2022 Sessions & Speakers

Gerard (Gerry) Wright, PhD; Professor, Department of Biochemistry and Biomedical Sciences & Executive Director Global Nexus for Pandemics and Biological Threats, McMaster University

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Bio: Gerard (Gerry) Wright is the Michael G. DeGroote Chair in Infection and Anti-Infective Research and Professor in the Department of Biochemistry and Biomedical Science at McMaster University, Hamilton, Ontario. He is the Executive Director of Canada’s Global Nexus for Pandemics and Biological Threats and was the founding director of the Michael G. DeGroote Institute for Infectious Disease Research (2007-2022) and the David Braley Centre for Antibiotic Discovery (2018-2022). Gerry was elected as a Fellow of the Royal Society of Canada (2012) and a fellow of the American Academy of Microbiology (2013). He is the recipient of a Killam Research Fellowship (2011-1012), the R.G.E. Murray Award for Career Achievement of the Canadian Society of Microbiologists (2013), the NRC Research Press Senior Investigator Award from the Canadian Society for Molecular Biosciences (2016), Premier’s Research Excellence (1999), and the Polanyi Prize (1993). Gerry is the co-founder of the Canadian Anti-Infective Innovation Network (www.cain-amr.ca). He has trained over 75 graduate students and postdocs, is the author of over 300 manuscripts, and is a member of the editorial boards of several peer-reviewed journals. In 2016 he was named a McMaster Distinguished University Professor, the highest academic honor at the university. His research interests are in the origins and mechanisms of antibiotic resistance and the discovery of new anti-infective strategies, particularly focusing on applying microbial natural products and synthetic biology to this goal.

Talk: Resistance-guided antibiotic discovery

Abstract: The selection for multidrug-resistant infectious pathogens and their global distribution isfuellingthe need for new antibiotics and their alternatives.Naturalproducts from bacteria and fungi are the traditionalsourcesoftheantibiotics used in human and animal health, however, the antibioticdiscoveryanddevelopmentsectorhas largely pivoted away from these molecules towards synthetic compounds over the past three decades with poor results. The move away fromnaturalproducts in antibioticdiscoverywas prompted, among other things, by the frequent rediscovery of known scaffolds, the chemical complexity and lowyieldsof manynaturalproductsthat is incompatible with modern high throughputdiscovery, and thechallengesof identifying new biological sources of compounds. The genomic era of the 21st Century offers creative opportunities to addressmany of these drawbacks. Genome sequencing of natural product-producing microbes is revealing genetic programs -biosynthetic gene clusters –representing a vast untapped source of new chemical matter that is often not accessible under laboratory conditions. By directly exploring microbial genomes and identifying biosynthetic programs that have not yet been fully explored for their antibiotic potential, it is possible to select organisms and pathways likely to yield novel chemistry and antimicrobial activity. One approach is to use resistance as a guide to identify which compounds to prioritize to refine this search. This approach, coupled with synthetic biology tools to exploit these pathways, can discover new antibiotics with new modes of action. Examples of our recent work using this genomes-first strategy coupled with a phylogenomic filter will be presented.

Wright Lab

Mary Dunlop, PhD; Associate Professor, Biomedical Engineering, Boston University

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Bio: Mary Dunlop is an Associate Professor of Biomedical Engineering at Boston University with additional appointments in Bioinformatics and in the Molecular Biology, Cell Biology & Biochemistry program. She graduated from Princeton University with a B.S.E. in Mechanical and Aerospace Engineering and a minor in Computer Science. She then received her Ph.D. from the California Institute of Technology, where she studied synthetic biology with a focus on dynamics and feedback in gene regulation. As a postdoctoral scholar, she conducted research on biofuel production at the Department of Energy's Joint BioEnergy Institute. Her lab engineers novel synthetic feedback control systems and also studies naturally occurring examples of feedback in gene regulation. In recognition of her outstanding research and service contributions, she has received many honors including a DOE Early Career Award, an NSF CAREER Award, the ACS Synthetic Biology Young Investigator Award, and an NSF Transitions Award. She is also the recipient of several teaching awards, including Boston University’s Biomedical Engineering Professor of the Year Award (2019) and the College of Engineering Teaching Excellence Award (2020).

Talk: Optogenetic Feedback Control of Gene Expression and Antibiotic Resistance in Single Cells

Abstract: Cell-to-cell heterogeneity in gene expression can elevate antibiotic resistance in one microbe while other cells remain susceptible. These transient forms of drug resistance are often stochastic and dynamic, leading to single-cell level differences in resistance that change with time. To date, methods for quantifying these effects have relied on careful observations of native expression patterns. In this talk, I will discuss a novel approach for controlling gene expression dynamics in single cells that can be used to precisely drive expression in thousands of cells in parallel. In support of this, I will discuss our recent advances in automated image processing of time-lapse microscopy data using deep learning models (DeLTA). Once trained, the DeLTA algorithm requires minimal input from the user and can rapidly segment, track, and reconstruct lineages for bacteria growing in microfluidic chips and on two dimensional surfaces. I will also discuss optogenetic control methods that allow us to use light-based feedback to regulate gene expression in real time. Using a combination of deep learning-based models and rapid image analysis, we can simultaneously control gene expression in thousands of cells in parallel. Together, these approaches offer powerful methods that can be used to quantify and control cell-to-cell heterogeneity in antibiotic resistance, providing a detailed view into strategies bacteria can use to evade drug treatment.

