Antibiotic resistance-the need for global solutions.

The causes of antibiotic resistance are complex and include human behaviour at many levels of society; the consequences affect everybody in the world. Similarities with climate change are evident. Many efforts have been made to describe the many different facets of antibiotic resistance and the interventions needed to meet the challenge. However, coordinated action is largely absent, especially at the political level, both nationally and internationally. Antibiotics paved the way for unprecedented medical and societal developments, and are today indispensible in all health systems. Achievements in modern medicine, such as major surgery, organ transplantation, treatment of preterm babies, and cancer chemotherapy, which we today take for granted, would not be possible without access to effective treatment for bacterial infections. Within just a few years, we might be faced with dire setbacks, medically, socially, and economically, unless real and unprecedented global coordinated actions are immediately taken. Here, we describe the global situation of antibiotic resistance, its major causes and consequences, and identify key areas in which action is urgently needed.

New business models for antibiotic innovation.

The increase in antibiotic resistance and the dearth of novel antibiotics have become a growing concern among policy-makers. A combination of financial, scientific, and regulatory challenges poses barriers to antibiotic innovation. However, each of these three challenges provides an opportunity to develop pathways for new business models to bring novel antibiotics to market. Pull-incentives that pay for the outputs of research and development (R&D) and push-incentives that pay for the inputs of R&D can be used to increase innovation for antibiotics. Financial incentives might be structured to promote delinkage of a company's return on investment from revenues of antibiotics. This delinkage strategy might not only increase innovation, but also reinforce rational use of antibiotics. Regulatory approval, however, should not and need not compromise safety and efficacy standards to bring antibiotics with novel mechanisms of action to market. Instead regulatory agencies could encourage development of companion diagnostics, test antibiotic combinations in parallel, and pool and make transparent clinical trial data to lower R&D costs. A tax on non-human use of antibiotics might also create a disincentive for non-therapeutic use of these drugs. Finally, the new business model for antibiotic innovation should apply the 3Rs strategy for encouraging collaborative approaches to R&D in innovating novel antibiotics: sharing resources, risks, and rewards.

Antisense Gene Silencing and Bacteriophages as Novel Disinfection Processes for Engineered Systems

The growth and proliferation of invasive bacteria in engineered systems is an ongoing problem. While there are a variety of physical and chemical processes to remove and inactivate bacterial pathogens, there are many situations in which these tools are no longer effective or appropriate for the treatment of a microbial target. For example, certain strains of bacteria are becoming resistant to commonly used disinfectants, such as chlorine and UV. Additionally, the overuse of antibiotics has contributed to the spread of antibiotic resistance, and there is concern that wastewater treatment processes are contributing to the spread of antibiotic resistant bacteria.

Due to the continually evolving nature of bacteria, it is difficult to develop methods for universal bacterial control in a wide range of engineered systems, as many of our treatment processes are static in nature. Still, invasive bacteria are present in many natural and engineered systems, where the application of broad acting disinfectants is impractical, because their use may inhibit the original desired bioprocesses. Therefore, to better control the growth of treatment resistant bacteria and to address limitations with the current disinfection processes, novel tools that are both specific and adaptable need to be developed and characterized.

In this dissertation, two possible biological disinfection processes were investigated for use in controlling invasive bacteria in engineered systems. First, antisense gene silencing, which is the specific use of oligonucleotides to silence gene expression, was investigated. This work was followed by the investigation of bacteriophages (phages), which are viruses that are specific to bacteria, in engineered systems.


