[Show abstract][Hide abstract]ABSTRACT: The human cornea is a tri-laminar structure composed of several cell types with substantial mitotic potential. Age-related changes in the cornea are associated with declining visual acuity and the onset of overt age-related corneal diseases. Corneal transplantation is commonly used to restore vision in patients with damaged or diseased corneas. However, the supply of donor tissue is limited, and thus there is considerable interest in the development of tissue-engineered alternatives. A major obstacle to these approaches is the short replicative lifespan of primary human corneal endothelial cells (HCEC). Accordingly, a comprehensive investigation of the signalling pathways and mechanisms underpinning proliferative lifespan and senescence in HCEC was undertaken. The effects of exogenous human telomerase reverse transcriptase expression, p53 knockdown, disruption of the pRb pathway by over-expression of CDK4 and reduced oxygen concentration on the lifespan of primary HCEC were evaluated. We provide proof-of-principle that forced expression of telomerase, when combined with either p53 knockdown or CDK4 over-expression, is sufficient to produce immortalized HCEC lines. The resultant cell lines express an HCEC-specific transcriptional fingerprint, and retain expression of the corneal endothelial temperature-sensitive potassium channel, suggesting that significant dedifferentiation does not occur as a result of these modes of immortalization. Exploiting these insights into proliferative lifespan barriers in HCEC will underpin the development of novel strategies for cell-based therapies in the human cornea.
[Show abstract][Hide abstract]ABSTRACT: Small-colony variants (SCVs) of Staphylococcus aureus are phenotypic variants characterised by their small colony size and improved intracellular survival and are associated with persistent and relapsing infections. XF drugs are membrane-active, porphyrin-based antibacterial agents for topical administration, exerting rapid bactericidal activity against actively growing or resting, antibiotic-susceptible and multidrug-resistant strains of S. aureus. In this study, minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of XF-70 against isogenic, electron-transport deficient, SCV hemB mutants of one meticillin-susceptible S. aureus (MSSA) strain and one meticillin-resistant S. aureus (MRSA) strain were evaluated. Macrodilution MICs of XF-70 for MSSA strain 8325-4 and its hemB(+)-complemented derivative (0.5-1mg/L) were reproducible and were slightly higher than that for the SCV hemB mutant (0.25-0.5mg/L) and were not influenced by increasing inoculum size from 10(6) to 10(8) colony-forming units (CFU)/mL. MICs for MRSA strain COL, its SCV hemB mutant and hemB(+)-complemented derivative were equivalent (0.25-1mg/L). MBCs of XF-70 were ≤ 2-fold higher than MICs for all isolates. Extensive killing (≥ 4 log reduction in CFU/mL) was produced by 2mg/L XF-70 within 30 min against SCV hemB mutants both of 8325-4 and COL as well as their respective parent or hemB(+)-complemented derivatives. Pre-incubation of 10(7)CFU/mL of 8325-4 and its SCV hemB mutant with 5 × 10(6) polymorphonuclear neutrophils for 30 min markedly protected phagocytised organisms from rapid extensive killing by bactericidal levels (2mg/L) of subsequently added XF-70. The rapid bactericidal activity of XF-70 at low concentrations both against SCV and normally growing S. aureus is remarkable and represents an attractive potential for the treatment of persistent localised infections.
