Discerning in vitro pharmacodynamics from OD measurements: A model-based approach.

Time-kill experiments can discern the pharmacodynamics of infectious bacteria exposed to antibiotics in vitro, and thus help guide the design of effective therapies for challenging clinical infections. This task is resource-limited, therefore typically bypassed in favor of empirical shortcuts. The resource limitation could be addressed by continuously assessing the size of a bacterial population under antibiotic exposure using optical density measurements. However, such measurements count both live and dead cells and are therefore unsuitable for declining populations of live cells. To fill this void, we develop here a model-based method that infers the count of live cells in a bacterial population exposed to antibiotics from continuous optical-density measurements of both live and dead cells combined. The method makes no assumptions about the underlying mechanisms that confer resistance and is widely applicable. Use of the method is demonstrated by an experimental study on Acinetobacter baumannii exposed to levofloxacin.

Prospective Evaluation of Light Scatter Technology Paired with Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Rapid Diagnosis of Urinary Tract Infections.

Urinary tract infections are one of the most common reasons for health care visits. Diagnosis and optimal treatment often require a urine culture, which takes an average of 1.5 to 2 days from urine collection to results, delaying optimal therapy. Faster, but accurate, alternatives are needed. Light scatter technology has been proposed for several years as a rapid screening tool, whereby negative specimens are excluded from culture. A commercially available light scatter device, BacterioScan 216Dx (BacterioScan, Inc.), has recently been advertised for this application. Paired use of mass spectrometry (MS) for bacterial identification and automated-system-based susceptibility testing straight from the light scatter suspension might provide dramatic improvement in times to a result. The present study prospectively evaluated the BacterioScan device, with culture as the reference standard. Positive light scatter specimens were used for downstream rapid matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS organism identification and automated-system-based antimicrobial susceptibility testing. Prospective evaluation of 439 urine samples showed a sensitivity of 96.5%, a specificity of 71.4%, and positive and negative predictive values of 45.1% and 98.8%, respectively. MALDI-TOF MS analysis of the suspension after density-based selection yielded a sensitivity of 72.1% and a specificity of 96.9%. Antimicrobial susceptibility testing of the samples identified by MALDI-TOF MS produced an overall categorical agreement of 99.2%. Given the high sensitivity and negative predictive value of results obtained, BacterioScan 216Dx is a reasonable approach for urine screening and might produce negative results in as few as 3 h, with no downstream workup. Paired rapid identification and susceptibility testing might be useful when MALDI-TOF MS results in an organism identification, and it might decrease the time to a result by more than 24 h.

Role of the carboxy terminus of SecA in iron acquisition, protein translocation, and virulence of the bacterial pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3' end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2'-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.

Potentiation of antibacterial activity of the MB-1 siderophore-monobactam conjugate using an efflux pump inhibitor.

Preliminary enthusiasm over the encouraging spectrum and in vitro activities of siderophore conjugates, such as MB-1, was recently tempered by unexpected variability in in vivo efficacy. The need for these conjugates to compete for iron with endogenously produced siderophores has exposed a significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux for siderophore secretion in Pseudomonas aeruginosa and provide evidence that efflux inhibition may circumvent this in vivo-relevant resistance liability.

Adaptation-based resistance to siderophore-conjugated antibacterial agents by Pseudomonas aeruginosa.

Multidrug resistance in Gram-negative bacteria has become so threatening to human health that new antibacterial platforms are desperately needed to combat these deadly infections. The concept of siderophore conjugation, which facilitates compound uptake across the outer membrane by hijacking bacterial iron acquisition systems, has received significant attention in recent years. While standard in vitro MIC and resistance frequency methods demonstrate that these compounds are potent, broad-spectrum antibacterial agents whose activity should not be threatened by unacceptably high spontaneous resistance rates, recapitulation of these results in animal models can prove unreliable, partially because of the differences in iron availability in these different methods. Here, we describe the characterization of MB-1, a novel siderophore-conjugated monobactam that demonstrates excellent in vitro activity against Pseudomonas aeruginosa when tested using standard assay conditions. Unfortunately, the in vitro findings did not correlate with the in vivo results we obtained, as multiple strains were not effectively treated by MB-1 despite having low MICs. To address this, we also describe the development of new in vitro assays that were predictive of efficacy in mouse models, and we provide evidence that competition with native siderophores could contribute to the recalcitrance of some P. aeruginosa isolates in vivo.

