Alex J. O'Neill

Targeting bacterial membrane function: an underexploited mechanism for treating persistent infections.

Persistent infections involving slow-growing or non-growing bacteria are hard to treat with antibiotics that target biosynthetic processes in growing cells. Consequently, there is a need for antimicrobials that can treat infections containing dormant bacteria. In this Review, we discuss the emerging concept that disrupting the bacterial membrane bilayer or proteins that are integral to membrane function (including membrane potential and energy metabolism) in dormant bacteria is a strategy for treating persistent infections. The clinical applicability of these approaches is exemplified by the efficacy of lipoglycopeptides that damage bacterial membranes and of the diarylquinoline TMC207, which inhibits membrane-bound ATP synthase. Despite some drawbacks, membrane-active agents form an important new means of eradicating recalcitrant, non-growing bacteria.

Treatment of health-care-associated infections caused by Gram-negative bacteria: a consensus statement

This consensus statement presents the conclusions of a group of academic and industrial experts who met in London in September, 2006, to consider the issues associated with the treatment of hospital infections caused by Gram-negative bacteria. The group discussed the severe clinical problems arising from the emergence of antibiotic resistance in these bacteria and the lack of new antibacterial agents to challenge the threat. The discovery of new drugs active against hospital-acquired Gram-negative bacteria is essential to prevent a future medical and social catastrophe. An important strategy to promote drug discovery will be the development of focused cooperations between academic institutions and small pharmaceutical companies.

Consequences of daptomycin-mediated membrane damage in Staphylococcus aureus

OBJECTIVES The proposed lethal action of daptomycin on Staphylococcus aureus results from the loss of K(+) and membrane depolarization. However, whether these events alone cause cell death has been questioned. We sought to determine whether other consequences of daptomycin-mediated membrane damage may contribute to cell death. METHODS Previously established assays were used to evaluate the membrane damaging activity of daptomycin at a single time-point of 10 min. More detailed time-course experiments were also performed to determine the kinetics of membrane depolarization and leakage of K(+), Mg(2+) and ATP. The kinetics of inhibition of macromolecular synthesis following exposure to daptomycin were also determined by assaying the incorporation of radioactive precursors into macromolecules. RESULTS Daptomycin exhibited no membrane damaging activity in single time-point assays following exposure to the antibiotic for 10 min. Kinetic analysis confirmed these results as leakage of intracellular components did not occur until 20-30 min, membrane depolarization was gradual and cells remained biosynthetically active for at least 30 min after exposure to daptomycin. Viability declined rapidly after exposure to daptomycin and appeared to precede other detectable changes. CONCLUSIONS These data show that daptomycin-induced loss of Mg(2+) and ATP occurs in conjunction with the previously reported leakage of K(+) and membrane depolarization. We propose that the lethal activity of daptomycin is not simply due to loss of K(+) and probably involves more general damage to the membrane.

Staphylococcus aureus biofilms promote horizontal transfer of antibiotic resistance

ABSTRACT Growth as a biofilm facilitates the emergence of antibiotic resistance by mutation in Staphylococcus aureus. Here we demonstrate that biofilm growth of this species also dramatically increases horizontal transfer of plasmid-borne antibiotic resistance determinants by conjugation/mobilization and that standard laboratory practices to induce conjugation in staphylococci achieve optimal efficiency owing to the presence of a biofilm.

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.

Silver resistance in Gram-negative bacteria: a dissection of endogenous and exogenous mechanisms

Objectives To gain a more detailed understanding of endogenous (mutational) and exogenous (horizontally acquired) resistance to silver in Gram-negative pathogens, with an emphasis on clarifying the genetic bases for resistance. Methods A suite of microbiological and molecular genetic techniques was employed to select and characterize endogenous and exogenous silver resistance in several Gram-negative species. Results In Escherichia coli, endogenous resistance arose after 6 days of exposure to silver, a consequence of two point mutations that were both necessary and sufficient for the phenotype. These mutations, in ompR and cusS, respectively conferred loss of the OmpC/F porins and derepression of the CusCFBA efflux transporter, both phenotypic changes previously linked to reduced intracellular accumulation of silver. Exogenous resistance involved derepression of the SilCFBA efflux transporter as a consequence of mutation in silS, but was additionally contingent on expression of the periplasmic silver-sequestration protein SilE. Silver resistance could be selected at high frequency (>10−9) from Enterobacteriaceae lacking OmpC/F porins or harbouring the sil operon and both endogenous and exogenous resistance were associated with modest fitness costs in vitro. Conclusions Both endogenous and exogenous silver resistance are dependent on the derepressed expression of closely related efflux transporters and are therefore mechanistically similar phenotypes. The ease with which silver resistance can become selected in some bacterial pathogens in vitro suggests that there would be benefit in improved surveillance for silver-resistant isolates in the clinic, along with greater control over use of silver-containing products, in order to best preserve the clinical utility of silver.

