Bioengineered Lysozyme Reduces Bacterial Burden and Inflammation in a Murine Model of Mucoid Pseudomonas aeruginosa Lung Infection

The spread of drug-resistant bacterial pathogens is a growing global concern and has prompted an effort to explore potential adjuvant and alternative therapies derived from nature''s repertoire of bactericidal proteins and peptides. In humans, the airway surface liquid layer is a rich source of antibiotics, and lysozyme represents one of the most abundant and effective antimicrobial components of airway secretions. Human lysozyme is active against both Gram-positive and Gram-negative bacteria, acting through several mechanisms, including catalytic degradation of cell wall peptidoglycan and subsequent bacterial lysis. In the infected lung, however, lysozyme''s dense cationic character can result in sequestration and inhibition by polyanions associated with airway inflammation. As a result, the efficacy of the native enzyme may be compromised in the infected and inflamed lung. To address this limitation, we previously constructed a charge-engineered variant of human lysozyme that was less prone to electrostatic-mediated inhibition in vitro. Here, we employ a murine model to show that this engineered enzyme is superior to wild-type human lysozyme as a treatment for mucoid Pseudomonas aeruginosa lung infections. The engineered enzyme effectively decreases the bacterial burden and reduces markers of inflammation and lung injury. Importantly, we found no evidence of acute toxicity or allergic hypersensitivity upon repeated administration of the engineered biotherapeutic. Thus, the charge-engineered lysozyme represents an interesting therapeutic candidate for P. aeruginosa lung infections.     

In silico optimization of targeted aerosol delivery in upper airways via Inhaled Volume Tracking

BackgroundDespite the widespread use of aerosol inhalation as a drug delivery method, targeted delivery to the upper airways remains an ongoing challenge in the quest for improved clinical response in respiratory disease.MethodsHere, we examine in silico flow and particle dynamics when using an oral Inhaled Volume Tracking manoeuvre. A short pulsed aerosol bolus is injected during slow inhalation flow rates followed by clean air, and a breath-hold is initiated once it reaches the desired depth. We explore the fate of a broad particle size range (1–40 μm) for both upright and supine positions.FindingsOur findings illustrate that despite attempts to mitigate dispersion using slower flow rates, the laryngeal jet disperses the aerosol bolus and thus remains a hurdle for efficient targeted delivery. Nevertheless, we show a decrease in extra-thoracic deposition; large aerosols in the range of 10–30 μm potentially outperform existing inhalation methods, showing deposition fractions of up to 80% in an upright orientation.InterpretationThe improved deposition during Inhaled Volume Tracking shows promise for clinical applications and could be leveraged to deliver larger payloads to the upper airways.     

Recent advances in the potential interconnection between antimicrobial resistance to biocides and antibiotics

Workshop on biocides: do they select for antimicrobial resistance?

Second International Conference on Antimicrobial Research

Lisbon, Portugal, 21–23 November 2012.

Interconnection between microbial resistance to biocides and antibiotics is a topic of increasing interest given the recent changes in European legislation and claims of a risk of biocide use on bacterial resistance. In the second International Conference on Antimicrobial Research held in Lisbon in November 2012, a workshop specifically addressed this topic, presentations included approaches to risk assessment and investigations into the molecular mechanisms of biocide resistance and co- and cross-resistance to antibiotics. The overall conclusion was that, even if each biocide represents a specific case, there is scientific evidence that biocides select for biocide resistance, but that there is, so far, no conclusive evidence that this also determined or will determine an increase in antibiotic resistance.     

Iclaprim: a differentiated option for the treatment of skin and skin structure infections

ABSTRACT

Introduction: Iclaprim is a selective bacterial dihydrofolate reductase (DHFR) inhibitor. Although there are alternative options for the treatment of acute bacterial skin and skin structure infections (ABSSSI), iclaprim is differentiated from other available antibiotics.

