Download Novel Antimicrobial Agents

Novel Antimicrobial Agents
Tuesday 15th June 2010
School of Pharmacy & Life Sciences, and Research Institute for Health
and Welfare, Robert Gordon University, Aberdeen
Conference Abstracts
Welcome
We welcome you to the Robert Gordon University today for
what promises to be a very interesting conference. Having
assembled the programme over the last few months, it is
pleasing to have gathered such a prestigious grouping of people
who will be discussing their work on novel antimicrobials. We
would like to take the opportunity to thank all of those who
accepted our invitation to speak today.
It is only very occasionally that we can enjoy such an event in
North-east Scotland and thanks must also be offered to the
Society for General Microbiology and the Society for Applied
Microbiology for their generous financial support.
We would like to thank all our colleagues within Robert Gordon
University who have contributed to the organisation of todays
event.
Finally, we hope you enjoy the conference.
Dr Andrew Lamb
Dr Derek Chapman
15th June 2010
Programme
9.45 – 10.25
Registration and Poster Assembly/Viewing
Session 1
Novel Antimicrobials
10.25 -10.30
Dr Andrew Lamb, Robert Gordon University
Welcome and Introduction
Chair – Dr Andrew Lamb
10.30 – 11.10 Professor Peter Taylor, University of London
‘Some alternatives to conventional antibiotics:
what's on offer?’
11.10 – 11.40 Dr Derry Mercer, NovaBiotics Ltd
‘Advances in antifungal drug discovery’
11.40 – 12.00 Dr Alan Bowman, University of Aberdeen
‘A novel GPI-linked antibacterial protein in the
sheep tick, Ixodes ricinus’
12.00 – 12.20 Ms Noelle O’Driscoll, Robert Gordon University
‘Cationic antimicrobial peptides: novel
alternatives to existing antibiotics’
12.20 – 12.55 Lunch
12.55 – 1.10
Poster Viewing Session
Session 2
Synthesis and Evaluation of
Antibacterials
Chair – Professor Don Cairns
1.10 – 1.50
Professor Colin Suckling, University of
Strathclyde
‘Antibacterial minor groove binders – the
Strathclyde Solution!’
Programme
1.50 – 2.20
Dr Geoff Coxon, University of Strathclyde
‘2-Aminothiazole-4-carboxylates (ATCs) as a
potential new class of anti-tuberculosis drug’
2.20 – 2.40
Dr Cedric Charrier, NovaBiotics Ltd
‘1 + 1 ≠ 2 : Novel synergistic antibacterials’
2.40 – 2.55
Tea/Coffee
Session 3
Antiparasitics and Drug Resistance
2.55 – 3.35
Dundee
Professor
3.35 – 4.05
Dr Paul Hunt, University of Edinburgh
‘Genetic and genomic identification of drug
resistance pathways in malaria’
4.05
Close
Chair – Dr Derek Chapman
Alan
Fairlamb,
University
of
‘Spinach leaves, butterfly wings, nutmeg oil and
antiparasitic drugs’
Speaker Abstracts
Some alternatives to conventional
antibiotics: what's on offer?
Professor Peter W. Taylor
School of Pharmacy, 29-39 Brunswick Square, London WC1N 1AX UK
Over the last two decades, there has been a steady decline in
the number of new antibacterial agents launched into the
market; this period has coincided with a substantial rise in the
frequency of isolation of multi-drug-resistant pathogens from
nosocomial and, increasingly, community-acquired infections.
Currently useful antibiotics impose enormous selective pressure
on bacterial populations and the emergence of resistance to
these drugs is all but inevitable; we need, therefore, to
constantly replenish our antibacterial arsenal. The hope that the
exploitation of genomics and target-based high throughput
screening would yield a new generation of novel drugs
addressing new molecular targets has not been realised in spite
of massive investment; furthermore, a recent molecular
analysis of protein expression in experimental infection raises
the possibility that we may have already exploited most of the
broad-spectrum targets essential for bacterial survival in vivo.
As academia and government agencies are being urged to
become more involved in the process of antibiotic development,
the time may be right to begin to exploit more unconventional
approaches to the eradication of infection. This contribution will
focus on alternatives to conventional antibiotic chemotherapy
for the resolution of troublesome infections - particularly those
due to multi-drug-resistant pathogens such as MRSA, XDR-TB
and the like. Thus, bacteriophage therapy, phage components
such as lysins, constructs that interact with previously
unexploited bacterial targets and agents that advantageously
modify the antibiotic resistance and virulence of bacterial
pathogens will be evaluated.
