Download 1 AMR - The global problem requiring global

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AMR - The global problem requiring global coordination and response
Sally Davies1
1. Department of Health - UK, London, LONDON, United Kingdom
Antimicrobial Resistance (AMR) is a complex problem requiring diverse and dynamic solutions. It is not a problem that any one
country can tackle in silo. Dame Sally will explore the factors which contribute to the development of drug resistant infections
and discuss the many different approaches that need to be considered and implemented to tackle this problem. She will
highlight why a coordinated response - globally, across a number of different sectors - is the only way to tackle drug resistance
infections and the steps we are taking to move this forward globally.
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From the prostate biopsy sepsis nexus to shrimp gate: A journalist’s AMR journey
Jason Gale1
1. Bloomberg News, Sydney, NSW, Australia
The media can be a powerful ally in creating awareness and effecting change in response to antimicrobial resistance.
Bloomberg’s Jason Gale discusses reporting on prostate biopsies, NDM-1 and commercial poultry production in India, and the
murky world of aquaculture.
Key publications:
How Antibiotic-Tainted Seafood From China Ends Up on Your Table (Dec 2016)
China's Five-Star Pig Pens Are Latest Weapon in the Superbug War (Sept 2016)
Antibiotic Apocalypse Fear Stoked by India’s Drugged Chickens (March 2016)
Superbugs spread to 40 nations threatening India medical tourism (May 2012)
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GARDP, a PDP Contributing to Address Public Health Priorities
Jean-Pierre Paccaud1
1. Global Antibiotic R&D Partnership, Geneva, Switzerland
The Global Antibiotic Research and Development Partnership (GARDP) is a not-for-profit research and development
organization that addresses global public health needs in the field of bacterial infectious diseases by developing and delivering
new antibiotic treatments, while endeavouring to ensure stewardship and affordable access to them. Initiated and incubated
through close collaboration between WHO and Drugs for Neglected Diseases initiative (DNDi), GARDP is part of the
implementation of the Global Action Plan on Antimicrobial Resistance that calls for new public-private partnerships for
encouraging research and development of new antimicrobial agents and diagnostics. GARDP has received seed funding from
the governments of the United Kingdom, Germany, the Netherlands, South Africa, and Switzerland, as well as from the medical
humanitarian organization Médecins Sans Frontières. GARDP is a joint WHO/DNDi initiative being currently hosted within DNDi
in its start-up phase.
GARDP’s mission is to work in partnership with the public and private sectors to develop and deliver new treatments for
bacterial infections wherever drug resistance is present or emerging, or for which inadequate treatment exists, and where it is
unlikely that they will be developed by the private sector.
GARDP will prioritize R&D projects by focusing on unaddressed global public health gaps, particularly for drug-resistant
bacterial infections. Through partnerships, collaborations, and coordination, GARDP will ensure that new antibiotic treatments
remain affordable subject to a global sustainable access agenda, including stewardship.
GARDP has, within its first 8 months, built up a team of 10 people with additional support staff from DNDi contributing directly to
the GARDP programmes and is actively fund-raising to start projects by end of 2017.
Priority setting methodology and R&D interventions considered to date will be presented and discussed.
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Using genomics to reveal the secrets of multi-drug resistant superbugs
Scott Beatson1
1. University of Queensland, Brisbane, QLD, Australia
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Multiple antimicrobial resistant Escherichia coli from humans and food animals – A One Health
Approach
Steven Djordjevic1, Cameron Reid1, Max Cummins1, Jessica McKinnon1, Ethan Wyrsch1, Toni A Chapman2, Michael
Liu1, Piklu Roy Chowdhury1, 2, Aaron E Darling1
1. The ithree institute, The University of Technology, Sydney, Broadway, NSW, Australia
2. NSW Department of Primary Industries, Elizabeth Macarthur Agricultural Institute, Menangle, NSW, Australia
The number of deaths from antimicrobial-resistant infections has been estimated to rise to 10 million p.a. by 2050, of which 3
million p.a. are predicted to succumb to infections caused by multi-drug resistant Escherichia coli (E. coli). E. coli pathogens are
broadly defined as intestinal pathogenic E. coli (IPEC) and extra-intestinal pathogenic E. coli (ExPEC) and outbreaks of severe
disease have been caused by both. Multiple antimicrobial resistance (MAR) has not garnered much attention in IPEC, but the
development of MAR in ExPEC poses a serious threat to human and food animal health. ExPEC represent the most common
Gram negative pathogen in humans and are responsible for a range of diseases including infections of the urinary tract (cystitis,
prostatitis, pyelonephritis), sepsis and meningitis, particularly in neonates. ExPEC infections incur an enormous cost to health
care budgets and often have a foodborne aetiology, but there are major gaps in knowledge. There is an urgent need to better
understand human, food animal and environmental resistomes. Over 500 E. coli that carry the class 1 integron integrase gene
intI1, a reliable proxy for a multiple drug resistance genotype were sequenced. Our collection includes uropathogenic E.coli
from humans and a combination of commensal and pathogenic E. coli from intensively reared food animals. We examined the
phylogenetic relationships these E. coli share and examined the repertoire of mobile genetic elements that play an important
role in mobilizing antimicrobial resistance genes. The datasets underpin a One Health approach to tackling the problem of
antimicrobial resistance.
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Reclaiming legacy drugs with antibiotic adjuvants
Gerry Wright1
1. McMaster University, Hamilton, ONTARIO, Canada
The challenges of addressing the crisis of antibiotic drug discovery are significant. Discovering new antibiotics with novel
chemical scaffold is proving to be difficult to achieve. A complimentary strategy is through the co-admininsteation of antibiotic
adjuvants that block resistance mechanisms directly or indirectly thereby rescuing the activity of existing drugs. The advantages
of this approach include the maintenance of proven drugs with excellent activity, known pharmacological properties and proven
(and often lengthy) experience in the clinic. Challenges to this approach include the fact that this approach requires
combinations of agents that need to be matched for pharmacological properties. Nevertheless, the strategy is well established
in infectious disease therapy in the form of beta-lactam/beta-lactamase inhibitor combinations. Recent work on identifying
agents that block resistance will be presented along with efforts to reverse resistance to a variety of antibiotic classes.
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Using in vitro evolution to define the resistome and the drug-able genome
Elizabeth Winzeler1
1. University of California San Diego, La Jolla, CALIFORNIA, United States
In order to achieve malaria elimination as well as to overcome parasite drug resistance, new classes of medicines are needed
that have the capacity to completely eliminate the parasites that cause malaria. To identify new leads for drug discovery we
are using two approaches. First we are developing and implementing phenotypic screens that can identify compounds that are
likely to provide symptomatic relief, protect against malaria and prevent malaria transmission. We are also working to identify
the targets of these compounds that we identified by black box screening with the goal of developing biochemical assays. To
this end we have performed in vitro evolution in the presence of 37 different, potent, small molecule inhibitors of parasite growth
using both the model organism, Saccharomyces cerevisiae, as well as Plasmodium falciparum. Whole genome sequencing of
more than 500 isogenic drug resistant clones has provided the identity of many new drug-able proteins as well as a
comprehensive list of the genes that contribute to multidrug resistance in eukaryotic pathogens.
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Early assessment of drug resistance to guide the development of next-generation
antimalarials
Didier Leroy1
1. Medicines for Malaria Venture, Geneva, GENEVA, Switzerland
Today, one of the major challenges to discover, develop and deliver new generations of effective antimalarial drugs concerns
the evolution of resistance. Not only should new drug candidates be efficacious and well tolerated but they should also, ideally,
be resistance proof when deployed in the field. However, given that resistance of some degree has emerged to almost all
antimalarials, the remaining question is how to evaluate and make decisions on compounds, regarding resistance, in the
discovery phase given uncertainty in how such findings will translate to the clinic.
To address this key question, Medicines for Malaria Venture (MMV) – a not-for-profit product development partnership prioritises the testing of portfolio compounds against panels of sensitive and multidrug-resistant P. falciparum strains (clinical
isolates adapted to culture and resistant lines generated in the laboratory) and clinical isolates freshly isolated from the field.
Also, the selection of drug resistance is studied in the laboratory and accompanied by sequencing any mutants to identify
markers. Furthermore, analysis of published parasite genomes to look for any identified resistance markers helps answer
whether pre-existing mutations are prevalent.
Altogether, this strategy provides a framework to guide the design and prioritisation of new antimalarials, and a risk assessment
of resistance before major investments are made in drug development. In addition, this approach is expected to facilitate the
selection of adequate partner drugs for combination therapies as drug candidates progress in the portfolio.
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Discovery of LFF571 as an investigational agent for C. difficile infection: lessons learned in the
opportunities and challenges of antimicrobial development
Jennifer Leeds1
1. Novartis Institutes for BioMedical Research, Emeryville, CALIFORNIA, United States
Clostridium difficile infection causes serious diarrheal disease. In the United States it is estimated that C. difficile caused
approximately 453,000 incident infections and was associated with approximately 29,000 deaths in the United States in 2011.
The US Centers for Disease Control categorizes C. difficile at a hazard level of “urgent” with respect to public health threats.
Several drugs are available for treatment of C. difficile infection including vancomycin, metronidazole and the newer agent
fidaxomicin. Nevertheless recurrences of disease remain a problem. LFF571 is a novel semisynthetic macrocyclic thiopeptide
antibiotic that was designed to achieve high aqueous solubility and excellent potency against a wide spectrum of Gram positive
pathogens including C. difficile. Following extensive pre-clinical profiling, LFF571 was shown in a phase 2 exploratory study to
be efficacious and well-tolerated for the treatment of C. difficile infections in adults with primary episodes or first recurrences of
moderate C. difficile infection. LFF571 represents a novel chemical class which inhibits a molecular target that is
unprecedented in human clinical therapy, and LFF571 has the potential to be a safe and efficacious addition to the treatment
options for C. difficile infection.
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Conjugation of an Acinetobacter selective siderophore to the Gram-positive active only
antibiotic, daptomycin, generates a sideromycin with potent activity against Gram-negative
Acinetobacter baumannii
Marvin Miller1
1. University of Notre Dame, Notre Dame, INDIANA, United States
Daptomycin, a lipopeptide antibiotic, is a valuable treatment against Gram-positive bacteria. It acts by perturbing the integrity of
the Gram-positive bacterial cell wall. It circumvents a number of bacterial defense mechanisms, such as efflux pumps. We
hypothesized that conjugation of daptomycin, to our previously reported Acinetobacter selective mixed ligand siderophore,
would allow us to determine if it is possible to target Gram negative bacteria with what are normally Gram-positive active
antibiotics using the siderophore-based iron transport mediated Trojan Horse concept. After synthesis, purification and full
physiochemical characterization of the conjugate, we subjected this novel sideromycin (HT-10) to in vitro and in vivo studies.
The results indicate that, as planned, we have demonstrated for the first time that, if properly derivatized, daptomycin can be a
powerful antibiotic against targeted multi-drug resistant strains of Acinetobacter baumannii.
MIC testing showed activity against several multidrug resistant strains of A. baumannii at MIC levels of 0.006 to 0.8 µM!
Daptomycin alone had no activity. HT-10 retained activity against Gram-positive S. aureus at 6 µM but was not as active as
daptomycin alone (0.8 µM). HT-10 showed no activity against Pseudomonas, Burkholderia, or E. coli, reflecting siderophoreinduced selectivity. HT-10 was well tolerated in mice and active in vivo in a mouse infection model.
Thus, HT-10, a synthetic sideromycin based on a mixed ligand daptomycin conjugate, is recognized by A. baumannii, and is
highly active in vitro and in vivo against different sensitive and resistant strains of this Gram negative bacterium including
multidrug resistant (MDR) strains.
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P01 - Discovery of new lead-like compounds targeting malaria chemoprotection
Kathy Andrews1
1. Griffith University, Herston, QLD, Australia
The World Health Organization estimates there were ~214 million clinical cases and 438,000 deaths due to malaria in 2015.
While this represents a decline in morbidity and mortality over the past decade, all currently used antimalarial drugs are now
under threat due to parasite drug resistance. This places recent gains in a precarious situation. With ~3.2 billion people at risk
of malaria and a global agenda focused on eradication, there is an urgent need to develop new drugs with novel modes of
action for both the treatment and prevention (chemoprotection) of malaria. We have screened ~18,000 compounds from the
CSIRO Compound Library (a chemically diverse small molecule library assembled from decades of research at the CSIRO) for
new chemotypes with potential as malaria chemoprotection drug leads. As a key criteria of chemoprotection drugs is a mode of
action different to treatment drugs, hit compounds were identified as those with increased activity against P. falciparum asexual
intraerythrocytic stage parasites in 96h assays (two developmental cycles) compared to activity in 48h assays (one
developmental cycle). This strategy has identified several novel chemotypes with drug-like predicted physicochemical
properties, sub-micromolar activity against drug-sensitive and multi-drug resistant P. falciparum parasites, >100-fold selectivity
for P. falciparum versus human cells and a potential mode of action different to slow action/delayed death antimicrobials with
antimalarial activity (e.g. clindamycin; determined using IPP rescue). Data will be presented on lead-like chemoprotection
compounds in the context of changing malaria drug discovery dynamics under a global agenda focused on eradication.
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P02 - Potential biocide mode of action and resistance determinants in an opportunistic human
pathogen Acinetobacter baumannii
Liping Li1, Karl Hassan1, Amy Cain2, Stephen Baker3, Julian Parkhill2, Ian T Paulsen1
1. Department of Chemistry and Biomolecular Sciences,, Macquarie University,, Sydney, NSW, Australia
2. Wellcome Trust Sanger Institute, Hinxton, Cambridge, United Kingdom
3. The Hospital for Tropical Diseases, Wellcome Trust Major Overseas Programme, Oxford University Clinical Research Unit,
Ho Chi Minh City, Vietnam
The Gram-negative opportunistic human pathogen A. baumannii has become a significant worldwide threat for immunecompromised patients who are hospitalized in Intensive Care Unit (ICU) wards. This is primarily due to the emergence of A.
baummannii clonal lineages with high level resistance to antibiotics, biocides and desiccation. The role antibiotic usage in the
evolution of MDR bacteria has been extensively studied. In contrast, little is known about the contribution of indiscriminate use
of biocides, the key antibacterials for infection control. To advance our knowledge of both biocide modes of action and
resistance mechanisms, we performed genome-wide identification of the fitness determinants of a MDR A. baumannii strain
against ten clinically important biocides through transposon-directed insertion-site sequencing (TraDIS). A Tn5 transposon
mutant library of more than 100,000 unique insertion mutants was used to identify mutants with a fitness advantage or defect
against the biocides. This revealed that the disruption of the genes involving in capsule polysaccharide biosynthesis,
lipooligosaccharide outer core biosynthesis, chaperone-usher pilus and genes encoding various membrane transporters can
affect the susceptibility of the A. baumannii host to multiple structurally distinct biocides. Additionally, we also revealed that the
host susceptibility to silver nitrate was also affected by the disruption of several hypothetical genes and genes involving in TCA
cycle and respiration.
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P03 - Reversible carbapenemase inhibitors bridging the serine–metallo divide
Adam Renslo1, Kyle DeFrees, Orville Pemberton, Priyadarshini Jaishankar, Nick Torelli, Xiujun Zhang, Yu Chen
1. University of California, San Francisco, San Francisco, CA, United States
The emergence of multi-drug resistant Gram-negative pathogens (especially Pseudomonas aeruginosa, Klebsiella
pneumoniae, Acinetobacter baumannii and the Enterobacteriaceae) represents a serious challenge for the treatment of serious
infections. Most of these resistant pathogens now possess extended spectrum beta-lactamases that confer resistance to thirdgeneration cephalosporins and mono-bactams. Even more troubling, many strains now express carbapenemases, threatening
the future utility of carbapenems.
We sought to apply fragment screening by SPR, computational docking, and synthetic/medicinal chemistry to identify and
optimize small molecule leads that reversibly inhibit clinically relevant carbapenemases. An SPR screen of ~3,000 small
molecule fragments across representative carbapenemases, including KPC-2 (Class A), NDM-1 (Class B), and OXA-48 (Class
D) was performed using a Biacore 4000. Interestingly, a number of the fragment hits bound across classes, despite the
divergent structures of these hydrolases. Similarly, two distinct inhibitor scaffolds identified by computational docking were
found to inhibit both serine- and metallo-carbapenemases and these became the subject of a medicinal chemistry optimization
effort. Structure-activity studies revealed a surprisingly robust correlation in the activity against KPC-2 and NDM-1 enzymes,
suggesting a shared “substrate envelope” and the potential to develop reversible, non-covalent, pan-carbapenemase inhibitors.
Co-crystal structures of selected analogs bound to KPC-2, VIM-2, and NDM-1 reveal how these new compounds are able to
bind and inhibit diverse carbapenemase
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Broken permeability barriers and their stories
Helen Zgurskaya1
1. University of Oklahoma, Norman, OKLAHOMA, United States
Gram-negative bacteria are intrinsically resistant to many antibiotics. Species that acquired multi-drug resistance and cause
infections that are effectively untreatable present a serious threat to public health. The problem is broadly recognized and
tackled at both the fundamental and applied levels. The major obstacle in discovery and development of antibiotics effective
against such pathogens is the low permeability barrier of Gram-negative cell envelopes. This presentation will discuss ongoing
efforts to understand the molecular bases of this barrier and specific strategies to break it in order to achieve potent activities
against difficult Gram-negative bacteria.
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A potpourri of polymxyins
Tony Velkov1
1. Monash Institute of Pharmaceutical Sciences, Monash University, Parkville , VIC, Australia
The world is facing an enormous and growing threat from the emergence of bacterial ‘superbugs’ that are resistant to all
available antibiotics. The healthcare cost of antibiotic resistance to society is staggering. In the US, antibiotic resistance costs
an estimated $20 billion per year in direct health care costs, $35 billion per year in other societal costs and >8 million additional
days of extended hospital stay. If bacteria continue developing resistance to multiple antibiotics at the present rate and at the
same time the antibiotic pipeline continues to dry up, there could be catastrophic costs to healthcare and society globally. New
antibiotics are urgently needed to treat infections caused by bacterial ‘superbugs’, in particular Gram-negative Pseudomonas
aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae. Building upon our systematic polymyxin pharmacology
research since 1999, we have employed a new structure-activity relationship model to develop a novel class of polymyxin
antibiotics against infections caused by Gram-negative ‘superbugs’.
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Development of new polymyxin antibiotics with improved safety and efficacy
Kade Roberts2, 1, Jiping Wang2, Heidi Yu2, Lv Wang2, Olga Lomovskaya3, David Griffith3, Scott Hecker3, Michael
Dudley3, Roger L Nation2, Philip E Thompson1, Tony Velkov2, Jian Li2
1. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
2. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Parkville, VIC, Australia
3. The Medicines Company, San Diego, California, United States of America
The emergence of multi-drug resistant (MDR) gram-negative bacterial pathogens such as Pseudomonas aeruginosa,
Acinetobacter baumannii and Klebsiella pneumoniae has become a major global health issue. This problem has been further
compounded by the lack of development of new antibiotics. The cyclic lipopeptide antibiotics, polymyxin B and colistin are used
as the last-line therapy against these problematic ‘superbugs’. However, the effective use of polymyxins in the clinic is
hampered by their potential for nephrotoxicity. Recent clinical studies have reported that polymyxin-associated nephrotoxicity
can occur in up to 60% of patients when administered intravenously and is the major dose-limiting factor for their optimal
clinical use. In this presentation we report on our pre-clinical polymyxin drug discovery program, a National Institutes of Health
(NIH) funded joint academic-industry collaboration between Monash University and The Medicines Company (USA). This
program aims to produce 1-2 new polymyxin clinical candidates with improved safety and efficacy profiles compared to
polymyxin B and colistin. Aspects of our novel drug design strategy and lead optimisation studies will be discussed. To date a
number of lead compounds have been identified with improved efficacy and significantly reduced nephrotoxicity compared to
polymyxin B and colistin.
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Bicyclic nitroimidazoles with antiparasitic and antitubercular activity
Angie M Jarrad1, Anjan Debnath2, Chee Wei Ang1, Kyra Woods3, Amy J Jones4, Ruby Pelingon1, Mark S Butler1,
Tomislav Karoli1, Vicky M Avery4, Nick P West3, Mark AT Blaskovich1, Matthew Cooper1
1. Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
2. Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California, San Diego, La Jolla, California, USA
3. School of Chemistry and Molecular Bioscience, University of Queensland, Brisbane, QLD, Australia
4. Eskitis Institute for Drug Discovery, Griffith University, Brisbane, Queensland, Australia
The antitubercular bicyclic nitroimidazoles, delamanid and pretomanid have potential to be repurposed for the treatment of the
parasitic disease visceral leishmaniasis.1,2 We have found pretomanid to have potent activity against the enteric parasites
Giardia lamblia and Entamoeba histolytica. These results inspired us to design novel bicyclic nitroimidazoles as possible new
antiparasitic compounds. An efficient synthetic approach utilising a one pot, two step alkylation/cyclisation was devised to
access the nitro-imidazopyrazinone scaffold. This scaffold was alkylated to yield N-alkylated nitroimidazopyrazinone and Oalkylated nitroimidazopyrazine derivatives. More than 30 analogues were prepared with variations at the R 1, R2 and R3 sites.
Structure activity relationships were determined against a broad range of pathogenic parasites, bacteria and fungi.
Promising activity was identified against G. lamblia, E. histolytica, Trypanosoma brucei brucei and Mycobacterium tuberculosis.
While clear, organism specific SAR was present, a number of derivatives had potent activity against more than two species,
with MCC_8399 having activity against each of these targets. The majority of the compounds had no cytotoxicity when counterscreened against mammalian cell lines (CC50 >100 μM). Selected derivatives were next evaluated for drug-like properties,
including protein plasma binding, microsome stability and Caco-2 permeability. Several compounds had comparable results to
pretomanid in these assays. Compounds that displayed high levels of plasma protein binding, metabolic stability and high
apparent permeability were identified. Taken together, these results support further evaluation of promising derivatives in in
vivo efficacy models of parasitic and tuberculosis infection.
1.
2.
Patterson, S., Wyllie, S., Stojanovski, L., Perry, M. R., Simeons, F. R. C., Norval, S., et al. (2013). The R enantiomer
of the antitubercular drug PA-824 as a potential oral treatment for visceral Leishmaniasis. Antimicrob. Agents Chemother.,
57(10), 4699–4706.
Patterson, S., Wyllie, S., Norval, S., Stojanovski, L., Simeons F. R. C., Auer, J. L., et al. (2016). The anti-tubercular
drug delamanid as a potential oral treatment for visceral leishmaniasis. eLife, 5, e09744.
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NO-donor cephalosporins as new agents against bacterial biofilms
Michael J Kelso1
1. University of Wollongong, Wollongong, NSW, Australia
Low concentrations of nitric oxide (NO) have been shown to act as a signal that induces some biofilm bacteria to disperse and
revert to the free-swimming (planktonic) form. This finding has unveiled an exciting new anti-biofilm paradigm; i.e. use of NOdonor compounds in combination with antibiotics to clear chronic biofilm infections, as it is known that planktonic bacteria are
up to 1000x more susceptible to antibiotics and host immune defences than their better-protected biofilm
counterparts. We are developing a novel class of biofilm-activated cephalosporin-based NO-donor prodrugs (cephalosporin-3’diazeniumdiolates, C3Ds) for clinical applications. The targeted NO signal released from C3Ds (triggered by biofilm βlactamases) can induce biofilms to disperse and when used in combination with antibiotics we've shown that the compounds
are highly active against Pseudomonas aeruginosa in vitro biofilms. The presentation will summarise key results to date and
highlight our recent efforts to establish whether it might be possible to create “all-in-one” bactericidal/anti-biofilm
cephalosporins; i.e. compounds that are activated to release NO through reactions with penicillin-binding proteins (PBPs,
transpeptidases), the molecular target of β-lactam antibiotics.