Areas covered: Iclaprim is under clinical development for ABSSSI. This review summarizes the mechanism of action, pharmacokinetics, microbiology, clinical development program, and the differentiation of iclaprim from other antibiotics.

Expert commentary: Iclaprim has a different mechanism of action (DHFR inhibitor) compared to most other antibiotics, is active and rapidly bactericidal against Gram-positive pathogens including antibiotic-resistant pathogens, and suppresses bacterial exotoxins (alpha hemolysin, Panton Valentine leukocidin, and toxic shock syndrome toxin-1). Compared to trimethoprim, iclaprim has lower MIC_90s, can be given without a sulfonamide, overcomes select trimethoprim resistance, and does not cause hyperkalemia. Iclaprim is administered as a fixed dose, does not require dose adjustment in renally-impaired or obese patients, and was not associated with nephrotoxicity in the Phase 3 pivotal REVIVE studies. Iclaprim represents a novel, alternative option for the treatment of severe skin and skin structure infections due to Gram-positive bacteria, particularly in patients at risk of acute kidney injury.     

Structure-activity relationships of antimicrobial and lipoteichoic acid-sequestering properties in polyamine sulfonamides

We have recently confirmed that lipoteichoic acid (LTA), a major constituent of the gram-positive bacterial surface, is the endotoxin of gram-positive bacteria that induces proinflammatory molecules in a Toll-like receptor 2 (TLR2)-dependent manner. LTA is an anionic amphipath whose physicochemical properties are similar to those of lipopolysaccharide (LPS), which is found on the outer leaflet of the outer membranes of gram-negative organisms. Hypothesizing that compounds that sequester LPS could also bind to and inhibit LTA-induced cellular activation, we screened congeneric series of polyamine sulfonamides which we had previously shown effectively neutralized LPS both in vitro and in animal models of endotoxemia. We observed that these compounds do bind to and neutralize LTA, as reflected by the inhibition of TLR2-mediated NF-κB induction in reporter gene assays. Structure-activity studies showed a clear dependence of the acyl chain length on activity against LTA in compounds with spermine and homospermine scaffolds. We then sought to examine possible correlations between the neutralizing potency toward LTA and antimicrobial activity in Staphylococcus aureus. A linear relationship between LTA sequestration activity and antimicrobial activity for compounds with a spermine backbone was observed, while all compounds with a homospermine backbone were equally active against S. aureus, regardless of their neutralizing potency toward LTA. These results suggest that the number of protonatable charges is a key determinant of the activity toward the membranes of gram-positive bacteria. The development of resistance to membrane-active antibiotics has been relatively slower than that to conventional antibiotics, and it is possible that compounds such as the acylpolyamines may be useful clinically, provided that they have an acceptable safety profile and margin of safety. A more detailed understanding of the mechanisms of interactions of these compounds with LPS and LTA, as well as the gram-negative and -positive bacterial cell surfaces, will be instructive and should allow the rational design of analogues which combine antisepsis and antibacterial properties.     

Carbapenem resistance in Acinetobacter baumannii: laboratory challenges,mechanistic insights and therapeutic strategies

Unprecedented levels of antimicrobial resistance in bacterial isolates have prompted great concerns globally. In 2012 the WHO released a publication outlining the evolving threat of antimicrobial resistance in order to raise awareness and to stimulate coordinated international efforts. The carbapenem class of antibiotics is largely considered as an antibiotic of last-resort when treating infections. Now carbapenem resistance further limits treatment options. In this article the authors discuss carbapenem resistance in Acinetobacter baumannii, a bacterial isolate often implicated in nosocomial infections. Virulence factors, intrinsic and acquired resistance mechanisms, together with laboratory challenges in the detection and antibiotic susceptibility testing of A. baumannii make this a truly problematic isolate. Therapeutic options are exceedingly limited, relying on polymyxins in combinations with other antibiotics, with few, if any, new active agents in the pipeline.     