Structural basis for effectiveness of siderophore-conjugated monocarbams against clinically relevant strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen that causes nosocomial infections for which there are limited treatment options. Penicillin-binding protein PBP3, a key therapeutic target, is an essential enzyme responsible for the final steps of peptidoglycan synthesis and is covalently inactivated by ß-lactam antibiotics. Here we disclose the first high resolution cocrystal structures of the P. aeruginosa PBP3 with both novel and marketed ß-lactams. These structures reveal a conformational rearrangement of Tyr532 and Phe533 and a ligand-induced conformational change of Tyr409 and Arg489. The well-known affinity of the monobactam aztreonam for P. aeruginosa PBP3 is due to a distinct hydrophobic aromatic wall composed of Tyr503, Tyr532, and Phe533 interacting with the gem-dimethyl group. The structure of MC-1, a new siderophore-conjugated monocarbam complexed with PBP3 provides molecular insights for lead optimization. Importantly, we have identified a novel conformation that is distinct to the high-molecular-weight class B PBP subfamily, which is identifiable by common features such as a hydrophobic aromatic wall formed by Tyr503, Tyr532, and Phe533 and the structural flexibility of Tyr409 flanked by two glycine residues. This is also the first example of a siderophore-conjugated triazolone-linked monocarbam complexed with any PBP. Energetic analysis of tightly and loosely held computed hydration sites indicates protein desolvation effects contribute significantly to PBP3 binding, and analysis of hydration site energies allows rank ordering of the second-order acylation rate constants. Taken together, these structural, biochemical, and computational studies provide a molecular basis for recognition of P. aeruginosa PBP3 and open avenues for future design of inhibitors of this class of PBPs.

Identification and evaluation of twin-arginine translocase inhibitors.

The twin-arginine translocase (TAT) in some bacterial pathogens, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis, contributes to pathogenesis by translocating extracellular virulence determinants across the inner membrane into the periplasm, thereby allowing access to the Xcp (type II) secretory system for further export in Gram-negative organisms, or directly to the outside surface of the cell, as in M. tuberculosis. TAT-mediated secretion appreciably contributes to virulence in both animal and plant models of bacterial infection. Consequently, TAT function is an attractive target for small-molecular-weight compounds that alone or in conjunction with extant antimicrobial agents could become novel therapeutics. The TAT-transported hemolytic phospholipase C (PlcH) of P. aeruginosa and its multiple orthologs produced by the above pathogens can be detected by an accurate and reproducible colorimetric assay using a synthetic substrate that detects phospholipase C activity. Such an assay could be an effective indicator of TAT function. Using carefully constructed recombinant strains to precisely control the expression of PlcH, we developed a high-throughput screening (HTS) assay to evaluate, in duplicate, >80,000 small-molecular-weight compounds as possible TAT inhibitors. Based on additional TAT-related functional assays, purified PlcH protein inhibition experiments, and repeat experiments of the initial screening assay, 39 compounds were selected from the 122 initial hits. Finally, to evaluate candidate inhibitors for TAT specificity, we developed a TAT titration assay that determines whether inhibition of TAT-mediated secretion can be overcome by increasing the levels of TAT expression. The compounds N-phenyl maleimide and Bay 11-7082 appear to directly affect TAT function based on this approach.

Clinically relevant Gram-negative resistance mechanisms have no effect on the efficacy of MC-1, a novel siderophore-conjugated monocarbam.

The incidence of hospital-acquired infections with multidrug-resistant (MDR) Gram-negative pathogens is increasing at an alarming rate. Equally alarming is the overall lack of efficacious therapeutic options for clinicians, which is due primarily to the acquisition and development of various antibiotic resistance mechanisms that render these drugs ineffective. Among these mechanisms is the reduced permeability of the outer membrane, which prevents many marketed antibiotics from traversing this barrier. To circumvent this, recent drug discovery efforts have focused on conjugating a siderophore moiety to a pharmacologically active compound that has been designed to hijack the bacterial siderophore transport system and trick cells into importing the active drug by recognizing it as a nutritionally beneficial compound. MC-1, a novel siderophore-conjugated ß-lactam that promotes its own uptake into bacteria, has exquisite activity against many Gram-negative pathogens. While the inclusion of the siderophore was originally designed to facilitate outer membrane penetration into Gram-negative cells, here we show that this structural moiety also renders other clinically relevant antibiotic resistance mechanisms unable to affect MC-1 efficacy. Resistance frequency determinations and subsequent characterization of first-step resistant mutants identified PiuA, a TonB-dependent outer membrane siderophore receptor, as the primary means of MC-1 entry into Pseudomonas aeruginosa. While the MICs of these mutants were increased 32-fold relative to the parental strain in vitro, we show that this resistance phenotype is not relevant in vivo, as alternative siderophore-mediated uptake mechanisms compensated for the loss of PiuA under iron-limiting conditions.