Dunlop Lab

Roman Manetsch, PhD; Associate Professor Department of Chemistry and Chemical Biology, and Department of Pharmaceutical Sciences, Northeastern University

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Bio: Roman Manetsch received his PhD degree in 2002 from the University of Basel (Switzerland) working on catalytic antibodies with Professor Wolf-Dietrich Woggon and Professor Jean-Louis Reymond (University of Bern, Switzerland). After a postdoctoral experience with Professor K. Barry Sharpless investigating click chemistry applications at the Scripps Research Institute in La Jolla (CA, USA), he started his independent career as an Assistant Professor at the Department of Chemistry at the University of South Florida (Tampa, Florida). In 2014, Associate Professor Manetsch moved to the Department of Chemistry and Chemical Biology and the Department of Pharmaceutical Sciences at Northeastern University (Boston, Massachusetts). His research focuses on the development of fragment-based lead discovery strategies, the development of bio-orthogonal chemical probes for the study of specific proteins in complex biological matrices, as well as hit-to-lead optimizations of antimalarial, antibacterial, and antiamoebic agents (www.manetschlab.com).

Talk: The antimicrobial activity of streptothricins and the convergent synthesis of streptothricin F

Abstract: Streptothricin F is a member of the streptothricin natural products class and is highly active against gram-positive and gram-negative pathogens ESKAPE pathogens. Common within each streptothricin are a carbamoylated gulosamine sugar core, a streptolidine lactam moiety, and a β-lysine homopolymer of varying unit length. We have recently completed a convergent, diversity-enabling total synthesis of streptothricin consisting of 35 total steps and an overall yield of 0.40%. Our convergent strategy relies on two key disconnections at the C7 and C8 amines on the gulosamine core. Deriving from these disconnections, we assembled three fragments resembling each of the three key moieties of streptothricin F. These fragments are joined together in a set of late-stage coupling reactions to form the streptothricin F backbone. Using this fully developed streptothricin F synthesis, we are pursuing the synthesis of both previously reported and novel analogs of the streptothricin class.

Complementary to our synthetic studies, we have expanded on a reported method to isolate pure streptothricin F and streptothricin D from the commercially available mixture of streptothricins called nourseothricin. With these purified streptothricins, we have characterized the inhibitory and bactericidal activity of streptothricin F across multidrug-resistant CRE pathogens, profiled its selectivity and toxicity, and defined its target through mutational resistance studies and solving the cryo-EM structures of streptothricin F and streptothricin D in complex with the 70S ribosome of Acinetobacter baumannii.

Manetsch Lab

Su Chiang, PhD; Senior Alliance Manager, CARB-X, Boston University School of Law

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Bio: Su joined CARB-X in 2018, bringing early-stage product development experience from previous roles at Harvard University as Senior Associate Director at the Blavatnik Biomedical Accelerator and Assistant Director of Small Molecule Screening at the New England Regional Center of Excellence in Biodefense and Emerging Infectious Diseases. She has a Ph.D. in Medical Sciences (Microbiology and Molecular Genetics) from Harvard.

Talk: CARB-X: Lessons Learned and Future Visions

Abstract: Combating Antibiotic-Resistant Bacteria Biopharmaceutical Accelerator (CARB-X) is a global non-profit partnership accelerating antibacterial products to address drug-resistant bacteria, a leading cause of death around the world. The CARB-X portfolio is the world’s most scientifically diverse, early development pipeline of new antibiotics, vaccines, rapid diagnostics, and other products. Funded by a global consortium of governments and foundations, CARB-X is the only global partnership that integrates solutions for the prevention, diagnosis and treatment of life-threatening bacterial infections, translating innovation from basic research to first-in-human clinical trials.  CARB-X headquarters are located at Boston University. CARB-X fills a critical gap to help stem the antibiotic resistance crisis. Beginning with competitive funding calls to address unmet medical needs, CARB-X provides non-dilutive funding as well as scientific, regulatory and business support to product developers. CARB-X focuses both on the most serious drug-resistant bacteria identified by the World Health Organization and the US Centers for Disease Control and Prevention and on the syndromes with the highest degrees of mortality and morbidity. Diagnostics are supported from feasibility through prototype development, while therapeutics and preventatives are supported from finding potential leads for new drugs through preclinical development and into a demonstration of safety in human clinical trials.

CARB-X

Kwangmin Son PhD; Co-founder and CEO, PhAST

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Bio: Dr. Kwangmin Son is the co-founder and CEO at PhAST. He is responsible for setting the vision of the company and for the overall execution of the company’s clinical development, business plan and strategic partnerships that align with the company’s strategy and product vision. Dr. Son is a recognized healthcare technology expert within the industry and was invited to present to the Presidential Advisory Council on Combating Antibiotic Resistant Bacteria (PACCARB) at the Department of Human and Health Services, at the European Congress of Clinical Microbiology & Infectious Diseases (ECCMID 2022), and at the Infectious Diseases Society of America (IDSA)’s IDWeek 2022. Dr. Son received a PhD in Engineering from Massachusetts Institute of Technology (MIT), and a Bachelor of Science and Master of Science degrees in Engineering from Seoul National University in South Korea.