For the antisense gene silencing work, a computational approach was used to quantify the number of off-targets and to determine the effects of off-targets in prokaryotic organisms. For the organisms of Escherichia coli K-12 MG1655 and Mycobacterium tuberculosis H37Rv the mean number of off-targets was found to be 15.0 + 13.2 and 38.2 + 61.4, respectively, which results in a reduction of greater than 90% of the effective oligonucleotide concentration. It was also demonstrated that there was a high variability in the number of off-targets over the length of a gene, but that on average, there was no general gene location that could be targeted to reduce off-targets. Therefore, this analysis needs to be performed for each gene in question. It was also demonstrated that the thermodynamic binding energy between the oligonucleotide and the mRNA accounted for 83% of the variation in the silencing efficiency, compared to the number of off-targets, which explained 43% of the variance of the silencing efficiency. This suggests that optimizing thermodynamic parameters must be prioritized over minimizing the number of off-targets. In conclusion for the antisense work, these results suggest that off-target hybrids can account for a greater than 90% reduction in the concentration of the silencing oligonucleotides, and that the effective concentration can be increased through the rational design of silencing targets by minimizing off-target hybrids.

Regarding the work with phages, the disinfection rates of bacteria in the presence of phages was determined. The disinfection rates of E. coli K12 MG1655 in the presence of coliphage Ec2 ranged up to 2 h-1, and were dependent on both the initial phage and bacterial concentrations. Increasing initial phage concentrations resulted in increasing disinfection rates, and generally, increasing initial bacterial concentrations resulted in increasing disinfection rates. However, disinfection rates were found to plateau at higher bacterial and phage concentrations. A multiple linear regression model was used to predict the disinfection rates as a function of the initial phage and bacterial concentrations, and this model was able to explain 93% of the variance in the disinfection rates. The disinfection rates were also modeled with a particle aggregation model. The results from these model simulations suggested that at lower phage and bacterial concentrations there are not enough collisions to support active disinfection rates, which therefore, limits the conditions and systems where phage based bacterial disinfection is possible. Additionally, the particle aggregation model over predicted the disinfection rates at higher phage and bacterial concentrations of 108 PFU/mL and 108 CFU/mL, suggesting other interactions were occurring at these higher concentrations. Overall, this work highlights the need for the development of alternative models to more accurately describe the dynamics of this system at a variety of phage and bacterial concentrations. Finally, the minimum required hydraulic residence time was calculated for a continuous stirred-tank reactor and a plug flow reactor (PFR) as a function of both the initial phage and bacterial concentrations, which suggested that phage treatment in a PFR is theoretically possible.

In addition to determining disinfection rates, the long-term bacterial growth inhibition potential was determined for a variety of phages with both Gram-negative and Gram-positive bacteria. It was determined, that on average, phages can be used to inhibit bacterial growth for up to 24 h, and that this effect was concentration dependent for various phages at specific time points. Additionally, it was found that a phage cocktail was no more effective at inhibiting bacterial growth over the long-term than the best performing phage in isolation.

Finally, for an industrial application, the use of phages to inhibit invasive Lactobacilli in ethanol fermentations was investigated. It was demonstrated that phage 8014-B2 can achieve a greater than 3-log inactivation of Lactobacillus plantarum during a 48 h fermentation. Additionally, it was shown that phages can be used to protect final product yields and maintain yeast viability. Through modeling the fermentation system with differential equations it was determined that there was a 10 h window in the beginning of the fermentation run, where the addition of phages can be used to protect final product yields, and after 20 h no additional benefit of the phage addition was observed.

In conclusion, this dissertation improved the current methods for designing antisense gene silencing targets for prokaryotic organisms, and characterized phages from an engineering perspective. First, the current design strategy for antisense targets in prokaryotic organisms was improved through the development of an algorithm that minimized the number of off-targets. For the phage work, a framework was developed to predict the disinfection rates in terms of the initial phage and bacterial concentrations. In addition, the long-term bacterial growth inhibition potential of multiple phages was determined for several bacteria. In regard to the phage application, phages were shown to protect both final product yields and yeast concentrations during fermentation. Taken together, this work suggests that the rational design of phage treatment is possible and further work is needed to expand on this foundation.

High-throughput identification of chemical inhibitors of E. coli Group 2 capsule biogenesis as anti-virulence agents.

