsible for stress response.

Caspase-1 inhibitor reduces severity of pseudomonas aeruginosa keratitis in mice.

PURPOSE: To test an inhibitor of IL-1beta converting enzyme (ICE), with or without ciprofloxacin, in a C57BL/6 mouse model of keratitis induced by Pseudomonas aeruginosa in which corneal perforation is expected. METHODS: Clinical score, histopathology, myeloperoxidase (MPO) activity, bacterial counts, and ELISA analysis were used to assess the efficacy of treatment initiated at 18 hours postinfection (p.i.) with ICE inhibitor versus placebo; and with ICE inhibitor plus ciprofloxacin versus placebo plus ciprofloxacin. Efficacy of the ICE inhibitor was also tested and evaluated for clinical score in experimental corneal infection induced by a clinical isolate and a ciprofloxacin-resistant bacterial strain. RESULTS: Clinical scores were reduced at 3, 5, and 7 days p.i. in ICE inhibitor versus placebo-treated mice; reduced scores also were observed with a combined treatment (ICE inhibitor and ciprofloxacin). Further testing (MPO assay) revealed reduced PMN number, particularly striking in ICE inhibitor and ciprofloxacin versus placebo and ciprofloxacin-treated mice. Corneal protein levels for IL-1beta and MIP-2 also were reduced in mice treated with the ICE inhibitor versus placebo and in ICE inhibitor and ciprofloxacin versus ciprofloxacin and placebo-treated mice. Treatment with ICE inhibitor also reduced clinical scores after corneal infection with a clinical isolate, KEI-1025, and with a ciprofloxacin-resistant P. aeruginosa strain. CONCLUSIONS: Downregulation of IL-1beta by ICE together with ciprofloxacin to kill bacteria may provide alternate therapy to current treatment.

Pseudomonas aeruginosa proteases and corneal virulence.

Pseudomonas aeruginosa is a common cause of corneal infections, particularly among users of soft contact lenses. Previous studies with chemically induced mutants deficient in alkaline protease (AP) or elastase (LasB) suggested that these proteases contributed to the rapid liquifactive stromal necrosis characteristic of P. aeruginosa corneal infections. Because these mutants might harbor other chromosomal changes that could affect virulence, the role of these proteases in the pathogenesis of corneal disease (as well as a second elastase, LasA protease) was reexamined by constructing isogenic mutants deficient only in these enzymes. Allelic exchange was used to construct mutants of P. aeruginosa PAO1-V deficient in AP (PAO1-V AP[ - ]), LasB and LasA protease (PDO801 LasB[ - ]), or all three proteases (PDO801 TM). These mutants were then evaluated for virulence using mouse scratch and rabbit intrastromal injection models of corneal disease. Loss of AP significantly increased disease scores in the rabbit (P < 0.030) but not the mouse (P > 0.060) model of infection. Loss of both elastases had no effect on ocular virulence in either animal model of corneal disease (P > 0.100). The loss of all three proteases significantly decreased disease scores in the rabbit (P < 0.035), but not in the mouse (P > 0.110). Taken together, these data suggest that AP, LasB, and LasA protease are not essential for initiating or maintaining a corneal infection. Furthermore, AP appears to be an important mediator of pathology depending on the location of the organism within the cornea and whether or not concomitant elastolytic activity is present.

Pseudomonas aeruginosa exotoxin A and keratitis in mice.

PURPOSE: To determine the importance of Pseudomonas aeruginosa exotoxin A (ETA) as a virulence factor in corneal disease. METHODS: Isogenic mutants deficient in ETA were constructed in P. aeruginosa strains PAO1 and ATCC 19660 by allelic exchange and then evaluated for virulence in a mouse model of bacterial keratitis. The effect of ETA on adherence to scarified corneal epithelium was assessed in an in vitro organ culture model. RESULTS: Mutants of either P. aeruginosa PAO1 or 19660, deficient in ETA, adhered to wounded corneal tissue and initiated ocular disease similar to that in wild-type strains. However, in contrast to wild-type strains, ETA mutants were quickly cleared from the eye, inflammation diminished, and the cornea healed. CONCLUSIONS: Although ETA has no effect on the ability of P. aeruginosa to adhere to corneal wounds or to initiate Pseudomonas keratitis, it is crucial for the organism to persist in the eye and ultimately cause disease.