No preview · Article · Mar 2011 · International journal of antimicrobial agents
[Show abstract][Hide abstract]ABSTRACT: XF-73 is a dicationic porphyrin drug with rapid Gram-positive antibacterial activity currently undergoing clinical trials for the nasal decolonization of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA). In multistep (55-passage) resistance selection studies in the presence of subinhibitory concentrations of XF-73, retapamulin, mupirocin, fusidic acid, and vancomycin against four Network on Antimicrobial Resistance in Staphylococcus aureus MRSA strains, there was no >4-fold increase in the MIC for XF-73 after 55 passages. In contrast, there was an increase in the MICs for retapamulin (from 0.25 μg/ml to 4 to 8 μg/ml), for mupirocin (from 0.12 μg/ml to 16 to 512 μg/ml), for fusidic acid (from 0.12 μg/ml to 256 μg/ml), and for vancomycin (from 1 μg/ml to 8 μg/ml in two of the four strains tested). Further investigations using S. aureus NRS384 (USA300) and daptomycin demonstrated a 64-fold increase in the MIC after 55 passages (from 0.5 μg/ml to 32 μg/ml) with a >4-fold increase in the MIC obtained after only five passages. Sequencing analysis of selected isolates confirmed previously reported point mutations associated with daptomycin resistance. No cross-resistance to XF-73 was observed with the daptomycin-resistant strains, suggesting that whereas the two drugs act on the bacterial cell membrane, their specific site of action differs. XF-73 thus represents the first in a new class of antibacterial drugs, which (unlike the comparator antibiotics) after 55 passages exhibited a ≤4-fold increase in MIC against the strains tested. Antibacterial drugs with a low propensity for inducing bacterial resistance are much needed for the prevention and treatment of multidrug-resistant bacteria both within and outside the hospital setting.
Full-text · Article · Dec 2010 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract]ABSTRACT: The authors report the findings of in vivo studies of XF-70 (a novel, dicationic porphyrin) against Staphylococcus aureus in a murine model of a burn wound infection. Mice received a 15% total body scald burn wound, which were inoculated with S. aureus (1.8 x 10 CFU). After 24 hours, escharectomies were performed and groups (n = 8) received single or two doses (6 hours apart) of XF-70* (100 microg/wound) or silver sulfadiazine, Acticoat, or saline applied topically. Viable bacteria were quantified from homogenized burn tissue biopsies and the spleen by plating dilutions onto agar plates and CFU determination. A single dose of XF-70 reduced bacterial burden by 98.77% (untreated: 2.78 +/- 2.96 x 10 CFU/g vs XF-70 treated: 3.4 +/- 0.19 x 10 CFU/g, P < .01). Two XF-70 doses reduced the growth of S. aureus by 99.96% (1.2 +/- 0.6 x 10 CFU/g, P < .01). These results were similar to the results obtained from commonly used topical antibacterials silver sulfadiazine and Acticoat. The spleens of mice treated with saline had a robust growth of S. aureus (7.0 +/- 1.97 x 10 CFU/g) whereas those treated with one or two XF-70 doses grew only 3.5 +/- 0.002 x 10 CFU/g and 5.7 +/- 0.002 x 10 CFU/g, respectively, a significant (P < .001) reduction in S. aureus dissemination. Single and multiple doses of XF-70 were effective in controlling S. aureus growth in burn wounds and inhibited systemic dissemination of S. aureus. Early treatment of burn wounds with XF-70 may be effective in slowing bacterial dissemination to other tissues.
No preview · Article · May 2010 · Journal of burn care & research: official publication of the American Burn Association
[Show abstract][Hide abstract]ABSTRACT: The antibacterial activity of XF-73, a dicationic porphyrin drug, was investigated against a range of Gram-positive and Gram-negative bacteria with known antibiotic resistance profiles, including resistance to cell wall synthesis, protein synthesis, and DNA and RNA synthesis inhibitors as well as cell membrane-active antibiotics. Antibiotic-sensitive strains for each of the bacterial species tested were also included for comparison purposes. XF-73 was active [minimum inhibitory concentration (MIC) 0.25-4 mg/L] against all of the Gram-positive bacteria tested, irrespective of the antibiotic resistance profile of the isolates, suggesting that the mechanism of action of XF-73 is unique compared with the major antibiotic classes. Gram-negative activity was lower (MIC 1 mg/L to > 64 mg/L). Minimum bactericidal concentration data confirmed that the activity of XF-73 was bactericidal. Time-kill kinetics against healthcare-associated and community-associated meticillin-resistant Staphylococcus aureus isolates demonstrated that XF-73 was rapidly bactericidal, with > 5 log(10) kill obtained after 15 min at 2 x MIC, the earliest time point sampled. The post-antibiotic effect (PAE) for XF-73 under conditions where the PAE for vancomycin was < 0.4h was found to be > 5.4 h. XF-73 represents a novel broad-spectrum Gram-positive antibacterial drug with potentially beneficial characteristics for the treatment and prevention of Gram-positive bacterial infections.