Early insights into the interactions of different ß-lactam antibiotics and ß-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3.

Acinetobacter baumannii is an increasingly problematic pathogen in United States hospitals. Antibiotics that can treat A. baumannii are becoming more limited. Little is known about the contributions of penicillin binding proteins (PBPs), the target of ß-lactam antibiotics, to ß-lactam-sulbactam susceptibility and ß-lactam resistance in A. baumannii. Decreased expression of PBPs as well as loss of binding of ß-lactams to PBPs was previously shown to promote ß-lactam resistance in A. baumannii. Using an in vitro assay with a reporter ß-lactam, Bocillin, we determined that the 50% inhibitory concentrations (IC(50)s) for PBP1a from A. baumannii and PBP3 from Acinetobacter sp. ranged from 1 to 5 µM for a series of ß-lactams. In contrast, PBP3 demonstrated a narrower range of IC(50)s against ß-lactamase inhibitors than PBP1a (ranges, 4 to 5 versus 8 to 144 µM, respectively). A molecular model with ampicillin and sulbactam positioned in the active site of PBP3 reveals that both compounds interact similarly with residues Thr526, Thr528, and Ser390. Accepting that many interactions with cell wall targets are possible with the ampicillin-sulbactam combination, the low IC(50)s of ampicillin and sulbactam for PBP3 may contribute to understanding why this combination is effective against A. baumannii. Unraveling the contribution of PBPs to ß-lactam susceptibility and resistance brings us one step closer to identifying which PBPs are the best targets for novel ß-lactams.

Heterocyclic methylsulfone hydroxamic acid LpxC inhibitors as Gram-negative antibacterial agents.

The synthesis and antibacterial activity of heterocyclic methylsulfone hydroxamates is presented. Compounds in this series are potent inhibitors of the LpxC enzyme, a key enzyme involved in the production of lipopolysaccharide (LPS) found in the outer membrane of Gram-negative bacteria. SAR evaluation of compounds in this series revealed analogs with potent antibacterial activity against challenging Gram-negative species such as Pseudomonas aeruginosa and Klebsiella pneumoniae.

Distinctive attributes of ß-lactam target proteins in Acinetobacter baumannii relevant to development of new antibiotics.

Multi-drug-resistant forms of the Gram-negative pathogen Acinetobacter baumannii are an emerging threat to human health and further complicate the general problem of treating serious bacterial infections. Meeting this challenge requires an improved understanding of the relationships between the structures of major therapeutic targets in this organism and the activity levels exhibited against it by different antibiotics. Here we report the first crystal structures of A. baumannii penicillin-binding proteins (PBPs) covalently inactivated by four ß-lactam antibiotics. We also relate the results to kinetic, biophysical, and computational data. The structure of the class A protein PBP1a was solved in apo form and for its covalent conjugates with benzyl penicillin, imipenem, aztreonam, and the siderophore-conjugated monocarbam MC-1. It included a novel domain genetically spliced into a surface loop of the transpeptidase domain that contains three conserved loops. Also reported here is the first high-resolution structure of the A. baumannii class B enzyme PBP3 in apo form. Comparison of this structure with that of MC-1-derivatized PBP3 of Pseudomonas aeruginosa identified differences between these orthologous proteins in A. baumannii and P. aeruginosa. Thermodynamic analyses indicated that desolvation effects in the PBP3 ligand-binding sites contributed significantly to the thermal stability of the enzyme-antibiotic covalent complexes. Across a significant range of values, they correlated well with results from studies of inactivation kinetics and the protein structures. The structural, biophysical, and computational data help rationalize differences in the functional performance of antibiotics against different protein targets and can be used to guide the design of future agents.

Evaluation of a Novel Light Scattering Methodology for the Detection of Pathogenic Bacteria in Urine.

