Sal-type ABC-F proteins: intrinsic and common mediators of pleuromutilin resistance by target protection in staphylococci.

The first member of the pleuromutilin (PLM) class suitable for systemic antibacterial chemotherapy in humans recently entered clinical use, underscoring the need to better understand mechanisms of PLM resistance in disease-causing bacterial genera. Of the proteins reported to mediate PLM resistance in staphylococci, the least-well studied to date is Sal(A), a putative ABC-F NTPase that-by analogy to other proteins of this type-may act to protect the ribosome from PLMs. Here, we establish the importance of Sal proteins as a common source of PLM resistance across multiple species of staphylococci. Sal(A) is revealed as but one member of a larger group of Sal-type ABC-F proteins that vary considerably in their ability to mediate resistance to PLMs and other antibiotics. We find that specific sal genes are intrinsic to particular staphylococcal species, and show that this gene family is likely ancestral to the genus Staphylococcus. Finally, we solve the cryo-EM structure of a representative Sal-type protein (Sal(B)) in complex with the staphylococcal 70S ribosome, revealing that Sal-type proteins bind into the E site to mediate target protection, likely by displacing PLMs and other antibiotics via an allosteric mechanism.

An extensively validated whole-cell biosensor for specific, sensitive and high-throughput detection of antibacterial inhibitors targeting cell-wall biosynthesis.

BACKGROUND: Whole-cell biosensor strains are powerful tools for antibacterial drug discovery, in principle allowing the identification of inhibitors acting on specific, high-value target pathways. Whilst a variety of biosensors have been described for detecting cell-wall biosynthesis inhibitors (CWBIs), these strains typically lack specificity and/or sensitivity, and have for the most part not been rigorously evaluated as primary screening tools. Here, we describe several Staphylococcus aureus CWBI biosensors and show that specific and sensitive biosensor-based discovery of CWBIs is achievable. METHODS: Biosensors comprised lacZ reporter fusions with S. aureus promoters (PgltB, PilvD, PmurZ, PoppB, PORF2768, PsgtB) that are subject to up-regulation following inhibition of cell-wall biosynthesis. Induction of biosensors was detected by measuring expression of ß-galactosidase using fluorogenic or luminogenic substrates. RESULTS: Three of the six biosensors tested (those based on PgltB, PmurZ, PsgtB) exhibited apparently specific induction of ß-galactosidase expression in the presence of CWBIs. Further validation of one of these (PmurZ) using an extensive array of positive and negative control compounds and conditional mutants established that it responded appropriately and uniquely to inhibition of cell-wall biosynthesis. Using this biosensor, we established, validated and deployed a high-throughput assay that identified a potentially novel CWBI from a screen of >9000 natural product extracts. CONCLUSIONS: Our extensively validated PmurZ biosensor strain offers specific and sensitive detection of CWBIs, and is well-suited for high-throughput screening; it therefore represents a valuable tool for antibacterial drug discovery.

Resistance to antibacterial antifolates in multidrug-resistant Staphylococcus aureus: prevalence estimates and genetic basis.

OBJECTIVES: Antibacterial antifolate drugs might have a wider role in the management of staphylococcal infection. One factor that could potentially limit their use in this context is pre-existing resistance. Here we explored the prevalence and genetic basis for resistance to these drugs in a large collection (n = 1470) of multidrug-resistant (MDR) Staphylococcus aureus. METHODS: Strains were subjected to susceptibility testing to detect resistance to trimethoprim, sulfamethoxazole, co-trimoxazole and the investigational drug, iclaprim. Whole-genome sequences were interrogated to establish the genetic basis for resistance. RESULTS: According to CLSI breakpoints, 15.2% of the strains were resistant to trimethoprim, 5.2% to sulfamethoxazole and 4.1% to co-trimoxazole. Using the proposed breakpoint for iclaprim, 89% of the trimethoprim-resistant strains exhibited non-susceptibility to this agent. Sulfamethozaxole resistance was exclusively the result of mutation in the drug target (dihydropteroate synthase). Resistance to trimethoprim and iclaprim also resulted from mutation in the target (dihydrofolate reductase; DHFR) but was more commonly associated with horizontal acquisition of genes encoding drug-insensitive DHFR proteins. Among the latter, we identified a novel gene (dfrL) encoding a DHFR with ∼35% identity to native and known resistant DHFRs, which was confirmed via molecular cloning to mediate high-level resistance. CONCLUSIONS: This study provides a detailed picture of the genotypes underlying staphylococcal resistance to antifolate drugs in clinical use and in development. Prevalence estimates suggest that resistance to the diaminopyrimidines (trimethoprim/iclaprim) is not uncommon among MDR S. aureus, and considerably higher than observed for sulfamethoxazole or co-trimoxazole.

Creating a framework to align antimicrobial resistance (AMR) research with the global guidance: a viewpoint.