Full-text · Article · Mar 2010 · International journal of antimicrobial agents
[Show abstract][Hide abstract]ABSTRACT: Slow-growing and non-dividing bacteria exhibit tolerance to many antibiotics. However, membrane-active agents may act against bacteria in all growth phases. We sought to examine whether the novel porphyrin antibacterial agents XF-70 and XF-73, which have rapid membrane-perturbing activity against Staphylococcus aureus, retained antistaphylococcal activity against growth-attenuated cells.
The killing kinetics of XF-70, XF-73 and various comparator agents against exponential phase cultures of S. aureus SH1000 were compared with effects on cells held at 4 degrees C, non-growing cultures expressing the stringent response induced by mupirocin and bacteria in the stationary phase. Biofilms of S. aureus SH1000 were generated with the Calgary device to examine the activities of XF-70 and XF-73 under a further system exhibiting diminished bacterial growth.
Cold culture, stringent response and stationary phase cultures remained susceptible to XF-70 and XF-73, which caused > or =5 log reductions in viability over 2 h. During this period the most active comparator agents (chlorhexidine and cetyltrimethylammonium bromide) only promoted a 3 log drop in viability. XF-70 and XF-73 were also highly active against biofilms, with both agents exhibiting low biofilm MICs (1 mg/L) and minimum biofilm eradication concentrations (2 mg/L).
XF-70 and XF-73 remained highly active against various forms of slow-growing or non-dividing S. aureus. The results support the hypothesis that membrane-active agents may be particularly effective in eradicating slow- or non-growing bacteria and suggest that XF-70 and XF-73 could be utilized to treat staphylococcal infections where the organisms are only dividing slowly, such as biofilm-associated infections of prosthetic devices.
Full-text · Article · Nov 2009 · Journal of Antimicrobial Chemotherapy
[Show abstract][Hide abstract]ABSTRACT: XF-73 is a novel porphyrin antibacterial agent previously reported to inhibit a range of gram-positive bacterial species, including Staphylococcus aureus. Its mode of action is unknown. Using S. aureus as a model organism we sought to examine the basis of its antibacterial activity.
The effects of XF-73 on the growth and survival of S. aureus SH1000 were investigated by viable count and culture absorbance techniques. Inhibition of macromolecular synthesis and disruption of membrane integrity after exposure to XF-73 were examined by radiolabelling experiments, the BacLight fluorescent dye assay and measurement of K(+) and ATP leakage from the cell. The effect of XF-73 on a staphylococcal coupled transcription-translation system was also investigated.
XF-73 was rapidly bactericidal against S. aureus SH1000 and demonstrated more rapid killing kinetics than all other comparator agents when tested at an equivalent multiple (4x) of the MIC. Exposure of S. aureus to XF-73 for 10 min completely inhibited DNA, RNA and protein synthesis. XF-73 had no effect on transcription and translation in vitro. Cells exposed to XF-73 gave a positive response in the BacLight assay, which detects membrane damage. The drug also caused substantial loss of K(+) and ATP from the cell, but did not promote bacterial lysis.
XF-73 exhibited rapid membrane-perturbing activity, which is likely to be responsible for inhibition of macromolecular synthesis and the death of staphylococci exposed to the drug.
