<p>The synthesis of novel oxadiazole antibiotics more water soluble than previously published analogues within the group will be discussed in this thesis as part of synthesizing a library with increased sp3 character.  These analogues were tested against S. aureus and MRSA strains to determine the most active compounds then percent hemolysis confirmed little RBC lysis among the compounds.  Synergistic activity was also tested for HSGN-2241 previously synthesized within the group.  Biofilm eradication assays were completed for HSD 1919 alongside time kill assays.</p>

Microbial natural products have been an invaluable resource for providing clinically relevant therapeutics for almost a century, including most of the commonly used antibiotics that are still in medical use today. In more recent decades, the need for new biotherapeutics has begun to grow, as multi-drug resistant pathogens continue to emerge, putting into question the long-term efficacy of many drugs that we routinely depend on to combat infectious diseases. To affect this growing medical crisis, new efforts are being applied to computationally mine the genomes of microorganisms for biosynthetic gene clusters that code for molecules possessing anti-microbial activities that circumvent known resistance mechanisms. To this end, cutting-edge software platforms have been developed that can identify, with high predictive accuracy, microbial genomes that code for natural products of potential interest. However, with such analyses comes the need to thoroughly vet each predicted gene cluster, to identify those high-value candidate molecules that are not associated with known resistance mechanisms. In this work, a new strategy was developed that involved cataloguing all known ‘self-resistance’ mechanisms encoded by natural product producing microorganisms, which protect the producer from the highly toxic effects of their secreted anti-microbial agents. This collection of resistance data was leveraged and used to engineer an automated software-based pipeline that interrogates biosynthetic gene clusters and relates them to previously identified resistance mechanisms. Gene clusters that are revealed to be independent of known resistance mechanisms can then be flagged for further chemical and biological study in the laboratory. Such in-depth interrogations of microbial genomes aim to help reveal the full biological repertoire of antibiotics yet to be discovered from microorganisms, and will lead to the development of the next generation of biotherapeutics to quell the growing medical crisis of antibiotic-resistance among human pathogenic organisms. / Thesis / Master of Science (MSc) / It would be hard to imagine a world where we could no longer use the antibiotics we are routinely being prescribed for common bacterial infections. Currently, we are in an era where this thought could become a reality. Although we have been able to discover antibiotics in the past from soil dwelling microbes, this approach to discovery is being constantly challenged. At the same time, the bacteria are getting smarter in their ways to evade antibiotics, in the form of resistance, or self-protection mechanisms. As such is it essential to devise methods which can predict the potential for resistance to the antibiotics we use early in the discovery and isolation process. By using what we have learned in the past about how bacteria protect themselves for antibiotics, we can to stay one step ahead of them as we continue to search for new sources of antibiotics from bacteria.

Multiple antibiotic resistant bacteria continue to be a threat to the health of the world's population. Glycopeptide antibiotics are one type of drug that are used to treat these serious pathogens. Increased usage over the years has led to the emergence of bacteria which are resistant to these glycopeptide antibiotics and now the need for altered antibiotics with an increased effectiveness has arisen. 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 𝘵𝘰𝘺𝘰𝘤𝘢𝘦𝘯𝘴𝘪𝘴 NRRL 15009 produces the glycopeptide antibiotic A47934. Here, the biosynthetic gene cluster for A47934 was sequenced in its entirety. All enzymes encoded by assigned open reading frames were analyzed and functions assigned where possible. The resulting biosynthesis cluster encodes all the enzymes necessary to produce A47934, as well as confer resistance and regulate the resistance response. In addition to sequencing the biosynthetic gene cluster, enzymatic studies were attempted on the two-component regulatory system (VanR and VanS) which confers resistance to A47934. Finally, inactivation of 𝘴𝘵𝘢𝘓 was attempted. Overall, the results presented here should help us to further understand how these chemically complex glycopeptide antibiotics are made and lend further insight into how we can attempt to produce new semi-synthetic versions. / Thesis / Master of Science (MSc)

Thesis / Master of Science (MS)

There is an urgent need to identify novel antibiotics for multidrug-resistant Gram-negative pathogens. These bacteria are intrinsically resistant to many antimicrobials due to a formidable outer membrane barrier. Herein we investigate the potential of perturbing the outer membrane to sensitize Gram-negative bacteria to otherwise inactive antibiotics. In chapter 2, we identify the ability of mcr-1 mediated resistance to confer protection from the lytic but not outer membrane-perturbing activity of colistin. Exploiting this sensitivity, we show that colistin and clarithromycin in combination are efficacious against mcr-1-expressing Klebsiella pneumoniae in murine infection models. This demonstrates the viability of colistin combination therapies against Gram-negative pathogens harbouring mcr-1, and points to a mechanism of mcr-1-mediated resistance extending beyond the predicted reduction in binding affinity of polymyxins to the outer membrane. We continue to investigate the potential of using outer membrane perturbants with otherwise inactive antimicrobials in chapter 3. In this work, we identify the ability of OM disruption to change the rules of Gram-negative entry, render pre-existing resistance ineffective, reduce the development of spontaneous resistance and attenuate biofilm formation. Together, these data suggest that OM disruption overcomes many traditional hurdles encountered during antibiotic treatment and is a high priority approach for further development. / Thesis / Doctor of Philosophy (PhD)