We describe here an initial analysis of national and international guidance documents on antimicrobial resistance (AMR) to propose a framework to align AMR research activities with global guidance. The framework provides a summary roadmap for core activities in AMR research and highlights the need for interdisciplinary and One Health collaboration. This analysis also revealed limitations in the current guidance, including a lack of explicit mention of some research activities highly relevant to AMR and a dearth of concrete objectives; consequently, an over-reliance on global guidance could be funnelling research efforts down a generic trajectory without regard to contextual factors. We suggest this framework be used by academics and policymakers to align AMR research and guidance. However, we recommend that deeper exploration be undertaken to fully contextualize the development of meaningful questions based on current knowledge, methodologies and gap analyses.

Algorithm-driven activity-directed expansion of a series of antibacterial quinazolinones.

Activity-directed synthesis (ADS) is a structure-blind, function driven approach that can drive the discovery of bioactive small molecules. In ADS, arrays of reactions are designed and executed, and the crude product mixtures are then directly screened to identify reactions that yield bioactive products. The design of subsequent reaction arrays is then informed by the hit reactions that are discovered. In this study, algorithms for reaction array design were developed in which the reactions to be executed were selected from a large set of virtual reactions; the reactions were selected on the basis of similarity to reactions known to yield bioactive products. The algorithms were harnessed to design arrays of photoredox-catalysed alkylation reactions whose crude products were then screened for inhibition of growth of S. aureus ATCC29213. It was demonstrated that the approach enabled expansion of a series of antibacterial quinazolinones. It is envisaged that such algorithms could ultimately enable fully autonomous activity-directed molecular discovery.

A platform for detecting cross-resistance in antibacterial drug discovery.

BACKGROUND: To address the growing antibiotic resistance problem, new antibacterial drugs must exert activity against pathogens resistant to agents already in use. With a view to providing a rapid means for deselecting antibacterial drug candidates that fail to meet this requirement, we report here the generation and application of a platform for detecting cross-resistance between established and novel antibacterial agents. METHODS: This first iteration of the cross-resistance platform (CRP) consists of 28 strains of defined resistance genotype, established in a uniform genetic background (the SH1000 strain of the clinically significant pathogen Staphylococcus aureus). Most CRP members were engineered through introduction of constitutively expressed resistance determinants on a low copy-number plasmid, with a smaller number selected as spontaneous resistant mutants. RESULTS: Members of the CRP collectively exhibit resistance to many of the major classes of antibacterial agent in use. We employed the CRP to test two antibiotics that have been proposed in the literature as potential drug candidates: γ-actinorhodin and batumin. No cross-resistance was detected for γ-actinorhodin, whilst a CRP member resistant to triclosan exhibited a 32-fold reduction in susceptibility to batumin. Thus, a resistance phenotype that already exists in clinical strains mediates profound resistance to batumin, implying that this compound is not a promising antibacterial drug candidate. CONCLUSIONS: By detecting cross-resistance between established and novel antibacterial agents, the CRP offers the ability to deselect compounds whose activity is substantially impaired by existing resistance mechanisms. The CRP therefore represents a useful addition to the antibacterial drug discovery toolbox.

Impaired Alanine Transport or Exposure to d-Cycloserine Increases the Susceptibility of MRSA to ß-lactam Antibiotics.

Prolonging the clinical effectiveness of ß-lactams, which remain first-line antibiotics for many infections, is an important part of efforts to address antimicrobial resistance. We report here that inactivation of the predicted d-cycloserine (DCS) transporter gene cycA resensitized methicillin-resistant Staphylococcus aureus (MRSA) to ß-lactam antibiotics. The cycA mutation also resulted in hypersusceptibility to DCS, an alanine analogue antibiotic that inhibits alanine racemase and d-alanine ligase required for d-alanine incorporation into cell wall peptidoglycan. Alanine transport was impaired in the cycA mutant, and this correlated with increased susceptibility to oxacillin and DCS. The cycA mutation or exposure to DCS were both associated with the accumulation of muropeptides with tripeptide stems lacking the terminal d-ala-d-ala and reduced peptidoglycan cross-linking, prompting us to investigate synergism between ß-lactams and DCS. DCS resensitized MRSA to ß-lactams in vitro and significantly enhanced MRSA eradication by oxacillin in a mouse bacteremia model. These findings reveal alanine transport as a new therapeutic target to enhance the susceptibility of MRSA to ß-lactam antibiotics.

Potential for repurposing the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents for topical application.

OBJECTIVES: Bacterial biofilms represent a major impediment to healing in chronic wounds and are largely refractory to the antibacterial agents currently used in wound management. From a repurposing screen of compounds considered safe for topical application in humans, we report the identification of the personal care product preservatives bronopol and bronidox as broad-spectrum antibiofilm agents and potential candidates for reducing biofilm burden in chronic wounds. METHODS: Antibiofilm activity was assessed by viable counting against single-species biofilms of Staphylococcus aureus and Pseudomonas aeruginosa in the Calgary Biofilm Device, and against mixed-species biofilms of the two organisms growing on nitrocellulose discs. RESULTS: Bronopol and bronidox exhibited broad-spectrum antibiofilm activity that encompassed the two major wound pathogens, S. aureus and P. aeruginosa. When impregnated into gauze dressings at their existing maximum authorized concentrations for safe use and placed onto an established mixed-species biofilm, bronopol and bronidox completely eradicated P. aeruginosa and achieved an ∼5 log10 reduction in the S. aureus population. The antibiofilm action of bronopol and bronidox was attributed to their ability to kill slow- or non-growing bacteria found in biofilms, and both compounds exhibited synergistic antibiofilm effects in combination with established wound-treatment agents. CONCLUSIONS: Bronopol and bronidox kill bacteria regardless of growth state, a property that endows them with broad-spectrum antibiofilm activity. As this effect is observed at concentrations authorized for use on human skin, these compounds represent promising candidates for the treatment of chronic wounds.