Antifungal Drug Discovery: Where next?
Dr Derry Mercer
NovaBiotics Ltd, Cruickshank Building, Craibstone, Aberdeen, AB21
9TR, UK
Fungal infections range from the superficial, causing
inconvenience rather than illness, to systemic infections with
high rates of associated morbidity and mortality. The everincreasing number of immunocompromised patients has led to
a dramatic increase in the number of life-threatening fungal
infections in the last two decades. Systemic fungal infections
with Candida spp. and Aspergillus spp. can have mortality rates
of up to 40% and 90%, respectively, if not diagnosed and
treated quickly.
The increasing incidence of fungal infections demands
improvements to the current antifungal armamentarium.
Current antifungal drugs are limited to four main drug classes
and therefore there is a significant requirement for novel
antifungal agents with disparate modes of action.
A number of interesting approaches are being used in
antifungal drug development including areas such as vaccine
and biologics-based approaches, as well as more traditional
approaches examining novel chemical entities interacting with
specific targets. Inevitably, expansion of existing drug classes
continues apace.
In this paper I shall present an overview of fungal infections
and currently available drugs before examining potential novel
drug classes and highlighting a few of the more promising
therapeutic candidates.
A novel GPI-linked antibacterial protein in
the sheep tick, Ixodes ricinus
Dr Alan Bowman
M.J. Burdin1, K. Forbes2, and A.S. Bowman1
1
School of Biological Sciences (Zoology) and 2Immunology & Infection,
Medical School, University of Aberdeen, Scotland, UK
Here we report on a new class of antimicrobial peptide (AMP) –
the novelty of which concerns a GPI-anchor to hold it on the cell
surface in contrast to all other reported AMPs that are directly
secreted into the circulation to counter microbial infections.
RicOrph17, found in the sheep tick, contains a putative 26amino acid signal peptide and a 26-coding amino acid GPIanchoring signal. The RicOrph17 protein shares some structural
features of three finger protein family, including a cysteine
skeleton responsible for the formation of 5 disulfide bonds.
RicOrph17 was equally expressed in all tick tissues tested.
Injection of RicOrph17-dsRNA into unfed ticks silenced the
target gene expression and caused the tick to be more sensitive
to bacterial challenge, resulting in a significantly higher
mortality rate. A recombinant protein (rRicOrph17) lacking the
secretion signal peptide and GPI-anchoring signal was
produced.
rRicOrph17
exhibited
antibacterial
activity
(bacteriostatic and bacteriocidal) against 10 clinically relevant
isolates (Gram +ve and –ve) and against 3 yeasts.
Fluorescently-tagged rRicOrph17 was observed to enter and
accumulate inside bacteria rather than remain associated with
the bacterial wall unlike the pore-forming magainin.
Additionally, rRicOrph17 associated with DNA.
Overall, we have identified a novel class of AMP, previously
unknown, but widely present throughout the arthropod phyla.
The GPI-linkage could serve as a rapid and localised release of
the protein after a bacterial infection.
Cationic antimicrobial peptides: novel
alternatives to existing antibiotics
Ms Noelle O’Driscoll
N. O’Driscoll1, K. Matthews1, D.K. Mercer2 and A. J. Lamb1
1
School of Pharmacy and Life Sciences, Robert Gordon University,
Schoolhill, Aberdeen. AB10 1FR 2NovaBiotics Ltd, Cruickshank
Building, Craibstone, Aberdeen, AB21 9TR, UK.
Despite the use of antimicrobial agents for nearly eight decades
since the introduction of sulphonamides in the 1930s, infectious
diseases continue to have an impact both on human morbidity
and mortality throughout the world and place a major burden
on healthcare services. The World Health Organisation
estimated 11 million deaths in 2002 from acute respiratory
infections and major infectious diseases including tuberculosis
and AIDS/HIV. These figures increase yearly. There is an
increasing urgency in the search for new antibacterials that are
active against a wide range of bacteria, possess minimal host
toxicity and do not readily select for resistant mutants. Cationic
antimicrobial peptides (CAPS) comprise a group of effective,
safe and chemically diverse antibacterials. These gene coded
CAPs are found in the tissues of most complex organisms,
comprising part of the innate immune system. Constitutively
expressed or induced, endogenous CAPs provide a fast
response to bacterial invasion, are mobilized quickly after
microbial infection and rapidly neutralise a broad range of
microbes.