[1] Barraud, N., Kardak, B. G., Yepuri, N. R., Howlin, R. P., Webb, J. S., Faust, S. N., Kjelleberg, S., Rice, S. A., Kelso, M. J.
(2012). Cephalosporin-3’-diazeniumdiolates as targeted NO-donor prodrugs for dispersing bacterial biofilms. Angewandte
Chem. Int. Ed, 51, 9057-9060.
[2] Barraud, N., Kelso, M. J., Rice, S. A., Kjelleberg, S. (2015). Nitric Oxide: A Key Mediator of Biofilm Dispersal with
Applications in Infectious Diseases. Curr. Pharm. Des. 21, 31-42.
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The coming crisis in antibiotics
Ramanan Laxminarayan1
1. Center for Disease Dynamics, Economics & Policy, Washington, DC, WASHINGTON, DC, United States
Global access to effective antimicrobials is under threat. Currently, insufficient access and delays in access to antibiotics cause
more deaths than antibiotic resistance, but more resistance-related deaths are being reported in all countries irrespective of
income level. The key is to promote universal provision of antimicrobials in appropriate situations while ensuring continued
effectiveness. This talk is about how we might leverage the momentum achieved through the UN General Assembly HighLevel Meeting of Heads of State on sustainable access to effective antimicrobials in September 2016 to develop realistic goals,
stimulate political will, mobilize resources, and agree on an accountability mechanism for global collective action on this issue.
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Wellcome Trust new strategic priority to fight antimicrobial resistance
Timothy Jinks1
1. Wellcome Trust, London, LONDON, United Kingdom
Drug-resistant infection is a global public health threat that undermines the progress made in the fight against infectious
disease in the last century. There have been longstanding calls to address AMR and the response has not yet been sufficient
to tackle the rising challenge. Recognizing this as pivotal time to convert appeals into action, particularly following the UN
declaration last year, Wellcome has developed a new strategic focus in support of a global agenda. The desired impact of
Wellcome’s focused strategy is to reduce the threat of drug-resistant infection which requires bold, ambitious action to
safeguard global health. This presentation will describe a core set of coordinated activities Wellcome will be implementing to
tackle AMR.
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EU funding to stimulate research and innovation in AMR
Barbara Kerstiens1
1. European Commission, Brussels, Belgium
Last year world leaders recognised antimicrobial resistance (AMR) as a threat to global public health, security and prosperity. In
that same year, the Council of the European Union called upon all EU members to reinforce and expand measures against
AMR, which is currently responsible for an estimated 700,000 deaths annually around the world. Working against pathogenic
microbes in a globalised world is a matter of self-interest, at least as much as it is a responsibility to our global well-being. The
EU has been committed to combatting AMR since 1999, with 1.3 billion Euro invested in research and innovation so far. It has
developed different funding instruments to address different needs to address this challenge, from collaborative research, to
public-public and public-private partnerships as well as inducement prizes. The European Commission will continue and scale
up its fight against AMR, with the launch in 2017 of a One Health Action Plan to support EU Member States in the fight against
AMR. Further promoting international collaboration to stimulate research and innovation will be an important element of that
plan.
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Combating Antimicrobial Resistance - Antibacterial Drug Discovery and Development
Programs and Preclinical Services at NIAID
Zuoyu Xu1
1. National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MARYLAND, United States
To combat antimicrobial resistance and support antibacterial drug discovery and development, the National Institute of Allergy
and Infectious Diseases (NIAID) has been funding/supporting programs/projects covering basic research, translational
research, preclinical development, and clinical studies/trials. NIAID also provides preclinical services to facilitate translational
research with resource and technology with the goal of lowering barriers to entering the product development pipeline. The
overall goal of the discovery and development effort is to control, prevent, and treat diseases caused by infectious pathogens
including antimicrobial resistant organisms.
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Opportunities for funding research on antimicrobial resistance through the National Health
and Medical Research Council
Anne Kelso1
1. NHMRC, Canberra, ACT, Australia
The National Health and Medical Research Council (NHMRC) is the Australian Government’s primary agency for funding
research to improve human health. A broad spectrum of research is supported, from discovery through to clinical, public health
and health services research, via a range of schemes that provide grants and fellowships to outstanding individual investigators
and teams for investigator-initiated and priority-driven research. NHMRC’s corporate plan for 2015 – 2019 identifies the need to
prepare for rapid and unpredictable change as one of the major health issues facing Australia today and notes that
antimicrobial resistance (AMR) is a known threat requiring response. Research on many different aspects of antimicrobial
resistance is currently supported through several funding schemes, notably the Centres of Research Excellence and Project
Grants schemes. These and other opportunities, together with potential mechanisms to link and translate AMR research, will be
discussed.
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Harnessing nature’s gift: conjugating antibody and natural product antibiotics to treat
bacterial infections
Man-Wah Tan1
1. Genentech Inc, South San Francisco, CA, United States
Antibody is a potent component of the human immune repertoire and has great potential as therapeutics for treatment of
infectious diseases. In addition, nature has also gifted us with many natural products that the scientific community has
developed into life-saving antibiotics. Here I will discuss the development of an antibody-antibiotic-conjugate (AAC) that
combines key advantages of antibody and antibiotic into a novel therapeutic platform to treat bacteria infections. An AAC has
three components: an antibiotic payload to kill bacteria, an antibody to target delivery of the payload to bacteria, and a linker
attaching the payload to the antibody. Using the AAC against S. aureus as an example, I will highlight key considerations and
principles in the design and engineering of an efficacious AAC.
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A novel Immunotherapy for malaria
Michelle Wykes1
1. QIMR Berghofer, Brisbane, QLD, Australia
Malaria is caused by Plasmodium parasites, which after introduction into the host by mosquitoes proceed to infect hepatocytes
followed by red blood cells. It is the blood-stage infection that causes the symptoms and lethality associated with malaria. Many
pathogens, including Plasmodium spp., exploit the programmed death-1 (PD-1)/PD-1 ligand-1 (PD-L1) pathway to 'deactivate'
T cell functions but the role of PD-1/PD-L2 remains unclear. We studied malarial infections to understand the contribution of
PD-L2 to immunity. Here we show that higher PD-L2 expression on blood dendritic cells (DC), from Plasmodium falciparuminfected individuals, correlated with lower parasitemia. Mechanistic studies in mice showed that PD-L2 was indispensable for
establishing effective CD4+ Th1 immunity. Importantly, administration of soluble PD-L2 to mice with lethal malaria was sufficient
to dramatically improve immunity and survival (92% vs 0%). These studies show a new function for PD-L2, which has potential
to be translated into an effective treatment for malaria and other diseases where T cell immunity is ineffective or short-lived due
to PD-1.
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Bacterial cytological profiling: a new platform for antibiotic discovery
Joseph Pogliano1
1. University of California, San Diego, La Jolla, CA, United States
The emergence of multi-drug resistant bacteria and the decline in the number of new antibiotics creates an urgent need to
discover antibiotics that act by novel mechanisms of action (MOA). We have developed a rapid and versatile platform for
identifying antibiotics and determining their MOAs called Bacterial Cytological Profiling (BCP). BCP uses fluorescence
microscopy to observe changes in cytological parameters of bacteria exposed to lethal concentrations of antibiotics. Antibiotics
that inhibit targets in different pathways generate different cytological profiles. To identify compounds hitting new targets, we
are using a combination of approaches, including Rapid Inhibition Profiling (RIP), which allows us to generate cytological
profiles for essential enzymes for which there are currently no small molecule inhibitors. We have screened several small
libraries of compounds to identify those with activity against gram negative bacteria. We then used BCP to determine the MOA
of the hits, allowing us to prioritize further development based on MOA. Our results demonstrate that BCP is a powerful
approach for identifying molecules that can be further developed as antibiotics for the treatment of infections caused by drug
resistant bacteria.
27
Targeting active metabolic pathways to interrupt artemisinin induced dormancy
Qin Cheng1
1. Drug Resistance and Diagnostic, Australian Army Malaria Institute , The AMI lab, QIMR-Berghofer Medical Research
Institute, Brisbane, Queensland, Australia
P. falciparum parasites arrest their growth and development after a short exposure to artemisinin derivatives in vitro. A small
population of dormant parasites resume growth several days later causing recrudescence. This artemisinin-induced dormancy
phenomenon likely evolved to escape damaging environmental factors including antimalarial drugs. Identification of metabolic
pathways that remain active in dormant parasites not only helps understand underlying mechanisms supporting dormant
parasites, but also reveals potential targets for killing dormant parasites. We investigated metabolic pathways in dormant
parasites and observed that despite a general down regulation of major metabolic pathways, several cyclin-dependent protein
kinases and pathways in apicoplast and mitochondrion are active in dormant parasites. Inhibiting cyclin-dependent protein
kinases or enzymes involved in metabolic pathways in the aplicoplast and mitochondria can completely or partially prevent the
recovery of dormant parasites. These findings highlight that targeting critical metabolic pathways that maintain the viability of
dormant parasites may lead to ways of reducing recrudescence after artemisinin treatment.
28
Uridylpeptide natural product analogues as potent and selective anti-mycobacterials
Richard J Payne1, Wendy Tran1, Anh T Tran1, Emma E Watson1, Venugopal Pujari2, David I Roper3, Susan A Charman4,
Nicholas P West5, Warwick J Britton6
1. The University of Sydney, Camperdown, NSW, Australia
2. Mycobacteria Research Laboratories, Colorado State University,, Fort Collins, Colorado, USA
3. School of Life Sciences , University of Warwick, Coventry, UK
4. Centre for Drug Candidate Optimisation, Monash University, Melbourne, VIC, Australia
5. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, QLD, Australia
6. Centenary Institute and Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
Tuberculosis (TB) is caused by infection with the bacterium Mycobacterium tuberculosis (Mtb) and is the leading cause of death
of any infectious disease (1.5 million people per year).1 Current treatment for TB relies on a six month quadruple therapy
comprising rifampicin, isoniazid, ethambutol and pyrazinamide. 2 Despite achieving a cure rate of >95% for drug-sensitive TB,
this regimen is not effective against multi-drug resistant (MDR) and extensively drug resistant (XDR) TB, infections which are
emerging at an alarming rate globally. To effectively combat these drug resistant cases, new TB drugs with novel modes of
action are desperately needed.
We have recently explored uridylpeptide natural products as inspiration for the development of TB drug leads that operate
through a novel mechanism of action to currently employed anti-mycobacterials.3 This presentation will outline our development
of a rapid synthetic platform for generating uridylpeptide natural product analogues. The potent and highly selective activity
against the virulent H37Rv strain of Mtb in both normoxic and hypoxic models will also be discussed. Finally, the mechanism of
antibacterial action of these analogues will be presented - via the inhibition of Mtb Phospho-MurNAc-pentapeptide translocase the integral membrane enzyme responsible for the biosynthesis of lipid I.
References
1. Organization, W. H. Global Tuberculosis Report 2014; Geneva: Switzerland, 2014
2. Zumla, A.; Nahid, P.; Cole, S., Nature reviews. Drug discovery 2013, 12 (5), 388-404
3. Payne et al. Nature Commun. 2017, DOI: 10.1038/ncomms14414
29
Alternatives to antibiotics
Robert Hancock1
1. University of British Columbia, Vancouver, BRITISH COLUMBIA, Canada
The inexorable increase in multidrug resistant infections combined with a decrease in new antibiotic discovery and a lack of
compounds for chronic biofilm infections is creating a potential crisis in human medicine. Thus it is imperative to consider
alternatives to conventional antibiotic strategies and particularly for infections that are recalcitrant to current therapies (e.g.
sepsis and chronic biofilm infections).
Cationic host defence (antimicrobial) peptides are produced by virtually all organisms, ranging from plants and insects to
humans, as a major part of their innate defences against infection. We and others have demonstrated that they are a key
component of innate immunity and have multiple mechanisms that enable them to deal with infections and inflammation
including an ability to favourably modulate the innate immune system, and distinct antibiotic and anti-biofilm activities.
We have defined a class of peptides that act against biofilms formed by multiple species of bacteria in a manner that is
independent of activity vs. planktonic bacteria. We have now developed novel anti-biofilm peptides that (i) kill multiple species
of bacteria in biofilms (MBEC <1 mg/ml), including the ESKAPE pathogens and other major clinically relevant Gram negative
and Gram positive bacteria, including, (ii) work synergistically with antibiotics in multiple species, and (iii) are effective in animal
models of biofilm and abscess infections. Structure activity relationships studies showed no major overlap between anti-biofilm
and antimicrobial (vs. planktonic bacteria) activities, and indeed organisms completely resistant to antibiotic peptides were still
able to be treated with anti-biofilm peptides. The action of such peptides is dependent on their ability to trigger the degradation
of the nucleotide stress signal ppGpp.
The manipulation of natural innate immunity represents a new adjunctive therapeutic strategy against antibiotic-resistant
infections. Cationic host defence peptides boost protective innate immunity while suppressing potentially harmful
inflammation/sepsis, and work synergistically with conventional therapies. Using the principle of selective boosting of innate
immunity we have developed novel small innate defence regulator (IDR) peptides with no direct antibacterial activity, that are
nevertheless able to protect in animal models against many different microbial infections, including antibiotic resistant infection
models against the superbug methicillin resistant Staph aureus (MRSA), E. coli, P. aeruginosa, MDR tuberculosis, as well as
cerebral malaria and inflammatory diseases, providing a new concept in anti-infective therapy. Good activity in models of
wound healing, pre-term birth and cystic fibrosis has also been achieved.
30
Role of microbiota metabolites in maintaining vaginal health
Gilda Tachedjian1, 2, 3, 4
1. Centre for Biomedical Research, Burnet Institute, Melbourne, VIC, Australia
2. Department of Microbiology, Monash University, Melbourne, VIC, Australia
3. School of Science, College of Science, Engineering and Health, RMIT University, Melbourne, VIC, Australia
4. Department of Microbiology and Immunology at the Doherty Institute for Infection and Immunity, The University of
Melbourne, Melbourne, VIC, Australia
Organic acid metabolites produced by the vaginal microbiota have reported antimicrobial and immune modulatory activities
suggesting their potential role in modulating susceptibility to sexually transmitted infections including HIV. In asymptomatic
women of reproductive age the vaginal microbiota is comprised of lactic-acid producing bacteria that are primarily responsible
for acidifying the vagina to pH ~3.5 by the production of ~110 mM of lactic acid. In contrast, women with bacterial vaginosis
(BV), a dysbiosis of the vaginal microbiota, is characterised by a decrease in lactic acid-producing microbiota and an increase
in polymicrobial bacteria accompanied with elevated pH>4.5. BV is associated with a sharp decrease in lactic acid and
increased levels of short chain fatty acids (SFCAs) including acetate, propionate and butyrate. Compared to women with
lactobacillus-dominated microbiota, women with polymicrobial bacteria (e.g. BV) have adverse reproductive health outcomes
and increased susceptibility to sexually transmitted infections (STIs) including HIV. Here, in vitro studies will be presented
demonstrating that lactic acid produced by the vaginal microbiota has microbicidal activities that may protect against STIs and
vaginal microbiota dysbiosis (e.g. BV) as well as immune modulatory properties on the cervicovaginal epithelium that could
decrease HIV susceptibility. These findings highlight the potential use of lactic acid and lactic acid producing probiotics in the
lower female reproductive tract as an adjunct to antivirals and antibiotics in maintaining vaginal health.
31
Optimized non-genetic β-lactam resistance dissemination due to outer membrane vesicle
secreted lipidated β-lactamase
Herbert P Schweizer1, Sunisa Chirakul1, Michael H Norris1, Apichai Tuanyok1
1. University of Florida, Gainesville, FLORIDA, USA
β-Lactams are broad-spectrum bactericidal antibiotics useful for treatment of infections caused by Gram-negative bacteria.
Resistance to β-lactam antibiotics in these bacteria is primarily mediated by periplasmic β-lactamases. Recent studies showed
that some types of β-lactamases are outer membrane (OM) associated lipoproteins. The two best-studied examples are NDM-1
carbapenemase1 and Burkholderia pseudomallei PenA β-lactamase2. In both instances conversion of the protein to a
periplasmic form does not affect enzyme activity or specificity. However, in the case of NDM-1 expressed in Escherichia coli
deacylation affects OM vesicle (OMV) localization. B. pseudomallei PenA is likewise located in the OM and associated with
OMVs. Although soluble periplasmic proteins, including β-lactamases, are found in OMVs, their vesicle localization is likely the
result of serendipitous entrapment. In contrast, lipoproteins are compartmentalized into OMVs by virtue of their OM localization.
OMV localization of β-lactamases provides protection of otherwise β-lactam susceptible bacteria in the immediate environment.
This either occurs by antibiotic degradation inside the vesicles where the enzyme is protected from proteolytic degradation or
by fusion of the β-lactamase containing OMVs with susceptible recipient cells placing the lipoprotein into the recipients OM. A
potential benefit of conferring resistance by protein transfer to an otherwise susceptible subpopulation is temporary survival
under antibiotic pressure until genetic resistance can be established. This may especially be significant in bacteria like B.
pseudomallei that rely primarily on chromosomally encoded genes for resistance development rather than horizontally
transferred resistance. Inhibitors of bacterial protein lipidation may provide a novel opportunity for combatting β-lactam
resistance.
1.
2.
1. Gonzalez LJ, Bahr G, Nakashige TG, Nolan EM, Bonomo RA, Vila AJ. 2016. Membrane anchoring stabilizes and
favors secretion of New Delhi metallo-beta-lactamase. Nat. Chem. Biol. 12:516-522
2. Randall LB, Dobos K, Papp-Wallace KM, Bonomo RA, Schweizer HP. 2016. Membrane bound PenA betalactamase of Burkholderia pseudomallei. Antimicrob. Agents Chemother. 60:1509-1514
33
Clinical bacteriophage therapy for the 21st century
Sandra Morales1
1. AmpliPhi, Sydney/Brookvale, NSW, Australia
It is now unanimously accepted that the world has entered a dangerous phase in the treatment of multidrug-resistant (MDR)
bacterial infections. The speed at which resistance develops and spreads among bacterial populations far exceeds the capacity
of the biomedical and scientific community to develop new antibiotics. After serving us so well for so long on their own,
antibiotics
now
need
help
from
new
classes
of
antibacterial
agents.
A new type of antibacterial would ideally be capable of matching, and possibly exceeding, the troublesome mutation rate of its
bacterial targets. In other words, be an “intelligent” agent that “out-mutates the mutators”. Bacteriophages (phages) are highly
specific bacterial viruses with the potential to achieve this. Historically, phages were widely used to treat bacterial infections
from the time of their discovery 100 years ago at the Pasteur Institute until the advent of small molecule antibiotics such as
penicillin. Phage therapy then faded into near-obscurity for 50 years, before a major renaissance in the late 1990s. With this
resurgence has come a wealth of data exploring the in vivo behaviour of phages and their potential for clinical application.
This talk will concentrate on results from pre-clinical and human clinical trials, and the issues facing the fledgling phage therapy
industry such as production, regulatory, and financial constraints. Finally, exciting new developments in the combined use of
phages and antibiotics will be described which offer the tantalising possibility that phage therapy might not only be able to
contain antibiotic resistant bacteria but even reverse it – an ideal partner for the MDR era, indeed!
34
Honey: a topical solution for drug-resistant infections
Elizabeth Harry1, Nural Cokcetin1, Shona Blair1, Dee Carter2
1. ithree institute, University of Technology Sydney, Broadway, NSW, Australia
2. School of Life and Environmental Sciences, , University of Sydney, Sydney, NSW, Australia
Antimicrobial resistance has been described as an ‘apocalyptic’ threat to human health requiring urgent action. Resistance to
antibiotics is exacerbated because bacteria generally exist in biofilms, which are recalcitrant to antibiotics. The need for new
infection treatment options has prompted interest in complex natural products as antimicrobials.
Honey has been used as a topical wound treatment throughout history, predominantly due to its antimicrobial activity. Manuka
honey particularly has potent broad-spectrum antibacterial activity, effective against antibiotic resistant pathogens and is
currently licensed for use in honey dressings to treat wounds. Importantly, unlike traditional topical antibiotics, bacterial
resistance to honey has not been reported. While an increasing number of health-care professionals genuinely include it in the
treatment of skin infections and wounds, honey still remains underutilised.
We have shown that manuka honey can prevent and eradicate established biofilms formed by Staphylococcus aureus and
Pseudomonas aeruginosa, at concentrations that can be maintained in wound dressings. We also demonstrate that biofilm cell
suspensions did not show resistance to honey. Through passaging experiments on culture-grown bacteria, we were not able to
isolate bacteria resistant to manuka honey under conditions that rapidly induced resistance to antibiotics. We are currently
investigating the mode of action of honey and its major components to better understand how it kills bacteria without resistance
developing. This project may lead to new avenues for antimicrobial development such as rational design of honey substitutes,
antibiotics, or antibiotic strategies, as well as encourage wider clinical use of this evolutionary answer to antibiotic resistance.
35
P04 - Host-directed antimicrobials: inducing host cell death to combat infections
Thomas Naderer1
1. Monash University, Clayton, VIC, Australia
Antibiotics can fail to clear bacterial pathogens that replicate within our own cells. We have now devised an innovative strategy
to combat intracellular infections, by focusing on host survival proteins. As a proof of principle, we exploited Legionella
pneumophila, which causes severe pneumonia by replicating in lung macrophages. L. pneumophila is either naturally resistant
or requires several fold higher concentrations of antibiotics for elimination from macrophages. Here we show L. pneumophila
depends on the host cell pro-survival protein, BCL-XL, to establish infections, both in vitro and in vivo. BCL-XL keeps the death
factors, BAX and BAK, in check to prevent apoptotic cell death. The related protein, MCL-1, contains redundant functions to
BCL-XL. However, L. pneumophila inhibits host protein synthesis to disarm macrophages, which leads to the loss of the shortlived MCL-1. This leaves BCL-XL as the sole protein to keep infected macrophages alive. Consequently, compounds that
inhibit BCL-XL, originally developed to kill cancer cells, induce cell death of infected macrophages. Remarkably, a single dose
of BCL-XL-targeted compound significantly reduces L. pneumophila burden in the lungs and prevents lethal bacterial infection
in mice, in the absence of antibiotic therapy. This demonstrates that it is possible to kill only infected cells, while uninfected
immune cells remain viable, which is a promising strategy for the treatment of intracellular pathogens.
36
Developing new antibacterial products: Perspectives on giving new therapies the best chance
John Rex1
1. CARB-X and Wellcome Trust, Wellesley Hills, MA, United States
Effective antibiotics underpin all of healthcare and it is critical that the global pipeline be vibrant and robust. Substantial
resources are available for antibiotic R&D but successfully developing a new antibiotic requires discipline & focus. Planning for
registration begins with initiation of R&D and must recognize the practical realities of development by remembering that “To
finish first, first you must finish.” Where permitted by the spectrum of the candidate, planning for a UDR-focused (Usual Drug
Resistance) Phase 3 non-inferiority study at a standard body site vs. a standard comparator is the most reliable path. Agents
with a very narrow spectrum are a special case where development paths are still being defined but recent global
conversations do point to a plausible path based on a combination of exhaustive PK-PD data plus at least some clinical data.
Pathways for agents that act via indirect mechanisms and that hence lack standalone efficacy (e.g., virulence inhibitors) require
either development as a preventative or a demonstration that their addition to standard of care produces a measurable
improvement in outcome, both of which are paths with substantial challenges that must be anticipated. Finally, reimbursement
of new antibiotics will increasingly depend substantially on demonstration of a novel basis for activity that offers the
metaphorical equivalent of a new class of fire extinguisher – small incremental improvements appear unlikely to be well
rewarded.