No preview · Article · Sep 2009 · Journal of Antimicrobial Chemotherapy
[Show abstract][Hide abstract]ABSTRACT: Little is known about the senescent phenotype of human vascular smooth muscle cells (VSMCs) and the potential involvement of senescent VSMCs in age-related vascular disease, such as atherosclerosis. As such, VSMCs were grown and characterised in vitro to generate senescent VSMCs needed for microarray analysis (Affymetrix). Comparative analysis of the transcriptome profiles of early (14 CPD) and late (39-42 CPD) passage VSMCs found a total of 327 probesets called as differentially expressed: 149 are up-regulated in senescence and 178 repressed (p-value<0.5%, minimum effect size of at least 2-fold differential regulation, explore data at http://www.madras.cf.ac.uk/vsmc). Data mining shows a differential regulation of genes at senescence associated with the development of atherosclerosis and vascular calcification. These included genes with roles in inflammation (IL1beta, IL8, ICAM1, TNFAP3, ESM1 and CCL2), tissue remodelling (VEGF, VEGFbeta, ADM and MMP14) and vascular calcification (MGP, BMP2, SPP1, OPG and DCN). The microarray data for IL1beta, IL8 and MGP were validated by either, ELISA, Western blot analysis or RT-PCR. These data thus provide the first evidence for a role of VSMC senescence in the development of vascular calcification and provides further support for the involvement of senescent VSMCs in the progression of atherosclerosis.
[Show abstract][Hide abstract]ABSTRACT: Background: A key feature of biofilm formation is the bacteria’s ability to resist antibiotic activity. The slower growth rate of bacteria in biofilms may be an important factor in increased resistance. We investigated the activity of XF-73, the lead compound in an entirely new class of antimicrobial agents, against biofilm and slow growing cultures of Staphylococcus aureus. Methods: Minimum inhibitory concentrations (MICs) were determined for planktonic cultures by broth microdilution according to BSAC guidelines. Biofilm MICs (bMIC) and minimum biofilm eradication concentrations (MBECs) were determined using the Calgary biofilm device. The effect of XF-73 on the viability of cold culture cells was determined by growing S. aureus SH1000 to early exponential phase at 37ºC and resuspending the cells in pre-chilled media where they were maintained in the presence and absence of XF-73 and controls. The effect of XF-73 on slow growing cells expressing the stringent response was determined by growing cultures to early log phase and the stringent response induced by the addition of the IRS inhibitor mupirocin. XF-73 and controls were then added and samples recovered for viable cell determinations. Results: XF-73 had excellent antibiofilm activity with a bMIC of 1 μg/ml and a MBEC of 2 μg/ml against S. aureus SH1000 compared with bMICs of 4, 0.5 and 0.03 μg/ml and MBECs of >256, >256 and 128 μg/ml for ciprofloxacin, fusidic acid and rifampin respectively. Cold culture and stringent response cultures remained susceptible to XF-73 with a 5 log drop in viability observed within 1 hour compared with no loss of viability for cultures treated with fosfomycin. Conclusions: The excellent activity of XF-73 against biofilms and slow growing S. aureus cultures demonstrates that its antibacterial activity is independent of the growth state of the bacteria and suggests additional utility for XF-73 in the treatment of biofilm associated infections.
[Show abstract][Hide abstract]ABSTRACT: Background: As a novel class of antibacterials, XF drugs possess a unique mechanism of action, and the likelihood for the development of resistance to them is remote. The lead compound of the series has entered into clinical development as a topical anti-staphylococcal agent. We herein report the findings of in vivo studies assessing the systemic administration of DPD-207, a new member of the XF drug family. Methods: CD-1 mice were immunocompromised and infected with 107 cfu/ml Staphylococcus aureus (SA) per thigh. DPD-207 was administered SC, IV or PO in dosages ranging from 0.25 to 40 mg/kg/day. Bacterial density in thigh tissue was assessed at 2-hour intervals from 0 to 8 hours and again at 24 hours in each treatment and control group. A one-way ANOVA was performed between groups at each time of sample collection with significance set at P <0.05. Results: Statistical evaluations demonstrated significant antibacterial effects over time for both IV and PO doses versus the control groups. IV administration of DPD-207 led to a significant (P<0.05) anti-SA effect, although mortality was observed at doses >10mg/kg. DPD-207 also demonstrated significant (P<0.05) anti-SA activity when given via the oral route, with no apparent acute toxicity at doses up to 40 mg/kg. No significant anti-staphylococcal activity or any apparent acute toxicity was observed with the subcutaneous administration of DPD-207. Conclusions: These results are the first in vivo studies of DPD-207 and show anti-SA activity of this XF drug after either IV or oral administration. While mortality was seen with the higher IV dosing regimens, none was observed after high oral doses. Further studies with DPD-207 are warranted, using formulations designed to enhance its activity profile. As the likelihood of resistance developing is remote, the XF compounds have the potential to be developed into anti-bacterial agents with a long lifetime of clinical utility.