BACKGROUND: Urine culture, the gold standard for detecting and identifying bacteria in urine, is one of the highest volume tests in many microbiology laboratories. The inability to accurately predict which patients would benefit from culture leads not only to monopolization of laboratory resources, but also to unnecessary antimicrobial exposure as patients receive empirical treatment for suspected or presumed urinary tract infections (UTI) while awaiting culture results. A common approach to decrease unnecessary urine culture is screening samples using urinalysis (UA) parameters to determine those that should proceed to culture (reflex). In this study, we compared the performance of a novel uropathogen detection method to urinalysis for purposes of UTI screening. METHODS: Urine specimens submitted for culture (n = 194) were evaluated by urinalysis and a novel light scattering device (BacterioScan 216Dx UTI System) capable of detecting the presence of bacteria in urine. Sensitivity and specificity for prediction of a positive urine culture by UA and 216Dx were determined relative to urine culture results. A positive urine culture was defined as growth in culture of one or two uropathogens at concentrations of ≥50,000 CFU/mL. RESULTS: 194 urine samples were evaluated by UA, 216Dx, and urine culture. The 216Dx demonstrated a 100% [95%CI: 88.43%-100.0%] sensitivity and 81.71% [95%CI: 74.93%-87.30%] specificity for the detection of bacteriuria, vs UA with a sensitivity of 86.67% [95%CI 69.28%-96.24%] and specificity of 71.95% [95%CI: 64.41%-78.68%] when compared to urine culture (diagnostic reference method). CONCLUSIONS: BacterioScan allows for an alternative method of screening with satisfactory sensitivity and improved specificity that may facilitate a reduction of unnecessary cultures. Additional studies are required to determine if a concomitant decrease in inappropriate antibiotic use can be realized with the 216Dx technology.

The Acinetobacter baumannii 19606 OmpA protein plays a role in biofilm formation on abiotic surfaces and in the interaction of this pathogen with eukaryotic cells.

The ability of Acinetobacter baumannii to adhere to and persist on surfaces as biofilms could be central to its pathogenicity. The production of pili and a biofilm-associated protein and the expression of antibiotic resistance are needed for robust biofilm formation on abiotic and biotic surfaces. This multistep process also depends on the expression of transcriptional regulatory functions, some of which could sense nutrients available to cells. This report extends previous observations by showing that although outer membrane protein A (OmpA) of A. baumannii 19606 plays a partial role in the development of robust biofilms on plastic, it is essential for bacterial attachment to Candida albicans filaments and A549 human alveolar epithelial cells. In contrast to abiotic surfaces, the interaction with biotic surfaces is independent of the CsuA/BABCDE-mediated pili. The interaction of A. baumannii 19606 with fungal and epithelial cells also results in their apoptotic death, a response that depends on the direct contact of bacteria with these two types of eukaryotic cells. Furthermore, the bacterial adhesion phenotype correlates with the ability of bacteria to invade A549 epithelial cells. Interestingly, the killing activity of cell-free culture supernatants proved to be protease and temperature sensitive, suggesting that its cytotoxic activity is due to secreted proteins, some of which are different from OmpA.

Iron acquisition functions expressed by the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is a gram-negative bacterium that causes serious infections in compromised patients. More recently, it has emerged as the causative agent of severe infections in military personnel wounded in Iraq and Afghanistan. This pathogen grows under a wide range of conditions including iron-limiting conditions imposed by natural and synthetic iron chelators. Initial studies using the type strain 19606 showed that the iron proficiency of this pathogen depends on the expression of the acinetobactin-mediated iron acquisition system. More recently, we have observed that hemin but not human hemoglobin serves as an iron source when 19606 isogenic derivatives affected in acinetobactin transport and biosynthesis were cultured under iron-limiting conditions. This finding is in agreement with the observation that the genome of the strain 17978 has a gene cluster coding for putative hemin-acquisition functions, which include genes coding for putative hemin utilization functions and a TonBExbBD energy transducing system. This system restored enterobactin biosynthesis in an E. coli ExbBD deficient strain but not when introduced into a TonB mutant. PCR and Southern blot analyses showed that this hemin-utilization gene cluster is also present in the 19606 strain. Analysis of the 17978 genome also showed that this strain harbors genes required for acinetobactin synthesis and transport as well as a gene cluster that could code for additional iron acquisition functions. This hypothesis is in agreement with the fact that the inactivation of the basD acinetobactin biosynthetic gene did not affect the growth of A. baumannii 17978 cells under iron-chelated conditions. Interestingly, this second iron uptake gene cluster is flanked by perfect inverted repeats and includes transposase genes that are expressed transcriptionally. Also interesting is the observation that this additional cluster could not be detected in the type strain 19606, an observation that suggests some significant differences in the iron uptake capacity between these two A. baumannii strains. Transposome mutagenesis of the strain 19606 resulted in the isolation of a derivative unable to grow under iron-chelated conditions. Gene mapping and protein analysis together with complementation assays showed that a protein related to SecA, which is a component of the Sec protein secretion system in a wide range of bacteria, is needed at least for the production of the BauA acinetobactin outer membrane receptor. Furthermore, this derivative was unable to use hemin as an iron source under limiting conditions. Taken together, these results indicate that A. baumannii expresses siderophore-mediated and hemin acquisition functions, although different isolates differ in their iron acquisition capacity. Unexpectedly, the ability of this pathogen to acquire iron depends on the expression of a SecA protein secretion function, which has not been associated with iron acquisition in bacteria.