37
Pathogen-directed strategies at Entasis Therapeutics
Alita Miller1
1. Entasis Therapeutics, Waltham, MA, United States
Antibacterial regulatory guidelines have been recently updated to allow for more streamlined development of narrow-spectrum,
precision therapies to treat highly problematic, multidrug-resistant bacterial infections. Accordingly, various strategies are being
pursued to develop candidate agents for narrower-than-usual or specific pathogen-directed indications. Entasis Therapeutics
has embraced this new paradigm as exemplified by its two current clinical programs: (1) zoliflodacin, a novel oral antibiotic for
the treatment of uncomplicated gonorrhea that recently successfully completed Phase II and (2) sulbactam-ETX2514, a novel
combination agent targeting carbapenem-resistant Acinetobacter baumannii which is currently in Phase I testing. Details
around the discovery and development of each of these candidates will be presented.
38
Drug discovery for neglected diseases
Ian Gilbert1
1. University of Dundee, Dundee, ANGUS, United Kingdom
The Drug Discovery Unit (DDU) was set up at the University of Dundee in 2006. It is a fully integrated drug discovery unit,
combining hit discovery, medicinal and computational chemistry, drug metabolism and pharmacokinetics. The key aims of the
unit are to tackle unmet medical need. We have two main therapeutic focuses: neglected tropical diseases such as malaria,
tuberculosis and kinetoplastid infections; and novel drug targets emerging from the academic sector. Drug resistance to
antimicrobials is a major issue and there is a need for new drugs which operate by new modes of action to tackle this problem.
In this presentation, I will outline some of the work that we have carried out on drug discovery for neglected infectious diseases.
In particular, I will focus on the development of a potential new compound for the treatment of malaria that works by a novel
mechanism of action.
39
Identifying strategies to target host cell remodeling pathways of the malaria parasite
Tania de Koning-Ward1, Kathryn Matthews1, Natalie A Counihan1, Scott A Chisholm1, Hayley E Bullen2, Paul R
Sanders2, Anubhay Srivastava3, Brendan S Crabb2, Darren J Creek3, Paul R Gibson2
1. School of Medicine, Deakin University, Waurn Ponds, VIC, Australia
2. Burnet Institute, Melbourne, VIC, Australia
3. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC,
Australia
Plasmodium parasites, the causative agents of malaria, drastically modify their host erythrocyte to avoid the immune system
and to prevent host cell breakdown whilst at the same time rendering the cell permeable to supplementary nutrient uptake from
the plasma and to provide a mechanism for waste disposal. To perform these modifications, Plasmodium spp. export hundreds
of effector proteins across an encasing vacuolar membrane into the cytoplasm of the erythrocyte. Many of these proteins then
continue their journey to the erythrocyte surface to exert their effector function. This complex trafficking pathway and many of
the effector proteins that remodel the host cell are unique to Plasmodium parasites. Since protein export and host cell
remodeling is essential for the parasite to thrive and survive, our research has focused on understanding the key steps involved
in these biological processes. We reveal using reverse genetics, biochemical and proteomic approaches, which steps in this
complex pathway provide new opportunities for drug targeting.
40
Meeting the challenge of eliminating Hepatitis C: Combining direct acting antivirals with
prophylactic vaccines
Heidi Drummer1
1. Burnet Institute, Melbourne, VIC, Australia
Hepatitis C infects more than 120 million people world-wide and causes approximately 700,000 deaths each year. The
availability of highly effective non-toxic direct acting antivirals (DAAs) that cure HCV infection in >95% of chronically infected
people will make an enormous impact on reducing the prevalence of HCV in Australia. To address the global Hepatitis C
epidemic, the World Health Organization has introduced the following targets for viral hepatitis C for 2030: An 80% reduction in
new infections, a 65% reduction in deaths and 90% of people diagnosed. However, these targets and the delivery of DAAs to
those infected with HCV presents a number of challenges. Direct acting antivirals are prohibitively expensive relative to GDP in
most countries, restricting their availability, and placing a major impost on health care payers. Approximately 50 million people
have undiagnosed HCV suggesting the number of people requiring treatment is underestimated. As HCV has a high rate of
mutation, there is a potential risk of drug resistance emerging if DAAs are used improperly. Even after a person has cleared
HCV, people remain at risk of contracting HCV if re-exposed, and reinfection rates amongst PWID is estimated to be between
8-12/100 person years. As a result, there remains an urgent need to develop a prophylactic vaccine that can prevent primary
infection and reinfection. HCV is an antigenically diverse pathogen classified into 7 genotypes and possesses immune evasion
mechanisms to restrict the generation of broadly neutralizing antibody responses (bNAbs), confounding vaccine design.
Neutralizing antibodies are key components of all currently licensed vaccines and bNAbs have been shown to prevent and cure
HCV in experimental animal models. To achieve the elimination of HCV will require the combined use of DAAs, prophylactic
vaccines, diagnosis, and harm reduction measures. Numerous vaccine candidates are at various stages of development.
Modelling shows that even a moderately effective vaccine (30% efficacy) can work together with DAAs to reduce the number of
treatments and halve prevalence. In a setting of 50% initial prevalence, 26 DAA treatments are required for every 1000 PWID
for 15 years to halve prevalence. A 50% reduction in prevalence over 15 years can be achieved by vaccinating 50/1000 PWID
and providing treatment to 13/1000 PWID per year. Significant cost savings to health care systems can be achieved through
the combined use of DAAs and vaccines to achieve elimination targets faster.
41
Imaging Dengue Virus Infection and Treatment Response
Subhash Vasudevan1
1. Duke-NUS Graduate Medical School, Singapore, SINGAPORE
The development of antiviral drugs and biotherapeutics for acute viral diseases like dengue fever require reliable and robust
biomarkers. In this talk I will discuss our recent exploration of using positron emission tomography in conjunction with a widely
used raidoligand, 18F-fluorodeoxyglucose (FDG), for assessing treatment response during drug efficacy testing in a mouse
model of dengue infection.
42
New defences against mosquito-vectors of disease – progress and prognoses
Greg Devine1
1. QIMR Berghofer, Herston, QLD, Australia
Where there are no vaccines or chemotherapeutants, mosquito management remains the mainstay of control for many vectorborne diseases. Despite this reliance, our existing control tools are compromised by issues of coverage, cost, insecticide
resistance and human compliance. In recent years, sequential epidemics and pandemics of dengue, chikungunya and Zika,
costing billions of dollars, have created an appetite for the critical evaluation of existing control tools and the development and
trial of innovative, operationally sustainable alternatives. These include: 1) the use of mosquitoes as vehicles for the transfer of
larvicides [“auto-dissemination”] 2) the rapid deployment of volatile pyrethroids to prevent mosquito bites in outbreak areas 3)
the replacement of natural mosquito populations with ones infected with a bacteria (Wolbachia) that blocks virus transmission
4) “crashing” the natural vector population by releasing Wolbachia-infected males whose subsequent matings result in nonviable eggs and, similarly, 5) releasing genetically modified male mosquitoes as a modern variant of the “sterile insect
technique”. The progress, operational feasibility and sustainability of these various approaches will be discussed.
43
P05 - Development of bioluminescent murine models of bacterial infection for preclinical
efficacy trials of novel drug classes.
Abiodun D Ogunniyi1, Manouchehr Khazandi1, Elizabeth E Hickey1, Zlatko Kopecki2, Allison Cowin2, Stephen W Page3,
Darren J Trott1
1. Australian Centre for Antimicrobial Resistance Ecology, School of Animal and Veterinary Sciences, The University of
Adelaide Roseworthy, SA 5371 AUSTRALIA, The University of Adelaide, Roseworthy, SA, Australia
2. Future Industries Institute, The University of South Australia, Mawson Lakes, SA 5095, Australia, The University of South
Australia, Mawson Lakes, SA, Australia
3. Neoculi Pty Ltd, Burwood, VIC 3125, Australia, Burwood, VIC, Australia
New drug classes are urgently needed to address the increase and global spread of antimicrobial resistance in humans and
emerging losses in animals worldwide. However, animal models for detailed investigation of disease progression that mimics
bacterial infection in animals and humans often involve the use of separate cohorts of animals at each stage of disease. We
have developed and optimised bioluminescent (light-emitting) models of dermal partial-thickness scald wounds and sepsis in
mice by infection with a recombinant luciferase-expressing Staphylococcus aureus strain (Xen29). For the skin wound model,
mice were infected with Xen29 two days post-wounding after which separate groups were treated twice daily with either a 2%
topical mupirocin ointment or PBS control for 7 days. For the sepsis model, mice were infected with a lethal dose of Xen29
intraperitoneally (IP) after which separate groups were treated IP with 6 mg/kg daptomycin or vehicle, and time to moribund
monitored for 72 h. We obtained consistent and reproducible bacterial burden data from individual mice by regular
quantification of photon intensities on a Xenogen IVIS Lumina XRMS Series III live animal biophotonic imaging system, with
concomitant significant reduction in photon intensities in drug-treated mice. The bacterial burden in wounds or blood correlated
strongly with the total flux obtained from the bioluminescent signals of Xen29. This bioluminescent model can potentially
replace laborious and costly techniques associated with harvesting, plating and counting of bacteria from infected mice, and
also allow preclinical efficacy testing of new drug classes for treating acute and chronic bacterial infections.
44
P06 - Efficacy of a novel echinocandin, CD101, in a mouse model of azole-resistant
disseminated candidiasis
Lynn Miesel1, Kun-Yuan Lin2, Jui-Che Chien2, Mei-Lee Hsieh2, Voon Ong3, Ken Bartizal3
1. Eurofins Panlabs Inc., St. Charles, MO, United States
2. In vivo Pharmacology, Eurofins Panlabs Taiwan, Taipei, Taiwan
3. Cidara Therapeutics, Inc., San Diego, CA, USA
CD101 is a novel echinocandin with long-acting pharmacokinetics and exceptional stability that is being developed for
treatment of serious fungal infections. This study objective was to evaluate the in vivo efficacy of CD101 in a neutropenic
mouse model of azole-resistant candidiasis.
An azole-resistant C. albicans human blood isolate, strain R357, was used for the mouse disseminated candidiasis model.
R357 is resistant to fluconazole (Flu), voriconazole, and posaconazole but is susceptible to amphotericin B (AmB) and
echinocandins. Mice were rendered neutropenic with cyclophosphamide then infected by intravenous (IV) injection with 10 5
CFU/mouse. Test articles were administered once, 2 hrs after infection, to mouse groups treated with AmB (3 mg/kg, IV
administration), Flu (20 mg/kg, oral (PO) administration), and CD101 (3, 10 or 30 mg/kg intraperitoneal (IP) administration).
Animals were euthanized at 72 hr after infection and C. albicans counts in kidney tissue were measured (CFU/g-tissue).
A single 3-mg/kg dose of CD101 resulted in a significant reduction in C. albicans bioburden relative to the vehicle control group
(>99.9% reduction in CFU; p<0.05). AmB (3 mg/kg) was also efficacious in this model (>99% CFU reduction p<0.05). Flu (20
mg/kg) was less efficacious (83.9% CFU reduction).
In conclusion, CD101 by IP administration was effective in a mouse model of disseminated C. albicans infection with an azoleresistant C. albicans strain. The efficacy supports the potential advancement of CD101 for human use against azole-resistant
Candida infections.
45
P07 - Improvement of antibiotics biopotency by complexing them with metal cations
Zyta M Ziora1, Alysha G Elliott1, Mark A T Blaskovich1, Matthew A Cooper1
1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
There is an urgent demand for antimicrobial agents effective against multi-drug resistant bacteria. Careless use of antibiotics
has led to untreatable infections, and this rise and spread of resistant bacteria is a serious threat for health systems across the
globe. With the ongoing increase in drug resistance, the treatment of bacterial diseases by antibiotics is becoming less
effective, and there are very few new antibiotics in the clinical pipeline, necessitating the development of alternative
approaches. The possibility of modifying existing commercially available drugs to overcome MDR is a favorable approach,
saving costs and time compared to the design and development of completely new drugs.
One possible tactic to increase the activity of antibiotics involves their complexation with metal cations. Metal ions such as
Zn(II), Cu(II), Ag(I) or silver nanoparticles are known both for their antimicrobial properties and for their ability to increase the
activity of different antibiotics. Coordination of drugs with certain transition metals is known to influence the drug´s antimicrobial
properties and biological activity. Isothermal titration calorimetry was used to examine the structure and interactions between
Cu(II), Ag(I) or Zn(II) and antibiotics. The metal complexes were also tested for antimicrobial activity showing a synergistic
increase in the activity of antibiotics.
At the current stage, our results add to the preliminary evidence supporting the application of silver ion as antibiotic adjuvant to
develop more efficient and sophisticated therapies and to offer a potential new approach towards treating the threat of
‘superbugs’.
46
P08 - The journey towards individualised treatment of Neisseria gonorrhoea utilising the
SpeeDx Rapid Dx technology.
Ella Trembizki1, Cameron Buckley1, David Whiley1, 2
1. The university of Queensland Centre for Clinical Research (UQCCR), Brisbane, QLD, Australia
2. Central Laboratory, Pathology Queensland , Brisbane, QLD
Neisseria gonorrhoeae (NG) antimicrobial resistance (AMR) is a major global concern. A proposed strategy to combat
resistance is the recycling of previously effective antibiotics via the use of molecular methods to predict susceptibility and inform
treatment. Ciprofloxacin is considered an ideal candidate for this purpose. As a part of a large nationwide study, we examined
the molecular basis of ciprofloxacin AMR in 3,028 NG isolates from throughout Australia (years 2012 to 2014). Mutation profiles
were correlated with minimum inhibitory concentrations. Through this screening we identified candidate sequences that predict
ciprofloxacin susceptibility. We then developed and validated a real-time PCR method to predict ciprofloxacin susceptibility
directly from clinical samples (Cipro-NAAT), and trialed it using NG-positive clinical samples (n=1,630; year 2014) from the
Northern Territory (NT) of Australia where bacterial culture is limited. Based on the 3,028 tested isolates, the gyrA S91F
provided 99% accuracy for predicting ciprofloxacin susceptibility and was used as the sequence target for the Cipro-NAAT.
When the Cipro-NAAT was applied to the 1,630 NT clinical samples, 75.4% (1,229/1,630) were successfully characterised; of
these, only 5.77% (CI 95%: 4.47-7.07%) were indicated to be ciprofloxacin resistant. This was notably lower than culture-based
testing at 13.53% (CI 95%: 9.1-17.96%). Overall these data highlight the feasibility of recycling ciprofloxacin for treatment of
gonorrhoea in Australia. We are now collaborating with SpeeDxTM to develop a rapid Dx NG treatment algorithm based on the
latter targets and progressing towards a clinical trial.
47
P09 - Development of monoclonal antibodies against uropathogenic Escherichia coli Antigen
43 impairing bacterial cell autoaggregation
Alvin W Lo1, Jason J Paxman 2, Martina Jones 3, Jeff Hou3, Kelvin Goh1, Begoña Heras4, Mark A Schembri1
1. Antibody Development National Biologics Facility, Australian Infectious Diseases Research Centre, School of Chemistry and
Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, , La Trobe University, Melbourne, VIC,
Australia
3. Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Antibody Development National
Biologics Facility, Brisbane, QLD , Australia
4. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC,
Australia
Uropathogenic Escherichia coli (UPEC) is the major etiological agent of urinary tract infection (UTI). UPEC are increasingly
associated with antibiotic resistance, leading to high rates of treatment failure and the need to develop alternative approaches
to treat and prevent UTI. Autotransporter proteins represent an important group of virulence factors that contribute to UPEC
pathogenesis through their ability to mediate adhesion, aggregation, invasion and biofilm formation. We have recently
determined the crystal structure of functional α-domain from the UPEC autotransporter adhesin Antigen 43 (Ag43) and
demonstrated that its unique L-shaped structure drives the formation of cell aggregates via a molecular Velcro-like mechanism
(1). In this study, we identified two monoclonal antibodies (mAbs; 7D10 and 10C12) that inhibited Ag43-mediated cell
autoaggregation. Recombinant fragment antigen-binding (Fab) domain of 10C12 (but not 7D10 Fab) exhibited similar cell
autoaggregation inhibition capacity as the 10C12 mAb. 10C12 Fab also inhibited Ag43-mediated UPEC biofilm formation.
Epitope mapping of 10C12 Fab binding using an ELISA against a set of Ag43 mutants that contain specific structure-targeted
deletions (1) revealed that the exposed b hairpins at the L-shaped bending of Ag43 are 10C12 Fab target, and thus required for
the maintenance of a cell autoaggregation competent state of Ag43. Further molecular characterization of the interaction
between Ag43 and 10C12 Fab is in progress, which will provide new insight into Ag43 function and could form the basis for the
development of novel therapeutics to prevent UPEC aggregation and biofilm formation.
1.
1. Heras, B., Totsika, M., Peters, K. M., Paxman, J. J., Gee, C. L., Jarrott, R. J., Perugini, M. A., Whitten, A. E., and
Schembri, M. A. (2014) The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated
bacterial clumping. Proceedings of the National Academy of Sciences of the United States of America 111, 457-462
48
P10 - Rapid molecular detection of antibiotic resistance mutations of using novel qPCR
technologies
Litty Tan1, Samantha Walker1, Rebecca Seehoo1, Simon Erskine1, Madeline Windsor1, Elisa Mokany1
1. SpeeDx, Sydney, NSW, Australia
Publish consent withheld
49
P11 - Exploring the potential of T7 bacteriophage protein Gp2 as a novel inhibitor of
mycobacterial RNA polymerase
Juanelle du Plessis1, Ruben Cloete2, Lynn Burchell3, Paramita Sarkar3, Robin M Warren1, Alan A Christoffels2, Ramesh
Wigneshweraraj3, Samatha L Sampson1
1. DST/NRF Centre of Excellence in Biomedical Tuberculosis Research, Stellenbosch University, Cape Town, Western Cape,
South Africa
2. South African National Bioinformatics Institute, University of the Western Cape, Cape Town, Western Cape, South Africa
3. MRC Center for Molecular Bacteriology and Infection, Imperial College, London, United Kingdom
Publish consent withheld
1.
2.
3.
4.
5.
6.
7.
Sheppard C, Cámara B, Shadrin A, Akulenko N, Liu M, Baldwin G, Severinov K, Cota E, Matthews S,
Wigneshweraraj SR. 2011. Reprint of: inhibition of Escherichia coli RNAp by T7 Gp2 protein: role of negatively charged
strip of amino acid residues in Gp2. J Mol Biol 412:832–841.
Mekler V, Minakhin L, Sheppard C, Wigneshweraraj S, Severinov K. 2011. Molecular Mechanism of Transcription
Inhibition by Phage T7 gp2 Protein. Journal of Molecular Biology 413:1016–1027.
Shadrin A, Sheppard C, Severinov K, Matthews S, Wigneshweraraj S. 2012. Substitutions in the Escherichia coli
RNA polymerase inhibitor T7 Gp2 that allow inhibition of transcription when the primary interaction interface between Gp2
and RNA polymerase becomes compromised. Microbiology (Reading, Engl) 158:2753–2764.
James E, Liu M, Sheppard C, Mekler V, Cámara B, Liu B, Simpson P, Cota E, Severinov K, Matthews S,
Wigneshweraraj S. 2012. Structural and mechanistic basis for the inhibition of Escherichia coli RNA polymerase by T7
Gp2. Mol Cell 47:755–766.
Shadrin A, Sheppard C, Savalia D, Severinov K, Wigneshweraraj S. 2013. Overexpression of Escherichia coli udk
mimics the absence of T7 Gp2 function and thereby abrogates successful infection by T7 phage. Microbiology (Reading,
Engl) 159:269–274.
Nechaev S, Severinov K. 1999. Inhibition of Escherichia coli RNA Polymerase by Bacteriophage T7 Gene 2 Protein.
Journal of Molecular Biology 289:815–826.
Cámara B, Liu M, Reynolds J, Shadrin A, Liu B, Kwok K, Simpson P, Weinzierl R, Severinov K, Cota E, Matthews S,
Wigneshweraraj SR. 2010. T7 phage protein Gp2 inhibits the Escherichia coli RNA polymerase by antagonizing stable
DNA strand separation near the transcription start site. PNAS 107:2247–2252.
50
P12 - Progressing antimicrobial sensitivity testing for fastidious bacterial species
Conny Turni1, Junyao Wang1, Sandra Prueller2, Martin Beyerbach3, Guenter Klein2, Lothar Kreienbrock2, Katrin
Strutzberg-Minder4, Heike Kaspar5, Diana Meemken2, Corinna Kehrenberg2, Pat Blackall1
1. Queensland Alliance for Agriculture and Food Innovation (QAAFI), The University of Queensland, Ecosciences Precinct,
Dutton Park, QLD 4102, Australia
2. Institute for Foodquality and Foodsafety, University of Veterinary Medicine Hanover, Hannover, Germany
3. Department of Biometry, Epidemiology and Information Processing, WHO Collaborating Centre for Research and Training in
Veterinary Public Health,, University of Veterinary Medicine Hannover, Hannover, Germany
4. Innovative Veterinary Diagnostics (IVD) Laboratory, Hannover, Germany
5. Federal Office of Consumer Protection and Food Safety, Berlin, Germany
Several fastidious bacteria have been associated with serious respiratory disease in livestock, including Haemophilus parasuis
in pigs and Avibacterium paragallinarum in poultry. Both these bacteria cause significant diseases (Glässer’s disease and
infectious coryza, respectively) that have a negative economic impact worldwide (Aragon et al. 2012; Blackall and Soriano
2005). None of the current CLSI approved media are suitable for the growth of these two bacterial species and breakpoints do
not exist. A recent study suggested that supplemented Test Medium Broth (TMB) and an agar form of the same medium are
ideal media for H. parasuis sensitivity testing (Dayao et al. 2014). However, these media are very complex and hence this
research has aimed to find less complex media that supports the growth of isolates of both species from diverse origins. For H.
parasuis, cation-adjusted Mueller-Hinton broth (CAMHB) plus NADH and sterile filtered heat-inactivated chicken serum, was
developed. CLSI quality control strains were first used to validate this medium. Then field strains representing 13 serovars
were tested with the supplemented CAMHB and the supplemented TMB. There was no significant difference in the results
from both media, suggesting that the new supplemented CAMHB is ideal for use in routine diagnostic laboratories. Our
research is currently looking at the suitability of blood agar with supplements compared to the agar version of CAMHB as a disk
diffusion medium and TMB compared to CAMHB as an MIC medium for Av. paragallinarum antimicrobial susceptibility testing.
1.
Aragon, V., Segales, J., Oliveira, S., 2012. Glasser’s disease. In: Zimmerman, J., Karrker, L., Ramirez, A., Schwarz,
K., Stevenson, G. (Eds.), Diseases of Swine. 10th edn. John Wiley & Sons, Inc, Iowa, USA, pp. 760–770. Blackall P.J.,
Soriano-Vargas E., 2013. Infectious coryza and related bacterial infections. In: Swayne DE, Glisson JR, McDougald LR,
Nolan, L.K., Suarez, D.L and Nair, V.L. (Eds.) Diseases of Poultry. 13th edn. Wiley-Blackwell, Ames, Iowa, pp. 859-874.
Dayao, D.A., Kienzle, M., Gibson, J.S., Blackall, P.J., Turni, C. 2014. Use of a proposed antimicrobial susceptibility testing
method for Haemophilus parasuis. Veterinary Microbiology, 172: 586-589.