The aim of this research was focussed on examining the effects
of novel CAPs on Escherichia coli NCTC 4174. Results will be
presented illustrating the effect of these novel CAPs on this
bacterium. From these results it will be evident that the
bactericidal effect of these polypeptides is not solely due to
membrane disruption. Cellular aggregation, gross deformation
of bacterial cells and inhibition of septation are some of the
effects observed.
Antibacterial minor groove binders – the
Strathclyde Solution
Professor Colin Suckling
Department of Pure and Applied Chemistry, University of Strathclyde,
Glasgow, UK.
The natural products distamycin and netropsin, which are
oligoamides of pyrrole amino acids that bind to the minor
groove of DNA, have been known for 50 years to have
antibacterial and antiviral activity. Only in the last 10 years,
however, have derivatives been obtained with sufficient
selectivity to be serious candidates for preclinical drug
development. For bacterial infections, we have discovered a
novel class of minor groove binder for DNA with potent
antibacterial activity against Gram positive bacteria (MIC ~ 
mol) and remarkably low toxicity to mammalian cells. Some
compounds are also active against Gram negative bacteria but
are less potent. The key strategy in developing these
compounds was to increase the hydrophobic content of the
minor groove binder to promote increased hydrophobic
interactions with the wall of the minor groove of double
stranded DNA. This has been achieved by introducing branched
alkyl substituents in place of methyl groups and alkenes in
place of amides. Not all substitutions lead to active compounds.
With the aid of spectroscopic and physical methods, we have
identified the structural requirements for binding to DNA; these
features also correlate with antibacterial activity. However
available information does not allow us to conclude that binding
to bacterial DNA is the only component of their mechanism of
action. This presentation will introduce the structures of our
compounds in the context of their biological properties and
describe what we know of their mechanism of action.
2-Aminothiazole-4-carboxylates (ATCs)
towards a potential new class of antituberculosis drug.
Dr Geoff Coxon
Institute of Pharmacy and Biomedical Sciences, University of
Strathclyde, The John Arbuthnott Building, 27 Taylor Street, Glasgow,
G4 0NR, UK.
Tuberculosis (TB) is a disease which kills two million people
every year and infects approximately over one-third of the
world's population. The difficulty in managing tuberculosis is the
prolonged treatment duration, the emergence of drug
resistance and co-infection with HIV/AIDS. Tuberculosis control
requires new drugs that act at novel drug targets to help
combat resistant forms of Mycobacterium tuberculosis and
reduce treatment duration. Our approach was to modify the
naturally occurring and synthetically challenging antibiotic
thiolactomycin (TLM) to the more tractable 2-aminothiazole-4carboxylate scaffold to generate compounds that mimic TLM's
novel mode of action. We report here the identification of a
series of compounds possessing excellent activity against M.
tuberculosis H37Rv and, dissociatively, against the β-ketoacyl
synthase enzyme mtFabH which is targeted by TLM.
Specifically, methyl 2-amino-5-benzylthiazole-4-carboxylate
was found to inhibit M. tuberculosis H37Rv with an MIC of 0.06
µg/ml (240 nM), but showed no activity against mtFabH,
whereas methyl 2-(2-bromoacetamido)-5-(3-chlorophenyl)t
hiazole-4-carboxylate inhibited mtFabH with an IC50 of
0.95±0.05 µg/ml (2.43±0.13 µM) but was not active against
the whole cell organism. These findings clearly identify the 2aminothiazole-4-carboxylate scaffold as a promising new
template towards the discovery of a new class of antitubercular agents.
1+1≠2
Novel Synergistic Antibacterials
Dr Cedric Charrier
C. Charrier, D.K. Mercer, S. Robertson, D. O’Neil.
NovaBiotics Ltd, Cruickshank Building, Craibstone, Aberdeen, AB21
9TR, UK
Objectives
The ever-increasing emergence and spread of multi-drug
resistant bacteria and the recurrence of microbial biofilm
infections is a serious concern for the treatment of all infectious
diseases, such as respiratory tract and skin infections. To
address this unmet medical need for safe and effective
therapeutics, NovaBiotics is developing novel antibacterials that
have the potential for activity against all cell types encountered
in bacterial biofilms, namely planktonic, sessile and persister
cells. The technology, originally based on naturally-occurring
antimicrobial peptides, has been developed to display a low
toxicity profile, a low risk for antimicrobial resistance
development and is compatible with currently available
therapies and anti-biofilm agents.