[Show abstract][Hide abstract]ABSTRACT: Background: XF-73, the lead compound in a new class of bactericidal antimicrobial agents, has previously been shown to be potent (MIC50 and MBC50 1 mg/L) against a range of Staphylococcus aureus strains including healthcare-associated methicillin-resistant S. aureus and community-associated methicillin-resistant S. aureus (CA-MRSA). The aim of this study was to compare the propensity of XF-73 and daptomycin to cause mutational resistance in USA300, the predominant CA-MRSA strain in the USA. Methods: MICs were determined for XF-73 and daptomycin using a macrodilution broth method. USA300-0114 was passaged 55X at 0.5X MIC (determined from the previous passage) to investigate if there was an increase in the MIC, suggestive of the development of mutational resistance. Daptomycin MICs were determined in broth supplemented with calcium to a final concentration of 50mg/L. Results: The initial MICs for XF-73 and daptomycin were 0.25 and 0.5 mg/L, respectively. A stepwise doubling in the MIC of daptomycin was observed during the course of the study, the MIC doubling being followed by a period of constant MIC before the next increase, with the MIC reaching 32 mg/L after 55 passages. A significant (≥8-fold) increase in the MIC was observed after only 5 passages. In comparison, the MIC of XF-73 did not increase significantly (<8-fold increase), even after 55 passages (final MIC 0.5mg/L). Conclusions: The emergence of stable resistance to daptomycin during passage studies has been reported previously. In this study, daptomycin resistance was found to emerge rapidly in USA300 while in contrast, no mutational resistance emerged for XF-73. Thus, XF-73 may have a significant advantage over daptomycin for clinical application.
[Show abstract][Hide abstract]ABSTRACT: Background: XF-73, the lead compound in a new class of bactericidal antimicrobial agents, has been shown to be potent (MIC50 1 mg/L) against a range of Staphylococcus aureus (SA) strains. XF-73 is in clinical development for nasal SA decolonization. The aim of this study was to investigate its propensity to cause mutational resistance in common methicillin-resistant SA clones compared with mupirocin, which is widely used for nasal SA decolonization. Methods: MICs were determined for XF-73 and mupirocin using a macrodilution broth method. Five “Network on Antimicrobial Resistance in Staphylococcus aureus” strains were tested. All strains were passaged 55X at 0.5X MIC (determined from the previous passage) to investigate if there was an increase in the MIC, suggestive of the development of mutational resistance. Results: For all 5 strains, a rapid increase in the MIC of mupirocin was observed after only 4-8 repeat passages, with the MIC reaching 8-512 mg/L after 55 passages. In comparison, the MIC of XF-73 did not increase significantly (<8-fold increase), even after 55 passages.
MIC Passage 0 (mg/L)
MIC Passage 55 (mg/L)
Conclusions: The use of mupirocin for nasal decolonization is limited by the rapid emergence of resistant strains. In this study, mupirocin resistance emerged rapidly while no mutational resistance emerged to XF-73. These data suggest the likelihood of mutational resistance development against XF-73 is remote and XF-73 may therefore have a significant advantage over mupirocin for intra-nasal decolonization. The widespread use of XF-73 for MRSA decolonization in hospitals as part of effective infection control can thus be contemplated.