abstract: Intrinsic antibiotic resistance is of growing concern in modern medical treatment. The primary action of multidrug resistant strains is through over-expression of active transporters which recognize a broad range of antibiotics. In Escherichia coli, the TolC-AcrAB complex has become a model system to understand antibiotic efflux. While the structures of these three proteins (and many of their homologs) are known, the exact mechanisms of interaction are still poorly understood. By mutational analysis of the TolC turn 1 residues, a drug hypersensitive mutant has been identified which is defective in functional interactions with AcrA and AcrB. Antibiotic resistant revertants carry alterations in both TolC and AcrA act by stabilizing functional complex assembly and opening of the TolC aperture, as monitored by stability of a labile TolC mutant and sensitivity to vancomycin, respectively. Alterations in the AcrB periplasmic hairpin loops lead to a similar antibiotic hypersensitivity phenotype and destabilized complex assembly. Likewise, alterations in TolC which constitutively open the aperture suppress this antibiotic sensitivity. Suppressor alterations in AcrA and AcrB partially restore antibiotic resistance by mediating stability of the complex. The AcrA suppressor alterations isolated in these studies map to the three crystallized domains and it is concluded they alter the AcrA conformation such that it is permanently fixed in an active state, which wild type only transiently goes through when activated by AcrB. Through this genetic evidence, a direct interaction between TolC and AcrB which is stabilized by AcrA has been proposed. In addition to stabilizing the interactions between TolC and AcrB, AcrA is also responsible for triggering opening of the TolC aperture by mediating energy flow from AcrB to TolC. By permanently altering the conformation of AcrA, suppressor mutants allow defective TolC or AcrB mutants to regain functional interactions lost by the initial mutations. The data provide the genetic proof for direct interaction between AcrB and that AcrA mediated opening of TolC requires AcrB as a scaffold. / Dissertation/Thesis / Ph.D. Microbiology 2012

Class of 2014 Abstract / Specific Aims: Determine time to appropriate therapy for S. maltophilia infection before implementation of mass spec. A second part of this project will evaluate the time to appropriate therapy after implementation of mass spec. The hypothesis is the time to appropriate therapy will decrease by > 2 days after implementation of mass spec. appropriate antibiotic therapy will be based on susceptibility data reported for each isolate of S. maltophilia. Potential appropriate therapy that will be evaluated includes high-dose sulfamethoxazole-trimethoprim (10-20 mg/kg/day based on TMP adjusted for renal function), ticarcillin- clavulanate plus aztreonam, moxifloxacin or levofloxacin, and ceftazidime. Methods: A retrospective chart review was done to evaluate time to identification and time to appropriate therapy for S. maltophilia as baseline data before implementation of mass spectrometry for earlier species identification. Subject selection included all patients between June 1, 2011 through May 31, 2012 with S. Maltophilia isolated from any source while admitted to the University of Arizona Medical Center-University Campus, Tucson, AZ. Patients with initial S. maltophilia isolated from a post-mortem sample or were colonizers were excluded. Main Results: There were 86 subjects included in the study based on inclusion and exclusion criteria. There were 60 subjects that received appropriate therapy for S. maltophilia coverage. The averaged time to initiation of appropriate antibiotic prior to the implementation of mass spectrometer was determined to be 6.5 days. Conclusion: Since S. maltophilia is not susceptible to many antibiotics used as empiric therapy, early identification of the pathogen via mass spectrometry, in addition to pharmacist intervention, may lead to initiation of appropriate antibiotics that is earlier than an average of 6.5 days found in this study.

Class of 2011 Abstract / OBJECTIVES: To determine the appropriateness of antimicrobial therapy in patients initiated on empiric meropenem therapy. METHODS: Adult patients prescribed empiric meropenem therapy between January 1, 2010 and March 31, 2010 at a tertiary care, academic medical center were included. Data collected included site of infection, culture and susceptibility data, risk factors for multi-drug resistant organisms, and changes in antimicrobial therapy during the first seven days after meropenem therapy was initiated. Demographic variables included age, sex, weight, and race. RESULTS: RESULTS: A total of 89 patients were included in the study analysis. Initial culture(s) was obtained before administration of antibiotics in only 58% of patients. During the first 24 hours of admission, four or more different antibiotics were prescribed in 26% of patients often with overlapping spectrums of activity. The majority of patients received meropenem for either less than 1 day or greater than 4 days. CONCLUSION: The primary issues identified with appropriate antibiotic prescribing involved the timing of cultures, and multiple changes in antibiotic therapy without culture-driven reasoning.

