The silver cation (Ag+): antistaphylococcal activity, mode of action and resistance studies.

OBJECTIVES: To examine several poorly understood or contentious aspects of the antibacterial activity of silver (Ag(+)), including its cidality, mode of action, the prevalence of resistance amongst clinical staphylococcal isolates and the propensity for Staphylococcus aureus to develop Ag(+) resistance. METHODS: The effects of Ag(+) on the viability, macromolecular synthesis and membrane integrity of S. aureus SH1000 were assessed using established methodology. Silver nitrate MICs were determined for a collection of staphylococcal isolates (n = 1006) collected from hospitals across Europe and Canada between 1997 and 2010. S. aureus biofilms were grown using the Calgary Biofilm Device. To examine the in vitro development of staphylococcal resistance to Ag(+), bacteria were subjected to continuous subculture in the presence of sub-MIC concentrations of Ag(+). RESULTS: Silver was bactericidal against S. aureus in buffered solution, but bacteriostatic in growth medium, and was unable to eradicate staphylococcal biofilms in vitro. Challenge of S. aureus with Ag(+) caused rapid loss of membrane integrity and inhibition of the major macromolecular synthetic pathways. All clinical staphylococcal isolates were susceptible to ≤ 16 mg/L silver nitrate and prolonged exposure (42 days) to Ag(+) in vitro failed to select resistant mutants. CONCLUSIONS: The rapid and extensive loss of membrane integrity observed upon challenge with Ag(+) suggests that the antibacterial activity results directly from damage to the bacterial membrane. The universal susceptibility of staphylococci to Ag(+), and failure to select for resistance to Ag(+), suggest that silver compounds remain a viable option for the prevention and treatment of topical staphylococcal infections.

The nature of Staphylococcus aureus MurA and MurZ and approaches for detection of peptidoglycan biosynthesis inhibitors.

Staphylococcus aureus and a number of other Gram-positive organisms harbour two genes (murA and murZ) encoding UDP-N-acetylglucosamine enolpyruvyl transferase activity for catalysing the first committed step of peptidoglycan biosynthesis. We independently inactivated murA and murZ in S. aureus and established that either can sustain viability. Purification and characterization of the MurA and MurZ enzymes indicated that they are biochemically similar in vitro, consistent with similar overall structures predicted for the isozymes by molecular modelling. Nevertheless, MurA appears to be the primary enzyme utilized in the staphylococcal cell. Accordingly, murA expression was approximately five times greater than murZ expression during exponential growth, and the peptidoglycan content of S. aureus was reduced by approximately 25% following inactivation of murA, but remained almost unchanged following inactivation of murZ. Despite low level expression during normal growth, murZ expression was strongly induced (up to sixfold) following exposure to inhibitors of peptidoglycan biosynthesis, which was not observed for murA. Strains generated in this study were validated as potential tools for identifying novel anti-staphylococcal agents targeting peptidoglycan biosynthesis using known inhibitors of the pathway.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Discovery of new inhibitors of D-alanine:D-alanine ligase by structure-based virtual screening.

The terminal dipeptide, D-Ala-D-Ala, of the peptidoglycan precursor UDPMurNAc-pentapetide is a crucial building block involved in peptidoglycan cross-linking. It is synthesized in the bacterial cytoplasm by the enzyme d-alanine:d-alanine ligase (Ddl). Structure-based virtual screening of the NCI diversity set of almost 2000 compounds was performed with a DdlB isoform from Escherichia coli using the computational tool AutoDock 4.0. The 130 best-ranked compounds from this screen were tested in an in vitro assay for their inhibition of E. coli DdlB. Three compounds were identified that inhibit the enzyme with K(i) values in micromolar range. Two of these also have promising antibacterial activities against Gram-positive and Gram-negative bacteria.

Diazenedicarboxamides as inhibitors of D-alanine-D-alanine ligase (Ddl).

D-Alanine-D-alanine ligase (Ddl) catalyzes the biosynthesis of an essential bacterial peptidoglycan precursor D-alanyl-D-alanine and it represents an important target for development of new antibacterial drugs. A series of semicarbazides, aminocarbonyldiazenecarboxylates, diazenedicarboxamides, and hydrazinedicarboxamides was synthesized and screened for inhibition of DdlB from Escherichia coli. Compounds with good inhibitory activity were identified, enabling us to deduce initial structure-activity relationships. Thirteen diazenedicarboxamides were better inhibitors than D-cycloserine and some of them also possess antibacterial activity, which makes them a promising starting point for further development.

Assessment of a microplate method for determining the post-antibiotic effect in Staphylococcus aureus and Escherichia coli.

OBJECTIVES: The post-antibiotic effect (PAE) is an important parameter of antibiotic action that is widely used as a predictor of pharmacodynamic activity. Traditionally, PAE has been determined by a labour-intensive method involving determination of viable cell numbers. New methods using spectrophotometric procedures could offer significant advantages for PAE determinations, particularly in terms of speed. A number of such methods have been described in the literature, but extensive comparison with the classical procedure for determining PAEs has not been carried out. We have now compared PAE values obtained using a rapid microplate method with those achieved by the classical viable count procedure. METHODS: We determined PAE values for a variety of antibiotics against Staphylococcus aureus and Escherichia coli following exposure to 5 x MIC drug concentrations for 60 min in Mueller-Hinton Broth (MHB). The duration of the PAE was obtained by following the recovery of bacterial growth in antibiotic-free MHB measured either as colony forming units on Mueller-Hinton agar, or as culture absorbance (600 nm) in a microplate reader. RESULTS: For bacteriolytic agents there was poor correlation between the two methods for both S. aureus (R2=0.096) and E. coli (R2=0.5456). However, when PAEs for bacteriostatic agents and non-lytic bactericidal agents were compared, correlation between the two methods was high for both S. aureus (R2=0.7529) and E. coli (R2=0.7687). CONCLUSIONS: The spectrophotometric microplate method for determining PAEs may be a suitable alternative to the classical method for those antibiotics that do not induce bacterial cell lysis.

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.