Pseudomonas aeruginosa twitching motility-mediated chemotaxis towards phospholipids and fatty acids: specificity and metabolic requirements.

Pseudomonas aeruginosa demonstrates type IV pilus-mediated directional twitching motility up a gradient of phosphatidylethanolamine (PE). Only one of four extracellular phospholipases C of P. aeruginosa (i.e., PlcB), while not required for twitching motility per se, is required for twitching-mediated migration up a gradient of PE or phosphatidylcholine. Whether other lipid metabolism genes are associated with this behavior was assessed by analysis of transcription during twitching up a PE gradient in comparison to transcription during twitching in the absence of any externally applied phospholipid. Data support the hypothesis that PE is further degraded and that the long-chain fatty acid (LCFA) moieties of PE are completely metabolized via beta-oxidation and the glyoxylate shunt. It was discovered that P. aeruginosa exhibits twitching-mediated chemotaxis toward unsaturated LCFAs (e.g., oleic acid), but not saturated LCFAs (e.g., stearic acid) of corresponding lengths. Analysis of mutants that are deficient in glyoxylate shunt enzymes, specifically isocitrate lyase (DeltaaceA) and malate synthase (DeltaaceB), suggested that the complete metabolism of LCFAs through this pathway was required for the migration of P. aeruginosa up a gradient of PE or unsaturated LCFAs. At this point, our data suggested that this process should be classified as energy taxis. However, further evaluation of the ability of the DeltaaceA and DeltaaceB mutants to migrate up a gradient of PE or unsaturated LCFAs in the presence of an alternative energy source clearly indicated that metabolism of LCFAs for energy is not required for chemotaxis toward these compounds.

Translational deficiencies in antibacterial discovery and new screening paradigms.

An impending disaster is currently developing in the infectious disease community: the combination of rapidly emerging multidrug-resistance among clinically relevant bacterial pathogens, together with an unprecedented withdrawal from industrial dedication to this disease area, is jeopardizing human health on a societal level. For those who remain focused and dedicated to identifying solutions to this growing problem, additional challenges await when in vitro activity does not correlate with in vivo efficacy. Thus the development of more effective translational assays will greatly improve and streamline the process of identifying novel antibacterial agents that can stand the test of preclinical and clinical development. Here we describe recent examples of research that justify the need for such assays.

Discovery and characterization of a novel class of pyrazolopyrimidinedione tRNA synthesis inhibitors.

A high-throughput phenotypic screen for novel antibacterial agents led to the discovery of a novel pyrazolopyrimidinedione, PPD-1, with preferential activity against methicillin-resistant Staphylococcus aureus (MRSA). Resistance mapping revealed the likely target of inhibition to be lysyl tRNA synthetase (LysRS). Preliminary structure-activity relationship (SAR) studies led to an analog, PPD-2, which gained Gram-negative antibacterial activity at the expense of MRSA activity and resistance to this compound mapped to prolyl tRNA synthetase (ProRS). These targets of inhibition were confirmed in vitro, with PPD-1 showing IC50s of 21.7 and 35 µM in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 µM, respectively. The highly attractive chemical properties of these compounds combined with intriguing preliminary SAR suggest that further exploration of this compelling novel series is warranted.

