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.

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.

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.

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.

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.

Structural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens.

Target protection proteins confer resistance to the host organism by directly binding to the antibiotic target. One class of such proteins are the antibiotic resistance (ARE) ATP-binding cassette (ABC) proteins of the F-subtype (ARE-ABCFs), which are widely distributed throughout Gram-positive bacteria and bind the ribosome to alleviate translational inhibition from antibiotics that target the large ribosomal subunit. Here, we present single-particle cryo-EM structures of ARE-ABCF-ribosome complexes from three Gram-positive pathogens: Enterococcus faecalis LsaA, Staphylococcus haemolyticus VgaALC and Listeria monocytogenes VgaL. Supported by extensive mutagenesis analysis, these structures enable a general model for antibiotic resistance mediated by these ARE-ABCFs to be proposed. In this model, ABCF binding to the antibiotic-stalled ribosome mediates antibiotic release via mechanistically diverse long-range conformational relays that converge on a few conserved ribosomal RNA nucleotides located at the peptidyltransferase center. These insights are important for the future development of antibiotics that overcome such target protection resistance mechanisms.

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.

Structure of the 70S Ribosome from the Human Pathogen Acinetobacter baumannii in Complex with Clinically Relevant Antibiotics.

Acinetobacter baumannii is a Gram-negative bacterium primarily associated with hospital-acquired, often multidrug-resistant (MDR) infections. The ribosome-targeting antibiotics amikacin and tigecycline are among the limited arsenal of drugs available for treatment of such infections. We present high-resolution structures of the 70S ribosome from A. baumannii in complex with these antibiotics, as determined by cryoelectron microscopy. Comparison with the ribosomes of other bacteria reveals several unique structural features at functionally important sites, including around the exit of the polypeptide tunnel and the periphery of the subunit interface. The structures also reveal the mode and site of interaction of these drugs with the ribosome. This work paves the way for the design of new inhibitors of translation to address infections caused by MDR A. baumannii.

Activity-directed expansion of a series of antibacterial agents.

The feasibility of using activity-directed synthesis to drive antibacterial discovery was investigated. An array of 220 Pd-catalysed microscale reactions was executed, and the crude product mixtures were evaluated for activity against Staphylococcus aureus. Scale-up of the hit reactions, purification and evaluation, enabled expansion of a class of antibacterial quinazolinones. The novel antibacterials had MICs from 0.016 µg mL-1 (i.e. 38 nM) to 2-4 µg mL-1 against S. aureus ATCC29213.

Target protection as a key antibiotic resistance mechanism.

Antibiotic resistance is mediated through several distinct mechanisms, most of which are relatively well understood and the clinical importance of which has long been recognized. Until very recently, neither of these statements was readily applicable to the class of resistance mechanism known as target protection, a phenomenon whereby a resistance protein physically associates with an antibiotic target to rescue it from antibiotic-mediated inhibition. In this Review, we summarize recent progress in understanding the nature and importance of target protection. In particular, we describe the molecular basis of the known target protection systems, emphasizing that target protection does not involve a single, uniform mechanism but is instead brought about in several mechanistically distinct ways.

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.

Transient Silencing of Antibiotic Resistance by Mutation Represents a Significant Potential Source of Unanticipated Therapeutic Failure.

Sporadic literature reports describe isolates of pathogenic bacteria that harbor an antibiotic resistance determinant but remain susceptible to the corresponding antibiotic as a consequence of a genetic defect. Such strains represent a source from which antibiotic resistance may reemerge to cause treatment failure in patients. Here, we report a systematic investigation into the prevalence and nature of this phenomenon, which we term silencing of antibiotic resistance by mutation (SARM). Instances of SARM were detected among 1,470 Staphylococcus aureus isolates through side-by-side comparison of antibiotic resistance genotype (as determined by whole-genome sequencing) versus phenotype (as assessed through susceptibility testing). Of the isolates analyzed, 152 (10.3%) harbored a silenced resistance gene, including 46 (3.1%) that exhibited SARM to currently deployed antistaphylococcal drugs. SARM resulted from diverse mutational events but most commonly through frameshift mutation of resistance determinants as a result of point deletion in poly(A) tracts. The majority (∼90%) of SARM strains reverted to antibiotic resistance at frequencies of ≥10-9; thus, while appearing antibiotic sensitive in the clinical microbiology laboratory, most S. aureus isolates exhibiting SARM will revert to antibiotic resistance at frequencies achievable in patients. In view of its prevalence in a major pathogen, SARM represents a significant potential threat to the therapeutic efficacy of antibiotics.IMPORTANCE Antibiotic resistance hinders the treatment of bacterial infection. To guide effective therapy, clinical microbiology laboratories routinely perform susceptibility testing to determine the antibiotic sensitivity of an infecting pathogen. This approach relies on the assumption that it can reliably distinguish bacteria capable of expressing antibiotic resistance in patients, an idea challenged by the present study. We report that the important human pathogen Staphylococcus aureus frequently carries antibiotic resistance genes that have become inactivated ("silenced") by mutation, leading strains to appear antibiotic sensitive. However, resistance can rapidly reemerge in most such cases, at frequencies readily achievable in infected patients. Silent antibiotic resistance is therefore prevalent, transient, and evades routine detection, rendering it a significant potential threat to antibacterial chemotherapy.

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.

Acquired Nisin Resistance in Staphylococcus aureus Involves Constitutive Activation of an Intrinsic Peptide Antibiotic Detoxification Module.

