Nicola Ooi

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.

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.

Redox-active compounds with a history of human use: antistaphylococcal action and potential for repurposing as topical antibiofilm agents

Objectives To investigate the antistaphylococcal/antibiofilm activity and mode of action (MOA) of a panel of redox-active (RA) compounds with a history of human use and to provide a preliminary preclinical assessment of their potential for topical treatment of staphylococcal infections, including those involving a biofilm component. Methods Antistaphylococcal activity was evaluated by broth microdilution and by time–kill studies with growing and slow- or non-growing cells. The antibiofilm activity of RA compounds, alone and in combination with established antibacterial agents, was assessed using the Calgary Biofilm Device. Established assays were used to examine the membrane-perturbing effects of RA compounds, to measure penetration into biofilms and physical disruption of biofilms and to assess resistance potential. A living skin equivalent model was used to assess the effects of RA compounds on human skin. Results All 15 RA compounds tested displayed antistaphylococcal activity against planktonic cultures (MIC 0.25–128 mg/L) and 7 eradicated staphylococcal biofilms (minimum biofilm eradication concentration 4–256 mg/L). The MOA of all compounds involved perturbation of the bacterial membrane, whilst selected compounds with antibiofilm activity caused destructuring of the biofilm matrix. The two most promising agents [celastrol and nordihydroguaiaretic acid (NDGA)] in respect of antibacterial potency and selective toxicity against bacterial membranes acted synergistically with gentamicin against biofilms, did not damage artificial skin following topical application and exhibited low resistance potential. Conclusions In contrast to established antibacterial drugs, some RA compounds are capable of eradicating staphylococcal biofilms. Of these, celastrol and NDGA represent particularly attractive candidates for development as topical antistaphylococcal biofilm treatments.

Further Characterization of Bacillus subtilis Antibiotic Biosensors and Their Use for Antibacterial Mode-of-Action Studies

ABSTRACT We further examined the usefulness of previously reported Bacillus subtilis biosensors for antibacterial mode-of-action studies. The biosensors could not detect the tRNA synthetase inhibitors mupirocin, indolmycin, and borrelidin, some inhibitors of peptidoglycan synthesis, and most membrane-damaging agents. However, the biosensors confirmed the modes of action of several RNA polymerase inhibitors and DNA intercalators and provided new insights into the possible modes of action of ciprofloxacin, anhydrotetracycline, corralopyronin, 8-hydroxyquinoline, and juglone.

Antibacterial activity and mode of action of tert-butylhydroquinone (TBHQ) and its oxidation product, tert-butylbenzoquinone (TBBQ)

OBJECTIVES The antioxidant tert-butylhydroquinone (TBHQ) is a food additive reported to have antibacterial activity, and may therefore have application in the healthcare setting. This study sought to characterize the antibacterial activity and mode of action of TBHQ and its oxidation product, tert-butylbenzoquinone (TBBQ). METHODS The stability of TBHQ/TBBQ was studied in buffer. Susceptibility testing was performed by broth microdilution, and killing and lytic activity were evaluated by viable counting and culture turbidity measurements. Mode of action studies included following the incorporation of radiolabelled precursors into macromolecules. The effect of TBHQ/TBBQ upon bacterial and mammalian membranes was assessed using the BacLight(TM) assay and by monitoring the haemolysis of equine erythrocytes. RESULTS TBHQ underwent oxidation in solution to form TBBQ. When oxidation was prevented, TBHQ lacked useful antibacterial activity, indicating that TBBQ is responsible for the antibacterial activity attributed to TBHQ. TBBQ demonstrated activity against Staphylococcus aureus SH1000 (MIC 8 mg/L) and against a panel of clinical S. aureus isolates (MIC90 16 mg/L). TBBQ at 4× MIC caused a >4 log10 drop in cell viability within 6 h without lysis, and eradicated staphylococcal biofilms at 8× MIC. TBBQ did not display preferential inhibition of any single macromolecular synthetic pathway, but caused loss of staphylococcal membrane integrity without haemolytic activity. CONCLUSIONS TBBQ is responsible for the antibacterial activity previously ascribed to TBHQ. TBBQ prompts loss of staphylococcal membrane integrity; it is rapidly and extensively bactericidal, but is non-lytic. In view of the potent and selective bactericidal activity of TBBQ, this compound warrants further investigation as a candidate antistaphylococcal agent.

Cryptic silver resistance is prevalent and readily activated in certain Gram-negative pathogens

Objectives To assess the prevalence of cryptic silver (Ag+) resistance amongst clinical isolates of Gram-negative bacteria, and to examine how overt Ag+ resistance becomes activated in such strains. Methods Established methods were used to determine the susceptibility of 444 recent clinical isolates to Ag+, and to evaluate the potential for overt Ag+ resistance to emerge in susceptible isolates by spontaneous mutation. The genetic basis for Ag+ resistance was investigated using PCR amplification and DNA sequencing. Results None of the isolates tested displayed overt Ag+ resistance. However, upon silver challenge, high-level Ag+ resistance (silver nitrate MIC >128 mg/L) was selected at high frequency (10-7 to 10-8) in 76% of isolates of Enterobacter spp., ∼58% of isolates of Klebsiella spp. and ∼0.7% of isolates of Escherichia coli. All strains in which Ag+ resistance could be selected harboured the sil operon, with resistance apparently resulting from activation of this system as a consequence of single missense mutations in silS. By contrast, Ag+ resistance was not selected in isolates lacking sil, which included all tested representatives of Pseudomonas aeruginosa, Acinetobacter spp., Citrobacter spp. and Proteus spp. Conclusions Whilst overt Ag+ resistance in Gram-negative pathogens is uncommon, cryptic Ag+ resistance pertaining to the sil operon is prevalent and readily activated in particular genera (Enterobacter and Klebsiella).