Objective — To describe the vascular distribution pattern of contrast medium during intraosseous regional perfusion (IORP) of the distal portion of the forelimb in horses. Sample Population — 13 cadaver forelimbs from 12 horses without vascular or orthopedic disease of the distal forelimb. Procedure — Serial lateromedial radiographs were taken of 10 heparinized cadaver distal forelimbs at 0, 1, 2, 6, 15, and 30 minutes during IORP of the third metacarpal bone (MCIII) using iodinated contrast medium and a tourniquet placed over the proximal MCIII. Vascular regions of interest (ROI) were created for each radiograph. Reviewers identified presence or absence of contrast in each ROI. This information was summarized to identify vessel-filling patterns over time. Vessel identification was verified using computed tomography angiography and latex perfusion studies on separate cadaver distal forelimbs. Results — During IORP, contrast medium filled the medullary cavity of the MCIII, exited via trans-cortical vessels and diffused distally to the remaining arteries and veins of the limb, distal to the tourniquet. Maximum vessel and soft tissue opacification occurred in most specimens at 6 and 30 minutes, respectively. Serial radiography vessel patterns matched those of CT images and dissected specimens. Conclusions and Clinical Relevance — Intraosseous regional perfusion provides a repeatable pattern of vascular distribution in the distal portion of the equine forelimb. This is the first documentation of arterial perfusion using this technique. Previous reports indicate that IORP only delivers medications to the venous vessels of the perfused limb. Maximum soft tissue perfusion was observed at 30 minutes. / Master of Science

There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï ­g/ml), chloramphenicol (Cm; 25 ï ­g/ml), streptomycin (Sm; 100 ï ­g/ml), and tetracycline (Tc; 25 ï ­g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter. / Master of Science / There is significant concern that agricultural use of antimicrobials leads to spill over of antimicrobial resistance (AMR) into the general population. Animal manures contain a large and diverse reservoir of AMR genes. While poultry litter is a rich source of nitrogen, it contains an abundance of AMR genes and genetic elements associated with its dissemination. Reduce this reservoir and its potential to transmit AMR to pathogens is diminished. Methods. The ability of litter microbiota to transmit AMR was assessed using a rifampicin (Rp)-resistant Salmonella Typhimurium LT2 strain, as the recipient, and an Escherichia coli pR100, plasmid donor strain. Autoclaved poultry litter served as a negative control in plasmid transfer studies. Abundance of phenotypic AMR was assessed for litter microbiota to the antibiotics: ampicillin (Ap; 25 ï ­g/ml), chloramphenicol (Cm; 25 ï ­g/ml), streptomycin (Sm; 100 ï ­g/ml), and tetracycline (Tc; 25 ï ­g/ml). qPCR was used to estimate gene load of streptomycin-resistance and sulfonamide-resistance genes aadA and sul, respectively in the poultry litter community. AMR gene load was determined relative to total population; using 16S rRNA qPCR to estimate the total bacterial load. Different growth conditions (temperature, media) and cell densities (filter vs liquid matings) were assessed to determine conditions optimal for AMR transfer from the litter community to the recipient Salmonella strain. Results. Poultry litter contained 108 CFU/g, with Gram-negative enterics representing a minor population (< 103 CFU/g). There was high abundance of resistance to Sm (106 CFU/g) and tetracycline (107 CFU/g); and a sizeable antimicrobial resistance gene (aadA- 2.63 E+6; sul- 7.01 E+10) load in poultry litter. Autoclaving reduced viable bacterial counts by >6 log10 and the antimicrobial resistance gene load was undetectable and by 4 log10 for aadA and sul, respectively. While plasmid transfer was observed between the Salmonella recipient with R100 plasmid, no AMR Salmonella were observed in matings with live or dead, poultry litter microbiota under conditions optimal for F-plasmid transfer. Conclusions. While poultry litter microbiota contains a significant AMR gene load, potential to transmit resistance is low, under conditions commonly used to assess plasmid conjugation. Autoclaving poultry litter significantly reduces the litter resistome. Several processes (ex. composting) are currently being examined to identify one that ameliorates poultry litter.