Mucosal biofilms: challenges and future directions

Francisella tularensis is a category A bioterrorism agent. It is the etiological agent of tularemia, a zoonotic disease found throughout the northern hemisphere. The intentional spread of F. tularensis aerosols would probably lead to severe and often fatal pneumonia cases, but also secondary cases from contaminated animals and environments. We are not ready to face such a situation. No vaccine is currently available. A few antibiotics are active against F. tularensis, but strains resistant to these antibiotics could be used in the context of bioterrorism. We need new therapeutic strategies to fight against category A bioterrorism agents, including development of new drugs inhibiting F. tularensis growth and/or virulence, or enhancing the host response to infection by this pathogen.     

Efficacy of Aerosol MP-376, a Levofloxacin Inhalation Solution,in Models of Mouse Lung Infection Due to Pseudomonas aeruginosa

Progressive respiratory failure due to Pseudomonas aeruginosa is the leading cause of morbidity and mortality in patients with cystic fibrosis. The pulmonary delivery of antimicrobial agents provides high concentrations of drug directly to the site of infection and attains pharmacokinetic-pharmacodynamic indices exceeding those which can be achieved with systemic dosing. MP-376 is a new formulation of levofloxacin that enables the safe aerosol delivery of high concentrations of drug to pulmonary tissues. In vivo studies were conducted to demonstrate the efficacy of MP-376 in models of mouse pulmonary infection. The superiority of aerosol dosing over systemic dosing was demonstrated in models of both acute and chronic lung infection. In a model of acute lung infection, aerosol treatment with MP-376 once or twice daily reduced the lung bacterial load to a greater extent than aerosol tobramycin or aztreonam did when they were administered at similar or higher doses. The bacterial killing by aerosol MP-376 observed in the lung in the model of acute pulmonary infection translated to improved survival (P < 0.05). In a model of chronic pulmonary infection, aerosol MP-376 had antimicrobial effects superior to those of aztreonam (P < 0.05) and effects similar to those of tobramycin (P > 0.05). In summary, these data show that aerosol MP-376 has in vivo activity when it is used to treat acute and chronic lung infections caused by P. aeruginosa.Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, remains an important cause of pulmonary infection in patients with cystic fibrosis (CF). A majority of patients with CF become infected with P. aeruginosa by the age of 18 years (5, 11). Chronic infection with P. aeruginosa is associated with morbidity and mortality in patients with CF (7, 8, 14, 17). In addition to chronic infection with P. aeruginosa in patients with CF, recent studies have also demonstrated an important role of chronic infection with P. aeruginosa in patients with chronic obstructive pulmonary disease (COPD) (16, 20).Since achievement of an adequate antimicrobial concentration is a requirement for the successful treatment of chronic pulmonary infections, aerosol antibiotic therapy has become a valuable approach to treatment in patients with CF. Compared to systemic dosing, aerosol treatment provides higher pulmonary concentrations of antibiotics and reduces the systemic level of exposure to the drug (3, 6, 27). Tobramycin inhalation solution is currently the only aerosol antibiotic approved for use for the treatment of bacterial infections in patients with CF. With the aerosol administration of tobramycin, the potential for systemic toxicity is reduced and a clinical benefit has been shown over several cycles of treatment (23); its long-term use, however, has been associated with multiple-antibiotic-resistant P. aeruginosa strains (19, 27). Thus, there is a need for the development of different classes of aerosol antibiotics for the treatment of chronic lung infections in patients with CF.The fluoroquinolone levofloxacin has potent activity against key pathogens in patients with CF, including P. aeruginosa, with no loss of potency in CF sputum (13). Pharmacokinetic (PK)-pharmacodynamic (PD) studies with systemically administered levofloxacin have shown that bactericidal activity and clinical efficacy are linked to the area under the concentration-time curve (AUC)/MIC and maximum concentration of drug in plasma (C_max)/MIC ratios (9, 12, 22). In addition, both in vitro and in vivo studies have shown that high levels of exposure (C_max) relative to the MIC can reduce the rate of selection of drug-resistant bacteria in vivo (12, 13).Since aerosol administration of antibiotics produces drug levels in pulmonary tissues that are higher than those that can be produced with systemic dosing and that these increased local levels are associated with improved efficacy, a novel aerosol formulation of levofloxacin, MP-376, was developed. MP-376 is a levofloxacin solution formulated with divalent cations and permeant ions for inhalational use. The purpose of the studies described here was to determine the efficacy of aerosol MP-376 in models of acute and chronic lung infection due to P. aeruginosa.(This work was presented in part at the 21st and 22nd Annual North American Cystic Fibrosis Conferences, October 2007 and 2008, respectively [25, 26].)     