Rising antibiotic resistance among Escherichia coli, the leading cause of urinary tract infections (UTIs), has placed a new focus on molecular pathogenesis studies, aiming to identify new therapeutic targets. Anti-virulence agents are attractive as chemotherapeutics to attenuate an organism during disease but not necessarily during benign commensalism, thus decreasing the stress on beneficial microbial communities and lessening the emergence of resistance. We and others have demonstrated that the K antigen capsule of E. coli is a preeminent virulence determinant during UTI and more invasive diseases. Components of assembly and export are highly conserved among the major K antigen capsular types associated with UTI-causing E. coli and are distinct from the capsule biogenesis machinery of many commensal E. coli, making these attractive therapeutic targets. We conducted a screen for anti-capsular small molecules and identified an agent designated "C7" that blocks the production of K1 and K5 capsules, unrelated polysaccharide types among the Group 2-3 capsules. Herein lies proof-of-concept that this screen may be implemented with larger chemical libraries to identify second-generation small-molecule inhibitors of capsule biogenesis. These inhibitors will lead to a better understanding of capsule biogenesis and may represent a new class of therapeutics.

Design of Antibacterial Prochelators to Target Drug-Resistant Bacteria

Transition metals such as iron and copper are valued in biology for their redox activities because they are able to access various oxidation states. However, these transition metals are also implicated in a number of human disease states and play a role in bacterial infections. The ability to manipulate and monitor metal ions has vast implications on the fields of biology and human health. As such, the research described here covers two related goals: to manipulate metals in specific biological circumstances and to visualize this disturbance in cellular metal homeostasis.

Antibiotic resistance necessitates the development of drugs that exploit new mechanisms of action such as the disruption of metal homeostasis. In order to manipulate metals at the site of bacterial infection, two prochelators were developed around a β-lactam core such that the active chelator is released in the presence of bacteria that produce the resistance-causing β-lactamase enzyme. Both prochelators display enhanced activity toward resistant bacteria compared to clinical antibiotics.

Fluorescent sensors are a powerful tool for detecting small concentrations of biological analytes. Two analogs of a ratiometric fluorescent sensor were designed and synthesized to monitor cellular concentrations of copper and iron. These sensors were found to operate as designed in vitro; however the fluorescence intensity necessary for quantification of cellular metal pools has not yet been achieved.

Host gene expression classifiers diagnose acute respiratory illness etiology.

Acute respiratory infections caused by bacterial or viral pathogens are among the most common reasons for seeking medical care. Despite improvements in pathogen-based diagnostics, most patients receive inappropriate antibiotics. Host response biomarkers offer an alternative diagnostic approach to direct antimicrobial use. This observational cohort study determined whether host gene expression patterns discriminate noninfectious from infectious illness and bacterial from viral causes of acute respiratory infection in the acute care setting. Peripheral whole blood gene expression from 273 subjects with community-onset acute respiratory infection (ARI) or noninfectious illness, as well as 44 healthy controls, was measured using microarrays. Sparse logistic regression was used to develop classifiers for bacterial ARI (71 probes), viral ARI (33 probes), or a noninfectious cause of illness (26 probes). Overall accuracy was 87% (238 of 273 concordant with clinical adjudication), which was more accurate than procalcitonin (78%, P < 0.03) and three published classifiers of bacterial versus viral infection (78 to 83%). The classifiers developed here externally validated in five publicly available data sets (AUC, 0.90 to 0.99). A sixth publicly available data set included 25 patients with co-identification of bacterial and viral pathogens. Applying the ARI classifiers defined four distinct groups: a host response to bacterial ARI, viral ARI, coinfection, and neither a bacterial nor a viral response. These findings create an opportunity to develop and use host gene expression classifiers as diagnostic platforms to combat inappropriate antibiotic use and emerging antibiotic resistance.

PKC signaling regulates drug resistance of the fungal pathogen Candida albicans via circuitry comprised of Mkc1, calcineurin, and Hsp90.