Talk: Rapid Phenotypic Antibiotic Susceptibility Test (PhAST) directly from patient samples based on Artificial Intelligence and Machine Learning (AI/ML)

Abstract: PhAST is a Boston-based Artificial Intelligence (AI) healthcare technology company developing a rapid diagnostic solution for Antibiotic Susceptibility Testing (AST) that is phenotypic, applicable to multiple sample types, and sufficiently economical for wide adoption. The technology is based on the acquisition of time-lapse images (i.e., videos) of patient samples and their analysis through advanced computer vision and AI algorithms. Patient samples loaded into a disposable cartridge are automatically imaged over time. Phenotypic information of all objects present in a sample is extracted from videos and used by AI algorithms to accurately determine the presence, abundance and characteristics of pathogens and other particulates, enabling PhAST’s technology to work directly from both urine and blood culture samples and to have a uniquely rapid time-to-result: detecting infection in 10 minutes and reporting AST in 90 minutes. The system requires less than 1 minute of hands-on-time and integrates seamlessly into the clinical workflow.
The technology has been developed and tested using over 35,000 patient samples from collaborating hospitals, including Brigham and Women’s Hospital in Boston and Seoul National University Hospital in South Korea. Active data gathering and diagnostic model developments are currently in progress with the goal to cover all targeted antibiotics for full clinical validations and regulatory submissions. For urine samples, PhAST’s technology to date provides, within 10 minutes, (1) bacteriuria, (2) pyuria, (3) funguria, and (4) Gram status of pathogens. For urine samples that are infection-positive and blood cultures that have been flagged as positive (i.e., positive blood cultures), PhAST additionally reports AST in 90 minutes with an initial focus on Gram negative pathogens. The current diagnostic performance based on prospective clinical tests will be presented with respect to the FDA criteria on sensitivity, specificity, categorical agreement (SIR- Susceptible/Intermediate/Resistant) and very-major/major/minor errors. The rapid diagnostic information provided by PhAST will help healthcare practitioners to (i) avoid prescribing unnecessary antibiotics, (ii) prescribe targeted antibiotics instead of broadspectrum empirical therapy, and (iii) avoid prescribing antibiotics to which pathogens are resistant. PhAST’s technology will thus improve patient outcomes, support antibiotic stewardship, and reduce healthcare costs.

PHAST Diagnostics

Maha R. Farhat, MD MSc; Gil Omenn Associate Professor of Biomedical Informatics, Harvard Medical School
Associate Physician, Massachusetts General Hospital

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Bio: Maha Farhat holds an MD from the McGill University Faculty of Medicine and an MSc in biostatistics from the Harvard Chan School of Public Health. She is also a practicing physician at the Massachusetts General Hospital Division of Pulmonary and Critical Care Medicine. Dr. Farhat's research focuses on the development and application of methods for associating genotype and phenotype in infectious disease pathogens, with a strong emphasis on translation to better diagnostics and surveillance in resource-poor settings. To date, Farhat's work has focused on the pathogen Mycobacterium tuberculosis and spans the spectrum from computational analysis to field studies. She is PI and Co-Investigator on several large projects funded by NIH including the NIAID and the BD2K initiative.

Talk: The power and perils of DNA based diagnostics for antimicrobial resistance

Abstract: Long diagnostic wait times hinder international efforts to address antibiotic resistance in M. tuberculosis. Pathogen whole genome sequencing, coupled with statistical and machine learning models, offers a promising solution. I will present our work on achieving high accuracy and generalizability of genome based resistance phenotype prediction, and discuss current gaps and challenges. I will also discuss policies that have aided the implementation of sequencing for resistance diagnosis in a global context.

The Farhat Lab

Eric Stern, PhD; CTO and co-founder, Selux Diagnostics

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Bio: Eric is the Chief Technology Officer of Selux Diagnostics and the father of two little girls. He earned his BS in Chemistry, his MS in Electrical Engineering, and his PhD in Biomedical Engineering from Yale University and performed his postdoctoral work at Harvard University. He worked as a Senior Engineer and Director at two alternative energy startups before co-founding Selux with Alek Vacic, his good friend and the smartest guy he knew in grad school. Eric is incredibly grateful to the Selux investors and advisors for making the company possible and the amazing Selux team that is dedicated to redefining AST and is a blast to work with.

Talk: AST: The original companion diagnostic

Abstract: Accurate, rapid antimicrobial susceptibility test (AST) results are critical for infectious diseases patient care and for combatting the antimicrobial resistance (AMR) epidemic. While AST in US clinical microbiology laboratories is synonymous with automated platforms, these legacy systems suffer from three serious shortcomings: slow speeds, incomplete menus, and an inability to update breakpoints despite FDA guidance and CAP requirements. These issues result from the core assay design of these systems, the determinition of minimum inhibitory concentrations (MICs) by growth curves. This design lies in sharp contrast to the endpoint determination of the gold-standard reference method for AST, broth microdiluton (BMD). Here we introduce a divide-and-conquer strategy that emulates BMD by separating the determination of sufficient microorganim growth from the MIC-determining assays. This design enables accurate, same-shift results to be obtained with 384-well consumable panels that support parallel testing of dozens of antimicrobials, all with updated breakpoints.

Daniel R. Vlock, MD; CEO, Alopexx, Inc.