51
P13 - Structure-activity (SAR) and structure-toxicity (STR) relationships of novel polymyxin
analogues
Jiping Wang1, Kade D Roberts1, 2, Heidi Yu3, Lv Wang1, Roger L Nation1, Philip E Thompson2, Tony Velcov1, Jian Li3, 1
1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC,
Australia
2. Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
3. Monash Biomedicine Discovery Institute, Department of Microbiology, Monash University, Clayton, VIC, Australia
Polymyxin B (PMB) and colistin (COL) are the last-line therapy for the treatment of multidrug-resistant (MDR) Gram-negative
pathogens. However, their effective use in the clinic is hampered by the dose-limiting nephrotoxicity. In order to develop novel,
safer polymyxins, we investigated the SAR and STR of substituting the residues at positions 6 and 7 of the polymyxin core with
less hydrophobic amino acids. Peptides were synthesized using solid-phase peptide synthesis. MICs were determined using
broth microdilution against a panel of 17 PM-susceptible clinical MDR Gram-negative isolates and ATCC strains. Nephrotoxicity
was investigated in mice and kidney histological examination was conducted. In vivo efficacy was evaluated using a mouse
blood infection model. Substitution of either position 6 (D-Phe/Leu) or position 7 (Leu) amino acid residues with Thr, Ala, Val,
Ser or Abu resulted in no histological kidney damage, compared to PMB and COL (mild to severe nephrotoxicity) in mice. While
changes at position 7 retained antimicrobial activity, a minimum level of hydrophobicity (D-Leu6) is required at position 6 to
maintain comparable in vitro (MIC <0.125-2 mg/mL) and in vivo (Dlog10 > -1 to -3) antibacterial activity to PMB and COL.
Further modification by changing the stereochemistry of position 3 to the D-configuration nullified the decrease in the
nephrotoxicity achieved by modifications at position 7. In conclusion, we found that polymyxin nephrotoxicity can be modulated
through substitution of position 7 with less hydrophobic residues without compromising antibacterial potency. Our lead peptides
show potential for obtaining safer polymyxins, and are undergoing further preclinical evaluation.
52
P14 - Liptins: a new class of antimicrobial small molecules that bind phosphatidylglycerol (pg)
at the bacterial plasma membrane
Dennis H Burns1
1. Wichita State University, Wichita, KANSAS, United States
Prior work in our group has demonstrated the PG lipid-binding ability of several small synthetic molecules we term liptins.1-3 A
liptin based on a picket-porphyrin scaffold (PPL) has been shown to selectively bind to PG in synthetic vesicles or when in
Gram-negative and Gram-positive bacterial plasma membranes. Unlike antimicrobial peptides (AMPs) that use non-specific
Coulombic interactions to bind to bacterial membranes, our liptin’s binding pockets are complementary to the PG
multifunctional head group, resulting in high binding affinity. Unlike AMPs that permeate the bacterial membrane, the PPL
appears to stay strongly bound to the lipid head group. We have found that PPL binds to the plasma membrane of Escherichia
coli, Staphylococcus aureus, and Enterococcus faecalis, resulting in the inhibition of bacterial growth and increased bacterial
killing at low µM concentrations. Binding PG causes depolarization of the plasma membrane in S. aureus at 2-3 µM PPL.
Serial passage experiments show the bacteria do not develop resistance to the compound’s inhibitory activity after 80-100
bacterial generations. Toxicity studies showed the PPL has no adverse effect on human hepatic cells. When the liptin-PG
complex forms in the plasma membrane it dramatically alters the effective lipid head group charge and size. This binding event
is hypothesized to disrupt the homeostasis of the bacterial lipidome and lower cell viability (graphical representation in figure
below). Due to the general nature of the membrane damage caused by liptins, the rate of resistance occurrence is expected to
be low.
53
P15 - Pharmacodynamics of aerosolized amikacin monotherapy against non-susceptible
clinical isolate of Pseudomonas aeruginosa
Fekade B Sime1, 2, Adam Johnson3, Sarah Whalley3, Anahi Santoyo-Castelazo3, Jeffrey Lipman2, William Hope3, Jason
A Roberts1, 2
1. Centre for Translational Anti-infective Pharmacodynamics , School of Pharmacy, The University of Queensland, Brisbane ,
Queensland , Australia
2. Burns Trauma and Critical Care Research Centre, School of Medicine, The Universtty of Queensland , Brisbane,
Queensland , Australia
3. Department of Molecular and Clinical Pharmacology, The University of Liverpool, Liverpool, Merseyside, United Kingdom
Introduction: There is an ongoing effort to develop aerosolized amikacin formulations, alone or in combination with other
antibiotics, for the management of multidrug resistant ventilator associated pneumonia. However, data are still limited on the
pharmacodynamics of antibiotics at the exposures achieved in the airway.
Objectives: The aim of this study was to describe the pharmacodynamics of simulated epithelial lining fluid exposures of
aerosolized amikacin against non-susceptible clinical isolate of P. aeruginosa.
Methods: A hollow fibre infection model (HFIM) was set up with an initial inoculum of 10 8 CFU/mL of P. aeruginosa (MIC = 16
mg/L). Peak epithelial lining fluid concentrations of 1500 mg/L, 300 mg/L and 60 mg/L were simulated with a twice daily dosing
schedule over 7 days of treatment. The change in the total and resistant bacterial population over the treatment period was
determined by quantitative cultures.
Results: The peak concentration of 1500 mg/L achieved rapid and sustained bactericidal effect with complete eradication
within 24 hours (Figure1). The 300 mg/L peak concentration resulted in rapid killing on day one followed by rapid regrowth to
initial inoculum concentration from day two through to the last day of treatment. Only a brief bacteriostatic effect (» 3 log kill)
was observed with 60 mg/L peak concentrations.
Conclusions: High concentrations of amikacin achieved by inhalational administration are required to maximize bactericidal
activity and reduce the risks of selective regrowth of resistant subpopulations.
55
P17 - Inspiration from natural peptidoglycan precursors to develop small molecules against
bacterial cell wall assembly
wei-chieh cheng
Publish consent withheld
56
P18 - Siderophore antibiotic conjugates with novel linkers
Poulami Talukder1, Brian Blank1, Patricia Miller2, Marvin Miller2, Adam Renslo1
1. University of California, San Francisco, San Francisco, CA, United States
2. University of Notre Dame, Notre Dame, IN, USA
Publish consent withheld
57
P19 - Antibacterial drug discovery using secondary metabolites from Australian native plants
Dane Lyddiard1, Ben W Greatrex1
1. School of Science & Technology, University of New England, Armidale, NSW, Australia
With an increase in antibacterial drug resistance and a lull in drug discovery, researchers are taking a variety of approaches in
an attempt to fill the growing need for novel antibacterial drugs. Traditional screening of bacterial and fungal species for active
metabolites has yielded the majority of clinically used antimicrobial agents, however, other compound reservoirs such as plants
have been less explored. To improve molecular diversity among antibacterial lead compounds, a platform has been
constructed whereby secondary metabolites are extracted from Australian native plants not previously investigated. Extracts
are screened for activity, TLC bioautographies are undertaken on hits to inform active compound isolation, compounds are
characterised, MIC assays back test isolated compounds, and further testing is performed as required. In its early stages, the
screening program has focused on genera rich in secondary metabolites and has demonstrated some promising results.
58
P20 - Inhibition of wound pathogens by garlic and manuka honey
Rowena Jenkins1, P Jones1, Danielle Williams2, Michael Graz2, Rose Cooper1
1. Department of Biomedical Sciences, Cardiff Metropolitan University, Cardiff, Glamorgan, United Kingdom
2. Neem Biotech Ltd, Abertillery, Blaenau Gwent, United Kingdom
With the continued emergence of antibiotic resistant pathogens, ancient wound remedies are being re-evaluated. The
antibacterial potential of garlic and manuka honey against wound pathogens was determined by adapted EUCAST broth
microdilution, synergistic combinations were investigated using chequerboards and FICI values were calculated. Two garlic
preparations were tested (NBR150075 and NBR150067-3) and one type of medical grade manuka honey (Medihoney™). Test
bacteria were Staphylococcus aureus NCTC 6571, Epidemic strain of methicillin-resistant S. aureus (MRSA NCTC 13142),
Staphylococcus epidermidis ATCC 12228, Pseudomonas aeruginosa PA01, Acinetobacter baumannii NCTC 1216 and a
clinical strain (E75U382259), Escherichia coli NCTC 10418, and a clinical isolate of vancomycin-resistant Enterococcus
faecalis. Effects on bacterial morphology and viability were determined using epifluorescence with LIVE/DEAD staining. Mode
of action against test bacteria as determined from MICs and MBCs indicated that each of manuka honey and NBR150067-3
were bactericidal, whereas NBR150075 was bacteriostatic. Of the 12 combinations tested, honey and garlic preparations acted
synergistically against two of the organisms and six other additive combinations. No antagonistic combinations were found.
Garlic and honey induced morphological changes such as cell elongation and lysis in some of the test organisms. This
suggests a possible role for these compounds in modern wound care in the future.
59
P21 - Investigating the potential therapeutic application of novel rumen bacterial-derived
antimicrobial peptides against Pseudomonas aeruginosa
Adam Mulkern1, Linda B Oyama1, Gareth Evans2, Danielle Williams2, Sharon A Huws1, Michael Graz2
1. Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom
2. Neem Biotech Ltd, Abertillery, Blaenau Gwent, United Kingdom
Within the European Union, approximately one in every 2500 babies is born with cystic fibrosis (CF). Sufferers of CF are
inadvertently more susceptible to opportunistic pathogens such as Pseudomonas aeruginosa. Persistent colonisation of the
lungs with P.aeruginosa can lead to the formation of drug resistant biofilms, facilitating the development of life-threatening
infection and limiting the effectiveness of antibiotics. Despite the wealth of knowledge on CF, there is still no known cure and
developing alternative biofilm control agents is important in controlling the lung infections associated with the disease. We
examined the potential of three novel, semi-synthetic antimicrobial peptides (AMPs), isolated from cow rumen bacteria as
antimicrobial agents against P.aeruginosa. The efficacy of these AMPs was tested against 8 strains of P.aeruginosa, isolated
from CF patients, including the Liverpool Epidemic strains. Minimum inhibitory concentrations of the strains ranged between 832 μg/ml. The AMPs were able to eradicate biofilms of P.aeruginosa PAO1 at concentrations close to the MIC for this strain (32
μg/ml). Time kill kinetics also showed that the AMPs have a rapid bactericidal effect, inducing ≥3 log10 CFU/ml decrease in
P.aeruginosa PAO1 CFU/mL within the first 2 h of exposure. Time kill kinetics and biofilm kill assays have only been completed
with P.aeruginosa PAO1 and further characterisation and mode of action studies are currently ongoing encompassing more
strains. Irrespective, these promising antimicrobial peptides derived from the cow microbiome may form new alternatives for the
effective control of P.aeruginosa infections and improve the quality of life for cystic fibrosis patients.
60
P22 - Ajoene, a novel therapeutic that shows enhanced activity in in vivo and in vitro models of
Pseudomonas aeruginosa infection
Laura Bricio-Moreno1, EM Waters1, Daniel Neill1, Gareth Evans2, Michael Graz2, Jo L Fothergill1, Aras Kadioglu1
1. Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom
2. Neem Biotech Ltd, Abertillery, Blaenau Gwent, United Kingdom
Pseudomonas aeruginosa is an opportunistic pathogen that causes a variety of infections in humans. It is one of the ESKAPE
pathogens described as clinically relevant and highly multidrug resistant. In this study we tested the antimicrobial properties of
Ajoene against the epidemic P. aeruginosa strain LESB65 in vivo and in vitro. Artificial Sputum Media was used to study the
antimicrobial properties of Ajoene against developing and mature P. aeruginosa biofilms to mimic both early and chronic
infections. Ajoene was able to significantly inhibit the growth of LESB65 during the first 48h of infection when administered in
combination with Tobramycin in the early infection in vitro model. Additionally, the production of pyocyanin was significantly
reduced in the combined presence of Tobramycin and Ajoene when compared to treatment with Tobramycin and Ajoene alone
during the 7 days of the experiment. Ajoene and Tobramycin in combination were more effective against pre-established
biofilms. Changes in gene expression of virulence-related genes were also studied. In the in vivo model of infection, we
observed a reduction in the number of bacteria colonising the upper respiratory tract of mice treated with Tobramycin alone or
with the combination of Tobramycin and Ajoene. By day 7, 80% of mice had cleared P. aeruginosa from the upper respiratory
tract using the Ajoene/Tobramycin combination compared to 0% of the controls. These results suggest that Ajoene, particularly
in combination with Tobramycin, could be used to treat lung infections with P. aeruginosa.
61
P23 - Polymyxin resistance in Pseudomonas aeruginosa: a transcriptomic approach
Mei-Ling Han1, Yan Zhu2, Alina D. Gutu3, Hsin-Hui Shen2, Darren J. Creek1, Samuel M. Moskowitz4, Tony Velkov1, Jian
Li2
1. Monash Institute of Pharmaceutical Sciences, Monash University, Melbourne, VIC, Australia
2. Faculty of Medicine, Nursing & Health Sciences, Monash University, Melbourne, VIC, Australia
3. Department of Molecular Biology, Massachusetts General Hospital, Boston, MA, United States
4. Vertex Pharmaceuticals, Boston, MA, United States
Polymyxins are often used as the last-line therapy for multidrug-resistant Pseudomonas aeruginosa (PA) infections. However,
polymyxin-resistant PA isolates have been increasingly reported, highlighting the urgency for the elucidation of the
comprehensive mechanisms of polymyxin resistance. Here, we employed transcriptomics to investigate the responses of
polymyxin-susceptible wild-type PAK and -resistant mutant PAKpmrB6 after exposure to polymyxin B (PMB). Transcriptomic
profiles of bacterial samples treated with 4 mg/L PMB at 1 and 4 h were obtained using Illumima RNA-Seq and pathway
analysis was conducted. PMB induced different transcriptomic responses in PAK and PAKpmrB6. Notably, PMB treatment led
to 558/226 (up/down) differentially expressed genes (DEGs) in PAK at 1 h, which decreased to 94/7 at 4 h. Whereas, in the
polymyxin-resistant PAKpmrB6, minimal transcriptomic response was induced by PMB, with only 8/1 and 11/1 DEGs observed
at 1 and 4 h, respectively. Up-regulation of two major operons, arnBCADTEF (responsible for lipid A modification, 47-, 48-, 35-,
32-, 35-, 16-, 12-fold) and speDE (spermidine synthesis, ~20-fold) was evident in PAK by PMB at 1 h; the two-component
regulatory systems, PmrAB, PhoPQ and CarRS were also significantly up-regulated (> 2-fold) [1,2]. Notably, these genes were
not differentially expressed after PMB treatment in PAKpmrB6, which displays 4-amino-L-arabinose modified lipid A. A novel
finding of our study is the up-regulation of pagL (responsible for lipid A deacylation, > 3-fold) and PRK_04467 (corresponding to
a ß-lactamase, > 20-fold) in both strains due to PMB treatment. This study provides important molecular insights into the
response of PA to polymyxins.
1.
[1] Fernández L, Alvarez-Ortega C, Wiegand I, Olivares J, Kocíncová D, Lam JS, Martínez JL, Hancock RE.
Characterization of the polymyxin B resistome of Pseudomonas aeruginosa. 2013. Antimicrob Agents Chemother. 57:1109. [2] Gutu AD, Sgambati N, Strasbourger P, Brannon MK, Jacobs MA, Haugen E, Kaul RK, Johansen HK, Høiby N,
Moskowitz SM. 2013. Polymyxin resistance of Pseudomonas aeruginosa phoQ mutants is dependent on additional twocomponent regulatory systems. Antimicrob Agents Chemother. 57:2204-15.
62
P24 - Antiplasmodial alkaloids from Australian Rutaceae species
Luke P Robertson1, Sandra Duffy2, Vicky M Avery2, Anthony R Carroll1, 2
1. Environmental Futures Research Institute, Griffith University, Gold Coast, QLD, Australia
2. Eskitis Institute, Griffith University, Brisbane, QLD, Australia
Malaria is a disease caused by the protozoan parasite Plasmodium. In 2014, it was responsible for ~438,000 deaths, nearly all
of which were caused by P. falciparum [1]. Although a number of highly successful antimalarials have been developed over the
past century, drug resistances have rendered the majority ineffective [2]. Our last line of defence against malaria, artemisininbased combination therapies (ACTs), are now also beginning to fail in South-East Asia. It is likely that artemisinin resistance
will intensify and spread throughout Asia and into Africa, potentially causing a public health disaster [3]. Resultantly, there is a
dire need for the development of new drugs to replace ACTs.
Natural products have historically been an important source of antimalarial drugs – the plant-derived natural products quinine
and artemisinin have formed the backbone of antimalarial chemotherapeutics for centuries [4]. With the aim of discovering new
antimalarial drug leads, we chemically investigated the Australian endemic plant genus Flindersia (Rutaceae). This genus is
particularly rich in alkaloids and is known to contain compounds that are active against chloroquine-resistant P. falciparum [5].
By using semi-preparative HPLC in combination with MS and NMR-guided interpretation, we isolated multiple new and
previously known indole alkaloids. We report the antiplasmodial activity of a number of previously untested bis-indole alkaloids
as <1 µM.
1.
2.
3.
4.
5.
World Malaria Report 2015. Geneva, Switzerland: World Health Organization; 2015
White NJ. Antimalarial drug resistance. J Clin Invest 2004; 113: 1084-1092
Ariey F, Witkowski B, Amaratunga C, Beghain J, Langlois A-C, Khim N, Kim S, Duru V, Bouchier C, Ma L, Lim P,
Leang R, Duong S, Sreng S, Suon S, Chuor CM, Bout DM, Menard S, Rogers WO, Genton B, Fandeur T, Miotto O,
Ringwald P, Le Bras J, Berry A, Barale J-C, Fairhurst RM, Benoit-Vical F, Mercereau-Puijalon O, Menard D. A molecular
marker of artemisinin-resistant Plasmodium falciparum Nature 2014; 505: 50-55
Wells TNC. Natural products as starting points for future anti-malarial therapies: Going back to our roots? Malar J
2011; 10(Suppl 1): S3
Fernandez LS, Buchanan MS, Carroll AR, Feng YJ, Quinn RJ, Avery VM. Flinderoles A− C: Antimalarial bis-indole
alkaloids from Flindersia Org Lett 2008; 11: 329-332
63
P25 - Uncovering novel susceptibility targets to enhance the efficacy of third-generation
cephalosporins against MDR E. coli
Minh Duy Phan1, Amy Bottomley2, Kate Peters1, Liz Harry2, Mark Schembri1
1. Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of
Queensland, Brisbane, QLD, Australia
2. The ithree Institute, University of Technology Sydney, Sydney, NSW, Australia
Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract infection (UTI), one of the most common infectious
diseases in humans. UPEC are increasingly associated with resistance to multiple antibiotics. This includes resistance to thirdgeneration cephalosporins, a common class of antibiotics frequently used in the treatment of UTI. We have utilized an
innovative high-throughput functional genomic screen (transposon directed insertion-site sequencing; TraDIS) to identify key
genes required for survival of the multidrug resistant (MDR) UPEC sequence type 131 (ST131) strain EC958 in the presence of
the third generation cephalosporin cefotaxime. We confirmed that blaCMY-23 is the major extended spectrum β-lactamase (ESBL)
gene in EC958 responsible for mediating resistance to cefotaxime. Strikingly, our screen also revealed that mutation of genes
involved in cell division sensitizes EC958 to cefotaxime. We explored the role of these cell division and protein secretion genes
to confirm their contribution to cefotaxime resistance, and test their role in resistance to other third-generation cephalosporins.
Overall, the work provided a novel exemplar for the use of TraDIS to define molecular mechanisms of antibiotic resistance, and
may lead to the discovery of novel targets for the development of new antibiotics to treat MDR pathogens.
64
P26 - Identifying the cellular targets of antibiotics using T7 phage display
Shalini Tirunagari1, Jenny Vo1, Peter Karuso1, Andrew M Piggott1
1. Department Of Chemistry And Biomolecular Sciences,, Macquarie University, Sydney, NSW 2109, Australia
Antibiotic resistance is currently one of the most significant global healthcare challenges. New resistance mechanisms are
evolving rapidly, imparting broad-spectrum antibiotic resistance to an ever-increasing microbial population. Consequently, a
major consideration in the development of next-generation of antibiotics must be their mode of action, as scaffolds targeting
known compromised targets are clearly less attractive. The challenge is to develop unbiased genome-wide methodologies that
can rapidly link new and old antibiotics with their cellular targets.
In this work, we have employed reverse chemical proteomics using T7 phage display 1 as a platform technology to link
antibiotics to their cellular targets, allowing their modes of action to be determined. This scalable approach allows antibiotics
targeting novel cellular/biochemical pathways to be identified and early prioritized for further development as chemotherapeutic
agents to address the current antibiotic resistance crisis.
Initially, two bacterial T7 phage display libraries were constructed from genomic DNA (gDNA) isolated from Klebsiella
pneumoniae and Bacillus subtilis. The gDNA was fragmented by restriction enzymes and cloned into the T7Select10B vector
with compatible restriction sites to create phage displayed libraries of proteins and peptides. As a proof-of-concept, these
libraries were then subjected to affinity selection (biopanning) using biotinylated analogues of valnemulin, telomycin and
bicyclomycin (Fig. 1). The rescued clones were sequenced and the identities of the displayed proteins determined through
BLAST searching. Finally, the affinities of the antibiotics for each recovered target were determined using on-phage binding
assays.
Fig. 1: Structures of valnemulin, telomycin and bicyclomycin.
1.
Piggott, A. M.; Karuso, P. “Identifying the cellular targets of natural products using T7 phage display", Nat. Prod.
Rep., 2016, 33, 626-636.
65
P27 - Amphipathic guanidine-embedded glyoxamide-based peptidomimetics as novel
antimicrobial agents and biofilm inhibitors
Shashidhar Nizalapur1, Naresh Kumar1, David StC Black1
1. School of Chemistry, University Of New South Wales, Sydney, NSW, Australia
Antimicrobial resistance is becoming increasingly prevalent due to the ability of bacteria to transfer genes encoding resistant
mechanisms. Furthermore, bacterial biofilms are also known to increase the development of antibiotic resistance. Antimicrobial
peptides (AMPs) are a key component of the mammalian immune system that provides protection against infections caused by
various pathogens. Unlike antibiotics, AMPs disrupt the bacterial membranes making it difficult for the bacteria to develop
resistance against AMPs. Thus, the development of novel peptidomimetics which can disrupt bacterial membranes and biofilm
formation is a viable strategy to counteract increasing antimicrobial resistance. In the present study, we report the design and
synthesis of novel amphipathic guanidine-embedded glyoxamide-based peptidomimetics via the ring-opening reactions of Nnaphthoylisatins with amines and amino acids. The antibacterial activity of these compounds by the determination of minimum
inhibitory concentration (MIC90) exhibited 6, 8 and 10 µg mL-1 against S. aureus; and 17 and 19 µg mL-1, against, E. coli. Biofilm
disruption and inhibition activities were also evaluated against various Gram-positive and Gram-negative bacteria. The most
active compounds exhibited greatest disruption of established biofilms by 65% in Staphylococcus aureus, 61% in
Pseudomonas aeruginosa, and 60% in Serratia marcescensa. Also, inhibit the formation of non-established biofilms of S.
marcescens and P. aeruginosa by 72% and 65% respectively. The in vitro toxicity of these compounds against MRC-5 human
lung fibroblast cells were shown to possess selective toxicity against bacterial cells over mammalian cells. Therefore, these
peptidomimetics represent a new cost-effective antimicrobial strategy.
66
P28 - Antifungal octapeptins
Jessica Chitty1, Mark S Butler1, Matthew A Cooper1, James Fraser1, Avril AB Robertson1
1. University of Queensland, St Lucia, QLD, Australia
Fungal pathogens cause significant morbitity and mortality in immunocompromised patients. The basidiomycete yeast
Cryptococcus neoformans is the most common of these, predominantly infecting individuals suffering from AIDS or patients
undergoing immune system-suppressing medical interventions. Current therapeutic options for treating disseminated fungal
infections are inadequate. Furthermore sustained use of the few available antifungal therapeutics is leading to emergence of
resistant fungal strains. New options in treatment of fungal pathogens are urgently needed.
