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.

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.

Staphylococcus aureus and Pseudomonas aeruginosa are two of the most common causes of bacterial keratitis and corresponding corneal blindness. Accordingly, such infections are predominantly treated with broad-spectrum fluoroquinolones, such as moxifloxacin. Yet, the rising fluoroquinolone resistance has necessitated the development of alternative therapeutic options. Herein, we describe the development of a polymyxin B-trimethoprim (PT) ophthalmic formulation containing the antibiotic rifampin, which exhibits synergistic antimicrobial activity toward a panel of contemporary ocular clinical S. aureus and P. aeruginosa isolates, low spontaneous resistance frequency, and in vitro bactericidal kinetics and antibiofilm activities equaling or exceeding the antimicrobial properties of moxifloxacin. The PT plus rifampin combination also demonstrated increased efficacy in comparison to those of either commercial PT or moxifloxacin in a murine keratitis model of infection, resulting in bacterial clearance of 70% in the animals treated. These results suggest that the combination of PT and rifampin may represent a novel antimicrobial agent in the treatment of bacterial keratitis.

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.

19 Preliminary enthusiasm over the encouraging spectrum and in vitro activity of siderophore conjugates 20 such as MB-1 has recently been tempered by unexpected variability in in vivo efficacy. The need for 21 these conjugates to compete for iron with endogenously-produced siderophores has exposed a 22 significant liability for this novel antibacterial strategy. Here, we have exploited dependence on efflux 23 for siderophore secretion in Pseudomonas aeruginosa, and provide evidence that efflux inhibition could 24 circumvent this in vivo-relevant resistance liability. 25 on S etem er 2, 2017 by gest httpaac.asm .rg/ D ow nladed fom

• Antibiotic resistance levels have reached critically high levels. • Antibiotic discovery efforts have reached critically low levels. • Governments are attempting to incentivize reinvestment in antibiotic development. • Federal funding of antibacterial research is dwarfed by that of other disease areas. Antibiotics have been the most transformative medicines in the history of human health. Although once considered a mainstay in the pipeline of most major players in the pharmaceutical industry, new antibiotic discovery and development efforts have been minimalized as a consequence of challenges attributed to the identification of new chemical matter, the increasing complexity of stringent regulatory requirements, and the dwindling economic incentives that justify the early-stage investments. Catastrophically, this discovery void comes at a time when antibiotic resistance emergence and spread has reached unprecedented levels, fueling the clinical desperation for novel molecules that are not impacted by relevant resistance mechanisms. This review will discuss recent economic and regulatory attempts to revitalize the antibiotic discovery space.

•Antibiotic resistance levels have reached critically high levels.•Antibiotic discovery efforts have reached critically low levels.•Governments are attempting to incentivize reinvestment in antibiotic development.•Federal funding of antibacterial research is dwarfed by that of other disease areas.

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.

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.

ABSTRACT 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.

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 β-lactam core structures. Results from drug sensitivity studies with β-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.

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 IC 50 s of 21.7 and 35 μ M in purified LysRS and ProRS enzyme assays, and PPD-2, 151 and 0.04 μ M , 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.

ABSTRACT 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.

ABSTRACT 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 .

Herein we describe the structure-aided design and synthesis of a series of pyridone-conjugated monobactam analogues with in vitro antibacterial activity against clinically relevant Gram-negative species including Pseudomonas aeruginosa , Klebsiella pneumoniae , and Escherichia coli . Rat pharmacokinetic studies with compound 17 demonstrate low clearance and low plasma protein binding. In addition, evidence is provided for a number of analogues suggesting that the siderophore receptors PiuA and PirA play a role in drug uptake in P. aeruginosa strain PAO1.

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.