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Bio: Dr. Vlock is CEO of Alopexx. Previously, Dr. Vlock held senior-level medical and research positions at GPC Biotech, Inc., Pharmacia Corporation, Ethicon Endo-Surgery (a subsidiary of Johnson & Johnson Company), and Boehringer Ingelheim Pharmaceuticals, Inc. where he was involved in the development of numerous oncology drugs, medical devices and drug-device combinations. While at Pharmacia he ran the Celebrex Oncology Group. While at other companies he ran programs which successfully developed and introduced monoclonal antibodies into clinical trials. Prior to moving to industry, Dr. Vlock was on the faculty at Harvard Medical School, University of Pittsburgh School of Medicine and Yale University School of Medicine, where he led laboratory and clinical programs in tumor immunology.

Dr. Vlock received his A.B. from Cornell University and an M.D. from Baylor College of Medicine. He completed a research fellowship in clinical immunology at Baylor College of Medicine, an internship and medical residency at Temple University Hospital and a senior medical residency and fellowship in medical oncology at Yale New Haven Hospital.

Talk: Targeting PNAG  (Poly N-Acetyl Glucosamine)  Development of Broad-Spectrum Immunotherapeutics for the Prevention and Treatment of a Wide Range of Microbial Infections

Abstract: For microbial pathogens, surface capsules, often composed of highly varied carbohydrate polymers, have been used successfully to vaccinate humans against Streptococcus pneumonia, Hemophilus influenzae type b, Neisseria meningitidis and Salmonella enterica serovar typhi. However, efficacy is limited to the capsule types included in the vaccines. The PNAG polysaccharide is different. It has been found to be a component of the microbial surfaces of a large and expanding number of bacterial, fungal and parasitic pathogens. The synthesis of PNAG by all of these types of pathogens suggests an important role for this molecule in microbial biology. This indicates that PNAG could serve as a therapeutic target against many different microbes.

Natural antibodies to PNAG are common in humans and many animal species but these antibodies do not routinely have robust opsonic killing or immune protection against PNAG-producing microbes. This is due to inadequate engagement of the serum complement co-factor system that is needed for the induction of protective immunity. Thus, broad-based immunity to this antigen rarely develops naturally in human or animal populations. Naturally occurring microbial colonization or infection does not usually induce effective antibody responses to PNAG.

Alopexx’s major proprietary advance was the recognition that this ineffective natural immunity was due to the high degree of acetylation of the native PNAG molecule. Those surface acetyl groups are responsible for inducing a natural but ineffective immune response. The acetyl groups can be removed by chemically altering PNAG. This results in a strong protective immunogen that now targets the backbone of PNAG and avoids participation from the acetyl groups.

Alopexx has developed two immune therapeutics directed at PNAG – a monoclonal antibody and a vaccine. The potential efficacy of these immune therapeutics has been evaluated in studies of 14 pathogens in 20 animal models of infection. In all of those infection models a positive therapeutic effect was observed in preventing or treating infections.

These anti-infective immune therapeutics represent a potential paradigm shift in the prevention, treatment, and amelioration of many microbial infections. Anti-bacterial antibiotics and anti-fungal drugs must be administered frequently and over periods of days to months, whereas antibodies induced by the vaccine or administered as a monoclonal antibody are injected once or twice months apart and remain at high levels for 2-12 months. The Alopexx immunotherapeutics would be used in the same settings that antibiotics are used such as the intensive care unit, individuals undergoing surgery, people with suppressed immune systems and persons at high risk for microbial infections. This paradigm shift would be driven by the increased efficacy in reducing infections and costs due to effective immunity to PNAG. The Alopexx vaccine and monoclonal antibody could dramatically alter the way many infections are prevented and treated and significantly reduce reliance on antibiotics and the development of anti-microbial resistance.

Alopexx

Katharina Ribbeck, PhD; Professor, Department of Biological Engineering, MIT

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Bio: Prof. Ribbeck obtained her Bachelor’s degree and her PhD in Biology from the University of Heidelberg, Germany. She continued her postdoctoral research at the European Molecular Biology Laboratory, Heidelberg, Germany, and the Department of Systems Biology, Harvard Medical School. Katharina Ribbeck established her independent research group as a Bauer Fellow at the FAS Center for Systems Biology, Harvard University in 2007, and joined the Department of Biological Engineering at MIT as an Assistant Professor in 2010. Her research is focused on mucus, the gel that lines all wet epithelia in the body. Her focus is on basic mechanisms by which mucus barriers exclude, or allow passage of different molecules and pathogens, and the mechanisms pathogens have evolved to penetrate mucus barriers. One mission of her lab is to implement the lessons learned from nature and create synthetic mucus gels that mimic the basic health promoting and protective properties of the biological material.

Talk: Mucin glycans regulate microbial virulence

Abstract: Mucus is a biological gel that lines all wet epithelia in the body, including the mouth, lungs, and digestive tracts, and has evolved to protect us from pathogenic invasion. Microbial pathogenesis in these mucosal systems, however, is often studied in mucus- free environments, which lack the geometric constraints and microbial interactions that are found in natural, three- dimensional mucus gels. My laboratory has developed model test systems to understand how the mucus barrier influences microbial virulence, and moreover, to elucidate strategies used by microbes to overcome the normal protective mucus barrier. We show that mucin polymers, and specifically their associated glycans, significantly impact the physiological behavior of microbes, including surface attachment, quorum sensing, the expression of virulence genes, and biofilm formation. The picture is emerging that mucin glycans are key host players in the regulation of microbial virulence and underscores the untapped therapeutic opportunities found in these host-derived molecules.