Results
The cationic antimicrobial peptide NP108 and the mucolytic
agent NM001 demonstrated synergistic activity against a
number of bacterial species responsible for causing skin and
respiratory tract infections. These synergistic compounds
demonstrated antibacterial activity against planktonic, sessile
as well as persister cells, which are the main cause of infection
recurrence. Additionally, this novel antimicrobial combination
demonstrated low cytotoxicity and haemolytic activity along
with a low risk of resistance development.
Conclusion
The synergistic activity of NovaBiotics novel antimicrobials
against the various cell types encountered in microbial biofilms
represents a novel therapeutic option to address the unmet
need for effective and safe treatments for the eradication of
microbial biofilms.
Spinach leaves, butterfly wings, nutmeg oil
and antiparasitic drugs
Professor Alan H. Fairlamb
Division of Biological Chemistry & Drug Discovery, College of Life
Sciences, University of Dundee, DD1 5EH.
New drugs are urgently required for the treatment of many
killer parasitic diseases, which, for economic reasons, are
“neglected” by the pharmaceutical industry. In 2006, the Drug
Discovery Unit was established in Dundee to fill the
translational gap between discovery and validation of novel
drug targets through to development of clinical drug candidates
for African sleeping sickness, visceral leishmaniasis and Chagas’
disease. The specific goal is to deliver at least one preclinical
candidate for human African trypanosomiasis by March 2011.
Some successes and failures, challenges and lessons learned
will be illustrated with enzyme targets related to metabolites
originally identified in spinach leaves, butterfly wings and
nutmeg oil.
Genetic and genomic identification of drug
resistance pathways in malaria
Dr Paul Hunt
Centre for Immunity, Infection and Evolution, University of Edinburgh,
Ashworth Laboratory, Kings Buildings, Edinburgh, EH9 3JT
Drug resistance continues to exert a major impact on the
treatment and control of malaria. A knowledge of the genetic
changes conferring drug resistance is essential for optimising
drug delivery and disease control.
A rapid proactive
identification of relevant genetic markers might aid efforts to
minimise the evolution of resistance.
One approach combines experimental evolution to generate
drug-resistant mutant parasites with whole genome resequencing to identify the mutations. We have used the rodent
malaria Plasmodium chabaudi to generate a lineage of mutant
parasites with in vivo resistance to a number of drugs and their
combinations. A comprehensive inventory of the mutations
arising in this lineage has been defined, using the Solexa
platform.
Importantly, the functional significance of these mutations can
be evaluated using a whole-genome genetics approach, called
Linkage Group Selection.
This has the features of a
quantitative genome-wide scan of selection, multiple natural
transfection and Quantitative Trait Loci analysis. In these
ways, the genetic architecture of resistance to drugs can be
elucidated.
I will describe how this model system has been used to
determine the precise mutations conferring resistance to
artemisinin, chloroquine, mefloquine and anti-folates, and
indicate possible novel pathways of parasite adaptation to
drugs.
Poster Presentations
The X-ray crystal structures of PfIspF and
PaPabC: Targets for the discovery of antimicrobial agents.
1
P.E.F. O’Rourke1, T.C. Eadsforth1, P.K. Fyfe1, J.
Kalinoskwa-Tlusckic1 and W.N. Hunter1
Division of Biological Chemistry and Drug Discovery, University of
Dundee, Wellcome Trust Building, Dow Street, Dundee, Scotland, DD1
5EH
The X-ray crystal structures of two essential microbial enzymes
were solved to assist early-stage drug discovery. The enzymes
selected were IspF (2C-methyl-D-erythritol cyclodiphosphate
synthase) from Plasmodium falciparum and PabC (4-amino-4deoxychorismate lyase) from Pseudomonas aeruginosa. IspF is
involved in isoprenoid biosynthesis and PabC is part of the
folate biosynthetic pathway. The proteins were recombinantly
expressed (E. coli) and suitable crystallisation conditions were
identified using an automated liquid-handling system.