LpxC inhibitors as new antibacterial agents and tools for studying regulation of lipid A biosynthesis in Gram-negative pathogens.

UNLABELLED: The problem of multidrug resistance in serious Gram-negative bacterial pathogens has escalated so severely that new cellular targets and pathways need to be exploited to avoid many of the preexisting antibiotic resistance mechanisms that are rapidly disseminating to new strains. The discovery of small-molecule inhibitors of LpxC, the enzyme responsible for the first committed step in the biosynthesis of lipid A, represents a clinically unprecedented strategy to specifically act against Gram-negative organisms such as Pseudomonas aeruginosa and members of the Enterobacteriaceae. In this report, we describe the microbiological characterization of LpxC-4, a recently disclosed inhibitor of this bacterial target, and demonstrate that its spectrum of activity extends to several of the pathogenic species that are most threatening to human health today. We also show that spontaneous generation of LpxC-4 resistance occurs at frequencies comparable to those seen with marketed antibiotics, and we provide an in-depth analysis of the mechanisms of resistance utilized by target pathogens. Interestingly, these isolates also served as tools to further our understanding of the regulation of lipid A biosynthesis and enabled the discovery that this process occurs very distinctly between P. aeruginosa and members of the Enterobacteriaceae. Finally, we demonstrate that LpxC-4 is efficacious in vivo against multiple strains in different models of bacterial infection and that the major first-step resistance mechanisms employed by the intended target organisms can still be effectively treated with this new inhibitor. IMPORTANCE: New antibiotics are needed for the effective treatment of serious infections caused by Gram-negative pathogens, and the responsibility of identifying new drug candidates rests squarely on the shoulders of the infectious disease community. The limited number of validated cellular targets and approaches, along with the increasing amount of antibiotic resistance that is spreading throughout the clinical environment, has prompted us to explore the utility of inhibitors of novel targets and pathways in these resistant organisms, since preexisting target-based resistance should be negligible. Lipid A biosynthesis is an essential process for the formation of lipopolysaccharide, which is a critical component of the Gram-negative outer membrane. In this report, we describe the in vitro and in vivo characterization of novel inhibitors of LpxC, an enzyme whose activity is required for proper lipid A biosynthesis, and demonstrate that our lead compound has the requisite attributes to warrant further consideration as a novel antibiotic.

Siderophore receptor-mediated uptake of lactivicin analogues in gram-negative bacteria.

Multidrug-resistant Gram-negative pathogens are an emerging threat to human health, and addressing this challenge will require development of new antibacterial agents. This can be achieved through an improved molecular understanding of drug-target interactions combined with enhanced delivery of these agents to the site of action. Herein we describe the first application of siderophore receptor-mediated drug uptake of lactivicin analogues as a strategy that enables the development of novel antibacterial agents against clinically relevant Gram-negative bacteria. We report the first crystal structures of several sideromimic conjugated compounds bound to penicillin binding proteins PBP3 and PBP1a from Pseudomonas aeruginosa and characterize the reactivity of lactivicin and ß-lactam core structures. Results from drug sensitivity studies with ß-lactamase enzymes are presented, as well as a structure-based hypothesis to reduce susceptibility to this enzyme class. Finally, mechanistic studies demonstrating that sideromimic modification alters the drug uptake process are discussed.

Discovery of Dap-3 polymyxin analogues for the treatment of multidrug-resistant Gram-negative nosocomial infections.

We report novel polymyxin analogues with improved antibacterial in vitro potency against polymyxin resistant recent clinical isolates of Acinetobacter baumannii and Pseudomonas aeruginosa . In addition, a human renal cell in vitro assay (hRPTEC) was used to inform structure-toxicity relationships and further differentiate analogues. Replacement of the Dab-3 residue with a Dap-3 in combination with a relatively polar 6-oxo-1-phenyl-1,6-dihydropyridine-3-carbonyl side chain as a fatty acyl replacement yielded analogue 5x, which demonstrated an improved in vitro antimicrobial and renal cytotoxicity profiles relative to polymyxin B (PMB). However, in vivo PK/PD comparison of 5x and PMB in a murine neutropenic thigh model against P. aeruginosa strains with matched MICs showed that 5x was inferior to PMB in vivo, suggesting a lack of improved therapeutic index in spite of apparent in vitro advantages.