Octapeptins were discovered in the 1970s as naturally occurring cationic cyclic lipopeptides with striking structural similarities to
the polymyxin antibiotics. Although they are structurally similar, octapeptins are functionally divergent as they retain activity
against polymyxins-resistant bacteria. Despite early reports of activity against bacteria, yeasts, fungi and protozoa, octapeptins
remain relatively unexplored with a surprisingly small number of publications surrounding the family of 18 naturally occurring
derivatives.
This report gives the first detailed study of antifungal activity of pure, synthetic octapeptin C4, and also provides direct
comparative data for polymyxin B and E. Furthermore, nine novel octapeptin C4 analogs were synthesized and used to
examine the structure activity relationships (SAR) of the pharmacophore. Understanding the SAR of these compounds provides
the first step in guiding design and synthesis of active efficacious derivatives suitable for drug candidate selection.
67
P29 - The challenge of exploiting microbial natural products to deliver novel antibiotics
Olga Genilloud1
1. Fundacion MEDINA, Granada, GRANADA, Spain
Microbial natural products (NPs) have been one of the most prolific sources of new leads for the discovery of novel drugs to
respond to unmet needs in infectious diseases with a large number of molecules and analogs today in the clinic. NPs present a
unique chemical space and architectural complexity, and their potency and selectivity is the result of an extended evolutionary
selection to create biologically active molecules with the required properties to interact and potentially inhibit bacterial targets.
MEDINA is a research center focused on the discovery of novel bioactive NPs with one of the richest and most diverse NPs
collections that are at the origin of our collaborative drug discovery research programs. To enrich our NPs libraries, new and
diverse strains from untapped environments are continuously added to the existing microbial collection after culture-based
approaches developed to succeed in maximizing the chemical diversity of our libraries. Our drug discovery programs are
focused in the identification of novel antibiotics with the required profile to be developed as potential new candidates to respond
to unmet needs in the management of infectious diseases and respond to the emerging antibiotic microbial resistance. As a
result of these screening programs we have identified different novel families of molecules with interesting new chemistry and
spectrum of activities currently in development that will be discussed in the context of current discovery approaches.
68
P30 - Mitigating the metabolism of antifungal aminothiazoles
Avril Robertson1, Nicholas Massey1
1. University of Queensland, Brisbane, QUEENSLAND, Australia
Current treatments for cryptococcal infections are associated with toxicity, selectivity and availability issues and are not optimal
for treating the growing threat of infection [1,2]. The 2-aminothiazole scaffold forms the basis of several successful therapeutics
and through a collaboration of the University of Cape Town and the Community for Open Antimicrobial Drug Discovery (CO-
ADD) a set of 2-aminothiazoles were tested against a panel of bacteria and fungi. A number of the compounds exhibited potent
antifungal activity against the pathogenic fungi Candida albicans and Cryptococcus neoformans. The set of 2-aminothiazoles
were reported to be rapidly metabolized when incubated with liver microsomes [3]. However, it was proposed that a carefully
designed library of heterocyclic variants could improve metabolic stability while maintain the antifungal activity of the parent
series. The heterocyclic variants were synthesized and resulted in up to 10-fold improvement of metabolic stability. Due to this
significant outcome, results of antifungal assays are awaited with enthusiasm and possibility of in vivo testing in the near future
is promising.
1.
St Jean, D. J.; Fotsch, C., Mitigating Heterocycle Metabolism in Drug Discovery. J. Med. Chem. 2012, 55 (13), 60026020
2.
3.
Odds, F. C.; Brown, A. J. P.; Gow, N. A. R., Antifungal agents: mechanisms of action. Trends Microbiol. 2003, 11 (6),
272-279
Mjambili, F. et al., Synthesis and Biological Evaluation of 2-Aminothiazole Derivatives as Antimycobacterial and
Antiplasmodial Agents. Bioorg. Med. Chem. Lett. 2014, 24 (2), 560-64
69
P31 - Antibacterial effects of copper(II) ionophores
Alastair G McEwan1, Karrera Y Djoko1, William M Shafer2
1. University of Queensland, ST LUCIA, QLD, Australia
2. Department of Microbiology, Emory University, Atlanta, Georgia, USA
The efficacy of copper in the treatment of infections has been recognised since antiquity but this metal ion not found use in a
contemporary clinical settings. There are limitations in the use of aqueous copper as a consequence of its poor cellular
permeability but there is increasing interest in the use of lipophilic copper (Cu)-containing complexes to overcome such
difficulties. In this work we assessed the potential of copper ionophores to combat bacterial infections. We showed that Cu
complexes with bis(thiosemicarbazone) ligands [Cu(btsc)] exert antibacterial activity against a range of medically significant
pathogens including Neisseria gonorrhoeae, Streptococcus pneumoniae and Haemophilus influenzae. The Cu(btsc) complexes
were effective against multi-drug resistant gonococci suggesting that they may have some utility against this 'urgent threat'
bacterial pathogen. Mechanistic studies indicate that the effect of Cu(btsc) complexes is complex and involves both the delivery
of toxic copper ions to the cell cytoplasm and also, in some cases, the inhibition of respiratory dehydrogenases of the electron
transport chain. Our results suggest that copper ionophores may be of utility in the treatment of infections by some important
mucosal pathogens.
70
P32 - Combating antibiotic resistance by the use of probiotics
Hanna E Sidjabat1, Amanda Bordin1, Kyra Cottrell1, Anders Cervin1, 2, 3
1. The University of Queensland Centre for Clinical Research, Brisbane, QLD, Australia
2. School of Medicine, The University of Queensland, Brisbane, Queensland, Australia
3. Department of Otolaryngology, Head and Neck Surgery, Royal Brisbane and Women's Hospital, Brisbane, Queensland,
Australia
Background: Probiotics have been mostly used to maintain gut health. Lactobacillus spp. is the most commonly used
probiotics. Here, we tested the activity of various Lactobacillus spp. strains against pathogens causing upper respiratory tract
infections and opportunistic pathogens causing nosocomial infections.
Materials and methods: A total of 21 strains of Lactobacillus spp. were isolated from 78 participants of study on upper airway
microbiota characterisation using conventional microbiology approach. The bactericidal activity of Lactobacillus spp. strains
were tested against 48 S. aureus and 48 antibiotic resistant Gram-negative bacteria, mainly carbapenemase-producing
Enterobacteriaceae (CPE). Agar overlay was used to determine the phenotype of bactericidal activity of the probiotic strains.
Results: The species of Lactobacillus spp. were L. rhamnosus (n=7), L. acidophilus (n=10), L. casei (n=1), L. fermentum (n=3).
The bactericidal activities were classified as excellent (n=12), moderate (n=4) and poor (n=5). S. aureus was generally more
difficult to be eliminated by Lactobacillus spp. than Gram-negative bacteria. Interestingly, there was no difference in the
phenotype of bactericidal activity of Lactobacillus spp. against CPE and non-CPE. In contrast, L. fermentum appeared to
enhance the growth of S. aureus.
Conclusions: Lactobacillus spp. especially L. rhamnosus and L. acidophilus had strong bactericidal activities in vitro to various
pathogens. Phenotypic testing of bactericidal activity of Lactobacillus spp. will provide preliminary screening of the probiotic
strains which can be used to combat antibiotic resistance.
71
P33 - Structural studies on the ribosomal binding mode of GE81112 reveal a novel target and
mechanism of inhibition of eubacterial protein synthesis
Tatsuya Kaminishi1, Jorge López-Alonso1, Attilio Fabbretti2, Andreas Schedlbauer1, Letizia Brandi2, Claudio Gualerzi2,
Sean Connell1, 3, Paola Fucini1, 3
1. CIC bioGUNE, Derio, Spain
2. Camerino University, Camerino, Italy
3. Ikerbasque, Bilbao, Spain
GE81112 is a natural product composed by four non proteinogenic amino acids, with a great potential to become a lead
compound for the development of a completely new class of antibiotics. In fact, GE81112 has a selective antimicrobial activity
and low/no toxicity toward eukaryotic cells for which, in addition, it does not display in vivo or in vitro any inhibition of the
translational process (1-3). Furthermore, GE81112 displays a rather broad spectrum of action towards several Gram positive
and Gram negative bacteria, such as Escherichia coli, Bacillus subtilis, Moraxella catarrhalis, Streptococcus pneumoniae and
Enterococcus faecalis, with minimal inhibitory concentrations (MICs) ranging from ~ 0.1 to ~ 64 μg/ml (1-4). Our biochemical
and structural studies on GE81112, based on X-ray crystallography and cryo electron microscopy (5, 6), further reveal that
GE81112 has a unique mechanism of action as it binds to the ribosome in an essential and distinct pocket, so far not exploited
by any of the other translational inhibitor discovered and it stabilizes the anticodon stem loop of the initiator tRNA and three
helices of the ribosome, h44/45/24, in an off-set conformation. The study further suggests that this switch in the conformation of
h44/45/24 is usually controlled by the action of two factors that intervene in the initiation process, IF1 and IF3, such as only in
the presence of a correct positioning of the initiation codon and initiator tRNA, IF1 and IF3 would allow the progression of the
initiation phase of protein synthesis.
1.
2.
3.
4.
5.
6.
(1) Brandi et al., 2006, Proc Natl Acad Sci USA, 103, 39–44.
(2) Brandi et al., 2006, Biochemistry, 45, 3692-3702.
(3) Binz et al., 2010, J Biol Chem, 285, 32710–32719.
(4) Maio et al.. 2016, Antibiotics, 5, E17.
(5) Fabbretti et al., 2016, Proc Natl Acad Sci USA, 113, E2286–E2295
(6) López-Alonso et al., 2016, under revision in NAR.
72
P34 - Adapting Gram-positive FabI inhibitors to Gram-negative pathogens
Michael E Johnson1, 2, Shahila Mehboob2
1. University of Illinois at Chicago, Chicago, IL, United States
2. Novalex Therapeutics, Inc., Chicago, IL, USA
In recent work, we have developed benzimadazole-based inhibitors of the bacterial enoyl reductase, FabI, as leads for
antibiotic development [J Med Chem (2012) 55, 268 & 5933 and ACS Infectious Diseases (2016), in press]. These inhibitors
show antibacterial activity against both wild type and methicillin resistant S. aureus, as well as B. anthracis and F. tularensis,
but are primarily active against gram-positive species for which FabI is essential. Targeting FabI has the advantage that the
majority of the gut microbiome bacteria utilize other enoyl reductase isoforms and will not be susceptible to FabI inhibitors,
providing pathogen selectivity. More recently, we have found that our FabI inhibitors exhibit synergy with polymyxin, extending
the spectrum of activity to gram-negative pathogens for which FabI is essential, including A. baumanii, and reducing the
effective concentrations needed for antimicrobial activity. Details of the activity spectrum will be discussed. (Supported in part
by NIAID grant AI110090.)
73
P35 - Using SPR to screen fragments against ESBLs and carbapenemases
Kyle DeFrees1, Afroza Akhtar2, Maurice Horton1, Xiujun Zhang2, Priya Jaishankar1, Derek Nichols2, Yu Chen2, Adam
Renslo1
1. University of California, San Francisco, San Francisco, California
2. University of South Florida, Tampa, Florida
A number of emergent serine- and metallo-beta lactamases are threatening the future effectiveness of cephalosporins and
carbapenems in treating serious Gram-negative infections. Here we applied surface plasmon resonance (SPR) methods to
screen a fragment library against the Class A expanded-spectrum beta-lactamase CTX-M and carbapenemases KPC-2 and
OXA-48. Reversible, non-covalent inhibitors of CTX-M developed previously by our group (e.g. SMDC-735487) were employed
as positive controls in developing and validating the SPR assay. Subsequently a library of ~3,000 fragments was screened
against the three proteins. Hits were further profiled in dose response by SPR to assess binding affinity and kinetics, and in
biochemical assays to afford Ki values. The preliminary results of this screening effort are described herein. Several promising
chemotypes displayed activity across multiple enzyme types. The long-term goal of this program is to identify and structurally
characterize fragments that bind across important enzyme families, and to use this information to design cell-active lead
compounds effective against key Class A, B, and D beta-lactamases.
74
P36 - Open source drug discovery: profiling of the pathogen box
Sandra Duffy1, Vicky M Avery1
1. Griffith University, Nathan, QLD, Australia
Open source drug discovery utilises the world’s research community to facilitate drug discovery. Experts across multiple
disciplines contribute and openly discuss current data and future directions of what is in essence a worldwide drug discovery
project, orchestrated by a single conductor in order to ensure continued momentum. This approach to drug discovery is gaining
credibility, particularly within the neglected diseases.
The Pathogen Box comprises 400 compounds selected for activity against a range of pathogens including malaria, which is
readily accessible from Medicines for Malaria Venture (MMV), on condition that any data generated from the testing these
compounds is made public. The objective being to continuously build upon an information platform in order to highlight
compounds with the greatest potential for quick progression through the drug discovery process for a number of diseases
primarily affecting the poor.
Herein we present the anti-malarial profile of the compounds, including asexual stage of effect, speed of kill and their
gametocyte activity profile.
75
P37 - Reversing antimicrobial resistance with efflux pump inhibitors
Rumana Mowla1, Shutao Ma2, Henrietta Venter1
1. University of South Australia, Adelaide, SA, Australia
2. Department of Medicinal Chemistry, Shandong University, Jinan, China
Drug efflux pumps confer multidrug resistance on bacteria by transporting a wide spectrum of structurally diverse antibiotics.
Moreover, organisms can only acquire resistance in the presence of an active efflux pump. The substrate range of drug efflux
pumps is not limited to antibiotics, but it also includes toxins, dyes, detergents, lipids and molecules involved in quorum
sensing; hence efflux pumps are also associated with virulence and biofilm formation. Inhibitors of efflux pumps (EPI) are
therefore attractive compounds to reverse multidrug resistance, reduce biofilm formation, attenuate the pathogen and to
prevent the development of resistance in clinically relevant pathogens 1.
We have recently produced the first structure of an active, intact, multiprotein drug efflux complex from a Gram-negative
bacterium2. Our group is now using in silico screening to translate this structural information into the development of EPIs.
Compounds identified are tested for (a) their antibacterial activity, (b) their ability to restore sensitivity to antibiotics and (c) the
inhibition of substrate efflux. Hit compounds are further characterised to ensure their effects are target-specific by measuring
their effect on cells lacking efflux pumps and by ensuring that they do not permeabilise the inner- or outer membranes.
We have identified several phytochemicals isolated from plants used in traditional medicine as EPIs. A range of derivatives
were synthesized from the original compounds and structure activity relationship optimisation was performed on all the
derivatives.
These novel compounds could be lead compounds for an antimicrobial drug discovery program aimed at stemming the tide of
untreatable, multidrug resistant infections.
1.
2.
1. Venter H, Mowla R, Ohene-Agyei T, Ma S (2015) RND-type Drug Efflux Pumps from Gram-negative Bacteria:
Molecular Mechanism and Inhibition Front. Microbiol. 6:377.
2. Du D, Wang Z, James NR, Voss, JE, Klimont E, Ohene-Agyei T, Venter H, Chiu W, Luisi BF (2014) Structure of
the AcrAB–TolC multidrug efflux pump. Nature. 509, 512-515.
76
P38 - Insights into the genomic features of Lactobacillus species with potential applications to
probiotic treatment in chronic rhinosinusitis
Amanda Bordin1, Anders Cervin3, 1, 2, Kyra Cottrell1, Hanna E Sidjabat1
1. UQ Centre for Clinical Research, University of Queensland, Brisbane, Queensland, Australia
2. Department of Otolaryngology, Head and Neck Surgery, Royal Brisbane and Women's Hospital, Brisbane, Queensland,
Australia
3. School of Medicine, University of Queensland, Brisbane, Queensland, Australia
Background: Chronic Rhinosinusitis (CRS) is a common chronic inflammatory disease. Sinus microbiome dysbiosis (i.e.
pathogen overabundance and commensal loss) is key to many CRS cases. This study focuses on opportunistic pathogen
Staphylococcus aureus and Upper Respiratory Tract (URT) commensal Lactobacillus species. In CRS, S. aureus often
dominate the sinus microbiome while Lactobacillus spp. are severely depleted. Here, we propose that probiotic Lactobacillus
spp. may be beneficial as a novel CRS treatment by reducing pathogen burden.
Methods: Bactericidal activity of Lactobacillus spp. was tested using agar-overlay assays against 48 strains of S. aureus, a
major pathogen of CRS. A total of 39 Lactobacillus spp. (including 13 L. casei/paracasei, 9 L. rhamnosus, 4 L. fermentum) were
genome sequenced using Illumina HiSeq2000. Genomes were analysed for probiotic features e.g. bacteriocins.
Results: Bactericidal activity varied between Lactobacillus species. Most L. casei/paracasei (n=12) and all L. rhamnosus
isolates showed strong bactericidal activity. L. fermentum isolates showed poor bactericidal activity; some even enhanced the
growth of the pathogens. Strong bactericidal activity correlated with the presence of bacteriocin-encoding genes and auxillary
bacteriocin genes. These genes were absent in L. fermentum isolates. The bacteriocin-encoding gene of the one L.
casei/paracasei isolate with poor bactericidal activity was translocated and extended.
Conclusions: Strong bactericidal activity and intact bacteriocin gene presence in L. rhamnosus and L. casei/paracasei
suggested that these warrant further investigation as potential CRS probiotics. The use of L. fermentum as an URT probiotic
was not recommended.
77
P39 - Investigating new treatment options for giardiasis
Tina S Skinner-Adams1
1. Griffith University, Brisbane, QLD, Australia
Giardia duodenalis is an intestinal parasite and the causative agent of giardiasis, a gastrointestinal disease that causes
significant worldwide morbidity with ~184 million cases annually. While the disease is more prevalent in the developing world it
is also a burden in developed countries. Australia has a prevalence rate of 2-7%, however rates of >60% have been
documented in indigenous communities. Giardiasis is commonly associated with nausea, vomiting and acute diarrhoea, but can
manifest as a chronic disease, causing malabsorption, weight loss and failure to thrive in children. Treatment is dependent on
drugs with limitations including side-effects and treatment failures. The search for new compounds with anti-Giardia activity is
limited by current assay methods which can be labour-intensive, expensive and restricted to measuring activity at a single timepoint. We have developed a new automated live-cell image-based assay to assess the activity of compounds against Giardia
parasites that eliminates the need for stains and permits the unbiased assessment of parasite growth at multiple time-points.
We have used this assay to screen for new compounds with anti-Giardia activity and have identified new chemical scaffolds
with nM in vitro activity and promising selectivity.
78
P40 - Utilising bismuth in the fight against antimicrobial resistance
Phil Andrews1
1. Monash University, Clayton, VIC, Australia
Metal-based antibiotics are not new and those based on silver still attract much attention; both as metal-organic compounds (eg
silver sulfadiazine) and more recently as nanoparticles (e.g. AgNPs). 1 However, there are significant problems; environmental
contamination and persistence, human toxicity, and emerging resistance have all been identified as future limiting factors which
may lead to greater regulation.2 Alternatives to silver which provide equal if not better benefits will need to be found.
In this presentation we describe our recent efforts in developing bismuth(III) compounds which display excellent antimicrobial
activity, particularly against multi-resistant strains of bacteria, while showing negligible toxicity towards mammalian cells.
In particular, we will focus on the development of homo- and heteroleptic bismuth(III) hydroxamates and thiolates,3-5 their
synthesis, stability and solubility, and structural chemistry. And in particular consider the ‘trojan horse’ approch to ion mimicry of
Fe(III).
We will present the results of assays against a range of Gram positive and Gram negative bacteria, including MRSA and VRE,
and discuss the implications for develoloping bismuth-based antibacterial drugs and new antimicrobial polymers, coatings and
materials.
1.
2.
3.
4.
5.
L. Rizzello, P. P. Pompa, Chem. Soc. Rev., (2015), 43, 1501.
J-Y. Maillard, P. Hartemann, Crit. Rev. Microbiol., (2013), 39, 373.
A. Pathak, V. L. Blair, R.Ferrero, P. C. Junk, R. F. Tabor and P. C. Andrews, Dalton Trans., (2015), 44, 16903.
A. Luqman, V. L. Blair, R. Brammananth, P. K. Crellin, R. L. Coppel, P. C. Andrews, Chem. Eur. J., (2014), 20,
14362.
A. Luqman, V. L. Blair, R. Brammananth, P. K. Crellin, R. L. Coppel and P. C. Andrews, Eur. J. Inorg. Chem. 2016,
2738.
79
P41 - Bismuth Vs. Bacteria: Antibacterial compounds for materials and surfaces
Melissa Werrett1, Phil Andrews1, Rajini Brammananth1, Paul Crellin1, Uthpala Garusinghe1, Warren Batchelor1
1. Monash University, Clayton, VIC, Australia
Antimicrobial resistance has been recognised by the World Health Organization as a global threat requiring urgent
governmental, administrative and scientific action.1 Alarmingly, antibiotic-resistant infections are common in hospitals and
healthcare facilities. Biofilms easily form on a range of surfaces providing easy mechanisms for the transfer of infection. One
method to reduce the risk associated with antimicrobial infections is to introduce antimicrobial function into a range surfaces
within hospitals and healthcare facilities.
The use of bismuth in medicine spans more than two centuries owing to its non-toxic nature as well as reported antibacterial
properties.2-4 Phosphinic acids have limited exploration as antibacterial agents however C-P natural products have been
extensively used in medicine.5 We have therefore synthesised a series of bismuth(III) phosphinate complexes of the type
BiPhL2 and BiL3 (Figure 1); where L are a series of aryl or alky phosphinates. Both series of bismuth(III) phosphinates are
insoluble in a range of common solvents and are also hydrolytically stable. These properties make them ideal candidates for
material incorporation, as they are unlikely to leach.
Antibacterial testing was conducted on both series of complexes, as insoluble powders. Results indicated that the BiPhL 2
complexes display antibacterial activity towards both Gram negative and Gram positive bacteria, including E. coli, S. aureus,
MRSA and VRE. Interestingly, the BiL3 were inactive. We have also incorporated the BiPhL2 complexes into different
matrices/supports, including agar and a cellulosic polymer matrix. The bismuth-containing paper (cellulose), at loadings of 5 to
20% BiPhL2, displayed antibacterial action toward all bacteria mentioned above.
1.
2.
3.
4.
World Health Organization, (2014), 1–232.
P. L. Russo; A. C. Cheng; M. Richards; N. Graves and L. Hall, Aust. Heal. Rev. (2015), 39, 37
M. Cruickshank and J. Ferguson, Reducing harm to patients from health care associated infection: the role of
surveillance, Australian Commission on Safety and Quality in Health Care (2008).
G. R. Nimmo and G. W. Coombs, Int. J. Antimicrob. Agents (2008), 31, 401.
5.
W.W. Metcalf; W.A. van der Donk, Annu. Rev. Biochem. (2009), 78, 65.
80
P42 - The biotin biocycle presents multiple new drug targets for anti-bacterial discovery
Steven W Polyak1, Matthew C Wilce2, Kate L Wegener1, John D Turnidge1, Grant W Booker1, Andrew D Abell3
1. School of Biological Science, University of Adelaide, Adelaide, SA, Australia
2. School of Biomedical Science, Monash University, Clayton, Victoria, Australia
3. School of Chemistry and Physics, University of Adelaide, Adelaide, SA, Australia
There is a well-recognized need to replenish the drug discovery pipeline with new products to combat the rise of antibioticresistant bacteria. Target based approaches remain appealing in early stage drug discovery due to the ability to rationally
optimize hits through medicinal chemistry in concert with biochemistry, microbiology and structural biology. Our group has
investigated the biotin biocycle as a source of promising new drug targets. Biotin is an essential micronutrient that functions as
a co-factor for key metabolic enzymes in important metabolic pathways, namely fatty acid biosynthesis and replenishment of
the Krebs cycle. Genetic studies have demonstrated that these pathways are required for both growth (fatty acid synthesis) and
virulence (Krebs cycle). Enzymes that participate intimately in the biocycle provide a number of discrete antimicrobial targets.