Fungal pathogens exploit diverse mechanisms to survive exposure to antifungal drugs. This poses concern given the limited number of clinically useful antifungals and the growing population of immunocompromised individuals vulnerable to life-threatening fungal infection. To identify molecules that abrogate resistance to the most widely deployed class of antifungals, the azoles, we conducted a screen of 1,280 pharmacologically active compounds. Three out of seven hits that abolished azole resistance of a resistant mutant of the model yeast Saccharomyces cerevisiae and a clinical isolate of the leading human fungal pathogen Candida albicans were inhibitors of protein kinase C (PKC), which regulates cell wall integrity during growth, morphogenesis, and response to cell wall stress. Pharmacological or genetic impairment of Pkc1 conferred hypersensitivity to multiple drugs that target synthesis of the key cell membrane sterol ergosterol, including azoles, allylamines, and morpholines. Pkc1 enabled survival of cell membrane stress at least in part via the mitogen activated protein kinase (MAPK) cascade in both species, though through distinct downstream effectors. Strikingly, inhibition of Pkc1 phenocopied inhibition of the molecular chaperone Hsp90 or its client protein calcineurin. PKC signaling was required for calcineurin activation in response to drug exposure in S. cerevisiae. In contrast, Pkc1 and calcineurin independently regulate drug resistance via a common target in C. albicans. We identified an additional level of regulatory control in the C. albicans circuitry linking PKC signaling, Hsp90, and calcineurin as genetic reduction of Hsp90 led to depletion of the terminal MAPK, Mkc1. Deletion of C. albicans PKC1 rendered fungistatic ergosterol biosynthesis inhibitors fungicidal and attenuated virulence in a murine model of systemic candidiasis. This work establishes a new role for PKC signaling in drug resistance, novel circuitry through which Hsp90 regulates drug resistance, and that targeting stress response signaling provides a promising strategy for treating life-threatening fungal infections.

Impact of a Point-of-Care Rapid Influenza Test on Antibiotic Prescribing Patterns in Southern Sri Lanka

Background: Acute febrile respiratory illnesses, including influenza, account for a large proportion of ambulatory care visits worldwide. In the developed world, these encounters commonly result in unwarranted antibiotic prescriptions; data from more resource-limited settings are lacking. The purpose of this study was to describe the epidemiology of influenza among outpatients in southern Sri Lanka and to determine if access to rapid influenza test results was associated with decreased antibiotic prescriptions.

Methods: In this pretest- posttest study, consecutive patients presenting from March 2013- April 2014 to the Outpatient Department of the largest tertiary care hospital in southern Sri Lanka were surveyed for influenza-like illness (ILI). Patients meeting World Health Organization criteria for ILI-- acute onset of fever ≥38.0°C and cough in the prior 7 days--were enrolled. Consenting patients were administered a structured questionnaire, physical examination, and nasal/nasopharyngeal sampling. Rapid influenza A/B testing (Veritor System, Becton Dickinson) was performed on all patients, but test results were only released to patients and clinicians during the second phase of the study (December 2013- April 2014).

Results: We enrolled 397 patients with ILI, with 217 (54.7%) adults ≥12 years and 188 (47.4%) females. A total of 179 (45.8%) tested positive for influenza by rapid testing, with April- July 2013 and September- November 2013 being the periods with the highest proportion of ILI due to influenza. A total of 310 (78.1%) patients with ILI received a prescription for an antibiotic from their outpatient provider. The proportion of patients prescribed antibiotics decreased from 81.4% in the first phase to 66.3% in the second phase (p=.005); among rapid influenza-positive patients, antibiotic prescriptions decreased from 83.7% in the first phase to 56.3% in the second phase (p=.001). On multivariable analysis, having a positive rapid influenza test available to clinicians was associated with decreased antibiotic use (OR 0.20, 95% CI 0.05- 0.82).

Conclusions: Influenza virus accounted for almost 50% of acute febrile respiratory illness in this study, but most patients were prescribed antibiotics. Providing rapid influenza test results to clinicians was associated with fewer antibiotic prescriptions, but overall prescription of antibiotics remained high. In this developing country setting, a multi-faceted approach that includes improved access to rapid diagnostic tests may help decrease antibiotic use and combat antimicrobial resistance.