Characteristics and antimicrobial choice of pediatric bacterial enteritis in the Kanto region of Japan: A multicenter retrospective observational study

IntroductionThere are few reports on the causative microorganisms of bacterial enteritis in children in Japan in recent years. The distribution of causative microorganisms is important for estimating pathogens and making decisions regarding the treatment plan, as antimicrobial agents are not required for mild bacterial enteritis cases but are used for severe cases or immunocompromised patients.MethodsWe retrospectively surveyed pediatric patients who underwent stool culture at eight hospitals in the Kanto region of Japan from 2014 to 2019 for patient characteristics, causative microorganisms, and prescribed antimicrobial agents.ResultsA total of 4,475 stool cultures were submitted, and the positivity rate for bacterial enteritis was 11%. The causative microorganisms were Campylobacter spp. in 338 cases (67.3%), Salmonella spp. in 85 cases (16.9%), enterohemorrhagic Escherichia coli O157 in 23 cases (4.6%), and Yersinia spp. in 45 cases (9.0%). Hospitals with pediatric infectious disease physicians had a lower rate of antimicrobial therapy for Campylobacter enteritis than hospitals without pediatric infectious disease physicians.ConclusionsCampylobacter spp. are the most common causative agent for bacterial enteritis in this study, and the presence of pediatric infectious disease physicians may promote the appropriate use of antimicrobial agents.     

Nurse Practitioners' Attitudes,Perceptions, and Knowledge About Antimicrobial Stewardship

BackgroundAntimicrobial resistance is an urgent public health problem. There is very limited information regarding nurse practitioners' attitudes, perceptions, and knowledge about antibiotic use and resistance.MethodsWeb-based anonymous survey in a university-affiliated hospital.ResultsMost respondents agreed that antimicrobial resistance is a problem locally and nationally, were concerned about resistance in the community when prescribing antibiotics, and agreed that more appropriate use of antibiotics would decrease resistance. There is a paucity of knowledge in the management of anaerobic infections and resistant gram-negative bacteremia.ConclusionIncorporating nurse practitioners into antimicrobial stewardship programs could improve evidence-based practices and antimicrobial use.     

Bactericidal Activity of Ceragenin CSA-13 in Cell Culture and in an Animal Model of Peritoneal Infection

Ceragenins constitute a novel family of cationic antibiotics characterized by a broad spectrum of antimicrobial activities, which have mostly been assessed in vitro. Using a polarized human lung epithelial cell culture system, we evaluated the antibacterial activities of the ceragenin CSA-13 against two strains of Pseudomonas aeruginosa (PAO1 and Xen5). Additionally, the biodistribution and bactericidal activity of a CSA-13–IRDye 800CW derivate were assessed using an animal model of peritoneal infection after PAO1 challenge. In cell culture, CSA-13 bactericidal activities against PAO1 and Xen5 were higher than the activities of the human cathelicidin peptide LL-37. Increased CSA-13 activity was observed in polarized human lung epithelial cell cultures subjected to butyric acid treatment, which is known to increase endogenous LL-37 production. Eight hours after intravenous or intraperitoneal injection, the greatest CSA-13–IRDye 800CW accumulation was observed in mouse liver and kidneys. CSA-13–IRDye 800CW administration resulted in decreased bacterial outgrowth from abdominal fluid collected from animals subjected to intraperitoneal PAO1 infection. These observations indicate that CSA-13 may synergistically interact with antibacterial factors that are naturally present at mucosal surfaces and it maintains its antibacterial activity in the infected abdominal cavity. Cationic lipids such as CSA-13 represent excellent candidates for the development of new antibacterial compounds.     