Protein biotinylation, catalyzed by biotin protein ligase (BPL), is one such example. We have employed rational drug design and
template guided synthesis to identify and develop BPL inhibitors with potential as new anti-bacterials. Significantly, as clinically
important Staphylococcus aureus is particularly sensitive to the inhibition of BPL, this presents an opportunity to develop
narrow spectrum agents for this challenging pathogen. Additionally, as de novo synthesis is the sole source of cellular biotin for
Mycobacterium tuberculosis, biotin biosynthetic enzymes represent promising targets for new anti-tubercular therapeutics. Our
group is now focused upon the development of small molecule chemotherapeutics for pre-clinical target validation experiments
that exploit the biotin biocycle.
81
P43 - Ethics, antimicrobial resistance, and the tragedy of the commons
Euzebiusz Jamrozik1
1. Monash University, VIC, Australia
Antimicrobial resistance threatens to undermine many of the benefits of modern medicine, in both the developed and the
developing world. A “post-antibiotic era” would lead to a dramatic rise in harms to patients and the costs of treatment. In this
talk I argue that antimicrobial existence is an example of the 'tragedy of the commons', at both the local and global level, and
outline implications for future policy. Solutions to the problem require not only new or improved treatments and diagnostic tests,
but also fair restrictions in the use of current treatments so that risks are equitably distributed. Public health ethics has a key
role to play at this critical moment. How should new drug development be incentivised and/or funded? Why should wealthy
countries support access and use in poorer regions? How should we distribute the scarce resource of effective antimicrobials?
What duties do we have to conserve treatments for future generations? How much risk and financial cost should patients and
doctors accept? What are the ethical implications of small individual contributions to a massive global problem? What moral
assumptions underpin antimicrobial stewardship? This presentation will provide critical analyses of such questions from an
ethical perspective.
82
P44 - Chemical modulation of anti-candida activity of oligomycin A against resistant strains
Olga A. Omelchuk1, 2, Lyudmila N. Lysenkova1, Natalia E. Grammatikova1, 3, Alexander M. Korolev1, Andrey E.
Shchekotikhin1, 2
1. Gause Institute of New Antibiotics, Moscow, Russian Federation
2. D. I. Mendeleev University of Chemical Technology of Russia, Moscow, Russian Federation
3. I.M. Sechenov First Moscow State Medical University, Moscow, Russian Federation
Yeasts of genus Candida, such as C. albicans, C. krusei, C. parapsilosis and C. tropicalis, are the cause of the most common
type of yeast infections - candidiasis. Frequently fluconazole and related triazole-based drugs are used for treatment of
candidiasis, but several Candida spp. strains exhibit multiple triazole resistance. Oligomycin A display a variety of biological
activities, including strong antifungal effect, due to inhibition of F 1F0 ATP-synthase by binding to F0 c-subunit and blocking
proton translocation.1
Oligomycin A showed high anti-candida activity, including resistant strains. However, clinical application of oligomycin A is
limited due to its high cytotoxicity for human cell. Our research is aimed to searching of semi-synthetic approach for the
modification of oligomycin A to develop of new derivatives with high selective anti-fungal activity and low human cell
cytotoxicity.
Recently biological studies of 2,3-dihydrooligomycin A showed its higher anti-fungal activity as compared with the native
antibiotic against S. cerevisiae.2 Based on these data, we have studied a catalytic reduction of double C-C bonds of oligomycin
A (1). Hydrogenation of antibiotic 1 in the presence of Pd/C in methanol lead to 2,3,16,17,18,19-hexahydrooligomycin A (2) in
good yield. 3
New derivative 2 showed high selective activity against both resistant (C. krusei) and non-resistant Candida spp. strains.
Hexahydrooligomycin 2 was also less toxic than oligomycin A (1) against human cell lines. Thus, reduction of three double C-C
bonds in macrocyclic moiety of 1 increases selectivity to Candida spp. with simultaneous decrease of human cells cytotoxicity.
This work was supported by RSF (№ 15-15-00141).
1.
2.
3.
Symersky J., Osowski D., Walters D. E., Mueller D. M. PNAS, 2012, 109 (35), 13961-13965.
Park J.W., Park S.R., Han A.R., Ban Y.-H., Yoo Y. J., Kim E. J. et al. J. Antibiot., 2011, 64, 155-157.
Omelchuk O.A., Belov N.M., Tsvetkov V.B., Grammatikova N.E., Lysenkova L.N. et al. Macroheterocycles, 2016,
9(4), 453-461.
83
P45 - Design and Synthesis of Lactams Derived from Mucochloric and Mucobromic acids as
Quorum Sensing Inhibitors of Pseudomonas aeruginosa
Basmah Almohaywi1, 2, Aditi Taunk 1, Shashidhar Nizalapur , Nripendra Biswas , George Iskander , Renate Griffith,
David Black, Naresh Kumar
1. School of Chemistry , University of New South Wales , Sydney , NSW, Australia
2. School of Medical Science , University of New South Wales , Sydney , NSW, Australia
Bacterial infections, particularly hospital-acquired infections mediated by Pseudomonas aeruginosa have become an alarming
global threat, leading to high mortality. New approaches to combat bacterial infections are urgently needed, especially against
emerging strains that are resistant to most traditional antibiotics. Gram-negative bacteria including P. aeruginosa utilise N-acyl
homoserine lactones (AHLs) as signalling molecules in a process known as quorum sensing (QS), to regulate the expression of
many phenotypes involved in pathogenicity of bacteria. Recent strategies targeting bacterial behaviours or QS have received
great attention to combat bacterial resistance due to their ability to disarm pathogenic bacteria, rather than killing bacteria that
applies selective evolutionary pressure on them.
In the present study, we report the design and synthesis of a range of dichloro- and dibromo-pyrrole-2-one derivatives based on
reductive amination of mucochloric and mucobromic acids with aryl and alkyl amines. The QS inhibition activity of the
synthesised compounds was determined by measuring the LasR expression in the Pseudomonas aeruginosa MH602 reporter
strain. Amongst the tested compounds, N-phenolic compounds exhibited the best QS inhibition activity of 78-83% reduction
compared to control at 250 µM. The activity was maintained with compounds incorporating an N-phenyl group and indole core
and was retained at lower concentrations (125 µM and 62.5 µM). In addition, computational docking studies were performed
using LasR receptor protein of P. aeruginosa, which highlighted the hydrogen bonding and hydrophobic interactions involved
between the compounds and the receptor protein.
1.
2.
J. T. Hodgkinson, M. Welch and D. R. Spring, ACS Chem. Biol., 2007, 2, 715-717.
M. E. Mattmann and H. E. Blackwell, J. Org. Chem., 2010, 75, 6737-6746.
84
P46 - Overcoming antibiotic resistance by co-evolutionary arms races
Andrew D Higginson1
1. University of Exeter, Exeter, UK, United Kingdom
Antibiotic-resistant pathogens arise from an evolutionary process in which genes that enhance survival in the presence of
antibiotics propagate through populations. Much research effort has been expended on chemically altering existing antibiotics
to restore their efficacy, with little consideration of possible solutions derived from ecology. Ecologists often study multi-species
communities with short generation times in the laboratory to better understand evolutionary processes, such as co-evolutionary
arms races between prey that evolve better antipredator defences and predators that evolve to overcome these defences. Such
a protocol could be used to select for mutations in antibiotic-producing agents that overcome the defensive adaptations of
resistant strains. For example, exposing Penicillium spp. to competition for resources from methicillin-resistant Staphylococcus
aureus should select for mutations that alter antibiotic production and thereby restore efficacy. In order to maximise the chance
of success researchers must carefully choose the particulars of these experiments, such as the amount and patterns of
resource availability, relative population sizes of competing species, and the particulars of dispersal between populations and
locations. Ecologists mimic the selective process in computer-based simulations in order to predict the outcome of evolutionary
arms races. Here, I present the results of evolutionary simulations that I use to identify the conditions that would maximise
selection on antibiotic producers and lead to the production of effective antibiotics. This approach could lead to the overcoming
of antibiotic resistance by allowing natural selection to identify the mechanisms, and so does not rely on researchers envisaging
these mechanisms in advance.
85
P47 - Have you got the next antibiotic? CO-ADD: Community for Open Antimicrobial Drug
Discovery
Alysha G Elliott1, Johannes Zuegg1, Mathilde Desselle1, Ruth Neale1, Karl A Hansford1, Mark AT Blaskovich1, Matt A
Cooper1
1. University of Queensland, St Lucia, QLD, Australia
The antibiotic pipeline is broken, with a dearth of new antibiotics, and the exhaustion of chemical diversity contained in pharma
libraries. We believe that there is an untapped resource contained in the laboratories of chemists; synthetic compounds
prepared for other projects and targets, or to develop methodologies, that have never been tested for their antimicrobial
potential. The global screening initiative The Community for Open Antimicrobial Drug Discovery [CO-ADD] will uncover this
significant and rich chemical diversity held outside of corporate screening collections by providing a low-barrier access to
testing.
Antibacterial drugs occupy a unique property space that is vastly different to drugs developed for other therapeutic areas,
suggesting that commercially available chemical compounds lack the physicochemical properties ideal for activity against
bacteria and therefore, a varied source of chemical diversity needs to be investigated. Built upon a suite of established in vitro
and in vivo assays, medicinal chemistry and core researcher expertise CO-ADD has developed and validated high-throughput
antimicrobial screening methodologies for bacteria and fungi in 384-well microtitre plate broth assays and has the current
capacity to analyse >10,000 compounds per week against a panel of nine microorganisms in duplicate.
With support from The Wellcome Trust and the University of Queensland, we have created a not-for-profit open access pipeline
to provide unencumbered free antimicrobial screening for any interested researcher. After only 24 months in operation COADD has screened >160,000 compounds for their antimicrobial potential, more than twice the number of any other screening
program so far disclosed.
1.
2.
3.
4.
5.
www.co-add.org
McGilvray A. (2016) Compound screening: Fresh hunting ground. Nat 533, S65-7.
Blaskovich M.A.T. et al., (2016) Helping chemists discover new antibiotics. ACS Infect Dis 1, 285-7.
Hansford K.A. et al., (2016) Chemistry philanthropy: a path forward for antibiotic discovery? Future Med Chem 9,
925-9.
Cooper M.A. (2015) A community-based approach to new antibiotic discovery. Nat Rev Drug Discov 14, 587.8.
86
P47 - Combining whole genome sequencing and in-vitro PK/PD to optimize drug dosage
regimen for carbapenem-resistant Acinetobacter baumannii
Xiaofen Liu1, Huajun Zheng2, Xingchen Bian3, Jing Zhang1
1. Huashan Hospital, Fudan University, Shanghai, SHANGHAI, China
2. Shanghai-MOST Key Laboratory of Health and Disease Genomics, Shanghai, China
3. Shanghai Normal University, Shanghai, China
Background:
Two isolates of A. baumannii, AB1845 and AB2092, were isolated from sputum of a HAP patient. The meropenem MICs of the
two isolates were 16 mg/L and 64 mg/L, respectively. Whole genome sequencing was applied to investigate the meropenem
resistance mechanism developed in-vivo. Meanwhile, in-vitro PK/PD dynamic system has been employed to optimize the
combinational dosage regimen to combat carbapenen-resistant A. baumannii.
Methods:
The Genomic DNA of the two isolates was extracted and sequenced by Illumina Hi-seq 2000 platform for the genomic
comparison. In the PK/PD model, the combination of meropenem and colistin was tested. Chinese healthy volunteers’
pharmacokinetic profiles of meropenem (1g or 2g, infusion for 3h) were simulated and colistin was maintained at 1mg/L at the
constant concentration to mimic the steady state in vivo in the PK/PD model.
Results:
Whole-genome sequencing resulted in more than 12,050,346 pairs of 150-bp reads, and 131 contigs larger than 500 bp were
assembled for both isolates.The two isolates showed 99% similarity with 33 synonymous SNPs. However, blaOXA-23 was
amplified three times in AB2092 compared to AB1845, which could be the resistance mechanism. Meanwhile, the in-vitro
PK/PD showed the meropenem dosage of 2g infusion for 3h combined with 1mg/L colistin could achieve 2 log10 decreases at
24h, despite blaOXA-23 was presented in the bacteria.
Conclusions:
The whole genome shows the amplification of blaOXA-23 could be the mechanism for meropenem resistance in vivo. Besides,
the in-vitro PK/PD showed the meropenem and colistin combination could exert the antibacterial effect against carbapenemresistance A. baumanii.
87
P49 - Targets For Resistance, Persistence, and Virulence: 1. Cell Membrane, 2. Metabolism of
Nucleotides, And 3. Bacterial Stringent Response
Dominik Rejman1, Libor Krásný2, Radek Pohl1, Eva Zborníková1, Dianne Keough3, Luke Guddat3, Vasili Hauryliuk4
1. IOCB Prague, Praha 6, CZECH REPUBLIC, Czech Republic
2. Institute of Microbiology, Czech Academy of Sciences v.v.i., Prague, Czech republic
3. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
4. Department of Molecular Biology, Umeå University, Umeå, Sweden
Three possible approaches to deal with resistance, persistence, and virulence are presented. 1) Targeting the cytoplasmic
membrane possess advantages: rapid action and potency against non-multiplying and multi-resistant bacteria. We report here
synthesis and properties of second generation of membrane targeting antimicrobials lipophosphonoxins (LPPOs)[i],[ii] 2)
Hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) is the key enzyme in the purine salvage
pathway. Its activity is essential for the survival of the parasites, Plasmodium falciparum (Pf) and vivax (Pv). We have
developed inhibitors of the Pf and Pv enzymes, based on pyrrolidine azanucleotide core, and have shown that their prodrugs
arrest the growth of Pf in cell culture. We have extended our studies to develop inhibitors that are effective against
Mycobacterium tuberculosis (Mt)HGPRT. Prodrugs of these compounds also arrest the growth of a virulent strain of
tuberculosis. 3) Another promising direction is targeting molecular systems involved in bacterial virulence. [iii] One such system is
the bacterial stringent response (SR).[iv],[v],[vi],[vii] We have focused on the design and synthesis of inhibitors of RelA - the key
enzyme of SR pathway.
The research has been supported by IOCB Prague and CSF-15-11711S.
1.
2.
3.
4.
5.
6.
7.
Rejman D, et al. J. Med. Chem. 54(22),7884-98, (2011).
Panova N, et al. Plos One 2015;10(12):e0145918.
Sun, H. Curr. Drug. Targets. 13, 294-296, (2012).
Wexselblatt, E. et al. Nucleic Acids Symp. Ser. (Oxf), 633-634, (2008).
Wexselblatt, E. et al. Bioorg. Med. Chem. 18, 4485-4497, (2010).
Wexselblatt, E. et al. PLoS Pathog. 8, e1002925, (2012).
Wexselblatt E. et al. Eur. J. Med. Chem. 70, 497-504, (2013).
88
P50 - Synthesis and antibacterial activity of benzoxaborole – azithromycin hybrid antibiotics
Anna N Tevyashova1, Alexander M Korolev1, Yury N Luzikov1, Elena P Mirchink1, Elena B Isakova1, Ilia A Osterman2
1. Laboratory of Chemical Transformation of Antibiotics, Gause Institute of New Antibiotics, Moscow, Russia
2. Department of Chemistry, M.V. Lomonosov Moscow State University, Moscow, Russia
Concept of dual acting (hybrid) antibiotics is one of a developing strategies for the search of new drugs which can overcome
multidrug resistance of bacteria, have a broader spectrum of action compared with the initial antibiotics and retard the
development of antibiotic resistance [1,2]. Azithromycin has served as a scaffold for different series of dual acting
antibacterials, including large number of azithromycin - fluoroquinolones hybrids (macrolones) described by GlaxoSmithKline
and Pliva [3, 4].
Benzoxaborole, a privileged structure in medicinal chemistry due to its desirable physicochemical and drug-like properties, was
used for the synthesis of azithromycin – benzoxaborole conjugates in which benzoxaborole fragment was attached to the 4'hydroxy
group
of
antibiotic
via
aminoalkylcarbomoyl
spacer.
Antibacterial activity of synthesized derivatives in comparison with azithromycin was tested on a wide panel of gram-positive
and gram-negative bacterial strains. New hybrid antibiotics demonstrated high activity against S. pyogenes ATCC 19615 and P.
acnes ATCC 6919 strains. Some of the novel derivatives were more active that azithromycin against S. pneumoniae ATCC
49619 and E. faecium strains. The longer spacer between benzoxaborole moiety and azithromycin moieties was preferable for
the activity against gram-negative strains, while the 11, 12-cyclic carbonate group was favorable for the high activity of the
studied compounds against S. pneumoniae ATCC 49619 and E. faecium strains. Newly synthesized benzoxaboroleazithromycin conjugates retained the ability of azithromycin to block the nascent peptide in ribosome tunnel.
The reported study was funded by the Russian Foundation for Basic Research according to the research project № 16-3460110.
1.
2.
3.
4.
Tevyashova A.N., Olsufyeva E.N. Preobrazhenskaya M.N. Design of dual action antibiotics as an approach to search
for new promising drugs. Russ. Chem. Rev., 2015, V. 84, P. 61-97.
Pokrovskaya V., Baasov T. Dual-acting hybrid antibiotics: a promising strategy to combat bacterial resistance. Expert
Opin Drug Discov., 2010, V. 5, P. 883-902.
Kapic S., Paljetak H.C., Alihodzic S., Antolovic R., Haber E.V., Jarvest R.L., Holmes D.J., Broskey J.P., Hunt E. 6Alkylquinolone-3-carboxylic acid tethered to macrolides synthesis and antimicrobial profile. Bioorg. Med. Chem., 2010, V.
18, P. 6569–6577.
Škugor M.M., Štimac V., Palej I., Lugaric D., Paljetak H.C., Filic D., Modric M., Ðilovic I., Gembarovski D, Mutak S.,
Haber V.E., Holmes D.J., Ivezic-Schoenfeld Z., Alihodzic S. Synthesis and biological activity of 4”-O-acyl derivatives of 14and 15-membered macrolides linked to ω-quinolone-carboxylic unit. Bioorg. Med. Chem., 2010, V. 18, P. 6547–6558.
89
P51 - Investigating the antiplasmodial activity of primary sulfonamide compounds identified in
open source malaria data
Gillian M Fisher1, Silvia Bua2, Sonia Del Prete2, 3, Megan SJ Arnold1, Clemente Capasso3, Claudiu T Supuran2, Katherine
T Andrews1, Poulsen Sally-Ann1
1. Griffith Institute for Drug Discovery, Griffith University, Nathan, QLD, Australia
2. Neurofarba Department,Sezione di Scienze Farmaceutiche Nutraceutiche, and Laboratorio di Chimica Bioinorganica,
Università degli Studi di Firenze, Sesto Fiorentino (Florence), Italy
3. Istituto di Bioscienze e Biorisorse, CNR, Via Pietro Castellino 111, Napoli, Italy
In the past decade we have seen significant reductions in deaths due to malaria, mainly due to the success of the gold
standard antimalarial treatment - artemisinin combination therapies (ACTs). However the potential threat of ACT failure and the
lack of a broadly effective malaria vaccine are driving efforts to discover new chemical entities (NCEs) to target this disease.
The primary sulfonamide (PS) moiety is a component of several clinical drugs, including those for treatment of kidney disease,
glaucoma and epilepsy, however this chemotype has not yet been exploited for malaria. In this study 31 PS compounds
sourced from the GlaxoSmithKline (GSK) Tres Cantos antimalarial set (TCAMS) were investigated for their ability to selectively
inhibit the in vitro growth of Plasmodium falciparum asexual stage malaria parasites. Of these, 14 compounds were found to
have submicromolar activity (IC50 0.16-0.89 µM) and a modest selectivity index (SI) for the parasite versus human cells (SI >12
to >43). As the PS moiety is known to inhibit carbonic anhydrase (CA) enzymes from many organisms, the PS compounds
were assessed for recombinant P. falciparum CA (PfCA) mediated inhibition of CO2 hydration. The PfCA inhibition activity did
not correlate with antiplasmodial potency. Furthermore, no significant difference in IC50 was observed for P. falciparum versus
P. knowlesi (P >0.05), a Plasmodium species that is not known to contain an annotated PfCA gene. Together these data
suggest that the asexual intraerythrocytic stage antiplasmodial activity of the PS compounds examined in this study is likely
unrelated to PfCA inhibition.
90
P52 - An in vitro study on antimicrobial effect of metallic silver colloidal against multispecies
oral biofilm
Helen He1, Zhihao Li2, Hans Laroo3, Zyta Ziora4, Laurence Walsh1
1. School of Dentistry, The University of Queensland, Brisbane, QLD, Australia
2. School of Chemistry and Molecular Biology, The University of Queensland, Brisbane, QLD, Australia
3. Silver-Colloids, Laroo Research, Ipswich, QLD, Australia
4. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
Background: Ions of silver (Ag+) are toxic to microorganisms, while metallic silver is inert in the presence of human tissues.
Colloidal silver has been reported to show strong bactericidal activity on single species bacteria in suspension. Bacteria in
biofilms have much higher resistance against antimicrobial agents than those in planktonic form, which makes infections more
difficult to control. The aims of this study was to assess the antimicrobial properties of colloidal metallic silver on a multispecies
oral biofilm. Materials and methods: 4-day-old multispecies oral biofilms were cultured from saliva with Brain-heart-infusion
broth supplemented with sheep blood in 96-well plates. The biofilms were exposed to colloidal silver, PBS, 0.4% NaOCl and
0.12% chlorhexidine (CHX) for 20 minutes or 24 hours. Cell viability of treated biofilms was assessed using the XTT assay. The
relative reduction of viable biomass was calculated versus a negative control (PBS). Results: After being exposed to
antimicrobial agents, bacteria in biofilms treated for 24 h were less viable than those treated for only 20 min. NaOCl and CHX
were effective on biofilms even with a short exposure, and showed stronger effects than colloidal silver, which had milder and
slower killing effects. There were variations in performance between the 4 different colloidal silver preparations tested.
Conclusions: The test preparations of colloidal silver gave moderate and prolonged antibacterial effects on multiple-species
oral biofilms. The potential use of colloidal silver in long-term control of infections should be explored
further.
91
P53 - Octapeptin C4 – enabling last resort antibiotics by dissecting the molecular determinants
of nephrotoxicity
Karl A Hansford1, Alysha G Elliot1, Miranda E Pitt1, Bernd Becker1, Johnny X Huang1, Mark S Butler1, Sonia Troeira
Henriques1, Minh Duc1, Devika Ganesamoorthy1, Mark AT Blaskovich1, Lachlan Coin1, Lawrence H Lash2, Matthew A
Cooper1
1. Institute for Molecular Bioscience, University of Queensland, St Lucia, QLD, Australia
2. Department of Pharmacology, Wayne State University School of Medicine, Detroit, MI, USA
The octapeptins are a family of closely related natural product lipopeptides consisting of a cyclic heptapeptide core with a
lipophilic acyl monopeptide tail. They bear structural similarity to polymyxin B (PmxB) and colistin (PmxE), two antibiotics
clinically used as “last resort” treatments for multidrug-resistant (MDR) and extensively drug resistant (XDR) Gram-negative (Gve) infections. Despite their “last resort” status, dose-limiting renal toxicity severely limits the clinical utility of Pmx antibiotics.
Pmx–resistant strains of G-ve bacteria have emerged, with the propensity for rapid dissemination via plasmid transmission. A
catastrophic scenario is developing where widespread resistance to Pmx, combined with a paucity of new G-ve treatments in
the pharmaceutical pipeline, will give rise to infections that are no longer treatable by any known antibiotic.