Resistance to the lantibiotic nisin (NIS) arises readily in Staphylococcus aureus as a consequence of mutations in the nsaS gene, which encodes the sensor kinase of the NsaRS two-component regulatory system. Here we present a series of studies to establish how these mutational changes result in reduced NIS susceptibility. Comparative transcriptomic analysis revealed upregulation of the NsaRS regulon in a NIS-resistant mutant of S. aureus versus its otherwise-isogenic progenitor, indicating that NIS resistance mutations prompt gain-of-function in NsaS. Two putative ABC transporters (BraDE and VraDE) encoded within the NsaRS regulon that have been reported to provide a degree of intrinsic protection against NIS were shown to be responsible for acquired NIS resistance; as is the case for intrinsic NIS resistance, NIS detoxification was ultimately mediated by VraDE, with BraDE participating in the signaling cascade underlying VraDE expression. Our study revealed new features of this signal transduction pathway, including that BraDE (but not VraDE) physically interacts with NsaRS. Furthermore, while BraDE has been shown to sense stimuli and signal to NsaS in a process that is contingent upon ATP hydrolysis, we established that this protein complex is also essential for onward transduction of the signal from NsaS through energy-independent means. NIS resistance in S. aureus therefore joins the small number of documented examples in which acquired antimicrobial resistance results from the unmasking of an intrinsic detoxification mechanism through gain-of-function mutation in a regulatory circuit.IMPORTANCE NIS and related bacteriocins are of interest as candidates for the treatment of human infections caused by Gram-positive pathogens such as Staphylococcus aureus An important liability of NIS in this regard is the ease with which S. aureus acquires resistance. Here we establish that this organism naturally possesses the cellular machinery to detoxify NIS but that the ABC transporter responsible (VraDE) is not ordinarily produced to a degree sufficient to yield substantial resistance. Acquired NIS resistance mutations prompt activation of the regulatory circuit controlling expression of vraDE, thereby unmasking an intrinsic resistance determinant. Our results provide new insights into the complex mechanism by which expression of vraDE is regulated and suggest that a potential route to overcoming the resistance liability of NIS could involve chemical modification of the molecule to prevent its recognition by the VraDE transporter.

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

BACKGROUND: Fitness 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. RESULTS: Epistasis 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. CONCLUSIONS: With 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.

1-((2,4-Dichlorophenethyl)Amino)-3-Phenoxypropan-2-ol Kills Pseudomonas aeruginosa through Extensive Membrane Damage.

The ever increasing multidrug-resistance of clinically important pathogens and the lack of novel antibiotics have resulted in a true antibiotic crisis where many antibiotics are no longer effective. Further complicating the treatment of bacterial infections are antibiotic-tolerant persister cells. Besides being responsible for the recalcitrant nature of chronic infections, persister cells greatly contribute to the observed antibiotic tolerance in biofilms and even facilitate the emergence of antibiotic resistance. Evidently, eradication of these persister cells could greatly improve patient outcomes and targeting persistence may provide an alternative approach in combatting chronic infections. We recently characterized 1-((2,4-dichlorophenethyl)amino)-3-phenoxypropan-2-ol (SPI009), a novel anti-persister molecule capable of directly killing persisters from both Gram-negative and Gram-positive pathogens. SPI009 potentiates antibiotic activity in several in vitro and in vivo infection models and possesses promising anti-biofilm activity. Strikingly, SPI009 restores antibiotic sensitivity even in resistant strains. In this study, we investigated the mode of action of this novel compound using several parallel approaches. Genetic analyses and a macromolecular synthesis assays suggest that SPI009 acts by causing extensive membrane damage. This hypothesis was confirmed by liposome leakage assay and membrane permeability studies, demonstrating that SPI009 rapidly impairs the bacterial outer and inner membranes. Evaluation of SPI009-resistant mutants, which only could be generated under severe selection pressure, suggested a possible role for the MexCD-OprJ efflux pump. Overall, our results demonstrate the extensive membrane-damaging activity of SPI009 and confirm its clinical potential in the development of novel anti-persister therapies.

N-Leucinyl Benzenesulfonamides as Structurally Simplified Leucyl-tRNA Synthetase Inhibitors.

N-Leucinyl benzenesulfonamides have been discovered as a novel class of potent inhibitors of E. coli leucyl-tRNA synthetase. The binding of inhibitors to the enzyme was measured by using isothermal titration calorimetry. This provided information on enthalpy and entropy contributions to binding, which, together with docking studies, were used for structure-activity relationship analysis. Enzymatic assays revealed that N-leucinyl benzenesulfonamides display remarkable selectivity for E. coli leucyl-tRNA synthetase compared to S. aureus and human orthologues. The simplest analogue of the series, N-leucinyl benzenesulfonamide (R = H), showed the highest affinity against E. coli leucyl-tRNA synthetase and also exhibited antibacterial activity against Gram-negative pathogens (the best MIC = 8 µg/mL, E. coli ATCC 25922), which renders it as a promising template for antibacterial drug discovery.

Antibiotic Resistance ABC-F Proteins: Bringing Target Protection into the Limelight.

Members of the ATP-binding cassette (ABC)-F protein subfamily collectively mediate resistance to a broader range of clinically important antibiotic classes than any other group of resistance proteins and are widespread in pathogenic bacteria. Following over 25 years' of controversy regarding the mechanism by which these proteins work, it has recently been established that they provide antibiotic resistance through the previously recognized but underappreciated phenomenon of target protection; they bind to the ribosome to effect the release of ribosome-targeted antibiotics, thereby rescuing the translation apparatus from antibiotic-mediated inhibition. Here we review the ABC-F resistance proteins with an emphasis on their mechanism of action, first exploring the history of the debate about how these proteins work and outlining our current state of knowledge and then considering key questions to be addressed in understanding the molecular detail of their function.