The Ribbeck Lab

Bernat Olle, PhD; CEO, Vedanta Biosciences

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Bio: Dr. Olle is a co-founder and Chief Executive Officer of Vedanta Biosciences. He has been a member of the founding teams of several companies of the PureTech portfolio and served as a member of the Board of Directors of Vedanta Biosciences and Follica Biosciences.

In 2013 Dr. Olle was named "Innovator of the Year" in MIT Technology Review Spain's "Innovators under 35" awards. He also received the 2019 Barry M. Portnoy Immigrant Entrepreneur Award from The Immigrant Learning Center.

He completed his doctoral work at the Chemical Engineering Department at MIT, where he developed a novel method for large-scale bacterial culture. During his graduate work, Dr. Olle was awarded the "la Caixa" fellowship. Dr. Olle received his B.S. in Chemical Engineering from Universitat Rovira i Virgili, in Catalonia, his M.S. and PhD. in Chemical Engineering Practice from MIT, and his M.B.A. from the MIT Sloan School of Management. He has published his work in journals including Nature and Nature Biotechnology.

Talk: Development of a defined bacterial consortium candidate for the prevention of Gram-negative infections

Abstract:

Infections with multidrug-resistant organisms (MDRO) are increasing at an alarming rate in hospitals worldwide. Due to the rapidly growing threat of antibiotic resistance, there is an urgent unmet need for novel therapies to tackle MDRO infections. Intestinal MDRO colonization, resulting from microbiota disruption, frequently precedes infection with the colonizing organism. As such, decolonization strategies such as non-absorbable antibiotics and fecal microbiota transplantation (FMT) have shown efficacy at preventing infection following decolonization. Despite the success of FMT at resolving intestinal MDRO colonization and dysbiosis without leading to resistance, FMT efficacy is variable and its safety profile questionable. This highlights the need for a uniform, well-characterized microbiome-based product with robust efficacy that can be produced and administered in a standardized manner.

Vedanta is developing VE707, a defined live biotherapeutic product (LBP) consisting of beneficial gut bacteria to reduce intestinal carriage of carbapenem-resistant and ESBL-producing Klebsiella pneumoniae (Kpn) and Escherichia coli (Eco), restore a healthy microbiota and prevent infection and colonization recurrence. Using a top-down approach, we first characterized fecal material from healthy individuals for their ability to suppress Kpn and Eco and identified a donor enriched for activity against both pathogens. Next, we used a series of  in vitro, in silico, and in vivo tools to assemble bacterial strains from this donor into LBPs and evaluated their ability to decolonize Kpn and Eco in a mouse co-colonization model. Of 70 LBPs screened, VE707 showed the greatest decolonization efficacy as demonstrated by a ≥3-log reduction in Kpn and Eco fecal levels. Furthermore, VE707 was active against several MDR Kpn and Eco clinical isolates. Our results show that VE707, a defined bacterial consortium with pathogen-antagonistic properties, was successful at decolonizing Kpn and Eco and it is currently in manufacturing phase.

Vedanta Biosciences

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Visiting Fellow, Massachusetts Eye and Ear, Harvard Medical School, Department of Ophthalmology

Poster Title: Genomic epidemiology and antimicrobial resistance of MRSA isolates causing eye infections.

Authors: C. André, M.S. Gilmore, P. J. M. Bispo

Abstract: Purpose: Staphylococcus aureus is a major cause of eye infections. Methicillin-resistant S. aureus (MRSA) is commonly associated with multidrug-resistant (MDR) infections, resulting in treatment failure and poor visual outcome. To assess the emergence of high-risk MDR clones of MRSA causing eye infections and the repertoire of antimicrobial resistance (AMR) genes circulating among this population, we have sequenced the genomes of all MRSA isolates recovered from patients presenting with eye infections between January 2014 and December 2021 at the Massachusetts Eye and Ear.

Methods: Whole Genome Sequencing was performed on 160 MRSA isolates causing eye infections using Illumina MiSeq. Molecular typing was performed by multilocus sequence typing. CARD and Pathogenwatch were used to identify genes and mutations that confer AMR. Routine antimicrobial susceptibility testing was performed by using the MicroScan WalkAway system.

Results: The population structure of MRSA isolates causing eye infections was dominated by lineages grouped within the clonal complex 5 (CC5) (40.0%) and CC8 (46.9%). Patterns of association of distinct genotypes have been noted with orbital abscess/cellulitis mainly caused by CC8 (64.8%) whereas keratitis were substantially enriched in the CC5 lineage (54.5%). 70% of MRSA CC5 isolates were MDR (resistance to ≥3 antimicrobial classes). In line with these observations, we found that the resistome of these CC5 strains was comprised of multiple acquired AMR genes that confer resistance to various clinically important antibiotics including aminoglycosides, macrolides, lincosamides, and streptograminB. Transference of some of these genes between strains is facilitated by horizontal gene transfer as they are found in mobile genetic elements such as transposons. In our CC5 population the mecA gene is integrated into a SCCmec cassette which also carries a full transposon (Tn554) containing the ermA and ant(9)-Ia genes, both present in 79.7% of our CC5 isolates. We found between 2 and 4 mutations in the quinolone resistance-determining regions (QRDR) of gyrA, parC and parE genes for MRSA isolates that were associated with resistance to fluoroquinolones.