Despite their structural similarity to Pmx, the octapeptins retain potent activity toward Pmx-resistant MDR and XDR Gram-ve
bacteria, but do not show cross resistance to this class following resistance induction. Intrigued by this “forgotten” class of
antibiotics, we initiated a research program to design octapeptin analogues with improved safety profiles and spectra of action
compared to Pmx. Here we detail our efforts to understand the unique behaviour of the octapeptins through a comprehensive
structure-activity and structure-toxicity study of octapeptin C4, a representative member of this class of lipopeptides. The study
has identified promising analogues that show reduced potential for nephrotoxicity in vitro, with corresponding efficacy in an in
vivo mouse thigh infection model.
92
P54 - Understanding the branch point enzymes - IMP dehydrogenase and adenylosuccinate
synthetase of de novo purine biosynthetic pathway in Cryptococcus neoformans - for the
design of selective inhibitors
Lalith K Kummari1, 2, Emily Furlong2, Zhenyao Luo3, Ross D Blundell1, Mark S Butler2, James A Fraser1, Avril A.B
Robertson2, Matthew A Cooper2, Bostjan Kobe1, 4
1. Australian Infectious Disease Research, School of Chemistry and Molecular Biosciences, University of Queensland,
Brisbane City, Queensland, Australia
2. Institute for Molecular Biosciences, University of Queensland, Brisbane City, Queensland, Australia
3. School of Chemistry and Molecular Bioscciences, University of Queensland, Brisbane City, Queensland, Australia
4. Institute for Molecular Biosciences, University of Queensland, Brisbane City, Queensland, Australia
Infections caused by fungi and bacteria are a major global problem. In humans, cryptococcosis is one of the life-threatening
infection caused by fungi Cryptococcus neoformans. The treatment is based on three antifungal agents: Amphotericin B,
Fluconazole and Flucytosine. However, there are significant side effects associated with these drugs and increased drug
resistance in some cases. The discovery of new antifungals is essential to fight against the rise of invasive infections caused by
fungal pathogens.
In all organisms, purine metabolic pathway is essential for the biosynthesis of adenosine triphosphate (ATP) and guanosine
triphosphate (GTP) nucleotides. In de novo purine biosynthetic pathway, Inosine monophosphate (IMP) acts as a branch point
substrate for the formation of ATP and GTP through two different directional pathways catalyzed by Adenylosuccinate
synthetase (ADSS) and Inosine monophosphate dehydrogense (IMPDH). To design inhibitors of C. neoformans IMPDH
(CnIMPDH) the high-throughput screening was conducted with 114,000 drug-like compounds from Walter and Eliza Hall
Compound Collection before to onset of my thesis work. One of the hit set is an interesting structural class comprising 1,3,4oxadiazole ring attached to benzothiophene-1,1-dioxide moiety via thioether group. By modification of aromatic group at fifth
position of 1,3,4-oxadiazole ring moiety we found two selective CnIMPDH inhibitors with potent antifungal activity. Also, the
structural elements of C. neoformans ADSS (CnADSS) are studied using protein crystallography technique to understand the
substrate (IMP) and cofactor (GTP) binding mechanism. These understandings are used for design of inhibitors through
computational studies.
93
P55 - Contribution of physicochemical parameters to the antimicrobial activity / toxicity index
of β-hairpin peptides.
Ingrid Edwards1, Alysha Elliott1, Mark Blaskovich1, Matt Cooper1
1. The University of Queensland, St Lucia, QLD, Australia
Increasing rates of bacterial resistance to current antibiotics has led to a dire need for discovery of new replacement drugs for
obsolete antibiotics. Antimicrobial peptides (AMPs) are ubiquitous in nature, being essential components of the innate immune
system of multicellular organisms. β-hairpin AMPs are cationic, amphipathic, membranolytic and exhibit a broad range of
activities against Gram-positive, Gram-negative and fungal pathogens which consequently offer low rate of resistance
development and make them great candidates as antibiotic replacements. However, the use of β-hairpin AMPs is still very
limited mainly due to their toxicity over mammalian cells. The examination of the physicochemical properties of select β-hairpin
AMPs help us better understand the correlation between antimicrobial activity and toxicity, membrane binding and membrane
permeability. Hydrophobicity, pI and net charge at physiological pH were correlated to our data collected from antimicrobial
activity over six key bacterial pathogens and two fungi, cytotoxicity against human cell lines, hemolytic activity in human
erythrocytes and membrane permeability assay against Escherichia coli. Our findings represent a good starting point for the
design of new therapeutically valuable AMPs. Yet further investigation is still required to uncover the reasons for increased
antimicrobial activity/toxicity indexes.
94
P56 - Screening of fungal extracts for antibiotic resistance alleviation
Leila Hilout1, Rohani Rahim1, Anis Low Muhammad Low1, Jean-Frédéric F. Weber1
1. Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), University Teknologi MARA (Selangor Branch), Puncak
Alam Campus, Bandar Puncak Alam, Selangor, Malaysia
Alleviating existing resistance to antibiotics appears as an attractive strategy to fight the global issue of multiresistant
“superbugs”. Due to their inherent druglikeness, natural products appear as a promising source. We undertook screening
extracts obtained from cultures of Malaysian endophytic microfungi. Endophytes are microorganisms hosted by plants in
symbiotic or commensalistic relationships and notably preventing pathogenic infections. Extracts from over 200 endophytic
fungal cultures were prepared, while several bacteria were made resistant to various antibiotics by subculturing them
repetitively in increasing concentrations of antibiotics. The screening is performed using the disc diffusion method (extract: 20
ug/disc). A positive result arises from the combination of growth inhibition in the presence of antibiotics (50% MIC) and normal
growth in the absence of antibiotics. Here we report on the first 88 extracts tested against Staphylococcus aureus made
resistant to ciprofloxacin (S.a.cip), rifampicin, and chloramphenicol (S.a.col), and Pseudomonas aeruginosa made resistant to
ciprofloxacin, rifampicin, and tetracycline. None of the extracts demonstrated any antiresistance activity on P. aeruginosa made
resistant to any of the three above antibiotics. Four extracts gave positive results towards S.a.cip and one towards S.a.col. It is
notable that each extract is active on a single resistant bacterium, hinting thus of a possibly quite specific mechanism of action.
This screening campaign shall continue using more extracts, including those from polar fungi, on a wider range of antibiotic
resistant bacteria. The most potent and/or selective extracts will be dereplicated and the active ingredients identified, while their
mechanisms of action will be examined.
95
P57 - Specific biochemical changes associated with PfKelch13-mutant artemisinin resistant
Plasmodium falciparum revealed by multi-omics analysis
Ghizal Siddiqui1, Anubhav Srivastava1, Adrian s Russell1, Darren J Creek1
1. Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville
Campus, Parkville, Victoria, Australia
Artemisinin resistance in the malaria parasite, Plasmodium falciparum, has recently emerged in South-East Asia, posing a
major threat to the efficacy of the first-line treatment for malaria. The artemisinin resistance phenotype is associated with
certain point mutations in the propeller domain of PfKelch13. However, not all PfKelch13 mutations are the sole determinant of
clinical resistance. The biochemical function of PfKelch13 is not known and the underlying mechanism associated with
artemisinin resistance is poorly understood. Therefore, this study aimed to identify biochemical changes associated with
artemisinin resistance using an integrative multi-omics approach, combining metabolomics, peptidomics and proteomics to
analyse PfKelch13-mutant artemisinin -resistant and -sensitive strains of P. falciparum.
When applied to P. falciparum-infected red blood cells, our multi-omics platform facilitated the identification of approximately
2824 proteins, 584 putative metabolites and 148 naturally abundant peptides. Proteomics analysis found PfKelch13 mutations
to be specifically associated with two-fold decreased abundance of PfKelch13 protein. As the PfKelch13 sequence is already a
genetic biomarker for artemisinin resistance, we show for the first time that the abundance of this protein is also associated with
the resistance phenotype. Metabolomics analysis demonstrated accumulation of the key antioxidant molecule, glutathione, and
its precursor, gamma-glutamylcysteine. Peptidomics analysis revealed lower abundance of several endogenous peptides
derived from haemoglobin (HBα and HBβ) in the artemisinin resistant strains.
In conclusion, PfKelch13 mutations associated with artemisinin resistance lead to enhanced glutathione production, decreased
haemoglobin digestion and decreased abundance of PfKelch13 protein.
96
P58 - Dual Action Antibacterial Biomaterials and Polymers Based on Quorum Sensing
Inhibitors and Nitric Oxide Donors
Aditi Taunk1, Edgar Wong2, Cyrille Boyer2, Mark DP Willcox3, Naresh Kumar1
1. School of Chemistry, The University of New South Wales, Sydney, NSW 2052, Australia
2. Centre for Advanced Macromolecular Design (CAMD), School of Chemical Engineering, The University of New South Wales,
Sydney, NSW 2052, Australia
3. School of Optometry and Vision Science, The University of New South Wales, Sydney, NSW 2052, Australia
Bacterial biofilms on implanted medical devices are one of the major causes of hospital-acquired infections. Treatment or
removal of these infected devices results in increased medical costs along with high mortality and morbidity 1. At present no
effective strategies are available to treat these infections. In addition, the emergence of multi-drug resistant bacteria has made
device-related infections extremely difficult to treat. A new strategy to inhibit biofilm formation is to disrupt the bacterial
communication mechanism known as quorum sensing (QS)2 by incorporating QS inhibiting compounds such as
dihydropyrrolones (DHPs) on biomaterial surfaces and polymers. Similarly, the biological signalling molecule nitric oxide (NO)
can disperse biofilms3. Therefore, this project focused on developing novel dual action biomaterial surfaces and polymers that
are functionalized by both QS inhibitors and a NO releasing moiety. The surfaces were found to inhibit Staphylococcus aureus
and Pseudomonas aeruginosa biofilm formation by up to 80% and 71% respectively, while the polymers reduced biofilm
biomass of P. aeruginosa by 80-86% (p < 0.001) at 10-40 µM concentration. The dual action surfaces and polymers have the
potential to reduce biofilm formation on medical implants.
1.
2.
3.
Hu, B. et al. Device-associated infection rates, device use, length of stay, and mortality in intensive care units of 4
Chinese hospitals: International Nosocomial Control Consortium findings. Am. J. Infect. Control 41, 301–306 (2013).
Davies, D. G. et al. The involvement of cell-to-cell signals in the development of a bacterial biofilm. Science 280,
295–298 (1998).
Kutty, S. K. et al. Hybrids of acylated homoserine lactone and nitric oxide donors as inhibitors of quorum sensing and
virulence factors in Pseudomonas aeruginosa. Org. Biomol. Chem. 13, 9850–9861 (2015).
97
P59 - The interactive antimicrobial activity of conventional antibiotics and native Australian
medicinal plants
Aishwarya Ilanko1, Ian Cock1, 2
1. School of Natural Sciences, Griffith University, Brisbane, QLD, Australia
2. Environmental Futures Research Institute, Griffith University , Brisbane, QLD, Australia
An increase in antibiotic resistance and a corresponding decrease in antimicrobial discovery have directed researchers towards
alternative therapies, including plant based medicine. However, synergistic combinations of plant extracts with conventional
antibiotics are a far more effective approach in overcoming resistance and potentiating the activity of antibiotics that are
otherwise resistant. In this study, Petalostigma spp., a native Australian medicinal plant, was combined with a range of
conventional antibiotics and tested against various microbial triggers of autoimmune diseases. The fruits and leaves of the plant
were extracted with solvents of varying polarity and investigated for the ability to inhibit bacterial growth using disc diffusion and
liquid dilution MIC techniques. Methanolic and water extracts showed moderate inhibitory activity against several microbes.
However, combinations of extracts and antibiotics proved significantly more effective in inhibiting the growth of Proteus mirabilis
and Acinetobacter baylyi (the respective bacterial triggers of rheumatoid arthritis and multiple sclerosis). In total, 14 different
combinations proved to be synergistic. Notably, two antibiotics with no inhibitory activity alone were shown to have substantial
activity when tested in combination with Petalostigma spp. extracts. Although the mechanisms of synergy are still unclear,
studies indicate that active compounds within Petalostigma spp. could potentially mimic the actions of resistance modifying
agents, thus potentiating the activity of several antibiotics that are relatively ineffective alone. Isolation of these agents can be
highly beneficial in drug design against the microbial triggers of rheumatoid arthritis and multiple sclerosis.
98
P60 - Inhibition of dihydrodipicolinate synthase, an essential enzyme involved in cell wall and
protein syntheses
Chamodi K Gardhi1, Rebecca M Christoff1, Tatiana P Soares de Costa2, Cody J Hall2, Rachael Impey2, Anthony R
Gendall3, Matthew A Perugini2, Belinda M Abbott1
1. Department of Chemistry and Physics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria,
Australia
2. Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe Univeristy, Melbourne,
Victoria, Australia
3. AgriBio, Centre for AgriBiosciences, La Trobe University, Melbourne, Victoria, Australia
Dihydrodipicolinate synthase (DHDPS) catalyses the rate limiting step in the diaminopimelate pathway of bacteria. This
pathway yields meso-diaminopimelate and lysine, which are essential building blocks for the synthesis of cell wall and
housekeeping proteins. Given the essentiality of DHDPS in bacteria, and its absence in humans, this enzyme represents a
promising, yet unexplored, antibiotic target. We have recently conducted a high throughput chemical screen (HTCS) targeting
bacterial DHDPS using a library of 87,648 drug-like compounds. The HTCS yielded a series of micromolar inhibitors, including
a promising hit that has subsequently been developed to generate a series of structurally-related analogues. Using a
combination of enzyme kinetics, microscale thermophoresis and viability assays, these analogues have been shown to bind
and inhibit both Gram-negative and Gram-positive bacterial DHDPS in vitro, and possess bactericidal activity in vivo without
showing toxicity to human cells. Interestingly, the analogues also have broad spectrum activity against plant DHDPS. This
study provides proof-of-concept that a new class of broad spectrum antimicrobials and herbicides can be developed targeting
the essential enzyme, DHDPS.
99
P61 - Critical analysis of antibiotics in the clinical pipeline
Mark S Butler1, Mark AT Blaskovich1, Matthew A Cooper1
1. University of Queensland, St Lucia, QLD, Australia
The lack of new classes of antibiotics, especially those that can treat Gram-negative bacterial infections, will soon leave the
world in a vulnerable position to widespread morbidity and mortality (Figure 1). Given that the drug discovery process takes
many years from discovery to approved drug, it is important that we have an understanding of what new antibiotics are in the
clinical pipeline to identify the pipeline’s strengths and weaknesses and, as a consequence, our future antibiotic
armamentarium. We will update our “Antibiotic in the Clinical Pipeline” review1-3 and provide a critical analysis of the current
state of the antibiotic pipeline.
Figure 1. Total number of new antibiotics and new antibiotic classes in clinical trials in 2011, 2013 and 2015.
1.
2.
3.
“Antibiotics in the clinical pipeline at the end of 2015”, Mark S. Butler, Mark A. T. Blaskovich and Matthew A. Cooper,
J. Antibiot., 2017, 70, 3–24.
“Antibiotics in the pipeline in 2013”, Mark S. Butler, Mark A. Blaskovich and Matthew. A Cooper, J. Antibiot., 2013,
66, 571–591
“Antibiotics in the clinical pipeline in 2011”, Mark S. Butler and Matthew A. Cooper, J. Antibiot., 2011, 64, 413–425
100
P62 - Observational study of erm A and erm C genes and prevalence of MLSB resistance in
Staphylococcus aureus isolates at a tertiary-care hospital
Avneet Kaur Heyar1, Amarjit Kaur Gill1, Prabhjot Kaur Gill1
1. Adesh University Bhatinda, PUNJAB INDIA, Bhatinda, PUNJAB, India
Background: Resiatance to macrolide, lincosamide and streptogramin B (MLSB) among Staphylococcus aureus (S. aureus)
isolates is becoming an increasing problem. Clindamycin is a preferable agent for treatment of S. aureus infections and works
well in patients that are allergic to penicillin. 1 But due to emergence of resistance to macrolide, lincosamide and streptogramin
B (MLSB) due to acquisition of erythromycin resistance methylase (erm) genes which encode enzymes that methylate the 23S
rRNA which is shared commonly by these three drugs resulted in therapeutic failure. 2
Objective: The aim of this investigation was to know the prevalence of inducible MLSB (iMLSB), constitutive MLSB (cMLSB)
and MS resistance and observation of erm A and erm C genes among MLSBi isolates.
Material and Methods: A total of 372 of S. aureus were isolated from OPD, IPD and ICU patients and they were detected by
Double disk approximation/Disk induction test (D-test). The isolates which were iMLSB resistance were randomly selected and
subjected to PCR for erm A and erm C genes observation.
Results: Prevalence of iMLSB, cMLSB and MS isolates was 33.94%, 26.60% and 39.44% respectively, however no isolates
were found to be resistant to Vancomycin, Tiecoplanin and Linezolid. The iMLSB resistant was mainly due to presence of erm
C gene.
Conclusion: This study concluded that D-test should be done as routine procedure to minimize the drug misuse and thereby to
minimize the risk of therapeutic failure. PCR is the most sensitive and quick method to detect genes responsible for inducible
clindamycin resistance.
1.
2.
1. Ajantha GS, Kulkarni RD, Shetty J, Shubhada C, Jain P. Phenotypic detection [1] of inducible clindamycin
resistance among Staphylococcus aureus isolates by using the lower limit of recommended inter-disk distance. Indian J
Pathol Microbiol. 2008; 51:376-78
2. Lewis JS, Jorgensen JH. Inducible clindamycin resistance in staphylococci: should [6] clinicians and
microbiologists be concerned? Clin Infect Dis.2005; 40:280-85
101
P63 - Enhanced antibiotic activity through complexation with metal ions: evaluated via ITC and
biological studies
Chris Caboche1, Yifang Zhao1, Shaoyang Zhang1, Hans Laroo2, Matt A Cooper3, Rink-Jan Lohman3, David P Fairlie3,
Alysha G Elliott3, Zyta M Ziora3
1. School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia
2. Laroo Research, Ipswich, Queensland, Australia
3. Institute for Molecular Bioscience, University of Queensland, Brisbane, Queensland, Australia
The growing problem of multi-drug resistant bacteria reduces the efficacy of antibacterial agents requiring alternative solutions
to be developed. Some metal ions are known to provide antimicrobial effects and have previously been shown to be effective
on open wounds and injured skin where uncontrolled microorganism growth can delay the normal process of wound healing.
An effective antibacterial agent is required to improve the wound healing process in order to avoid open wound complications
for patients. This study explores the possibility of increasing the activity of antibiotics by creating silver complexes of various
antibiotics. Silver (I) complexes of nitrate, sulphate, and acetate, as well as colloidal silver, were also assessed as potential
antimicrobial agents. Isothermal titration calorimetry was used to examine complex formation between antibiotics and metal
cations, such as Cu(II), Zn(II) or Ag(I), and the antimicrobial potential of the metal complexes was assessed by microbroth
dilution assays. The Cu(II) and Zn(II) complexes showed greater activity than antibiotic alone only in the case of Methicillinresistant S. aureus, except for the Zn(II) complex with penicillin G which did not. Most of the antibiotic complexes of Ag(I)
demonstrated synergistic effects, when the complex exhibits better potency than antibiotic or metal alone against tested
bacterial strains. The silver complexes have also been tested ex vivo in experiments on rat skin and have shown an enhanced
ability over the antibiotics alone to inhibit the growth of microbes.
102
P64 - Transposon Directed Insertion Sequencing (TraDIS) to investigate genes required for
survival during antimicrobial synergy in multidrug-resistant Klebsiella pneumoniae
Amy K Cain1, 2, Bimal Jana3, 4, Christine J Boinett1, 5, Julian Parkhill1, Luca Guardabassi3, 4
1. Pathogen Genomics, Wellcome Trust Sanger Institute, Cambridge, Cambridgeshire, UK
2. Chemistry and BioMolecular Sciences, Macquarie University, Sydney, NSW, Australia
3. Department of Veterinary Disease Biology, University of Copenhagen, Copenhagen, Denmark
4. Department of Biomedical Sciences, Ross University School of Veterinary Medicine, Basseterre, St Kitts
5. Hospital for Tropical Diseases, Oxford University Clinical Research Unit, Ho Chi Minh City, Vietnam
Klebsiella pneumoniae is a deadly, nosocomial pathogen that is often Multi-Drug Resistant (MDR) meaning that infections can
be difficult to treat. Thus, combination therapy using synergistic antibiotics is becoming an attractive last-line treatment option.
Here, we used Transposon Directed Insertion Sequencing (TraDIS) to assay each chromosomal gene in a MDR K.
pneumoniae hospital ST258 clone for involvement in survival during synergistic antibiotic exposure. A dense TraDIS library of
300,000 unique Tn5 mutants (see Jana and Cain et al., 2017) was exposed to subinhibitory concentrations of ciprofloxacin
(cip), amikacin (amk) or imipenem (imp), and in synergistic combinations of these (cip+amk or cip+imp). After sequencing and
TraDIS analysis, levels of insertions were determined for each gene and compared to an untreated control. We examined
genes important for survival only during exposure to antimicrobial combinations.
Five genes displayed a significant reduction in insertion mutants in the presence of both antimicrobial combinations, including a
core transcription-translation regulator in antibiotic resistance (ihfB) and an uncharacterized sRNA. We confirmed the
involvement of ihfB only in synergistic resistance, not for the individual antibiotics, using a single knockout mutant. We also
identified genes involved in sensitivity only during synergy, including zntR, a zinc efflux regulator.
This study pushes forward the emerging field of antibiotic combination therapy by providing insights into the molecular action of
antibiotic synergy as well as survival strategies of MDR K. pneumoniae during exposure to specific antimicrobial combinations.
The information generated by the study also opens future avenues to finding novel antimicrobial targets.
1.
Jana and Cain et al. The secondary resistome of multidrug-resistant Klebsiella pneumoniae. Sci. Rep. 7, 42483; doi:
10.1038/srep42483 (2017).
103
P65 - Comparison of drug resistance profile of clinical isolates of Pseudomonas aeruginosa
Dinesh Subedi1, Ajay Kumar Vijay1, Scott A Rice2, 3, Mark Willcox1
1. School of Optometry and Vision Science , University Of New South Wales, Kensington, NSW, Australia
2. The School of Biological Sciences, Nanyang Technological University, Singapore
3. Centre for Marine Bio-Innovation, University of New South Wales, Sydney, NSW, Australia
Infections with antibiotic resistance bacteria are becoming increasingly prevalent. The aim of the study is to determine the
antibiotic and disinfectant resistance of P. aeruginosa strains collected from keratitis and cystic fibrosis patients from two
different continents. Altogether, 22 strains were used in this study, of them, 10 were lung isolates from Australia and 12 were
ocular. Half of the eye isolates were from India and a half from Australia. Bacterial resistance was determined by MIC using 10
different anti-pseudomonal antibiotics and 4 commercial disinfectants for contact lens care. All Australian eye isolates were
sensitive to all antibiotics tested except for two which were resistant to one of the fluoroquinolones, a common empirical
therapy for microbial keratitis. On the other hand, all Indian eye isolates were resistant to at least two antibiotics. Unlike
Australian eye isolates, 92% of lung isolates were resistance to at least one antibiotic. Compared to ocular isolates, a larger
percentage of lung isolates were resistant to beta-lactams and aminoglycosides, while the resistance rate for fluoroquinolones
was similar (Figure). All four commercial disinfectant solutions were effective against eye isolates at 100% concentration with
some variations in sensitivity at different dilutions. There was no association between drug resistance and disinfectant
susceptibility observed in this study. This study will help understand how antibiotic resistance pattern is distributed in P.
aeruginosa for two different infections. Further study on the molecular mechanism of resistance will elucidate factors
responsible for resistance transfer in this bacterium.