Optimisation of these initial conditions produced crystals that
diffracted x-rays. Screening chemical fragment libraries against
these proteins is in progress. Any fragments that are identified
will be used in co-crystallisation trials.
Antimicrobial activity of Bog myrtle (Myrica
Gale) essential oil against Staphylococcus
epidermidis and Staphylococcus aureus.
1
N. Picamilho-Rua1, D. Hogan2, A.J. Lamb2 and M.C.E.
McFadyen2
Universite Blaise Pascal, Polytech Clermont-Ferrand, 63173 Aubiere,
France. 2School of Pharmacy and Life Sciences, Robert Gordon
University, Schoolhill, Aberdeen, AB10 1FR.
Bog myrtle (Myrica gale), an aromatic shrub widely distributed
in Northern wetlands is known to possess antimicrobial
properties. The essential oil component is a complex mixture of
mono and sesquiterpenes, with considerable variation in
chemical composition between different populations. The aim of
this study was to assess the antibacterial activity of European
and North American essential oil of Myrica gale against the
Gram positive bacteria Staphylococcus epidermidis NCTC 8558
and Staphylococcus aureus NCTC 6571 (MSSA).
Broth
macrodilution and microdilution assays were performed to
establish bacterial susceptibility to the antimicrobial agents.
Potential synergy between the antimicrobials was evaluated via
the chequerboard method and broth macrodilution antimicrobial
combination studies. Antimicrobial activity of Myrica gale oil
vapour against MSSA was investigated via a vapour activity
assay.
Myrica gale inhibited S. epidermidis in the broth
macrodilution assay, with minimum inhibitory concentration
(MICs) of 0.25 % v/v and 0.5 %v/v for the European and North
American oils respectively. Both oils had MICs of 1% v/v in the
broth microdilution assay. Combining chlorhexidine with Myrica
gale produced neither synergy nor antagonism against S.
epidermidis.
Myrica gale, inhibited MSSA in the broth
microdilution assay with MICs of 0.5% v/v and 0.125% v/v for
the European and North American oils respectively. In addition,
clear inhibition of growth was observed with the vapour phase
of Myrica gale against MSSA.
These finding highlight the
potential of Myrica gale essential as putative novel topical
antimicrobial against S. epidermidis and MSSA.
Antimicrobial effect of Patchouli essential oil
(Pogostemon cablin), alone and in
combination with chlorhexidine.
1
D. Hogan1, A.J. Lamb1 and M.C.E. McFadyen1
School of Pharmacy and Life Sciences, Robert Gordon University,
Schoolhill, Aberdeen, AB10 1FR.
Essential oils are naturally occurring organic compounds used
since the middle ages for their antibacterial, antiviral and
antifungal properties. The aim of this current study was to
investigate the antimicrobial activity of one such oil patchouli
(Pogostemon cablin), alone and in combination with a
conventional antimicrobial; chlorhexidine, against S.epidermidis
8558. The antimicrobial activity of this combination therapy
against methicillin-resistant S. aureus 11940 (MRSA) was also
established. Broth macrodilution and microdilution assays were
performed to establish bacterial susceptibility to the
antimicrobial
agents.
Potential
synergy
between
the
antimicrobials was evaluated via the chequerboard method and
broth
macrodilution
antimicrobial
combination
studies.
Chlorhexidine demonstrated antimicrobial activity against
S.epidermidis and MRSA in broth macrodilution and
microdilution assays, yielding MIC (minimum inhibitory
concentration) values of 0.5 mg/ml against S. epidermidis and
2mg/ml against MRSA. Patchouli exhibited MICs of 0.031% v/v
and 0.125% v/v against S. epidermidis, in the macrodilution
and microdilution assays, respectively, and an MIC of 0.5% v/v
against MRSA in the microdilution assay. MICs of chlorhexidine
and patchouli oil were reduced when the two were combined
against S. epidermidis (MIC of 0.5mg/ml reduced to 0.25mg/ml
and MIC of 0.031% v/v reduced to 7.8 x 10-3 % v/v, for
chlorhexidine and patchouli oil, respectively). These findings
highlight the potential use of patchouli alone and in combination
with chlorhexidine as a novel topical antimicrobial agent against
S. epidermidis and MRSA.
In vitro antibacterial evaluation of synthetic
flavone analogues against quinolone- and
methicillin-resistant Staphylococcus aureus.