Our battle against pathogens has become a challenge due to the rise in antibiotic resistance and the dwindling number of new antibiotics entering the clinic. Most antibiotics owe their origins to soil bacteria, which have been producing these natural products for millennia. The rifamycins are products of actinomycetes and semisynthetic derivatives of these have been very successful in the clinic. Rifampin (RIF) has been a cornerstone agent against tuberculosis for over 50 years. In the clinic, pathogens typically develop RIF resistance by mutation of the drug. Nonetheless, a number of diverse RIF resistance mechanisms have been described, including enzymatic inactivation. Environmental bacteria are multidrug resistant, likely due to sharing the same niche as antibiotic producers and represent a reservoir of ancient resistance determinants. Furthermore, these resistance determinants have been linked to pathogens. Exploring the antibiotic resistome, the collection of all antibiotic resistance determinants from the global microbiota, reveals the diversity and evolution of resistance and provides insight on vulnerabilities of our current antibiotics. Herein, I describe a diverse collection of RIF-inactivating mechanisms from soil actinomycetes. I identified heretofore unknown RIF glycosyltransferase and RIF phosphotransferase genes (rgt and rph, respectively). RGT and RPH enzymes display broad rifamycin specificity and contribute to high-level resistance. Interestingly, RIF-sensitive Gram-positive pathogens are carriers of RPH, highlighting the existence of a ‘silent’ resistome in clinically relevant bacteria and emphasize the importance of studying resistance from environmental bacteria. Furthermore, I identified a conserved upstream DNA motif associated with RIF-inactivating genes from actinomycetes and demonstrate its role in RIF-responsive gene regulation. Finally, I explore the use of a RIF-resistance guided approach to identify novel rifamycin producing bacteria. This study expands the rifamycin resistome, provides evidence of vulnerabilities of our current arsenal of rifamycin antibiotics, and offers a strategy to identify new members of this family natural product family. / Thesis / Doctor of Science (PhD)

Antibiotics are one of the most important advances in medical science, but today, antibiotic-resistant bacteria threaten this legacy. We risk losing our ability to treat acute infections, perform invasive surgeries, and exploit immunosuppressive therapies like transplantation and cancer chemotherapy. The antibiotics we use today have ancient roots and have been produced by microbial denizens of the soil for millions of years before we adopted them in the 20th century. This history has modern consequences, as strategies to resist these compounds have evolved in concert for millions of years. The result is a vast reservoir of antimicrobial resistance that exists in environmental bacteria, which have the potential to be mobilized into human pathogens and cripple our antibiotic arsenal. Here, I set out to deepen our understanding of the environmental resistome, focusing on the rifamycin antibiotics. These compounds inhibit bacterial RNA polymerase and are frontline agents for treating tuberculosis. Environmental bacteria from the phylum Actinobacteria induce the production of resistance enzymes in response to these compounds. Although mechanistic questions remain, we demonstrate that this induction stems from the inhibition of RNA polymerase by rifamycins. The induction process is known to require a specific DNA motif; here, I identify additional sequences as part of this motif and use this information to map inducible rifamycin resistance across the entire phylum. The most common rifamycin-inducible gene was an uncharacterized family of proteins annotated as DNA helicases. I investigated these proteins and discovered that they bind to RNA polymerase and displace rifamycin antibiotics, a novel mechanism of rifamycin resistance. Lastly, we repurposed this inducible system to develop an assay to screen for novel RNA polymerase inhibitors. From this screen, we identified a rifamycin immune to a common environmental resistance enzyme and a new family of rifamycin antibiotics. / Thesis / Doctor of Philosophy (PhD) / Our antibiotic arsenal consists primarily of metabolites produced by soil microbes, which humanity repurposed into life-saving medicines in the 20th century. As a direct result of the natural origin of antibiotics, resistant bacteria exist in these same environments, independent of human use. Individual genetic determinants from this reservoir can emerge in pathogenic bacteria without warning and render antibiotics ineffective. The aim of this work was to understand how environmental bacteria resist the rifamycin class of antibiotics. Firstly, I investigated the ability of some bacteria to sense the presence of rifamycins, and in response produce proteins to protect themselves. I discovered that this process requires specific DNA sequences nearby resistance genes. Using this DNA sequence as a guide I cataloged resistance genes in thousands of bacterial genomes and discovered a new mechanism of rifamycin resistance. Lastly, I exploited this rifamycin sensing system to discover new antibiotics from soil microbes.

Yes / During January 15th – 17th, 2017 a group of scientists met, under the auspices of the UK-India Centre for Advanced Technology for Minimizing Indiscriminate Use of Antibiotics, to discuss the further developments and potential solutions to antimicrobial resistance. This was the third work shop under this funding stream held in Hyderabad. The presentations and outcomes of the workshop are released here. Key out comes included the need to address improved treatment and detection of TB, delivery of antimicrobial peptides, potential strategies for combating beta-lactam resistance. / Medical Research Council