Intratracheal catheters as drug delivery systems

Medication delivery into the lungs can be used to provide a high therapeutic index for agents targeted to specific lung diseases. In addition, the lung can be used as a portal of entry for other agents targeted to systemic diseases. Delivery of medications into the lung can be accomplished by either instillation or aerosolization. Instillation approaches are limited by the fluid volume that can be given safely, and instilled liquids distribute according to gravity. In contrast, aerosolization approaches can deliver larger volumes over longer periods and aerosols distribute according to ventilation. In the mechanically ventilated patient, externally generated aerosols have very poor lung delivery because the endotracheal tube functions as a barrier to aerosol passage. Novel aerosol generating systems at the ends of small-diameter catheters that can be placed into the trachea (or beyond) are being developed to address this. In vitro testing has shown these systems to be capable of producing appropriately sized particles, with high rates of lung deposition. These catheters could be coupled with tracheal gas insufflation systems, not only to deliver therapeutic aerosols but also to create water aerosols to supply necessary humidification during tracheal gas insufflation.     

Nouveautés et perspectives thérapeutiques des pneumonies acquises sous ventilation mécanique à Pseudomonas aeruginosa

Pseudomonas aeruginosa is frequently responsible for hospital-acquired pneumonia, especially in intensive care unit (ICU) patients. A foreseeable increase in antibiotic resistance, evolving faster than development of new antibiotics, favors the high morbidity and mortality of these infections. While former molecules are reemerging due to their sustained efficacy like colistin, new antibiotics, new inhibitors of resistance as well as topical antibiotics by aerosol are currently developed to improve the available therapeutic options. Innovative research has emerged to develop alternatives for prevention and treatment of P. aeruginosa pneumonia. Antimicrobial peptides, carbon monoxidereleasing molecules, and pyocins should widen the field of antimicrobial molecules. New treatments aim to modulate P. aeruginosa phenotype by inhibiting bacterial adhesion and communication including macrolides to inhibit the quorum-sensing. Moreover, rediscovery of lytic bacteriophages, which are specific viruses inducing bacterial lysis, is recent in Western countries despite large-scale use in Eastern Europe following the Second World War. Bacteriophages may be suitable for the treatment of ventilator-associated pneumonia, especially if related to multidrug resistant P. aeruginosa. Finally, epidemiological and experimental data may lead us to focus on the role played by fungal colonization in the pathogenesis of bacterial pneumonia.     

Comparative in vitro efficacies and antimicrobial durabilities of novel antimicrobial central venous catheters

We investigated the efficacies and durability of novel antimicrobial central venous catheters (CVCs) in preventing the adherence of microbial organisms to the surfaces of the CVCs. Novel antimicrobial CVCs investigated in this in vitro study were impregnated with antibiotics (minocycline and rifampin), with Oligon agent (silver, platinum, and carbon black), with approved antiseptics (chlorhexidine and silver sulfadiazine), or with a novel antiseptic agent, gendine, which contains gentian violet and chlorhexidine. When tested against methicillin-resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa, gendine-coated CVC segments provided protection against bacterial adherence significantly more than all other types of tested CVCs (P < 0.05). Gendine-coated CVCs also provided better protection against Candida albicans and Candida parapsilosis than CVCs impregnated with antibiotics or with silver, platinum, and carbon (P < 0.02). After 28 days of being soaked in serum, the CVCs impregnated with chlorhexidine and silver sulfadiazine and the CVCs impregnated with silver, platinum, and carbon had lost antimicrobial activity against MRSA, P. aeruginosa, and C. parapsilosis, and the CVCs impregnated with minocycline and rifampin had lost activity against P. aeruginosa and C. parapsilosis. The CVCs impregnated with gendine maintained antimicrobial activities against MRSA, P. aeruginosa, and C. parapsilosis after 28 days of being soaked in serum. Central venous catheters impregnated with the novel investigational antiseptic gendine showed in vitro efficacy and provided protection against bacterial adherence more than other approved novel antimicrobial-coated CVCs.     