1
E. Medu1, R. Brown2 and A.J. Lamb1
School of Pharmacy and Life Sciences, Robert Gordon University,
Schoolhill, Aberdeen. AB10 1FR. 2School of Chemistry, University of
Southampton, Highfield, Southampton. SO17 1BJ.
Objectives: This study was undertaken to screen for in vitro
antibacterial activity of a series of synthetic flavones and
chromones, and examine any potential synergism in
combination with polymyxin.
Methods: Microbroth MIC/MBC determination and modified agar
dilution methods were used to screen the compounds for in
vitro antibacterial activity. A chequerboard technique and timekill assays were used to examine synergism with polymyxin.
Result: The compound, 2-(cyclohexylmethyl)-6-methoxy-4Hchromen-4-one (F1), gave MIC/MBC value of 32µg/ml against
quinolone-resistant Staphylococcus aureus (QRSA) and MIC and
MBC values of 128 and 512µg/ml against methicillin-resistant
Staphylococcus aureus (MRSA). When evaluated using
chequerboard technique, MIC/MBC of F1 reduced to 8 and
16µg/ml against QRSA and MRSA respectively. The
chequerboard combination reduced MIC/MBC of polymyxin
against both species from 128 to 32µg/ml. In time-kill viability
assays, either 32µg/ml (MIC) F1 or 128µg/ml (MIC) polymyxin
demonstrated bacteriostatic activity with 2 log reductions in
density of exposed bacterial population within 24 hours.
Increased F1 concentration up to 32 × MIC maintained
bacteriostatic activity. But, combination of 32µg/ml (MIC) F1
with 128µg/ml (MIC) polymyxin was bactericidal with 7 log
reductions of exposed bacterial population within 8 hours.
Conclusion: The antibacterial potency of 2-(cyclohexylmethyl)6-methoxy-4H-chromen-4-one and synergism exhibited with
polymyxin demonstrate this compound to have potential
antibacterial activity against these resistant staphylococcal
species.
Antifungal effect of lactic acid bacteria on
Rhizopus stolonifer
1
A.D. Bayliss1 and E. Cowie1
School of Pharmacy and Life Sciences, Robert Gordon University, St
Andrews Street, Aberdeen, UK, AB25 1HG.
The ability of lactic acid bacteria (LAB) to inhibit the main
fungal contaminants of bread including Aspergillus, Fusarium
and Penicillium, has previously been reported. Introduction of
antifungal LAB isolated from these studies into bread dough
allowed for a reduction of the preservative calcium propionate
by 50%. An improvement in bread volume was also found.
The aim of this study was to identify LAB with antifungal
activity against Rhizopus stolonifer (R. stolonifer) which could
potentially be used in the preservation of bakery products. A
total of 19 presumptive LAB isolates (Gram-positive and
catalase-negative) were obtained from 8 samples of
commercially available flours. Antifungal activity was analysed
by testing the bacteria against target mould using the dualoverlay assay method.
Nine LAB strains from culture
collections were also tested. Results indicated low inhibitory
activity of 10 isolates and 5 LAB strains against conidial
germination (no fungal growth on 0.1-3% of the plate area per
bacterial streak). One isolate and 1 LAB strain (Lactobacillus
cremoris NIZO B40) exhibited medium inhibitory activity (no
fungal growth on 3-8% of the plate area per bacterial streak).
Lactobacillus cremoris NIZO B40 has not previously been
reported to have antifungal activity against this mould species.
The strain related antifungal ability of LAB isolates was
demonstrated here.
LAB may have a role in maintaining
microbial safety and shelf-life whilst reducing the need for
chemical preservation of baked products. Future work will
include further characterising LAB isolates and the antifungal
agents they produce. Isolates of LAB could be incorporated into
bread production and the preservation effect measured.
Structural confirmation by NMR of 3-Ooctanoyl-(-)-epicatechin, a novel
antibacterial
1
A. Di Salvo1, E. Medu1 and A.J.Lamb1
School of Pharmacy and Life Sciences, The Robert Gordon University,
Schoolhill, Aberdeen, AB10 1FR.
