William Rhys-Williams

XF-70 and XF-73, novel antibacterial agents active against slow-growing and non-dividing cultures of Staphylococcus aureus including biofilms

OBJECTIVES 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. METHODS 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. RESULTS 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). CONCLUSIONS 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.

Investigation of the Potential for Mutational Resistance to XF-73, Retapamulin, Mupirocin, Fusidic Acid, Daptomycin, and Vancomycin in Methicillin-Resistant Staphylococcus aureus Isolates during a 55-Passage Study

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.

XF-73, a novel antistaphylococcal membrane-active agent with rapid bactericidal activity

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Calcium-binding phospholipids as a coating material for implant osteointegration

Among the many biomolecules involved in the bone mineralization processes, anionic phospholipids play an important role because of their ability to bind calcium. In particular, phosphatidylserine is a natural component of the plasmalemma and of the matrix vesicles generated from the osteoblast membrane to create nucleation centres for calcium phosphate crystal precipitation. In the present work, we demonstrate that calcium-binding phospholipids can be used as biomimetic coating materials for improving the osteointegration of metal implants. Relatively thick phosphatidylserine-based coatings were deposited on titanium coupons by dip-coating. Upon dehydration in a simulated body fluid phospholipids were quickly crosslinked by calcium and re-arranged into a three-dimensional matrix able to induce rapid formation of a calcium phosphate mineral phase. The rate of mineralization was shown to be dependent on the adopted coating formulation. In the attempt to closely mimic the cell membrane composition, heterogeneous formulations based on the mixing of anionic phospholipids (either phosphatidylserine or phosphatidylinositol) with phosphatidylcholine and cholesterol were synthesized. However, surface plasmon resonance studies as well as scanning electron microscopy and elemental analysis demonstrated that the homogeneous phosphatidylserine coating was a more effective calcification environment than the heterogeneous formulations.

In vitro activity of XF-73, a novel antibacterial agent, against antibiotic-sensitive and -resistant Gram-positive and Gram-negative bacterial species

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.

A mechanistic investigation into the microbial chiral inversion of 2-arylpropionic acids using deuterated derivatives of 2-phenylpropionic acid

Abstract Cordyceps militaris has been previously shown to invert the chirality of ( r )-2-phenylpropionic acid to its antipode in the absence of any other biotransformation. To investigate the mechanism of this unusual biotransformation. ( r , s )-[2- 2 H 1 ]-2-phenylpropionic acid, ( r , s )-[2- 2 H,3,3,3- 2 H 3 ]-2-phenylpropionic acid, and ( r , s )-[3,3,3- 2 H 3 ]-2-phenylpropionic acid were synthesized and incubated with C. militaris . NMR spectroscopy showed that deuterium exchange of the α-methine proton occurred during the inversion process but there was no exchange of the β-methyl protons. There were no significant differences in the rates of chiral inversion of the three deuterated derivatives and the undeuterated compound, and with all the compounds attaining 84% enantiomeric excess of the ( s )-enantiomer after 48 h incubation. The deuterium exchange is not therefore the rate limiting process in this biotransformation. The recovery profiles demonstrated that there was rapid metabolism of the β-methyl deuterated derivatives which did not occur for the undeuterated 2-phenylpropionic acid or the derivative which was only deuterated at the α-carbon. These studies clearly illustrate that C. militaris is a useful in vitro model of the chiral inversion of 2-arylpropionic acid derivatives in mammalian systems.

Indirect enantiomeric separation of 2-arylpropionic acids and structurally related compounds by reversed phase HPLC

A reversed-phase high-performance liquid chromatographic method, using an organic modifier-phosphate buffered mobile phase, for the determination of the enantiomeric composition of 2-arylpropionic acids and other structurally related compounds in microbial media is described. The method is based on the resolution of diastereoisomeric amides formed from the reaction of the arylpropionic acid with either (-)-(S)-alpha-methylbenzylamine or (-)-(S)-1-(naphthen-1-yl)ethylamine in the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCl and 1-hydroxybenzotriazole and incorporating an internal standard. The addition of sodium pentanesulphonate to the mobile phase as an ion-pairing agent was necessary to remove unreacted amine to avoid rapid column deterioration. The method provides and efficient, rapid and reproducible means of monitoring the microbial chiral inversion of 2-arylpropionic acids and other structurally related molecules.

A mechanistic investigation of the microbial chiral inversion of 2‐phenylpropionic acid by Verticillium lecanii

Previous investigations have described the development of nongrowing suspensions of Verticillium lecanii as a microbial model of the mammalian chiral inversion of the 2-arylpropionic acids (2-APAs). Mechanistic studies in mammals have shown that inversion involves loss of the alpha-methine proton but retention of the original atoms at the beta-methyl position, and a mechanism has been proposed involving enzymatic epimerisation of acyl-CoA thioester derivatives of the substrate. Inversion of the 2-APAs by V. lecanii exhibits extensive intersubstrate variation in the presence, rate, extent, and direction of inversion, which are different from those observed in mammalian systems, possibly indicating differences in the mechanism of inversion between mammalian and microbial cells. This study involved the investigation of proton/deuterium exchange by 1H-nuclear magnetic resonance following incubation of deuterated derivatives of 2-phenylpropionic acid (2-PPA), a model compound, in cell suspensions of V. lecanii and incubation of undeuterated 2-PPA in cell suspensions containing D2O. The results indicated that the inversion of 2-PPA by V. lecanii also involved exchange of the alpha-methine proton but complete retention of the original atoms at the beta-methyl position. No kinetic deuterium isotope effect was observed, indicating that loss of the alpha-methine proton is not the rate-limiting step of the inversion process. This suggests that the observed differences between microbial and mammalian systems probably involve the stereoselective acyl-CoA thioester formation step and not the subsequent epimerisation of the resultant diastereomers.

Extracellular and intracellular bactericidal activities of XF-70 against small-colony variant hemB mutants of meticillin-susceptible and meticillin-resistant Staphylococcus aureus

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.

Demonstration of the chiral inversion of ibuprofen by Verticillium lecanii in non‐growing cultures

Racemic ibuprofen has previously been shown to undergo metabolism by Verticillium lecanii to yield (S)‐2‐[4‐(2‐hydroxy‐2‐methylpropyl)phenyl] propionic acid which is enriched in the (S)‐enantiomer. However, it had not been possible to elucidate whether ibuprofen or the metabolite was inverted. This paper describes the incubation of ibuprofen in non‐growing cultures of V. lecanii, the results of which indicate that racemic ibuprofen is converted into a 70 : 30 (S : R) enantiomeric mixture after 6 d incubation in the absence of any metabolism. This suggests that V. lecanii could be a useful model for the investigation of the mechanism of mammalian chiral inversion of iburprofen.