104
P66 - Efficient drug delivery by layered double hydroxide nanoparticles
LI LI1
1. UNIVERSITY OF QUEENSLAND, ST LUCIA, QLD, Australia
Layered double hydroxide (LDH) nanoparticles have great potentials in drug and siRNA delivery. However, the functionalization
and a random agglomeration of LDHs in phosphate buffer solution (PBS) and cell culture medium limit their applications. To
circumvent this issue, silica (SiO2) nanodot coated LDH nanocomposites have been developed via a nanodot-coating strategy.
The characteristization results demonstrated that LDH-SiO2 nanocomposites retained the morphology and size of LDH
nanoparticles and SiO2 nanodots with the size of 10 nm were evenly distributed on the surface of LDH nanoparticles. After
coating with SiO2 nanodots, LDH-SiO2 nanocomposites showed good dispersion in aqueous solution and cell culture medium.
We further employed LDH-SiO2 nanocomposites as a nanocarrier to deliver methotrexate (MTX), a chemotherapy drug, to the
human osteosarcoma cell. The anticancer delivery test demonstrated significant inhibition of cancer cell proliferation using
LDH-SiO2 nanocomposites to deliver MTX.
105
P67 - Understanding FtsZ as an antibacterial target
Kennardy Kusuma1, Alison Ung2, Elizabeth Harry1
1. i3 Institute, UTS, Sydney, NSW, Australia
2. School of Mathematical and Physical Sciences, UTS, Sydney, NSW, Australia
In April 2014, the first comprehensive and up to date analysis of antimicrobial resistance (AMR) was written by the World
Health Organisation (WHO). The report outlined the shifting of society into a post-antibiotic era where common and minor
infections will once again be lethal. In addition to that, many medical therapies and surgeries that are considered routine or
operable may soon cease to be viable because of the high risk or complication associated with untreatable infections. One way
to tackle this problem is to develop new antimicrobials with a novel mechanism of action. One process that is yet to be targeted
by any clinically approved antimicrobials is bacterial cell division.
Bacterial cell division is an essential process whereby all genetic material is duplicated and passed down to the two newly
formed daughter cells. Central to this process is the protein called FtsZ. There are many studies into compounds which inhibits
FtsZ but, many do not show concrete evidence for the inhibition of FtsZ. One possible reason is that, regardless understanding
FtsZ structurally, we are yet to understand the protein molecularly. Using Discovery Studio 4.5, we compared published FtsZ Xray crystal structures from various bacterial species and found the primary different to be within the central core H7 helix and
the organisation of the β-strands on the C-terminal of the protein. Molecular dynamics showed that the β-strands on the Cterminus moves like a sliding door upon binding to nucleotide. This allow less or more access to the interdomain cleft of FtsZ.
106
P68 - Identification of novel antimicrobial compounds from Australian Myrtaceae species
Malin A Olsson1, Peter R Brooks2, Roy M Robins-Browne3, Steven M Ogbourne2, Stephen J Trueman2, Ji Yang3, David J
McMillan1
1. School of Health and Sports Sciences, University of the Sunshine Coast, Maroochydore, Queensland , Australia
2. Faculty of Science, Health, Engineering and Education, University of the Sunshine Coast, Maroochydore, Queensland,
Australia
3. Peter Doherty Institute, University of Melbourne, Parkville, Victoria, Australia
Plants have been used for their medicinal properties for thousands of years. They produce thousands of structurally diverse
secondary metabolites, including aromatic compounds, many of which have antimicrobial activity. However, it has been
estimated that only 15% of higher plant species have been phyto-chemically analysed, and even a smaller percentage
evaluated for their antibacterial properties.
With the emergence of multiple antibiotic resistant bacterial species, the discovery of antimicrobial compounds from natural
sources has regained prominence. The major aim of this study is to comprehensively identify and characterise novel
compounds in Australian Myrtaceae species (Eucalyptus, Corymbia, Angophora and Syncarpia) that have antimicrobial activity
against multiple ESKAPE pathogens.
Uniquely, three distinct bioactivity assays will be used to assess antimicrobial activity in extracts from these species. Firstly,
standard high-throughput MIC/MBC assays will be used to assess bactericidal and bacteriostatic activity. Secondly, biofilm
specific screens will be used to determine if extracts inhibit biofilm formation of selected species. Thirdly, we will use a reportergene assay to identify inhibitors of the RegA response regulator of Citrobacter rodentium. Response regulators are exciting
antimicrobial targets because the inhibition of key virulence regulators may attenuate the pathogenicity of the target bacteria
while leaving the commensal microflora intact. Selected fractions with bioactivity will be further purified, and individual
compounds subjected to additional functional assays.
107
P69 - From sepsis to sequencing; a culture-free bacterial sepsis detection approach
Marwa M Hassan1, Andrea Ranzoni1, Devika Ganesamoorthy Ganesamoorthy1, Minh Duc Cao1, Mark A Blaskovich1,
Lachlan JM Coin1, Matthew A Cooper1
1. The University of Queensland, Institute for Molecular Bioscience, Brisbane, QLD, Australia
Sepsis is a complicated syndrome that causes high hospital associated morbidity and mortality rates worldwide. Diagnosis of
bacterial sepsis necessitates the quick detection from at least 6-10 mL blood samples concurrent with a sensitive detection of 1
cfu/mL. The large sample volume, complex sample and low CFU leaves culture and molecular amplification techniques as the
only detection methods. However, time of detection remains the key challenge. In this study, blood samples were treated with
polymer solution to induce aggregation of interfering cells without the need for a centrifugation step. Vancomycin magneticnanoparticles were synthesized, quantified and used to capture bacteria from blood samples. Captured-bacterial cells were
processed for DNA extraction and detection. The processing methodology resulted in a remarkable decrease in the content of
interfering human cells of up to 99% of whole blood cells with minimal effect on spiked bacteria. The DNA-extraction method is
quick and proved efficiency for all tested Gram-positive bacteria including capsulated strain. Further, we showed sensitive
qPCR detection of the extracted S. aureus bacterial DNA with a detection limit of 5 cfu/mL from blood samples with at least 20
times faster than conventional culture-based approaches. Our Bac-ID method showed comparable efficiency in DNA
enrichment from 1 and 10 mL samples owing to its triple enrichment approach. We successfully detected extracted bacterial
DNA from spiked-platelet samples using MinION sequencing. The Bac-ID method can start as soon as the blood sample is
collected allowing for quick detection and can be fully integrated into clinical practice for sepsis
detection.
108
P70 - Product development strategies for challenging molecules
Sanjay Garg1, May Song1, Stephen Page2, Darren Trott3
1. Centre for Pharmaceutical Innovation and Development (CPID), University of South Australia, Adelaide, SA, Australia
2. Neoculi Pty Limited, Hawthorn, Victoria, Australia
3. Australian Centre for Antimicrobial Resistance Ecology (ACARE), The University of Adelaide, Roseworthy, SA, Australia
Solubility, stability, and bio-availability are key characteristics that dictate the "druggability" of a new active ingredient. A
common feature of novel antimicrobial compounds that transition from drug discovery to development stage is poor aqueous
solubility, which limits their progression. Approximately 85% of the new compounds under development in our laboratory are
water insoluble. In addition to chemical modifications, there are a range of pharmaceutical techniques such as nano-sizing, cosolvents, cyclodexrin complexation, lipid based formulations, and solid dispersion (SD), which can be highly valuable in dealing
with the challenge. These approaches will be discussed with the help of a case study.
NCL812 and NCL099 are antiprotozoals, being repurposed and developed as antibacterial compounds. These compounds
have been shown to have potent activity against MDR bacteria. In common with most newly discovered drug candidates, they
are poorly water soluble. Two technologies, including nanosizing and solid dispersion, were employed and optimized for the
solubility improvement. A range of solvents and buffers (pH 1.8 to pH 8) were used for solubility assessment. Commonly used
polymers and surfactants, such as PVPP, SLS, Tween 80, PVP K30, HPC, Kollidon ®, Soluplus® and Pluronic F-68 were
screened for initial aqueous solubility improvement. The samples were analyzed by validated HPLC methods. PEG 4000,
Soluplus®, Kollidon® and PVP K30 were selected as carriers for SDs. Different ratios of the drugs and carriers were optimized
using solvent evaporation method. Crystallinity and thermal properties of actives and SDs were investigated. Soluplus® SD
increased the solubility of NCL812 by 220 fold and that of NCL099 by 177824 times.
1.
Page, S., and Garg, S. (Jan 10, 2017). Methods for Treating Bacterial Infections. USA Patent No. US 9539223 B2.
109
P71 - Antibiotic-metal constructs to combat Gram-positive and Gram-negative bacteria
resistance
Zyta M Ziora1, Shaoyang Zhang1, 2, Keisuke Uenishi1, Mark S Butler1, James Cameron3, Alan White3, Michael
Whitehouse3, Brett Collins1, Matthew A Cooper1
1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD, Australia
2. School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, Australia
3. Instrumental Chemistry Laboratory, Griffith University, Nathan Campus, QLD, Australia
The globalization of the whole world causes the wide spread of bacterial infectious diseases which generate a no negligible
threat. Antibiotics currently used to fight against bacteria can be rendered useless as treatments for multi-drug resistant (MDR)
bacterial infections. One possible solution is to form metal ion complexes to enhance their antimicrobial effectiveness and
overcome issues with MDR.
In this study, Vancomycin was complexed with metal ions, such zinc, copper and silver; and silver ion was identified as the
most potent antimicrobial adjuvant and was complexed with the other antibiotics, Colistin and Gentamicin. Inductively coupled
plasma optical emission spectroscopy, ICP-OES, was used to quantitatively determine the amount of metal in the formulated
complexes and isothermal titration calorimetry, ITC, was used to investigate interactions between metals and the antibiotics,
and to provide thermodynamics and stoichiometry helpful in the preparation of complexes for in vitro assays.
Analysis of ICP-OES indicated that the amount of metals in their salts were in the expected range for all silver forms, while in
case of chlorides, ZnCl2 contained much less zinc than calculated. It also showed that there was no metal contamination in
tested antibiotics. From ITC results, a weak interaction with Ag(I) and Colistin was found while strong binding was observed for
Gentamicin only with silver from acetate salt. Strong binding affinity and great energy change was reported for Vancomycin and
Ag(I) from three salts.
In conclusion, silver-Vancomycin constructs demonstrated the potential to enhance antimicrobial effectiveness which
consequently can help to overcome the MDR.
110
P72 - Characteristics of Induced Resistance against Polymyxin B, Colistin and Octapeptin C4
in Extensively Drug-Resistant Klebsiella pneumoniae
Miranda E Pitt1, Minh Duc Cao1, Soumya Ramu1, Mark S Butler 1, Devika Ganesamoorthy1, Mark AT Blaskovich1,
Lachlan Coin1, Matthew A Cooper1
1. Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, Australia
Infections facilitated by extensively drug-resistant (XDR) bacteria are perpetually emerging with a lack of effective antibiotics
and high mortality. This is increasingly evident for Klebsiella pneumoniae (KP) in which polymyxins, including polymyxin B and
E (colistin), are commonly utilised for treatment (1). Polymyxin resistance is rapidly arising, however, a structurally similar class
of antibiotic, the octapeptins, retain potency against these bacteria (2, 3).
In order to resolve the potential mechanism of action and development of resistance towards octapeptins, a polymyxinsusceptible XDR-KP clinical isolate was passaged in increasing concentrations of octapeptin C4 (OctC4) over 20 days. As a
comparison of resistance acquisition, polymyxin B and colistin were also assayed. Over the 20 day time course, all polymyxin
exposed replicates harboured a resistant phenotype (MIC: >2 μg/ml) with the majority possessing a substantial 1000-fold MIC
elevation. OctC4 resistance induced replicates only reached a 4-fold MIC increase. Day 20 replicates harboured no crossreactivity between polymyxins and OctC4 which correlated with the vast deviations in lipid A profile. Sequencing of polymyxinresistant replicates detected mutations in genes previously associated with lipid A modifications including crrB, mgrB, phoP,
phoQ and pmrB alongside several other genes yet to be reported. OctC4 replicates incurred mutations impacting pathways
never associated with any other class of antibiotic and the involvement was discerned through complementation assays.
The slow progression of resistance, lack of cross-reactivity with polymyxins and mutations in new pathways implies OctC4 is
infiltrating XDR-KP via a novel mechanism.
1.
2.
3.
Karaiskos I, Giamarellou H. 2014. Multidrug-resistant and extensively drug-resistant Gram-negative pathogens:
current and emerging therapeutic approaches. Expert Opin Pharmacother. 15:1351-1370.
Giamarellou H. 2016. Epidemiology of infections caused by polymyxin-resistant pathogens. Int J Antimicrob Agents.
48:614-621.
Velkov T, Roberts KD, Li J. 2017. Rediscovering the octapeptins. Nat Prod Rep. doi: 10.1039/c6np00113k.
111
P73 - An open real-time intelligent platform for genetic analysis of the acquisition of drug
resistance
Michael B Hall1, Miranda E Pitt1, Alysha G Elliott1, Mark AT Blaskovich1, Matthew A Cooper1, Lachlan Coin1, Minh Duc
Cao1
1. Institute for Molecular Bioscience, University of Queensland, Saint Lucia, QLD, Australia
It is of critical importance to understand the molecular mechanisms of antibiotic resistance in order to i. implement strategies for
minimising acquisition of antibiotic resistance to existing and new drugs; ii. design drug combinations capable of subverting
resistance mechanisms and iii. develop rapid diagnostics for identification and tracking of drug-resistant bacteria.
Genetics is a powerful and affordable tool to elucidate the molecular mechanisms of resistance. Whole-genome sequencing of
clinical isolates has successfully detected acquired resistance genes and novel chromosomal variants in pathways associated
with the antibiotics mechanism of action. Furthermore, several mechanisms have been uncovered through resistance induction
experiments with susceptible strains or by disruption of resistance mechanisms using random transposon insertion. However,
analysis of these datasets remains challenging, and moreover to maximise their informativeness they need to be analysed in
the context of existing data of resistance to related antibiotics in related strains and species.
We are developing a web-based platform to provide a free, automatic and real-time analysis of the genetic mechanisms of
antibiotic resistance to laboratories around the world. The platform delivers a dynamic, real-time visualisation of the results as
the samples are processed. The platform will aggregate a large number of bacterial samples and build a comprehensive
database of genomic variation coupled to phenotypic resistance information. Importantly, this platform will 'learn' models of
antibiotic resistance as sequence and phenotypic resistance data accumulate, allowing re-analysis of existing data in the
context of new information.
112
P74 - Characterization of M. tuberculosis clinical isolates from the Torres Strait using whole
genome sequencing: insights into transmission and emergence of drug resistance
Arnold Bainomugisa1, 2, Evelyn Lavu3, Sushil Pandey4, Ellen Donnan5, Ben Marais6, Chris Coulter4, 5, Lachlan Coin2
1. School of Medicine, The University of Queensland, Brisbane, QLD, Australia
2. Institute for Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
3. Central Public Health Laboratory, Port Moresby, Papua New Guinea
4. Queensland Mycobacterium Reference Laboratory, Brisbane, QLD, Australia
5. Communicable Diseases Unit, Queensland Department of Health, Brisbane, QLD, Australia
6. Marie Bashir Institute for Infectious diseases and Biosecurity, Sydney, NSW, Australia
Abstract
Increased transmission of drug resistant strains of M. tuberculosis pose a major challenge to regional tuberculosis (TB) control.
In order to understand the transmission dynamics and evolution of drug resistant M. tuberculosis strains in the Torres Strait, we
performed whole genome sequencing (WGS) on strains identified over a 10 year period. In total, 107 strains were sequenced;
83 (77.5%) were identified as Lineage 2 and 24 (22.5%) as Lineage 4 (Euro-American). Using 7 single nucleotide
polymorphism (SNP) difference, we identified 10 SNP clusters among Beijing and 2 SNP clusters among Euro-American
lineages. We constructed plausible transmission links among the clusters using genomic data. The most common rpoB
mutation was associated fitness compensatory mutations in the rpoC, and fabG1 promoter mutation was commonest among
isoniazid resistant isolates. All of the multi-drug resistant (MDR) isolates (n=30), were from the Beijing sub-lineage and they
posed a new frame shift mutation in ndh gene. Molecular clock analysis of Beijing strain revealed a mutation rate of 0.36
SNP/genome/year and its most recent ancestor was established to be in the 1940s. A timeline for resistance acquisition among
the Beijing strains was constructed and the progenitor to have acquired isoniazid and streptomycin resistance was established
to be around 1969. Routine WGS based identification of transmission clusters and evolution of drug resistance could provide
valuable real-time surveillance and guidance for better target public health responses.
113
P75 - Antimicrobial activity of a modified biopolymer combined with silver
Izabela Zawisza2, 1, Wojciech Bal1, Malgorzata Popko2, Rink-Jan Lohman3, Theresa Kolmar3, Soumya Ramu3, Ekaterina
Strounina4, David P. Fairlie3, Mark A. T. Blaskovich3, Matthew A. Cooper3, Zyta Ziora3
1. Institute of Biochemistry and Biophysics Polish Academy of Sciences, Warsaw, Poland
2. KF Niccolum sp. z o.o., Marki, MAZOVIA, Poland
3. The University of Queensland, Institute for Molecular Bioscience, St Lucia 4072, Australia, Brisbane
4. The University of Queensland, Centre for Advanced Imaging, St Lucia 4072, Australia, Brisbane
The growing threat of untreatable bacterial infections due to increasing bacterial strains resistant to current clinical antibiotics is
resulting in a demand for new antimicrobial substances. Some researchers propose that combination therapy using silver with
antimicrobial agents could synergistically enhance the biocidal efficiency of these agents. A related concept is to combine the
silver ions with polymers for enhanced antimicrobial efficacy.
We have prepared new antimicrobial substances by modifying biocompatible polymers in combination with silver (KF Niccolum
R&D Laboratory). The structure and chemical composition of these complexes were studied using NMR spectroscopy and
scanning electron microscopy (SEM). We determined the minimal inhibitory concentration (MIC) of the modified biopolymers
combined with silver against selected bacteria and tested their antibacterial properties on rat skin. Our studies showed that this
combination has strong antimicrobial activity against Gram-negative and Gram-positive strains, including Methicillin-resistant
Staphylococcus aureus (MRSA). This material has great potential as a topical therapeutic for infected wound healing, as
demonstrated ex vivo on rat skin (Fig. 1). Future studies will test combinations in vivo in skin infection assays to determine
efficacy and safety.
Fig. 1. Bacterial growth from skin scrapes of rat skin treated with compounds (2 and 12 hours of compound treatment) after 24
h on agar plates
114
P76 - A Phase 1 study to evaluate the safety, tolerability and preliminary effectiveness of ABSA01 in patients with chronic rhinosinusitis associated with Staphylococcus aureus infection
Sandra Morales1, Mian Ooi2, Amanda Drilling2, Sophia Moraitis2, Stephanie Fong2, Sarah Vreugde2, Alkis Psaltis2, Peter
J Wormald2
1. AmpliPhi, Sydney/Brookvale, NSW, Australia
2. ENT, Queen Elizabeth Hospital , Adelaide, SA, Australia
Chronic rhinosinusitis (CRS) is a debilitating inflammatory and infection based condition, affecting up to 9% of the Australian
population. Currently available therapies to treat this condition include steroids, antibiotics and surgical intervention.
Unfortunately, there remains a cohort of patients that are resistant to both medical and surgical interventions who experience
persistent CRS symptoms, termed recalcitrant CRS (rCRS). Thus, the safety and preliminary effectiveness of a novel
bacteriophage-based
treatment,
AB-SA01,
was
evaluated
in
rCRS
patient.
Three patient cohorts (n=3 patients/ cohort) were dosed with twice daily (i) lower concentration phage for 7 days; (ii) lower
concentration phage for 14 days; and (iii) higher concentration phage for 14 days. Safety observations included vital signs,
physical examinations, clinical laboratory tests and adverse event (AEs) reporting. Preliminary efficacy was assessed
comparing pre- and post-treatment microbiology results, endoscopic Lund Kennedy Scores (LKS) and symptom scores using
Visual Analogue Scale (VAS) and Sino-Nasal Outcome Test (SNOT-22).
AB-SA01 was safe and well tolerated when administered to participants. Preliminary efficacy data were suggestive of proof-ofconcept. Results were subject to inter-individual variation but support decreased bacterial load following treatment, and
improvements in endoscopic findings and symptoms in participant-reported outcomes. A phase 2a, multicenter, randomized,
double-blind, parallel-group, placebo-controlled study to evaluate the safety and efficacy of AB-SA01 is currently being planned.
115
P77 - Antimicrobial activities of the trunk exudates from Araucaria heterophylla, Norfolk Island
Pine
Mohammad Sharifi Sharifi1, Renata Andrzejewska1
1. Australian Food and Pharmaceutical Industries, Bomaderry, NSW, Australia
Leaves of Araucaria angustifoliais has been traditionally used as emollient, antiseptic, and for rheumatism and respiratory
infection. The resin of Araucaria araucana is used to treat contusions, ulcers, wounds and to ease cauterization. The bark of
Araucaria bidwillii, native to South Africa, is used to treat amenorrhea by indigenous people. In Papua New Guinea Araucaria
cunninghamii is used in rituals and Araucaria heterophylla has been traditionally used for toothache.
In this work the trunk exudates of Araucaria heterophylla was used to evaluate its antimicrobial activity against Gram-positive
bacteria: Staphylococcus aureus, Bacillus subtilis, Sarcina ventriculi and Gram negative: Escherichia coli, and Proteus vulgaris.
Materials and methods: The composite trunk exudates of Araucaria heterophylla (known as: Norfolk Island Pine), collected
from Sydney beach, (Brighton Le Sands) was dissolved in methanol. The solution was then screened for antimicrobial activity
against Gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis, Sarcina ventriculi and Gram-negative: Escherichia
coli, Proteus vulgaris.
A mixture of methanol and water was served as a blank with the same concentration as the test solution.
Results: The gum demonstrated activities against both Gram-positive and Gram-negative bacteria ranging from 5µg/ml50µg/ml. The highest activity was against Staphylococcus aureus and lowest one against Bacillus subtilis.
Conclusion: The results are very promising and warrant screening of the chemical entities of the gum against a variety of
microorganisms and further investigation into the toxicity and mode of action.
Keywords: Antimicrobial; Araucaria heterophylla; Staphylococcus aureus; Escherichia coli
116
P78 - Phage-bacteria evolutionary dynamics and how this affects phage therapy for
Pseudomonas aeruginosa
Zeinab ZHD Hosseini-Doust1
1. Mcmaster University, Hamilton, ONTARIO, Canada
The rise of bacterial variants in the presence of lytic phage has been one of the basic grounds for evolution studies. However
there are incongruent results among different studies investigating the effect of acquiring phage resistance on bacterial fitness
and virulence. We employed an in vitro burn wound model to study the development of phage resistance and associated
variations in virulence and biofilm formation for the resulting phage-resistant PA phenotypes. Experimental evolution was used
to generate Pseudomonas aeruginosa PAO1 variants under selective pressure from different homogeneous and one
heterogeneous phage environment. The phages were chosen to target different receptors to decrease the chance of crossresistance. Investigation of phenotypic traits of the variants revealed significant changes in various fitness and virulence
determinants such as growth, motilities, biofilm formation, resistance to oxidative stress and production of siderophores and
chromophores compared to the control. The choice of therapeutic phage (or phage cocktail) allowed for the control of evolution
of resistant phenotypes (i.e., selective pressure). This work is currently being followed up by in vivo experiments on various
models and also using microfabricated microenvironments for a more mechanistic investigation. The knowledge gained from
this study will fundamentally contribute to our understanding of the evolutionary dynamics of bacteria under phage selective
pressure, which is crucial for the efficient utilization of bacteriophages for phage therapy.