The discovery of antibiotics and their subsequent clinical use has had a tremendous and beneficial impact on human health. The β-lactam antibiotics, which include penicillins, cephalosporins, carbapenems, and monobactams, constitute over half of the global antibiotic market. However, like all antibiotics, the β-lactams are susceptible to bacterial antibiotic resistance. One of the most disconcerting manifestations of bacterial resistance to β-lactam antibiotics is the evolution and dissemination of β-lactamases, enzymes able to chemically inactivate β-lactam antibiotics. These resistance determinants are the key contributing factor to extensively-drug resistant Gram-negative pathogens, for which we are already bereft of chemotherapeutic treatment options in some cases. The coadministration of a β-lactamase inhibitor (BLI) with a β-lactam antibiotic is a proven therapeutic strategy to counter β-lactamase expression. Unfortunately, the emergence of both serine β-lactamases (SBLs) that are resistant to BLIs and metallo-β-lactamases (MBLs), which are intrinsically resistant to BLIs due to a discrete mechanism of β-lactam hydrolysis, threaten the efficacy of combination therapy. Notwithstanding this bacterial adaptation, the discovery and development of novel BLIs is an attractive strategy to evade resistance, as evidenced by the recent clinical approval of the diazabicyclooctane (DBO) SBL inhibitor, avibactam. Herein, I describe efforts directed at understanding the mechanism of avibactam SBL inhibition. Furthermore, DBO derivatives are shown to display bifunctional properties in inhibiting both β-lactamases and the targets of β-lactam antibiotics, the penicillin-binding proteins. In addition to understanding the enzymology and chemical biology of DBOs, I describe two screening campaigns directed towards discovering inhibitors of MBLs, an unmet clinical need. Using target and cell-based screening of both synthetic and natural product chemical libraries, a fungal natural product inhibitor of clinically relevant MBLs was discovered and characterized. This study expands our understanding of the mechanisms by which DBOs can be used to combat extensively drug-resistant Gram-negative pathogens. It also describes the discovery of a new natural product MBL inhibitor using a workflow that should be amenable to other resistance determinants. It’s hoped that these studies can contribute meaningfully to countering antibiotic resistance observed in clinical settings. / Thesis / Doctor of Philosophy (PhD) / Beta-lactam antibiotics like penicillin are a mainstay for treatment of bacterial infections. Bacterial resistance to these antibiotics threatens their utility and therefore new strategies are required to counter this phenomenon. Herein I describe efforts aimed at understanding new drugs and candidate drugs that act by inhibiting the function of enzymes produced by bacteria that are able to degrade beta-lactam antibiotics. Through the discovery of new molecules and an understanding of their chemical mechanism of inhibition it is believed that bacterial resistance to beta-lactam antibiotics can be reversed.

The rifamycins are a class of antibiotics which were once used almost exclusively to treat tuberculosis, but are currently receiving renewed interest. Resistance to rifamycins is most commonly attributed to mutations in the drug target, RNA polymerase. Yet environmental isolates are also able to enzymatically inactivate rifamycins in a number of ways. Recently, rifamycin resistance determinants from the environment were found to be closely associated with a so called rifamycin associated element (RAE). The region containing the RAE from an environmental strain was shown to induce gene expression in the presence of rifamycins, hinting at an inducible system for rifamycin resistance. In this work, we examine the RAE from a model organism for Streptomyces genetics, Streptomyces venezuelae. We confirm that the promoter region containing the RAE upstream of a rifamycin monooxygenase rox is inducible by rifamycins. The strains of S. venezuelae generated in this work can be used in future genetic studies on the RAE. As well, the rifamycin monooxygenase Rox was purified for the first time and characterized biochemically. The structure of Rox was obtained with and without the substrate rifampin. Steady state kinetics for the enzyme were determined with a number of substrates, and its ability to confer resistance to rifamycins was examined. Monooxygenated rifamycin SV compound was purified and structurally characterized by NMR analysis. We proposed an aromatic hydroxylase type mechanism for Rox, in which the enzyme hydroxylates the aromatic core of the rifamycin scaffold and causes a non-enzymatic C-N bond cleavage of the macrolactam ring. This is a new mechanism of rifamycin resistance, and sheds some light on the decomposition of rifamycins mediated by monooxygenation, which is still poorly understood. / Thesis / Master of Science (MSc) / Antibiotic resistance represents a major threat to global health. Infections that were once readily treatable are no longer so due to the rise in multidrug resistant bacteria. As our arsenal of effective antibiotics is depleted, new drugs are being discovered less and less frequently. This has caused the scientific community to get creative in coming up with treatments: trying combinations of antibiotics, using antibiotics which were once considered too toxic, and repurposing antibiotics for different bacteria. Rifamycins are a class of antibiotics most commonly used in the treatment of tuberculosis. However, they are becoming more widely used as a result of antibiotic resistance. There are a number of different ways bacteria can become resistant to the harmful effects of rifamycins: by modifying the target so the drug can no longer bind to it, actively pumping the drug out of the cell, or by changing the drug in some way so it is no longer effective. Bacteria in the environment use antibiotics as a form of chemical warfare to gain an advantage over their neighbours; therefore, they have had millions of years to evolve very effective methods of antibiotic resistance. By surveying what kinds of antibiotic resistance are in the environment, we can predict what we might see one day in a medical setting. In this thesis, I have studied a protein that bacteria make to inactivate rifamycins. The rifamycin monooxygenase Rox adds an oxygen to the rifamycin scaffold; this causes spontaneous cleavage of the rifamycin backbone and changes the conformation of the drug so it can no longer bind to its target. I have also investigated the regulation of this and other genes in the bacterial strain Streptomyces venezuelae. By understanding how this process works, we can potentially design inhibitors to stop this from happening, should this method of resistance ever become clinically prevalent.

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