Tert-butyl benzoquinone: mechanism of biofilm eradication and potential for use as a topical antibiofilm agent

Objectives Tert-butyl benzoquinone (TBBQ) is the oxidation product of tert-butyl hydroquinone (TBHQ), an antimicrobial food additive with >40 years of safe use. TBBQ displays potent activity against Staphylococcus aureus biofilms in vitro. Here, we report on studies to further explore the action of TBBQ on staphylococcal biofilms, and provide a preliminary preclinical assessment of its potential for use as a topical treatment for staphylococcal infections involving a biofilm component. Methods The antibacterial properties of TBBQ were assessed against staphylococci growing in planktonic culture and as biofilms in the Calgary Biofilm Device. Established assays were employed to measure the effects of TBBQ on biofilm structure and bacterial membranes, and to assess resistance potential. A living-skin equivalent was used to evaluate the effects of TBBQ on human skin. Results TBBQ eradicated biofilms of S. aureus and other staphylococcal species at concentrations ≤64 mg/L. In contrast to other redox-active agents exhibiting activity against biofilms, TBBQ did not cause substantial destructuring of the biofilm matrix; instead, the antibiofilm activity of the compound was attributed to its ability to kill slow- and non-growing cells via membrane perturbation. TBBQ acted synergistically with gentamicin, did not damage a living-skin equivalent following topical application and exhibited low resistance potential. Conclusions The ability of TBBQ to eradicate biofilms appears to result from its ability to kill bacteria regardless of growth state. Preliminary evaluation suggests that TBBQ represents a promising candidate for development as a topical antibiofilm agent.

Revisiting unexploited antibiotics in search of new antibacterial drug candidates: the case of MSD-819 (6-chloro-2-quinoxalinecarboxylic acid 1,4-dioxide)

Revisiting unexploited antibiotics in search of new antibacterial drug candidates: the case of MSD-819 (6-chloro-2-quinoxalinecarboxylic acid 1,4-dioxide)

In vitro biological evaluation of novel broad-spectrum isothiazolone inhibitors of bacterial type II topoisomerases.

OBJECTIVES To evaluate the in vitro biological properties of a novel class of isothiazolone inhibitors of the bacterial type II topoisomerases. METHODS Inhibition of DNA gyrase and topoisomerase IV activity was assessed using DNA supercoiling and decatenation assays. MIC and MBC were determined according to CLSI guidelines. Antibacterial combinations were assessed using a two-dimensional chequerboard MIC method. Spontaneous frequency of resistance was measured at various multiples of the MIC. Resistant mutants were generated by serial passage at subinhibitory concentrations of antibacterials and genetic mutations were determined through whole genome sequencing. Mammalian cytotoxicity was evaluated using the HepG2 cell line. RESULTS Representative isothiazolone compound REDX04957 and its enantiomers (REDX05967 and REDX05990) showed broad-spectrum bactericidal activity against the ESKAPE organisms, with the exception of Enterococcus spp., as well as against a variety of other human bacterial pathogens. Compounds retained activity against quinolone-resistant strains harbouring GyrA S83L and D87G mutations (MIC ≤4 mg/L). Compounds inhibited the supercoiling activity of wild-type DNA gyrase and the decatenation function of topoisomerase IV. Frequency of resistance of REDX04957 at 4× MIC was <9.1 × 10(-9). Against a panel of recent MDR isolates, REDX05967 demonstrated activity against Acinetobacter baumannii with MIC50 and MIC90 of 16 and 64 mg/L, respectively. Compounds showed a lack of cytotoxicity against HepG2 cells at 128 mg/L. CONCLUSIONS Isothiazolone compounds show potent activity against Gram-positive and -negative pathogens with a dual targeting mechanism-of-action and a low potential for resistance development, meriting their continued investigation as broad-spectrum antibacterial agents.

Epistasis analysis uncovers hidden antibiotic resistance-associated fitness costs hampering the evolution of MRSA

BackgroundFitness costs imposed on bacteria by antibiotic resistance mechanisms are believed to hamper their dissemination. The scale of these costs is highly variable. Some, including resistance of Staphylococcus aureus to the clinically important antibiotic mupirocin, have been reported as being cost-free, which suggests that there are few barriers preventing their global spread. However, this is not supported by surveillance data in healthy communities, which indicate that this resistance mechanism is relatively unsuccessful.ResultsEpistasis analysis on two collections of MRSA provides an explanation for this discord, where the mupirocin resistance-conferring mutation of the ileS gene appears to affect the levels of toxins produced by S. aureus when combined with specific polymorphisms at other loci. Proteomic analysis demonstrates that the activity of the secretory apparatus of the PSM family of toxins is affected by mupirocin resistance. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production becomes necessary. This hidden fitness cost provides a likely explanation for why this mupirocin-resistance mechanism is not more prevalent, given the widespread use of this antibiotic.ConclusionsWith dwindling pools of antibiotics available for use, information on the fitness consequences of the acquisition of resistance may need to be considered when designing antibiotic prescribing policies. However, this study suggests there are levels of depth that we do not understand, and that holistic, surveillance and functional genomics approaches are required to gain this crucial information.