The Target of Daptomycin is Absent from Escherichia coli and other Gram-Negative Pathogens

ABSTRACT Antistaphylococcal agents commonly lack activity against Gram-negative bacteria like Escherichia coli owing to the permeability barrier presented by the outer membrane and/or the action of efflux transporters. When these intrinsic resistance mechanisms are artificially compromised, such agents almost invariably demonstrate antibacterial activity against Gram negatives. Here we show that this is not the case for the antibiotic daptomycin, whose target appears to be absent from E. coli and other Gram-negative pathogens.

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.

In Vitro Studies Indicate a High Resistance Potential for the Lantibiotic Nisin in Staphylococcus aureus and Define a Genetic Basis for Nisin Resistance

ABSTRACT Lantibiotics such as nisin (NIS) are peptide antibiotics that may have a role in the chemotherapy of bacterial infections. A perceived benefit of lantibiotics for clinical use is their low propensity to select resistance, although detailed resistance studies with relevant bacterial pathogens are lacking. Here we examined the development of resistance to NIS in Staphylococcus aureus, establishing that mutants, including small-colony variants, exhibiting substantial (4- to 32-fold) reductions in NIS susceptibility could be selected readily. Comparative genome sequencing of a single NISr mutant exhibiting a 32-fold increase in NIS MIC revealed the presence of only two mutations, leading to the substitutions V229G in the purine operon repressor, PurR, and A208E in an uncharacterized protein encoded by SAOUHSC_02955. Independently selected NISr mutants also harbored mutations in the genes encoding these products. Reintroduction of these mutations into the S. aureus chromosome alone and in combination revealed that SAOUHSC_02955(A208E) made the primary contribution to the resistance phenotype, conferring up to a 16-fold decrease in NIS susceptibility. Bioinformatic analyses suggested that this gene encodes a sensor histidine kinase, leading us to designate it “nisin susceptibility-associated sensor (nsaS).” Doubling-time determinations and mixed-culture competition assays between NISr and NISs strains indicated that NIS resistance had little impact on bacterial fitness, and resistance was stable in the absence of selection. The apparent ease with which S. aureus can develop and maintain NIS resistance in vitro suggests that resistance to NIS and other lantibiotics with similar modes of action would arise in the clinic if these agents are employed as chemotherapeutic drugs.

Transposon library screening for identification of genetic loci participating in intrinsic susceptibility and acquired resistance to antistaphylococcal agents

OBJECTIVES To establish an experimental platform in Staphylococcus aureus for identifying genetic loci that determine intrinsic antibiotic susceptibility and/or that have the potential to contribute to acquired antibiotic resistance. A near-saturation S. aureus transposon (Tn) library was screened for mutants exhibiting altered susceptibility to the antistaphylococcal agents daptomycin, vancomycin and nisin. METHODS S. aureus SH1000 was mutagenized with Tn InsTet(G+)2(Cm) by electroporation of transposomes. Approximately 20500 transposants were screened for increased or reduced susceptibility to the three antistaphylococcal agents and Tn insertion sites were mapped by DNA sequencing in mutants of interest. RESULTS Transposants exhibiting hypersusceptibility or reduced susceptibility were identified for all three antibacterial agents; mapping of Tn insertion sites in these mutants identified genetic determinants of intrinsic susceptibility and potential contributors to acquired resistance, respectively. Tn insertions in the dlt operon caused cross-hypersusceptibility to vancomycin, daptomycin and nisin. Daptomycin hypersusceptibility was also associated with disruption of genes directing lipoteichoic acid and riboflavin biosynthesis, apparent inactivation of a putative membrane protein encoded by SAOUHSC_00957 and truncation of the cell-division gene ezrA. Tn-mediated disruption of the vraDE- and SAOUHSC_02953/4-encoded ABC transporters conferred hypersusceptibility to nisin. Reduced susceptibility to both daptomycin and vancomycin was associated with Tn insertions in rpsU and upstream of yycFG. Several loci were associated with reduced susceptibility to nisin, including two genes encoding putative glycosyltransferases. CONCLUSIONS Tn library screening identified both known and novel modulators of antibacterial susceptibility in S. aureus and therefore represents a useful approach towards delineating the staphylococcal resistome.