An overview of antimicrobial peptides and the latest advances in their development

Introduction: The recent dramatic increase in the incidence of antimicrobial resistance has been recognized by organizations such as the United Nations and World Health Organization as well as the governments of the USA and several European countries. A relatively new weapon in the fight against severe infections caused by multi-drug resistant bacteria is antimicrobial peptides (AMPs). These include colistin, currently regarded as the last line of antimicrobial therapy against multi-drug resistant microorganisms.

Areas covered: Here, the authors provide an overview of the current research on AMPs. The focus is AMPs currently being developed for the treatment of recalcitrant bacterial infections, the synergies of AMPs and antibiotics, and the activity of AMPs against biofilm. This review also includes a brief introduction into the use of AMPs in infections caused by Mycobacterium, fungi, and parasites.

Expert opinion: In research into new antimicrobials, AMPs are gaining increasing attention. While many are natural and are produced by a wide variety of organisms, others are being newly designed and chemically synthesized in the laboratory to achieve novel antimicrobial agents. The same strategy to fight infections in nature is thus being effectively exploited to safeguard human and animal health.     

Empirical antibiotic choice alters microbiological outcomes: Findings from comparative antibiograms in a trauma intensive care unit

Background Inappropriate empirical antibiotics promote antibiotic resistance. Antibiograms guide empirical antibiotic therapy by outlining the percentage susceptibility of each pathogen to individual antibiotics. In 2016, the Trauma Intensive Care Unit at Charlotte Maxeke Johannesburg Academic Hospital escalated empirical antibiotic therapy for nosocomial infections from piperacillin-tazobactam to imipenem plus amikacin. Objectives This study assessed the impact of escalation in empirical antimicrobial treatment on organism prevalence and resistance profile. Methods A retrospective analysis of bacterial and fungal microscopy, culture and susceptibility reports from the laboratory information system of the National Health Laboratory Services, from 1 January 2015 to 31 December 2015 and 1 January 2017 to 31 December 2017, was conducted. Data were de-duplicated according to standard guidelines. Fisher’s exact test was used to determine p-values. Results Organism prevalence shifted between the years, with a 2.7% increase in streptococci (p=0.0199), 1.7% increase in Candida auris (p=0.0031) and 4.6% and 4.4% reduction in Acinetobacter baumannii (p=0.0508) and Pseudomonas aeruginosa (p=0.0196), respectively. Similarly, there was a change in the resistance profile, with a 28.9% reduction in multi-drug resistant (MDR) A. baumannii (p=0.0001), 60.4% reduction in MDR P. aeruginosa (p=0.0001) and a 6.5% increase in carbapenem-resistant Enterobacterales (p=0.007). The predominant specimen type differed between the years, with significantly more pus, tissue and fluid samples and fewer respiratory samples sent for investigation in 2017 than 2015. Conclusion Escalation in the use of empirical antibiotics showed a change in organism prevalence and an improvement in the susceptibility profile of MDR non-fermenters. Contributions of the study Current literature on the effects of antibiogram-guided empirical antibiotics is scarce within the South African context. This study shows how antibiograms are an effective antimicrobial stewardship strategy to reduce antimicrobial resistance rates by guiding appropriate choice of empirical antibiotics.     