Conclusions: MRSA isolates causing eye infections mainly clustered within the CC8 and CC5, with patterns of disease/tissue enrichment. CC5 strains carried a constellation of AMR genes and mutations that result in high levels of phenotypic resistance to first-line topical therapies.

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Postdoctoral Research Fellow, Department of Pathology, Beth Israel Deaconess Medical Center

Poster Title: Apramycin-based Antimicrobial Combinations: More Efficacious and Less Toxic Treatment Options against M. abscessus Infections

Authors: Yanqin Huang, Katherine Truelson, Isabella A. Stewart, James E. Kirby

Abstract: Background: Combining amikacin with other antimicrobials is a common strategy for treatment of Mycobacterium abscessus infections, a leading cause of chronic progressive pulmonary disease. However, the associated kidney damage and permanent hearing loss from long-term use of amikacin in these regimens is highly problematic. Apramycin, a bicyclic aminoglycoside with altered ribosomal binding, may have an improved safety profile, supported by early clinical trial data and in vitro studies. Therefore, this study aimed to define efficacy of apramycin- in comparison with amikacin-based combination therapy as a potentially less toxic therapeutic option for M. abscessus infections.

Methods: MICs of apramycin, amikacin, linezolid, clofazimine, and tigecycline were tested against 17 M. abscessus clinical isolates by broth microdilution. Checkboard synergy testing by innovative inkjet printer technology was used to explore combinations of apramycin and amikacin respectively with linezolid, clofazimine, and tigecycline against three clinical isolates (#18, #27, and #28). Time kill assays were performed for apramycin or amikacin at 1xMIC, 2xMIC, and 4xMIC in combination with clofazimine at 2xMIC for isolate #27.

Results: The modal MICs for apramycin and amikacin were 2 mg/L and 16 mg/L, respectively. The checkboard assays showed that fractional inhibitory concentration index (FIC) values for apramycin combinations ranged from 1.5 to 3 (mode of 1.5), while FIC values for amikacin combinations ranged from 1.5 to 5.5 (mode of 3). In time-kill assays, clofazimine at 2x MIC did not show antimicrobial activity. However, in combination with apramycin 1xMIC, 2xMIC, or 4xMIC, there was a 3.31, 3.67, and 4.80 log10CFU reduction, respectively; while in combination with amikacin at 1xMIC, 2xMIC, or 4xMIC there was a reduction of 0.42, 0.89, and 2.15 log10CFU, respectively, compared to clofazimine 2xMIC monotherapy.

Conclusions: Apramycin-based combinations were more potent than amikacin-based combinations against M. abcessus while both provide similar in vivo exposure based on available PK data. Therefore, apramycin may offer a safer and more efficacious treatment option for M. abscessus infections worthy of further investigation.

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Senior Research Fellow, Broad Institute of MIT and Harvard

Poster Title: Mechanism-dependent phenotypic responses of carbapenem-resistant Enterobacterales (CRE)

Authors: Alexis Jaramillo Cartagena, Kyra Taylor, Joshua Smith, Abigail Manson, Ashlee Earl, Virginia Pierce, Roby Bhattacharyya

Abstract: The global spread of carbapenem-resistant Enterobacterales (CRE) presents a major threat to public health as these pathogens are resistant to some of our best antibiotics and are responsible for thousands of deaths annually in the Unites States. CRE employ two molecular mechanisms of resistance: 1) expression carbapenemases (CPases), which efficiently hydrolyze carbapenems or 2) disruption of porins, which reduces carbapenem influx.

We measured carbapenem minimum inhibitory concentrations (MICs) for 103 Enterobacterales isolated from hospitals in Massachusetts and California using broth microdilution assays at 14 inocula spanning four orders of magnitude. We observed the two mechanisms result in distinct profiles; the MICs of CPase-encoding isolates show strong inoculum dependence, whereas the MICs of porin deficient isolates remain largely constant at all inocula. The synergistic action of these mechanisms leads to high-level resistance that we termed “hyper-CRE”. To validate the hyper-CRE phenotype, we employed CRISPR-based gene editing to knock out the major porin in CPase-producing strains, which elevated their carbapenem resistance to hyper-CRE levels. We also discovered that in some Klebsiella pneumoniae isolates the upregulation of a transcription factor increases resistance to multiple antibiotics including quinolones, tetracyclines, nitrofurans, sulfonamides, and carbapenems.

We also determined 18% of our isolates changed susceptibility classification. This is worrisome for the treatment of infections with strains that are deemed “susceptible” via in vitro MIC assays but are truly resistant. Overall, our approach demonstrates that measuring MICs at different inoculum can yield crucial diagnostic information about mechanisms of resistance which has important implications for patient care, infection control, and surveillance of emerging CPases.

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Graduate Research Fellow, Boston University Department of Biomedical Engineering

Poster Title: Near-Infrared Antimicrobial Activity Against MRSA

Authors: Sebastian Jusuf, Yuewei Zhan, Jiyang Chen, Ramprasath Rajagopal, Shyamsunder Erramilli, Ji-Xin Cheng

Abstract: Among antibiotic resistant bacterial strains, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as remained one of the leading pathogens responsible for deaths associated with resistance, causing more than 100,000 deaths worldwide in 2019 alone. The proliferation of this strain has triggered the need for alternative, non-drug reliant methods of treating MRSA infections. While Cheng Lab has discovered the capability of blue light to sensitize MRSA infections to antimicrobial agents through light induced photolysis of the MRSA staphyloxanthin (STX) pigment, the usage of blue light at all limits clinical translation due to the high absorbance of blue light through skin. To address this issue, the Cheng Lab has further innovated on the applications of phototherapy by demonstrating the ability for near-infrared (NIR) light to photobleaching the STX pigment present in MRSA, allowing for the increased effectiveness of reactive oxygen species (ROS) sources and re-sensitization of conventional antibiotics against MRSA. Through two-photon excitation of STX, NIR treatment of MRSA can allow for greater clinical applicability of phototherapy against deep tissue MRSA wounds, bypassing several of the current limitations with blue light.