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

Thesis advisor: Roche P. John / The growing threat of antibiotic resistance makes it extremely important that citizens be informed about the risks posed by antibiotic-resistant bacteria, and measures with which they can reduce these risks. The print media are major sources of such information for members of the public. In the present study, articles from major newspapers in the United States and Canada appearing between 1998 and 2002 were surveyed to determine the extent to which mention was made of antibiotic resistance and the risks associated with antibiotic resistance, the contextual precision with which this information was communicated, and the extent to which information was presented about causes, and risk-reduction measures, associated with antibiotic resistance. The majority of articles surveyed mentioned antibiotic resistance, but most failed to mention associated risks (i.e., the risk of illness and/or the risk of mortality). Articles that did report risks, did so only at a low level of contextual precision. A relatively low percentage of articles mentioned causes of antibiotic resistance, and even fewer mentioned risk reduction measures. These findings suggest that the print media could improve the efficacy with which they inform the public about issues associated with antibiotic resistance. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

The ubiquitous nature of antibiotic resistance and antibiotic resistance genes (ARGs) among environmental pathogens from a variety of wastewater effluents, suggests that the aquatic environment, and specifically alternative wastewater treatment systems, may act as reservoirs for drug resistant bacteria and ARGs, thereby contributing to the widespread dissemination of antibiotic resistance. More research is necessary to contribute to our understanding of the occurrence, fate, and mobility of antibiotic resistance and ARGs among bacterial indicators of faecal contamination as well as pathogenic bacteria within Canadian wastewater treatment systems. The primary objective of this research was to determine the prevalence, fate, and potential transfer of bacterial resistance and ARGs among selected environmental pathogens exposed to alternative wastewater treatment systems, while considering the impact of treatment strategies on the expression of antibiotic resistance. A detailed analysis was initially conducted with respect to the characterization and quantification of microbial populations (including antibiotic resistant bacteria) in a variety of treatment systems and waste effluent sources. Traditional culture-based screening techniques in combination with molecular characterization (through colony or multiplex PCR), and molecular quantification using real-time quantitative PCR were utilized in order to help establish a preliminary environmental assessment of selected pathogens (Escherichia coli, Enterococcus spp., Salmonella spp.) and ARGs (tetA, blaSHV, & ampC) within a variety of wastewater treatment systems (lab-scale mesocosms, constructed wetland, constructed lagoon system, and pilot-scale biological nutrient removal (BNR) system). Overall, the level of multiple antibiotic resistance (MAR) among culturable indicator (E. coli & Enterococcus spp.) and environmental bacteria was high (reaching 100% in several instances) within different types of wastewater treatment systems and effluent sources (poultry waste effluent, municipal wastewater, aquaculture wastewater). Common antibiotic resistance profiles among E. coli isolates included simultaneous resistance to between three and five antimicrobials, whereas common MAR profiles among Enterococcus spp. isolates showed resistance to ten or more antibiotics. Real time quantitative PCR was used to determine the concentration of three bacterial pathogens; E. coli, Enterococcus faecalis, and Salmonella spp., and three ARGs; tetA, ampC, and blaSHV, within a variety of wastewater samples. Based on the results, it was concluded that high concentrations of ARGs were present in the treated effluent (10⁴- 10⁶ target gene copies/100 mL), regardless of system type (i.e. constructed lagoon, pilot-scale BNR, or constructed wetland), which may ultimately serve as a potential route for entry of ARGs and antibiotic resistant bacteria into the natural environment. Water is considered an important medium for transfer of resistance genes and resistant bacteria to the broader environment. Few studies have examined the transferability via conjugation of ARGs in E. coli and Salmonella spp. isolated from wastewater. Identification of three resistance determinants (tetA, strA, strB) conferring resistance to tetracycline and streptomycin was performed on selected multi-drug resistant Salmonella spp. and E. coli isolates. The potential for transfer of tetracycline and streptomycin resistance genes was demonstrated through broth conjugation experiments using multi-drug resistant Salmonella spp. and E. coli isolates as donors, and E. coli K12 as the recipient. Conjugation was successfully observed in 75% (9/12) of donor isolates, occurring in both Salmonella spp. and E. coli isolates. Six strains (50%) were capable of transferring their tetA, strA, and strB genes to the recipient strain, resulting in 58.5% (38/65) of total transconjugant strains acquiring all three resistance determinants. The results confirm the role of environmental bacteria (isolated from wastewater treatment utilities) as a reservoir of antibiotic resistance and ARGs, containing mobile genetic elements, which are capable of disseminating and transferring ARGs. As concerns about water quality and environmental contamination by human and agricultural effluents have increased, it has become increasingly more important to consider the prevalence and transferability of ARGs to opportunistic and human pathogens. As observed in this research, the ubiquitous nature of multi-drug resistant bacteria in water and wastewater effluents, the presence of diverse ARGs of human and veterinary health significance, as well as the transfer of resistance determinants through conjugative plasmids to recipient bacteria, suggests that environmental exposure through contact or consumption with contaminated water is probable. However, a lack of critical information still exists regarding the movement of resistance genes within and between microbial populations in the environment. In addition, the extent of human exposure to ARGs and antibiotic resistant bacteria is still not well understood, and future studies on human exposure to these resistant contaminants are necessary.