Design, Synthesis and Microbiological Evaluation of Novel Compounds as Potential Staphylococcus aureus Phenylalanine tRNA Synthetase Inhibitors.

AS THE RESISTANCE of Staphylococcus aureus to antibiotics represents a major threat to global health, anti-infectives with novel mechanisms must be developed. Novel compounds were generated as potential phenylalanine tRNA synthetase (PheRS) inhibitors based on the published homology model of S. aureus PheRS to aid the design process using Molecular Operating Environment (MOE) software. PheRS was selected as it is structurally unique enzyme among the aminoacyl-tRNA synthetases (aaRS), it is considerably different from human cytosolic and human mitochondrial aaRS and it is essential and conserved across bacterial species. The designed compounds were synthesized according to different clear schemes. The compounds were confirmed by 1H NMR, 13C NMR, HRMS and/or microanalysis, and they were microbiologically evaluated.

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).

Targeting Multiple Aminoacyl-tRNA Synthetases Overcomes the Resistance Liabilities Associated with Antibacterial Inhibitors Acting on a Single Such Enzyme.

Bacterial aminoacyl-tRNA synthetases (aaRSs) represent promising antibacterial drug targets. Unfortunately, the aaRS inhibitors that have to date reached clinical trials are subject to rapid resistance development through mutation, a phenomenon that limits their potential clinical utility. Here, we confirm the intuitively correct idea that simultaneous targeting of two different aaRS enzymes prevents the emergence of spontaneous bacterial resistance at high frequency, a finding that supports the development of multitargeted anti-aaRS therapies.

A Polymorphism in leuS Confers Reduced Susceptibility to GSK2251052 in a Clinical Isolate of Staphylococcus aureus.

GSK2251052 is a broad-spectrum antibacterial inhibitor of leucyl tRNA-synthetase (LeuRS) that has been evaluated in phase II clinical trials. Here, we report the identification of a clinical isolate of Staphylococcus aureus that exhibits reduced susceptibility to GSK2251052 without prior exposure to the compound and demonstrate that this phenotype is attributable to a single amino acid polymorphism (P329) within the editing domain of LeuRS.

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.

Identification and characterization of an anti-pseudomonal dichlorocarbazol derivative displaying anti-biofilm activity.

Pseudomonas aeruginosa strains resistant towards all currently available antibiotics are increasingly encountered, raising the need for new anti-pseudomonal drugs. We therefore conducted a medium-throughput screen of a small-molecule collection resulting in the identification of the N-alkylated 3,6-dihalogenocarbazol 1-(sec-butylamino)-3-(3,6-dichloro-9H-carbazol-9-yl)propan-2-ol (MIC = 18.5 µg mL⁻¹). This compound, compound 1, is bacteriostatic towards a broad spectrum of Gram-positive and Gram-negative pathogens, including P. aeruginosa. Importantly, 1 also eradicates mature biofilms of P. aeruginosa. 1 displays no cytotoxicity against various human cell types, pointing to its potential for further development as a novel antibacterial drug.

Mutagenesis mapping of the protein-protein interaction underlying FusB-type fusidic acid resistance.

FusB-type proteins represent the predominant mechanism of resistance to fusidic acid in staphylococci and act by binding to and modulating the function of the drug target (elongation factor G [EF-G]). To gain further insight into this antibiotic resistance mechanism, we sought to identify residues important for the interaction of FusB with EF-G and thereby delineate the binding interface within the FusB-EF-G complex. Replacement with alanine of any one of four conserved residues within the C-terminal domain of FusB (F156, K184, Y187, and F208) abrogated the ability of the protein to confer resistance to fusidic acid; the purified mutant proteins also lost the ability to bind S. aureus EF-G in vitro. E. coli EF-G, which is not ordinarily able to bind FusB-type proteins, was rendered competent for binding to FusB following deletion of a 3-residue tract (529SNP531) from domain IV of the protein. This study has identified key regions of both FusB and EF-G that are important for the interaction between the proteins, findings which corroborate our previous in silico prediction for the architecture of the complex formed between the resistance protein and the drug target (G. Cox, G. S. Thompson, H. T. Jenkins, F. Peske, A. Savelsbergh, M. V. Rodnina, W. Wintermeyer, S. W. Homans, T. A. Edwards, and A. J. O'Neill, Proc. Natl. Acad. Sci. U. S. A. 109:2102-2107, 2012).

Staphylococcus aureus biofilms promote horizontal transfer of antibiotic resistance.

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 target of daptomycin is absent from Escherichia coli and other gram-negative pathogens.

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.

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.