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PhD Student, Northeastern University Department of Chemistry and Chemical Biology

Poster Title:Antimicrobial and structural characterization of streptothricin F, and the convergent synthesis of streptothricin analogs

Authors: Yuliya A. Marusyk, Brandon C. Miller, Matthew G. Dowgiallo, Mintesinot Kassu, Kenneth P. Smith, Christopher E. Morgan, Hanna R. Warinner, Andrew Fetigan, Edward W. Yu, James E. Kirby, Roman Manetsch

Abstract: Streptothricin F is a member of the streptothricin natural products class and is highly active against Gram-positive and Gram-negative pathogens of resistance-concern. Natural isolates of varying streptothricin homolog compositions are referred to as nourseothricin, and common within each streptothricin are a carbamoylated gulosamine sugar core, a streptolidine lactam moiety, and a β-lysine homopolymer of varying length.
We have recently completed a convergent, diversity-enabling total synthesis of streptothricin F consisting of 35 total steps and an overall yield of 0.40%. Using this fully developed streptothricin F synthesis, we are pursuing the synthesis of both previously reported and novel analogs of the streptothricin class. Complementary to our synthetic studies, we have expanded on a reported method to purify the nourseothricin mixture and enable access to streptothricin F and streptothricin D. We have characterized the inhibitory and bactericidal activity of streptothricin F across multidrug-resistant CRE pathogens, profiled the selectivity and toxicity, and confirmed the efficacy of streptothrcin F in the presence of a representative set of Gram-negative antibiotic resistance mechanisms. Additionally, we have solved the bound cryo-EM structures of streptothricin F and streptothricin D in complex with the 70S ribosome of Gram-negative Acinetobacter baumannii.

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Postdoctoral Research Fellow at the Dept. of Pathology at BIDMC & Harvard Medical School

Poster Title: Avibactam as a Model Diazabicyclooctane: Activity against Highly Resistant Gram-Negative Pathogens

Authors: Michelle Naegeli, Shade Rodriguez, James E. Kirby, Thea Brennan-Krohn

Abstract: Introduction: Gram-negative bacteria are increasingly resistant to existing beta-lactam antibiotics. Avibactam, a diazabicyclooctane (DBO) beta-lactamase inhibitor, was the first non-beta-lactam beta-lactamase inhibitor approved for clinical use, in combination with ceftazidime. Ceftazidime-avibactam is not active against metallo-β-lactamase (MBL)-producing isolates strains and has limited activity against carbapenem-resistant Acinetobacter baumannii (CRAB). New DBOs in development offer possibilities for treatment of these organisms, either through direct antibacterial activity against MBL-producing Enterbacterales (zidebactam, nacubactam) or restoration of sulbactam activity against CRAB (durlobactam). We investigated the activity of avibactam as a prototype for these newer DBOs, which are not yet clinically available.
Methods: To evaluate the spectrum of direct avibactam activity, we determined the minimal inhibitory concentration (MIC) of avibactam against 74 bacterial strains (92% carbapenemase producers) using an inkjet printer-assisted digital dispensing method and determined baseline avibactam resistance mutation frequency of two avibactam-susceptible strains. We then evaluated synergistic activity of avibactam in combination with sulbactam, meropenem, aztreonam, and ceftazidime against 15 CRAB strains.
Results: Avibactam showed direct activity against most Enterobacterales strains (MIC50 = 16 ug/mL) but not against Pseudomonas or Acinetobacter isolates (MIC50 >64 ug/mL). The baseline rate of resistance in two strains with MICs of 8 ug/mL was 6.7-8.1x10-6. Avibactam at 4 ug/mL lowered the MIC50 of sulbactam from 16 to 4 ug/mL and the MIC50 of ceftazidime from >64 to 32 ug/mL; there was little effect when avibactam was combined with meropenem or aztreonam.
Conclusion: Avibactam had direct antimicrobial activity against most Enterobacterales isolates, and while it is less potent than newer DBOs, it may be useful as a tool for in vitro study of novel DBOs. The combination of avibactam with sulbactam may be a potential treatment option for highly resistant CRAB isolates, especially considering the fact that there are very few effective therapy options available for these infections.