Recent widespread reports of high bacterial resistance to
common antimicrobial agents have resulted in studies aimed at
the production of more sophisticated molecular agents to
address the problem. 3-O-octanoyl-(-)-epicatechin is the semi
synthetic derivative of a well know flavonoid with antibacterial
properties. Acylation of the hydroxyl group in C6 position has
been preliminarily associated with anchoring of the derivative
onto the bacterial cell surface, resulting in more efficient
biological activity. Therefore the need has arisen to streamline
the synthetic procedure and obtain viable quantities of the
compound for mechanistic antimicrobial studies. This work
presents full structural elucidation of the molecular target by
NMR for quality checking purposes and to extend the knowledge
base of this class of compounds for further synthetic
development.
Investigation of the activity of Triclosan on
growth, morphology and cell integrity of
bacteria
1
N. O’Driscoll1, K. Matthews1 and A. J. Lamb1
School of Pharmacy and Life Sciences, Robert Gordon University,
Schoolhill, Aberdeen, AB10 1FR.
Triclosan (2,4,4`-trichloro-2`-hydroxydiphenyl ether) is a broad
spectrum antibacterial agent known to act upon enoyl-ACPreductase as the primary target thereby inhibiting fatty acid
synthesis. This investigation was undertaken to further examine
the antibacterial activity of Triclosan. Bacteriostatic and
bactericidal effects of Triclosan were established by measuring
viability of E. coli NCTC 4174 and S. aureus NCTC 6571 using
standard microbroth assays. The effect of antimicrobial
exposure on bacterial viability was also examined by flow
cytometry from cultures treated with propidium iodide and Syto
9.
Scanning
electron
microscopy
monitored
bacterial
morphology for possible changes manifest by bacteria in
response to Triclosan incubation. Damage to the cytoplasmic
membrane was examined by measuring amount of internal
potassium lost. Triclosan inhibits onset of the log phase of
bacterial growth in a concentration dependent manner.
Significant changes in dynamics of bacterial growth, dependant
on length of exposure to/concentration of Triclosan, were
identified via flow cytometry. Incubation of E. coli or S. aureus
with both sub-MIC and MIC of Triclosan revealed, via SEM,
presence of cellular aggregates. Incubation of bacteria with
Triclosan induced minimal potassium loss, suggesting that
structural integrity of the bacterial membrane is not
compromised by this agent. Whilst previous investigations have
reported Triclosan to induce potassium loss, our investigation
clearly demonstrates potassium leakage does not occur, even
at 100xMIC. This data confirms the finding of Villalain et al
(2001) that structural integrity of the membrane is not
compromised by Triclosan.
Modified Franz diffusion for the in vitro
determination of the efficacy of antimicrobial
wafers against methicillin-resistant
Staphylococcus aureus (MRSA)
1
O. Labovitiadi1, K. H. Matthews1 and A.J. Lamb1
School of Pharmacy and Life Sciences, Robert Gordon University,
Schoolhill, AB10 1FR, Aberdeen, UK
Antimicrobial wafers offer great potential for sustained topical
drug delivery directly to an infected wound bed. It is necessary
to verify the efficacy of these lyophilised delivery systems,
containing a range of broad spectrum antimicrobial compounds,
against methicillin-resistant Staphylococcus aureus (MRSA).
Lyophilised wafers were produced by casting karaya gels
containing clinical concentrations of either neomycin sulphate
(NS), povidone iodine (PVP-I), chlorhexidine digluconate (ChD)
or silver sulfadiazine (SS) into polystyrene plates and freezedried. The effect of the released antimicrobials against MRSA
was determined in vitro using a modified Franz diffusion cell.
The dissolution medium of the receptor chamber was inoculated
with 5×105 cfu/mL and antimicrobial wafers placed on top of a
cellulose membrane (12-14 kDa) in contact with the inoculated
medium. Time course samples were diluted to subinhibitory
levels and plated onto nutrient agar plates.
Activity was
examined in relation to an established disc diffusion method.
Compared to control wafer MRSA population density decreased
dramatically over a 24-hour period. PVP-I and ChD displayed a
four-log reduction of MRSA within 5-6 hrs and 7-8 hours
respectively, whereas NS took between 8 and 24 hours. The
insoluble nature of SS limited its passage through the
membrane from wafer to dissolution medium. In conclusion, the
release of antimicrobials from impregnated lyophilised wafers
has been clearly demonstrated . Such antimicrobial release has
a profound effect upon the viability of planktonic MRSA. The
modified Franz diffusion cell represented an effective in vitro
method, compared to disc diffusion, for evaluating efficacy of
antimicrobial wafers.