Smart nebulizers

Physicians are familiar with conventional nebulizers, which deliver aerosols in a relatively uncontrolled manner. As aerosol medications evolve beyond bronchodilators, the need for control of dose variability, the possibility of overdose, and the need for efficient delivery have provoked the industry to redesign aerosol delivery systems. The need to target aerosol delivery to specific lung regions has focused efforts to coordinate aerosol delivery with defined breathing maneuvers. This review summarizes the major factors affecting aerosol deposition, discusses how those factors are guiding new designs for aerosol delivery systems, and describes some examples of the improved precision and efficiency of those systems.     

A comparison of linezolid with glycopeptides in severe MRSA pneumonia

Evaluation of: Luna CM, Bruno DA, García-Morato J et al. Effect of linezolid compared with glycopeptides in methicillin-resistant Staphylococcus aureus severe pneumonia in piglets. Chest 135(6), 1564–1571 (2009).

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a major pathogen in nosocomial infections and accounts for a large proportion of nosocomial pneumonia. However, there are limited antibiotics available for the treatment of this serious and potentially lethal infection. Until recently, the only effective antibiotic was vancomycin, but the oxazolidinones, such as linezolid, have been shown to be a valuable addition to the arsenal of antimicrobial agents that can be used for MRSA pneumonia. Clinical trials have been conducted to compare vancomycin and linezolid head-to-head in pneumonia and, in post hoc subgroup analyses, showed that linezolid use was associated with improved survival. The ensuing debate over these results was dominated by two opinions; there were those who speculated on the mechanism by which linezolid achieved this benefit, namely attributing it to pharmacodynamics and pharmacokinetics, and others who criticized the methodology of the studies and questioned the validity of the results altogether. This study by Luna and colleagues was designed with several goals in mind. The first was to attempt to generate an animal model of MRSA pneumonia in piglets by duplicating techniques used in animal models of Gram-negative pneumonia. Then they studied the effect of three antibiotics (vancomycin, linezolid and teicoplanin) on outcomes in the same model, while simultaneously measuring antibiotic levels in the serum, bronchoalveolar lavage fluid and lung tissue, in an attempt to attribute differences in survival to pharmacological properties of the drugs used. Their results showed a survival benefit only for linezolid, despite the fact that all three antibiotics had levels above MIC in all the compartments sampled, leading them to speculate that linezolid may have improved outcomes by mechanisms not directly related to its antimicrobial actions.     

Adenylate Kinase Release as a High-Throughput-Screening-Compatible Reporter of Bacterial Lysis for Identification of Antibacterial Agents

Adenylate kinase (AK) is a ubiquitous intracellular enzyme that is released into the extracellular space upon cell lysis. We have shown that AK release serves as a useful reporter of bactericidal agent activity and can be exploited for antimicrobial screening purposes. The AK assay exhibits improved sensitivity over that of growth-based assays and can detect agents that are active against bacteria in clinically relevant growth states that are difficult to screen using conventional approaches, such as small colony variants (SCV) and bacteria within established biofilms. The usefulness of the AK assay was validated by screening a library of off-patent drugs for agents that exhibit antimicrobial properties toward a variety of bacterial species, including Escherichia coli and all members of the “ESKAPE” pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). The assay detected antibiotics within the library that were expected to be active against the organism screened. Moreover, 38 drugs with no previously reported antibacterial activity elicited AK release. Four of these were acquired, and all were verified to exhibit antimicrobial activity by standard susceptibility testing. Two of these molecules were further characterized. The antihistamine, terfenadine, was active against S. aureus planktonic, SCV population, and biofilm-associated cells. Tamoxifen, an estrogen receptor antagonist, was active toward E. faecium in vitro and also reduced E. faecium pathogenesis in a Galleria mellonella infection model. Our data demonstrate that the AK assay provides an attractive screening approach for identifying new antimicrobial agents. Further, terfenadine and tamoxifen may represent novel antimicrobial drug development scaffolds.