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Research Assistant, Beth Israel Deaconess Medical Center Department of Pathology

Poster Title: Antibiotic activity on Burkholderia cepacia complex grown in Artificial Sputum Media

Authors: Anusha Shukla, Shade Rodriguez, Dr. James Kirby, Dr. Thea Brennan-Krohn

Abstract: Burkholderia cepacia complex (Bcc), a collection of Gram-negative bacteria of at least nine species, has emerged as a highly problematic opportunistic pathogen for Cystic Fibrosis (CF) and immunocompromised patients. Bcc’s intrinsic resistance to many antibiotics and its ability to rapidly acquire additional resistance mechanisms limit treatment options. The standard method of antibiotic susceptibility testing (AST) is limited in its ability to predict clinical outcomes for CF patients. In this study, an artificial sputum media (ASM) that includes mucin, amino acids, free DNA and albumin, was prepared that better represents the microaerophlic mucus layer in which bacteria grow in the lungs of people with CF. Two strains of Bcc, Burkholderia cenocepacia and Burkhoderia multivorans, were grown in ASM and treated with antibiotics with activity against Bcc. Patterns of bacterial growth and antibiotic activity were tested and validated over 72 hours with two complementary assays: a time-kill study, and a fluorescence-based growth detection assay with resazurin. Furthermore, a preliminary microcalorimetry assay was preformed to compare growth patterns of different strains of Bcc and other bacteria grown in cation-adjusted Mueller-Hinton broth (CAMHB) and ASM. Differences between growth in standard media and in ASM, and between minimal inhibitory concentration (MIC) results and activity of antibiotics in ASM, underscored the importance of identifying assays that more accurately predict Bcc growth and antibiotic activity in vivo. In vitro susceptibility testing in ASM may have a future role in predicting clinical efficacy of Bcc treatment regimens for people with CF.

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Graduate Student, Harvard University Department of Chemistry and Chemical Biology

Poster Title: Discovery Through Chemical Synthesis of Antibiotics Effective Against Multidrug-resistant Bacterial Pathogens

Authors: Kelvin J. Y. Wu, Ben I. C. Tresco, Elena V. Aleksandrova, Egor A. Syroegin, Antonio Ramkissoon, Amy Benedetto, Dominic N. See, Maxim S. Svetlov, Yury S. Polikanov, Andrew G. Myers

Abstract: We disclose a fully-synthetic class of antibiotics that combines macrocyclic thiolincosamines with an oxepanoprolinamide motif to successfully overcome acquired and intrinsic lincosamide resistance in high priority ESKAPE pathogens. We demonstrate the power of preorganization as a molecular design strategy to overcome multiple modes of bacterial antimicrobial resistance. Furthermore, these studies highlight the ability of total synthesis to elucidate drivers of antibacterial potency through single atom substitutions, uncovering the previously unappreciated importance of the thioglycoside moiety common to lincosamide antibiotics.

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PhD Student, Boston College, Beth Israel Deaconess Medical Center

Poster Title: Amotosalen is a Bacterial Multidrug-Resistance Efflux Pump Substrate Potentially Affecting its Ability to Inactivate Pathogens in Blood Transfusion Products

Authors: Alex B. Green, Lucius Chiaraviglio, Katherine A. Truelson, Katelyn E. Zulauf, Meng Cui, Zhemin Zhang, Matthew P. Ware, Isabella Stewart, Willy A. Flegel, Richard L. Haspel, Ed Yu, James E. Kirby

Abstract: Background. Bacterial contamination of transfusion products is a leading cause of transfusion-associated mortality. Culture-confirmed sepsis is estimated to occur in at least 1 in 100,000 platelet transfusions without the use of pre-emptive pathogen reduction technology. The INTERCEPT Blood System is approved in the US to pre-emptively inactivate pathogens in platelets and plasma units. It uses amotosalen, a derivatized psoralen, in combination with UVA light to irreversibly crosslink nucleic acid leading to pathogen killing. We noted structural similarity of amotosalen to efflux pump substrates and therefore hypothesized that contemporary multidrug-resistant pathogens might be resistant to amotosalen based on expression of RND efflux pumps.

Methods. A. baumannii adeAB was cloned under a pBAD promoter; adeC was cloned under an IPTG-inducible promoter; P. aeruginosa mexAB and mexXY, and E. coli acrAB were also cloned under a pBAD promoter. Constructs were transformed into ΔacrAB, ΔacrEF-deficient E. coli, AG100AX. We determined the amotosalen minimal inhibitor concentration (MIC) for these E. coli strains; for clinical strains; and for strains with isogenic knockouts of RND pumps, after exposure to UVA light and overnight incubation. A fluorescence polarization assay was used to determine the binding affinity of amotosalen for AdeB.

Results. A majority of contemporary clinical A. baumannii, E. coli, K. pneumoniae, P. aeruginosa, S. maltophila, and Burkholderia spp. isolates had MICs exceeding or similar to the 150 µM concentration of amotosalen used during pathogen inactivation. Heterologous expression of AdeABC, AcrAB, and MexXY in E. coli resulted in substantial increases in MIC (up to 32-fold) compared with vector controls, while E. coli tolC and lptD mutants were notably susceptible. We tested isolates’ susceptibility in the presence of an efflux pump inhibitor, PaβN, and MICs decreased for amotosalen 64X, indicating efflux pump activity. To estimate the amount of free drug available in the media, we conducted serum- and albumin-binding competition experiments, and the MIC of amotosalen increased 2-fold, and 32-fold with 4% human serum albumin, potentially indicating binding site occupation in the presence of human serum. Additionally, the KD of amotosalen for major efflux pump subunit AdeB was 27.9±1.8 µM, a similar binding dissociation constant of other known efflux substrates to this pump.

Conclusion. Amotosalen is a substrate for major efflux pumps in gram-negative bacteria. Our data may guide studies on pathogen inactivation technology in transfusion medicine and the potential resistance in contemporary MDR pathogens.